TB-Drugome May Pave Way for Anti-TB Drug Discovery

A study by researchers at the University of Leeds, United Kingdom, and the University of California, San Diego, School of Medicine, United States, showed that one third of the existing drugs examined have potential to be repurposed to treat TB. The scientists used a novel computational strategy to investigate whether any existing drugs could bind to any of the approximately 50 percent of proteins in the M. tuberculosis (Mtb) proteome with decipherable three-dimensional structures. The results linked hundreds of federally approved drugs to more than 1,000 proteins in Mtb. The investigators then constructed a complex network of drug-target interactions, a TB-drugome, available to all scientists. Although the new process of drug discovery is promising, one of the research scientists, Lei Xie of the Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California, San Diego, warned that only experimentation can validate the most promising drug-target combinations, and there will be many failures along the way. Also, Sarah Kinnings of the University of Leeds noted that drugs confirmed to bind to Mtb proteins might need to be modified, to increase their ability to penetrate the bacterial cell membrane, reduce the required dosage, and improve other pharmacological properties. This article was published in the journal PLoS Computational Biology 6(11): e1000976. doi:10.1371/journal.pcbi.1000976

Prevalence and Genetic Structures of Streptococcus pnuemoniae Serotype 6D, South Korea

EID Journal Home>Volume 16 Number 11 November 2010

Abstract:
To determine prevalence and genetic structures of new serotype 6D strains of pneumococci, author examinded isolates from diverse clinical specimens in South Korea during 1991 to 2008. Fourteen serotype 6D strains accounted for 10.4% of serogroup 6 pneumococci from blood, sputum, nasopharynx and throat samples. Serotype 6 D strains consisted of 3 sequence types.
Streptococcus pneumoniae is a common cause of invasice infection in infants, children and adults. The polysaccharide capsule of S.pneumoniae is the major virulence factor that protects the organism from host phagocytosis. Recently, 2 new serotypes of serogroup 6 pneumococci, 6C and 6D were genetically and biochemically characterized. Serotype 6C was identified in 2007 on the basis of its distinct binding patterns with 2 monoclonal antibodies; serotype 6C had previously been typed as 6A according to the standard quellung reaction.Serotype 6C produces glucose in the place of galactose in the 6A capsular polysaccharide and has the wciNbeta gene, which is about 200 bp shorter than the corresponding wciN gene in 6A. After the discovery and characterization of 6C through genetic and biochemical studies, a new experimental serotype, 6X1(later named 6D) was created by mutating the critical nucleotide in the wciP gene of the 6C capsule gene locus or by inserting the wciNbeta gene into the 6B capsule gene locus. However, this putative serotype, 6D was thought to not exist in nature until recently, when 2 studies found 6D strains in nasopharyngeal aspirates from children in Fiji during 2004 to 2007 and in 2 nasopharyngeal aspirates from children in South Korea in 2008. Although serotype 6C has only recently been described, several studies indicate that serotype 6C pneumococci have been circulating in many countries, including the US, the Netherlands, Australia, Israel and South Africa. However, reports of naturally occurring serotype 6D pneumococci are limited.
Author investigated the prevalence of serotypes of 6C and 6D in 2 collections of pneumococci isolated from clinical specimens in South Korea, we compared the genetic diversity and antimicrobial drug susceptibility patterns of the 4 serotypes, 6a, 6B, 6C and 6D.

The Study:
Of the 2 collections of pneumococcal isolates, the first consisted of 587 clinical specimens obtained from infants and children at Seoul National University Children's Hospital, Seoul, South Korea, from May 1991 to May 2008. The second collection consisted of 225 clinical specimens obtained from adults at 2 participating hospitals in Seoul from March 2004 through August 2007. When >1 isolate was recovered from the same person, only the initial isolate was included in the study. From these 2 sample collections (n=812), author redetermined serotypes for 134 isolates previously assigned to serogroup 6.
Serotyping was performed by uisng the quellung reaction with antiserum for serogroup 6, factor 6b and factor 6c(Statens Serum Institute,Copenhagen, Denmark).To assign serotypes 6C and 6D we screened all strain for wciNbeta and wciP-6B by using two simple PCRs and subsequent sequencing analysis. The wciN gene was amplified with the forward primer (5106) 5-TAC CAT GGA GGG TGG AAT GT-3 and reverse primer (3101) 5-CCA TCC TTC GAG TAT TGC-3, resulting in product size of 1.8 kb for serotypes 6C and 6D for the wciNbeta gene. The wciP gene was amplified by using the forward primer 5-AAT TTG TAT TTT ATT CAT GCC TAT ATC TGG-3 and the reverse primer 5-TTA GCG GAG ATA ATT TAA AAT GAT GAC TA-3. Presence of wciNbeta and wciP-6B was confirmed by sequencing analysis. A characteristic of 6B wciP is the presence of an A at nucleotide position 584 (according to the sequence of wciP), which creates a codon for asparagine at residue 195 of the 6B wciP protein. Antimicrobial drug susceptibility testing, multilocus sequence typing -MLST and eBURST analyses were performed.

Conclusions:
We identified 14 naturally occuring serotype 6D strains among 134 serogroup 6 pneumococci collected from diverse clinical specimens in South Korea during 1991 to 2008. The prevalence rate of serotype 6D among serogroup 6 isolates was 10.4%, slightly higher than that of serotype 6C(4.5%). Although serotype 6D was only recently discovered, we demonstrated that serotype 6D strains have been circulating since at least 1996. Serotype 6D was identified from various clinical sources, including blood, sputum, throat swab and nasopharynx specimens, contrasting with findings of 2 previous studies.
The genetic structures of serotype 6D pneumococci in the MLST database http://www.mlst.net/ were single isolates o f ST4241(Australia); ST982, ST4190, ST5085 and ST5086 (China) and 2 isolates of ST282 (South Korea). Of those, 3 strains from China (ST982, ST5085 and ST5086) were closely related to the ST3171 strain from South Korea. This cluster of serotype 6D strains was associated with serotype 6A and 6B isolates from 3 countries in Asia. A single isolate of ST4241 was related to STs associated mostly with serotype 6B, but the ST4170 strain did not seem to be linked to other STs. This study demonstrated that 7 serotype 6 D strains of ST189 and 3 serotype 6D stains of ST282 were related to clonal complex 81, which had previiously been associated with many other global serotypes, such as 23F, 19F and 19A. Although the mechanism is not completely clear, available data indicate that capsular switching from serotypes 6A, 23F, 19F or 19A to serotype 6D is possible, this switching could occur in addition to replacement of the wciNbeta gene into the 6B capsule gene locus. A previous study indicated capsular switching as the possible event for formation of serotype 6C isolates.
In a recent study, factor 6D antiserum was validated for accurate serotyping of 6C and is now commercially available, but antiserum for detection of 6D has not yet been developed. Further studies will be required to investigate the prevalence and genetic relatedness of serotype 6D pneumococci in different countries and to evaluate the effect of pneumococcal conjugate vaccine on serotype distribution.

QUELLUNG REACTION:
The Quellung reaction is a biochemical reaction in which antibodies bind to the Bacterial capsule of Streptococcus pneumoniae, Klebsiella pneumoniae, Neisseria meningitidis and Haemophilus influenzae and thus allow them to be visualized under a microscope. If the reaction is positive, the capsule becomes opaque and appears to enlarge.
Quellung is the German word for "swelling" and describes the microscopic appearance of pneumococcal or other bacterial capsules after their polysaccharide antigen has combined with a specific antibody. The antibody usually comes from a bit of serum taken from an immunized laboratory animal. As a result of this combination, and precipitation of the large, complex molecule formed, the capsule appears to swell, because of increased surface tension, and its outlines become clearly demarcated.

The pneumococcal Quellung reaction was first described in 1902  by the scientist Fred Neufeld, both as microscopic capsular swelling and macroscopic agglutination (clumping visible with the naked eye). It has been used to identify the 90 known capsular serotypes of Streptococcus pneumoniae in a diagnostic setting, but in recent years has been challenged by molecular typing techniques which target genetic differences.

Protein Pathologies: Amyloid guided by glycans

Functional Glycomics (11 November 2010) | doi:10.1038/fg.2010.36

Mutational analysis and tracking of glycosylated isoforms suggest a functional role for glycans in amyloid processing.

 

Amyloid-beta peptides have been closely linked to Alzheimer’s disease, but identifying the mechanism of pathogenicity and even which species is most important in the process, has proved difficult.In addition, it is now common to study amyloid deposition associated with neurons, Amyloid-beta is also deposited in the blood vessels of the brain. By turning their attention to the relevant brain microvascular endothelial cells-BMECs, Taniguchi and colleagues now identify an alternate amyloid-beta precursor and its glycosylation status, as having an unexpected role in the process.

Amyloid precursor protein-APP has three alternative isoforms:APP695 is primarily expressed in neurons, whereas APP751 and APP770 are expressed in several cell types. In addition to the APP695 sequence,APP751 contains the Kunitz-type protease inhibitor-KPI domain, where APP770 includes the KPI domain in tandem with an OX2 domain. Each sequence can be cleaved by Beta-secretase and other enzymes to yield the disease-associated peptides Amyloid beta40 and Amyloid beta 42.

Reporting in the Journal of Biological Chemistry, the authors first describe western blotting and other experiments that indicate that BMECs express more APP than do neurons. Furthermore, neurons express only APP695 as expected whereas BMECs express a substantial amount of APP770. The western blot analysis also revealed a high molecular weight band in the BMEC sample; as previous work has suggested that APP might be glycosylated, the authors used a series of enzymatic treatments and lectin pull down assays to identify N-glycan chains, including high mannose or hybrid glycans and sialylated O-glycans as likely APP modifications, the authors further investigated the OX2 domain, which is unique to this isoform. Mutation of each serine or threonine residue in this region identified Thr353 as a probable point of attachment for an O-glycan but mutation of four additional sites outside this domain guided by previous research was required to completely abrogate O-glycosylation.

These results demonstrated that APP770 is modified by several sugar chains, but what is the role of these carbohydrates? Although several glycoforms could be detected in the cell lysate, the authors detected only a single processed sequence in the cell media. The molecular weight of this processed ‘sAPP’ showed that the polypetide was derived from the high molecular weight APP770 , this origin was explained by a combination of biotinylation experiments.

intermittent fasting on prostate cancer tumor

Prostate Cancer and Prostatic Diseases (2010) 13, 350–355; doi:10.1038/pcan.2010.24; published online 24 August 2010

Effect of intermittent fasting on prostate cancer tumor growth in a mouse model

Abstract:

Caloric restriction (CR) has been shown to have anti-cancer properties. However, CR may be difficult to apply in humans secondary to compliance and potentially deleterious effects. An alternative is intermittent CR, or in the extreme case intermittent fasting (IF). In a previous small pilot study, we found 2 days per week of IF with ad libitum feeding on the other days resulted in trends toward prolonged survival of mice bearing prostate cancer xenografts. We sought to confirm these findings in a larger study. A total of 100 (7- to 8-week-old) male severe combined immunodeficiency mice were injected subcutaneously with 1 × 10⁵ LAPC-4 prostate cancer cells. Mice were randomized to either ad libitum Western Diet (44%carbohydrates, 40% fat and 16% protein) or ad libitum Western Diet with twice-weekly 24 h fasts (IF). Tumor volumes and mouse bodyweights were measured twice weekly. Mice were killed when tumor volumes reached 1000 mm³. Serum and tumor were collected for analysis of the insulin/insulin-like growth factor 1 (IGF-1) hormonal axis. Overall, there was no difference in mouse survival (P=0.37) or tumor volumes (P0.10) between groups. Mouse body weights were similar between arms (P=0.84). IF mice had significantly higher serum IGF-1 levels and IGF-1/IGFBP-3 ratios at killing (P<0.001). However, no difference was observed in serum insulin, IGFBP-3 or tumor phospho-Akt levels (P0.39). IF did not improve mouse survival nor did it delay prostate tumor growth. This may be secondary to metabolic adaptations to the 24 h fasting periods. Future studies are required to optimize CR for application in humans.

Turmeric (Curcuma longa) has protective effect on Prostrate Cancer

Prostate Cancer and Prostatic Diseases (2010) 13, 343–349; doi:10.1038/pcan.2010.26; published online 3 August 2010

Curcumin interrupts the interaction between the androgen receptor and Wnt/β-catenin signaling pathway in LNCaP prostate cancer cells

 

Abstract:

Recently, studies have investigated the significance of the Wnt/β-catenin pathway in prostate cancer. The transcriptional activity of the androgen receptor (AR) is modulated by interaction with coregulators, one of which is β-catenin. Curcumin, a dietary yellow pigment of Curcuma longa, has emerged as having a chemopreventive role. Although curcumin has been shown to inhibit AR expression, its molecular mechanism has not been fully elucidated. In this study, whether curcumin mediates the Wnt/β-catenin signaling pathway with regard to AR/β-catenin interactions was studied. Curcumin was shown to induce significant inhibition of AR expression in a dose-dependent manner. Marked curcumin-induced suppression of β-catenin was shown in the nuclear and cytoplasmic extracts as well as whole cell lysates. Further analysis revealed that phosphorylation of Akt and glycogen synthase kinase-3β were attenuated, but phosphorylated β-catenin was increased after curcumin treatment. Finally, cyclin D1 and c-myc, the target gene of the β-catenin/T-cell factor transcriptional complex, were also decreased. These findings suggest that curcumin modulates the Wnt/β-catenin signaling pathway and might have a significant role in mediating inhibitory effects on LNCaP prostate cancer cells.

What Alcohol Actually Does to Your Brain and Body

Lifehacker.com

Alcohol like caffeine, has an enormous reputation but loose understanding in popular culture.

Everyone, it seems, takes their cues on how alcohol affects the mind and body from an eclectic mix of knowledge: personal experience, pop culture, tall tales of long nights, the latest studies to make the health news wires and second hand tips.You might have gathered that alcohol is a depressant, that it’s dehydrating, that you can drink about one drink an hour and stay relatively sober. Some of that is true. But much of it depends on a large number of factors.

Taken from “Buzz: The Science and Lore of Alchol and Caffeine”.

Your body sees alcohol as a poison  or at least as something it doesn’t actually want inside it. To fight back and sober you up, humans have evolved to produce Alcohol Dehydrogenase.

That enzyme gets its shot at your alcohol when it attempts to pass through the stomach lining and when it reaches your liver, primarily.  On contact, it snatches a hydrogen atom off the ethanol molecules in your drink, rendering it into intoxicating acetaldehyde.

Human can then use aldehyde dehydrogenase as a kind of clean up crew, breaking down the aldehyde that’s sometimes considered a cause of hangovers, along with dehydration.

It’s a fight between how much you can drink, versus how fast your enzymes can bust down your indulgences and their by products. But many factors affect certain people’s production of the two alcohol- crushing compounds.

Alcohol Dehydrogenase: more effective in men than in women. Young men, in fact, may have up to 70 to 80 percent greater enzyme activity in the presence of alcohol. But men’s AD effectiveness also drops off with age at a faster rate than in women, such that , by around 55 or 60 , men may find themselves able to handle less alcohol than their female counterparts.

Full stomach:helps break down alcohol, but not because your food “soaks up” the alcohol. When you eat a big mean, your stomach’s pyloric sphincter, a kind of release valve into the small intestine, closes tightly. Your body knows that you’ve got food that should get a good going over in your stomach before it heads straight to the high absorption small intestine, so it keep it there and the AD in your stomach has more time to work on the alcohol.

Drink on an empty stomach and the liquid quickly makes it into the small intestine, where there’s more than 200 square meters of surface area for absorption into your body.

Genetics: Your great-great-grandparents have a say in how buzzed your Friday night gets, for sure, but for roughly on third to half of Asian drinkers, it’s more than a slight variance. Alcohol flush reaction, a flushing of the face when drinking, occurs because the enzyme “clean-up-crew”, AD is mutated by just one amino acid. That changes how effective its molecules are in bonding with and busting up, acetaldehyde. With excess acetaldehyde in their system, those with a flush reaction get red-faced and can experience heart palpitations, dizziness & severe nausea in extreme cases.

Aspirin: Don’t take aspirin before drinking. Aspirin seriously cuts the effectiveness of your bod’s AD enzymes. In one  1990 study, the average blood alcohol levels of those who took two maximum strength aspirin tablets before drinking were an average of 26 percent higher than those who were aspirin free. Other studies have suggested even more impact on your body’s ability to break down alcohol. That also means more acetaldehyde in your system down the line, so you will learn your lesson quickly if you are considering aspirin as a helper.

Absorption and Elimination Is a Curve, Not a Straight Line: Your Blood Alcohol Calculator moves through plateaus, responds differently to drinks higher than 20- 25 percent alcohol by volume and eliminates some alcohol in pure form- which is how police can measure it on your breath.

It Extends Your Life—Kind Of: Alcoholism:Clinical and Experimental Research, researchers followed 1824 people over a total of 20 years, as they aged between 55 and 65. Of those who abstained entirely, 69 percent died. Among those who drank in “moderate” amounts, 41 percent died- which was 23 percent less than the “light” drinkers. Even “heavy drinkers” fared better than abstainers, with just 61 percent passing away during the study period.

Popular theories center on the antioxidants and reservatrol compounds found in wines or on the studies showing alcohol as increasing levels of HDL(“good”) cholesterol.

It Doesn’t “Kill” Brain Cells, but Does Inhibit Them: It’s true that a high concentrations, like the nearly 100% pure alcohol used in sterilizing solutions, alcohol can indeed kill cells and neurons. But given that the blood reaching your brain is only at 0.08% percent alcohol if you are legally intoxicated.

What alcohol can and does do to your brain is affect the way your neurons get their firing triggers from glutamate.It infiltrates the glutamate receptors in your synapses, hurting their ability to send off their normal “fire” messages. Alcohol has this impact all  across your brain- the parts that control muscles, speech, coordination, judgment and so on.

That’s Also Why it, Uh, “Inhibits” Sex: The studies and implications are numerous, to say the least, but if you want a thumbnail understanding of how alcohol, as Shakespeare put it, “provokes the desire, but.. takes away the performance,” it has to do with the firing of nerves , in the brain and elsewhere, that would relax the arteries enough to get both parties moving. It’s a bit more complex than that and drinking in moderation can be a net benefit in some cases, but alcohol, paradoxically, doesn’t help one specific region of your self to “relax”.

Alcohol is Particularly Effective at Inhibiting Memories: N-Methyl-D-aspartic acid or NMDA, the receptor for which alcohol seems particularly  adept at interfering with, studies have shown that while subjects under alcohol’s influence can recall existing memories, events happening during inebriation are regularly hard to remember. It varies with the amount consumed and seems to top out at a serious 0.2% blood alcohol content.

It Makes Other People Seems More “Intentional” :If you’d never been raised to think things through, you’d assume that most actions people took were fairly intentional and possibly pointed at causing you harm.

It’s a Terrible Sleep Aid:Ever heard the term “nightcap?” People have long believed that alcohol helps you get to sleep and that part can be true, for some. Once you’re asleep, though, alcohol’s interaction with your brain can lead to some fitful sleep and no sleep at all, especially if you consumed caffeine anytime close to hitting the pillow. Caffeine, take up to 5 Hours to break down half a dose.