Molecular Identification and Evolutionary Divergence of the Sri Lankan Sambar Deer, Rusa unicolor (Kerr 1792).

The Sambar is one of the largest deer species distributed mainly in Asia, and it has been listed as a vulnerable species. Taxonomy based on morphological characterization has been the gold standard method used to identify the Sambar deer species. Yet, morphological identification is challenging and requires expertise. To conduct species identification and taxonomic decisions, we performed the molecular identification of R. unicolor found in Sri Lanka using DNA barcodes, COI, and Cyt b to compare the Sri Lankan R. unicolor with the Indian R. unicolor and other R. unicolor subspecies. We obtained mitochondrial DNA sequences from COI and Cyt b from blood samples collected from the wet zone in Sri Lanka. A phylogenetic tree was constructed based on the Bayesian analyses using MrBayes 3.2.7. Molecular dating was implemented in Bayesian Evolutionary Analysis Sampling Trees (BEAST v1.8.2) on the concatenated sequence using a log-normal relaxed clock and Yule species tree prior, with four categories. The results showed that the Sri Lankan R. unicolor is genetically different from the Indian R. unicolor and other R. unicolor subspecies. The divergence occurred approximately 1.1 MYA (million years ago) in the Pleistocene era. The results are essential for designing new conservation platforms for these Sambar deer species.

In silico study of SARS-CoV-2 spike protein RBD and human ACE-2 affinity dynamics across variants and Omicron subvariants.

The coronavirus disease 2019 virus outbreak continues worldwide, with many variants emerging, some of which are considered variants of concern (VOCs). The WHO designated Omicron as a VOC and assigned it under variant B.1.1.529. Here, we used computational studies to examine the VOCs, including Omicron subvariants, and one variant of interest.  Here we found that the binding affinity of human receptor angiotensin-converting enzyme 2 (hACE2) and receptor-binding domain (RBDs) increased in the order of wild type (Wuhan-strain) < Beta < Alpha < OmicronBA.5 < Gamma < Delta < Omicron BA.2.75 < BA.1 < BA.3 < BA.2. Interactions between docked complexes revealed that the RBD residue positions like 452, 478, 493, 498, 501, and 505 are crucial in creating strong interactions with hACE2. Omicron BA.2 shows the highest binding capacity to the hACE2 receptor among all the mutant complexes. The BA.5's L452R, F486V, and T478K mutation significantly impact the interaction network in the BA.5 RBD-hACE2 interface. Here for the first time, we report the His505, an active residue on the RBD forming a salt bridge in the BA.2, leading to increased mutation stability. When the active RBD residues are mutated, binding affinity and intermolecular interactions increase across all mutant complexes. By examining the differences in different variants, this study may provide a solid foundation for structure-based drug design for newly emerging variants.

Antimicrobial activity of Plumbago indica and ligand screening of plumbagin against methicillin-resistant Staphylococcus aureus.

In this study, the antimicrobial properties of Plumbago indica root bark against bacterial strains and a fungal strain were investigatedusing the disc diffusion and minimum inhibitory concentration assays. Gas chromatography/mass spectrometry, nuclear magnetic resonance spectrometry, and column chromatography analyses were conducted to identify and isolate the active compounds. A docking study was performed to identify possible interactions between the active compound and DNA gyrase using the Schrödinger Glide docking program. Both methanol extract and the ethyl acetate fraction of the root bark showed significant antimicrobial activity against the gram-positive bacteria than against the gram-negative bacteria and the fungal strain. The active compound was identified as plumbagin. A disc diffusion assay of plumbagin revealed potent antimicrobial activity against methicillin-resistant Staphylococcus aureus. Molecular docking of plumbagin revealed high specificity towards the DNA gyrase binding site with a high fitness score and a minimum energy barrier of -7.651 kcal/mol. These findings indicate that P. indica exhibits significant antimicrobial activity, primarily due to the presence of plumbagin. The specificity of plumbagin toward DNA gyrase in S. aureus indicates the feasibility of utilizing P. indica for developing new drug leads against drug resistant microbial strain. Communicated by Ramaswamy H. Sarma.

Mechanistic Insight into Apoptotic Induction in Human Rhabdomyosarcoma and Breast Adenocarcinoma Cells by Chnoospora minima: A Sri Lankan Brown Seaweed.

The current study determined the cytotoxic and apoptotic potential of the polyphenol-rich methanol extract of Chnoospora minima (C. minima) and its fractions against human breast adenocarcinoma (MCF-7) and rhabdomyosarcoma (RMS) cells. MTT and neutral red assays were used to determine cytotoxicity. The clonogenic assay evaluated the antineoplastic activity, while the apoptotic activity was determined by cellular morphological changes, caspase 3/7 activity, and DNA fragmentation. Morphological alterations in apoptosis were observed by an inverted phase-contrast microscope and Hoechst 33342 staining methods. The total phenolic, flavonoids, alkaloids, and antioxidant activity in the hexane and chloroform fractions were determined, based on their cytotoxic activity. The hexane fraction of C. minima effectively reduced the cell growth that is concentration-dependent in human RMS and MCF-7 cell lines. It also exhibited low cytotoxicity on Vero cells. The characteristic cellular and nuclear apoptotic morphological features were observed. A noticeable caspase 3/7 activation and the fragmented DNA were detected only in the hexane fraction treated RMS cells, whereas MCF-7 cells showed low caspase 3/7 activation due to a lack of caspase 3 and no evidence of having a typical ladder pattern of apoptosis. Further analysis revealed that the hexane fraction-treated RMS cells upregulated the p53 gene twofold (2.72) compared to the p21 (0.77) gene, whereas in the MCF-7 cells, a 2.21-fold upregulation of p53 was observed compared to the p21 (0.64) gene. The hexane fraction exhibited moderate total phenolics, flavonoids, alkaloids content, and antioxidant activity. According to the different antioxidant mechanisms, hexane and chloroform fractions showed the highest antioxidant activities by FRAP and ORAC assays, respectively. GC-MS analysis of hexane fraction revealed the presence of methyl tetradecanoate (38.314%) as the most abundant compound. The study's findings highlighted that the non-polar compounds present in the hexane fraction of C. minima suppressed cell proliferation and induced apoptosis-mediated cell death in RMS and MCF-7 cells, mainly via the activation of the p53 gene. Hence, the isolation of compounds is warranted. However, more studies are required to understand the mechanistic insights of these observations.

Antidiabetic Potential of Marine Brown Algae-a Mini Review.

Marine algae are an important source of bioactive metabolites in drug development and nutraceuticals. Diabetes mellitus is a metabolic disorder and the third leading cause of death worldwide due to lifestyle changes associated with rapid urbanization. Due to the adverse side effects of currently available antidiabetic drugs, search for an effective natural-based antidiabetic drug is important to combat diabetes and its complications. Therefore, in lieu with herbal drug development, it is important to find the potential benefits of seaweeds for the management of type 2 diabetes as they are underexplored yet in Sri Lanka. Among the marine seaweeds, natural bioactive compounds are abundant in brown algae with potentials in application as active ingredients in drug leads and nutraceuticals. Bioactive secondary metabolites are derived from numerous biosynthetic pathways of marine algae which contribute to various chemical and biological properties. Phlorotannins present in marine brown algae exhibited antidiabetic activities through different mechanisms such as the inhibitory effect of enzyme targets mainly by inhibiting the enzymes such as α-amylase, α-glucosidase, angiotensin-converting enzymes (ACE), aldose reductase, dipeptidyl peptidase-4, and protein tyrosine phosphatase 1B (PTP 1B) enzyme. In addition, phlorotannins derived from brown algae have the ability to reduce diabetic complications. Hence, the present review focuses on the different antidiabetic mechanisms of secondary bioactive compounds present in marine brown algae.

Commercial Formulation of Chlorpyrifos Alters Neurological Behaviors and Fertility.

Pesticides are known to result in toxic insult. We aimed to evaluate Judo 40, the commercial formulation of chlorpyrifos on the neurological activities, fertility, and hormone levels of male rats. Male Wistar rats were treated orally with 1 mL of 20 or 50 mg/kg Judo 40. The doses were administered four times, twice a day. Sexual and exploratory behavior indices, fertility indices, serum androgen levels, blood acetylcholinesterase (BChE) levels, and neurological and muscular effects were evaluated. Serum testosterone and luteinizing hormone were significantly reduced in the rats receiving 50 mg/kg Judo 40. A reduction in viable implantation sites and live pups born were evident in the female rats mated with the male rats treated with the highest dose. Similarly, in the rats treated with the highest dose of Judo 40, a significant reduction in plasma BChE enzyme was observed. According to the results, prolonged Judo 40 exposure can cause impairment of the neurological alterations and sex hormones leading to impaired fertility. Therefore, chemical handlers should be educated on protection and risk minimization.

In-Vitro Antioxidant, Hypoglycemic Activity, and Identification of Bioactive Compounds in Phenol-Rich Extract from the Marine Red Algae Gracilaria edulis (Gmelin) Silva.

Obesity and diabetes are major metabolic disorders which are prevalent worldwide. Algae has played an important role in managing these disorders. In this study, Gracilaria edulis, a marine red algae, was investigated for antioxidant and hypoglycemic potential using in vitro models. De-polysaccharide methanol extract of G. edulis was sequentially partitioned with hexane, chloroform, ethyl acetate, and antioxidants, and hypoglycemic potentials were evaluated using multiple methods. High antioxidant potential was observed in the ethyl acetate fraction in terms of ferric reducing antioxidant power, iron chelating, and DPPH and ABTS radical scavenging activities, while the crude methanol extract exhibited potent oxygen radical-absorbance capacity. Potent α-amylase inhibitory activity was observed in the ethyl acetate fraction, while the ethyl acetate fraction was effective against α-glucosidase inhibition. Glucose diffusion was inhibited by the ethyl acetate fraction at 180 min, and the highest antiglycation activity was observed in both chloroform and ethyl acetate fractions. Additionally, gas chromatography-mass spectrometry analysis of the ethyl acetate fraction revealed the presence of several potent anti-diabetic compounds. In conclusion, G. edulis exhibited promising antidiabetic potential via multiple mechanisms. The ethyl acetate fraction exhibited the strongest hypoglycemic and antiglycation potential among the four fractions, and hence the isolation of active compounds is required to develop leads for new drugs to treat diabetes.

1,3-Dinitrobenze-Induced Genotoxicity Through Altering Nuclear Integrity of Diploid and Polyploidy Germ Cells.

1,3-Dinitrobenzene (mDNB) is a widely used intermediate in commercial products and causes testicular injury. However, genotoxic effects upon low-level exposure are poorly understood. The present study evaluated the effects of very low-chronic doses of mDNB on sperm nuclear integrity. Male hamsters were treated with 1.5 mg/kg/d/4 wks (group A), 1.5 mg/kg/mDNB/d/week/4 weeks (group B), 1.0 mg/kg/mDNB/3 d/wk/4 wks (group C), or polyethylene glycol 600 (control). Nuclear integrity of distal cauda epididymal sperm was determined using the sperm chromatin structure assay and acridine orange staining (AOS). The germ cell nuclear integrity was assessed by the comet assay. Testicular histopathology was conducted to evaluate the sensitive stages. The comet assay revealed denatured nuclear DNA in group A (in diploid and polyploid cells from weeks 2-5); respectively at week 4 and weeks 3 to 4 in groups B and C. According to AOS, only group A animals exhibited denatured sperm DNA (weeks 1 and 3). The effective sperm count declined from weeks 1 to 6. Mean sperm DNA denaturation extent, percentage cells outside the main population, and standard deviation indicated altered sperm nuclear integrity in group A. Same animals exhibited progressive disruption of the Sertoli cells, while groups B and C exhibited damages on germ cells. The results suggest that mDNB affects sperm nuclear integrity at very low chronic doses targeting cell-specific testicular damage.

Cytotoxic and Genotoxic Effects of Acephate on Human Sperm.

Extensive use of organophosphorus pesticides (OPs) could alter semen quality and sperm DNA at different stages of spermatogenesis. Acephate is a highly toxic extensively used OP and, therefore, we aimed to evaluate the effects of acephate on human semen quality and sperm DNA integrity. Sperm collected from healthy males were exposed to 0, 50, 100, and 200 µg/mL of acephate and incubated for 1 h, 2 h, and 3 h. Subsequently, sperm motility, vitality, functional integrity of plasma membrane, sperm capacitation, and DNA damage were examined. Result showed a significant decline of the motility at 100 µg/mL after 3 h and with 200 µg/mL after 1 h, 2 h, and 3 h. Viability was significantly reduced at 200 µg/mL after 2 h and 3 h. Functional integrity was significantly affected at 100 µg/mL after 3h and in 200 µg/mL dose after 2 h and 3h. Similarly, sperm capacitation was significantly affected at 200 µg/mL after 1 h, 2 h, and 3 h and at 100 µg/mL at 3 h. DNA damage was significantly increased only in 200 µg/mL dose after 3 h. The study suggests that exposure to acephate may result in alterations of sperm structure and function thus contributing towards deteriorating in human semen quality triggering infertility.

Evaluation of Aqueous Leaf Extract of Cardiospermum halicacabum (L.) on Fertility of Male Rats.

Treatment with 100 mg/kg and 200 mg/kg body weight of aqueous leaf extract (ALE) of Cardiospermum halicacabum for 30 days produced a significant dose dependent increase in the sperm counts and sperm motility in both caput and cauda regions. Further, significant increase in serum testosterone level was evident at all applied doses. However, no significant changes in the weight of sex organs were observed. Aqueous leaf extract also increased the number of females impregnated, number of implantations, and number of viable fetuses while decreasing the total number of resorption sites in the pregnant females. However, the total cholesterol level in the serum remained unchanged and there were no records on renotoxicity; nevertheless ALE exhibited a hepatoprotective effect. It was concluded that aqueous leaf extract of Cardiospermum halicacabum enhanced sperm concentration, motility, and testosterone, leading to positive results in fertility.