Clinico-microbiological spectrum of anaerobic pyogenic infections in an Indian tertiary care teaching hospital: A two-year study.

INTRODUCTION: Anaerobes are important however the most neglected pathogens. Timely isolation of anaerobes can guide the clinician about the correct course of clinical treatment and thus reduce the mortality and also the problem of antimicrobial resistance. MATERIALS AND METHODS: Tissue and/or pus aspirates were collected aseptically from infectious sites in the Robertson's cooked meat medium (RCM) and sent to anaerobic bacteriology laboratory for culture. Subcultures from RCM for each sample were done on neomycin blood agar and 5% sheep blood agar along with metronidazole disc (5µg). The plates were incubated in an anaerobic jar using GasPak for 72 hrs. The preliminary identification was performed by standard biochemical tests for both obligate and facultative anaerobic isolates. Speciations of obligate anaerobes were performed by Vitek 2 automated system. RESULTS: Obligate anaerobes either single or polymicrobial were obtained in 38/216 (14.5 %) samples processed during the study period. Polymicrobial infections were reported in 21/216 (55.26%) samples and most commonly with obligate anaerobic gramnegative bacilli i.e. Prevotella-Porphyromonas and Bacteroides fragilis group. Most common monomicrobial anaerobic infections were observed with Veillonella spp. (n=4) and Porphyromonas spp. (n=4) followed by Bacteroides fragilis (n=3). Obligate anaerobes were predominantly isolated from skin and soft tissue infections (n=14) followed by surgical site infections (n=8). CONCLUSION: Although most of the infections are polymicrobial, a rise in the incidence of monomicrobial anaerobic infections has been noticed. Therefore, the performance of anaerobic cultures along with aerobic cultures is much needed for complete bacterial work-up of specimens from infectious sites and better patient management.

Biocompatible nanocrystalline natural bonelike carbonated hydroxyapatite synthesized by mechanical alloying in a record minimum time.

Single phase nanocrystalline biocompatible A-type carbonated hydroxyapatite (A-cHAp) powder has been synthesized by mechanical alloying the stoichiometric mixture of CaCO3 and CaHPO4.2H2O powders in open air at room temperature within 2h of milling. The A-type carbonation in HAp is confirmed by FTIR analysis. Structural and microstructure parameters of as-milled powders are obtained from both Rietveld's powder structure refinement analysis and transmission electron microscopy. Size and lattice strain of nanocrystalline HAp particles are found to be anisotropic in nature. Mechanical alloying causes amorphization of a part of crystalline A-cHAp which is analogous to native bone mineral. Some primary bond lengths of as-milled samples are critically measured. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay test reveals high percentage of cell viability and hence confirms the biocompatibility of the sample. The overall results indicate that the processed A-cHAp has a chemical composition very close to that of biological apatite.

Towards new business models for R&D for novel antibiotics.

In the face of a growing global burden of resistance to existing antibiotics, a combination of scientific and economic challenges has posed significant barriers to the development of novel antibacterials over the past few decades. Yet the bottlenecks at each stage of the pharmaceutical value chain-from discovery to post-marketing-present opportunities to reengineer an innovation pipeline that has fallen short. The upstream hurdles to lead identification and optimization may be eased with greater multi-sectoral collaboration, a growing array of alternatives to high-throughput screening, and the application of open source approaches. Product development partnerships and South-South innovation platforms have shown promise in bolstering the R&D efforts to tackle neglected diseases. Strategies that delink product sales from the firms' return on investment can help ensure that the twin goals of innovation and access are met. To effect these changes, both public and private sector stakeholders must show greater commitment to an R&D agenda that will address this problem, not only for industrialized countries but also globally.

Role of cytokine p40 family in multiple sclerosis.

Over the last couple of decades of neuro-immunological research, the p40 family of cytokines has emerged out as one of the most intriguing areas of interest because of multi-faceted roles of these cytokine in immune-modulation and inflammation. The IL-12, the most widely studied cytokine of this family, is well-characterized for its Th1-favoring activity, and therefore plays a key role in the pathophysiology of Th1-mediated autoimmune diseases like multiple sclerosis (MS). On the other hand, the IL-23, another member of the p40 family with shared p40 subunit, drives polarization of Th17, a subset of T cell suspected to have a key role in the pathophysiology of MS and experimental allergic encephalomyelitis (EAE), poses a challenge to our current understanding of Th1/Th2 hypotheses in autoimmune diseases. However, the more puzzling issues, the researchers are currently confronted with, are the biological roles of other two members of this family, the p40 monomer and the p40(2), the homodimer. Predominance of the mRNA level of p40 over p35 in the central nervous system of EAE and MS suggests a possible involvement of p40 in the pathogenesis of MS. However, the distinctive biological role of monomeric and dimeric form of p40 is not clearly understood yet. Initially, it was thought that p402 does not have any biological activity and only involved in antagonizing bioactive IL-12 but according to recent evidences, both p402 and p40 appear to have a proinflammatory role, therefore might be a crucial molecule in the pathogenesis of MS. The current review focuses on biological function of p40 family of cytokines with particular emphasis on MS.

Evidence of linkage and association on 18p11.2 for psychosis.

The genetic basis of bipolar disorder (BPD) and schizophrenia (SCZ) has been established through numerous clinical and molecular studies. Although often considered separate nosological entities, evidence now suggests that the two syndromes may share some genetic liability. Recent studies have used a composite phenotype (psychosis) that includes BPD, SCZ, psychosis not otherwise specified, and schizoaffective disorder, to identify shared susceptibility loci. Several chromosomal regions are reported to be shared between these syndromes (18p, 6q, 10p, 13q, 22q). As a part of our endeavor to scan these regions, we report a positive linkage and association finding at 18p11.2 for psychosis. Two-point linkage analysis performed on a series of 52 multiplex pedigrees with 23 polymorphic markers yielded a LOD score of 2.02 at D18S37. An independent set of 159 parent offspring trios was used to confirm this suggestive finding. The TDT analysis yielded support for association between the marker D18S453 and the disease allele (chi2 = 4.829, P < 0.028). This region has been implicated by several studies on BPD [Sjoholt et al. (2004); Mol Psychiatry 9(6):621-629; Washizuka et al. (2004); Biol Psychiatry 56(7):483-489; Pickard et al. (2005); Psychiatr Genet 15(1):37-44], SCZ [Kikuchi et al. (2003); J Med Dent Sci 50(3):225-229; Babovic-Vuksanovic et al. (2004); Am J Med Genet 124(3):318-322] and also as a shared region between the two diseases [Ishiguro et al. (2001); J Neural Transm 108(7):849-854; Reyes et al. (2002); Mol Psychiatry 7(4):337-339; Craddock et al. (2005); J Med Genet 42(3):193-204]. Our findings provide an independent validation of the above reports, and suggest the presence of susceptibility loci for psychoses in this region.

Characterization of parallel and antiparallel G-tetraplex structures by vibrational spectroscopy.

A series of G-rich oligonucleotides able to form tetraplexes has been studied by FTIR spectroscopy. Characteristic markers of the formation of guanine tetrads are given. Moreover, we propose a new marker discriminating between parallel and antiparallel tetraplexes: the position of the C6O6 guanine carbonyl stretching vibration. In intermolecular parallel tetrameric structures formed by four separate strands this absorption is observed at 1693 cm-1 while for antiparallel tetrameric structures, either intramolecular or formed by dimerization of hairpins, this vibrational mode is observed at 1682 cm-1. These shifts to higher wavenumbers, when compared to the position of a free guanine C6O6 carbonyl stretching vibration observed at 1666 cm-1(Deltanu=27 cm-1 for parallel tetraplexes and Deltanu=16 cm-1 for antiparallel tetraplexes) reflect different strand orientations in the structures. This marker has been used to evidence the possibility of an antiparallel-parallel tetraplex reorganization for Oxytricha nova d(G4T4G4) and d((G4T4)3G4) and human d(G3T2AG3) telomeric sequences induced by Na+/K+ or Na+/Ca2+ ion exchange. Formation of the guanine tetrads, characterization of the phosphate geometries and of the sugar conformations have also been obtained by FTIR for the different tetraplexes.

Molecular analysis of Huntington's disease and linked polymorphisms in the Indian population.

OBJECTIVES: To understand the population variation and haplotypes of Huntington's disease (HD) in India we have analysed CAG repeats at the HD locus together with closely linked polymorphisms in both HD patients and normal controls. MATERIALS AND METHODS: The CAG repeat and linked polymorphisms were analysed in 30 Indian HD families together with 250 ethnically matched controls using fluorescent polymerase chain reaction (PCR) based size estimation. RESULTS: CAG repeats at the HD locus in the normal population showed a mean size of 17.99 +/- 2.66 repeats (range nine to 33 repeats). The HD mutation in our families did not show any significant association with either the (CCG)7 or (CCG)10 allele while haplotype analysis suggested the over-representation of the 7-2-I (CCG-D4s127-Delta 2642 loci) haplotype in a subset of families. CONCLUSION: The distribution of CAG repeats in the normal population suggests a higher prevalence of HD, closer to that seen in Western Europe. Haplotype analysis suggests the presence of a founder mutation in a subset of families and provides evidence for multiple and geographically distinct origins for the HD mutation in India.

Common psychiatric diseases and human genetic variation.

OBJECTIVE: A better understanding of human genetic variation is important in assessing disease epidemiology and phenotypic variation, and may be critical in evaluating genetic aspects of common genetic diseases, such as schizophrenia, bipolar disease and Parkinson's. These diseases are particularly difficult to investigate as there are few peripheral markers, and although a genetic aetiology has long been suspected, robust findings have been hard to establish. METHODS: Variations in alleles at 13 tri-nucleotide gene loci expressed in the brain and implicated in several neurodegenerative diseases, as well as certain other loci, were examined in the Indian population for comparison with other major ethnic groups. RESULTS AND CONCLUSION: In the Indian population, the distribution of alleles at the Machado-Joseph disease locus was similar to the Western European pattern of distribution. Analysis of haplotypes at the locus for Huntington's disease suggested multiple origins, and possible effects of population admixture because of the recent history of the country. At other alleles of neuropsychiatric interest (dopamine receptor, serotonin receptor, serotonin transporter, alcohol dehydrogenase), allele frequencies in the Indian population differed from other populations. Interspecies comparison suggests a gradual expansion in repeat size, with the exception of the CLOCK gene, which displays a contraction of CAG repeat numbers. World-wide differences in disease phenotypes need to be explored, and an appreciation of their genetic basis may provide a window of opportunity for improving our knowledge of the underlying genetic mechanisms.

Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae.

BACKGROUND: Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. RESULTS: A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the beta-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10 Upstream Activating Sequences (UASg). Upon derepression with galactose, beta-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. CONCLUSION: We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

CAG repeat instability at SCA2 locus: anchoring CAA interruptions and linked single nucleotide polymorphisms.

Spinocerebellar ataxia 2 (SCA2) is an autosomal dominant neurodegenerative disorder that results from the expansion of a cryptic CAG repeat within the exon 1 of the SCA2 gene. The CAG repeat in normal individuals varies in length from 14 to 31 repeats and is frequently interrupted by one or more CAA triplets, whereas the expanded alleles contain a pure uninterrupted stretch of 34 to 59 CAG repeats. We have previously reported the presence of a limited pool of 'ancestral' or 'at risk' haplotypes for the expanded SCA2 alleles in the Indian population. We now report the identification of two novel single nucleotide polymorphisms (SNPs) in exon 1 of the SCA2 gene and their characterization in 215 normal and 64 expanded chromosomes. The two biallelic SNPs distinguished two haplotypes, GT and CC, each of which formed a predominant haplotype associated with normal and expanded SCA2 alleles. All the expanded alleles segregated with CC haplotype, which otherwise was associated with only 29.3% of the normal chromosomes. CAA interspersion analysis revealed that majority of the normal alleles with CC haplotype were either pure or lacked the most proximal 5' CAA interruption. The repeat length variation at SCA2 locus also appeared to be polar with changes occurring mostly at the 5' end of the repeat. Our results demonstrate that CAA interruptions play an important role in conferring stability to SCA2 repeat and their absence predisposes alleles towards instability and pathological expansion. Our study also provides new haplotypes associated with SCA2 that should prove useful in further understanding the mutational history and mechanism of repeat instability at the SCA2 locus.

Progressive myoclonus epilepsy [EPM1] repeat d(CCCCGCCCCGCG)n forms folded hairpin structures at physiological pH.

The secondary structure of DNA has been shown to be an important component in the mechanism of expansion of the trinucleotide repeats that are associated with many neurodegenerative disorders. Recently, expansion of a dodecamer repeat, (CCCCGCCCCGCG)n upstream of cystatin B gene has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundborg type. We have investigated structure of oligonucleotides containing one, two and three copies of the EPM1 repeat sequences at physiological pH. CD spectra and anomalous faster gel electrophoretic mobilty indicates formation of intramolecularly folded structures that are formed independent of concentration. Hydroxylamine probing allowed us to identify the C residues that are involved in C.G base pairing. P1 nuclease studies elucidated the presence of unpaired regions in the folded back structures. UV melting studies show biphasic melting curves for the oligonucleotides containing two and three EPM1 repeats. Our data suggests multiple hairpin structures for two and three repeat containing oligonucleotides. In this paper we show that oligonucleotides containing EPM1 repeat adopt secondary structures that may facilitate strand slippage thereby causing the expansion.

Intramolecular i-motif structure at acidic pH for progressive myoclonus epilepsy (EPM1) repeat d(CCCCGCCCCGCG)n.

The most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type is the expansion of a dodecamer repeat, d(CCCCGCCCCGCG)n. We show that the C-rich strand of this repeat (2-3 copies) forms intercalated i-motif structure at acidic pH as judged by CD spectroscopy and anomalous gel electrophoretic mobility. The stability of the structure increases with the increase in the length of the repeat. Transient formation of stable, folded back structure like i-motif could play an important role in the mechanism of expansion of this repeat.

Association of CAG repeat loci on chromosome 22 with schizophrenia and bipolar disorder.

Chromosome 22 has been implicated in schizophrenia and bipolar disorder in a number of linkage, association and cytogenetic studies. Recent evidence has also implicated CAG repeat tract expansion in these diseases. In order to explore the involvement of CAG repeats on chromosome 22 in these diseases, we have created an integrated map of all CAG repeats > or =5 on this chromosome together with microsatellite markers associated with these diseases using the recently completed nucleotide sequence of chromosome 22. Of the 52 CAG repeat loci identified in this manner, four of the longest repeat stretches in regions previously implicated by linkage analyses were chosen for further study. Three of the four repeat containing loci, were found in the coding region with the CAG repeats coding for glutamine and were expressed in the brain. All the loci studied showed varying degrees of polymorphism with one of the loci exhibiting two alleles of 7 and 8 CAG repeats. The 8-repeat allele at this locus was significantly overrepresented in both schizophrenia and bipolar patient groups when compared to ethnically matched controls, while alleles at the other three loci did not show any such difference. The repeat lies within a gene which shows homology to an androgen receptor related apoptosis protein in rat. We have also identified other candidate genes in the vicinity of this locus. Our results suggest that the repeats within this gene or other genes in the vicinity of this locus are likely to be implicated in bipolar disorder and schizophrenia.

A clinical study of patients with genetically confirmed Huntington's disease from India.

BACKGROUND: Clinical data across the globe especially in genetic diseases like Huntington's disease (HD) is most helpful when collected using standardized formats. This helps in proper comparison of clinical and genetic data. METHODS: Herein, we report clinical data on 26 genetically confirmed HD patients from 19 Indian families predominantly from South India. Clinical data and evaluation was performed using standardized formats used by the Huntington Disease Study Group. RESULTS: Adult onset HD was commonest while Juvenile HD (onset <20 years) was observed in approximately 15% of patients. Chorea was the commonest presenting symptom (n=23, 88.5%) while remaining presented with psychiatric symptoms (n=3, 11.5%). Impairment of saccades was observed in approximately 75% of patients. Mean (SD) CAG repeats in the abnormal allele was 48.4 (8.7). Total motor score but not the total behavioral score worsens with duration of symptoms. The functional checklist score correlates with total motor score rather than with duration of symptoms. CONCLUSIONS: We detail clinical characteristics in genetically confirmed HD patients from a predominantly South Indian cohort. We observed a slightly higher occurrence of Juvenile HD. Functional disabilities in our patients correlate with worsening of motor rather than behavioral symptoms.

The polyglutamine motif is highly conserved at the Clock locus in various organisms and is not polymorphic in humans.

Circadian rhythms play a central role in diverse physiological phenomena and the recent years have witnessed the identification of a number of genes responsible for the maintenance of these rhythms. One of these is the Clock gene, which was first identified in mouse and subsequently in a large number of organisms, including humans. The human Clock gene has been proposed as a possible candidate for disorders affected by alterations of circadian rhythm, including bipolar disorder and schizophrenia. This gene contains a highly conserved polyglutamine motif, that in humans is coded for by CAG repeats. In view of the involvement of CAG repeat expansion in a number of neuro-psychiatric disorders, we have sought to determine the polymorphism status of CAG repeats at the Clock locus in humans. Our analysis of 190 unrelated individuals, who included patients suffering from bipolar disorder and schizophrenia, indicated that the repeat, which consisted of 6 CAG triplets, was not polymorphic in humans. An analysis of the repeat in non-human primates and other organisms revealed that the glutamine stretch is shortest in humans and baboons, and longest in Drosophila and zebrafish. A study of various Drosophila species revealed that the repeat number is highly polymorphic, ranging from 25 to 33 pure glutamine repeats. Unlike most other microsatellites, the CAG repeat stretch at the Clock locus in humans is smaller than its homologues in non-human primates. We propose that glutamine repeat size is functionally important in this gene and thus tightly regulated. The variation in repeat number is probably deleterious to the individual, resulting in the maintenance of a short and invariable repeat structure in the human population.

Angiotensin converting enzyme insertion allele in relation to high altitude adaptation.

Angiotensin converting enzyme (ACE) gene I/D polymorphism has been associated with high altitude (HA) disorders as well as physical performance. We, however, envisage that the polymorphism may be associated with adaptation to the hypobaric hypoxia of altitude, thus facilitating physical performance. For this purpose, three unrelated adult male groups, namely (1) the Ladakhis (HLs), who reside at and above a height of 3600 m, (2) lowlanders, who migrated to Ladakh (MLLs), and (3) resident lowlanders (LLs), have been investigated. The HLs had significantly (p & 0.001) greater numbers of the II homozygotes and the ID heterozygotes than the DD homozygotes, the genotype distribution being 0.46, 0.43 and 0.11 for II, ID and DD genotypes respectively. The MLLs comprised 60% II homozygotes, which was higher (p & 0.001) than the HLs (46%). In the LLs, the heterozygotes were greater (p & 0.001) in number than the II and DD homozygotes. The I allele frequency was 0.72 in the MLLs, 0.67 in the HLs and 0.55 in the LLs. Polymorphism study suggested that the II genotype could be associated with altitude adaptation, which might influence physical efficiency.

Molecular and clinical correlation in five Indian families with spinocerebellar ataxia 12.

Spinocerebellar ataxia 12 (SCA12) is a recently identified form of autosomal dominant cerebellar ataxia associated with the expansion of an unstable CAG repeat in the 5' untranslated region of the gene PPP2R2B. We analyzed 77 Indian families with autosomal dominant cerebellar ataxia phenotype and confirmed the diagnosis of SCA12 in 5 families, which included a total of 6 patients and 21 family members. The sizes of the expanded alleles ranged from 55 to 69 CAG repeats, and the sizes of the normal alleles ranged from 7 to 31 repeats. We believe our study is the first to demonstrate that SCA12 may not be as rare in some populations as previously thought.

Association analysis of CAG repeats at the KCNN3 locus in Indian patients with bipolar disorder and schizophrenia.

Bipolar affective disorder and schizophrenia are severe behavioral disorders with a lifetime risk of approximately 1% in the population worldwide. There is evidence that these diseases may manifest the phenomenon of anticipation similar to that seen in diseases caused by trinucleotide repeat expansions. A recent report has implicated a potassium channel-coding gene, KCNN3, which contains a polymorphic CAG repeat in its coding region, in schizophrenia and bipolar disorder. We have tried to confirm these findings in Indian patients suffering from bipolar disorder and schizophrenia. No statistically significant evidence for the presence of an excess of longer alleles in the patient population, as compared to ethnically matched controls, was found. However, an analysis of the difference of allele sizes revealed a significantly greater number of patients with schizophrenia having differences of allele sizes > or = 5 when compared to normal controls. This finding may be of functional significance as the KCNN3 protein is thought to act as a tetramer, and a large difference in allele sizes would result in an asymmetric molecule with a different number of glutamine residues in each monomer. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:744-748, 2000.

Association analysis of 5HT transporter gene in bipolar disorder in the Indian population.

A variable number tandem repeat (VNTR) polymorphism consisting of multiple copies of a 17-bp repeat in the second intron of the serotonin transporter gene (SERT) has been reported. Different alleles of this VNTR have been found to be associated with bipolar disorder and schizophrenia. These findings have been confirmed in some populations, but disconfirmed in others. Furthermore, significant ethnic variations in the distribution of these alleles both in normal and patient populations also have been reported. We analyzed the VNTR polymorphism in 50 Indian patients with bipolar disorder and in ethnically matched controls. Two alleles corresponding to 10 and 12 repeats of the VNTR were found in both groups. There were no significant differences either in allele frequency or genotype frequency between the two groups. The nine-repeat allele that has been reported in Japanese and Caucasian populations was absent in our sample. Although it will be important to extend the present study in a larger sample, our initial results do not suggest any large association with alleles of the VNTR in the SERT gene and bipolar disorder in Indian patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:170-172, 2000.