What is low testosterone (male hypogonadism)?

Low testosterone (male hypogonadism) is a condition in which the testes (testicles, the male reproductive glands) do not produce enough testosterone (a male sex hormone).

In men, testosterone helps maintain and develop:

Low testosterone affects almost 40% of men aged 45 and older. It is difficult to define normal testosterone levels, because levels vary throughout the day and are affected by body mass index (BMI), nutrition, alcohol consumption, certain medications, age and illness.

As a man ages, the amount of testosterone in his body gradually drops. This natural decline starts after age 30 and continues (about 1% per year) throughout his life.

There are many other potential causes of low testosterone, including the following:

Symptoms of low testosterone depend on the age of person, and include the following:

Other changes that occur with low testosterone include:

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Low Testosterone (Male Hypogonadism) | Cleveland Clinic

Recommendation and review posted by simmons

Dear Just Wondering,

Hypogonadism and hypergonadism are syndromes that result from abnormal levels of testosterone and estrogen. They affect the reproductive systems in both sexes, permanantly causing the testes and ovaries to not function properly. Hypogonadism acts by lowering the production and quality of testosterone and sperm in men and estrogen and eggs in women. This imbalance in the body's chemistry can result in a lowered sex drive in both men and women. Hypogonadism can also cause infertility.

Hypogonadism can appear either before or after puberty. If it occurs before puberty, the symptoms can include:

Hypogonadism after puberty can cause:

Treatment of hypogonadism usually comes through hormone replacement therapy. In men, testosterone is replaced, and in women, estradiol (a precursor to estrogen) and progesterone. While this therapy has improved the chances of many couples trying to have children, it does not help everybody.

On the other end of the spectrum is hypergonadism. As you most likely guessed, those with hypergonadism have higher levels of testosterone and estrogen in their systems. While this may sound great for the dating scene, the extra hormones are not as fun as they may seem.

Hypergonadism is rarer than hypogondism. But, like hypogonadism it can appear either before or after puberty.

Hypergonadism occuring before puberty actually prods puberty into action. After puberty those diagnosed exhibit the same affects as the prepubescent. Hypergonadism causes the same changes in both men and women, including:

Like hypogonadism, hormonal treatments are needed to correct hypergonadism. But since there are higher levels of estrogen and testosterone coursing through the body, a delicately balanced hormonal cocktail is needed. Treating hypergonadism is much more difficult because it is tougher to lower an excess of hormones than it is to add them to the body.

The latest research points towards many different sources as the cause of hypogonadism and hypergonadism in both males and females including:

It appears that the best course of action for treating both hypogonadism and hypergonadism is hormonal therapy. An endocrinologist, a medical provider specializing in the body's hormones, can make certain that hormonal balance is achieved in the safest possible way.

Read more:
Hypogonadism? | Go Ask Alice!

Recommendation and review posted by Bethany Smith

Common baldness in women, also called female pattern alopecia, is genetically inherited and can come from either the mothers or fathers side of the family. Female alopecia most commonly presents in a diffuse pattern, where hair loss occurs over the entire scalp. Less commonly, women exhibit a patterned distribution where most of the thinning occurs on the front and top of the scalp with relative sparing of the back and sides.

The type of hair loss, diffuse or patterned, has important implications for treatment. Women with diffuse hair loss are generally best treated medically, whereas women with patterned hair loss may be good candidates for hair transplant surgery. Interestingly, patterned hair loss is the most common type seen in men and accounts for why a greater proportion of men are candidates for surgery compared to women.

In women who are genetically predisposed to hair loss, both diffuse and patterned distributions are caused by the actions of two enzymes: aromatase (which is found predominantly in women) and 5-a reductase (which is found in both women and men). Diffuse hair loss is most often hereditary, but it can also be caused by underlying medical conditions, medications, and other factors; therefore, a thorough medical evaluation is an important part of the management.

In the next sections, we will take a closer look at both the mechanisms of genetically induced female hair loss as well as the medical conditions and drugs that can cause diffuse hair loss in women.

As with hair loss in men, female genetic hair loss largely stems from a complex stew of genes, hormones, and age. However, in women, there are even more players. In addition to 5-a reductase, testosterone, and dihydrotestosterone (DHT); which are also found in mens hair loss; also present in women are the enzyme aromatase and the female hormones estrone and estradiol. So lets break down the process that leads to common hair loss in women.

In both men and women, 5-a reductase reacts with testosterone to produce DHT, the hormone responsible for the miniaturization (shrinking) and the gradual disappearance of affected hair follicles. This explains why both men and women lose their hair. But one of the reasons why women seldom have the conspicuous bald areas that men do is because women naturally have only half the amount of 5-a reductase compared to men.

Adding to this complexity, in women, the enzyme aromatase is responsible for the formation of the female hormones, estrone, and estradiol, counteract the action of DHT. Women have higher levels of aromatase than men, especially at the frontal hairline. It is this presence of aromatase which may help explain why hair loss in women looks so different than in men, particularly with respect to the preservation of the frontal hairline. It may also explain why women have a poor response to the drug finasteride (Propecia), a medication widely used to treat hair loss in men that works by blocking the formation of DHT.

The following is a schematic chart of how the female hormones estrone and estradiol are produced and their relationship to DHT:

Womens hair seems to be particularly sensitive to underlying medical conditions. Since systemic medical conditions often cause a diffuse type of hair loss pattern that can be confused with genetic balding, it is important that women with undiagnosed alopecia be properly evaluated by a doctor specializing in hair loss (i.e., a dermatologist).

Below is a list of medical conditions that can lead to a diffuse pattern of hair loss:

A relatively large number of drugs can cause telogen effluvium, a condition where hair is shifted into a resting stage and then several months later shed. Fortunately, this shedding is reversible if the medication is stopped, but the reaction can be confused with genetic female hair loss if not properly diagnosed. Chemotherapy and radiotherapy can cause a diffuse type of hair loss called anagen effluvium that can be very extensive. This hair loss is also reversible when the therapy is over, but the hair does not always return to its pre-treatment thickness.

Causes of Telogen Effluvium

Causes of Anagen Effluvium

A host of dermatologic conditions can cause localized hair loss in women. The pattern that they produce is usually quite different from the diffuse pattern of female genetic hair loss and is easily differentiated from it by an experienced dermatologist. Occasionally, the diagnosis is difficult to make and tests, such as a scalp biopsy are necessary.

Localized hair loss in women may be sub-divided into scarring and non-scarring types.

Non-Scarring Alopecias

Alopecia Areata is a genetic, auto-immune disease that typifies the non-scarring type. It manifests with the sudden onset of discrete, round patches of hair loss associated with normal underlying skin. It usually responds quite well to local injections of corticosteroids.

Localized hair loss can be also be caused by constant pulling on scalp hair, either through braiding, tight clips or hair systems. Traction alopecia, the medical term for this condition, often causes reversible thinning but, if the tugging on the follicles persists for an extended period of time, the hair loss can be permanent. The most common presentation is thinning, or complete hair loss, at the frontal hairline and in the temples of women who wear their hair pulled tightly back. Early traction alopecia can reverse itself by simply wearing the hair loose. A hair transplant may be needed to restore the hair that is permanently lost from sustained traction.

Scarring Alopecias

Scarring hair loss can be caused by a variety of medical or dermatologic conditions such as Discoid Lupus, Lichen Planus, and infections. It can also be caused by thermal burns or local radiation therapy. Face-lift surgery may result in permanent localized hair loss that can be particularly bothersome if it occurs at the frontal hairline or around the temples. Fortunately, localized hair loss from injury or from medical problems are often amenable to hair transplantation.

Many of the factors that cause the rate of loss to speed up or slow down are unknown, but we do know that with age, a persons total hair volume will decrease. This is referred to as senile alopecia. Even when there is no predisposition to genetic balding, hair across the entire scalp will thin over time resulting in the appearance of less density. The age at which these effects finally manifest themselves varies from one individual to another and is mainly related to a persons genetic makeup.

See the article here:
Causes of Hair Loss in Women | Bernstein Medical

Recommendation and review posted by simmons

Generating a Knockout Using CRISPR

You can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cpf1 and a gRNA specific to the gene to be targeted. The genomic target can be any 20 nucleotide DNA sequence, provided it meets two conditions:

The PAM sequence is essential for target binding, but the exact sequence depends on which Cas protein you use. We'll use the popular S. pyogenes Cas9 (SpCas9) as an example, but check out our list of additional Cas proteins and PAM sequences. Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation. Importantly, the spacer region of the gRNA remains free to interact with target DNA.

Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3 end of the gRNA targeting sequence) will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3 to 5 direction. The zipper-like annealing mechanics of Cas9 may explain why mismatches between the target sequence in the 3 seed sequence completely abolish target cleavage, whereas mismatches toward the 5 end distal to the PAM often still permit target cleavage.

The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (3-4 nucleotides upstream of the PAM sequence).

The resulting DSB is then repaired by one of two general repair pathways:

The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations (for more information, jump to Plan Your Experiment.) In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. However, the strength of the knockout phenotype for a given mutant cell must be validated experimentally. Learn more about non-homologous end joining (NHEJ).

Browse Plasmids: Double-Strand Break (Cut)

CRISPR specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in the genome. Realistically, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing a gRNA for your experiment (see the Plan Your Experiment section below).

In addition to optimizing gRNA design, CRISPR specificity can also be increased through modifications to Cas9. As discussed previously, Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB.

Thus, two nickases targeting opposite DNA strands are required to generate a DSB within the target DNA (often referred to as a double nick or dual nickase CRISPR system). This requirement dramatically increases target specificity, since it is unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. Therefore, if high specificity is crucial to your experiment, you might consider using the dual nickase approach to create a double nick-induced DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene edits.

In 2015, researchers used rational mutagenesis to develop two high fidelity Cas9s: eSpCas9(1.1) and SpCas9-HF1. eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9's interactions with the DNA phosphate backbone. HypaCas9, developed in 2017, contains mutations in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wildtype Cas9.

Browse Plasmids: Single-Strand Break (Nick)

While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag.

In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence must be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template must contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms.) The length of each homology arm is dependent on the size of the change being introduced, with larger insertions requiring longer homology arms.

Depending on the application, the repair template may be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double-stranded DNA plasmid. When designing the repair template, do not include the PAM sequence present in the genomic DNA. This step prevents the repair template from being a suitable target for Cas9 cleavage. For example, you could alter the DNA sequence of the PAM with a silent mutation that does not change the amino acid sequence.

The efficiency of HDR is generally low (<10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. For this reason, many laboratories try to enhance HDR by synchronizing the cells, since HDR takes place during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ may also increase HDR frequency.

Since the efficiency of Cas9 cleavage is relatively high and the efficiency of HDR is relatively low, a large portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, the resulting population of cells will contain some combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele. Therefore, it is important to confirm the presence of the desired edit experimentally and to isolate clones containing the desired edit (see the validation section in Plan Your Experiment). Learn more about homology directed repair (HDR).

Browse Plasmids: Endogenous Tagging

As discussed above, the efficiency of HDR is very low due to the number of DSBs repaired by NHEJ. To make point mutations without using HDR, researchers have developed CRISPR base editors that fuse Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC1. Base editors are targeted to a specific locus by a gRNA, and they can convert cytidine to uridine within a small editing window near the PAM site. Uridine is subsequently converted to thymidine through base excision repair, creating a C->T change (or G->A on the opposite strand.) This class of base editors is available with multiple Cas9 variants and using high fidelity Cas9s. In addition, new base editors have been engineered to convert adenosine to inosine, which is treated like guanosine by the cell, creating an A->G (or T->C) change. Learn more about CRISPR DNA base editors.

Browse Plasmids: Base Edit

Type VI CRISPR systems, including the enzymes Cas13a/C2c2 and Cas13b, target RNA rather than DNA. Fusing an ADAR2(E488Q) adenosine deaminase to catalytically dead Cas13b creates a programmable RNA base editor that converts adenosine to inosine in RNA (termed REPAIR.) Since inosine is functionally equivalent to guanosine, the result is an A->G change in RNA. dPspCas13b does not appear to require a specific sequence adjacent to the RNA target, making this a very flexible editing system. Editors based on ADAR2(E488Q/T375G) display improved specificity, and editors carrying the delta-984-1090 ADAR truncation retain RNA editing capabilities and are small enough to be packaged in AAV particles.

Browse Plasmids: RNA Editing

CRISPR is a remarkably flexible tool for genome manipulation, as Cas enzymes bind target DNA independently of their ability to cleave target DNA. Specifically, both RuvC and HNH nuclease domains can be rendered inactive by point mutations (D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule retains the ability to bind to target DNA based on the gRNA targeting sequence.

Early experiments demonstrated that targeting dCas9 to transcription start sites was sufficient to repress transcription by blocking initiation. dCas9 can also be tagged with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcriptional repression or activation of downstream target genes. The simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator, A (e.g. VP64) or transcriptional repressor, R (e.g. KRAB; see panel A to the right).

Additionally, more elaborate activation strategies have been developed for more potent activation of target genes in mammalian cells. These include: co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g. SunTag system, panel B), dCas9 fused to several different activation domains in series (e.g. dCas9-VPR, panel C) or co-expression of dCas9-VP64 with a modified scaffold gRNA and additional RNA-binding helper activators (e.g. SAM activators, panel D). Importantly, unlike the genome modifications induced by Cas9 or Cas9 nickase, dCas9-mediated gene activation or repression is reversible, since it does not permanently modify the genomic DNA.

Browse Plasmids: Activate, Repress/Interfere

Cas enzymes can be fused to epigenetic modifiers like p300 and TET1 to create programmable epigenome-engineering tools. Like CRISPR activators and repressors, these tools alter gene expression without inducing double-strand breaks. However, they are much more specific for particular chromatin and DNA modifications, allowing researchers to isolate the effects of a single epigenetic mark.

Another potential advantage of CRISPR epigenetic tools is their persistence and inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated/removed from the system. In contrast, epigenetic marks left by targeted epigenetic modifiers may be more frequently inherited by daughter cells. In certain cases, epigenetic modifiers may work better than activators/repressors in modulating transcription. However, since the effects of these tools are likely cell type- and context-dependent, it might be beneficial to try multiple CRISPR strategies when setting up your experimental system.

Browse Plasmids: Epigenetics

Expressing several gRNAs from the same plasmid ensures that each cell containing the plasmid will express all of the desired gRNAs, thus increasing the likelihood that all desired genomic edits will be carried out by Cas9. Such multiplex CRISPR applications include:

Current multiplex CRISPR systems enable researchers to target anywhere from 2 to 7 genetic loci by cloning multiple gRNAs into a single plasmid. These multiplex gRNA vectors can conceivably be combined with any of the aforementioned CRISPR derivatives to not only knock out target genes, but activate or repress target genes as well. Read more about Cas9 multiplexing and Cpf1 multiplexing.

Browse Plasmids: Multiplex gRNA vectors

The ease of gRNA design and synthesis, as well as the ability to target almost any genomic locus, make CRISPR the ideal genome engineering system for large-scale forward genetic screening. Forward genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known. In general, the goal of a genetic screen is to generate a large population of cells with mutations in a wide variety of genes and then use these mutant cells to identify the genetic perturbations that result in a desired phenotype.

Before CRISPR, genetic screens relied heavily on shRNA technology, which is prone to off-target effects and false negatives due to incomplete knockdown of target genes. In contrast, CRISPR is capable of making highly specific, permanent genetic modifications that are more likely to ablate target gene function. CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis. Currently, the most popular method for conducting genome-wide screens using CRISPR involves the use of pooled lentiviral CRISPR libraries.

Pooled lentiviral CRISPR libraries (often referred to as CRISPR libraries) are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.

Guide RNAs are designed in silico and synthesized (see panel A below), then cloned in a pooled format into lentiviral transfer vectors (panel B). CRISPR libraries have been designed for common CRISPR applications including genetic knockout, activation, and repression for human and mouse genes.

Each CRISPR library is different, as libraries can target anywhere from a single class of genes up to every gene in the genome. However, there are several features that are common across most CRISPR libraries. First, each library typically contains 3-6 gRNAs per gene to ensure modification of every target gene, so CRISPR libraries contain thousands of unique gRNAs targeting a wide variety of genes. Guide RNA design for CRISPR libraries is usually optimized to select for guide RNAs with high on-target activity and low off-target activity.

Keep in mind that the exact region of the gene to be targeted varies depending on the specific application. For example, knockout libraries often target 5 constitutively expressed exons, but activation and repression libraries will target promoter or enhancer regions. Be sure to check the library page/original publication to see if a library is suitable for your experiment. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.

CRISPR libraries from Addgene are available in two formats: as DNA, or in select cases, as pre-made lentivirus.

In the case of DNA libraries, the CRISPR library will be shipped at a concentration that is too low to be used in experiments. Thus, the first step in using your library is to amplify the library (panel C) to increase the total amount of DNA. When amplifying the library, it is important to maintain good representation of gRNAs so that the composition of your amplified library matches that of the original library. You'll use next-generation sequencing (NGS) to verify that this is the case. Learn more about library verification.

Once the library has been amplified/verified, the next step is to generate lentivirus containing the entire CRISPR library (panel D). Then, you will transduce cells with the lentiviral library (panel E). Remember - if you are using a 2-vector system, you will transduce cells that are already expressing Cas9. After applying your screening conditions, you will look for relevant genes (hits) using NGS technology. For more detail on using CRISPR for both positive and negative screens, see our pooled library guide.

As noted above, forward genetic screens are most useful for situations in which the physiology or cell biology behind a particular phenotype or disease is well understood, but the underlying genetic causes are unknown. Therefore, genome-wide screens using CRISPR libraries are a great way to gather unbiased information regarding which genes play a causal role in a given phenotype. With any experiment, it is important to verify that the hits you identify are actually important for your phenotype of interest. You can individually test the gRNAs identified in your screen to ensure that they reproduce the phenotype of interest.

Find more information on factors to consider before starting your pooled library experiment in Practical Considerations for Using Pooled Lentiviral CRISPR Libraries (McDade et al., 2016).

Browse Libraries: CRISPR Pooled Libraries

Using catalytically inactive Cas9 (dCas9) fused to a fluorescent marker like GFP, researchers have turned dCas9 into a customizable DNA labeler compatible with fluorescence microscopy in living cells. Alternatively, gRNAs can be fused to protein-interacting RNA aptamers, which recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize targeted genomic loci.

CRISPR imaging has numerous advantages over other imaging techniques, including that it is easy to implement due to the simplicity of gRNA design, programmable for different genomic loci, capable of detecting multiple genomic loci, and compatible with live cell imaging. Compared to techniques like fluorescence in situ hybridization (FISH), CRISPR imaging offers a unique method for detecting the chromatin dynamics in living cells.

Multicolor CRISPR imaging allows for simultaneous tracking of multiple genomic loci in living cells. One method uses orthogonal dCas9s (e.g., S. pyogenes dCas9 and S. aureus dCas9) tagged with different fluorescent proteins. Alternatively, one can fuse gRNAs to orthogonal protein-interacting RNA aptamers, which recruit specific orthogonal RNA-binding proteins (RBPs) tagged with different fluorescent proteins, as seen in the popular CRISPRainbow kit.

The fluorescent CRISPR system has been used for dynamic tracking of repetitive and non-repetitive genomic loci, as well as chromosome painting in living cells. Visualizing a specific genomic locus requires recruitment of many copies of labeled proteins to the given region. For example, chromosome-specific repetitive loci can be efficiently visualized in living cells using a single gRNA that has multiple targeted sequences in close proximity. A non-repetitive genomic locus can also be labeled by co-delivering multiple gRNAs that tile the locus. Chromosome painting requires delivery of hundreds of gRNAs with target sites throughout the chromosome.

Browse Plasmids: Label

Identifying molecules associated with a genomic region of interest in vivo is essential to understanding locus function. Using CRISPR, researchers have expanded chromatin immunoprecipitation (ChIP) to allow purification of any genomic sequence specified by a particular gRNA.

In the enChIP (engineered DNA-binding molecule-mediated ChIP) system, catalytically inactive dCas9 is used to purify genomic DNA bound by the gRNA. An epitope tag(s) can be fused to dCas9 or gRNA for efficient purification. Various epitope tags including 3xFLAG-tag, PA, and biotin tags, can be used for enChIP, as well as an anti-Cas9 antibody. Biotin tagging of dCas9 can be achieved by fusing a biotin acceptor site to dCas9 and co-expressing BirA biotin ligase, as seen in the CAPTURE system. The locus is subsequently isolated by affinity purification against the epitope tag.

After purification of the locus, molecules associated with the locus can be identified by mass spectrometry (proteins), RNA-sequencing (RNAs), and next-generation sequencing (NGS) (other genomic regions). Compared to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward and enable direct identification of molecules associated with a genomic region of interest in vivo.

Browse Plasmids: Purify

In bacteria, type VI CRISPR systems recognize ssRNA rather than dsDNA. Many type VI enzymes also have the ability to process crRNA precursors to mature crRNAs. Upon ssRNA recognition by the crRNA, the target RNA is degraded. In bacteria, Cas13 enzymes can also cleave RNAs nonspecifically after the initial crRNA-guided cleavage. This promiscuous cleavage activity slows bacterial cell growth and may further protect bacteria from viral pathogens. Non-specific cleavage does not occur in mammalian cells. Similar to Cas9 and Cpf1, Cas13 can be converted to an RNA-binding protein through mutation of its catalytic domain. Learn more about Cas13a.

Browse Plasmids: RNA Targeting

While S. pyogenes Cas9 (SpCas9) is certainly the most commonly used CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every application. For example, the PAM sequence for SpCas9 (5 NGG 3) is abundant throughout the human genome, but an NGG sequence may not be positioned correctly to target your desired genes for modification. This limitation is of particular concern when trying to edit a gene using homology directed repair (HDR), which requires PAM sequences in very close proximity to the region to be edited. Kleinstiver et al. generated synthetic SpCas9-derived variants with non-NGG PAM sequences. Gao et al. subsequently engineered Cpf1 PAM variants. The inclusion of these variants into the CRISPR arsenal effectively doubles the targeting range of CRISPR in the human genome. Read more about Cas9 variants.

Additional Cas9 orthologs from various species bind a variety of PAM sequences. These enzymes may have other characteristics that make them more useful than SpCas9 for specific applications. For example, the relatively large size of SpCas9 (4kb coding sequence) means that plasmids carrying the SpCas9 cDNA cannot be efficiently packaged into adeno-associated virus (AAV). Since the coding sequence for Staphylococcus aureus Cas9 (SaCas9) is 1 kilobase shorter than SpCas9, SaCas9 can be efficiently packaged into AAV. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo.

Another limitation of SpCas9 is the low efficiency of making specific genetic edits via HDR. For specific point edits, CRISPR base editing is a useful alternative to HDR. For larger edits, Cpf1, first described by Zetsche et al., may be a better option. Unlike Cas9 nucleases, which create blunt DSBs, Cpf1-mediated DNA cleavage creates DSBs with a short 3 overhang. Cpf1s staggered cleavage pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which may increase the efficiency of gene editing. Like the Cas9 variants and orthologs described above, Cpf1 also expands the range of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9.

CRISPR is a powerful system that enables researchers to manipulate the genome like never before. This section will provide a general framework to get you started using CRISPR in your research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR in other organisms. First, consider the genetic manipulation that is necessary to model your specific disease or process of interest. Do you want to:

Once you have a clear understanding of your experimental goal, you are ready to start navigating the different reagents that are available for your particular experiment.

Different genetic manipulations require different CRISPR components. Selecting a specific genetic manipulation can be a good way to narrow down which reagents are appropriate for a given experiment. Make sure to check whether reagents are available to carry out your experiment in your particular model organism. There may not be a perfect plasmid for your specific application, and in such cases, it may be necessary to customize an existing reagent to suit your needs.

To use CRISPR, you will need both Cas9 and a gRNA expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may be sufficient to express the CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may be more appropriate. In cases where off-target editing is a major concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression.

The table below summarizes the major expression systems and variables for using CRISPR in mammalian cells. Some of the variables include:

Once you have selected your CRISPR components and method of delivery, you are ready to select a target sequence and design your gRNA.

When possible, you should sequence the region you are planning to modify prior to designing your gRNA, as sequence variation between your gRNA targeting sequence and target DNA may result in reduced cleavage. The number of alleles for each gene may vary depending on the specific cell line or organism, which may affect the observed efficiency of CRISPR knockout or knockin.

In order to manipulate a given gene using CRISPR, you will have to identify the genomic sequence for the gene you are trying to target. However, the exact region of the gene you target will depend on your specific application. For example:

A PAM sequence is absolutely necessary for Cas9 to bind target DNA. As such, one can start by identifying all PAM sequences within the genetic region to be targeted. If there are no PAM sequences for your chosen enzyme within your desired sequence, you may want to consider alternative Cas enzymes (see Cas9 variants and PAM sequences). Once possible PAM sequences and putative target sites have been identified, it is time to choose which site is likely to result in the most efficient on-target cleavage.

The gRNA target sequence needs to match the target locus, but it is also critical to ensure that the gRNA target sequence does NOT match additional sites within the genome. In a perfect world, your gRNA target sequence would have perfect homology to your target with no homology elsewhere in the genome. Realistically, a given gRNA target sequence will have partial homology to additional sites throughout the genome. These sites are called off-targets and should be examined during gRNA design. In general, off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. To increase specificity, you can also consider using a high-fidelity Cas enzyme.

In addition to off-target activity, it is also important to consider factors that maximize cleavage of the desired target sequence or on-target activity. Two gRNA targeting sequences with 100% homology to their DNA targets may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. For example, gRNA targeting sequences containing a G nucleotide at position 20 (1 bp upstream of the PAM) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the target sequence.

Many gRNA design programs can locate potential PAM and target sequences and rank the associated gRNAs based on their predicted on-target and off-target activity (see gRNA design software). Additionally, many plasmids containing validated gRNAs are now available through Addgene. These plasmids contain gRNAs that have been used successfully in genome engineering experiments. Using validated gRNAs can save your lab valuable time and resources when carrying out CRISPR experiments. Read more about how to design your gRNA.

Browse Plasmids: Validated gRNAs

Once you have selected your target sequences it is time to design your gRNA oligos and clone these oligos into your desired vector. In many cases, targeting oligos are synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors).

Choose a delivery method that is compatible with your experimental system. CRISPR efficiency will vary based on the method of delivery and the cell type. Before proceeding with your experiment, it may be necessary to optimize your delivery conditions. Learn more about CRISPR delivery in mammalian systems.

Once you have successfully delivered the gRNA and Cas enzyme to your target cells, it is time to validate your genome edit. CRISPR editing produces several possible genotypes within the resulting cell population. Some cells may be wild-type due to either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA.

Edited cells may be homozygous or heterozygous for edits at your target locus. Furthermore, in cells containing two mutated alleles, each mutated allele may be different owing to the error-prone nature of NHEJ. In HDR gene editing experiments, most mutated alleles will not contain the desired edit, as a large percentage of DSBs are still repaired by NHEJ.

How do you determine that your desired edit has occurred? The exact method necessary to validate your edit will depend upon your specific application. However, there are several common ways to verify that your cells contain your desired edit, including but not limited to:

More information on each of these techniques can be found in our blog post CRISPR 101: Validating your Genome Edit.

The majority of the CRISPR plasmids in Addgenes collection are from S. pyogenes unless otherwise noted.

Engineered Cpf1 variants with altered PAM specificities. 2017. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F. Nat Biotechnol. 35(8):789-792. PMID: 28581492

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. 2017. Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA. Nature. 550(7676):407-410. PMID: 28931002

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. 2017. Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR. Sci Adv. 3(8):eaao4774. PMID: 28875174

Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. 2017. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, Liu DR. Nat Commun. 8:15790. PMID: 28585549

Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. 2017. Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Nat Biotechnol. 35(4):371-376. PMID: 28191901

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. 2017. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F. Nat Biotechnol. 35(1):31-34. PMID: 27918548

Nucleic acid detection with CRISPR-Cas13a/C2c2. 2017. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science. 356(6336):438-442. PMID: 28408723

Programmable base editing of AT to GC in genomic DNA without DNA cleavage. 2017. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. Nature. 551(7681):464-471. PMID: 29160308

RNA editing with CRISPR-Cas13. 2017. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. Science. pii: eaaq0180. PMID: 29070703

RNA targeting with CRISPR-Cas13. 2017. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F. . 550(7675):280-284. PMID: 28976959

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. 2016. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. Nature. 529(7587):490-5. PMID: 26735016

Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. 2016. Ma H, Tu LC, Naseri A, Huisman M, Zhang S, Grunwald D, Pederson T. Nat Biotechnol. . PMID: 27088723

Naturally occurring off-switches for CRISPR-Cas9. 2016. Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR. Cell. 167(7):1829-1838. PMID: 27984730

Practical Considerations for Using Pooled Lentiviral CRISPR Libraries. 2016. McDade JR, Waxmonsky NC, Swanson LE, Fan M. Curr Protoc Mol Biol. 115:31.5.1-31.5.13. PMID: 27366891

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. 2016. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Nature. 533(7603):420-4. PMID: 27096365

Rationally engineered Cas9 nucleases with improved specificity. 2016. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Science. 351(6268):84-8. PMID: 26628643

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells. 2015. Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Ct J, Doyon Y. Cell Rep. 13(3):621-33. PMID: 26456817

An updated evolutionary classification of CRISPR-Cas systems. 2015. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J, Backofen R, Koonin EV. Nat Rev Microbiol. 13(11):722-36. PMID: 26411297

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. 2015. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR. Nat Biotechnol. 33(1):73-80. PMID: 25357182

CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins. 2015. Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts BS, Mackiewicz M, Mendenhall EM, Myers RM. Genome Res. 25(10):1581-9. PMID: 26355004

Read this article:
Addgene: CRISPR Guide

Recommendation and review posted by simmons

Definition: Hypopituitarism is a condition caused by low levels of pituitary hormones.

Alternative Names: Pituitary insufficiency

Causes, incidence, and risk factors: The pituitary gland is a small structure that is located just below the brain. It is attached by a stalk to the hypothalamus, the area of the brain that controls its function.

The hormones secreted by the pituitary gland (and their functions) are:

In hypopituitarism, there is an absence of one or more pituitary hormones. Lack of the hormone leads to loss of function in the gland or organ that it controls. For example, loss of thyroid stimulating hormone leads to loss of function in the thyroid gland.

Hypopituitarism may be caused by tumors of the pituitary gland or hypothalamus, head trauma, brain tumor, radiation, brain surgery, stroke, subarachnoid hemorrhage (from a burst aneurysm), or infections of the brain and the tissues that support the brain. Occasionally, hypopituitarism is due to uncommon immune system or metabolic diseases, such as sarcoidosis, histiocytosis X, and hemochromatosis.

Hypopituitarism is also a rare complication following pregnancy, a condition called Sheehan's syndrome. The cause of this type of hypopituitarism is unknown.

Symptoms:

Note: Symptoms may develop slowly and may vary greatly, depending upon the severity of the disorder, the number of deficient hormones, and their target organs.

Additional symptoms that may be associated with this disease:

Signs and tests:

Diagnosis of hypopituitarism must confirm low hormone levels due to an abnormality of the pituitary gland. The diagnosis must also rule out disease of the organ affected by this hormone.

In some cases, one of the hormones produced by the pituitary may be elevated in the blood stream if a patient has a pituitary tumor which is producing an excessive amount of that hormone. The tumor itself may be crushing the rest of the cells of the pituitary, leading to low levels of other hormones.

Treatment: If hypopituitarism is caused by a tumor, treatment by surgical removal, with or without radiation therapy, may be indicated. Replacement of deficient hormones is often required even after successful treatment of a pituitary tumor.

Hormone therapy is needed to replace hormones that are no longer made by the organs under the control of the pituitary gland. These may include corticosteroids (cortisol), thyroid hormone, sex hormones (testosterone for men and estrogen for women), and growth hormone. Drugs are also available to treat associated infertility in men and women.

Support Groups:

Expectations (prognosis): Hypopituitarism is usually permanent and requires life-long treatment; however, a normal life span can be expected.

Complications: Side effects of drug therapy can develop.

Calling your health care provider: Call your health care provider if symptoms of hypopituitarism develop.

Prevention: In most cases, the disorder is not preventable. Awareness of risk may allow early diagnosis and treatment.

Read more:
Hypopituitarism | UCLA Health

Recommendation and review posted by sam

Between five and 10% of all cancers are hereditary, which means that changes (or mutations) in specific genes are passed from one blood relative to another. People who inherit one of these gene changes will have a higher risk of developing cancer at some point in their life. Genetic counseling can help people understand this risk.

Genetic counseling is not for everyone. In most cases, people who need genetic counseling fit into one of two groups.

Group one includes people who are cancer-free but, due to other medical conditions or family history, may have an increased risk for developing the disease. This includes people with:

Group two includes people who have a cancer diagnosis and want to learn if it is genetic. Not everyone with cancer needs genetic counseling, though. Instead, it is usually recommended for patients who have:

If you fit into one of these categories, it's a good idea to meet with a genetic counselor.

The first step to understanding your genetic cancer risk is a genetic counseling session. There are several steps to these sessions.

The genetic counselor will take your medical history, as well as a cancer-focused family tree going back generations. Based on this information, the counselor will discuss how your familys cancer history may be hereditary and what that means for you.

Genetic testsuse a patients blood sample to look for genetic mutations that may lead to an increased risk for some cancers. After the medical and family history review, the counselor will discuss whether genetic testing is right for you. You will also cover the ethical and legal issues of genetic testing. If the counselor recommends genetic testing, you will be given information about the appropriate test or tests.

Based on your family history and/or genetic test results, you will discuss ways to reduce your cancer risk. This discussion may cover cancer screening strategies, chemoprevention or even preventative surgery. You also may be referred to a high-risk screening clinic for further discussion and long-term cancer screening and monitoring.

Patients are often given the chance to join clinical research trials and registries. These can improve cancer care in many ways. For example, they can help doctors understand cancer risk factors and learn what screening and prevention methods work best.

Originally posted here:
Genetic Testing | MD Anderson Cancer Center

Recommendation and review posted by Bethany Smith

Aurora Hereditary Cancer Prevention and Management Center (HCPMC)

Have genetic counseling and DNA testing determined that you or your family members have a hereditary cancer syndrome? Families with hereditary cancer syndromes are at high risk for multiple types of cancer. Even families whose genetic testing results are normal may be at increased risk for multiple cancers if they have complex cancer histories.

If your family history of cancer has been determined to be hereditary, or if your complex family history cant be explained by genetic testing, you deserve comprehensive care from a multidisciplinary team of experts in a single, convenient location.

The Aurora Hereditary Cancer Prevention and Management Center (HCPMC) specializes in testing and monitoring individuals and families with complex or difficult hereditary cancer conditions.

Through the HCPMC, you can:

If youre at risk for multiple types of cancer, ask your doctor for a referral to the Aurora Hereditary Cancer Prevention and Management Center in Milwaukee or Green Bay.

The HCPMC in Milwaukee is located within the Vince Lombardi Cancer Clinic at St. Lukes Medical Center. The HCPMC in Green Bay is located within the Vince Lombardi Cancer Clinic at Aurora BayCare Medical Center.

Call 877-647-2502 for more information.

Continued here:
Genetic Counseling | DNA Testing | Aurora Health Care

Recommendation and review posted by Bethany Smith

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Recommendation and review posted by simmons

Medically reviewed on August 22, 2017

Hypopituitarism is a rare disorder in which your pituitary gland either fails to produce one or more of its hormones or doesn't produce enough of them.

The pituitary gland is a small bean-shaped gland situated at the base of your brain, behind your nose and between your ears. Despite its size, this gland secretes hormones that influence nearly every part of your body.

In hypopituitarism, you have a short supply of one or more of these pituitary hormones. This deficiency can affect any number of your body's routine functions, such as growth, blood pressure and reproduction.

You'll likely need medications for the rest of your life to treat hypopituitarism, but your symptoms can be controlled.

Hypopituitarism is often progressive. Although the signs and symptoms can occur suddenly, they more often develop gradually. They are sometimes subtle and may be overlooked for months or even years.

Signs and symptoms of hypopituitarism vary, depending on which pituitary hormones are deficient and how severe the deficiency is. They may include:

See your doctor if you develop signs and symptoms associated with hypopituitarism.

Contact your doctor immediately if certain signs or symptoms of hypopituitarism develop suddenly or are associated with a severe headache, visual disturbances, confusion or a drop in blood pressure. Such signs and symptoms could represent sudden bleeding into the pituitary gland (pituitary apoplexy), which requires prompt medical attention.

Hypopituitarism may be the result of inherited disorders, but more often it's acquired. Hypopituitarism frequently is triggered by a tumor of the pituitary gland. As a pituitary tumor increases in size, it can compress and damage pituitary tissue, interfering with hormone production. A tumor can also compress the optic nerves, causing visual disturbances.

The cause of hypopituitarism can also be other diseases and events that damage the pituitary, such as:

Diseases of the hypothalamus, a portion of the brain situated just above the pituitary, also can cause hypopituitarism. The hypothalamus produces hormones of its own that directly affect the activity of the pituitary.

In some cases, the cause of hypopituitarism is unknown.

The pituitary gland and the hypothalamus are situated within the brain and control hormone production.

The endocrine system includes the pituitary gland, thyroid gland, parathyroid glands, adrenal glands, pancreas, ovaries (in females) and testicles (in males).

If your doctor suspects a pituitary disorder, he or she will likely order several tests to check levels of various hormones in your body. Your doctor may also want to check for hypopituitarism if you've had a recent head injury or radiation treatment that might have put you at risk of damage to your pituitary gland.

Tests your doctor may order include:

Successful treatment of the underlying condition causing hypopituitarism may lead to a complete or partial recovery of your body's normal production of pituitary hormones. Treatment with the appropriate hormones is often the first step. These drugs are considered as "replacement," rather than treatment, because the dosages are set to match the amounts that your body would normally manufacture if it didn't have a pituitary problem. Treatment may be lifelong.

Treatment for pituitary tumors may involve surgery to remove the growth. In some instances, doctors also recommend radiation treatment.

Hormone replacement medications may include:

If you've become infertile, LH and FSH (gonadotropins) can be administered by injection to stimulate ovulation in women and sperm production in men.

A doctor who specializes in endocrine disorders (endocrinologist) may monitor the levels of these hormones in your blood to ensure you're getting adequate but not excessive amounts.

Your doctor will advise you to adjust your dosage of corticosteroids if you become seriously ill or experience major physical stress. During these times, your body would ordinarily produce extra cortisol hormone. The same kind of fine-tuning of dosage may be necessary when you have the flu, experience diarrhea or vomiting, or have surgery or dental procedures. Adjustments in dosage may also be necessary during pregnancy or with marked changes in weight. You may need periodic CT or MRI scans as well to monitor a pituitary tumor or other diseases causing the hypopituitarism.

Wear a medical alert bracelet or pendant, and carry a special card, notifying others in emergency situations, for example that you're taking corticosteroids and other medications.

You're likely to start by seeing your family doctor or a general practitioner. However, in some cases, when you call to set up an appointment, you may be referred to a specialist called an endocrinologist.

Here's some information to help you prepare for your appointment.

Create a list of questions before your appointment so that you can make the most of your time with your doctor. For hypopituitarism, some basic questions to ask your doctor include:

Don't hesitate to ask any questions you have during your appointment.

Your doctor is likely to ask you some questions, such as:

1998-2018 Mayo Foundation for Medical Education and Research (MFMER). All rights reserved. Terms of use

Read the original post:
Hypopituitarism Disease Reference Guide - Drugs.com

Recommendation and review posted by sam

Hypopituitarism (an underactive pituitary gland) is rare in children. When a child has hypopituitarism, the pituitary gland has lost its ability to make one, some or all pituitary hormones. The condition is often permanent, but very treatable.

The pituitary gland in the middle of the head and brain is the bodys master gland. The table below describes what each hormone made by the pituitary gland does, what happens when each hormone is missing and medication that can replace each hormone.

In children, hypopituitarism is usually caused by something congenital (the child is born with the problem) or by a pituitary tumor. The tumor interferes with the gland. Sometimes, the cause cannot be determined.

Sometimes, the cause was present before the child was born. Often, we see that the pituitary gland is under-developed. A genetic error may be the reason the gland doesnt work well.

Children withsepto-optic dysplasia have varying degrees of hypopituitarism. Their vision is usually impaired because the optic nerves are under-developed. The eyes can move irregularly or wander. This disorder can affect the pituitary gland and other structures in the brain. Often, these children have diabetes insipidus and not enough growth hormone.

There are other forms of congenital hypopituitarism. Sometimes, the pituitary gland doesnt make enough growth hormone. Sometimes, the thyroid gland is underactive, or the adrenal gland doesnt work well. In Kallman syndrome, not enough of the hormones that stimulate the testes or ovaries are made, puberty is late or doesnt happen, and the sense of smell is affected.

A tumor can cause hypopituitarism. The tumor may grow in the pituitary gland or outside the gland, compressing the normal tissue. Parents worry that the tumor may be cancer, but thats unlikely.

Rarely, one of these conditions leads to this hormone problem:

Some hormone deficiencies cause complications over time. Our experienced doctors help you understand any long-term or serious effects of the missing hormones. For example:

Signs and symptoms vary, depending on which hormones are lacking and the childs age.

Common symptoms in newborns:

Commonsymptomsin older infants and children:

The symptoms you see may be due to other conditions and medical problems. Always talk to your childs doctor if you have a concern.

Our approach to diagnosing hypopituitarism is very thorough. We take one step at a time andminimizeinvasive procedures.

We can use other tests as needed. For example:

We know that the weeks of waiting for the full picture can be difficult. Our compassionate team and family-friendly environment support your family while we progress toward the answers and plan the right treatment. As soon as possible, youll receive a call from a doctor or nurse about what weve found and the next steps to take.

We treat the cause of the condition and replace the hormones the body isnt making.

Hormone replacement therapy mimics the bodys natural production. The medicines can be continued as long as needed, during childhood and adulthood. These medications are tolerated very well when the right amounts of hormones are replaced. The following are examples of hormone replacement therapy:

Some tumors respond to medicine that is swallowed. Other tumors need to be removed with surgery. Usually, the hormone deficiencies remain after a tumor is removed. Hormone therapy works for this.

To be effective, hormone replacement must be supported with ongoing care. Throughout childhood, we need to adjust the hormone doses to accommodate the growing childs needs and changes in symptoms. We evaluate the childs growth and development frequently and develop a working relationship with parent and child.

Our resources help bring the right specialists into your childs care to make sure the child gets the best treatment possible. Our endocrinologists and neurosurgeons co-manage patients in our hospital. Tools such as our electronic health record help nurses and doctors throughout the team work closely together.

We care for children with all forms of hypopituitarism. We treat each hormone deficiency to maintain the childs health and normal development. With the right care plan, children with hypopituitarism usually enjoy a normal life. We help the child to develop normally, interact with peers and feel well.

Learn more about pituitary disorders by visiting these physician-recommended websites:

Go here to read the rest:
Hypopituitarism Treatment & Causes | Lurie Children's

Recommendation and review posted by sam

Stem cells: Cells that are able to (1) self-renew (can create more stem cells indefinitely) and (2) differentiate into (become) specialized, mature cell types.

Embryonic stem cells: Stem cells that are harvested from a blastocyst. These cells are pluripotent, meaning they can differentiate into cells from all three germ layers.

Embryonic stem cells are isolated from cells in a blastocyst, a very early stage embryo. Once isolated from the blastocyst, these cells form colonies in culture (closely packed groups of cells) and can become cells of the three germ layers, which later make up the adult body.

Adult stem cells (or Somatic Stem Cell): Stem cells that are harvested from tissues in an adult body. These cells are usually multipotent, meaning they can differentiate into cells from some, but not all, of the three germ layers. They are thought to act to repair and regenerate the tissue in which they are found in, but usually they can differentiate into cells of completely different tissue types.

Adult stem cells can be found in a wide variety of tissues throughout the body; shown here are only a few examples.

The Three Germ Layers: These are three different tissue types that exist during development in the embryo and that, together, will later make up the body. These layers include the mesoderm, endoderm, and ectoderm.

The three germ layers form during the gastrula stage of development. The layers are determined by their physical position in the gastrula. This stage follows the zygote and blastocyst stages; the gastrula forms when the embryo is approximately 14-16 days old in humans.

Endoderm: One of the three germ layers. Specifically, this is the inner layer of cells in the embryo and it will develop into lungs, digestive organs, the liver, the pancreas, and other organs.

Mesoderm: One of the three germ layers. Specifically, this is the middle layer of cells in the embryo and it will develop into muscle, bone, blood, kidneys, connective tissue, and related structures.

Ectoderm: One of the three germ layers. Specifically, this is the outer layer of cells in the embryo and it will develop into skin, the nervous system, sensory organs, tooth enamel, eye lens, and other structures.

Differentiation, Differentiated: The process by which a stem cell turns into a different, mature cell. When a stem cell has become the mature cell type, it is called differentiated and has lost the ability to turn into multiple different cell types; it is also no longer undifferentiated.

Directed differentiation: To tightly control a stem cell to become a specific mature cell type. This can be done by regulating the conditions the cell is exposed to (i.e. specific media supplemented with different factors can be used).

The differentiation of stem cells can be controlled by exposing the cells to specific conditions. This regulation can cause the cells to become a specific, desired mature cell type, such as neurons in this example.

Undifferentiated: A stem cell that has not become a specific mature cell type. The stem cell holds the potential to differentiate, to become different cell types.

Potential, potency: The number of different kinds of mature cells a given stem cell can become, or differentiate into.

Totipotent: The ability to turn into all the mature cell types of the body as well as embryonic components that are required for development but do not become tissues of the adult body (i.e. the placenta).

A totipotent cell has the ability to become all the cells in the adult body; such cells could theoretically create a complete embryo, such as is shown here in the early stages.

Pluripotent: The ability to turn into all the mature cell types of the body. This is shown by differentiating these stem cells into cell types of the three different germ layers.

Embryonic stem cells are pluripotent cells isolated from an early stage embryo, called the blastocyst. These isolated cells can turn into cells representative of the three germ layers, all the mature cell types of the body.

Multipotent: The ability to turn into more than one mature cell type of the body, usually a restricted and related group of different cell types.

Mesenchymal stem cells are an example of multipotent stem cells; these stem cells can become a wide variety, but related group, of mature cell types (bone, cartilage, connective tissue, adipose tissue, and others).

Unipotent: The ability to give rise to a single mature cell type of the body.

Tissue Type: A group of cells that are similar in morphology and function, and function together as a unit.

Mesenchyme Tissue: Connective tissue from all three germ layers in the embryo. This tissue can become cells that make up connective tissue, cartilage, adipose tissue, the lymphatic system, and bone in the adult body.

Mesenchyme tissue can come from all three of the germ layers (ectoderm, mesoderm, and endoderm) in the developing embryo, shown here at the gastrula stage. The mesenchyme can become bone, cartilage, connective tissue, adipose tissue, and other components of the adult body.

Hematopoietic Stem Cells: Stem cells that can create all the blood cells (red blood cells, white blood cells, and platelets). These stem cells reside within bone marrow in adults and different organs in the fetus.

Hematopoietic stem cells can become, or differentiate into, all the different blood cell types. This process is referred to as hematopoiesis.

Bone marrow: Tissue within the hollow inside of bones that contains hematopoietic stem cells and mesenchymal stem cells.

Development: The process by which a fertilized egg (from the union of a sperm and egg) becomes an adult organism.

Zygote: The single cell that results from a sperm and egg uniting during fertilization. The zygote undergoes several rounds of cell division before it becomes an embryo (after about four days in humans).

When an egg is fertilized by a sperm, the resultant single cell is referred to as a zygote.

Blastocyst: A very early embryo (containing approximately 150 cells) that has not yet implanted into the uterus. The blastocyst is a fluid-filled sphere that contains a group of cells inside it (called the inner cell mass) and is surrounded by an outer layer of cells (the trophoblast, which forms the placenta).

The blastocyst contains three primary components: the inner cell mass, which can become the adult organism, the trophoblast, which becomes the placenta, and the blastocoele, which is a fluid-filled space. The blastocyst develops into the gastrula, a later stage embryo.

Inner Cell Mass: A small group of cells that are attached inside the blastocyst. Human embryonic stem cells are created from these cells in blastocysts that are four or five days post-fertilization. The cells from the inner cell mass have the potential to develop into an embryo, then later the fetus, and eventually the entire body of the adult organism.

Cells taken from the inner cell mass of the blastocyst (a very early stage embryo) can become embryonic stem cells.

Embryo: The developing organism from the end of the zygote stage (after about four days in humans) until it becomes a fetus (until 7 to 8 weeks after conception in humans).

Models: A biological system that is easy to study and similar enough to another, more complex system of interest so that knowledge of the model system can be used to better understand the more complex system. Such systems can include cells and whole organisms.

Model organism: An organism that is easy to study and manipulate and is similar enough to another organism of interest so that by understanding the model organism, a greater understanding of the other organism may be gained. For example, rats and mice can be used as model organisms to better understand humans.

Shown are several different model organisms frequently used in laboratory studies.

Severe Combined Immune-Deficient (SCID) mouse: A mouse lacking a functional immune system, specifically lacking or abnormal T and B lymphocytes. This is due to inbreeding or genetic engineering. They are extensively used for tissue transplants, because they lack an immune system to reject foreign substances, and for studying an immunocompromised system.

Cellular models: A cell system that can be used to understand normal, or diseased, functions that the cell has within the body. By taking cells from the body and studying them outside of the body, in culture, different conditions can be manipulated and the results studied, whereas this can be much more difficult, or impossible, to do within the body.

Stem cells obtained from different tissues of the body can be used as models to study normal, or diseased, cells in these tissues.

Cell Types:

Somatic Cell: Any cell in the body, developing or adult, other than the germline cells (the gametes, or sperm and eggs).

Gametes: The cells in the body that carry the genetic information that will be passed to the offspring. In other words, these are the germline cells: an egg (for females) or sperm (for males) cell.

Other terms:

Regenerative Medicine: A field of research that investigates how to repair or replace damaged tissues, usually by using stem cells. In this manner, stem cells may be differentiated into, or made to become, the type of cell that is damaged and then used in transplants. Also see clinical trials.

Clinical trials: A controlled test of a new drug or treatment on human subjects, normally performed after successful trials with model organisms. ClinicalTrials.gov lists many stem cell clinical trials.

Stem cells have great potential to treat a wide variety of human diseases and medical conditions.

Cell Surface Marker proteins, or simply Cell Markers: A protein on the surface of a cell that identifies the cell as a certain cell type.

Somatic Cell Nuclear Transfer (SCNT): A technique that uses an egg and a somatic cell (a non-germline cell). The nucleus, which contains the genetic material, is removed from the egg and the nucleus from the somatic cell is removed and combined with the egg. The resultant cell contains the genetic material of the nucleus donor, and is turned into a totipotent state by the egg. This cell has the potential to develop into an organism, a clone of the nucleus donor.

Dolly the sheep was cloned through somatic cell nuclear transfer (SCNT). An adult cell from the mammary gland of a Finn-Dorset ewe acted as the nuclear donor; it was fused with an enucleated egg from a Scottish Blackface ewe, which acted as the cytoplasmic (or egg) donor. An electrical pulse acted to fuse the cells and activate the oocyte after injection into the surrogate mother ewe. A successfully implanted oocyte developed into the lamb Dolly, a clone of the nuclear donor, the Finn-Dorset ewe.

Clone: A genetic, identical copy of an individual organism through asexual methods. A clone can be created through somatic cell nuclear transfer.

Other stem cell glossaries:

Image creditsImages of Endoderm, Mesoderm, Ectoderm, Bone Marrow, Neurons, Cartilage, Hand Skeleton, Connective and Adipose Tissue, Gastrula, Clinical Trials, Mouse, Rat, Drosophila, C. Elegans, Arabidopsis, Sea Urchin, Xenopus, Somatic Cell Nuclear Transfer to Create Dolly and other images were taken from the Wikimedia Commons and redistributed and altered freely as they are all in the public domain. The image of Hematopoiesis was also taken from the Wikimedia Commons and redistributed according to the GNU Free Documentation License.

2009. Teisha Rowland. All rights reserved.

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Recommendation and review posted by Bethany Smith

The Kotton labs goal is advancing our understanding of lung disease and developmental biology with a focus on stem cell biology and gene therapy. We believe that novel treatments for many lung diseases can be realized based on a better understanding of how the lung develops as well as regenerates after lung injury.

We are particularly interested in understanding how lung cells decide and remember who they are. To this end, one focus of our group is defining the genomic and epigenomic programs that regulate lung cell fate. A longer term goal is the de novo generation of the full diversity of lung lineages and transplantable 3D lung tissues from pluripotent stem cells. Our Principal Investigator, Dr. Darrell Kotton, also serves as the founding Director of the Center for Regenerative Medicine (CReM). Take a full tour of the CReM by clicking on our logo above.

Click on the menu to learn more about our research areas and our team

Have forty five minutes for an overview of our last decade? Listen here to Darrells ATS Discovery Series Lecture, Lung Regeneration: An Achievable Mission.

Open Source Works! Click here to access our:iPS Cell Lines, Lentiviral Vectors, Bioinformatics Datasets, or Detailed Protocols!

or read more about our Open Source Biology Philosophyor a recent interview on Darrells approach to sharing our cells

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Recommendation and review posted by simmons

More than 55% of VHL-affected individuals develop only multiple renal cell cysts. The VHL-associated RCCs that occur are characteristically multifocal and bilateral and present as a combined cystic and solid mass.[66] Among individuals with VHL, the cumulative RCC risk has been reported as 24% to 45% overall. RCCs smaller than 3 cm in this disease tend to be low grade (Fuhrman nuclear grade 2) and minimally invasive,[67] and their rate of growth varies widely.[68] An investigation of 228 renal lesions in 28 patients who were followed up for at least 1 year showed that transition from a simple cyst to a solid lesion was infrequent.[66] Complex cystic and solid lesions contained neoplastic tissue that uniformly enlarged. These data may be used to help predict the progression of renal lesions in VHL. Figure 1 depicts bilateral renal tumors in a patient with VHL.

Enlarge Figure 1. von Hippel-Lindau diseaseassociated renal cell cancers are characteristically multifocal and bilateral and present as a combined cystic and solid mass. Red arrow indicates a lesion with a solid and cystic component, and white arrow indicates a predominantly solid lesion.

Tumors larger than 3 cm may increase in grade as they grow, and metastasis may occur.[68,69] RCCs often remain asymptomatic for long intervals.

Patients can also develop pancreatic cysts, cystadenomas, and pancreatic NETs.[2] Pancreatic cysts and cystadenomas are not malignant, but pancreatic NETs possess malignant characteristics and are typically resected if they are 3 cm or larger (2 cm if located in the head of the pancreas).[70] A review of the natural history of pancreatic NETs shows that these tumors may demonstrate nonlinear growth characteristics.[71]

Retinal manifestations, first reported more than a century ago, were one of the first recognized aspects of VHL. Retinal hemangioblastomas (also known as capillary retinal angiomas) are one of the most frequent manifestations of VHL and are present in more than 50% of patients.[72] Retinal involvement is one of the earliest manifestations of VHL, with a mean age at onset of 25 years.[1,2] These tumors are the first manifestation of VHL in nearly 80% of affected individuals and may occur in children as young as 1 year.[2,73,74]

Retinal hemangioblastomas occur most frequently in the periphery of the retina but can occur in other locations such as the optic nerve, a location much more difficult to treat. Retinal hemangioblastomas appear as a bright orange spherical tumor supplied by a tortuous vascular supply. Nearly 50% of patients have bilateral retinal hemangioblastomas.[72] The median number of lesions per affected eye is approximately six.[75] Other retinal lesions in VHL can include retinal vascular hamartomas, flat vascular tumors located in the superficial aspect of the retina.[76]

Longitudinal studies are important for the understanding of the natural history of these tumors. Left untreated, retinal hemangioblastomas can be a major source of morbidity in VHL, with approximately 8% of patients [72] having blindness caused by various mechanisms, including secondary maculopathy, contributing to retinal detachment, or possibly directly causing retinal neurodegeneration.[77] Patients with symptomatic lesions generally have larger and more numerous retinal hemangioblastomas. Long-term follow-up studies demonstrate that most lesions grow slowly and that new lesions do not develop frequently.[75,78]

Hemangioblastomas are the most common disease manifestation in patients with VHL, affecting more than 70% of individuals. A prospective study assessed the natural history of hemangioblastomas.[79] The mean age at onset of CNS hemangioblastomas is 29.1 years (range, 773 y).[80] After a mean follow-up of 7 years, 72% of the 225 patients studied developed new lesions.[81] Fifty-one percent of existing hemangioblastomas remained stable. The remaining lesions exhibited heterogeneous growth rates, with cerebellar and brainstem lesions growing faster than those in the spinal cord or cauda equina. Approximately 12% of hemangioblastomas developed either peritumoral or intratumoral cysts, and 6.4% were symptomatic and required treatment. Increased tumor burden or total tumor number detected was associated with male sex, longer follow-up, and genotype (all P < .01). Partial germline deletions were associated with more tumors per patient than were missense variants (P < .01). Younger patients developed more tumors per year. Hemangioblastoma growth rate was higher in men than in women (P < .01). Figures 2 and 3 depict cerebellar and spinal hemangioblastomas, respectively, in patients with VHL.

Enlarge Figure 2. Hemangioblastomas are the most common disease manifestation in patients with von Hippel-Lindau disease. The left panel shows a sagittal view of brainstem and cerebellar lesions. The middle panel shows an axial view of a brainstem lesion. The right panel shows a cerebellar lesion (red arrow) with a dominant cystic component (white arrow).

Enlarge Figure 3. Hemangioblastomas are the most common disease manifestation in patients with von Hippel-Lindau disease. Multiple spinal cord hemangioblastomas are shown.

The rate of pheochromocytoma formation in the VHL patient population is 25% to 30%.[82,83] Of patients with VHL-associated pheochromocytomas, 44% developed disease in both adrenal glands.[84] The rate of malignant transformation is very low. Levels of plasma and urine normetanephrine are typically elevated in patients with VHL,[85] and approximately two-thirds will experience physical manifestations such as hypertension, tachycardia, and palpitations.[82] Patients with a partial loss of VHL function (Type 2 disease) are at higher risk of pheochromocytoma than are VHL patients with a complete loss of VHL function (Type 1 disease); the latter develop pheochromocytoma very rarely.[13,14,82,86] The rate of VHL germline pathogenic variants in nonsyndromic pheochromocytomas and paragangliomas was very low in a cohort of 182 patients, with only 1 of 182 patients ultimately diagnosed with VHL.[87]

Paragangliomas are rare in VHL patients but can occur in the head and neck or abdomen.[88] A review of VHL patients who developed pheochromocytomas and/or paragangliomas revealed that 90% of patients manifested pheochromocytomas and 19% presented with a paraganglioma.[84]

The mean age at diagnosis of VHL-related pheochromocytomas and paragangliomas is approximately 30 years,[83,89] and patients with multiple tumors were diagnosed more than a decade earlier than patients with solitary lesions in one series (19 vs. 34 y; P < .001).[89] Diagnosis of pheochromocytoma was made in patients as young as 5 years in one cohort,[83] providing a rationale for early testing. All 21 pediatric patients with pheochromocytomas in this 273-patient cohort had elevated plasma normetanephrines.[83]

VHL patients may develop multiple serous cystadenomas, pancreatic NETs, and simple pancreatic cysts.[1] VHL patients do not have an increased risk of pancreatic adenocarcinoma. Serous cystadenomas are benign tumors and warrant no intervention. Simple pancreatic cysts can be numerous and rarely cause symptomatic biliary duct obstruction. Endocrine function is nearly always maintained; occasionally, however, patients with extensive cystic disease requiring pancreatic surgery may ultimately require pancreatic exocrine supplementation.

Pancreatic NETs are usually nonfunctional but can metastasize (to lymph nodes and the liver). The risk of pancreatic NET metastasis was analyzed in a large cohort of patients, in which the mean age at diagnosis of a pancreatic NET was 38 years (range, 1668 y).[90] The risk of metastasis was lower in patients with small primary lesions (3 cm), in patients without an exon 3 pathogenic variant, and in patients whose tumor had a slow doubling time (>500 days). Nonfunctional pancreatic NETs can be followed by imaging surveillance with intervention when tumors reach 3 cm. Lesions in the head of the pancreas can be considered for surgery at a smaller size to limit operative complexity.

ELSTs are adenomatous tumors arising from the endolymphatic duct or sac within the posterior part of the petrous bone.[91] ELSTs are rare in the sporadic setting, but are apparent on imaging in 11% to 16% of patients with VHL. Although these tumors do not metastasize, they are locally invasive, eroding through the petrous bone and the inner ear structures.[91,92] Approximately 30% of VHL patients with ELSTs have bilateral lesions.[91,93]

ELSTs are an important cause of morbidity in VHL patients. ELSTs evident on imaging are associated with a variety of symptoms, including hearing loss (95% of patients), tinnitus (92%), vestibular symptoms (such as vertigo or disequilibrium) (62%), aural fullness (29%), and facial paresis (8%).[91,92] In approximately half of patients, symptoms (particularly hearing loss) can occur suddenly, probably as a result of acute intralabyrinthine hemorrhage.[92] Hearing loss or vestibular dysfunction in VHL patients can also present in the absence of radiologically evident ELSTs (approximately 60% of all symptomatic patients) and is believed to be a consequence of microscopic ELSTs.[91]

Hearing loss related to ELSTs is typically irreversible; serial imaging to enable early detection of ELSTs in asymptomatic patients and resection of radiologically evident lesions are important components in the management of VHL patients.[94,95] Surgical resection by retrolabyrinthine posterior petrosectomy is usually curative and can prevent onset or worsening of hearing loss and improve vestibular symptoms.[92,94]

Tumors of the broad ligament can occur in females with VHL and are known as papillary cystadenomas. These tumors are extremely rare, and fewer than 20 have been reported in the literature.[96] Papillary cystadenomas are histologically identical to epididymal cystadenomas commonly observed in males with VHL.[97] One important difference is that papillary cystadenomas are almost exclusively observed in patients with VHL, whereas epididymal cystadenomas in men can occur sporadically.[98] These tumors are frequently cystic, and although they become large, they generally have a fairly indolent behavior.

More than one-third of all cases of epididymal cystadenomas reported in the literature and most cases of bilateral cystadenomas have been reported in patients with VHL.[99] Among symptomatic patients, the most common presentation is a painless, slow-growing scrotal swelling. The differential diagnoses of epididymal tumors include adenomatoid tumor (which is the most common tumor in this site), metastatic ccRCC, and papillary mesothelioma.[100]

In a small series, histological analysis did not reveal features typically associated with malignancy, such as mitotic figures, nuclear pleomorphism, and necrosis. Lesions were strongly positive for CK7 and negative for RCC. Carbonic anhydrase IX (CAIX) was positive in all tumors. PAX8 was positive in most cases. These features were reminiscent of clear cell papillary RCC, a relatively benign form of RCC without known metastatic potential.[97]

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Recommendation and review posted by Bethany Smith

A frequent need to get up and go to the bathroom to urinate at night is called nocturia. It differs from enuresis, or bedwetting, in which the person does not arouse from sleep, but the bladder empties anyway. Nocturia is a common cause of sleep loss, especially among older adults.

Most people without nocturia can sleep for 6 to 8 hours without having to urinate. Some researchers believe that one event per night is within normal limits; two or more events per night may be associated with daytime tiredness. Patients with severe nocturia may get up five or six times during the night to go to the bathroom.

Nocturia is often a symptom of other medical conditions including urological infection, a tumor of the bladder or prostate, a condition called bladder prolapse, or disorders affecting sphincter control. It is also common in people with heart failure, liver failure, poorly controlled diabetes mellitus, or diabetes insipidus. Diabetes, pregnancy and diuretic medications are also associated with nocturia.

Until recently, nocturia was thought to be caused by a full bladder, but it is also a symptom of sleep apnea.

Nocturia becomes more common as we age. As we get older, our bodies produce less of an anti-diuretic hormone that enables us to retain fluid. With decreased concentrations of this hormone, we produce more urine at night. Another reason for nocturia among the elderly is that the bladder tends to lose holding capacity as we age. Finally, older people are more likely to suffer from medical problems that may have an effect on the bladder.

In fact, nearly two-thirds (65%) of those responding to NSF's 2003 Sleep in America poll of adults between the ages of 55 and 84 reported this disturbance at least a few nights per week.

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Nocturia or Frequent Urination at Night - National Sleep ...

Recommendation and review posted by Rebecca Evans

The adrenal glands (also known as suprarenal glands) are endocrine glands that produce a variety of hormones including adrenaline and the steroids aldosterone and cortisol.[1][2] They are found above the kidneys. Each gland has an outer cortex which produces steroid hormones and an inner medulla. The adrenal cortex itself is divided into three zones: the zona glomerulosa, the zona fasciculata and the zona reticularis.[3]

The adrenal cortex produces three main types of steroid hormones: mineralocorticoids, glucocorticoids, and androgens. Mineralocorticoids (such as aldosterone) produced in the zona glomerulosa help in the regulation of blood pressure and electrolyte balance. The glucocorticoids cortisol and corticosterone are synthesized in the zona fasciculata; their functions include the regulation of metabolism and immune system suppression. The innermost layer of the cortex, the zona reticularis, produces androgens that are converted to fully functional sex hormones in the gonads and other target organs.[4] The production of steroid hormones is called steroidogenesis, and involves a number of reactions and processes that take place in cortical cells.[5] The medulla produces the catecholamines adrenaline and noradrenaline, which function to produce a rapid response throughout the body in stress situations.[4]

A number of endocrine diseases involve dysfunctions of the adrenal gland. Overproduction of cortisol leads to Cushing's syndrome, whereas insufficient production is associated with Addison's disease. Congenital adrenal hyperplasia is a genetic disease produced by dysregulation of endocrine control mechanisms.[4][6] A variety of tumors can arise from adrenal tissue and are commonly found in medical imaging when searching for other diseases.[7]

The adrenal glands are located on both sides of the body in the retroperitoneum, above and slightly medial to the kidneys. In humans, the right adrenal gland is pyramidal in shape, whereas the left is semilunar or crescent shaped and somewhat larger.[8] The adrenal glands measure approximately 3cm in width, 5.0cm in length, and up to 1.0cm in thickness.[9] Their combined weight in an adult human ranges from 7 to 10grams.[10] The glands are yellowish in colour.[8]

The adrenal glands are surrounded by a fatty capsule and lie within the renal fascia, which also surrounds the kidneys. A weak septum (wall) of connective tissue separates the glands from the kidneys.[11] The adrenal glands are directly below the diaphragm, and are attached to the crura of the diaphragm by the renal fascia.[11]

Each adrenal gland has two distinct parts, each with a unique function, the outer adrenal cortex and the inner medulla, both of which produce hormones.[12]

The adrenal cortex is the outermost layer of the adrenal gland. Within the cortex are three layers, called "zones". When viewed under a microscope each layer has a distinct appearance, and each has a different function.[13] The adrenal cortex is devoted to production of hormones, namely aldosterone, cortisol, and androgens.[14]

The outermost zone of the adrenal cortex is the zona glomerulosa. It lies immediately under the fibrous capsule of the gland. Cells in this layer form oval groups, separated by thin strands of connective tissue from the fibrous capsule of the gland and carry wide capillaries.[15]

This layer is the main site for production of aldosterone, a mineralocorticoid, by the action of the enzyme aldosterone synthase.[16][17] Aldosterone plays an important role in the long-term regulation of blood pressure.[18]

The zona fasciculata is situated between the zona glomerulosa and zona reticularis. Cells in this layer are responsible for producing glucocorticoids such as cortisol.[19] It is the largest of the three layers, accounting for nearly 80% of the volume of the cortex.[3] In the zona fasciculata, cells are arranged in columns radially oriented towards the medulla. Cells contain numerous lipid droplets, abundant mitochondria and a complex smooth endoplasmic reticulum.[15]

The innermost cortical layer, the zona reticularis, lies directly adjacent to the medulla. It produces androgens, mainly dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), and androstenedione (the precursor to testosterone) in humans.[19] Its small cells form irregular cords and clusters, separated by capillaries and connective tissue. The cells contain relatively small quantities of cytoplasm and lipid droplets, and sometimes display brown lipofuscin pigment.[15]

The adrenal medulla is at the centre of each adrenal gland, and is surrounded by the adrenal cortex. The chromaffin cells of the medulla are the body's main source of the catecholamines adrenaline and noradrenaline, released by the medulla. Approximately 20% noradrenaline (norepinephrine) and 80% adrenaline (epinephrine) are secreted here.[19]

The adrenal medulla is driven by the sympathetic nervous system via preganglionic fibers originating in the thoracic spinal cord, from vertebrae T5T11.[20] Because it is innervated by preganglionic nerve fibers, the adrenal medulla can be considered as a specialized sympathetic ganglion.[20] Unlike other sympathetic ganglia, however, the adrenal medulla lacks distinct synapses and releases its secretions directly into the blood.

The adrenal glands have one of the greatest blood supply rates per gram of tissue of any organ: up to 60 small arteries may enter each gland.[21] Three arteries usually supply each adrenal gland:[8]

These blood vessels supply a network of small arteries within the capsule of the adrenal glands. Thin strands of the capsule enter the glands, carrying blood to them.[8]

Venous blood is drained from the glands by the suprarenal veins, usually one for each gland:[8]

The central adrenomedullary vein, in the adrenal medulla, is an unusual type of blood vessel. Its structure is different from the other veins in that the smooth muscle in its tunica media (the middle layer of the vessel) is arranged in conspicuous, longitudinally oriented bundles.[3]

The adrenal glands may not develop at all, or may be fused in the midline behind the aorta.[12] These are associated with other congenital abnormalities, such as failure of the kidneys to develop, or fused kidneys.[12] The gland may develop with a partial or complete absence of the cortex, or may develop in an unusual location.[12]

The adrenal gland secretes a number of different hormones which are metabolised by enzymes either within the gland or in other parts of the body. These hormones are involved in a number of essential biological functions.[23]

Corticosteroids are a group of steroid hormones produced from the cortex of the adrenal gland, from which they are named.[24] Corticosteroids are named according to their actions:

The adrenal gland produces aldosterone, a mineralocorticoid, which is important in the regulation of salt ("mineral") balance and blood volume. In the kidneys, aldosterone acts on the distal convoluted tubules and the collecting ducts by increasing the reabsorption of sodium and the excretion of both potassium and hydrogen ions.[18] Aldosterone is responsible for the reabsorption of about 2% of filtered glomerular filtration rates.[27] Sodium retention is also a response of the distal colon and sweat glands to aldosterone receptor stimulation. Angiotensin II and extracellular potassium are the two main regulators of aldosterone production.[19] The amount of sodium present in the body affects the extracellular volume, which in turn influences blood pressure. Therefore, the effects of aldosterone in sodium retention are important for the regulation of blood pressure.[28]

Cortisol is the main glucocorticoid in humans. In species that do not create cortisol, this role is played by corticosterone instead. Glucocorticoids have many effects on metabolism. As their name suggests, they increase the circulating level of glucose. This is the result of an increase in the mobilization of amino acids from protein and the stimulation of synthesis of glucose from these amino acids in the liver. In addition, they increase the levels of free fatty acids, which cells can use as an alternative to glucose to obtain energy. Glucocorticoids also have effects unrelated to the regulation of blood sugar levels, including the suppression of the immune system and a potent anti-inflammatory effect. Cortisol reduces the capacity of osteoblasts to produce new bone tissue and decreases the absorption of calcium in the gastrointestinal tract.[28]

The adrenal gland secretes a basal level of cortisol but can also produce bursts of the hormone in response to adrenocorticotropic hormone (ACTH) from the anterior pituitary. Cortisol is not evenly released during the day its concentrations in the blood are highest in the early morning and lowest in the evening as a result of the circadian rhythm of ACTH secretion.[28] Cortisone is an inactive product of the action of the enzyme 11-HSD on cortisol. The reaction catalyzed by 11-HSD is reversible, which means that it can turn administered cortisone into cortisol, the biologically active hormone.[28]

All corticosteroid hormones share cholesterol as a common precursor. Therefore, the first step in steroidogenesis is cholesterol uptake or synthesis. Cells that produce steroid hormones can acquire cholesterol through two paths. The main source is through dietary cholesterol transported via the blood as cholesterol esters within low density lipoproteins (LDL). LDL enters the cells through receptor-mediated endocytosis. The other source of cholesterol is synthesis in the cell's endoplasmic reticulum. Synthesis can compensate when LDL levels are abnormally low.[4] In the lysosome, cholesterol esters are converted to free cholesterol, which is then used for steroidogenesis or stored in the cell.[29]

The initial part of conversion of cholesterol into steroid hormones involves a number of enzymes of the cytochrome P450 family that are located in the inner membrane of mitochondria. Transport of cholesterol from the outer to the inner membrane is facilitated by steroidogenic acute regulatory protein and is the rate-limiting step of steroid synthesis.[29]

The layers of the adrenal gland differ by function, with each layer having distinct enzymes that produce different hormones from a common precursor.[4] The first enzymatic step in the production of all steroid hormones is cleavage of the cholesterol side chain, a reaction that forms pregnenolone as a product and is catalyzed by the enzyme P450scc, also known as cholesterol desmolase. After the production of pregnenolone, specific enzymes of each cortical layer further modify it. Enzymes involved in this process include both mitochondrial and microsomal P450s and hydroxysteroid dehydrogenases. Usually a number of intermediate steps in which pregnenolone is modified several times are required to form the functional hormones.[5] Enzymes that catalyze reactions in these metabolic pathways are involved in a number of endocrine diseases. For example, the most common form of congenital adrenal hyperplasia develops as a result of deficiency of 21-hydroxylase, an enzyme involved in an intermediate step of cortisol production.[30]

Glucocorticoids are under the regulatory influence of the hypothalamus-pituitary-adrenal (HPA) axis. Glucocorticoid synthesis is stimulated by adrenocorticotropic hormone (ACTH), a hormone released into the bloodstream by the anterior pituitary. In turn, production of ACTH is stimulated by the presence of corticotropin-releasing hormone (CRH), which is released by neurons of the hypothalamus. ACTH acts on the adrenal cells first by increasing the levels of StAR within the cells, and then of all steroidogenic P450 enzymes. The HPA axis is an example of a negative feedback system, in which cortisol itself acts as a direct inhibitor of both CRH and ACTH synthesis. The HPA axis also interacts with the immune system through increased secretion of ACTH at the presence of certain molecules of the inflammatory response.[4]

Mineralocorticoid secretion is regulated mainly by the reninangiotensinaldosterone system (RAAS), the concentration of potassium, and to a lesser extent the concentration of ACTH.[4] Sensors of blood pressure in the juxtaglomerular apparatus of the kidneys release the enzyme renin into the blood, which starts a cascade of reactions that lead to formation of angiotensin II. Angiotensin receptors in cells of the zona glomerulosa recognize the substance, and upon binding they stimulate the release of aldosterone.[31]

Primarily referred to in the United States as epinephrine and norepinephrine, adrenaline and noradrenaline are catecholamines, water-soluble compounds that have a structure made of a catechol group and an amine group. The adrenal glands are responsible for most of the adrenaline that circulates in the body, but only for a small amount of circulating noradrenaline.[23] These hormones are released by the adrenal medulla, which contains a dense network of blood vessels. Adrenaline and noradrenaline act at adrenoreceptors throughout the body, with effects that include an increase in blood pressure and heart rate.[23] actions of adrenaline and noradrenaline are responsible for the fight or flight response, characterised by a quickening of breathing and heart rate, an increase in blood pressure, and constriction of blood vessels in many parts of the body.[32]

Catecholamines are produced in chromaffin cells in the medulla of the adrenal gland, from tyrosine, a non-essential amino acid derived from food or produced from phenylalanine in the liver. The enzyme tyrosine hydroxylase converts tyrosine to L-DOPA in the first step of catecholamine synthesis. L-DOPA is then converted to dopamine before it can be turned into noradrenaline. In the cytosol, noradrenaline is converted to epinephrine by the enzyme phenylethanolamine N-methyltransferase (PNMT) and stored in granules. Glucocorticoids produced in the adrenal cortex stimulate the synthesis of catecholamines by increasing the levels of tyrosine hydroxylase and PNMT.[4][13]

Catecholamine release is stimulated by the activation of the sympathetic nervous system. Splanchnic nerves of the sympathetic nervous system innervate the medulla of the adrenal gland. When activated, it evokes the release of catecholamines from the storage granules by stimulating the opening of calcium channels in the cell membrane.[33]

Cells in zona reticularis of the adrenal glands produce male sex hormones, or androgens, the most important of which is DHEA. In general, these hormones do not have an overall effect in the male body, and are converted to more potent androgens such as testosterone and DHT or to estrogens (female sex hormones) in the gonads, acting in this way as a metabolic intermediate.[34]

Thehuman genomeincludes approximately 20,000 protein coding genes and 70% of thesegenes are expressedin the normal, adult adrenal glands.[35][36]Only some 250 genes are more specifically expressed in the adrenal glands compared to other organs and tissues.The adrenal gland specific genes with highest level of expression include members of the cytochrome P450 superfamily of enzymes. Corresponding proteins are expressed in the different compartments of the adrenal gland, such as CYP11A1, HSD3B2 and FDX1 involved in steroid hormone synthesis and expressed in cortical cell layers, and PNMT and DBH involved in noradrenalin and adrenalin synthesis and expressed in the medulla.[37]

The adrenal glands are composed of two heterogenous types of tissue. In the center is the adrenal medulla, which produces adrenaline and noradrenaline and releases them into the bloodstream, as part of the sympathetic nervous system. Surrounding the medulla is the cortex, which produces a variety of steroid hormones. These tissues come from different embryological precursors and have distinct prenatal development paths. The cortex of the adrenal gland is derived from mesoderm, whereas the medulla is derived from the neural crest, which is of ectodermal origin.[12]

The adrenal glands in a newborn baby are much larger as a proportion of the body size than in an adult.[38] For example, at age three months the glands are four times the size of the kidneys. The size of the glands decreases relatively after birth, mainly because of shrinkage of the cortex. The cortex, which almost completely disappears by age 1, develops again from age 45. The glands weigh about 1 g at birth[12] and develop to an adult weight of about 4 grams each.[28] In a fetus the glands are first detectable after the sixth week of development.[12]

Adrenal cortex tissue is derived from the intermediate mesoderm. It first appears 33 days after fertilisation, shows steroid hormone production capabilities by the eighth week and undergoes rapid growth during the first trimester of pregnancy. The fetal adrenal cortex is different from its adult counterpart, as it is composed of two distinct zones: the inner "fetal" zone, which carries most of the hormone-producing activity, and the outer "definitive" zone, which is in a proliferative phase. The fetal zone produces large amounts of adrenal androgens (male sex hormones) that are used by the placenta for estrogen biosynthesis.[39] Cortical development of the adrenal gland is regulated mostly by ACTH, a hormone produced by the pituitary gland that stimulates cortisol synthesis.[40] During midgestation, the fetal zone occupies most of the cortical volume and produces 100200mg/day of DHEA-S, an androgen and precursor of both androgens and estrogens (female sex hormones).[41] Adrenal hormones, especially glucocorticoids such as cortisol, are essential for prenatal development of organs, particularly for the maturation of the lungs. The adrenal gland decreases in size after birth because of the rapid disappearance of the fetal zone, with a corresponding decrease in androgen secretion.[39]

During early childhood androgen synthesis and secretion remain low, but several years before puberty (from 68 years of age) changes occur in both anatomical and functional aspects of cortical androgen production that lead to increased secretion of the steroids DHEA and DHEA-S. These changes are part of a process called adrenarche, which has only been described in humans and some other primates. Adrenarche is independent of ACTH or gonadotropins and correlates with a progressive thickening of the zona reticularis layer of the cortex. Functionally, adrenarche provides a source of androgens for the development of axillary and pubic hair before the beginning of puberty.[42][43]

The adrenal medulla is derived from neural crest cells, which come from the ectoderm layer of the embryo. These cells migrate from their initial position and aggregate in the vicinity of the dorsal aorta, a primitive blood vessel, which activates the differentiation of these cells through the release of proteins known as BMPs. These cells then undergo a second migration from the dorsal aorta to form the adrenal medulla and other organs of the sympathetic nervous system.[44] Cells of the adrenal medulla are called chromaffin cells because they contain granules that stain with chromium salts, a characteristic not present in all sympathetic organs. Glucocorticoids produced in the adrenal cortex were once thought to be responsible for the differentiation of chromaffin cells. More recent research suggests that BMP-4 secreted in adrenal tissue is the main responsible for this, and that glucocorticoids only play a role in the subsequent development of the cells.[45]

The normal function of the adrenal gland may be impaired by conditions such as infections, tumors, genetic disorders and autoimmune diseases, or as a side effect of medical therapy. These disorders affect the gland either directly (as with infections or autoimmune diseases) or as a result of the dysregulation of hormone production (as in some types of Cushing's syndrome) leading to an excess or insufficiency of adrenal hormones and the related symptoms.

Cushing's syndrome is the manifestation of glucocorticoid excess. It can be the result of a prolonged treatment with glucocorticoids or be caused by an underlying disease which produces alterations in the HPA axis or the production of cortisol. Causes can be further classified into ACTH-dependent or ACTH-independent. The most common cause of endogenous Cushing's syndrome is a pituitary adenoma which causes an excessive production of ACTH. The disease produces a wide variety of signs and symptoms which include obesity, diabetes, increased blood pressure, excessive body hair (hirsutism), osteoporosis, depression, and most distinctively, stretch marks in the skin, caused by its progressive thinning.[4][6]

When the zona glomerulosa produces excess aldosterone, the result is primary aldosteronism. Causes for this condition are bilateral hyperplasia (excessive tissue growth) of the glands, or aldosterone-producing adenomas (a condition called Conn's syndrome). Primary aldosteronism produces hypertension and electrolyte imbalance, increasing potassium depletion and sodium retention.[6]

Adrenal insufficiency (the deficiency of glucocorticoids) occurs in about 5 in 10,000 in the general population.[6] Diseases classified as primary adrenal insufficiency (including Addison's disease and genetic causes) directly affect the adrenal cortex. If a problem that affects the hypothalamic-pituitary-adrenal axis arises outside the gland, it is a secondary adrenal insufficiency.

Addison's disease refers to primary hypoadrenalism, which is a deficiency in glucocorticoid and mineralocorticoid production by the adrenal gland. In the Western world, Addison's disease is most commonly an autoimmune condition, in which the body produces antibodies against cells of the adrenal cortex. Worldwide, the disease is more frequently caused by infection, especially from tuberculosis. A distinctive feature of Addison's disease is hyperpigmentation of the skin, which presents with other nonspecific symptoms such as fatigue.[4]

A complication seen in untreated Addison's disease and other types of primary adrenal insufficiency is the adrenal crisis, a medical emergency in which low glucocorticoid and mineralocorticoid levels result in hypovolemic shock and symptoms such as vomiting and fever. An adrenal crisis can progressively lead to stupor and coma.[4] The management of adrenal crises includes the application of hydrocortisone injections.[46]

In secondary adrenal insufficiency, a dysfunction of the hypothalamic-pituitary-adrenal axis leads to decreased stimulation of the adrenal cortex. Apart from suppression of the axis by glucocorticoid therapy, the most common cause of secondary adrenal insufficiency are tumors that affect the production of adrenocorticotropic hormone (ACTH) by the pituitary gland.[6] This type of adrenal insufficiency usually does not affect the production of mineralocorticoids, which are under regulation of the reninangiotensin system instead.[4]

Congenital adrenal hyperplasia is a congenital disease in which mutations of enzymes that produce steroid hormones result in a glucocorticoid deficiency and malfunction of the negative feedback loop of the HPA axis. In the HPA axis, cortisol (a glucocorticoid) inhibits the release of CRH and ACTH, hormones that in turn stimulate corticosteroid synthesis. As cortisol cannot be synthesized, these hormones are released in high quantities and stimulate production of other adrenal steroids instead. The most common form of congenital adrenal hyperplasia is due to 21-hydroxylase deficiency. 21-hydroxylase is necessary for production of both mineralocorticoids and glucocorticoids, but not androgens. Therefore, ACTH stimulation of the adrenal cortex induces the release of excessive amounts of adrenal androgens, which can lead to the development of ambiguous genitalia and secondary sex characteristics.[30]

Adrenal tumors are commonly found as incidentalomas, unexpected asymptomatic tumors found during medical imaging. They are seen in around 3.4% of CT scans,[7] and in most cases they are benign adenomas.[47] Adrenal carcinomas are very rare, with an incidence of 1 case per million per year.[4]

Pheochromocytomas are tumors of the adrenal medulla that arise from chromaffin cells. They can produce a variety of nonspecific symptoms, which include headaches, sweating, anxiety and palpitations. Common signs include hypertension and tachycardia. Surgery, especially adrenal laparoscopy, is the most common treatment for small pheochromocytomas.[48]

Bartolomeo Eustachi, an Italian anatomist, is credited with the first description of the adrenal glands in 1563-4.[49][50] However, these publications were part of the papal library and did not receive public attention, which was first received with Caspar Bartholin the Elder's illustrations in 1611.[50]

The adrenal glands are named for their location relative to the kidneys. The term "adrenal" comes from ad- (Latin, "near") and renes (Latin, "kidney").[51] Similarly, "suprarenal", as termed by Jean Riolan the Younger in 1629, is derived from the Latin supra (Latin: "above") and renes (Latin: kidney). The suprarenal nature of the glands was not truly accepted until the 19th century, as anatomists clarified the ductless nature of the glands and their likely secretory role prior to this, there was some debate as to whether the glands were indeed suprarenal or part of the kidney.[50]

One of the most recognized works on the adrenal glands came in 1855 with the publication of On the Constitutional and Local Effects of Disease of the Suprarenal Capsule, by the English physician Thomas Addison. In his monography, Addison described what the French physician George Trousseau would later name Addison's disease, an eponym still used today for a condition of adrenal insufficiency and its related clinical manifestations.[52] In 1894, English physiologists George Oliver and Edward Schafer studied the action of adrenal extracts and observed their pressor effects. In the following decades several physicians experimented with extracts from the adrenal cortex to treat Addison's disease.[49] Edward Calvin Kendall, Philip Hench and Tadeusz Reichstein were then awarded the 1950 Nobel Prize in Physiology or Medicine for their discoveries on the structure and effects of the adrenal hormones.[53]

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Adrenal gland - Wikipedia

Recommendation and review posted by simmons

Get Paid to be an Apartment Mystery Shopper

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Ellis contracts with individuals to conduct over 8,000 apartment mystery shops monthly. Were fair to our shoppers, and our staff is available to answer questions and help with challenges.

Become an Ellis apartment mystery shop contractor today.

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BEWARE of Email Shopping ScamsEllis has been made aware of an email scam regarding shop contracts for our company. Please be advised you can verify the legitimacy of ALL Ellis shop contracts that are available by logging into your Ellis shopper account or contacting us by email or phone. Ellis does not offer apartment shop contract opportunities by mail. If you have reason to believe you have received a fraudulent email or other type of communication involving Ellis shop contract opportunities (especially for any type of assignment other than a multifamily housing mystery shop), please notify us immediately so we can take proper action.

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Get Paid to be an Apartment Mystery Shopper - Ellis Partners

Recommendation and review posted by Rebecca Evans

Cambridge Healthtech Institute s 3rd AnnualAugust 16-17, 2018

It is an exciting time for gene therapy therapies on the market, encouraging clinical data and a long list of pharma collaborations. Pricing and reimbursement takes a majority of the headlines but equally important is producing these therapies in a scalable, cost-effective and robust way, all the while developing a clear CMC and characterization profile that satisfies the regulators.

Cambridge Healthtech Institutes Gene Therapy Manufacturing meeting takes a practical, case study driven approach to the process development, scale-up and production of gene therapies, tackling key topics such as AAV, lentivirus and retrovirus process development and scale-up, CMO management from early to late-stage development.

Final Agenda

Day 1 | Day 2 | Speaker Biographies

Thursday, August 16

11:30 am Registration Open (Grand Ballroom Foyer)

12:15 pm Enjoy Lunch on Your Own

1:15 10th Anniversary Cake Break in the Exhibit Hall with Last Chance for Poster Viewing (Grand Ballroom)

1:55 Chairpersons Remarks

John Pieracci, PhD, Director, Purification, Biogen

2:00 KEYNOTE PRESENTATION: Challenges and Strategies for the Development of a Robust, Scalable, Cost-Effective Biomanufacturing Process

Sadettin Ozturk, PhD, Senior Vice President, Process and Analytical Development, MassBiologics

The use of viral vectors has increased in recent years, both as gene therapies and as vectors for ex vivo cell therapy products. Industrialization of viral vector manufacturing is maturing as companies tackle problems in process control, scale-up, facility design, characterization and quality, and regulatory considerations. This presentation will examine the current state of the art, emerging technologies and challenges.

2:45 Enabling Industrial Scale Production of Lentiviral Vectors for Gene Therapy

Kelly Kral, PhD, Associate Director, Vector Process Development and Manufacturing, bluebird bio

Lentiviral vectors are an ideal platform for indications requiring long-term, stable expression, but the production processes have historically been limited by scale. As the field has now entered commercialization, there is demand for larger quantities of vector, driving the need for more scalable processes. This presentation will review the development, scale-up, and tech transfer of our suspension-based lentiviral vector process.

3:15 Strategies to Deliver Scalable and Reliable Lentiviral Vector Biomanufacturing

Jeffrey Bartlett, PhD, CSO, Calimmune, Inc.

Large-scale clinical production of lentiviral vectors (LV) using current good manufacturing practice (cGMP) methods comes with significant challenges. We have established the Cytegrity stable cell line system for LV bioproduction and have defined key process, quality and regulatory parameters needed to achieve desired productivity and quality across multiples scales and different bioproduction systems. This approach has allowed the production of LV required for Phase I and II clinical trials, while paving the way for future commercialization.

3:45Evolving Process-Centric Facility Design

Mike Sheehan, MSc, MBA, PMP, Senior Project Manager, DPS Group

Increasingly gene therapy products transitioning from clinical phase to commercial manufacture is driving demand for companies to provide additional capacity. Bringing products to market requires exploring opportunities for leading edge facility design, implementing new & evolving technologies, responding to scalability, speed to market and financial considerations.

4:00 Refreshment Break (Foyer)

4:15 Scalable Lentiviral Vector Production Using HEK293 Suspension Cells

Parminder S Chahal, Research Officer, Human Health Therapeutics Research Centre, National Research Council Canada

We have developed expertise in the production of lentiviral vectors (LV) using packaging cell lines and stable producers. Both grow in suspension and in serum-free conditions. Using a stable producer cell line that produces LV expressing GFP, we have compared different modes of operation in bench-scale bioreactors (batch, fed-batch and perfusion). Next, a battery of filters and supplements were evaluated for clarification. A maximal recovery of 78% was obtained.

4:45 Development and Characterization of Novel Micro-RNA Attenuated Oncolytic Herpes Simplex Viruses

Jonathan Platt, PhD., Senior Research Scientist, CMC Operations, Oncorus

Oncorus is developing next generation HSV-based oncolytic virus with enhanced potency for tumor cell killing and recruitment of the immune system. Our innovative miR-attenuation strategy enables robust viral replication in tumor cells, while preventing replication in healthy tissue. The development and characterization of therapeutic oHSV requires thorough product understanding gained through process characterization. Strategies for development and characterization of manufacturing processes centered around a strong organizational infrastructure will be presented.

5:15 End of Day

Day 1 | Day 2 | Speaker Biographies

FRIDAY, AUGUST 17

8:00 am Registration Open and Morning Coffee (Grand Ballroom Foyer)

8:25 Chairpersons Remarks

Nathalie Clment, PhD, Associate Director and Associate Professor, Powell Gene Therapy Center, Pediatrics, University of Florida

8:30 FEATURED PRESENTATION: rAAV Vector Design, Capsid Directed Evolution and Scale Up Activities Using the BEVS System

Jacek Lubelski, PhD., VP, Global Pharmaceutical Development, uniQure

9:00 Towards a Pivotal Process for AAV Manufacture with HSV

David Knop, PhD, Executive Director, Process Development, AGTC

9:30 Large-Scale Manufacturing of Clinical Grade AAV in the Academic Setting

Nathalie Clment, PhD, Associate Director and Associate Professor, Powell Gene Therapy Center, Pediatrics, University of Florida

The talk will present our current methods for the production of research and clinical-grade rAAV with a special emphasis on the HSV-based suspension method capable of generating high titers of improved rAAV quality. Up-to-date in vitro, in vivo, and clinical data will be shown, and pros and cons of the method will be discussed in comparison to the two other most common methods, transfection and the baculovirus system.

10:00 Networking Coffee Break (Foyer)

10:30 Scale-Up Approach to AAV Manufacturing

Johannes C.M. van der Loo, PhD, Director, Clinical Vector Core, The Raymond G. Perelman Center for Molecular and Cellular Therapies, Childrens Hospital of Philadelphia

The Clinical Vector Core at the Childrens Hospital of Philadelphia manufactures preclinical- and clinical-grade AAV for academia and industry-sponsored clinical trials. With the field of gene therapy maturing, there is a growing need for larger scale products. We will discuss a strategy for scale-up that builds on our existing mammalian adherent cell-based manufacturing platform.

11:00 Virus-Like Particles and Other Extracellular Particles from Insect and Mammalian Cells

Alois Jungbauer, PhD, Professor, Institute of Biotechnology, University of Natural Resources and Life Sciences (BOKU)

Virus-like particles and other extra cellular particles are a next generation of biopharmaceuticals. They can be produced by a wide variety of host cells. The challenge is the production of high titers and downstream processing. The particle of interest are contaminated with other particles with similar biophysical properties and therefore difficult to separate. Examples will be given for 3 different cell types.

11:30 Considerations for the Purification Process Characterization of an AAV from Recovery to Drug Substance

Ratish Krishnan, PhD, Scientist, Bioprocessing Research & Development, Pfizer

Smart and efficient approaches for lab-scale characterization are required to ensure a robust adeno-associated manufacturing process. Specific challenges related to the uniqueness of characterizing an AAV manufacturing process will be discussed. Focus will be given to working with limited quantities of material and employing assays that are still being defined.

12:00 pm Next Generation AAV Viral Vector Manufacturing: Proven Technologies with a Modern Twist

Sandhya Buchanan, Director, Upstream Process Development, FUJIFILM Diosynth Biotechnologies

Current approaches to commercial-scale manufacture of viral vectors have been successful for many early phase trials and some late phase trials. Unique challenges/limitations arising for AAV manufacturing include quantities sufficient for patient needs and consumables for manufacturing. We discuss proven technologies blended with modern advancements to meet the needs of the advancing field of gene therapy.

12:30 Enjoy Lunch on Your Own

1:25 Chairpersons Remarks

Chia Chu, Senior Principal Scientist, Bioprocess Research & Development, Pfizer

1:30 FEATURED PRESENTATION: Separation of Full and Empty AAV Particles Using Scalable Isocratic Elution Chromatography

Meisam Bakhshayeshi, PhD, Head, Purification Development, Gene Therapy, Biogen

Robust and efficient removal of AAV empty particles is a critical part of the AAV manufacturing process. In this study, we present a scalable ion exchange chromatography process with isocratic wash and elution to separate full and empty particles. A combination of mono- and di-valent salts were used as eluents to achieve the high degree of resolution required for this separation. High product purity and recovery was achieved from this process.

2:00 Lyophilisation of AAV Gene Therapy Product

Tanvir Tabish, PhD, Head, Drug Product Development for Gene Therapy, Device and Combination Products, Shire

The gene therapy adeno-associated virus (AAV) subtype 8 containing Factor IX (FIX)(BAX335) was formulated in a new proprietary buffer and lyophilized. A stability study was established with the lyophilized material to determine its stability profile at the accelerated temperature of +5C over a 10 month period. The freeze-dried product displayed an improved stability profile when stored at a temperature of +5C. We demonstrated the feasibility of lyophilisation of the AAV viral drug product in the formulation buffer.

2:30 AAV Manufacturing at 2,000L Scale

Alex Fotopoulos, PhD., Senior Vice President, Technical Operations, Ultragenyx.

Changing the manufacturing site (tech transfer) should always include an assessment of comparability, however the ability to demonstrate this varies between early and late development. This talk will discuss common pitfalls and mistakes and highlight key aspects of the comparability exercise.

3:00 CMO Selection for Cell & Gene Therapy

Chad Green, PhD, Principal & Senior Consultant, Dark Horse

As the diversity of CMOs available for cell and gene therapies continues to grow worldwide, identifying the most suitable to engage is becoming an increasingly complex challenge. This presentation will address fundamental questions, such as whether a CMO is even the best choice for manufacturing before progressing to provide concrete guidance on the critical questions to ask prospective CMOs (and yourself), how to ask them and how to analyze the answers and make an optimal, rational choice.

3:30 Close of Conference

Day 1 | Day 2 | Speaker Biographies

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Gene Therapy Manufacturing - The Bioprocessing Summit

Recommendation and review posted by Jack Burke

The Oncotype DX test is a genomic test that analyzes the activity of a group of genes that can affect how a cancer is likely to behave and respond to treatment. The Oncotype DX is used in two ways:

Of all the breast cancer genomic tests, the Oncotype DX test hasthe strongest research behind it.

The results of the Oncotype DX test, combined with other features of the cancer, can help you make a more informed decision about whether or not to have chemotherapy to treat early-stage, hormone-receptor-positive breast cancer or radiation therapy to treat DCIS.

Genomic tests analyze a sample of a cancer tumor to see how active certain genes are. The activity level of these genes affects the behavior of the cancer, including how likely it is to grow and spread. Genomic tests are used to help make decisions about whether more treatments after surgery would be beneficial.

While their names sound similar, genomic testing and genetic testing are very different.

Genetic testing is done on a sample of your blood, saliva, or other tissue and can tell if you have an abnormal change (also called a mutation) in a gene that is linked to a higher risk of breast cancer. See the Genetic Testing pages for more information.

You may be a candidate for the Oncotype DX test if:

Most early-stage (stage I or II), estrogen-receptor-positive breast cancers that havent spread to the lymph nodes are considered to be at low risk for recurrence. After surgery, hormonal therapies such as an aromatase inhibitor or tamoxifen are prescribed to reduce the risk that the cancer will come back in the future. Whether or not chemotherapy is also necessary has been an area of uncertainty for patients and their doctors.

If youve been diagnosed with early-stage, estrogen-receptor-positive breast cancer, the Oncotype DX test can help you and your doctor make a more informed decision about whether or not you need chemotherapy. (Some research also suggests the test may help postmenopausal women with estrogen-receptor-positive breast cancer that has spread to the lymph nodes make chemotherapy decisions. Talk to your doctor if you are in this group.)

You also may be a candidate for the Oncotype DX test if:

DCIS is the most common form of non-invasive breast cancer. DCIS usually is treated by surgically removing the cancer (lumpectomy in most cases). After surgery, hormonal therapy may be recommended if the DCIS is hormone-receptor-positive. Radiation therapy may be recommended for some women. Doctors arent always sure which women will benefit from radiation therapy.

If youve been diagnosed with DCIS, the Oncotype DX test can help you and your doctor make a more informed decision about whether or not you need radiation therapy.

The Oncotype DX genomic test analyzes the activity of 21 genes that can influence how likely a cancer is to grow and respond to treatment.

Looking at these 21 genes can provide specific information on:

So, the Oncotype DX test is both a prognostic test, since it provides more information about how likely (or unlikely) the breast cancer is to come back, and a predictive test, since it predicts the likelihood of benefit from chemotherapy or radiation therapy treatment. Studies have shown that Oncotype DX is useful for both purposes.

Oncotype DX test results assign a Recurrence Score a number between 0 and 100 to the early-stage breast cancer or DCIS. You and your doctor can use the following ranges to interpret your results for early-stage invasive cancer:

The Oncotype DX DCIS score analyzes the activity of 12 genes. You and your doctor can use the following ranges to interpret your results for DCIS:

You and your doctor will consider the Recurrence Score in combination with other factors, such as the size and grade of the cancer, the number of hormone receptors the cancer cells have (many versus few), and your age. Together, you can make a decision about whether or not you should have chemotherapy or radiation therapy.

The Medicare program and several other major insurance companies have agreed to cover the Oncotype DX test. According to Genomic Health, about 90% of insured people in the U.S. are members of a plan that covers the test. If you discover that your plan does not cover the Oncotype DX test, talk to your doctor: he or she may be able to work with your insurance company to get coverage. If you have a low Recurrence Score and you and your doctor decide you do not need to have chemotherapy or radiation, your insurance company can save much more than the cost of the test.

Genomic Health also has started the Genomic Access Program to assist you with verifying insurance coverage and obtaining reimbursement. If you do not have or cannot secure insurance coverage, the Genomic Access Program still may be able to help. Various forms of financial assistance and payment plans are available for people facing financial hardships or those who are uninsured or underinsured. The Oncotype DX test costs about $4,000. For insurance- and payment-related questions, call 1-866-ONCOTYPE (1-866-662-6897) or by email at customerservice@genomichealth.com.

There are other genomics tests used to analyze breast cancer tumors. To learn more, click on the links below.

More here:
Oncotype DX: Genomic Test to Inform Breast Cancer Treatment

Recommendation and review posted by simmons

As is often seen on heritage railways, it is possible to keep old rail vehicles in service virtually indefinitely, although to do so often involves extensive repair and restoration work. Sometimes, circumstances are such that it is necessary for trains in front line operation to undergo similar extensive work.

It was with this thought in mind that Rail Engineer recently visited London Undergrounds project team to view some of the work taking place on the forty-year-old Bakerloo line trains to keep them in service for at least another 10 years.

Background

The Bakerloo line (Baker Street to Waterloo Railway) opened just over 110 years ago in 1906. Since then, it has been extended, had a branch opened, been truncated and eventually settled on its current route from Elephant and Castle in south east London to Harrow and Wealdstone in north east London. From Queens Park to Harrow and Wealdstone, it runs over Network Rails tracks, shared with London Overgrounds Class 378 trains.

Stabling sidings are provided at London Road, Lambeth, and at Queens Park. The main depot at Stonebridge Park is unique in that it is connected to Network Rails track and not to London Undergrounds.

The Bakerloo line is operated by a fleet of 36, seven-car, 1972 tube stock trains originally delivered in 1973/74. These trains are made up of a four-car unit and a three-car unit coupled together. They were designed for a nominal life of 36 years.

At 42 years, the Bakerloo trains are the oldest on the Underground, and amongst the oldest operating anywhere in the UK (other than heritage railways). Their design was based on the original Victoria line fleet, and has an aluminium-framed body with aluminium cladding mounted on a steel underframe.

They have four motor cars, each with four DC motors controlled by a camshaft-operated resistance controller and fitted with rheostatic braking. In addition, the entire train has electro-pneumatic brakes with a Westinghouse emergency brake, and there are electro-pneumatic sliding doors, and train protection is provided by tripcocks.

The fleet of 36 trains is made up of 33 Mk II and three Mk I units. The differences are superficial, and have mostly been eradicated over the years, but there are still some left to catch out anyone thinking they are all the same. They last had major work in the mid-1990s when they were refurbished an extensive visual modernisation whilst eliminating materials that were a fire hazard. For this work they were hauled over the National Rail network to the dockyard at Rosyth, which included travelling over the historic Forth Bridge.

The trains were originally planned for replacement by 2019 as part of the former PPP contracts, and then as the first use of the New Tube for London project. However, in 2013, London Underground decided to extend the life of the Bakerloo line trains to at least 2026.

Current projects

It was to understand more about what it takes to extend the life of a Tube train that Rail Engineer visited London Underground to talk to the project team and see the works over two days in May 2016.

The life extension project is just one of many projects that LU is carrying out on its older trains. LU has set up a Rolling Stock Renewals programme team to manage them all. The teams head, David Caulfield, outlined the various projects being carried out by his team. These include significant modifications to the Central line trains, upgrading 1960s and 1970s battery locomotives, and creating a Rail Adhesion Train (RAT) from some old District line cars to apply Sandite during the autumn leaf fall season.

The aim with all these projects is to keep older trains going to help Keep London Moving (from the Mayors Transport Strategy). David explained how LU is approaching these works.

LU has always carried out modifications to trains and has generally determined the sourcing strategy for each project on a one-off basis. For the future, LU has carried out a strategic review and has decided that it will invest in facilities to manage and execute work in house, bringing in specialist design and implementation resources or using in-house labour as appropriate.

This approach delivers a number of benefits including not having to send trains off site, which can add a week to each trains time out of service. LU train fleets achieve high utilisation and few trains are available to be taken out of service for modifications. An extra week in transit could add a year or more to a programme for fleets the size of LUs.

Bakerloo line

Back to the 42-year old Bakerloo line trains. One of the reasons that life extension was considered was, perversely, because extensive work was already under way to repair cracks and corrosion on the underframe and body. One might imagine these problems would hasten their demise, but the work was essential simply to keep the trains in service until the earliest date that new trains could be delivered.

In designing repairs, it is usually easiest to restore the original strength of the structure. It would be harder, and almost certainly no cheaper, to try and design repairs that would last just, say, five years. Thus the repair works deliver bodies that are structurally as good as new. As such, the work will easily last for the additional time required. Anything else necessary in sub-systems and components can and will be dealt with during routine maintenance, following proper engineering assessment of those components not normally replaced but being required to last beyond their normal lifespan.

The main consequence of extending the life beyond 2020 is the need to carry out modifications to comply with the Rail Vehicle Accessibility Regulations (2010). This contains similar requirements to those in the Technical Specification for Interoperability for People of Reduced Mobility TSIs do not apply to LU.

The RVAR work was explained by Paul Summers, project sponsor from the Asset Strategy and Investment team, and Zoe Dobell, RVAR project engineer (yes, my daughter!). The RVAR requires a number of features that make it easier to use such as handholds, passenger information displays, priority seats and provision for wheelchairs. Compliance is mandatory by 2020.

However, the Regulations recognise that strict compliance may not be possible for older trains. LU has therefore carried out extensive feasibility studies on the RVAR elements. These studies were then discussed with the Department for Transport with the aim of maximising the degree of compliance whilstnot incurring excessive cost for minimal benefit; DfT has been really supportive.

The main elements that will be installed are the wheelchair spaces (which will be in the trailer car of the three car unit), and an audio/visual passenger information system. The biggest challenge of all is the gap between the train and the platform. LUs practice on other lines is to use a mixture of platform humps and manual boarding ramps depending on the curvature and other factors. For the Bakerloo, LU has agreed with the DfT that no boarding aids will be provided where there is no interchange and no foreseeable prospect of providing street to platform step free access.

With agreement on all these features, the scope of the works is now frozen and work will start in mid-2018 for completion early in 2020, based on having two trains out of service at a time. To provide the wheelchair positions involves removing the seats on one side of the middle seat bay of the designated trailer cars. In common with all LU tube gauge cars, there is equipment under the seats this will have to be relocated and new flooring fitted to match the new floors being fitted as part of the body repairs (see below). Installing the passenger information system will involve work on all cars, and, although mandated by RVAR, will be of benefit to all passengers.

Acton Works

It was with considerable nostalgia that I set off from Acton Town station towards the large Acton Works complex, having first made that journey nearly 47 years ago.

The purpose was to see some of the repair works under way, a programme that will cost LU some 60 million or just over 200,000 per car. I was met by the underframe and body repairs project engineer Rob Bonarski, who is charged, inter alia, with making sure there is an approved repair system for every structural fault found.

Rob took me to shop AC15, which old timers like me will recall as the Heavy Repair shop. On the way, we visited some of the other workshops in which we saw Central line bogies being overhauled, Bakerloo line bogies being repaired, some battery locomotives being refurbished, D stock cars being converted for the new RAT and some 1938 tube stock cars being overhauled for the London Transport Museum.

Since I was last at Acton, AC15 shop has had extensive work carried out to prepare it for the Bakerloo line repairs. In former times, cars would have been lifted in Actons lifting shop and moved by traverser to the relevant workshop. This is no longer possible because the lifting shop was demolished many years ago to make way for LUs Railway Equipment overhaul Workshop (REW). The old wood block floor has been replaced with reinforced concrete to support the Mechan jacks that LU bought to lift the cars (four sets of 4 x 10 tonne jacks for passenger vehicles and one set of 4 x 20 tonne jacks that can also lift battery locomotives). There are nine roads, most of which can accommodate two cars. There has also been extensive work to improve lighting, and provide services for electric and pneumatic power tools.

Incompatible Train Movements

Bakerloo line trains start their journey for repairs at Stonebridge Park Depot in northwest London. From here, they make an overnight journey to Acton via Baker Street, Elephant and Castle, back to Baker Street, onto the Jubilee line to Wembley Park, onto the Metropolitan line to Rayners Lane, where they reverse and then travel via the Piccadilly line to Acton Town (see map right).

They travel overnight because there is no signalling nor train protection on the Jubilee line for Bakerloo line trains (Jubilee line trains use in-cab signalling with ATO and ATP). They travel over the Jubilee line section under special rules called an Incompatible Train Movement Plan.

On arrival at Acton Works, the cars are uncoupled on the reception road next to AC15 and moved via a traverser into AC15 where they are lifted. Here the real work starts.

Swan necks, floor traps and fasteners

Rob explained the voyage of discovery on the first few trains as they discovered the true extent of repairs required and the differences between apparently identical cars. Even he had been surprised by the extent of the work required, despite being involved since the beginning of the job. It soon became clear that what had to be done could only be confirmed, individually on each car, once they were stripped. During myvisit, they were working on train five, and Rob was confident that most of the problems had been discovered. Underframe swan neck repairs: Sole bars are straight, but the underframe also has two steel girders, approximately 300mm deep and 12mm thick, running the length of the car. In the main, as one would expect, the girders are under the floor but, over the bogies, this structure is above floor level and forms the seat risers for the longitudinal seats. The joint that connects the underfloor frame to the above floor frame is known as the swan neck. They are all cracked along the welds. The metal forming the joint is being cut out and replaced by a steel bracket of exactly the right shape machined from solid by WECS Precision of Epsom.

This allows welding to be carried out in locations where stresses are somewhat lower than they were in the original weld locations. The photo of the cut out section shows the cracks; anyone used to welding will not be surprised that they cracked.

Body pillars: Despite coatings applied during manufacture to protect against electrolytic corrosion between aluminium panels and the steel frame and underframes, the accumulation of moisture and cleaning fluids over 40 years has led to corrosion and cracks. These are being cut out and repaired. One of the challenges has been finding fittings that can be used in place of the hot rivets used on the original construction, especially where access is only available on one side. Fortunately, Alcoa Huck BOM fittings (rather like giant pop rivets) came to the rescue.

Body ends: Some of the body end brackets connecting the body end to the underframe have cracked. Investigations showed that many of the underframes were slightly distorted as a result of welding during manufacture and the brackets were adjusted to fit. They have cracked at the point of the adjustment. Rob explained that the replacements are being refitted with a metal putty being used to level the headstock plate.

Floors: The floor fitted during the 1990s refurbishment is a composite of polymer cladding and fire retardant ply on top of stainless steel in doorways and mild steel in seating bays. When the vehicles were stripped, it was found that the cladding was hiding a multitude of sins. The covering and ply is all stripped and the mild steel floor plates in the end seat bays are being replaced. From here, the entire floor is rebuilt with new fire retardant ply and a covering of Tiflex Treadmaster TM7 (see below). A feature of this era of tube train is trapdoors in the floor to access equipment on the underframe. One of the improvements made has been to rationalise the different designs of trapdoors used from 21 to seven.

Roofs: Over 40 years, some of the roof fasteners have become loose and these are being replaced by heavy duty blind fixings and fire retardant Terostat sealant (formerly Sikaflex).

Asbestos: Most of the materials containing asbestos are being replaced. Heat-barrier material is being replaced by Promat DURASTEEL, and the saloon heaters are being replaced by AmTecs low voltage heaters connected in series across the 600V traction supply.

Compressors: The three Mk I trains use a different, less reliable compressor than the remainder of the fleet, and the opportunity is being taken to replace them with compressors recovered from D stock trains (which are being replaced by S stock). This involves welding new mounts onto the underframe.

Drawings: As-built drawings lacked most of the detail necessary to source new parts and, as a result, over 600 new drawings have been produced.

The next challenge is to replace all the removed equipment, including the doors. The doors are a particular issue. Despite putting each door back in the same position from which it was removed, the scale of works on the vehicle has introduced small distortions that necessitate adjustments to each door so that it runs freely without binding.

From here its a case of testing each car, reassembling the vehicles into trains (in the right order!), testing as a train, and returning the trainto Stonebridge Park, from which it can enter service more or less immediately.

Rob told me that the plan is to increase the number of trains in work from one to two. This will have a great benefit in terms of both getting the work done more quickly and in terms of utilisation of the specialist teams who work on the trains. The repair work is due to be completed in 2018.

It was evident that the very high quality work being carried out will, in all probability, provide a structure that is stronger than new. The Bakerloo line structural repairs team are to be congratulated on what they have achieved.

Interior refresh

In parallel with the repair works, the interiors of the Bakerloo trains are being refreshed at Stonebridge Park Depot. Even things as apparently simple as new seat and floor coverings needed significant engineering input from the engineering team based in the LU operations department.

The seats, supplied by Pro Style, Coventry, had both to comply with modern fire standards and be comfortable. The floor had to be cleanable and slip resistant, and there is also a requirement to have a colour contrast between doorways and seating areas, to comply with the RVAR. Conventional wisdom was that the doorways had a higher footfall, would be more prone to dirt and so should be darker than seating areas.

In practice, cleaning around nooks and crannies in seating areas meant that the seated areas were not as clean as they ought to be, so following a trial, the lighter floor was specified for the doorway areas. In addition, to improve slip resistance, a new groove pattern was specified which also contributes to draining water from the floor to the outside.

On a final point, the observant reader might be wondering why the RVAR works were not merged with the weld repairs. It is simply a matter of urgency and timing. The structural repairs were urgent, couldnt be delayed and were under way before the decision was made to extend the life. In contrast, the RVAR works only became necessary as a result of the life-extension decision and a lot of feasibility work had to be completed before the scope could be decided and the works authorised. The teams are making every effort to make these two works streams as integrated as possible.

Thanks to LUs David Caulfield and his team, especially Guy Harris and Rob Bonarski, to Paul Summers from the Asset Strategy and Investment team, and to Sean Long from Operations LU Engineering for their assistance in preparing this article.

Written by Malcolm Dobell

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London Underground train life extension Rail Engineer

Recommendation and review posted by Jack Burke

Be it through literature, film, or television, the idea of life extension has been nothing short of prolific. The concept has become so ingrained in our cultural psyche that most give its presence little thought. In North America, the average life expectancy today is 78 years of age. Even though our current life expectancy is much higher in the West than in other parts of the world, we nonetheless continue to be fascinated, and in some cases, obsessed with the idea of extending our lives beyond what is currently possible. Today when we hear of someone living to 100, it is considered almost miraculous. But as scientific developments continue to progress, the idea of life extension well beyond 100 may become a reality.

The average age a person could live until would increase to roughly 115 years old.

Scientific studies and technology have since developed even further, and brought hope to those seeking a way to extend human life. That being said, there a lot of questions that are raised when we think about life extension. Will everyone have an equal opportunity to benefit from these scientific discoveries? How will this affect the planet? Or society? Because of these questions, the pursuit of life extension is a highly controversial debate that will only become more important with the growth of technological advancements.

via http://www.viralnovelty.net

One of the underlying sentiments behindlife extension is the idea the life isgoodanddeath isbad. For those who are pro-life extension (life extensionists), this perspective is a response to our current experiences and expectations given our limited maximum lifespans. From their perspective, if we were able to live longer lives (and perhaps have better health throughout), this would change how, and if, we perceive deaths as tragic. If we couldlive to 150, woulddying at 90 make us feel the same sadness as it does today?

Another argument amongst life extensionists is thatdeath is a waste sincewe loseaccumulated knowledge, experiences, and memories. Scientist Victoria Stevenswas quoted as saying, "I think the prospect of death it just seems like an awful waste after people spend their lives learning and progressing" (source). For some life extensionists, prolonging human life allows us topreserve the memories and accomplishments of humankind, resulting in positive social consequences. For instance, people may feel a greater sense ofpersonal responsibility and accountabilityfor their actions if they lived longer. If we think about the current state of the environment, this point definitely strikes a chord. If we expect to live longer, we may be more likely to care about how our actions and behaviours influence others, ourselves, and the planet (no more of that, "let the next generation figure it out" mentality).

via http://www.img.rt.com

Not only wouldonly certainpeople in society be able to afford life extension, but certain societieswill be unable to afford itat all.

If humans were to somehow have indefinite life spans, the question of life's meaning may become even more complex and confoundingthan it already is. And what would we do with the time that we have? Though it may seem to open us up to endless possibilities, the reality is that our lives would be similar to how they are now - just longer. We would have the same joys, but also the same struggles.

via http://www.iacpublishinglabs.com

There is also the argument that life extension technologies and treatments will createsocial problemsdue to the likely cost of these services. At first, they will undoubtedly be very expensive, essentially meaning that they wouldonly beaccessible to higher-incomeindividuals.This presents society with a wholemyriad of issues, as only certainpeople in society would be able to afford life extension, and certain societies (such as third worldcountries, for instance) would be unable to afford itat all. This couldcause greater social inequality, and greater social unrest. Disparities between rich and poor individuals, communities, and countries wouldgrow - the implications of which we cannot possibly know or predict. But it's likely safe to say that whatever these implications would be, they would not be positive.

There are alsoenvironmentalconcerns to consider. Our planet is suffering greatly from climate change. Earth is over-populated, and does not have enough natural resources to continue to support the current population (that is growing exponentially each year!) So, if life extension is thrown into the mix, what does this mean? If everyone is able to live longer lives, there would have to be entire generations of human beings that were unable to reproduce in order to avoid further overcrowding our world. We would also have to reevaluate how our resources are distributed and preserved. Needless to say, there would have to be a great deal of thinking and rethinking regarding our planet's population and use of resources in order for life extension to be at all a reasonable pursuit.

via lamcraft.wordpress.com

According to scholar Shai Lavi, one of the biggest changes in the 20th century was the way that death came to be seen as a failure, while medicine and science offered an intelligible hope in the face of a hopeless existence. While life extensionists want to showcase a highly optimistic future, the arguments against extending life are worthy of serious consideration. Our new will to master death goes hand-in-hand with the ways in which we avoid death. But as those in the Death Positive movement have tried to argue, death acceptance can bring us a long way towards fulfillment in life, and even hope in death (to say nothing of the role of religion in this respect).

A shift in our values and ethics will be unavoidable in the face of such a dramatic change in the way we live. Additionally, even if we live until 178 instead of 78, human beings are still just that: humans. Radical life-extensionist Aubrey de Grey acknowledges that humans will always be subject to violence, war, suicide, and accidents (Source). Life extension is not the same as invincibility. The extension of our human lives may makeus feel more than human, but that is what we will remain all the same.

With these arguments in mind, and regardless of which side of the debate you are on, it is important to consider how life extension will affect how human beings think about themselves and each other.

Originally posted here:
The Ethics of Life Extension | TalkDeath

Recommendation and review posted by simmons

Praise for The Gene Therapy PlanA guide to harnessing the power hidden in food to subvert a genetic predisposition for disease. . . . Gaynors informative tome is worth reading. Publishers Weekly

The Gene Therapy Plan identifies how the lives we lead, and in particular, the foods and nutritional supplements we ingest, are a key determining factor in whether latent disease (which most people have to some degree) materialize or stay dormant. By identifying researched nutritional protocols that target specific conditions, and by providing a range of rich case studies from his practice as a leading oncologist and internist, Dr. Gaynor provides insight and an action plan into how the body operates that will benefit medical practitioners and patients alike. Deepak Chopra, M.D.The Human Genome Project promised to create a new era of genetic medicine, new drugs, and therapies to advance human health. But the real awakening has been the understanding of foodreal whole foods, herbs, phytonutrientsas medicine and how it can literally upgrade your biologic software by improving the expression of your genes.In The Gene Therapy Plan Dr. Gaynor makes the healthcare of the future available to you today. If you want to learn how to use food and nutrients to prevent and even reverse most chronic disease, read this book! Mark Hyman, M.D., Director of the Cleveland Clinic Center for Functional Medicine and author of the #1 New York Times bestseller The Blood Sugar SolutionThe Gene Therapy Plan is a comprehensive and practical approach to the science of epigeneticsand how to apply it to your life right now. This book is a godsend that could save your life. Christiane Northrup, M.D., author of the New York Times bestseller Womens Bodies, Womens WisdomA brilliant and important piece of work from one of our most distinguished and creative medical thinkers. Do yourself and your family a huge favor: Read this phenomenally important book and learn why and how you can live a healthier life. Devra Davis, Ph.D., M.P.H., founder and president of the Environmental Health Trust, author of The Secret History of the War on CancerDr. Gaynor is a visionary healer. This is a comprehensive, coherent, practical, and easily digestible resource for all who wish to tip the balance away from disease toward health and wellness. Sheldon Marc Feldman, M.D., Vivian L. Milstein Associate Professor of Clinical Surgery, Columbia University College of Physicians and SurgeonsDr. Gaynor presents a comprehensive strategy for readers to re-orient their diet and lifestyle using everyday activities that can help one live longer, and live better. With The Gene Therapy Plan, Dr. Gaynor brings his own integrative philosophy and practice to readers in an engaging and actionable way. William Li, M.D., president and medical director of The Angiogenesis FoundationDr. Gaynor has and always will be at the forefront of integrative medicine. The Gene Therapy Plan empowers you to take control of your health and life. Mimi Guarneri, M.D., president of the Academy of Integrative Health and Medicine

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The Gene Therapy Plan: Taking Control of Your Genetic ...

Recommendation and review posted by Bethany Smith

What is Gene Therapy?

Certain diseases are caused byfaulty genes which produce defective proteins. The symptoms of genetic disease are the result of subsequent disrupted vital cell processes caused by missing or defective proteins. In theBio Building Blockssection of this web-site, protein synthesisis outlined as the process whereby,genesultimately give rise toproteinswhich are responsible for important cell processes. If a particular gene is defective, its protein product may not be made at all, may work poorly or may behave too aggressively.

For example:Cystic Fibrosis(CF) is caused by amissing or mutated genethat results in adefective cell membrane transport protein. This ultimately results in a build-up of thick mucus in the lungs and the body's airways.As another example,cancersare caused by cells that divide and grow uncontrollably.Particular genes can cause such cell growth to occur if they are defective. Such defective genes are calledoncogenes.

Are we treating the symptom or treating the cause? Historically, genetic disorders have been treated byaddressing the biological eventsthat result from the genetic mutation, as opposed tofixing a defective gene(or genes) the ultimate source of the problem.For example, the treatment of diabetes has historically involved the administration of insulin (a protein), instead of fixing the defective genes in pancreatic cells that actually prevent these cells from producing insulin in the proper amounts, on their own.

Gene therapy is an alternative approach whereby a genetic disorder is treated by inserting or integrating new genes into human cells. Many attempts at gene therapy aim to add a useful gene into a selected cell type to compensate for a missing or defective version. Other efforts aim to instill new properties in the target cell. This latter method is often employed in the treatment of cancer, where toxic genes are added to cancer cells in an effort to eliminate them.For an overview of how a specific gene is located and isolated from its source (so that it can be introduced into the patient) see ourGenetic Engineeringsection.

It should be noted that even the most advanced somatic cell therapy techniques are still in clinical trials, and are not yet approved for general application. Much more research is required to develop safe, reliable gene therapy techniques.

Depending on the cell types affected, gene therapy can be classified into two broad categories: germ-line gene therapy and somatic cell gene therapy.Germ-line therapyoccurs when germ cells (reproductive cells) are altered, meaning that the resultinggenetic changes will be passed on to the patient's offspring. Alternatively,somatic cell gene therapyinvolves the alteration of somatic cells (non-reproductive body cells, like skin, brain or muscle cells). This genetic manipulation willonly affect the individualto which the changes were made. Somatic cell gene therapy is the only type presently being considered in humans.

Suppose a patient is afflicted with a genetic disorder that affected only certain cells in her or his brain. How could she or he be treated using gene therapy so that the therapeutic gene targets only those cells affected by the disorder? One solution is through the use of avector. A vector is simply a "transporter" for the genetic material that allows it to enter the target cell and, depending on the vector type, can cause new genes to be integrated into the host cell genome. Vectors must be administered totarget specific cell types.

There are three principal ways in which vectors can be administered to carry new genes into target cells. The first is calledex vivosomatic gene therapy, wherethe target cells are removed from the body, cultured in the laboratory with a vector, and re-inserted into the body. This process is usually carried out using blood cells because they are the easiest to remove and return.

The second option,in situsomatic gene therapy, occurs when thevector is placed directly into the affected tissue. This process is being developed for the treatment of cystic fibrosis (by direct infusion of the vector into the bronchi of the lungs), to destroy tumours (eg: brain cancer), and for the treatment of muscular dystrophy.

The third option isin vivosomatic gene therapy, where thevector is injected into the bloodstream, and is able to find and insert new genes only into the cells for which it was specifically designed. Although there are presently noin vivotreatments available, a breakthrough in this area will make gene therapy a very attractive option for treatment.In this case the vector designed to treat our hypothetical patient could be injected into a blood vessel in her or his arm and would find its way to the affected brain cells!

Vectors used in gene therapy can be classified as eitherviralornon-viral.

BothDNAandRNAviruses are being developed as vectors for use in gene therapy. Viruses are an excellent choice for use as vectors, because they have gained, through long periods of evolution, the ability to avoid destruction by the human immune system, and the capacity to get their own genetic material inside human cells. As discussed in theBio Building Blockssection, viruses consist of genetic material (DNA or RNA) surrounded by a protective coat made of proteins and occasionally other molecule types as well.

Normally, a virus infects a cell when its genetic material enters it. Once the viral genetic material is inside, it "hijacks" the cell's DNA- and protein-making machinery, causing it to produce new viruses. Some viruses are even capable of integrating their own genetic material into the host cell's genome.

It is the outer protective viral coat that allows the inner genetic material to penetrate the cell. This outer coat also determines the type of cell that a given virus will infect. Once inside, it is the harmful viral genes that actually hijack the cell and eventually cause it to die.

To trick the virus, scientists retain the outer viral coat, but modify the inner genetic material. They remove the harmful genes and replace them with therapeutic ones. Now the virus ispathogenically disabled(it is no longer harmful to the cell it infects) and incapable of reproducing itself. However, it retains its capability to transfer its genetic material to the cells for which its outer coat was designed.The transfer of genetic material by way of a viral vector is calledtransduction.

The structure and mode of infection of retroviruses is discussed in theBio Building Blockssection. Briefly, retroviruses have RNA as their genetic material. These viruses also carry a specialenzymethat, once inside a cell, makes double-stranded DNA from the virus' RNA template. The new DNA becomes incorporated into the host cell's genome. When the "new" chromosomal genes are transcribed, new virus particles are made, which will leave the cell to infect other cells.

Most types of retroviruses are not very harmful to the cell. Even though allviruses to be used as vectors are deactivated,' meaning that their harmful genes are removed, the fact that the types of retroviruses presently being used as vectors are not very harmful in their natural forms means that their use poses less risk than the use of some other viruses. Even if something goes wrong and some of the original retrovirus particles are administered to the patient, they will not cause serious problems.

Themurine leukaemia virus(MuLV) is one of the more popular retroviruses used as a retroviral vector. The reproductive genes in the retrovirus are replaced with the therapeutic gene. When the virus infects the cell,the therapeutic gene gets incorporated into the cell chromosomes. The new gene causes a protein to be produced which is hoped to have some positive therapeutic effects, either providing an otherwise missing protein, or causing the destruction of harmful cells.

There are several challenges that scientists must overcome for effectivein vivotreatment of disease using retroviral vectors. For example, theviruses must be capable of targeting only those cells affected by the disorder. If this were the case, they could be injected directly into the bloodstream (in vivogene therapy) where they would become dispersed throughout the body, but would only transduce those cells for which they were designed. Presently, retroviral vectors are not terribly specific, meaning that many cells not intended for the transfer of the gene are transduced by the virus, which reduces the transfer to the targeted cell population.

To understand how viruses can be made to be more specific, we should considerhow viruses "choose" the cells they infect. A virus must bind to specific surface receptor molecules to gain entry into a cell. To this end, retroviruses have outer envelope proteins that fit perfectly into certain receptors on specific cells. The MuLV virus binds to cells containing a receptor called theamphotropic receptor. The problem is that a broad range of cell types possess the amphotropic receptor. This means that the MuLV virus, in its natural form, can infect all of these cell types, most of which are likely not the target of the therapy!

To make retroviral vectors more specific about the cells they invade, scientists are experimenting with ways ofreplacing or modifying the outer viral proteins, so that they fit into more rare receptors that appear only on specific cell types being targeted for therapy.Another approach has been toadd new proteinsto the outer viral envelope which either better recognize the target cell, or better recognize the region of the body where the target cells are located.

Another challenge is toengineer retroviral vectors to transducenon-dividingcells. Most retroviruses target actively dividing cells, which makes them ideal for the treatment of rapidly dividing tumour cells, but not in situations where a therapeutic gene is to be introduced into a non-dividing cell, like in the treatment of cystic fibrosis mentioned above. Those few retroviruses that have the ability to infect non-dividing cells are the harmful ones (HIV, the virus that results in AIDS, is one of them). HIV viruses (with their harmful genes removed) cannot be used as vectors, because even with the removal of these genes, there is still a possibility that the virus might become harmful again through a process called recombination. To virtually eliminate the possibility that harmful viruses are produced in this way, while still harnessing the capability of HIV to transduce non-dividing cells, scientists are experimenting with the development of hybrid vectors, made up mostly of other retroviruses and which contain very small and harmless parts of the HIV virus.

As of April, 1998, there was only one vector-based therapeutic technique in the final clinical trial stage(called Phase III). This technique employs a retroviral vector called G1TkSvNa for the treatment ofglioblastoma multiforma, a malignant brain tumour. The treatment is an in situ therapeutic technique, where mouse cells capable of producing and secreting the vector are injected into the tumour.The secreted vectors infect only those cells that are rapidly dividing, meaning only the tumour cells and the vessels supplying blood to the tumour are transduced. The gene transduced into the tumour cells gives rise to a protein (calledHerpes Simplex Thymidine Kinaseor HSTk).Fourteen days later, a drug called ganciclovir is injected into the patient, which is toxic to any cell that incorporates it into its DNA. Only the cells containing HSTk (the tumour cells) are capable of incorporating ganciclovir into their DNA and these cells are therefore selectively killed off.

Adenoviruses are DNA viruses that are able to transduce a large number of cell types, including non-dividing cells. Adenoviruses also have the capacity to carry long segments of added genetic information. In addition, it is fairly easy to produce large amounts of adenoviruses in culture. Adenoviruses, in their natural form, are not very harmful, typically causing nothing more serious than a chest cold in otherwise healthy people. This means that their use as vectors is quite safe. For all these reasons, adenoviruses are currently the most widely used DNA vectors for experiments inin situgene therapy.Research is currently under way using adenoviral vectors for the treatment of several cancers and cystic fibrosis.

The size of the adenovirus protein coat is just large enough to fit the original viral DNA inside. As a result, for every new therapeutic gene to be inserted into the viral genome, a corresponding piece of the old viral DNA must be removed.To make room for the new therapeutic DNA, a region of the old viral DNA called E3 is sometimes removed. However, removing the E3 region has drawbacks, because it codes for a protein that suppresses the human immune response against the vector. Without the E3 region, the virus is more susceptible to the immune system and is more likely to be destroyed before it has served its purpose.

Adenoviral vectors send their DNA to the nucleus, butthe DNA does not get incorporated into the host cell's chromosomes. For this reason, the viral DNA has a finite lifetime within the cell before it is degraded, meaning that the added genes are effective only temporarily. Treatments for chronic conditions like cystic fibrosis, therefore, would need to be repeated periodically, perhaps on a monthly or yearly basis. On the other hand, the transient nature of therapeutic gene expression is useful when the added genes are needed temporarily to induce an immune response to a cancer or pathogen.

Among the other virus types being explored as vectors are theadeno-associated virus(AAV) and theherpes simplex virus(HSV). Both are DNA-based viruses. AAV integrates its genetic material into a host chromosome and cause no diseases in humans. However, because AAV are small, they cannot accommodate large genes. HSV vectors do not integrate their genes into the host genome. They tend to target neurons and thus have the potential for use in the treatment of neurological disorders.

The use of non-viral vectors can involve a direct injection ofplasmid DNAor mixing plasmid DNA with compounds that allow it to cross the cell membrane and protect the DNA from degradation. These methods are currently less efficient than the use of viral vectors. However, unlike disabled viruses which have the possibility of changing spontaneously and causing disease, non-viral vectors possess no viral genes and therefore cannot cause disease.

Liposomes are small, hollow spheres of fatty molecules that are capable of carrying DNA inside of them.A liposome can fuse with the cell membrane, releasing its contents into the cell interior.

Plasmid DNA containing the therapeutic gene is incubated with the empty liposomes under specific conditions. The negatively charged DNA binds to the positively charged (calledcationic) liposomes and the plasmids are absorbed. Liposomes containing plasmid DNA are calledlipoplexes.The lipoplexes can subsequently enter the cells of interest, and thus introduce the therapeutic DNA into the cells.

Experiments have been carried out where lipoplexes have been injected into tumours. The lipolexes contained a gene that gives rise to a protein that is recognized by the human immune system. Theoretically, thesegenes should cause the tumour cells to express the recognizable protein on their surface, which will mark the cells for destructionby the immune system.

The use of lipoplexes for the treatment of cystic fibrosis is currently being studied as well. The cause of the illness is a defective gene which causes a particular protein in the patient's lung cells to be defective. The lipoplexes that are administered using an aerosol spray into the patient's lungs, contain the gene for a functional version of the protein.

Lipoplexes are not as efficient as viral vectors in introducing genes into cells. To improve their efficiency, scientists are attempting to incorporate some viral proteins into the outer surfaces of lipoplexes. In particular, the viral proteins that recognize and bind to specific molecules on the host cell's surface, are being incorporated.

Muscle cells have been shown to be capable of taking up and expressing plasmid DNA. This raises the possibility that plasmid DNA injected into muscles could stimulate the production by muscle cells of a therapeutic protein. This protein could then be secreted into the bloodstream and to the rest of the body. For example, the gene coding for erythropoietin (a protein which helps stimulate the production of red blood cells) has been experimentally injected into animal muscles with some success. Such a treatment would be useful to patients after chemotherapy or radiation therapy.

In addition,plasmid DNA shows promise for use in vaccines, stimulating protective immune responses against diseases like herpes, AIDS or malaria. When the plasmid DNA is injected into muscles, it enters muscle cells and as a result, causes the cells to produce the proteins that correspond to the genes the plasmids contain. The immune system will then learn to recognize the new proteins and will destroy them if they are encountered in the future. Experiments are currently under way where plasmids containing genes for viral coat proteins are injected, in attempt to make the immune system recognize these viruses, so that it will attack and destroy them if they are ever encountered.

As discussed in theBio Building Blockssection, viruses hijack cellular machinery to produce their own proteins and to replicate their genetic material, which results in the production of new viruses.One of the potential uses of antisense technology is to prevent viruses that infect a host cell from producing their own proteins. This would, in turn, prevent their replication.

Recall that proteins are constructed through atwo step process. In the first step,DNA is transcribed to produce messenger RNA(mRNA). The second step involves thetranslation of the mRNA to make a protein. Antisense drugs interact with mRNA, preventing them from being translated into their corresponding protein.

An mRNA molecule is a chain of nucleotides, that gets "read" by a ribosome in the synthesis of a protein. An antisense drug is anoligonucleotide(a relatively small, single stranded chain of nucleotides) that iscomplementaryto a small segment of a target mRNA molecule. When the drug comes into contact with its complementary mRNA, it binds to the mRNA in the same way as the two strands of a DNA molecule bind together.This makes the mRNA "unreadable" by the ribosome, and so no protein is produced.

Because an antisense drug is designed to be complementary to a particular mRNA sequence that is specific to a particular virus' mRNA, it will not interfere with any of the host cell's naturally produced mRNA, meaning that the side effects of the drug are minimal.

At the end of August, 1998, the US Food and Drug Administration (FDA) approved a drug calledformivirsenfor the treatment of cytomegalovirus (CMV) retinitis in patients with AIDS.This makes formivirsen the first antisense drug on the market.Formivirsen blocks the replication ofcytomegalovirus(CMV) which causesretinitis, an eye infection leading to blindness that mainly affects AIDS patients. The drug is periodically injected into the patient's eye, and is claimed to cause only mild side-effects as compared to some other antiviral drugs.

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GeoGene: Gene Therapy, What it is, The process and Vectors ...

Recommendation and review posted by sam

Bone marrow suppressionSynonymMyelotoxicity, myelosuppression

Bone marrow suppression also known as myelotoxicity or myelosuppression, is the decrease in production of cells responsible for providing immunity (leukocytes), carrying oxygen (erythrocytes), and/or those responsible for normal blood clotting (thrombocytes).[1] Bone marrow suppression is a serious side effect of chemotherapy and certain drugs affecting the immune system such as azathioprine.[2] The risk is especially high in cytotoxic chemotherapy for leukemia.

Nonsteroidal anti-inflammatory drugs (NSAIDs), in some rare instances, may also cause bone marrow suppression. The decrease in blood cell counts does not occur right at the start of chemotherapy because the drugs do not destroy the cells already in the bloodstream (these are not dividing rapidly). Instead, the drugs affect new blood cells that are being made by the bone marrow.[3] When myelosuppression is severe, it is called myeloablation.[4]

Many other drugs including common antibiotics may cause bone marrow suppression. Unlike chemotherapy the affects may not be due to direct destruction of stem cells but the results may be equally serious. The treatment may mirror that of chemotherapy-induced myelosuppression or may be to change to an alternate drug or to temporarily suspend treatment.

Because the bone marrow is the manufacturing center of blood cells, the suppression of bone marrow activity causes a deficiency of blood cells. This condition can rapidly lead to life-threatening infection, as the body cannot produce leukocytes in response to invading bacteria and viruses, as well as leading to anaemia due to a lack of red blood cells and spontaneous severe bleeding due to deficiency of platelets.

Parvovirus B19 inhibits erythropoiesis by lytically infecting RBC precursors in the bone marrow and is associated with a number of different diseases ranging from benign to severe. In immunocompromised patients, B19 infection may persist for months, leading to chronic anemia with B19 viremia due to chronic marrow suppression.[5]

Bone marrow suppression due to azathioprine can be treated by changing to another medication such as mycophenolate mofetil (for organ transplants) or other disease-modifying drugs in rheumatoid arthritis or Crohn's disease.

Bone marrow suppression due to anti-cancer chemotherapy is much harder to treat and often involves hospital admission, strict infection control, and aggressive use of intravenous antibiotics at the first sign of infection.[citation needed]

G-CSF is used clinically (see Neutropenia) but tests in mice suggest it may lead to bone loss.[6][7]

GM-CSF has been compared to G-CSF as a treatment of chemotherapy-induced myelosuppression/Neutropenia.[8]

In developing new chemotherapeutics, the efficacy of the drug against the disease is often balanced against the likely level of myelotoxicity the drug will cause. In-vitro colony forming cell (CFC) assays using normal human bone marrow grown in appropriate semi-solid media such as ColonyGEL have been shown to be useful in predicting the level of clinical myelotoxicity a certain compound might cause if administered to humans.[9] These predictive in-vitro assays reveal effects the administered compounds have on the bone marrow progenitor cells that produce the various mature cells in the blood and can be used to test the effects of single drugs or the effects of drugs administered in combination with others.

See more here:
Bone marrow suppression - Wikipedia

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CRISPR. What is it? And why is the scientific community so fascinated by its potential applications? Starting with its definition, we explain how this technology harnesses an ancient bacteria-based defense system and how it will impact the world around us today.

Imagine a future where parents can create bespoke babies, selecting the height and eye color of their yet unborn children.In fact, all traits can be customized to ones preferences: the size of domestic pets, the longevity of plants, etc.

It soundslike the backdrop of a dystopian science fiction novel. Yet some of this isalready happening.

Since its initial discovery in 2012, scientists have marveled at the applications of CRISPR (also known as Cas9 orCRISPR-Cas9).

And with a Jennifer Lopez-produced bio-terror TV drama called C.R.I.S.P.R. on the horizon, CRISPR has reached a new peak in interest from outside the scientific community.

CRISPR may revolutionize howwe tackle some of the worlds biggest problems, like cancer, food shortages, and organ transplant needs.Recent reports even examineits useasa highly efficient disease diagnostics tool. But, as with any new technology, it may also cause new unintended problems.

Changing DNA the code of life will inevitably come with a host ofimportant consequences. But society and industry cant have this conversation without understanding the basics of CRISPR.

In this explainer, we dive into CRISPR, from a simple explanation of what exactly it is to its applications and limitations.

CRISPR is adefining feature of the bacterial genetic code andits immune system,functioningas a defense system that bacteria use to protect themselves against attacks from viruses. Its also used by organisms in the Archaea kingdom (single-celled microorganisms).

The acronym CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. Essentially, it is a series ofshort repeating DNA sequences withspacers sitting in between them.

In short,bacteria usethese geneticsequences to remember each specific virus that attacks them.

They do this byincorporatingthe virus DNA into their own bacterial genome. Thisviral DNA ends up as the spacers in the CRISPR sequence.This method then gives thebacteria protection or immunity when a specific virus tries to attack again.

Accompanying CRISPR are genes that are always located nearby, called Cas (CRISPR-associated) genes.

Once activated, these genes make special proteins known as enzymes that seem to have co-evolved with CRISPR. The significance of these Cas enzymes is their ability to act as molecular scissors that can cut into DNA.

To recap: in nature,when a virus invades bacteria, its unique DNA is integrated into a CRISPR sequence in the bacterial genome. This means that the next time the virus attacks, the bacteria will remember it and sendRNA and Cas to locate and destroy the virus.

While there are other Cas enzymes derived from bacteria that cut out viruses when they attack bacteria, Cas9 is the best enzyme at doing this in animals. The widely-known term CRISPR-Cas9 refers to a Cas variety beingused to cut animal (and human) DNA.

Inharnessing this technology, researchers have added a new step: after DNA is cut by CRISPR-Cas9, a new DNA sequence carrying a fixed version of a gene can nestle into the new space. Alternatively, the cut can altogether knock out ofa particular unwanted gene for example, a gene that causes diseases.

Oneway to think about CRISPR-Cas9 isto compare it to theFind & Replace function in Word: itfinds thegenetic data (or word)you want to correct and replaces it with new material. Or, as CRISPR pioneer Jennifer Doudna puts it in her book A Crack In Creation: Gene Editing and the Unthinkable Power to Control Evolution, CRISPR is likea Swiss army knife, with different functions depending on how we want to use it.

CRISPR research has moved so fast that its already gone beyond basic DNA editing. In December 2017, the Salk Institute designed a handicapped version of the CRISPR-Cas9 system, capable of turninga targeted gene on or off without editing the genome at all. Going forward, this kind of process could ease the concerns surrounding the permanent nature of gene editing.

These are the 3 key players that help theCRISPR-Cas9 tech do its work:

Below, we illustrate how these parts come together to create a potential therapy.

Please click to enlarge.

The guide RNAserves as the GPS coordinates for finding the piece of DNA you want toedit and zeroes in on the targeted part of the gene. Once located, Cas9, the scissors, makes a double stranded break in the DNA, and the DNAyou want to insert takes its place.

The implications for this are vast.

Yes, this technology will disrupt medical treatment. But beyond that, it could also transform everything from the food we eat to the chemicals we use as fuel, since these may be engineered through gene technology as well.

Feng Zhang, PhD, from the Broad Institute of MIT and Harvard, describedCRISPR using a helpful nursery rhyme. We can imagine a certainDNA sequence that is fixed in this way:

Twinkle Twinkle Big Star Twinkle Twinkle Little Star

In this process:

The CRISPR sequence was first discovered in 1987. But its function would not be discovered until 2012.

Keypeople involved in the initial discovery of the bacterial CRISPR-Cas9 systems function include Jennifer Doudna, PhD at University of California, Berkeley, and French scientist Emmanuelle Charpentier, PhD. Through their strategic collaboration, they ushered in a new era of biotechnology.

Another important figure is Feng Zhang, PhD, who was instrumental in figuring out CRISPRs therapeutic applications using mice and human cells in 2013.Harvard geneticist George Church, PhDalso contributed to early CRISPR research with Zhang.

All four researchers went on to play crucial roles in setting up someof the most well-funded CRISPR therapeutic startups, includingEditas Medicine, CRISPR Therapeutics, and Intellia Therapeutics.All 3 of these companiesIPOed in 2016 and are in the drug discovery/pre-clinical stage of testing their respective CRISPR therapeutic candidates for various human diseases.

Before CRISPR was heralded asthegene editing method, two other gene-editing techniques dominated the field: Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). Research efforts using these tools are still ongoing.

Like CRISPR, these toolscan each cut DNA. Thought they are generally more difficult to make and use, these tools do offer their own advantages:

Each also has vital therapeutic applications.

Biotech companyCellectis uses the TALEN gene editing technology to make CAR-T therapies for leukemia, whileSangamo BioSciencesmakes ZFNs that can disable a gene known to be key in HIV infection.Notably, each of these companies hold key IP rights to these specific gene-editing methods, which could make it difficult for other biotech companiesto use these tools.

Meanwhile, CRISPR has certainly stolen the spotlight as of late, due to its efficiency, flexibility, and cheap price tag. Itsplausible that CRISPR could face similar IP issues and there are already some IP controversies going on but with such vast applications for this system, research on multiple fronts seems to be moving forward fast.

Every industry can harnessCRISPR as a tool: itcan create new drug therapies for human diseases, help farmers grow pathogen-resistant crops, create new species of plants and animals and maybe even bring back old ones.

Since the initial discovery of CRISPR as a gene-editing mechanism, the list of applications has grown rapidly. Though still in early stages, animal models (i.e. lab animals) have provided key insights into how we may be able to manipulate CRISPR.

Mice have been especially telling when it comes to CRISPRs therapeutic potential. As mammals sharing more than 90% of our human genes, mice have been used as ideal test subjects.

Experiments on mice haveshown that CRISPR can disable a defective gene associated with Duchenne muscular dystrophy (DMD), inhibit the formation of deadly proteins involved in Huntingtons disease, and eliminate HIV infection.

In 2015, Chinese scientists created two super muscular beagles by disabling the myostatin gene, which directs normal muscle development. In the absence of thegene, the beagles displayed muscular hypertrophy, creating dogs which were visibly much more muscular than non-genetically modified ones.

Other CRISPR animal trials haveranged from genetically engineering long-haired goats for higher production of cashmere to breeding hornless cows to avoid the painful process of shearing horns off.

Compared to research involving animals, CRISPR trialsthat edit human DNA have movedmore slowly, largely due to the ethical and regulatory issues at play.

Given the permanent nature of altering a humans genome, the FDA is approaching CRISPR cautiously. Some scientists have even proposed a moratorium on CRISPR trials untilwe have more information on the potentialimpact on humans.

In the US and Europe, 2018 will be the year for CRISPR human trials.

Currently (as of 2/13/18),the University of Pennsylvania is awaiting the FDAs final approval to start a study that would evaluate the safety of using CRISPR for patients with multiple myeloma, melanoma, and sarcoma.

Europe may also see its first human CRISPR study in 2018 withCRISPR Therapeutics study focused on a blood disorder known as beta-thalassemia,which results in abnormal red blood cell production.

While clinical trials involving patient participation are still awaiting regulatory approval, CRISPR has already been applied to both viable and non-viable human embryos.

For example, in August 2017, a team lead by reproductive biologist Shoukhrat Mitalipov of Oregon Health and Science University received private funding to use CRISPR-Cas9 to target a mutation in viable human embryos that causes the thickening of heart muscles. The altered embryos came back 72% mutation-free in the lab (higher than theusual 50% chance of inheritance).

Some critics say the gene editing of embryos is unethical, even if the edited embryos are not destined for transfer and implantation. This type of testing currently does not receive federal funding, but instead relies on private donor funding.

On the other side of the world, Chinese researchersoperate under a different regulatory framework. Some hospital ethics committees can approve studies in as little as one day, with no need to seek approval from a federal agency.

Since 2015, China has been conductinghuman trials using CRISPRto combat various cancers, HIV, and HPV. It is the only country in the world toconduct human trials thus far.

According to ClinicalTrials.Gov, there are 10 active or upcoming CRISPR therapy trials in China, targeting advanced cancers like stage 4 gastric and nasopharyngeal carcinomas. So far results are only anecdotal, and while some participants tumors shrank, no formal results have been made available.

Although possible long-term side effectsarent fully understood,CRISPR is already an option for some patients in China who have exhausted all of the conventional treatments.

Potential high impact industries for CRISPR include medicine, food, agriculture, and the industrial biotech space. BecausetheCRISPR-Cas9 gene-editing system issoeasy to make and use, researchers from a range of scientific disciplines can access it to genetically engineer the organism of their choice.

The future of medicine will be written with CRISPR.

The current drug discovery process is long, given the need to ensure patientsafety and gain a thorough understanding of unintended effects.Moreover, current US regulatory policies often result in a decades-long development process.

However, teamsusing CRISPR can bringcustomized therapies to market more quickly than was previously dreamed, speeding upthe traditional drug discovery process.

Timeline of drug development. Credit: PhRMA

CRISPRscheap price tag and flexibilityallows accurate and fast identification of potential gene targets for efficient pre-clinical testing. Because itcan be used to knock out different genes, CRISPR givesresearchers a faster and more affordableway to study hundreds of thousands of genes to see which ones are affected by a particular disease.

Of course, alongwith providing a more streamlined drug development process, CRISPR offers the possibility of new ways to treat patients.

For example,monogenic diseasesdiseases caused by a mutation ina single gene present an attractive starting point for CRISPR trials. The nature of these illnesses provides an exact target for the treatment: the problematic mutation on a single gene.

Blood-based, single-gene diseases like beta-thalassemia or sickle cell are alsogreat candidates for CRISPR therapy, because of their ability to be treated outside of the body (known as ex-vivo therapy). A patients blood cells can be taken out, treated with the CRISPR system, then put back into the body.

An earlyapplication of CRISPR was pioneered by yogurt company Danisco in the 2000s, when scientists used an early version of CRISPR to combat a key bacterium found inmilk and yogurt cultures (streptococcus thermophilus) that kept getting infected by viruses. At that point, the ins and outs of CRISPRwere still unclear.

Fast forward to today, when climate change will further increasethe need to use CRISPR to protect the food and agriculture industries against new bacteria.For example, cacao is becoming difficult to farm as growing regions get hotter and drier. This environmental change will further exacerbate the damage done by pathogens.

If youve eaten yogurt or cheese, chances are youve eaten CRISPR-ized cells.

Rodolphe Barrangou, former Daniscoscientist & Editor-in-Chief of The CRISPR Journal

To combat this issue, the Innovative Genomics Institute (IGI) at UC Berkeley is applying CRISPR to create disease-resistant cacao. Leading chocolate supplier MARS Inc. is supporting this effort.Gene editing can make farming more efficient. It can curb global food shortages for staple crops like potatoes and tomatoes. And it can create resilient crops, impervious to droughts and other environmental impacts.Regulators have shown little resistanceto gene-edited crops, and the United States Department of Agriculture (USDA) in particular is not regulating the space. This is largely because when CRISPR is applied to crops, theres no foreign DNA being added: CRISPR is simply used to edit a crops own genetics to select for desirable traits.In 2016, the white button mushroom, modified to beresistant to browning, became the first CRISPR-edited organism to bypass USDA. In October 2017, it was announced that agriculture company DuPont Pioneer and the Broad Institute would collaborate for agriculture researchusing their CRISPR-Cas9 intellectual property.

InSeptember 2017, biotech company Yield10 Bioscience got approval for its CRISPR-edited plantCamelina sativa (false flax), which hasenhanced omega-3 oil and is used to make vegetable oil and animal feed.

These are indicationsthat newbreeds of crops could reachmarketsmuch faster than previously thought. Without USDA oversight, these items and other food products could go into production relatively quickly.

This will impact the food we eat, as food items are edited tocarry more nutrients or to last longer on grocery shelves.

Another area currently generating buzz isthe production of leaner livestock.

In October 2017, scientists at the Chinese Academy of Sciences in Beijing used CRISPR to genetically engineer pig meat that had 24% less body fat.

Researchersdid this by inserting a mouse gene into pig cellsin order tobetter regulate body temperature.Although this example technically makes the result a GMO product, it may not be too long before pigs genes are used for the same purpose.

Future versionsof this technology applied to human nutrition will be one area to look out for.

Another key, but less obvious, use of CRISPR lies is in the industrial biotech space. By re-engineering microbes using CRISPR,researcher can create new materials.

How is this relevant to society at large?

From an industrial standpoint, this is big for modifying and creating new chemical products. We can alter microbes to increase diversity, create new bio-based materials, and make more efficient biofuels.From active chemicals in fragrances to those involved in industrial cleaning, CRISPR could have agreat impact here by creating new and more efficientbiological materials.

Jennifer Doudnas first CRISPR startup, Caribou Biosciences, was founded in 2011 for non-therapeutic research purposes across industries. It is one of the key companies providing various industries with the tools to use CRISPR fora range of purposes.

CRISPRs list of potential benefits is a long one. But the technology also brings with it a number of limitations.

Possible unintended effects and all the unknown variables are some of the drawbacks to this newtechnology, while newethicalquestions and controversies are also emerging as human trials near.

When using CRISPRfor human therapies, safety is the biggest issue. As with any new form of technology, researchers are unsure of the entire range of CRISPRs effects.Off-target activity is the main concern here. A single gene editcould cause unintended activity somewhere else in the genome. A possible consequence of this is abnormal growth of tissues, leading to cancer. As more research uncovers new details, this could result in more refined, precise gene targeting.

Another issue is the possibility of mosaic generation.After a CRISPR treatment, a patient could have a mix of both edited and unedited cells a mosaic. As cells continue to divide and replicate, some cells may get repaired, while others wont.

Finally, immune systemcomplications mean that these interventions and therapies may trigger an undesired response froma patients immune system.Early research shows theimmune system may dispose of Cas enzymes before they achieve their purpose, or may have an averse reaction resulting in side effects like inflammation. (In 1999, a patient in the US died of a severe immune reaction, instilling more caution in researchers when it comes to CRISPR trials.)

However, all three of these limitations have some possible solutions.

Different enzymes (molecular scissors) or more precise delivery vehicles can reduce off-target activity. If modified stem cells in egg or sperm (i.e. cells that can become every cell in the human body) are edited, mosaics can be avoided.

With the immune system issue, researchers can isolate different Cas proteins from more obscure bacterial strains that humans dont already have an adaptive immunity to in order to circumvent an unwanted immune response. Meanwhile, ex-vivo therapies, wherescientists take a patients blood cells out of the body and treat them before infusing them back in, can also helpbypass the immune system.

One potential big limitation for CRISPR isthat CRISPR-Cas9 system lacks surgical precision. The Cas enzyme cuts both strands of the DNA double helix, and this double-stranded breakcreates worries over the precision of the cut.

Repairing a defective gene would be like finding a needle in a haystack and then removing that needle without disturbing a single strand of hay in the process.-Jennifer Doudna

While currently the Cas9 enzyme gets the most attentionas the enzyme doing the cutting, scientists are actively pursuing alternatives to find better candidates.

Alternative options include asmaller version of Cas9, or a different enzyme entirely: Cpf1, whichhas become popular due to its easy transport to the targeted DNA location.

Besides using other Cas enzymes, alternate delivery vehiclesfor therapeutic genes are another option. Harmless engineered viruses can carry therapeutic genes to the site of mutation, while lipid nanoparticles can avoid immune system detection, avoiding an immune reaction. Both options present promising avenues of research.

Whentechnology can alter the code of life, its implications are far-reaching as are its controversies. Here we outlinea few of the main controversies surroundingCRISPR.

Originally posted here:
What Is CRISPR? - CB Insights

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Genetic testing is a type of medical test that identifies changes in chromosomes, genes, or proteins. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a persons chance of developing or passing on a genetic disorder. More than 1,000 genetic tests are currently in use, and more are being developed.

Several methods can be used for genetic testing:

Chromosomal genetic tests analyze whole chromosomes or long lengths of DNA to see if there are large genetic changes, such as an extra copy of a chromosome, that cause a genetic condition.

Genetic testing is voluntary. Because testing has benefits as well as limitations and risks, the decision about whether to be tested is a personal and complex one. A geneticist or genetic counselor can help by providing information about the pros and cons of the test and discussing the social and emotional aspects of testing.

See the original post:
What is genetic testing? - Genetics Home Reference - NIH

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