Biology subjects

Ahmad, W.

Publications and source records attributed to Ahmad, W..

9 recordsLinked to original sources

bioRxiv2024

Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger

The current study highlights the effect of mutagenesis on endoglucanase B activity of indigenous strain of Aspergillus niger and its heterologous expression studies in the pET28a+ vector. The physical and chemical mutagens were employed to incorporate mutations in A. niger. For determination of mutations, mRNA was isolated followed by cDNA synthesis and cellulase gene was amplified, purified and sequenced both from native and mutant A. niger. On comparison of gene sequences, it was observed that 5 nucleotide base pairs have been replaced in the mutant cellulase. The mutant recombinant enzyme showed 4.5 times higher activity (428.5 {micro}mol/mL/min) as compared to activity of native enzyme (94 {micro}mol/mL/min). The mutant gene was further investigated using Phyre2 and I-Tesser tools which exhibited 71% structural homology with Endoglucanase B of Thermoascus aurantiacus. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bonds analysis were carried at 35 {degrees}C and 50 {degrees}C to explore the integrity of structure of recombinant mutant endoglucanase B which corresponded to its optimal temperature. Hydrogen bonds analysis showed more stability of recombinant mutant endoglucanase B as compared to native enzyme. Both native and mutant endoglucanase B genes were expressed in pET 28a+ and purified with nickel affinity chromatography. Theoretical masses determined through ExPaSy Protparam were found 38.7 and 38.5 kDa for native and mutant enzymes, respectively. The optimal pH and temperature values for the mutant were 5.0 and 50 {degrees}C while for native these were found 4.0 and 35 {degrees}C, respectively. On reacting with carboxy methyl cellulose (CMC) as substrate, the mutant enzyme exhibited less Km (0.452 mg/mL) and more Vmax (50.25 {micro}mol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 {micro}mol/ml/min as Vmax. Among metal ions, Mg2+ showed maximum inducing effect (200 %) on cellulase activity at 50 mM concentration followed by Ca2+ (140%) at 100 mM concentration. Hence, expression of a recombinant mutant cellulase from A. niger significantly enhanced its cellulytic potential which could be employed for further industrial applications at pilot scale.

biochemistry↗

bioRxiv2023

The Host miR-17-92 Cluster Negatively Regulates Mouse Mammary Tumor Virus (MMTV) Replication via miR-92a by Targeting the Viral Genome

The mouse mammary tumor virus (MMTV) induces mammary tumors in mice. Recently, we have shown that MMTV perturbs expression of the host miR-17-92 cluster in MMTV-infected mammary glands and MMTV-induced mammary tumors. Known as oncomiR-1, this cluster is often dysregulated in a number of cancers, especially breast cancer. Therefore, we investigated whether there was a functional interaction between MMTV and the cluster. Our results reveal that MMTV expression led to dysregulation of the cluster in both mammary epithelial HC11 and HEK293T cells with the expression of miR-92a cluster member being affected the most. Conversely, over-expression of the whole or partial cluster significantly repressed MMTV expression. Interestingly, overexpression of miR-92a alone resulted in MMTV repression to the same extent as observed with the whole or partial cluster. Inhibition of miR-92a led to nearly a complete recovery of MMTV gene expression. Deletion/substitution of the miR-92a seed sequence within the cluster rescued MMTV expression. Dual luciferase assays identified MMTV genomic RNA as the target of miR-92a. These results show that the miR-17-92 cluster acts as part of the cells well-known miRNA-based anti-viral response to thwart incoming MMTV infection. Interestingly, miR-92a expression was the least amongst cluster members in MMTV-induced breast tumors, suggesting that the elevated level of MMTV expression in tumors is due perhaps to MMTV subverting miR-92a overexpression, restricting its ability to control MMTV replication during tumorigenesis. Thus, our study provides the first evidence demonstrating the biological significance of host miRNAs in controlling MMTV replication and influencing the process of tumorigenesis. ImportanceMMTV is a non-acute, slow-transforming retrovirus that causes breast cancer and lymphomas in mice. Consequently, understanding how MMTV interacts with its host can reveal critical aspects of cellular anti-viral responses and the process of tumorigenesis. This study reveals that the host miR-17-92 cluster plays an anti-viral role during MMTV infection which seems to be counteracted by MMTV during tumorigenesis. This observation is significant since MMTV-induced tumors share similarity with human breast cancer and the MMTV/mouse model is the best mammalian animal model to study breast cancer initiation, progression, and therapy. Moreover, MMTV is increasingly (though controversial) being implicated in human breast cancer, leukemias/lymphomas, and biliary cirrhosis due to its potential zoonosis into humans. Thus, if proven true, understanding how miRNAs modulate MMTV replication will be critical in appreciating how hosts miRNAs affect virus replication/tumorigenesis, and towards the development of novel miRNA-based therapeutics (e.g., anti-miRs) to thwart MMTV replication in humans.

microbiology↗

bioRxiv2023

Spatial autocorrelation dimension as a potential determinant for the temporal persistence of human atrial and ventricular fibrillation.

Background: Despite being central to atrial fibrillation (AF) and ventricular fibrillation (VF) mechanisms and therapy, the factors governing AF and VF termination are poorly understood. It has been noted that ratio of system size (L) and the two-point spatial correlation length ({xi}2) are associated with time until termination in transient spatiotemporally chaotic systems, but the relationship between these characteristics and termination has not been systematically studied in human AF and VF. Objective: We aimed assess whether the time to cardiac fibrillation termination can be predicted using a novel estimator, the spatial autocorrelation dimension (Di), defined as the ratio of L and {xi}2, in human AF and VF. Methods: Di was computed and compared in a multi-centre, multi-system study with data for sustained versus spontaneously terminating human AF/VF. VF data was collected during coronary-bypass surgery; and AF data during clinically indicated AF ablation. We analyzed: i) VF mapped using a 256-electrode epicardial sock (n=12pts); ii) AF mapped using a 64-electrode constellation basket-catheter (n=15pts); iii) AF mapped using a 16-electrode HD-grid catheter (n=42pts). To investigate temporal fibrillation persistence, the response of AF-episodes to flecainide (n=7pts) was also studied. Results: Spontaneously terminating fibrillation demonstrated a lower Di (P<0.001 all systems). Lower Di was also seen in paroxysmal compared to persistent AF (P=0.002). Post-flecainide, Di decreased over time (P<0.001). Lower Di was also associated with longer-lasting episodes of AF/VF (R2>0.90, P<0.05 in all cases). Using k-means clustering, two distinct clusters and their centroids were identified i) a cluster of spontaneously terminating episodes, and ii) a cluster of sustained epochs. Conclusion: Di predicts the temporal persistence of cardiac fibrillation. This finding provides potentially important insights into a possible common pathway to termination and therapeutic approaches.

physiology↗

bioRxiv2023

LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.

cell biology↗

bioRxiv2023

Indigenous Reduction of Multi-drug Resistant Bacterial Pathogens in a Natural Uncontrolled Fermented Milk Ecosystem

Raw milk and its products have been questioned for microbiological safety. However, Dahi: an artisanally fermented milk product that is yet microbiologically unexplored is popularly known for therapeutic uses in public. Aiming safety and therapeutic assessment of dahi, samples of raw milk and dahi were analysed for microbiology and bacterial pathogens. The pathogens were also evaluated for antibiotic resistance. Further dahi samples were assessed for antipathogen effect. The total bacterial count of raw milk was between 3.80 x 1002 to 2.97 x 1007 and total fungal count was 2.50 x 1001 to 4.90 x 1002 whereas total bacterial and fungal count of Dahi was 3.00 x 1001 to 4.05 x 1009 and 2.00 x 1001 to 5.30 x 1007. Bacterial population of the Dahi was found dominated by lactic acid bacteria. In milk samples, E. coli was detected as 38%, S. aureus 28%, L. monocytogenes 3%, Salmonella 42% and Pseudomonas spp 31%. In Dahi samples, their percentages were 7%, 10%, 7%, 8% and 2% respectively. The detected pathogens were found resistant against different antibiotics especially to third and fourth generations of cephalosporin and other beta-lactam drugs. When assessed invitro, the low incidence of pathogens in the Dahi samples were associated to its inhibitory effect against pathogens. Dahi samples also inhibited the growth of antibiotic resistance ATCC strains. The inhibitory activity of the Dahi is due to the changes occurs during fermentation but not corelated to the pH of the Dahi.

microbiology↗

bioRxiv2017

Suppression of the dihydrolipoamide dehydrogenase gene (dld-1) protects against the toxicity of human amyloid beta in C. elegans model of Alzheimer’s disease

Declines in energy metabolism and associated mitochondrial enzymes are linked to the progression of Alzheimers disease (AD). Dihydrolipoamide dehydrogenase (dld) and two of its enzyme complexes namely, pyruvate dehydrogenase and -ketoglutarate dehydrogenase are associated with AD and have a significant role in energy metabolism. Interestingly, dld gene variants are genetically linked to late-onset AD; and reduced activity of DLD-containing enzyme complexes has been observed in AD patients. To understand how energy metabolism influences AD progression, we suppressed the dld-1 gene in C. elegans expressing the human A{beta} peptide. dld-1 gene suppression improved many aspects of vitality and function directly affected by A{beta} pathology in C. elegans. This includes protection against paralysis, improved fecundity and improved egg hatching rates. Suppression of the dld-1 gene restores normal sensitivity to aldicarb, levamisole and serotonin, and improves chemotaxis. Suppression of dld-1 does not decrease levels of the A{beta} peptide, but does reduce the formation of toxic A{beta} oligomers. The mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) acts synergistically with A{beta} to overcome the protective effect of dld-1 gene suppression. Another metabolic toxin, phosphine, acted additively with A{beta}. Our work supports the hypothesis that lowering energy metabolism may protect against A{beta} pathogenicity, but that this may increase susceptibility to other metabolic disturbances.

neuroscience↗

bioRxiv2017

Glucose facilitates Aβ oligomerization and tau phosphorylation in C. elegans model of Alzheimer’s disease

Formation of A{beta} plaques from peptide oligomers and development of neurofibrillary tangles from hyperphosphorylated tau are hallmarks of Alzheimers disease (AD). These markers of AD severity are further associated with impaired glucose metabolism. However, the exact role of glucose metabolism on disease progression has not been elucidated. In this study, the effects of glucose on A{beta} and tau-mediated toxicity are investigated using a C. elegans model system. We find that addition of glucose or 2-deoxy-d-glucose (2DOG) to the growth medium delayed A{beta}-associated paralysis, though it was unable to restore previously impaired acetylcholine neurotransmission in pre-existing A{beta}-mediated pathology. Glucose also inhibited egg laying and hatching in the worms that express A{beta}. The harmful effects of glucose were associated with an increase in toxic A{beta} oligomers. Increased phosphorylation of tau is associated with formation of neurofibrillary tangles (NFTs) and increased severity of AD, but O-{beta}-GlcNAcylation can inhibit phosphorylation of adjacent phosphorylation sites. We reasoned that high glucose levels might induce tau O-{beta}-GlcNAcylation, thereby protecting against tau phosphorylation. Contrary to our expectation, glucose increased tau phosphorylation but not O-{beta}-GlcNAcylation. Increasing O-{beta}-GlcNAcylation, either with Thiamet-G (TMG) or by suppressing the O-GlcNAcase (oga-1) gene does interfere with and therefore reduce tau phosphorylation. Furthermore, reducing O-{beta}-GlcNAcylation by suppressing O-GlcNAc transferase (ogt-1) gene causes an increase in tau phosphorylation. These results suggest that protective O-{beta}-GlcNAcylation is not induced by glucose. Instead, as with vertebrates, we demonstrate that high levels of glucose exacerbate disease progression by promoting A{beta} aggregation and tau hyperphosphorylation, resulting in disease symptoms of increased severity. The effects of glucose cannot be effectively managed by manipulating O-{beta}-GlcNAcylation in the tau models of AD in C. elegans. Our observations suggest that glucose enrichment is unlikely to be an appropriate therapy to minimize AD progression.

neuroscience↗

bioRxiv2017

Effect of dihydrolipoamide dehydrogenase (dld-1) on human tau phosphorylation in a C. elegans model of Alzheimer’s Disease

The microtubule associated tau protein becomes hyperphosphorylated in Alzheimers disease (AD). While tau hyperphosphorylation promotes neurodegeneration, the cause and consequences of this abnormal modification are poorly understood. As impaired energy metabolism is an important hallmark of AD progression, we tested whether it could trigger phosphorylation of human tau protein in a transgenic C. elegans model of AD. We found that suppression or inhibition of a mitochondrial enzyme of energy metabolism, dihydrolipoamide dehydrogenase (DLD) either by RNAi or 2-methoxyindole-2-carboxylic acid (MICA) respectively, resulted in elevated whole-body glucose levels as well as increased phosphorylation of tau. Although the calcium ionophore A23187 could reduce tau phosphorylation induced by either chemical inhibition or genetic suppression of DLD, it was unable to reduce tau phosphorylation induced by hyperglycemia. While inhibition of the dld-1 gene or treatment with MICA partially reversed the inhibition of acetylcholine neurotransmission by tau, neither treatment affected tau inhibited mobility.

neuroscience↗

bioRxiv2017

Acute and chronic toxicity assessment of benzylpenicillin G residue in cooked meat

The current level of penicillin use and its persisting residues in livestock is potentially concerning; the toxicity of penicillin residues in heat-treated animal food products (HAFP) is yet to be elucidated. In this study, the acute and chronic toxicity of benzylpenicillin G (BPG) residues in HAFP was investigated in a mouse model. The calculated LD50 of BPG heated to cooking temperature (BPHCT) was 933.04 mg kg-1 [b.w.] intraperitoneally corresponding to 3.75 times lower than its prototype. Mice fed on the experimental diet containing heat-treated beef with high BPG levels for 6 months displayed a reduction in body weight and altered serum values indicating for liver and renal function. Further, the organ ratios of intestinal and spleen were increased. Histopathological changes were observed in the liver, lung and parenchyma testis tissue. BPHCT residue induced sperm aberration and micronucleated polychromatic erythrocytes formation. Present results indicate that prolonged exposure of BPHCT at higher levels of residue might have an impact on public health. Importantly the toxic concentrations of BPHCT are relatively high compared with levels that would result from the degradation of antibiotic residues in meat from animals that have received a therapeutic dose of BPG.

pharmacology and toxicology↗