Green synthesized silver nanoparticles from Phoenix dactylifera synergistically interact with bioactive extract of Punica granatum against bacterial virulence and biofilm development.

The global rise of antibiotic resistance poses a substantial risk to mankind, underscoring the necessity for alternative antimicrobial options. Developing novel drugs has become challenging in matching the pace at which microbial resistance is evolving. Recently, nanotechnology, coupled with natural compounds, has emerged as a promising solution to combat multidrug-resistant bacteria. In the present study, silver nanoparticles were green-synthesized using aqueous extract of Phoenix dactylifera (variety Ajwa) fruits and characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) coupled with Energy dispersive X-ray analysis (EDX), Transmission electron microscopy (TEM) and Thermogravimetric-differential thermal analysis (TGA-DTA). The in-vitro synergy of green synthesized P. dactylifera silver nanoparticle (PD-AgNPs) with selected antibiotics and bioactive extract of Punica granatum, i.e., ethyl acetate fraction (PGEF), was investigated using checkerboard assays. The most effective synergistic combination was evaluated against the QS-regulated virulence factors production and biofilm of Pseudomonas aeruginosa PAO1 by spectroscopic assays and electron microscopy. In-vivo anti-infective efficacy was examined in Caenorhabditis elegans N2 worms. PD-AgNPs were characterized as spherical in shape with an average diameter of 28.9 nm. FTIR analysis revealed the presence of functional groups responsible for the decrease and stabilization of PD-AgNPs. The signals produced by TGA-DTA analysis indicated the generation of thermally stable and pure crystallite AgNPs. Key phytocompounds detected in bioactive fractions include gulonic acid, dihydrocaffeic acid 3-O-glucuronide, and various fatty acids. The MIC of PD-AgNPs and PGEF ranged from 32 to 128 µg/mL and 250-500 µg/mL, respectively, against test bacterial strains. In-vitro, PD-AgNPs showed additive interaction with selected antibiotics (FICI 0.625-0.75) and synergy with PGEF (FICI 0.25-0.375). This combination inhibited virulence factors by up to 75 % and biofilm formation by 84.87 % in P. aeruginosa PAO1. Infected C. elegans worms with P. aeruginosa PAO1 had a 92.55 % survival rate when treated with PD-AgNPs and PGEF. The combination also reduced the reactive oxygen species (ROS) level in C. elegans N2 compared to the untreated control. Overall, these findings highlight that biosynthesized PD-AgNPs and bioactive P. granatum extract may be used as a potential therapeutic formulation against MDR bacteria.

Efflux Pump Inhibition-Based Screening and Anti-Infective Evaluation of Punica granatum Against Bacterial Pathogens.

Drug efflux pumps contribute to bacterial multidrug resistance (MDR), reducing antibiotic effectiveness and causing treatment failures. Besides their role in MDR, efflux pumps also assist in the transportation of quorum sensing (QS) signal molecules and increased the tolerance of biofilms. Recently, the search for efflux pump inhibitors from natural sources, including anti-infective plants, has gained attention as a potential therapy against drug-resistant bacteria. In this study, 19 traditional Indian medicinal plants were screened for their efflux pump inhibitory activity against Escherichia coli TGI. The promising extract, i.e., Punica granatum was subsequently fractioned in the solvents of increasing polarity. Among them, at sub-MIC active EPI fraction was PGEF (P. granatum ethyl acetate fraction), further investigated for anti-infective potential against Chromobacterium violaceum 12,472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. PGEF was also evaluated for in vivo efficacy in Caenorhabditis elegans model. Major phytocompounds were analyzed by mass spectroscopic techniques. At respective Sub-MIC, PGEF reduced violacein production by 71.14% in C. violaceum 12,472. Moreover, PGEF inhibited pyocyanin (64.72%), pyoverdine (48.17%), protease (51.35%), and swarming motility (44.82%) of P. aeruginosa PAO1. Furthermore, PGEF reduced the production of prodigiosin and exoprotease by 64.73% and 61.80%, respectively. Similarly, at sub-MIC, PGEF inhibited (≥ 50%) biofilm development in all test pathogens. The key phytocompounds detected in active fraction include 5-hydroxymethylfurfural, trans-p-coumaric acid 4- glucoside, (-)-Epicatechin 3'-O-glucuronide, and ellagic acid. Interestingly, PGEF also demonstrated anti-infective efficacy against the PAO1-infected C. elegans test model and highlighting its therapeutic potential as an anti-infective agent to combat drug-resistant problems.

Human microglia-derived proinflammatory cytokines facilitate human retinal ganglion cell development and regeneration.

Microglia (µG), the resident immune cells in the central nervous system, surveil the parenchyma to maintain the structural and functional homeostasis of neurons. Besides, they influence neurogenesis and synaptogenesis through complement-mediated phagocytosis. Emerging evidence suggests that µG may also influence development through proinflammatory cytokines. Here, we examined the premise that tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), the two most prominent components of the µG secretome, influence retinal development, specifically the morphological and functional differentiation of human retinal ganglion cells (hRGCs). Using controlled generation of hRGCs and human µG (hµG) from pluripotent stem cells, we demonstrate that TNF-α and IL-1ß secreted by unchallenged hµG did not influence hRGC generation. However, their presence significantly facilitated neuritogenesis along with the basal function of hRGCs, which involved the recruitment of the AKT/mTOR pathway. We present ex vivo evidence that proinflammatory cytokines may play an important role in the morphological and physiological maturation of hRGCs, which may be recapitulated for regeneration.

Exploring the mechanism of interaction of glipizide with DNA: Combined in vitro and bioinformatics approach.

DNA, vital for biological processes, encodes hereditary data for protein synthesis, shaping cell structure and function. Since revealing its structure, DNA has become a target for various therapeutically vital molecules, spanning antidiabetic to anticancer drugs. These agents engage with DNA-associated proteins, DNA-RNA hybrids, or bind directly to the DNA helix, triggering diverse downstream effects. These interactions disrupt vital enzymes and proteins essential for maintaining cell structure and function. Analysing drug-DNA interactions has significantly advanced our understanding of drug mechanisms. Glipizide, an antidiabetic drug, is known to cause DNA damage in adipocytes. However, its extract mechanism of DNA interaction is unknown. This study delves into the interaction between glipizide and DNA utilizing various biophysical tools and computational technique to gain insights into the interaction mechanism. Analysis of UV-visible and fluorescence data reveals the formation of complex between DNA and glipizide. The binding affinity of glipizide to DNA was of moderate strength. Examination of thermodynamic parameters at different temperatures suggests that the binding was entropically spontaneous and energetically favourable. Various experiments such as thermal melting assays, viscosity measurement, and dye displacement assays confirmed the minor grove nature of binding of glipizide with DNA. Molecular dynamics studies confirmed the glipizide forms stable complex with DNA when simulated by mimicking the physiological conditions. The binding was mainly favoured by hydrogen bonds and glipizide slightly reduced nucleotide fluctuations of DNA. The study deciphers the mechanism of interaction of glipizide with DNA at molecular levels.

Modulation of quorum sensing and biofilm of Gram-negative bacterial pathogens by Cinnamomum zeylanicum L.

The development of antibiotic resistant microbial pathogens has become a global health threat and a major concern in modern medicine. The problem of antimicrobial resistance (AMR) has majorly arisen due to sub-judicious use of antibiotics in health care and livestock industry. A slow progress has been made in last two decades in discovery of new antibiotics. A new strategy in combatting AMR is to modulate or disarm the microbes for their virulence and pathogenicity. Plants are considered as promising source for new drugs against AMR pathogens. In this study, fraction-based screening of the Cinnamomum zeylanicum extract was performed followed by detailed investigation of antiquorum sensing and antibiofilm activities of the most active fraction that is, C. zeylanicum hexane fraction (CZHF). More than 75% reduction in violacein pigment of C. violaceum 12472 was overserved. CZHF successfully modulated the virulence of Pseudomonas aeruginosa PAO1 by 60.46%-78.35%. A similar effect was recorded against Serratia marcescens MTCC 97. A broad-spectrum inhibition of biofilm development was found in presence of sub-MICs of CZHF. The colonization of bacteria onto the glass coverslips was remarkably reduced apart from the reduction in exopolymeric substances. Alkaloids and terpenoids were found in CZHF. GC/MS analysis revealed the presence of cinnamaldehyde dimethyl acetal, 2-propenal, coumarin, and α-copaene as major phytocompounds. This study provides enough evidence to support potency of C. zeylanicum extract in targeting the virulence of Gram -ve pathogenic bacteria. The plant extract or active compounds can be developed as successful drugs after careful in vivo examination to target microbial infections. RESEARCH HIGHLIGHTS: Hexane fraction of Cinnamomum zeylanicum is active against QS and biofilms. The broad-spectrum antibiofilm activity was further confirmed by microscopic analysis. Dimethyl acetal, 2-propenal, coumarin, α-copaene, and so forth are major phytocompounds.

Beyond the ordinary: exploring the synergistic effect of iodine and nickel doping in cobalt hydroxide for superior energy storage applications.

This study explores the iodine and nickel-doped cobalt hydroxide (I & Ni-co-doped-Co(OH)2) as a potential material for energy storage and conversion applications owing to its excellent electrochemical characteristics. According to our analysis, it was revealed that this material exhibits pseudocapacitive-like behavior, as evident from distinct redox peaks observed in cyclic voltammetry, which confirms its ability to store charges. The diffusion coefficient analysis reveals that this material possesses conductivity and rapid diffusion kinetics, making it particularly advantageous compared to materials synthesized in previous studies. Charge-discharge measurements were performed to analyze the charge storage capacity and stability of this material after 3000 consecutive cycles, showing its excellent stability with minimum loss of capacitance. Furthermore, its anodic and cathodic linear sweep voltammetry curves were measured to evaluate its oxygen evolution and hydrogen evolution reaction performance. The results showed that the material exhibited an excellent water splitting performance, which suggests its potential practical application for hydrogen production. This increased activity was attributed to the doping of α-Co(OH)2, which improved its structural stability, electrical conductivity, and charge transfer efficiency. Thus, I & Ni-co-doped-Co(OH)2 possesses enhanced properties that make it an excellent material for both energy storage and hydrogen generation applications.

Furin Egress from the TGN is Regulated by Membrane-Associated RING-CH Finger (MARCHF) Proteins and Ubiquitin-Specific Protease 32 (USP32) via Nondegradable K33-Polyubiquitination.

Furin primarily localizes to the trans-Golgi network (TGN), where it cleaves and activates a broad range of immature proproteins that play critical roles in cellular homeostasis, disease progression, and infection. Furin is retrieved from endosomes to the TGN after being phosphorylated, but it is still unclear how furin exits the TGN to initiate the post-Golgi trafficking and how its activity is regulated in the TGN. Here three membrane-associated RING-CH finger (MARCHF) proteins (2, 8, 9) are identified as furin E3 ubiquitin ligases, which catalyze furin K33-polyubiquitination. Polyubiquitination prevents furin from maturation by blocking its ectodomain cleavage inside cells but promotes its egress from the TGN and shedding. Further ubiquitin-specific protease 32 (USP32) is identified as the furin deubiquitinase in the TGN that counteracts the MARCHF inhibitory activity on furin. Thus, the furin post-Golgi trafficking is regulated by an interplay between polyubiquitination and phosphorylation. Polyubiquitination is required for furin anterograde transport but inhibits its proprotein convertase activity, and phosphorylation is required for furin retrograde transport to produce fully active furin inside cells.

Riboflavin (vitamin B2) sensitized photooxidation of ascorbic acid (vitamin C): A kinetic study.

Ascorbic acid (AH2) photoxidation sensitized by riboflavin (RF) has been studied between pH 2.0 and 12.0 in ambient air and anaerobic environment using UV and visible irradiation sources. The kinetics of AH2 degradation in aqueous medium along with RF is found to be first-order for its photodegradation. AH2 photolysis rate constants in aerobic and anaerobic conditions with RF (1.0-5.0 × 10-5 M) are 0.14-3.89 × 10-2 and 0.026-0.740 × 10-2 min-1, respectively. The rate constants (k2) of second-order kinetics for AH2 and RF photochemical interaction in aerobic and anaerobic conditions are in the range of 0.24-3.70 to 0.05-0.70 × 10-3 M-1 min-1, respectively, which manifests that increasing the RF concentration also increases the rate of photodegradation (photooxidation) of AH2. The k2 versus pH graph is bell-shaped which indicates that increasing the pH increases photolytic degradation rate of AH2 with RF. Increasing the pH results in the increased ionization of AH2 (ascorbyl anion, AH-) and redox potential which leads to the higher rates of photodegradation of AH2. Two-component spectrophotometric (243 and 266 nm, AH2 and RF, respectively) and high-performance liquid chromatography (HPLC) methods have been used to determine the concentration of AH2 and RF in pure and degraded solutions. The results obtained from these two methods are compared using a student t-test which showed no noteworthy difference between them.

Ascorbic acid assisted photodegradation of methylcobalamin using corrective irrelevant absorption spectrophotometric assay: A kinetic study.

Photodegradation of drug substances leads to the formation of known and unknown degradation products. These unknown degradation products interfere and give erroneous results because of absorption on analytical wavelengths. This interference could be eliminated using the correction of irrelevant absorbancies. This study is based on the application of linear and non-linear correction of irrelevant absorption for the determination of methylcobalamin (MC) and hydroxocobalamin in the photolytic degradation assisted by ascorbic acid (AH2). MC follows first-order degradation kinetics and the rate of degradation (kobs) ranges from 1.99-2.34 × 10-2, min-1 at pH 2.0-12.0. The second-order rate constants (k2) for the photochemical interaction of MC and AH2 are in the range of 17.9-60.3 × 10-2 M-1, min-1 (acidic region) and 10.3-24.6 × 10-2 M-1, min-1 (alkaline region). The k2-pH profile was found to be bell-shaped and the maximum rate of degradation in the presence of AH2 is at pH 5.0 (60.3 × 10-2 M-1, min-1) due to the protonation of MC. However, in alkaline pH, the rate of photodegradation decreases due to the ionization form of AH2 which is AH- species.

Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1.

SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides' antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 µg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Incidence and transferability of antibiotic resistance in the enteric bacteria isolated from hospital wastewater

This study reports the occurrence of antibiotic resistance and production of β-lactamases including extended spectrum beta-lactamases (ESβL) in enteric bacteria isolated from hospital wastewater. Among sixty-nine isolates, tested for antibiotic sensitivity, 73.9% strains were resistant to ampicillin followed by nalidixic acid (72.5%), penicillin (63.8%), co-trimoxazole (55.1%), norfloxacin (53.6%), methicillin (52.7%), cefuroxime (39.1%), cefotaxime (23.2%) and cefixime (20.3%). Resistance to streptomycin, chloramphenicol, nitrofurantoin, tetracycline, and doxycycline was recorded in less than 13% of the strains. The minimum inhibitory concentration (MIC) showed a high level of resistance (800-1600 µg/mL) to one or more antibiotics. Sixty three (91%) isolates produced β-lactamases as determined by rapid iodometric test. Multiple antibiotic resistances were noted in both among ESβL and non-ESβL producers. The β-lactamases hydrolyzed multiple substrates including penicillin (78.8% isolates), ampicillin (62.3%), cefodroxil (52.2%), cefotoxime (21.7%) and cefuroxime (18.8%). Fifteen isolates producing ESβLs were found multidrug resistant. Four ESβL producing isolates could transfer their R-plasmid to the recipient strain E. coli K-12 with conjugation frequency ranging from 7.0 x 10-3 to 8.8 x 10-4. The findings indicated that ESβL producing enteric bacteria are common in the waste water. Such isolates may disseminate the multiple antibiotic resistance traits among bacterial community through genetic exchange mechanisms and thus requires immediate attention.