REASONS FOR PATIENT DELAYS & HEALTH SYSTEM DELAYS FOR TUBERCULOSIS IN SOUTH INDIA

Background: Globally, the burden of Tuberculosis is escalating. Early diagnosis and prompt initiation of tuberculosis treatment is essential for an effective tuberculosis control programme. Objectives: To study the self reported reasons for patient and health system (diagnosis & treatment) delays in Tuberculosis patients. Methods: A community based cross sectional study was conducted among 98 new sputum positive TB cases aged > 15 years registered under RNTCP from Oct 2006 to June 2007 & receiving treatment under DOTS in Udupi taluk by interviewing them. Results: Total 98 patients were recruited and 68% were males. Out of 17 patients with patient delays, 82% felt that their symptoms were not severe, 71% felt that patient delay was due to lack of awareness and 71% did not take it seriously. Out of 86 patients with health system delays, 82.6% of patients mentioned that doctor has not advised for sputum examination, 76.7% of patients told that they first consulted a private doctor, 21% of them mentioned that doctor was unaware to diagnose TB. Conclusion: Symptoms not severe is the main reason for the patient delay and doctor didn’t advise for sputum examination is the main reason for health system delays

Molecular cloning of KS, a novel rat gene expressed exclusively in the kidney

Molecular cloning of xKSx, a novel rat gene expressed exclusively in the kidney.BackgroundWe aimed to identify genes with kidney specific, developmentally regulated expression. Here we report the cDNA sequence and expression pattern of KS, a novel kidney-specific rat gene.MethodsA partial cDNA was identified by differential display polymerase chain reaction (PCR) of a renal cell fraction enriched for proximal tubular and renin-expressing cells. Using the partial cDNA as a probe, a rat kidney cDNA library was screened. The full-length KS sequence was obtained by PCR amplification of cDNA ends. The expression pattern of KS was investigated by Northern blot. RNA was extracted from several organs of newborn and adult rats, as well as from the kidneys of rats with altered tubular function, that is, rats that had undergone unilateral nephrectomy, unilateral ureteral obstruction, neonatal losartan treatment, and the appropriate control animals. The expression of KS was also investigated in the kidneys of rats with spontaneous or renovascular hypertension.ResultsThe KS cDNA (2426bp) contained one open reading frame encoding a predicted 572 amino acid protein. The derived peptide sequence displayed approximately 70% similarity to the hypertension-related SA gene product and approximately 50% similarity to prokaryotic and eukaryotic acetyl-CoA synthases (EC 6.2.1.1). KS was expressed in the kidney and not in any other organ assayed. KS RNA was not detected in fetal and newborn rat kidney but became apparent after one week of postnatal life. Gene expression was downregulated in rat models of altered tubular function. KS expression was decreased in spontaneously hypertensive rats but not in renovascular hypertension.ConclusionKS, a novel rat gene, exhibits a unique tissue-specific expression exclusively in mature kidneys. The data suggest KS may encode an adenosine monophosphate binding enzyme

Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses

Rare variants in tenascin genes in a cohort of children with primary vesicoureteric reflux

Primary vesicoureteral reflux (PVUR) is the most common malformation of the kidney and urinary tract and reflux nephropathy is a major cause of chronic kidney disease in children. Recently, we reported mutations in tenascin XB (TNXB) as a cause of PVUR with joint hypermobility

Psychosocial Assessment of Candidates for Transplantation (PACT) Score Identifies High Risk Patients in Pediatric Renal Transplantation

Background: Currently, there is no standardized approach for determining psychosocial readiness in pediatric transplantation. We examined the utility of the Psychosocial Assessment of Candidates for Transplantation (PACT) to identify pediatric kidney transplant recipients at risk for adverse clinical outcomes.Methods: Kidney transplant patients <21-years-old transplanted at Duke University Medical Center between 2005 and 2015 underwent psychosocial assessment by a social worker with either PACT or unstructured interview, which were used to determine transplant candidacy. PACT assessed candidates on a scale of 0 (poor candidate) to 4 (excellent candidate) in areas of social support, psychological health, lifestyle factors, and understanding. Demographics and clinical outcomes were analyzed by presence or absence of PACT and further characterized by high (≥3) and low (≤2) scores.Results: Of 54 pediatric patients, 25 (46.3%) patients underwent pre-transplant evaluation utilizing PACT, while 29 (53.7%) were not evaluated with PACT. Patients assessed with PACT had a significantly lower percentage of acute rejection (16.0 vs. 55.2%, p = 0.007). After adjusting for HLA mismatch, a pre-transplant PACT score was persistently associated with lower odds of acute rejection (Odds Ratio 0.119, 95% Confidence Interval 0.027–0.52, p = 0.005). In PACT subsection analysis, the lack of family availability (OR 0.08, 95% CI 0.01–0.97, p = 0.047) and risk for psychopathology (OR 0.34, 95% CI 0.13–0.87, p = 0.025) were associated with a low PACT score and post-transplant non-adherence.Conclusions: Our study highlights the importance of standardized psychosocial assessments and the potential use of PACT in risk stratifying pre-transplant candidates

Antibacterial activity of linear peptides spanning the carboxy-terminal β-sheet domain of arthropod defensins

The antibacterial activity of peptides without disulfide bridges, spanning the carboxy-terminal segment of arthropod defensins, has been investigated. Although all the peptides have net positive charges, they exhibited varying antibacterial potencies and spectra. Atomic force and fluorescence microscopic analyses indicate that the peptides exert their activity by permeabilizing the outer and inner membranes of Gram-negative bacteria such as Escherichia coli. It appears that the plasticity observed in the activity of mammalian defensins with respect to sequence, number of disulfide bridges or net positive charge, is also observed in insect defensins

Organic solvent mediated self-association of an amyloid forming peptide from &#946;<SUB>2</SUB>-microglobulin: an atomic force microscopy study

Human &#946;2-microglobulin (&#946;2m) forms amyloid fibrils in hemodialysis related amyloidosis. Peptides spanning the &#946; strands of &#946;2m have been shown to form amyloid fibrils in isolation. We have studied the self-association of a 13-residue peptide Ac-DWSFYLLYYTEFT-am (P&#946;2m) spanning one of the &#946;-strands of human &#946;2-microglobulin when dissolved in various organic solvents such as methanol (MeOH), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and dimethylsulfoxide. We have observed that P&#946;2m forms amyloid fibrils when diluted from organic solvents into aqueous buffer at pH 7.0 as judged by increase in thioflavin T fluorescence. Fibril formation was observed to depend on the solvents in which peptide stock solutions were prepared. Circular dichroism spectra indicated propensity for helical conformation in MeOH, TFE, and HFIP. In buffer, &#946;-structure was observed irrespective of the solvent in which the peptide stock solutions were prepared. Atomic force microscopy images obtained by drying the peptide on mica from organic solvents indicated the ability of P&#946;2m to self-associate to form nonfibrillar structures. Morphology of the structures was dependent on the solvent in which the peptide was dissolved. Peptides that have the ability to self-associate such as amyloid-forming peptides would be attractive candidates for the generation of self-assembled structures with varying morphologies by appropriate choice of surfaces and solvents for dissolution

Morphology of self-assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac-VQIVYK-amide (AcPHF6) and Ac-QIVYK-amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self-association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly &#946;-conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for &#946;-structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and &#946;-structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides

Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides

We demonstrate that antimicrobial peptides induce an autophagic cell death in the protozoan pathogen, Leishmania donovani. In our study, three antimicrobial peptides, Indolicidin, and two peptides derived from Seminalplasmin exhibit antileishmanial activity with a 50% lethal dose of 3.5&#215;10-5, 3.8&#215;10-4 and 1.7&#215;10-8 &#956;M, respectively. The action of these antimicrobial peptides on the Leishmania cell involves ionic interactions, which are modulated by lipophosphoglycan on the parasite's surface. Peptide treatment caused dissipation of membrane potential and equilibration of intracellular pH with extracellular environment. However, there was no release of intracellular GFP molecules upon peptide treatment of a GFP expressing Leishmania clone. Transmission electron microscopic studies show extensive intracellular damage including cytoplasmic vacuolization and degeneration of cellular organization without disruption of the plasma membrane. These peptides induce cell death via a non-apoptotic process as shown by lack of nuclear fragmentation or DNA laddering and independent of caspase-like activity. Instead, Monodansylcadaverine (MDC), a biochemical marker of autophagy specifically labels the vacuoles induced by peptides. Collectively, these results indicate that in addition to their effects on the leishmanial membrane, these antimicrobial peptides induce pathway(s) for autophagic cell death in L. donovani