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.     

中国鳞翅目新物种2021年年度报告

本文总结了2021年国内外学者发表的中国鳞翅目新分类单元的情况。经过统计, 2021年共发表与中国鳞翅目物种相关的论文104篇, 专著1部, 涉及新分类群230个, 其中新属7个, 新种215个, 新亚种8个; 发表中国新记录属9个、中国新记录种78个(包括2亚种)、新组合31个; 提出4个种的异名, 2个种被赋予新名。新种(亚种)隶属于34科。云南省发表的新物种数和新记录物种数明显高于其他地区, 分别占全国发表总数的36.8%和39.7%。2021年我国南方新物种和新记录种发表数量仍然显著高于北方。     

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.     

m6A‐mediated alternative splicing coupled with nonsense‐mediated mRNA decay regulates SAM synthetase homeostasis

Alternative splicing of pre‐mRNAs can regulate gene expression levels by coupling with nonsense‐mediated mRNA decay (NMD). In order to elucidate a repertoire of mRNAs regulated by alternative splicing coupled with NMD (AS‐NMD) in an organism, we performed long‐read RNA sequencing of poly(A)⁺ RNAs from an NMD‐deficient mutant strain of Caenorhabditis elegans, and obtained full‐length sequences for mRNA isoforms from 259 high‐confidence AS‐NMD genes. Among them are the S‐adenosyl‐L‐methionine (SAM) synthetase (sams) genes sams‐3 and sams‐4. SAM synthetase activity autoregulates sams gene expression through AS‐NMD in a negative feedback loop. We furthermore find that METT‐10, the orthologue of human U6 snRNA methyltransferase METTL16, is required for the splicing regulation in␣vivo, and specifically methylates the invariant AG dinucleotide at the distal 3′ splice site (3′SS) in␣vitro. Direct RNA sequencing coupled with machine learning confirms m⁶A modification of endogenous sams mRNAs. Overall, these results indicate that homeostasis of SAM synthetase in C. elegans is maintained by alternative splicing regulation through m⁶A modification at the 3′SS of the sams genes.     

Cost effective surface functionalization of silver nanoparticles for high yield immobilization of Aspergillus oryzae β-galactosidase and its application in lactose hydrolysis

The present study demonstrates synthesis, characterization and surface functionalization of silver nanoparticles (AgNPs) via glutaraldehyde for high yield immobilization of Aspergillus oryzae β-galactosidase. Soluble β-galactosidase (SβG), enzyme adsorbed on unmodified AgNPs (UβG) and surface modified AgNPs (MβG) showed same pH-optima at pH 4.5. However, it was observed that MβG exhibited enhanced pH stability toward acidic and alkaline sides, and increased temperature resistance as compared to SβG and UβG. Michaelis constant, K_m was increased nearly three-folds for MβG while V_max for soluble and MβG was 0.515 mM/min and 0.495 mM/min, respectively. Furthermore, MβG showed greater resistance to product inhibition mediated by galactose as compared to it soluble counterpart and exhibited excellent catalytic activity even after its fourth successive reuse. The remarkable bioconversion rates of lactose from milk in batch reactors further revealed an attractive catalytic efficiency of β-galactosidase adsorbed on surface functionalized AgNPs thereby promoting its use in the production of lactose free dairy products.