DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation

Prevalence of peripheral neuropathy in newly diagnosed type 2 diabetics

ABSTRACT Our objective was to determine the prevalence and risk factors of peripheral neuropathy in newly diagnosed type 2 diabetes mellitus. One hundred newly diagnosed type 2 diabetic patients attending Diabetes Clinic, Regional Institute of Medical Sciences, Imphal were randomly selected for clinical and electrophysiological studies for diagnosis of peripheral neuropathy. Peripheral neuropathy was evaluated by using Neuropathy Symptoms Score (NSS), Neuropathy Disability Score (NDS) and Nerve Conduction Studies (NCV) and the diagnosis of peripheral neuropathy was made when two or more of the three abnormalities of NSS, NDS and NCV were present. 29 patients (29%), 17 males (28%) and 12 females (31%) of the 100 newly diagnosed type 2 diabetic patients had peripheral neuropathy. Multiple logistic regression analysis shows that duration of diabetes has maximum contribution and age, systolic blood pressure and blood glucose have some contribution to the development of diabetic peripheral neuropathy

A Multi-centre Study to Evaluate the Long-Term Efficacy and Safety of Biosimilar Infliximab (Infimab™) in Ankylosing Spondylitis in Real-world Clinical Settings - A perspective from Eastern India

Introduction: Owing to dearth of data on infliximab biosimilars in Indian patients, a pan-India case database-based study with infliximab biosimilar BOW015 (Infimab™) was carried out to capture its efficacy and safety in real world clinical settings in India. Here, we assessed its efficacy and safety in ankylosing spondylitis (AS) among patients in the East India cohort. Materials and methods: Data were collected from multiple centers across the eastern region of India. Patients diagnosed with AS, within the preceding 4-6 months during the preceding one year were included in the study. Patients who were given BOW015 for other indications, prior innovator infliximab or other biologics were excluded from the study. Primary variable was Ankylosing Spondylitis Disease Activity Scale (ASDAS) response defined as change of > 2 in the ASDAS score from the baseline by 4-6 months of follow up. Results: The cohort consisted of 149 patients, predominantly male (69.8%), with mean (±SD) age of 36.75 (±11.11) years and mean (±SD) body weight of 58.26 (±15.4) kgs. Of the treated patients, 91 (61.1%) patients were administered four doses, 10 (6.7%) patients were administered three doses, 37 (24.8%) patients were administered two doses and 11 (7.4%) patients were administered only a single dose of BOW015. In the final analysis set, 81 patients had data at baseline and 4th visit. Among the 81 patients, 74 (91%) patients achieved major improvement, 5 (6%) patients achieved clinically important improvement and 2 (3%) were non-responders at 4th visit. Secondarily, cross categorization of the cohort into disease activity categories by number of infusions administered from baseline to 4th visit and assessment of trends in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) scores were also carried out and these too confirmed the efficacy of BOW015. Conclusion: Infimab™ (BOW015) showed significant improvement in ASDAS and BASDAI in patients with AS at the end of 4-6 months of follow up with its clinical benefits being apparent as early as first dose of BOW015

The Bordetella bacteriophage DGR employs similar mechanisms for retrotransposition in heterologous species

Diversity-generating retroelements (DGRs) are a unique group of retroelements found in bacteria, archaea and their viruses. They mediate hyperdiversification of protein-encoding DNA sequences in facilitate the adaptation of their hosts to changing environments. The prototype DGR was discovered in the Bordetella bacteriophage BPP-1 and consists of three genes, mtd (major tropism determinant), avd (accessory variability determinant) and brt (Bordetella reverse transcriptase), and two imperfect repeats, variable repeat (VR) and template repeat (TR). VR is located at the 3' end of mtd, which encodes the phage distal tail fiber protein responsible for receptor recognition. Diversification of mtd results from unidirectional transfer of sequence information from TR to VR during which adenine residues in TR are converted into random nucleotides in VR, leading to phage tropic variants that recognize different receptor molecules. Here, we show that the BPP-1 DGR is also functional in heterologous bacterial species - Escherichia coli and Pseudomonas aeruginosa, and uses a similar mechanism for cDNA synthesis. However, efficiency of DGR mutagenic homing is affected by target sequence orientation in plasmids. Interestingly, overexpression of Avd and bRT has differential effects on DGR homing into targets inserted in different vectors. Surprisingly, homing into plasmid targets in E. coli is found to be largely independent of IMH (initiation of mutagenic homing) and the DNA stem-loop, elements important for its homing into native phage targets.Santa S. Naorem, Jin Han, Christa Jackson, Bingyue Zhang, James Guo and Huatao Guo ; Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri 6521

Substitution of glutamate residue by lysine in the dimerization domain affects DNA binding ability of HapR by inducing structural deformity in the DNA binding domain.

HapR has been given the status of a high cell density master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of the wild type protein. This work aims at investigating the nature of functional inertness of a HapR natural variant harboring a substitution of a conserved glutamate residue at position 117 which participates in forming a salt bridge by lysine (HapRV2G-E(117)K). Experimental evidence presented here reveals the inability of this variant to interact with various cognate promoters by in vitro gel shift assay. Furthermore, the elution profiles of HapRV2G-E(117)K protein along with the wild type functional HapRV2G in size-exclusion chromatography as well as circular dichroism spectra did not reflect any significant differences in its structure, thereby indicating the intactness of dimer in the variant protein. To gain further insight into the global shape of the proteins, small angle X-ray scattering analysis (SAXS) was performed. Intriguingly, increased radius of gyration of HapRV2G-E(117)K of 27.5 Å in comparison to the wild type protein from SAXS data analyses implied a significant alteration in the global shape of the dimeric HapRV2G-E(117)K protein. Structure reconstruction brought forth that the DNA binding domains were substantially "parted away" in this variant. Taken together, our data illustrates that substitution of the conserved glutamate residue by lysine in the dimerization domain induces separation of the two DNA binding domains from their native-like positioning without altering the dimeric status of HapR variant

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation

Association of HbE Haemoglobinopathy in Patients with Systemic Lupus Erythematosus: A Cross-sectional Study

Introduction: Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disease of autoimmune origin that affects multiple systems, with the haematologic system being commonly involved. However, the co-existence of haemoglobinopathies and connective tissue disorders has rarely been investigated, and the available data on this matter are primarily anecdotal. Aim: To determine the prevalence of Haemoglobin E (HbE) haemoglobinopathy in adult SLE patients and to assess the association of HbE haemoglobinopathy and SLE with disease activity. Materials and Methods: A hospital-based cross-sectional study was conducted at the Department of General Medicine, Regional Institute of Medical Sciences (RIMS) Hospital, Imphal, Manipur, India, from April 2021 to July 2022. The study included SLE patients diagnosed during the study period who attended the rheumatology Outpatient Department (OPD). The independent variables were age, gender, occupation, religion, and family history, while HbE haemoglobinopathy, Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Red Blood Cell (RBC) count, and Systemic Lupus Activity Measure Revised Index (SLAM-R index) score were the dependent variables. Data were analysed using Statistical Package for Social Sciences (SPSS) version 21.0, with proportions analysed using the Chi-square test and Fisher’s-exact test, and means compared using Analysis of Variance (ANOVA) and independent t-test. A p-value of <0.05 was considered significant. Results: Of the 105 SLE patients included in the study, 93% were females. The majority of participants (36.2%) were in the age group of 21-30 years. Twenty-five patients (23.8%) had HbE haemoglobinopathy. Anaemia and MCV were significantly associated with HbE patients. Among the 25 HbE patients, 24 (96%) had active SLE disease. Among the HbE negative patients, 55 (68.7%) had active disease, while 25 (31.3%) did not have any active disease. Active SLE disease was significantly associated with HbE haemoglobinopathy (p-value=0.002). Conclusion: The overall prevalence of HbE haemoglobinopathy in SLE patients was found to be 24%. Hb levels and MCV levels were significantly lower in HbE patients. There was a significant association between active SLE disease and HbE haemoglobinopathy

SAXS data analysis from the samples of HapR_V2G and HapR_V2G-E¹¹⁷K.

<p>(A) SAXS I(Q) profiles are plotted <i>versus</i> Q for both the samples (HapR_V2G, magenta; HapR_V2G-E¹¹⁷K, gray). Inset shows the linear region of the Guinier analysis done presuming globular nature of the protein molecules in solution. (B) Kratky analysis from individual SAXS data sets confirmed the globular nature of proteins in solution. (C) Real space information of the predominant scattering species computed by indirect Fourier transformation are plotted between P(r) and R.</p

Multiple sequence alignment of HapR homologues in <i>Vibrio</i> species.

<p>Multiple sequence alignment of the deduced amino acid sequences of HapR homologues, from different <i>Vibrio species</i>. The regions shadowed in yellow and turquoise color represents the DNA binding domain and dimerization domain respectively. The conserved glutamate 117 residue in different HapR homologues is highlighted in red. Abbreviations are as follows: [V. h.] -<i>Vibrio harveyi</i>; [V.p.] –<i>Vibrio parahaemolyticus</i>; [V.v.] -<i>Vibrio vulnificus</i>; [V.a.] -<i>Vibrio anguillarum</i>; [V.c.] -<i>Vibrio cholerae</i> (C6706); [V.f.] -<i>Vibrio fischeri</i>.</p