A Novel Synergistic Association of Variants in PTRH2 and KIF1A Relates to a Syndrome of Hereditary Axonopathy, Outer Hair Cell Dysfunction, Intellectual Disability, Pancreatic Lipomatosis, Diabetes, Cerebellar Atrophy, and Vertebral Artery Hypoplasia.

The gene PTRH2 encodes a protein with peptidyl-tRNA hydrolase activity and is involved in the translation process in protein synthesis. The kinesin family member 1-A (KIF1A) gene encodes a molecular motor involved in axonal transport along microtubules. Mutations in these genes lead to respective phenotypical conditions that have been reported in the literature. In this paper, we present a novel syndrome of concurrent occurrence of mutations in the PTRH2 and KIF1A genes in a 19-year-old girl of Dravidian-Tamil descent from the Southern part of India. The girl presented with global developmental delay, intellectual disability, weakness of upper and lower limbs, and diabetes. On workup, she was found to have severe peripheral axonopathy, outer hair cell (OHC) dysfunction, severe bilateral sensorineural hearing loss (SNHL), total pancreatic lipomatosis, exocrine pancreatic insufficiency, cerebellar atrophy, vertebral artery hypoplasia, and scoliosis. The patient had a deceased elder sibling who also had had a similar phenotype. Whole exome sequencing (WES) revealed a novel variant in the PTRH2 gene and a rare variant in the KIF1A gene. The predominant axonal involvement seen in our patient, which was attributable to KIF1A involvement, distinguishes this syndrome from the infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD) caused by PTRH2 involvement alone. To the best of our knowledge, this is the first report in the medical literature of a syndrome caused by the synergistic occurrence of mutations in the PTRH2 and KIF1A genes. In order to provide more clarity on the genetic and clinical features of such syndromes and to aid the treating clinician to recognize the existence of such syndromes, we propose the broader umbrella term "neuro-pancreatic syndromes" (NPS). Presently, under NPS, we include two entities: the syndrome described by us in this paper and the IMNEPD. Prompt and effective recognition and management of such NPS would immensely benefit the patient in terms of treatment and prognosis. Furthermore, we hope that this paper will promote further understanding of NPS and foster more research, both clinical and genetic, which would widen the spectrum of NPS. Eventually, this would throw more light on treatment options and ultimately benefit patients with NPS.

Diabetes in the young - a case of Alström syndrome with myopathy.

Alström syndrome is a rare ciliopathy affecting about 1 in 1,000,000 individuals. It is characterised by cone-rod dystrophy, insulin resistance, diabetes mellitus, cardiomyopathy, renal failure and hypogonadism. Progressive multi-organ dysfunction eventually leads to death. Only about 800 patients with this disorder have been identified so far. The diagnosis of Alström syndrome is critical as it can easily be overlooked because of the many features it shares with metabolic syndrome. The gene affected in this autosomal recessive disease is ALMS1, the protein product of which is involved in intracellular trafficking and ciliary function. Alström syndrome is being studied as a model which would potentially shed light on the pathophysiology of diabetes mellitus. In this report, we describe a patient with features of Alström syndrome and a clinical picture suggestive of a recurrent, severe, steroid responsive myopathy which, to the best of our knowledge, has not been reported so far.

Compound heterozygous mutations in TTC7A cause familial multiple intestinal atresias and severe combined immunodeficiency.

Familial multiple intestinal atresias is an autosomal recessive disease with or without combined immunodeficiency. In the last year, several reports have described mutations in the gene TTC7A as causal to the disease in different populations. However, exact correlation between different genotypes and various phenotypes are not clear. In this study, we report identification of novel compound heterozygous mutations in TTC7A gene in a Malay girl with familial multiple intestinal atresias and severe combined immunodeficiency (MIA-SCID) by whole exome sequencing. We found two mutations in TTC7A: one that destroyed a putative splicing acceptor at the junction of intron 17/exon 18 and one that introduced a stop codon that would truncate the last two amino acids of the encoded protein. Reviewing the recent reports on TTC7A mutations reveals correlation between the position and nature of the mutations with patient survival and clinical manifestations. Examination of public databases also suggests carrier status for healthy individuals, making a case for population screening on this gene, especially in populations with suspected frequent founder mutations.

Bilateral psoas and bilateral perinephric abscesses complicating acute pyelonephritis in pregnancy.

Acute pyelonephritis complicates 1-2% of pregnancies and causes significant maternal and fetal morbidity and mortality. The diagnosis of renal tuberculosis (TB) is often delayed and commonly presents with sterile pyuria or along with other pyogenic organisms. We report a case where the diagnosis of renal TB was missed in a pregnant woman when she presented with acute pyelonephritis, septic shock, and acute renal failure. There was clinical recovery with antibiotics, but bilateral psoas and perinephric abscesses (TB, Enterococcus sp., and E. coli) were diagnosed when she presented with loin pain and palpable left renal angle swelling. Bilateral psoas abscess due to TB in the absence of skeletal TB and human immunodeficiency virus infection is rare. The presentation of renal TB in pregnancy, its complications, and its management are discussed.

Experimental studies on the effect of different metallic substrates on marine biofouling.

In the wake of adoption of the resolution by the International Maritime Organization to control biofouling on vessels, which is recognized as a major vector for transfer of invasive species, this study attempts to create a baseline data on major hard-shelled biofouling organisms in the harbour waters. This study was primarily focused towards understanding the biofouling and corrosion pattern on various metals and their performance under immersed condition in a marine environment, at 0.3 and 3.0m depths. Furthermore, the study attempts to understand the surface dependent characteristics of barnacle base plate and its adhesion strength. Barnacle, mussels and oysters were the major fouling organisms accounting for 72.33% of the variation. Stainless steel and Titanium panels showed the highest average biofouling load of 176.36 and 168.35 g/300 cm(2), respectively. The variance in biofouling between metals and depths was highly significant at p<0.001 and p<0.01, respectively. Morphology of barnacle base plate interfacial surface varied between metals. Barnacles with 8-9 mm base diameter showed the maximum adhesion strength in shear of 6.86±0.95 kPa.

Glycosaminoglycans from marine clam Meretrix meretrix (Linne.) are an anticoagulant.

The extracted unfractionated glycosaminoglycans (GAGs) obtained from the marine clam Meretrix meretrix were fractionated by ion-exchange (Amberlite IRA-900 and 120) chromatography. The fractionated sample activity was determined through azure-A by metachromatic activity and agarose gel electrophoresis. The active fractionated sample molecular mass was determined through gradient polyacrylamide gel electrophoresis (PAGE). The structural characterization of low-molecular-weight GAGs was analyzed by Fourier transform infrared (FT-IR) and ¹H-nuclear magnetic resonance (NMR) spectroscopy. The Activated partial thromboplastin time (APTT) of fractionated heparin-like glycosminoglycan is 72 IU/mg and it has a molecular mass of 15,000 Da. The disaccharide profiles, such as uronic acid 15.31%, hexosamine 24.61%, and sulfate content 11.7%, were also determined. The results of this study suggest that the GAGs from M. meretrix could be an alternative source of heparin.

Modelling head losses in granular bed anaerobic baffled reactors at high flows during start-up.

Anaerobic treatment of low strength, high flow wastewaters can only be effective if the technology employed can meet key hydrodynamic requirements: maximising the contact surface area and contact period between the influent substrate and the biomass solids, minimising solid washout from the reactor and minimising the backpressure across the system. Backpressure or head loss is an important hydrodynamic property of gravity-flow packed bed reactors, where the flow is the resultant of frictional forces between the incoming fluid and the solid packing material through which the wastewater percolates. Excessive backpressure caused by high influent flow-rates can reduce the contact surface area and increase the influent head on the upstream side of the biomass bed leading to overflow spills, unstable performance and process failure. This study investigates the factors affecting backpressure across a Granular bed baffled reactor (GRABBR) with variable baffle positions. Experimental results were used to develop a mathematical model to quantify backpressure based on physical characteristics of the seed biomass, fluid-flow conditions and reactor geometry. Results have shown that for a constant flow rate the anaerobic baffled reactor exhibits the least backpressure characteristics when both the upflow and downflow areas are roughly 50% of the total compartmental width.

Preventive effect of low molecular weight glycosaminoglycan from Amussium pleuronectus (Linne) on oxidative injury and cellular abnormalities in isoproterenol-induced cardiotoxicity in Wistar rats.

The present work explores suspicious consequence of low molecular weight glycosaminoglycan (LMW-GAG) on oxidative stress and cellular abnormalities in isoproterenol (ISO)-induced myocardial infarction in an experimental model. Group-III male Wistar rats (140 +/- 10 g) were administrated by ISO (85 mg ISO/ml subcutaneously (SC) injected at the last two days of a 2-week period). Group-IV rats were treated LMW-GAG plus ISO (300 microg/day per rat SC for 1 week followed by 85 mg/kg ISO on the end last two days of the 2 - weeks). Untreated control (Group-I) and LMW-GAG drug control (Group-II) were also included. Serum and tissue lactate dehydrogenase, aminotransferases, and creatine kinase activities were increased in ISO group, which were normalized by LMW-GAG pretreatment rats. Antioxidant enzymes - superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities and non-enzymatic enzyme reduced glutathione (GSH) were decreased in the ISO induced rats, and this was increased by LMW-GAG pretreatment. Increased level of thiobarbituric acid reactive substances (TBARS) in plasma and the heart of ISO treated rats; pre s.c. injected with LMW-GAG to ISO-induced rats decreased the levels of TBARS. Histological examination revealed that the ISO-induced deleterious changes in the heart tissues were offset by LMW-GAG treatment. LMW-GAG affords considerable protection to the tissues challenged by cardiotoxicity, evidenced by its correction and restoration of serum and tissue indices of injury, to normalcy.

Isolation and characterization of low molecular weight glycosaminoglycans from marine mollusc Amussium pleuronectus (linne) using chromatography.

The glycosaminoglycan (GAG) heparin is a polyanionic sulfated polysaccharide most recognized for its anticoagulant activity. In the present study, the GAGs were extracted from bivalve mollusc Amussium pleuronectus. The crude GAGs were fractionated by ion-exchange (DEAE-cellulose and Amberlite IRA-900 & 120) chromatography. The recovered active fractions (as determined by metachromatic assay) were confirmed by agarose gel electrophoresis and the active fractions were purified in Sephadex G-100 column. Fractionated and purified GAG molecular weight was determined through gradient polyacrylamide gel electrophoresis. The structural characterization of low molecular weight GAG was analyzed by Fourier transform infrared spectroscopy. The activated partial thromboplastin time of purified GAG is 95 IU/mg and has molecular weight 6,500-7,500 Da. The disaccharide compositional analysis on the GAG sample was sulfated like porcine intestinal mucosal heparan sulfate, and it contains equivalent amount of uronic acid and hexosamine. The results of this study suggest that the GAG from A. pleuronectus could be an alternative source of heparin.

Comparing the performance of UASB and GRABBR treating low strength wastewaters.

Anaerobic technologies have proved successful in the treatment of various high strength wastewaters with perceptible advantages over aerobic systems. The applicability of anaerobic processes to treat low strength wastewaters has been increasing with the evolution of high-rate reactors capable of achieving high sludge retention time (SRT) when operating at low HRT. However, the performance of these systems can be affected by high variations in flow and wastewater composition. This paper reports on the comparative study carried out with two such high rate reactors systems to evaluate their performances when used for the treatment of low strength wastewaters at high hydraulic rates. One of the two systems is the most commonly used upflow anaerobic sludge blanket (UASB) reactor in which all reactions occur within a single vessel. The other is the granular bed baffled reactor (GRABBR) that encourages different stages of anaerobic digestion in separate vessels longitudinally across the reactor. The reactors, with equal capacity of 10 litres, were subjected to increasing organic loading rates (OLRs) and hydraulic retention times (HRTs) of up to 60 kg COD m(-3) d(-1) and 1 h respectively. Results show that the GRABBR has greater processes stability at relatively low HRTs, whilst the UASB seems to be better equipped to cope with organic overloads or shockloads. The study also shows that the GRABBR enables the harvesting of biogas with greater energetic value and hence greater re-use potential than the UASB. Biogas of up to 86% methane content is obtainable with GRABBR treating low strength wastewaters.

Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer's disease and related disorders.

Several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, are characterized pathognomonically by the presence of intra- and/or extracellular lesions containing proteinaceous aggregates, and by extensive neuronal loss in selective brain regions. Related non-neuropathic systemic diseases, e.g., light-chain and senile systemic amyloidoses, and other organ-specific diseases, such as dialysis-related amyloidosis and type-2 diabetes mellitus, also are characterized by deposition of aberrantly folded, insoluble proteins. It is debated whether the hallmark pathologic lesions are causative. Substantial evidence suggests that these aggregates are the end state of aberrant protein folding whereas the actual culprits likely are transient, pre-fibrillar assemblies preceding the aggregates. In the context of neurodegenerative amyloidoses, the proteinaceous aggregates may eventuate as potentially neuroprotective sinks for the neurotoxic, oligomeric protein assemblies. The pre-fibrillar, oligomeric assemblies are believed to initiate the pathogenic mechanisms that lead to synaptic dysfunction, neuronal loss, and disease-specific regional brain atrophy. The amyloid beta-protein (Abeta), which is believed to cause Alzheimer's disease (AD), is considered an archetypal amyloidogenic protein. Intense studies have led to nominal, functional, and structural descriptions of oligomeric Abeta assemblies. However, the dynamic and metastable nature of Abeta oligomers renders their study difficult. Different results generated using different methodologies under different experimental settings further complicate this complex area of research and identification of the exact pathogenic assemblies in vivo seems daunting. Here we review structural, functional, and biological experiments used to produce and study pre-fibrillar Abeta assemblies, and highlight similar studies of proteins involved in related diseases. We discuss challenges that contemporary researchers are facing and future research prospects in this demanding yet highly important field.

Extraction of heparin and heparin-like substance from marine mesogastropod mollusc Turritella attenuata (Lamarck, 1779).

Heparin was extracted from marine gastropod T. attenuata through the sequential precipitation with methanol and ethanol. The metachromatic dye method using toluidine blue was used to estimate colorimetrically the amount of heparin present in the sample. The anticoagulant activity of the sample was calculated as per United States of Pharmacopoeia standard procedure using sheep blood. After the purification, samples were analyzed, for the presence of heparin, with agarose-gel electrophoresis and HPLC and the mobility of the sample and the peak respectively were compared with standard heparin. The results of the present study shall help in finding out alternate source.

Tuberculosis among healthcare workers in a tertiary-care hospital in South India.

It is possible that tuberculosis is transmitted from patients to healthcare workers (HCWs). However, there are few data on this from developing countries. The object of this study was to document the incidence of tuberculosis among HCWs in the Christian Medical College (CMC), Vellore, India during a 10-year period (January 1992-December 2001). Data were collected from records maintained in the staff and students health services of CMC. A total of 125 cases were diagnosed during the period of study. The overall incidence of sputum positive cases was similar to that observed in the general population, during most years. However, it appears that focal outbreaks occur with transmission between HCWs. The chance of developing extra-pulmonary tuberculosis was higher in HCWs compared with the general population.

Evidence for class-specific factors in immunoglobulin isotype switching.

Immunoglobulin class switch recombination (SR) occurs by a B cell-specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate mu-->alpha and mu-->gamma3 SR.

Switch recombination in a transfected plasmid occurs preferentially in a B cell line that undergoes switch recombination of its chromosomal Ig heavy chain genes.

Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e. g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29 mu), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of S mu-S alpha recombination in the plasmid. Recombination of the plasmid in I.29 mu cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29 mu cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.

Analysis of immunoglobulin Sgamma3 recombination breakpoints by PCR: implications for the mechanism of isotype switching.

The molecular mechanism of immunoglobulin switch recombination is poorly understood. Switch recombination occurs between pairs of switch regions located upstream of the constant heavy chain genes. Previously we showed that switch recombination breakpoints cluster to a defined subregion in the Sgamma3, Sgamma1 and Sgamma2b tandem repeats. We have developed a strategy for direct amplification of Smu/Sgamma3 composite fragments as well as Smu and Sgamma3 regions by PCR. This assay has been used to analyze the organization of Smu, Sgamma3 and a series of Smu/Sgamma3 recombination breakpoints from hybridomas and normal mitogen-activated splenic B cells. DNA sequence analysis of the switch fragments showed direct joining of Smu and Sgamma3 without deletions or duplications. Mutations were found in two switch junctions on both sides of the crossover point, suggesting that template switching is the most likely model for the mechanism of switch recombination. Statistical analysis of the positions of the recombination breakpoints in the Sgamma3 tandem repeat indicates the presence of two sub-clusters, suggesting non-random usage of DNA substrate in the recombination reaction.

TCR alpha beta gene usage for myelin basic protein recognition in healthy monozygous twins.

The pathogenic role of myelin basic protein (MBP)-specific T lymphocytes in multiple sclerosis (MS) has been suggested by the encephalitogenicity of MBP-specific T cells in experimental allergic encephalomyelitis (EAE). In humans, extensive analysis of TCRs involved in MBP recognition has led to conflicting results, varying from an intra- and/or interindividual restriction to high diversity in TCRAV/TCRBV gene usage. We previously established MBP-specific T cell lines (TCLs) from healthy monozygous twins and characterized their fine epitope specificity. In this study, we report on the TCR alpha beta gene usage of 52 of these MBP TCLs that are specific for epitopes recognized by both co-twins within the same pair. High overall diversity in the TCR alpha and TCR beta genes used for recognition of this self-Ag, MBP, was observed. Variable genes belonging to 19 different TCRAV and 16 different TCRBV subfamilies are expressed by the 52 TCLs herein studied. In co-twins, TCLs utilized genes belonging to common TCRAV and/or TCRBV gene subfamilies in 7 of 13 instances of shared epitope recognition. Statistical analysis of intrapair concordance for TCR gene usage for the recognition of a given peptide did not show any significant deviation from values that would be anticipated in the absence of genetic background effect.

In vivo clonal expression of T lymphocytes specific for an immunodominant N-terminal myelin basic protein epitope in healthy individuals.

The role of myelin basic protein (MBP) T cell recognition in the induction of experimental allergic encephalomyelitis (EAE) has been well established in mice and rats. A remarkable restriction has been observed in T cell receptor (TCR) genes utilized by encephalitogenic T cell lines (TCLs) specific for immunodominant epitopes in these species. Pathological similarities between this animal model and multiple sclerosis (MS) has led to consider MBP as a major candidate autoantigen in this human disorder. Unlike in inbred strains of animals, the T cell response to MBP in humans is quite heterogenous with regard to fine epitope specificity. The existence of V alpha and/or V beta restriction in MBP-specific T cells, from MS patients and healthy controls, is still a matter of debate. In this study we generated 77 MBP-specific TCLs from nine healthy donors and showed that peptide 7-27 is one of the most frequently recognized epitopes. 37% of all epitope-specific TCLs recognized this peptide and p7-27 specific TCLs were generated from seven out of the nine subjects studied. A high level of in vivo clonal expansion was observed in p7-27-specific TCLs in several subjects, which however is not specific of this epitope since this phenomenon was also observed in p85-104- and 149-162-specific TCLs.

Healthy monozygous twins do not recognize identical T cell epitopes on the myelin basic protein autoantigen.

The T cell response against myelin basic protein (MBP) has been extensively studied in humans because of its putative role in the pathophysiology of multiple sclerosis (MS). Higher concordance rates in monozygous twins as well as an increased risk in relatives suggest the role of genetic factors in MS susceptibility. Very little is known about the shaping of T cell repertoire towards self antigens in humans and their contribution to disease susceptibility in autoimmune disorders. Here we report the comparative T cell epitope recognition patterns towards the MBP auto-antigen in healthy identical twins. We have established MBP-specific T cell lines from eight sets of twins and characterized their fine epitope specificity. Intra-pair comparison showed the co-existence of shared as well as distinct epitopes in six of eight pairs and a complete absence of concordant epitope recognition within two other pairs. These findings indicate that important differences in T cell repertoires against a self antigen may be observed between genetically identical healthy individuals, rendering difficult the interpretation of the differences which may be observed between identical twins discordant for an autoimmune disease.

Hypocalcemia in patients with prostate cancer.

We report a case of severe hypocalcemia in a patient with prostate cancer and extensive metastatic bone disease. The hypocalcemia in this patient was most likely on the basis of extensive accretion of calcium into the bones. We further studied 112 patients with prostate cancer, 15 of whom were discovered to be hypocalcemic on the basis of serum total calcium measurement. Fourteen of these 15 patients had bone metastases. Serum total calcium, total protein, and albumin levels were significantly lower in patients with bone metastases (n = 61) than those without (n = 51). Hypocalcemia could be explained on the basis of hypoalbuminemia or renal failure in these patients. Plasma ionized Ca measurements were made in 47 of the total 112 patients. Only one patient with extensive bone metastases was found to be hypocalcemic on the basis of ionized calcium measurement. Therefore, apparent hypocalcemia based on total calcium measurement is common in patients with prostate cancer (14% of all and 23% of those with bone metastases), whereas true hypocalcemia based on ionized calcium determinations is unusual.