Major Challenges in Clinical Management of TB/HIV Coinfected Patients in Eastern Europe Compared with Western Europe and Latin America

Objectives

Rates of TB/HIV coinfection and multi-drug resistant (MDR)-TB are increasing in Eastern Europe (EE). We aimed to study clinical characteristics, factors associated with MDR-TB and predicted activity of empiric anti-TB treatment at time of TB diagnosis among TB/HIV coinfected patients in EE, Western Europe (WE) and Latin America (LA).

Design and Methods

Between January 1, 2011, and December 31, 2013, 1413 TB/HIV patients (62 clinics in 19 countries in EE, WE, Southern Europe (SE), and LA) were enrolled.

Results

Significant differences were observed between EE (N = 844), WE (N = 152), SE (N = 164), and LA (N = 253) in the proportion of patients with a definite TB diagnosis (47%, 71%, 72% and 40%, p<0.0001), MDR-TB (40%, 5%, 3% and 15%, p<0.0001), and use of combination antiretroviral therapy (cART) (17%, 40%, 44% and 35%, p<0.0001). Injecting drug use (adjusted OR (aOR) = 2.03 (95% CI 1.00–4.09), prior anti-TB treatment (3.42 (1.88–6.22)), and living in EE (7.19 (3.28–15.78)) were associated with MDR-TB. Among 585 patients with drug susceptibility test (DST) results, the empiric (i.e. without knowledge of the DST results) anti-TB treatment included ≥3 active drugs in 66% of participants in EE compared with 90–96% in other regions (p<0.0001).

Conclusions

In EE, TB/HIV patients were less likely to receive a definite TB diagnosis, more likely to house MDR-TB and commonly received empiric anti-TB treatment with reduced activity. Improved management of TB/HIV patients in EE requires better access to TB diagnostics including DSTs, empiric anti-TB therapy directed at both susceptible and MDR-TB, and more widespread use of cART.     

Fe(2+)-tetracycline-mediated cleavage of the Tn10 tetracycline efflux protein TetA reveals a substrate binding site near glutamine 225 in transmembrane helix 7

TetA specified by Tn10 is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that, when chelated to tetracycline, Fe(2+) cleaved the backbone of TetA predominantly at a single position, glutamine 225 in transmembrane helix 7. The related class D TetA protein from plasmid RA1 was cut at exactly the same position. There was no cleavage with glycylcycline, an analog of tetracycline that does not bind to TetA. The Fe(2+)-tetracycline complex was not detectably transported by TetA. However, cleavage products of the same size as with Fe(2+) occurred with Co(2+), known to be cotransported with tetracycline. The known substrate Mg (2+)-tetracycline interfered with cleavage by Fe(2+). These findings suggest that cleavage results from binding at a substrate-specific site. Fe(2+) is known to be able to cleave amide bonds in proteins at distances up to approximately 12 A. We conclude that the alpha carbon of glutamine 225 is probably within 12 A of the position of the Fe(2+) ion in the Fe(2+)-tetracycline complex bound to the protein.     

QTL analysis of potato tuberization

Quantitative trait loci (QTLs) affecting tuberization were detected in reciprocal backcrosses between Solanum tuberosum and S. berthaultii. Linkage analyses were performed between traits and RFLP alleles segregating from both the hybrid and the recurrent parent using a set of framework markers from the potato map. Eleven distinct loci on seven chromosomes were associated with variation in tuberization. Most of the loci had small effects, but a QTL explaining 27% of the variance was found on chromosome 5. More QTLs were detected while following alleles segregating from the recurrent S. tuberosum parent used to make the backcross than were detected by following alleles segregating from the hybrid parent. More than half of the alleles favoring tuberization were at least partly dominant. Tuberization was favored by an allele from S. berthaultii at 3 of the 5 QTLs detected by segregation from the hybrid parent. The additive effects of the QTLs for tuberization explained up to 53% of the phenotypic variance, and inclusion of epistatic effects increased this figure to 60%. The most common form of epistasis was that in which presence of an allele at each of 2 loci favoring tuberization was no more effective than the presence of a favorable allele at 1 of the 2 loci. The QTLs detected for tuberization traits are discussed in relationship to those previously detected for trichome-mediated insect resistance derived from the unadapted wild species.Paper number 54 of the Department of Fruit and Vegetable Science, Cornell University     

The catecholic metal sequestering agent 1,2-dihydroxybenzene-3,5-disulfonate confers protection against oxidative cell damage.

Tiron (1,2-dihydroxybenzene-3,5-disulfonate), a nontoxic chelator of a variety of metals, is used to alleviate acute metal overload in animals. It is also oxidized to the EPR-detectable semiquinone radical by various biologically relevant oxidants, such as .OH, O2-., alkyl, and alkoxyl radicals. Since Tiron reacts with potentially toxic intracellular species and is also a metal chelator, we evaluated its protective effects in V79 cells subjected to various types of oxidative damage and attempted to distinguish the protection due to direct detoxification of intracellular radicals from that resulting from chelation of redox-active transition metals. We found that Tiron protects Chinese hamster V79 cells against both O2.(-)-induced (and H2O2 via dismutation of O2.-) and H2O2-induced cytotoxicity as measured by clonogenic assays. In experiments where Tiron was incubated with V79 cells and rinsed prior to exposure to HX/XO or H2O2, cytoprotection was observed, indicating that it protects against intracellular oxidative damage. On the other hand, Tiron did not protect V79 cells against the damage caused by ionizing radiation under aerobic conditions, which is predominantly mediated by H., .OH, and hydrated electrons in a metal-independent fashion. We demonstrate also that in in vitro studies, Tiron protects supercoiled DNA from metal-mediated superoxide-dependent strand breaks. We conclude that Tiron is a potentially useful protecting agent against the lethal effects of oxidative stress and suggest that it offers protection by chelating redox-active transition metal ions, in contrast to earlier reports where the protection by this compound in cellular systems subjected to oxidative damage has been interpreted as due to radical scavenging alone.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Membrane-Associated R Factor Proteins

R factor proteins are synthesized in R factor-containing Escherichia coli minicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.     

Dermatoglyphics in hypertension: a review

Hypertension is a major contributor to the global burden of disease and mortality. A major medical advancement would be a better means to ascertain which persons are at higher risk for becoming hypertensive beforehand. To that end, there have been a number of studies showing that certain dermatoglyphic markers are associated with hypertension. This association could be explained if the risk toward developing hypertension later on in life is somehow connected with fetal development of dermatoglyphics. It would be highly valuable from a clinical standpoint if this conjecture could be substantiated since dermatoglyphic markers could then be used for screening out individuals who might be at an elevated risk of becoming hypertensive. The aim of this review was to search for and appraise available studies that pertain to the association between hypertension and dermatoglyphics.A systematic literature search conducted using articles from MEDLINE (PubMed), Trip, Cochran, Google scholar, and gray literature until December 2014. Of the 37 relevant publications, 17 were included in the review. The review performed according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement.This review showed a fairly consistent finding of an increased frequency of whorl patterns along with a higher mean total ridge count in digital dermatoglyphic results in hypertensive samples compared to controls. However, it was imperative to discuss several limitations found in the studies that could make this association as yet unsettled.     

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

Background

Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.

Methodology

Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4°C, −20°C & −80°C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.

Conclusion

Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.     

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin

Using interferometry-based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation-independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM-binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM-binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca(2+) binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca(2+) dependence of CaM binding. Hence, changes in the Ca(2+) sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca(2+) sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.