Regulation of miR-61 and col-19 via TGF-ß and Notch signalling in Caenorhabditis elegans against Klebsiella aerogenes infection.

Klebsiella aerogenes, previously known as Enterobacter aerogenes, is a gram-negative bacterium typically present in the gastrointestinal tract. While numerous studies reported the pathogenicity and drug resistance of this bacterium there remains a lack of comprehensive research on K. aerogenes induced alterations in the host cellular mechanisms. In this study, we identify a previously uncharacterized C. elegans miR-61 that defines an evolutionarily conserved miRNA important for development and innate immunity regulation through Notch and TGF-ß signaling pathway. We employed C. elegans wild-type (N2) as well as mutant strains, such as TGF-ß (sma-6) and notch-signaling pathway mutants (adm-4 and mir-61). Our results have demonstrated that the K. aerogenes infected mutants exhibited significantly reduced survival rate, reduced pharyngeal pumping, altered swimming and chemotactic behavior. Moreover, K. aerogenes affects the healthspan by increasing ROS level in the mutants. The gene expression analysis revealed that K. aerogenes upregulated egl-30, tph-1 and sod-1 in adm-4, mir-61 mutants not in sma-6. The in-silico analysis indicated an interaction between mir-61 and col-19, which was confirmed by the upregulation of miR-61 expression and the downregulation of col-19 in sma-6, adm-4, and wild-type strains. These findings suggest that C. elegans activates mir-61 and col-19 regulation through the Notch and TGF-ß signaling pathway against K. aerogenes infection.

Rapid-killing efficacy substantiates the antiseptic property of the synergistic combination of carvacrol and nerol against nosocomial pathogens.

Globally, new classes of synthetic and natural antibiotics and antivirulents have continuously been validated for their potential broad-spectrum antagonistic activity with the aim of identifying an effective active molecule to prevent the spread of infectious agents in both food industry and medical field. In view of this, present study is aimed at evaluating the rapid killing efficacy of bioactive molecules Carvacrol (C) and Nerol (N) through British Standard European Norm 1276: phase2/step1 (EN1276) protocol. Active molecules C and N showed broad-spectrum antimicrobial activity against the test strains Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus hirae at concentration range of 78.125, 625, 156.25 and 312.5 µg/mL, respectively, for C, and 625 µg/mL for N. Whereas, combinatorial approach showed efficient activity with four times reduced concentration of C and N at 78.125 and 156.25 µg/mL, respectively, against test strains. Further, EN1276 results proved the rapid killing efficacy of test strains in 1 min of contact time with significant (> 5 log) growth reduction at 100X concentration of actives. SEM analysis and reduced concentration of protease, lipids and carbohydrate contents of treated group biofilm components ascertained preformed biofilm disruption potential of C + N on polystyrene and nail surfaces. C + N at synergistic concentration exhibited no adverse effect on HaCaT cells at 78.125 µg/mL (C) + 156.25 µg/mL (N). Taken together, based on the observed experimental results, present study evidence the antiseptic/disinfectant ability of C + N and suggest that the combination can preferentially be used in foam-based hand wash formulations.

Evaluation of selected Indian medicinal plants for antagonistic potential against Malassezia spp. and the synergistic effect of embelin in combination with ketoconazole.

The genus Malassezia comprises of extremely lipophilic yeasts secreting lipases as a vital factor for survival. They are emerging as opportunistic pathogens in medical microbiology and dermatology by causing recurring and recalcitrant infection. Combinatorial therapy is a constructive way to combat infectious diseases. In that prospect, totally 16 Indian medicinal plants were screened, among which a maximum degree of antimicrobial activity was ascertained in Embelia ribes. Subsequently embelin was identified as the bioactive principle with antagonistic potential by comparative antimicrobial assay and FTIR analysis. The MIC of embelin was determined as 400 µg/ml exhibiting ∼75% of growth inhibition. Further, a fungistatic activity based on anti-lipase potential (65-89%) of embelin has been clearly substantiated by XTT and lipase assay. In addition, embelin exhibited a synergistic effect with the antifungal drug ketoconazole (KTZ) against four different Malassezia spp. with FIC index of 0.5. Therefore, the combinations of embelin and KTZ may represent a promising therapeutic regimen to treat Malassezia infections with subjugated clinical and environmental toxicity. To the best of our knowledge, this is the first report delineating the anti-lipase activity of embelin and in vitro synergistic interaction between embelin and KTZ against Malassezia spp.

A systematic review on impact of SARS-CoV-2 infection.

Innumerable pathogens including RNA viruses have catastrophic pandemic propensity, in turn, SARS-CoV-2 infection is highly contagious. Emergence of SARS-CoV-2 variants with high mutation rate additionally codifies infectious ability of virus and arisen clinical imputations to human health. Although, our knowledge of mechanism of virus infection and its impact on host system has been substantially demystified, uncertainties about the emergence of virus are still not fully understood. To date, there are no potentially curative drugs are identified against the viral infection. Even though, drugs are repurposed in the initial period of infection, many are significantly negative in clinical trials. Moreover, the infection is dependent on organ status, co-morbid conditions, variant of virus and geographic region. This review article aims to comprehensively describe the SARS-CoV-2 infection and the impacts in the host cellular system. This review also briefly provides an overview of genome, proteome and metabolome associated risk to infection and the advancement of therapeutics in SARS-CoV-2 infection management.

Wounding Caenorhabditis elegans with Glass Wool.

Research on wound healing majorly relies on rat, mice and other animal models. However, an alternative animal model ought to be brought in the field, pertaining to the stringent ethical issues owing to the use of animals in research. In this regard, Caenorhabdits elegans, a miniature model nematode gains the great attention of the researchers in wound healing. Though, the model is being explored in wound research for more than a decade, the existing protocols lack the acquisition of large wound population that in turn could enable the utility of global genomics (G), proteomics (P) and metabolomics (M) based approaches. In order to overcome the inadequacy of the existing protocols, the protocol described here affords the acquisition of voluminous wound population in C. elegans using truncated glasswool pieces to enable the utility of high throughput analytical techniques. Graphic abstract: Steps involved in glass wool wounding protocol.

Proteomic analysis of Caenorhabditis elegans wound model reveals novel molecular players involved in repair.

Wound repair is a multistep process which involves coordination of multiple molecular players from different cell types and pathways. Though the cellular processes that are taking place in order to repair damage is already known, molecular players involved in crucial pathways are still scarce. In this regard, the present study intends to uncover crucial players that are involved in the central repair events through proteomics approach which included 2-D GE and LC-MS/MS using Caenorhabditis elegans wound model. Initial gel-based 2-D GE and following protein-protein interaction (PPI) network analyses revealed active role of calcium signaling, acetylcholine transport and serotonergic neurotransmitter pathways. Further, gel-free LC-MS/MS and following PPI network analyses revealed the incidence of actin nucleation at the initial hours immediately after injury. Further by visualizing the PPI network and the interacting players, pink-1, a mitochondrial Serine/threonine-protein kinase which is known to regulate mitochondrial dynamics, was found to be the central player in facilitating the mitochondrial fission and its role was further verified using qPCR analysis and pink-1 transgenic worms. Overall, the study delivers new insights from crucial regulatory pathways and central players involved in wound repair using high throughput proteomic approaches and the mass spectrometry Data (PXD024629/PXD024744) are available via ProteomeXchange. SIGNIFICANCE.

Sapindus mukorossi Gaertn. and its bioactive metabolite oleic acid impedes methicillin-resistant Staphylococcus aureus biofilm formation by down regulating adhesion genes expression.

Plants are boon to the mankind due to plenty of metabolites with medicinal values. Though plants have traditionally been used to treat various diseases, their biological values are not completely explored yet. Sapindus mukorossi is one such ethnobotanical plant identified for various biological activities. As biofilm formation and biofilm mediated drug resistance of methicillin-resistant Staphylococcus aureus (MRSA) have raised as serious global issue, search for antibiofilm agents has gained greater importance. Notably, antibiofilm potential of S. mukorossi is still unexplored. The aim of the study is to explore the effect of S. mukorossi methanolic extract (SMME) on MRSA biofilm formation and adhesive molecules production. Significantly, SMME exhibited 82 % of biofilm inhibition at 250 µg/mL without affecting the growth and microscopic analyses evidenced the concentration dependent antibiofilm activity of SMME. In vitro assays exhibited the reduction in slime, cell surface hydrophobicity, autoaggregation, extracellular polysaccharides substance and extracellular DNA synthesis upon SMME treatment. Further, qPCR analysis confirmed the ability of SMME to interfere with the expression of adhesion genes associated with biofilm formation such as icaA, icaD, fnbA, fnbB, clfA, cna, and altA. GC-MS analysis and molecular docking study revealed that oleic acid is responsible for the antibiofilm activity. FT-IR analysis validated the presence of oleic acid in SMME. These results suggest that SMME can be used as a promising therapeutic agent against MRSA biofilm-associated infections.

Understanding the role of p38 and JNK mediated MAPK pathway in response to UV-A induced photoaging in Caenorhabditis elegans.

Premature aging of the skin, principally induced by the UV radiations is called as photoaging, characterized by an increase in the level of ROS and the damage of the collagen layer leading to the damage of the cells. Mitogen activated Protein kinase (MAPK) pathway is known to mediate photoaging by controlling the level of ROS and initiating detoxification. Caenorhabditis elegans, a known model to analyze photoaging was used to understand the role of MAPK pathway (p38 and JNK) during UV-A mediated photoaging. Gene specific mutants of p38 MAPK pathway showed reduced survival when exposed to UV-A suggesting that UV-A mediated photoaging was dependent on this pathway. Also, the role of SKN-1 in eliciting response against UV-A was analyzed with the help of GFP tagged strains and qPCR analysis. Further, UV-A did not have any impact on the lifespan of JNK pathway mutants suggesting the importance of the pathway in eliciting a response against UV-A exposure, which was further validated by Western blot analysis. Overall, this study suggests that MAPK pathway could play an important part in initiating and eliciting a response by the host against UV-A exposure, by which it could be used as a marker to analyze the effects of photoaging.

Analyzing the Synergistic Effects of Antioxidants in Combating Photoaging Using Model Nematode, Caenorhabditis elegans.

Aging, a universal and unique process, occurs both intrinsically (chronological) and extrinsically (photoaging). Ultraviolet-A (UV-A)-mediated stress is a growing health hazard to mankind as it is the major cause of photoaging, which could lead to much damage of skin cells and tissues ranging from tan, burn, or even cancer. The present study focuses on the role of antioxidants and other natural compounds which have been widely used in oral/topical applications to combat and delay the effects of photoaging using model nematode Caenorhabditis elegans. Compounds like green tea extract, naringenin, and naringin, which are known for their antioxidant properties, were able to extend life span and healthspan of the nematode in normal as well as under UV-A-mediated stress conditions. Regulation of both the stress-responsive genes (skn-1 and sir-2.1) and the aging-regulating genes (daf-2 and age-1) was attributable for these conditions. Interestingly, it was observed that these compounds when combined in equal ratios by weight worked synergistically to combat the aging process. Pronounced synergistic effects were observed during UV-A-mediated stress conditions, suggesting that these could be used as potential antiphotoaging compounds which will be of greater significance for health-based research.

Inhibition of biofilm and biofilm-associated virulence factor production in methicillin-resistant Staphylococcus aureus by docosanol.

Antimicrobial resistance is a major public health concern in infection control. Hence, a multi-pronged approach is necessary to curb the severity of infections. The present study entails the identification of docosanol (fatty alcohol) from Streptomyces as a novel antibiofilm agent which can target the virulence factors of MRSA. Results showed that docosanol as a potent antibiofilm agent and found to inhibit several virulence factors of MRSA. The antibiofilm efficacy of docosanol analyzed through light and scanning electron microscopy showed a significant reduction in adherent cells. Moreover, analysis of three-dimensional structure of biofilm matrix by confocal laser scanning microscope demonstrated effective antibiofilm potential of docosanol. In addition, docosanol reduced the survival rate of MRSA in healthy human blood and enhanced the neutrophil-mediated killing by interfering with hemolysin production. RT-qPCR analysis revealed the down regulation of several virulence genes, possibly by affecting the expression of the accessory gene regulator (agr) system and transcriptional regulator sarA. These findings suggest that docosanol could effectively reduce the biofilm phenotype and virulence production, and thus becomes a promising candidate to treat MRSA infections.

Understanding the role of DAF-16 mediated pathway in Caenorhabditis elegans during UV-A mediated photoaging process.

Even though Sun is the major source of energy to all living beings in the universe, continuous and prolonged exposure to sunlight will lead to detrimental effects. Human skin will undergo extrinsic aging, known as photoaging upon prolonged exposure to sunlight which is characterized by wrinkles, dryness, loss of elasticity, and so on. The model nematode Caenorhabditis elegans which is widely used in aging studies, could be used to study photoaging also. Transcription factor DAF-16, which regulates longevity, stress resistance and many other physiological events, mediates the photoaging mechanism in C. elegans. Elevation in extracellular ROS and altered expression of SGK-1 indicates the role of DAF-16 during UV-A exposure. Further, the role of daf-2, the receptor gene and lys-7, an effector gene of DAF-16 were characterized through mutant based studies. The long lived daf-2 mutants upon UV-A exposure showed reduction in lifespan, but the upregulation of daf-16 allowed the other molecular mechanisms like healthspan, antimicrobial and stress resistance to be active. In the case of lys-7 mutants, the lifespan was reduced and all other molecular mechanisms were also downregulated. However, the daf-16 mutants showed no change in lifespan irrespective of UV-A exposure. This signifies the role of DAF-16 during UV-A mediated photoaging in C. elegans. The present study helps in understanding the role of daf-16 in UV-A mediated stress response which will be of considerable importance in the field of pharmacy in designing targets for specific agents against photoaging.

5-Dodecanolide interferes with biofilm formation and reduces the virulence of Methicillin-resistant Staphylococcus aureus (MRSA) through up regulation of agr system.

Methicillin resistant Staphylococcus aureus (MRSA) is a predominant human pathogen with high morbidity that is listed in the WHO high priority pathogen list. Being a primary cause of persistent human infections, biofilm forming ability of S. aureus plays a pivotal role in the development of antibiotic resistance. Hence, targeting biofilm is an alternative strategy to fight bacterial infections. The present study for the first time demonstrates the non-antibacterial biofilm inhibitory efficacy of 5-Dodecanolide (DD) against ATCC strain and clinical isolates of S. aureus. In addition, DD is able to inhibit adherence of MRSA on human plasma coated Titanium surface. Further, treatment with DD significantly reduced the eDNA synthesis, autoaggregation, staphyloxanthin biosynthesis and ring biofilm formation. Reduction in staphyloxanthin in turn increased the susceptibility of MRSA to healthy human blood and H2O2 exposure. Quantitative PCR analysis revealed the induced expression of agrA and agrC upon DD treatment. This resulted down regulation of genes involved in biofilm formation such as fnbA and fnbB and up regulation of RNAIII, hld, psmα and genes involved in biofilm matrix degradation such as aur and nuc. Inefficacy of DD on the biofilm formation of agr mutant further validated the agr mediated antibiofilm potential of DD. Notably, DD was efficient in reducing the in vivo colonization of MRSA in Caenorhabditis elegans. Results of gene expression studies and physiological assays unveiled the agr mediated antibiofilm efficacy of DD.

Unravelling the wound healing ability and mode of action of pyridine carboxamide oxime using Caenorhabditis elegans as potential prescreen wound model.

AIM: In the current scenario of ethical issues related to animal usage in research, the present study was intended to explore the proficient utility of nematode, Caenorhabditis elegans as wound model in preliminary screening of wound healing therapeutics. MAIN METHODS: In this study, a new wounding protocol and quantitative assessment strategies for various healing parameters [survival, Reactive Oxygen Species (ROS), calcium signals, F-actin dynamics, new collagen synthesis and wound induced anti-microbial peptides] were developed and used for preliminary screening of wound healing actives from natural sources. Wound healing ability of positive lead Tridax procumbens (TP) and its major phytocompounds [Octa decenoic acid (ODA), Pyridine carboxamide oxime, known as Nicotinamide (NA) and Dimethyl Benz[c]acridine (DMB)] were assessed using C. elegans wound model and cell lines scratch wound healing assay. Mode of action of active lead was elucidated using metabolome analysis coupled with MALDI-MS followed by molecular docking. KEY FINDINGS: From the four tested methanolic extracts, TP was chosen as positive lead compared to control, Benzalkonium chloride (BKC) based on survival and new collagen synthesis analyses. Results indicated that the wound healing ability of TP was majorly contributed by NA. Further, it was found that NA acts in chloromethyl nicotinamide derivative form by interacting with the known wound healing biomarker, glycogen synthase kinase 3 (GSK-3) to exert wound healing ability. SIGNIFICANCE: The study evidenced that C. elegans, could be a reliable wound model for high-throughput screening of wound healing actives and to identify their possible mode of action.

Ultraviolet-A triggers photoaging in model nematode Caenorhabditis elegans in a DAF-16 dependent pathway.

Ultraviolet radiations (UV) are the primary causative agent for skin aging (photoaging) and cancer, especially UV-A. The mode of action and the molecular mechanism behind the damages caused by UV-A is not well studied, in vivo. The current study was employed to investigate the impact of UV-A exposure using the model organism, Caenorhabditis elegans. Analysis of lifespan, healthspan, and other cognitive behaviors were done which was supported by the molecular mechanism. UV-A exposure on collagen damages the synthesis and functioning which has been monitored kinetically using engineered strain, col-19:: GFP. The study results suggested that UV-A accelerated the aging process in an insulin-like signaling pathway dependent manner. Mutant (daf-2)-based analysis concrete the observations of the current study. The UV-A exposure affected the usual behavior of the worms like pharyngeal movements and brood size. Quantitative PCR profile of the candidate genes during UV-A exposure suggested that continuous exposure has damaged the neural network of the worms, but the mitochondrial signaling and dietary restriction pathway remain unaffected. Western blot analysis of HSF-1 evidenced the alteration in protein homeostasis in UV-A exposed worms. Outcome of the current study supports our view that C. elegans can be used as a model to study photoaging, and the mode of action of UV-A-mediated damages can be elucidated which will pave the way for drug developments against photoaging.