The Importance of Tryptophan B28 in H2 Relaxin for RXFP2 Binding and Activation

H2 relaxin (relaxin) is a member of the insulin–relaxin superfamily and exhibits several non-reproductive functions in addition to its well-known properties as a pregnancy hormone. Over the years, the therapeutic potential of relaxin has been examined for a number of conditions. It is currently in phase III clinical trials for the treatment of acute heart failure. The 53 amino acid peptide hormone consists of two polypeptide chains (A and B) which are cross-linked by two inter-chains and one intra-A chain disulfide bridge. Although its cognate receptor is relaxin family peptide receptor (RXFP) 1, relaxin is also able to cross-react with RXFP2, for which the native ligand is INSL3. The “RXXXRXXI” motif in the B-chain of H2 relaxin is responsible for primary binding to LRR of the RXFP1 receptor (Büllesbach and Schwabe, J Biol Chem 280:14051–14056, 2005). Previous RXFP2 receptor mutation and molecular modelling studies strongly suggest that, in addition to this motif, the Trp-B28 residue in the B-chain is responsible for H2–RXFP2 interaction. To confirm this finding, here we have mutated H2 relaxin in which Trp-B28 was replaced with alanine. The synthetic relaxin analogue was then tested on cells expressing either RXFP1 or 2 to determine the affinity and potency for the respective receptors. Our results confirm that Trp-B28 in the B-chain is crucial for binding and activating RXFP2, but not for RXFP1.     

Preliminary Structure–Function Relationship Studies on Insulin-Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide bonded member of insulin/relaxin superfamily of peptides that includes insulin, insulin-like growth factor I and II (IGFI and IGFII), insulin-like peptide 3, 4, 5 and 6 (INSL3, 4, 5 and 6), relaxin-1 (H1 relaxin), -2 (H2 relaxin) and -3 (H3 relaxin). Although it is expressed in relatively high levels in the gut, its biological function remains unclear. However, recent reports suggest a significant orexigenic action and a role in the regulation of insulin secretion and β-cell homeostasis, which implies that both agonists and antagonists of the peptide may have significant therapeutic applications. Modern solid phase synthesis techniques together with regioselective disulfide bond formation were employed for a preliminary structure–function relationship study of mouse INSL5. Two point mutated analogues, mouse INSL5 A-B(R24A, W25A) and mouse INSL5 A-B(K6A, R14A, Y18A) were chemically prepared, where the residues in the B-chain that may be involved in receptor activation and affinity binding, were respectively mutated. Synthetic mouse INSL5 A-B(R24A, W25A) analogue was inactive on RXFP4, the native receptor for INSL5, suggesting Arg^B24 and Trp^B25 are probably directly involved in INSL5 receptor activation. Mouse INSL5 A-B(K6A, R14A, Y18A) analogue had both decreased affinity and potency on RXFP4 (pIC₅₀ 7.7 ± 0.2, pEC₅₀ 7.87 ± 0.18) which indicated that one or more of these residues are critical for the binding to the receptor.     

Association of multiple drug resistance-1 gene polymorphism with multiple drug resistance in breast cancer patients from an ethnic Saudi Arabian population

Chemotherapy has been used widely to treat cancer, both as a systemic therapy and as a local treatment. Unfortunately, many types of cancer are still refractory to chemotherapy. The mechanisms of anticancer drug resistance have been extensively explored but have not been fully characterized. This study analyzed the occurrences of polymorphism (SNP) in the MDR1 gene in breast cancer patients and determined a possible association with chemotherapy. The study group included one hundred breast carcinoma patients who subsequently received chemotherapy (the regimen generally consisted of commonly used drugs such as cyclophosphamide, adriamycin, 5-fluorouracil, docetaxel and their combinations). Blood samples from 100 healthy individuals are used, as controls were also genotyped for the MDR1 gene. This investigation revealed a significant correlation with response to various regimens of chemotherapy showing a low response to therapy with the CT/TT genotype at (exon 12) 1236 codon (p < 0.001). These findings demonstrate, for the first time, that the polymorphisms in (exon 12) 1236 codon of the MDR1 gene greatly influence the drug response in patients from the Arab population of Saudi Arabia.     

Adherence to Antiretroviral Therapy and Its Effect on Survival of HIV-Infected Individuals in Jharkhand,India

Introduction

Research in India has extensively examined the factors associated with non-adherence to antiretroviral therapy (ART) with limited focus on examining the relationship between adherence to ART regimen and survival status of HIV infected patients. This study examines the effect of optimal adherence to ART on survival status of HIV infected patients attending ART centers in Jharkhand, India.

Materials and Methods

Data from a cohort of 239 HIV infected individuals who were initiated ART in 2007 were compiled from medical records retrospectively for 36 months. Socio-demographic characteristics, CD4 T cell count, presence of opportunistic infections at the time of ART initiation and ART regimen intake and survival status was collected periodically. Optimal adherence was assessed using pill count methods; patients who took <95% of the specified regimens were identified as non-adherent. Cox-proportional hazard model was used to determine the relative hazards of mortality.

Results

More than three-fourths of the patients were male, on an average 34 year old and median CD4 T cell count was 118 cells/cmm at the time of ART registration. About 57% of the patients registered for ART were found to be adherent to ART. A total of 104 patients died in 358.5 patient-years of observation resulting in a mortality rate of 29 per 100 patient-years (95% confidence interval (CI): 23.9–35.2) and median survival time of 6.5 months (CI: 2.7–10.9). The mortality rate was higher among patients who were non-adherent to ART (64.5, CI: 50.5–82.4) than who were adherent (15.4, CI: 11.3–21.0). The risk of mortality was fourfold higher among individuals who were non-adherent to ART than who were adherent (Adjusted hazard ratio: 3.9, CI: 2.6–6.0).

Conclusion

Adherence to ART is associated with a higher chance of survival of HIV infected patients, ascertaining the need for interventions to improve the ART adherence and early initiation of ART.     

The Suitability of P. falciparum Merozoite Surface Proteins 1 and 2 as Genetic Markers for In Vivo Drug Trials in Yemen

Background

The accuracy of the conclusions from in vivo efficacy anti-malarial drug trials depends on distinguishing between recrudescences and re-infections which is accomplished by genotyping genes coding P. falciparum merozoite surface 1 (MSP1) and MSP2. However, the reliability of the PCR analysis depends on the genetic markers’ allelic diversity and variant frequency. In this study the genetic diversity of the genes coding for MSP1 and MSP2 was obtained for P. falciparum parasites circulating in Yemen.

Methods

Blood samples were collected from 511 patients with fever and screened for malaria parasites using Giemsa-stained blood films. A total 74 samples were infected with P. falciparum, and the genetic diversity was assessed by nested PCR targeting Pfmsp1 (Block2) and Pfmsp2 (block 3).

Results

Overall, 58%, 28% and 54% of the isolates harboured parasites of the Pfmsp1 K1, MAD20 and RO33 allelic families, and 55% and 89% harboured those of the Pfmsp2 FC27 and 3D7 allelic families, respectively. For both genetic makers, the multiplicity of the infection (MOI) was significantly higher in the isolates from the foothills/coastland areas as compared to those from the highland (P<0.05). Pfmsp2 had higher number of distinct allelic variants than Pfmsp1 (20 vs 11). The expected heterozygosity (HE) for Pfmsp1 and Pfmsp2 were 0.82 and 0.94, respectively. Nonetheless, a bias in the frequency distribution of the Pfmsp1 allelic variants was noted from all areas, and of those of Pfmsp2 in the samples collected from the highland areas.

Conclusions

Significant differences in the complexity and allelic diversity of Pfmsp1 and Pfmsp2 genes between areas probably reflect differences in the intensity of malaria transmission. The biased distribution of allelic variants suggests that in Yemen Pfmsp1 should not be used for PCR correction of in vivo clinical trials outcomes, and that caution should be exercised when employing Pfmsp2.     

Changing Emergence of Shigella Sero-Groups in Bangladesh: Observation from Four Different Diarrheal Disease Hospitals

Background

Shigellosis continues to be a public health challenge for developing countries, including Bangladesh. The aim of the study is to demonstrate recent changes in Shigella sero-groups and their geographical diversity.

Methods

Data were extracted from data archive of four diarrheal disease surveillance systems. A 2% sub sample from urban Dhaka Hospital (2008–2011; n = 10,650), and 10% from urban Mirpur Treatment Centre (2009–2011; n = 3,585), were enrolled systematically; whereas, all patients coming from the Health and Demographic Surveillance System area in rural Matlab (2008–2011; n = 6,399) and rural Mirzapur (2010–2011; n = 2,812) were included irrespective of age, sex, and disease severity. A fresh stool specimen was collected for identification of Shigella spp. Of them, 315 (3%) were positive for Shigella in Dhaka, 490 (8%) from Matlab, 109 (3%) from Mirpur and 369 (13%) from Mirzapur and considered as analyzable sample size.

Results

Among all Shigella isolates regardless of age, significant decreases in percentage of S. flexneri over time was observed in Mirpur (55→29%; p value of χ²-for trend = 0.019) and Mirzapur (59→47%; p = 0.025). A non-significant decrease was also seen in Dhaka (58→48%), while in Matlab there was a non-significant increase (73→81%). Similar patterns were observed among under-5 children at all sites. Emergence of S. sonnei was found in Dhaka (8→25%; p<0.001) and Mirpur (10→33%; p = 0.015), whereas it decreased in Mirzapur (32→23%; p = 0.056). The emergence of S. boydii was seen in all ages in Mirzapur [(3→28%; p<0.001); (3→27%; p<0.001)]. On the other hand, we saw non-significant percent reductions in S. boydii in Dhaka [overall (25→16%); under-5 (16→9%)]. Decreasing rates of Shigella dysenteriae were observed in Matlab, Mirpur and Mirzapur; whereas, in Dhaka it remained unchanged.

Conclusion and Significance

Emergence of S. sonnei and S. boydii as important infectious diarrhea etiologies and variations in geographical diversity underscore the need for monitoring, with possible implications for vaccine development.     

A Simple Insightful Approach to Investigating a Hospital Standardised Mortality Ratio: An Illustrative Case-Study

Background

Despite methodological concerns Hospital Standardised Mortality Ratios (HSMRs) are promoted as measures of performance. Hospitals that experience an increase in their HSMR are presented with a serious challenge but with little guidance on how to investigate this complex phenomenon. We illustrate a simple penetrating approach.

Methods

Retrospective analysis of routinely collected hospital admissions data comparing observed and expected deaths predicted by the Dr Foster Unit case mix adjustment method over three years (n = 74,860 admissions) in Shropshire and Telford NHS Trust Hospital (SaTH) constituting PRH (Princess Royal Hospital) and RSH (Royal Shrewsbury Hospital); whose HSMR increased from 99 in the year 2008/09 to 118 in the year 2009/10.

Results

The step up in HSMR was primarily located in PRH (109 to 130 vs. 105 to 118 RSH). Disentangling the HSMR by plotting run charts of observed and expected deaths showed that observed deaths were stable in RSH and PRH but expected deaths, especially at PRH, had fallen. The fall in expected deaths has two possible explanations–genuinely lower risk admissions or that the case-mix adjustment model is underestimating the risk of admissions perhaps because of inadequate clinical coding. There was no evidence that the case-mix profile of admissions had changed but there was considerable evidence that clinical coding process at PRH was producing a lower depth of coding resulting in lower expected mortality.

Conclusion

Knowing whether the change (increase/decrease) in HSMR is driven by the numerator or the denominator is a crucial pivotal first step in understanding a given HSMR and so such information should be an integral part of the HSMR reporting methodology.     

Effect of Simulated Microgravity on E. coli K12 MG1655 Growth and Gene Expression

This study demonstrates the effects of simulated microgravity on E. coli K 12 MG1655 grown on LB medium supplemented with glycerol. Global gene expression analysis indicated that the expressions of hundred genes were significantly altered in simulated microgravity conditions compared to that of normal gravity conditions. Under these conditions genes coding for adaptation to stress are up regulated (sufE and ssrA) and simultaneously genes coding for membrane transporters (ompC, exbB, actP, mgtA, cysW and nikB) and carbohydrate catabolic processes (ldcC, ptsA, rhaD and rhaS) are down regulated. The enhanced growth in simulated gravity conditions may be because of the adequate supply of energy/reducing equivalents and up regulation of genes involved in DNA replication (srmB) and repression of the genes encoding for nucleoside metabolism (dfp, pyrD and spoT). In addition, E. coli cultured in LB medium supplemented with glycerol (so as to protect the cells from freezing temperatures) do not exhibit multiple stress responses that are normally observed when cells are exposed to microgravity in LB medium without glycerol.     

Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

Background

Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP^TSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP^TSE concentrations in the femtomolar range.

Methodology/Principal Findings

We have developed a three-step assay that firstly captures PrP^TSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP^TSE detection by western blot. We achieved a PrP^TSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP^TSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP^TSE in human plasma spiked with a 10⁻⁸ dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP^TSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/Significance

We have developed a sensitive and specific amplification assay allowing the detection of PrP^TSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP^TSE in blood of patients displaying positivity in large scale screening tests.