N6-Methyladenine Progressively Accumulates in Mitochondrial DNA during Aging.

N6-methyladenine (6mA) in the DNA is a conserved epigenetic mark with various cellular, physiological and developmental functions. Although the presence of 6mA was discovered a few years ago in the nuclear genome of distantly related animal taxa and just recently in mammalian mitochondrial DNA (mtDNA), accumulating evidence at present seriously questions the presence of N6-adenine methylation in these genetic systems, attributing it to methodological errors. In this paper, we present a reliable, PCR-based method to determine accurately the relative 6mA levels in the mtDNA of Caenorhabditis elegans, Drosophila melanogaster and dogs, and show that these levels gradually increase with age. Furthermore, daf-2(-)-mutant worms, which are defective for insulin/IGF-1 (insulin-like growth factor) signaling and live twice as long as the wild type, display a half rate at which 6mA progressively accumulates in the mtDNA as compared to normal values. Together, these results suggest a fundamental role for mtDNA N6-adenine methylation in aging and reveal an efficient diagnostic technique to determine age using DNA.

In vitro antimutagenic, antioxidant activities and total phenolics of clove (Syzygium aromaticum L.) seed extracts.

The present work explores antimutagenic and antioxidant potential as well as total phenolics of aqueous and acidified methanol extractable components from clove (Syzygium aromaticum L.) seed. The magnitude of antimutagenic activity of clove seed extracts (CSE) against two mutant bacterial strains: S. typhimurium TA98 and S. typhimurium TA100 (Ames bacterial test) ranged from 34.11-79.74%. Antioxidant activity in terms of measurement of DPPH radical scavenging capacity and inhibition of linoleic acid peroxidation was noted to be 71.16-94.58% and 54.96-86.89%, respectively. CSE also exhibited an appreciable amount of total phenolics with contribution between 22.80 and 115.33 GAE mg/100g. A strong correlation between total phenolics and tested biological activities were recorded. The results of this study advocate that clove seed can be explored as a viable source of bioactives for the development of chemotherapeutic drugs against cancer in addition to acting as nutraceutical and functional food ingredient.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Effect of storage temperature and duration on direct PCR amplification of various feather types and DBS matrices.

The use of direct PCR has been pioneered over the last decade for DNA analysis of biological specimens of distinct origins. The information on how longer these specimens can be stored and amplified by direct PCR is however scanty. Such a piece of information could expedite research and diagnostic studies without compromising the reliability of results. The current study was therefore designed to analyze the effect of storage temperature and duration on direct PCR amplification of biological specimens having either low quantity or high quantity of DNA. Whole blood, dried blood spots (DBS), and feathers from chicken were stored for five years at three different temperatures, viz. room temperature (∼25 °C), 4 °C, and -20 °C. These samples were subjected to crude DNA extraction by diluting them in PBS buffer and heating at 98 °C after 1 day, 7 days, 15 days, 1 month, 3 months, 6 months, 1 year, 3 years and 5 years of storage. The crude DNA was PCR-amplified with the use of DNA sexing primers as well as DNA barcoding primers. Incubation at 98 °C for 10 min of any type of sample in PBS buffer was sufficient for crude DNA extraction. There was irrelevant impact of feather type, DBS matrix nature and storage temperature on amplification success over the period of analysis. It was possible to successfully accomplish the amplification of 96 samples with the use of routine PCR reagents within 3.5-6.0 hrs. In short, economical and fast genetic analysis of commonly used avian samples is feasible after their storage for longer time at room temperature.

DIVERISTY and enzymatic potential of indigenous bacteria from unexplored contaminted soils in Faisalabad.

Bacteria residing in contaminated waste soil degrade and utilize organic and inorganic material as a source of nutrients as well as reduce environmental contamination through their enzymatic machinery. This enzymatic potential of indigenous bacteria can be exploited at industrial level through detailed screening, characterization, optimization and purification. In present study, diversity and enzymatic potential of indigenous bacteria was investigated through qualitative and quantitative screening methods from unexplored contaminated soil waste sites in Faisalabad. Shannon diversity (H') index revealed that twenty-eight soil samples from four contaminated sites were highly diverse of amylase, protease and lipase producing bacteria. Maximum protease producing bacteria were detected in fruit waste (1.929 × 107), whereas amylase and lipase producing bacteria were found in industrial (1.475 × 107) and (5.38 × 106), in household waste soil samples. Most of the indigenous bacterial isolates showed potential for multiple enzymes. An isolate OC5 exhibited capability for amylase production and optimization at a wider range of cultural conditions; pH (6-8), temperature (25 °C, 37 °C, 45 °C), incubation time (24-72 h), and NaCl concentrations 0.5-13%, using (1%) starch and lactose as substrates. An isolate OC5 was identified by molecular identification and phylogenetic analysis showed 99% sequence similarity with Bacillus spp. ANOVA was used to analyzed all data statistically. This study enhances the importance of initial screening and reporting of industrially potent indigenous bacteria from unexplored contaminated waste soils. In future, indigenous bacteria in contaminated wastes may be good candidates to solve various environmental pollution problems.

Plant-Based Bioactive Phthalates Derived from Hibiscus rosa-sinensis: As In Vitro and In Silico Enzyme Inhibition.

Hibiscus rosa-sinensis is an attractive, ever-blossoming, and effortlessly available plant around the globe. The fabulous flowers of H. rosa-sinensis enjoy a significant status in folk medicine throughout the world and comprise a range of phyto constituents due to which this splendid flower owns numerous biological and pharmaceutical activities like antioxidant, antifungal, antimicrobial, anti-inflammatory, antipyretic, antidiabetic, and antifertility activity. Considering this, column chromatographic isolation of the phytoconstituents of ethyl acetate fraction of the flowers of H. rosa-sinensis was performed. A series of five phthalates including Di-n-octyl phthalate (HR1), ditridecyl phthalate (HR2), 1-allyl 2-ethyl phthalate (HR3), diethyl phthalate (HR4), and bis (6-methylheptyl) phthalate (HR5) were isolated. The structures of the isolated phthalates were elucidated by gas chromatography-mass spectrometry, 1H NMR, and 13C NMR. In silico and in vitro antidiabetic and antioxidant potential and DFT studies of isolated phthalates were carried out. In our study, isolated ligands were explored as potent antidiabetic as well as antioxidant agents as they exhibited good binding affinity (in in vitro and in silico experiments) against all selected protein targets. Compounds HR1-HR5 showed that the binding affinity value ranged from -5.9 to -5.2 kcal/mol, -5.5 to -4.3 kcal/mol, and -5.0 to -4.1 kcal/mol for target proteins 1HNY, 2I3Y, and 5O40, respectively. Among all isolated phthalates, HR5 can be a lead compound as it showed the best binding affinity with human pancreatic α-amylase (ΔG = -5.9 kcal/mol) and displayed a minimum inhibition concentration (IC50) of 11.69 µM among all phthalates. Compound HR1 was the best docked and scored compound for inhibiting glutathione peroxidase; however, HR2 possessed the lowest binding score of -5.0 kcal/mol, thus indicating the highest potential among isolated phthalates for inhibiting the superoxide dismutase. Furthermore, the top-ranked docked ligand-protein complex for each protein was assessed for stability of protein and complex mobility by molecular dynamics simulation using the IMOD server.

Knowledge of Human Monkeypox Infection among Final Year Medical, Pharmacy, and Nursing Students: A Multicenter, Cross-Sectional Analysis from Pakistan.

The World Health Organization (WHO) declared the monkeypox outbreak a public health emergency in June 2022. In Pakistan, positive cases of monkeypox were reported in April 2023. Healthcare workers (HCWs) are considered as a front-line force to combat such outbreaks. A questionnaire-based cross-sectional study was conducted among 11 public sector educational institutions in Punjab, Pakistan, during May and June 2023 among final year medical, pharmacy, and nursing students concerning their knowledge of monkeypox. This included the signs/symptoms of monkeypox. Healthcare students were chosen as they are the HCWs of tomorrow. A total of 389 healthcare students participated in the study, with a mean age of 23.17 ± 1.72 years, and the majority were female. The mean knowledge score was 17.69 ± 4.55 (95% CI 17.24-18.14) out of a maximum total knowledge score of 26 (each correct answer was given a score of 1). The proportion of students with good, moderate, and poor knowledge was 21.6%, 43.2%, and 35.2%, respectively. Age (p = 0.017), gender (p < 0.001), and education (p < 0.001) had a significant impact on the knowledge score. In the multivariate linear regression model, education was the only significant factor linked to knowledge scores. Overall, the majority of future HCWs had moderate knowledge of monkeypox. Consequently, educational activities are needed to improve monkeypox-related knowledge among future HCWs. Furthermore, emerging infectious diseases should be routinely incorporated into HCW curricula.

Machine Learning Reveals Synovial Fibroblast Genes Associated with Pain Affect Sensory Nerve Growth in Rheumatoid Arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We identified a module of 815 genes associated with pain, using a novel machine learning approach, Graph-based Gene expression Module Identification (GbGMI), in samples from patients with longstanding RA, but limited synovial inflammation at arthroplasty, and validated this finding in an independent cohort of synovial biopsy samples from early, untreated RA patients. Single-cell RNA-seq analyses indicated these genes were most robustly expressed by lining layer fibroblasts and receptor-ligand interaction analysis predicted robust lining layer fibroblast crosstalk with pain sensitive CGRP+ dorsal root ganglion sensory neurons. Netrin-4, which is abundantly expressed by lining fibroblasts and associated with pain, significantly increased the branching of pain-sensitive CGRP+ neurons in vitro . We conclude GbGMI is a useful method for identifying a module of genes that associate with a clinical feature of interest. Using this approach, we find that Netrin-4 is produced by synovial fibroblasts in the absence of inflammation and can enhance the outgrowth of CGRP+ pain sensitive nerve fibers. One Sentence Summary: Machine Learning reveals synovial fibroblast genes related to pain affect sensory nerve growth in Rheumatoid Arthritis addresses unmet clinical need.

Optimizing biocatalytic potential of Dipodascus australiensis M-2 for degrading lignin under laboratory conditions.

In present research, a potent fungal strain was isolated from paper mill effluent (black liquor) in order to investigate its potential for the biodegradation of lignin. Two step strategy was used to screen most efficient fungal strain having ability to growin MSM-black liquor medium and to degrade alkali lignin.The results of initial screening indicated that the strain M-2 produced comparatively higher ligninolytic zone on MSN agar plates supplemented with black liquor (BL) and alkali ligninase compared to the other isolates.The results of 18S rRNA gene sequencing revealed that strain M-2 showed ≥ 99% sequence homology with Dipodasceus australiansis.The process for the biodegradation of lignin was optimized using Taguchi Orthogonal Array design. Under optimized conditions of pH 9, 40 °C and 4% inoculum, a maximum of 89% lignin was degraded with 41% color reduction after 8 days of incubation period by Dipodasceus australiansis M-2. The pH and temperature were found to be significant terms with the p-values of 0.002 and 0.001 respectively. The laccase activity of the Dipodascus australiensis was found to be maximum of 1.511 U/mL. The HPLC analysis of lignin biodegradation indicated sharp transformation of peaks as compared to the control. Our results suggested that the strain Dipodascus australiensis M-2 possess excellent lignin degradation and color reduction capability and can be applied in waste treatment systems for pulp and paper mill effluent. In present work we are reporting first hand information regarding biodegradation of lignin by a potent strain of Dipodascus australiensis and statistical optimization of the bioprocess.