Phylogenetic Analysis of Methanogenic Enrichment Cultures Obtained from Lonar Lake in India: Isolation of <Emphasis Type="Italic">Methanocalculus</Emphasis> sp. and <Emphasis Type="Italic">Methanoculleus</Emphasis> sp.

The diversity of methanogenic archaea in enrichment cultures established from the sediments of Lonar Lake (India), a soda lake having pH ≈ 10, was investigated using 16S rDNA molecular phylogenetic approach. Methanogenic enrichment cultures were developed in a medium that simulated conditions of soda lake with three different substrates viz., H₂:CO₂, sodium acetate, and trimethylamine (TMA), at alkaline pH. Archaeal 16S rRNA clone libraries were generated from enrichment cultures and 13 RFLP groups were obtained. Representative sequence analysis of each RFLP group indicated that the majority of the 16S rRNA gene sequences were phylogenetically affiliated with uncultured Archaea. Some of the groups may belong to new archaeal genera or families. Three RFLP groups were related to Methanoculleus sp, while two related to Methanocalculus sp. 16S rRNA gene sequences found in Lonar Lake were different from sequences reported from other soda lakes and more similar to those of oil reservoirs, palm oil waste treatment digesters, and paddy fields. In culture-based studies, three isolates were obtained. Two of these were related to Methanoculleus sp. IIE1 and one to Methanocalculus sp. 01F97C. These results clearly show that the Lonar Lake ecosystem harbors unexplored methanogens.     

Ubiquitylation-independent degradation of Xeroderma pigmentosum group C protein is required for efficient nucleotide excision repair

The Xeroderma Pigmentosum group C (XPC) protein is indispensable to global genomic repair (GGR), a subpathway of nucleotide excision repair (NER), and plays an important role in the initial damage recognition. XPC can be modified by both ubiquitin and SUMO in response to UV irradiation of cells. Here, we show that XPC undergoes degradation upon UV irradiation, and this is independent of protein ubiquitylation. The subunits of DDB-Cul4A E3 ligase differentially regulate UV-induced XPC degradation, e.g DDB2 is required and promotes, whereas DDB1 and Cul4A protect the protein degradation. Mutation of XPC K655 to alanine abolishes both UV-induced XPC modification and degradation. XPC degradation is necessary for recruiting XPG and efficient NER. The overall results provide crucial insights regarding the fate and role of XPC protein in the initiation of excision repair.     

Variants of eae and stx genes of atypical enteropathogenic Escherichia coli and non-O157 Shiga toxin-producing Escherichia coli from calves

AIMS: To determine the subtypes of stx and eae genes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and to ascertain the typical and atypical nature of EPEC. METHODS AND RESULTS: One hundred and eighty-seven faecal samples from 134 diarrhoeic and 53 healthy calves were investigated for the presence of stx, eae and ehxA virulence genes by polymerase chain reaction and enzyme-linked immunosorbent assay. Subtype analysis of stx(1) exhibited stx(1c) in 13 (31.70%) isolates, while that of stx(2) revealed stx(2c) in eight (24.24%) and stx(2d) in two (6.06%) isolates. Subtyping of eae gene showed the presence of eae-beta, eae-eta and eae-zeta in two, three and four isolates respectively. None of the E. coli isolates possessed stx(2e), stx(2f), eae-alpha, eae-delta, eae-epsilon and eae-xi. All EPEC isolates were atypical. CONCLUSIONS: stx(1), stx(1c), stx(2), stx(2c), stx(2d), eae-beta, eae-eta and eae-zeta subtypes are prevalent in STEC and EPEC isolates in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first subtype analysis of stx(2) and eae genes of animal E. coli isolates in India and emphasizes the need to investigate their transmission to humans.     

Primary cutaneous mucormycosis in an immunocompetent host

Mucormycosis in immunocompetent hosts is rare, and is often related to trauma. We report a case of primary cutaneous mucormycosis in a 26 year old immunocompetent female due to Mucor species. A combination of Amphotericin B and surgical debridement completely eradicated the infection.     

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis

Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.     

Association of single nucleotide polymorphisms of CACNA1A gene in migraine

INTRODUCTION:

Migraine is a chronic, neurovascular polygenic disease where genetic and environmental factors are involved in its etiology. Dysfunction of neuronal ion transportation can provide a model for predisposition for common forms of migraine. Mutations in genes encoding ion channels disturb the rhythmic function of exposed tissue that may also explain the episodic nature of migraine. Our aim was to study the single nucleotide polymorphisms of CACNA1A gene in migraine patients.

MATERIALS AND METHODS:

The subjects were the patients of migraine, in the age range of 18-80 years, diagnosed by a Neurologist, as per the diagnostic criteria of International Headache Society (IHS) Classification 2004 after excluding other causes of headache by clinical examination and relevant investigations.The controls were the age and sex matched healthy persons from the same population excluding the relatives of patients. Only those patients and the controls, who voluntarily participated in the study, were taken and their blood samples were taken for the study. Deoxyribonucleic acid (DNA) extraction was performed according to the manufacturer''s protocol for Qiagen DNA extraction kits (Qiagen, Hilden, NRW, Germany). DNA content was quantified by spectrophotometric absorption (Nanodrop Spectrophotometer, BioLab, Scoresby, VIC, Australia). Polymerase chain reaction was performed using an iCycler Thermal Cycler (Bio.Rad, Hercules, CA, USA). The polymorphic analysis of CACNA1A gene was carried out by two methods: Restriction fragment length polymorphism and sequencing.

RESULTS:

The study included a total of 25 patients of migraine, diagnosed on out-patient department basis as per IHS Classification 2004 and compared with age and sex matched 25 healthy controls. Most of the patients 23 (92%) were below the age of 50 years. 20 of the patients (80%) were females and 5 (20%) were males. The polymorphic analysis of CACNA1A gene revealed the presence of only the wild form of the gene for the codon E993V in both case and control groups.

CONCLUSION:

In our study, we could not find any polymorphism of CACNA1A gene in the selected patients. Instead the wild type of genotype was found in both patients and controls. This negative result presented here, implies that if the CACNA1A gene is involved in typical migraine (with and without aura), its contribution is very modest and therefore difficult to discern. Nevertheless, there are other genes that could be considered potential candidates for typical migraine susceptibility for which further research is needed.     

The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.