Linkage analysis of X-linked cone-rod dystrophy: localization to Xp11.4 and definition of a locus distinct from RP2 and RP3.

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.     

TB Treatment Delays in Odisha,India: Is It Expected Even after These Many Years of RNTCP Implementation?

Background

In India, the Revised National TB Control Programme (RNTCP) envisages initiation of TB treatment within seven days of diagnosis among smear-positive patients. After nearly two decades of RNTCP implementation, treatment delays are usually not expected.

Objectives

To determine the proportion of sputum smear-positive TB patients who were initiated on treatment after seven days and their associated risk factors.

Methods

The study was conducted in Cuttack and Rayagada districts of Odisha. It was a retrospective cohort study that involves review of TB treatment registers and laboratory registers for 2013.

Results

Among 1,800 pulmonary TB (PTB) patients, 1,074 (60%) had been initiated on treatment within seven days of diagnosis, 721 (40%) had been initiated on treatment more than seven days, and 354 (20%) had delays of more than 15 days. The mean duration between TB diagnosis and treatment initiation was 21 days with a range of 8–207 days (median = 14 days). Odds of treatment delay of more than seven days were 4.9 times (95% confidence interval [CI] 3.3-6.6) among those who had been previously treated, 6.2 times (95% CI 1.3-29.7) among those infected with HIV, and 1.8 times (95% CI 1.1-2.9) among those diagnosed outside district DMC.

Conclusion

Delay in initiation of TB treatment occurred in majority of the smear-positive patients. The RNTCP should focus on core areas of providing quality TB services with time-tested strategies. To have real-time monitoring mechanisms for diagnosed smear-positive TB patients is expected to be the way forward.     

A Retrospective Evaluation of Critical Care Blood Culture Yield – Do Support Services Contribute to the “Weekend Effect”?

BackgroundThe “weekend effect” describes an increase in adverse outcomes for patients admitted at the weekend. Critical care units have moved to higher intensity working patterns to address this with some improved outcomes. However, support services have persisted with traditional working patterns. Blood cultures are an essential diagnostic tool for patients with sepsis but yield is dependent on sampling technique and processing. We therefore used blood culture yield as a surrogate for the quality of support service provision.We hypothesized that blood culture yields would be lower over the weekend as a consequence of reduced support services.MethodsWe performed a retrospective observational study examining 1575 blood culture samples in a university hospital critical care unit over a one-year period.ResultsPatients with positive cultures had, on average, higher APACHE II scores (p = 0.015), longer durations of stay (p = 0.03), required more renal replacement therapy (p<0.001) and had higher mortality (p = 0.024). Blood culture yield decreased with repeated sampling with an increased proportion of contaminants. Blood cultures were 26.7% less likely to be positive if taken at the weekend (p = 0.0402). This effect size is the equivalent to the impact of sampling before and after antibiotic administration.ConclusionsOur study demonstrates that blood culture yield is lower at the weekend. This is likely caused by delays or errors in incubation and processing, reflecting the reduced provision of support services at the weekend. Reorganization of services to address the “weekend effect” should acknowledge the interdependent nature of healthcare service delivery.     

Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10^-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

HupB,a Nucleoid-Associated Protein of Mycobacterium tuberculosis,Is Modified by Serine/Threonine Protein Kinases In Vivo

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H₃₇Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr⁶⁵ and Thr⁷⁴ in the DNA-embracing β-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg⁵⁵ is also identified as an important residue for N-HupB–DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H₃₇Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.     

Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.The rumen is a complex microbial ecosystem that is composed of an immense variety of bacteria, protozoa, fungi, and viruses (5). Among these microorganisms, bacteria are the most investigated population and have a significant effect on the animal''s performance. However, our understanding of how rumen bacteria change and adapt to different ruminal environments is in its infancy.In the feedlot cattle industry, when animals on a forage diet are directly put on a high-grain diet, a decrease in ruminal pH due to lactate production has been observed (23, 31, 42), which leads to the possibility of digestive disorders, which can cause a decrease in the animal''s performance (23, 45). Therefore, feeding programs have been implemented to adapt feedlot cattle from a high-forage diet to a high-concentrate diet by gradually increasing the concentration of grain in the diet and decreasing the fiber content (2, 35). During this adaptation to high-grain diets, significant changes in the ruminal environment and rumen bacterial population structure have been reported (17, 46, 48). However, the microbial changes that occur during this transition phase are poorly understood (17, 21, 26, 46). Studies performed to date have utilized culture-based techniques or have looked at the fluctuation of a few indicator bacteria (48, 47) to evaluate bacterial population changes. Due to limitations in culturing rumen bacteria, the use of culture-based techniques to evaluate bacterial populations substantially underestimates the diversity of microorganisms within the rumen. In this study, we have utilized culture-independent approaches to evaluate bacterial population structure and diversity using terminal restriction fragment length polymorphisms (T-RFLPs) and sequence analysis of 16S rRNA gene libraries to compare the rumen bacterial population structure in animals on prairie hay against that in animals adapting to a high-concentrate (high-grain) diet. We have also quantified the fluctuations in the populations of previously reported indicator bacterial species using quantitative real-time PCR (qRT-PCR) to assess the role of these organisms during adaptation to a high-concentrate diet.     

Evaluation of culture methods to identify bovine feces with high concentrations of Escherichia coli O157

Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.     

Dual role for Zn2+ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease

We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.