How a natural antibiotic uses oxidative stress to kill oxidant-resistant bacteria.

Natural products that possess antibiotic and antitumor qualities are often suspected of working through oxidative mechanisms. In this study, two quinone-based small molecules were compared. Menadione, a classic redox-cycling compound, was confirmed to generate high levels of reactive oxygen species inside Escherichia coli. It inactivated iron-cofactored enzymes and blocked growth. However, despite the substantial levels of oxidants that it produced, it was unable to generate significant DNA damage and was not lethal. Streptonigrin, in contrast, was poorer at redox cycling and did not inactivate enzymes or block growth; however, even in low doses, it damaged DNA and killed cells. Its activity required iron and oxygen, and in vitro experiments indicated that its quinone moiety transferred electrons through the adjacent iron atom to oxygen. Additionally, in vitro experiments revealed that streptonigrin was able to damage DNA without inhibition by catalase, indicating that hydrogen peroxide was not involved. We infer that streptonigrin can reduce bound oxygen directly to a ferryl species, which then oxidizes the adjacent DNA, without release of superoxide or hydrogen peroxide intermediates. This scheme allows streptonigrin to kill a bacterial cell without interference by scavenging enzymes. Moreover, its minimal redox-cycling behavior avoids alerting either the OxyR or the SoxRS systems, which otherwise would block killing. This example highlights qualities that may be important in the design of oxidative drugs. These results also cast doubt on proposals that bacteria can be killed by stressors that merely stimulate intracellular O2- and H2O2 formation.

Discovering targeted inhibitors for Escherichia coli efflux pump fusion proteins using computational and structure-guided approaches.

Multidrug resistance pathogens causing infections and illness remain largely untreated clinically. Efflux pumps are one of the primary processes through which bacteria develop resistance by transferring antibiotics from the interior of their cells to the outside environment. Inhibiting these pumps by developing efficient derivatives appears to be a promising strategy for restoring antibiotic potency. This investigation explores literature-reported inhibitors of E. coli efflux pump fusion proteins AcrB-AcrA and identify potential chemical derivatives of these inhibitors to overcome the limitations. Using computational and structure-guided approaches, a study was conducted with the selected inhibitors (AcrA:25-AcrB:59) obtained by data mining and their derivatives (AcrA:857-AcrB:3891) to identify their inhibitory effect on efflux pump using virtual screening, molecular docking and density functional theory (DFT) calculations. The finding indicates that Compound 2 (ZINC000072136376) has shown better binding and a significant inhibitory effect on AcrA, while Compound 3 (ZINC000072266819) has shown stronger binding and substantial inhibition effect on both non-mutant and mutated AcrB subunits. The identified derivatives could exhibit a better inhibitor and provide a potential approach for restoring the actions of resistant antibiotics.

Escherichia coli induces DNA repair enzymes to protect itself from low-grade hydrogen peroxide stress.

Escherichia coli responds to hydrogen peroxide (H2 O2 ) by inducing defenses that protect H2 O2 -sensitive enzymes. DNA is believed to be another important target of oxidation, and E. coli contains enzymes that can repair oxidative lesions in vitro. However, those enzymes are not known to be induced by H2 O2 , and experiments have indicated that they are not necessary for the cell to withstand natural (low-micromolar) concentrations. In this study, we used H2 O2 -scavenging mutants to impose controlled doses of H2 O2 for extended time. Transcriptomic analysis revealed that in the presence of 1 µM cytoplasmic H2 O2 , the OxyR transcription factor-induced xthA, encoding exonuclease III. The xthA mutants survived a conventional 15-min exposure to even 100 times this level of H2 O2 . However, when these mutants were exposed to 1 µM H2 O2 for hours, they accumulated DNA lesions, failed to propagate, and eventually died. Although endonuclease III (nth) was not induced, nth mutants struggled to grow. Low-grade H2 O2 stress also activated the SOS regulon, and when this induction was blocked, cell replication stopped. Collectively, these data indicate that physiological levels of H2 O2 are a real threat to DNA, and the engagement of the base-excision-repair and SOS systems is necessary to enable propagation during protracted stress.

A comparative analysis of exopolysaccharide and phytohormone secretions by four drought-tolerant rhizobacterial strains and their impact on osmotic-stress mitigation in Arabidopsis thaliana.

The ability of plant growth promoting rhizobacteria (PGPR) for imparting abiotic stress tolerance to plants has been widely explored in recent years; however, the diversity and potential of these microbes have not been maximally exploited. In this study, we characterized four bacterial strains, namely, Pseudomonas aeruginosa PM389, Pseudomonas aeruginosa ZNP1, Bacillus endophyticus J13 and Bacillus tequilensis J12, for potential plant growth promoting (PGP) traits under osmotic-stress, induced by 25% polyethylene glycol (PEG) in the growth medium. Growth curve analysis was performed in LB medium with or without PEG, in order to understand the growth patterns of these bacteria under osmotic-stress. All strains were able to grow and proliferate under osmotic-stress, although their growth rate was slower than that under non-stressed conditions (LB without PEG). Bacterial secretions were analyzed for the presence of exopolysaccharides and phytohormones and it was observed that all four strains released these compounds into the media, both, under stressed and non-stressed conditions. In the Pseudomonas strains, osmotic stress caused a decrease in the levels of auxin (IAA) and cytokinin (tZ), but an increase in the levels of gibberellic acid. The Bacillus strains on the other hand showed a stress-induced increase in the levels of all three phytohormones. P. aeruginosa ZNP1 and B. endophyticus J13 exhibited increased EPS production under osmotic-stress. While osmotic stress caused a decrease in the levels of EPS in P. aeruginosa PM389, B. tequilensis J12 showed no change in EPS quantities released into the media under osmotic stress when compared to non-stressed conditions. Upon inoculating Arabidopsis thaliana seedlings with these strains individually, it was observed that all four strains were able to ameliorate the adverse effects of osmotic-stress (induced by 25% PEG in MS-Agar medium) in the plants, as evidenced by their enhanced fresh weight, dry weight and plant water content, as opposed to osmotic-stressed, non-inoculated plants.

Applicability of the International League Against Epilepsy (ILAE) 2022 diagnostic criteria for epilepsy syndromes in children: A retrospective review of 1550 children with epilepsy.

OBJECTIVE: Recently, the ILAE Nosology and Definitions Task Force defined diagnostic criteria for epilepsy syndromes. There is paucity of data on the use of these new diagnostic criteria in children with epilepsy, and how these criteria may lead to changes from previous practice. METHODS: This was a retrospective chart review of data of children attending the epilepsy clinic in a tertiary care children's hospital from January 2011 to January 2023. The clinical details such as age at onset, types of seizures, co-morbidities, and results of EEG, MRI and genetic testing were reviewed. Epilepsy syndrome diagnosis was made as per the ILAE 2022 criteria, and compared with the previous syndrome diagnosis as per records. RESULTS: Data from 1550 children (63 % boys) with epilepsy were analysed, and 55.4 % children were classified to have epilepsy syndromes as per the new ILAE 2022 diagnostic criteria. Application of the new 2022 ILAE diagnostic criteria was associated with a change in name alone in 676 (77.8 %) children. Hundred (11.5 %) children were newly classified under an epilepsy syndrome who had previously remained unclassified. Eleven (1.3 %) children who were previously classified into an epilepsy syndrome could not be classified using the new diagnostic criteria. Eight (0.9 %) were shifted to a new syndromic category. Overall, change in diagnosis occurred in 13.7 (11.5 + 1.3 + 0.9)%. No change in epilepsy syndrome classification/nomenclature occurred in 74 (8.5 %) children. SIGNIFICANCE: The new diagnostic criteria led to an overall change in diagnosis in 13.7 % of children with epilepsy. These criteria will hopefully lead to uniformity in diagnosis of epilepsy syndromes across diverse settings.

Cytokines inhibitory mechanism of Prunus domestica L. (Plum) peptides as potential immunomodulators against systemic lupus erythematosus: an in-silico screening.

Natural bioactive peptides exhibit various chemical and structural properties to enhance the immune response against multiple inflammatory and autoimmune related disorders. The immunomodulatory function and bioactivity of seed peptides show the capability for the development of biotherapeutics that could prevent autoimmune diseases. The aim of current study is to determine the immunomodulatory function of bioactive peptides derived from the seed of plum (Prunus domestica L.) by applying various immunoinformatic approaches. A thorough analysis of forty-one peptides was performed including drug likeliness, pharmacokinetic, and bioactivity profiling studies. Further, molecular docking and molecular dynamics (MD) simulations of screened peptides were carried out with the two interleukin targets (IL-17A and IL-23) of systemic lupus erythematosus (SLE). After the systematic screening, four peptides, namely HLLP, LPLL, LPAGV, and NLPL, were found as potential inhibitors against SLE. Additionally, site-directed mutagenesis analysis was conducted to explore the role of essential amino acid residues in the binding pattern/energy change. Computational alanine screening analysis found that CYS123, CYS121 of IL-17A and ASP270, and SER249 of IL-23 as hot spot residues that could play an important role in the inhibition property of screened peptides. Overall, the methodology described in the study can be utilized for developing unique peptide inhibitors that have a preventative role against SLE. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00188-8.

Predictors of Toxicity in a Randomized Study of Consolidation Chemoradiation Versus Observation After First Line Chemotherapy in Advanced Gall Bladder Cancers.

Purpose: Gall bladder cancers (GBC) usually presents in advanced stage. First-line chemotherapy (CT) is the standard of care, and there is no other option for responders than to wait for disease progression. We conducted a randomized study of consolidation chemoradiation (CTRT) versus observation in responders to first line CT (NCT05493956), which showed an improvement in overall survival by 6 months and therefore is practice changing. We are reporting the toxicity and factors predicting toxicity due to CTRT so that it informs appropriate patient selection. Methods and Materials: Responders to first line CT (partial response, stable disease) were randomized to CTRT versus observation after 4 cycles. CTRT was delivered by 3D conformal radiotherapy (along-with concurrent capecitabine at 1250 mg/m2) to a dose of 45 Gy in 25 fractions to GBC and lymphatics followed by a boost of 9 Gy in 5 fractions to the GBC. Toxicities documented during CTRT were recorded using the Radiation Therapy Oncology Group criteria. Dose volume data were correlated with the radiation induced side effects. Results: Among 135 patients enrolled both arms are well balanced demographically, and 58% patients had T4 tumors, 42% had N2 and 15% had paraaortic lymph node, and 27% underwent upfront stenting. Grade 3 adverse events, such as anemia, dyspepsia, hepatotoxicity (Child Pugh B), and gastrointestinal bleed due to CTRT was observed in 9%, 1.5%, 13%, and 5.8%, respectively. Age >58 years (P = .02), planning target volume (PTV) 1 volume (>919 cc, P = .02), PTV2 volume (>380 cc, P = .01), mean liver dose (>28 Gy, P = .07), and liver V40 (>50%, P = .02) predicted radiation-induced liver disease. A receiver operating curve analysis revealed a cut-off value of PTV1 volume of 800 cc (sensitivity and specificity of 75% and 54%) and PTV2 volume of 300 cc (sensitivity and specificity of 81% and 65%) for prediction of hepatotoxicity. Duodenum V45 >45% (P = .02) predicted grade 3 anemia. Numerically high V15 duodenum (98%, P = .11), large PTV2 volume >484 cc (P = .06) and prior stenting had predilection for gastrointestinal bleed. Conclusions: Consolidation CTRT is tolerable in those with PTV1 volume less than 800 cc.

Identifying the mediators of intracellular E. coli inactivation under UVA light: The (photo) Fenton process and singlet oxygen.

Solar disinfection (SODIS) was probed for its underlying mechanism. When Escherichia coli was exposed to UVA irradiation, the dominant solar fraction acting in SODIS process, cells exhibited a shoulder before death ensued. This profile resembles cell killing by hydrogen peroxide (H2O2). Indeed, the use of specialized strains revealed that UVA exposure triggers intracellular H2O2 formation. The resultant H2O2 stress was especially impactful because UVA also inactivated the processes that degrade H2O2-peroxidases through the suppression of metabolism, and catalases through direct enzyme damage. Cell killing was enhanced when water was replaced with D2O, suggesting that singlet oxygen plays a role, possibly as a precursor to H2O2 and/or as the mediator of catalase damage. UVA was especially toxic to mutants lacking miniferritin (dps) or recombinational DNA repair (recA) enzymes, indicating that reactions between ferrous iron and UVA-generated H2O2 lead to lethal DNA damage. Importantly, experiments showed that the intracellular accumulation of H2O2 alone is insufficient to kill cells; therefore, UVA must do something more to enable death. A possibility is that UVA stimulates the reduction of intracellular ferric iron to its ferrous form, either by stimulating O2•- formation or by generating photoexcited electron donors. These observations and methods open the door to follow-up experiments that can probe the mechanisms of H2O2 formation, catalase inactivation, and iron reduction. Of immediate utility, the data highlight the intracellular pathways formed under UVA light during SODIS, and that the presence of micromolar iron accelerates the rate at which radiation disinfects water.

Computational screening for investigating the synergistic regulatory potential of drugs and phytochemicals in combination with 2-deoxy-D-glucose against SARS-CoV-2.

COVID-19 disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was declared a global pandemic by the World Health Organization (WHO) in March 2020. Since then, the SARS-CoV-2 virus has impacted millions of lives worldwide. Various preclinical and clinical trials on the treatment of COVID-19 disease have revealed that the drugs that work in combination are more likely to reduce reinfection and multi-organ failure. Considering the combination drug therapy, herein, we performed a systematic computational study starting with the formation of sixty-two combinations of drugs and phytochemicals with 2-deoxy-D-glucose (2-DG). The top nineteen combinations resulting from Drug-Drug Interaction (DDI) analysis were selected for individual and multiple-ligand-simultaneous docking (MLSD) study with a host target Serine Protease (TMPRSS2; PDB ID: 7MEQ) and two viral targets, Main Protease (3CLpro; PDB ID: 6LU7) and Uridylate-Specific Endoribonuclease (NSP15; PDB ID: 6VWW). We found that the resulting drugs and phytochemicals in combination with 2-DG shows better binding than the individual compounds. We performed the re-docking of the top three drug combinations by utilizing the polypharmacology approach to validate the binding patterns of drug combinations with multiple targets for verifying the best drug combinatorial output obtained by blind docking. A strong binding affinity pattern was observed for 2-DG + Ruxolitinib (NIH-recommended drug), 2-DG + Telmisartan (phase 4 clinical trial drug), and 2-DG + Punicalagin (phytochemical) for all the selected targets. Additionally, we conducted multiple-ligand-simultaneous molecular dynamics (MLS-MD) simulations on the selected targets with the 2-DG + Ruxolitinib combination. The MLS-MD analysis of the drug combinations shows that stabilization of the interaction complexes could have significant inhibition potential against SARS CoV-2. This study provides an insight into developing drug combinations utilizing integrated computational approaches to uncover their potential in synergistic drug therapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02049-0.

The dual role of phytochemicals on SARS-CoV-2 inhibition by targeting host and viral proteins.

BACKGROUND: The severe acute respiratory syndrome-2019 has affected more than 190 million people around the world and caused severe crises throughout the globe. Due to rapid mutation in the viral genome, its became important to simultaneously improvise the host immunity while targeting viral proteins to reduce the severity of infection. AIM: The current computational work focuses on multi-level rigorous screening of 47 medicinal plant-based phytochemicals for discovering effective phytochemical inhibitors against the host and viral targets. EXPERIMENTAL PROCEDURE: A total of 586 phytochemicals were analyzed in detail based on their drug-likeness, pharmacological properties, and structure-based activity against the viral proteins (Spike glycoprotein, Papain-like protease, and Main protease) and host proteins (ACE2, Importin-subunit α-5, and ß-1). Phytochemicals showing higher binding affinity with the dual capacity to target both the categories of proteins were further analyzed by profiling of their chemical reactivity using Density-Functional Theory (DFT) based quantum chemical methods. Finally, detailed molecular dynamics simulations were performed to analyze the interactions of the complexes. RESULTS AND CONCLUSION: The results revealed that the selected phytochemicals from Andrographis paniculata, Aconitum heterophyllum, Costus speciosus and Inula racemosa may have the capacity to act with prominent affinity towards the host and viral proteins. Therefore, the combination of active phytochemicals of these plants may prove to be more beneficial and can be used for developing the potential phytotherapeutic intervention.

Phone Camera Nano-Biosensor Using Mighty Sensitive Transparent Reusable Upconversion Paper.

Lycopene, a natural colorant and antioxidant with a huge growing market, is highly susceptible to photo/thermal degradation, which demands real-time sensors. Hence, here a transparent upconversion nanoparticles (UCNPs) strip having 30 mol % Yb, 0.1 mol % Tm, and ß-NaYF4 UCNPs, which shows an intense emission at 475 nm, has been developed. This strip has been found to be sensitive to lycopene with a detection limit as low as 10 nM using a smartphone camera, which is due to static quenching that is confirmed by the lifetime study. In comparison to previous paper strips, here the transparent strip has minimal scattering with maximum sensitivity in spite of not using any metal quenchers. An increase in strip hydrophobicity during the fabrication process complements the strip to selectively permeate and present an extraction-free substitute analysis for chromatography. Hydrophobicity endows the strip with the capability to reuse the strip with ∼100% luminescence recovery.

Targeting mitochondrial bioenergetics as a promising therapeutic strategy in metabolic and neurodegenerative diseases.

Mitochondria are the organelles that generate energy for the cells and act as biosynthetic and bioenergetic factories, vital for normal cell functioning and human health. Mitochondrial bioenergetics is considered an important measure to assess the pathogenesis of various diseases. Dysfunctional mitochondria affect or cause several conditions involving the most energy-intensive organs, including the brain, muscles, heart, and liver. This dysfunction may be attributed to an alteration in mitochondrial enzymes, increased oxidative stress, impairment of electron transport chain and oxidative phosphorylation, or mutations in mitochondrial DNA that leads to the pathophysiology of various pathological conditions, including neurological and metabolic disorders. The drugs or compounds targeting mitochondria are considered more effective and safer for treating these diseases. In this review, we make an effort to concise the available literature on mitochondrial bioenergetics in various conditions and the therapeutic potential of various drugs/compounds targeting mitochondrial bioenergetics in metabolic and neurodegenerative diseases.

Analogue discovery of safer alternatives to HCQ and CQ drugs for SAR-CoV-2 by computational design.

COVID-19 outbreak poses a severe health emergency to the global community. Due to availability of limited data, the selection of an effective treatment is a challenge. Hydroxychloroquine (HCQ), a chloroquine (CQ) derivative administered for malaria and autoimmune diseases, has been shown to be effective against both Severe Acute Respiratory Syndrome (SARS-CoV-1) and SARS-CoV-2. Apart from the known adverse effects of these drugs, recently the use of CQ and HCQ as a potential treatment for COVID-19 is under flux globally. In this study, we focused on identifying a more potent analogue of HCQ and CQ against the spike protein of SAR-CoV-2 that can act as an effective antiviral agent for COVID-19 treatment. Systematic pharmacokinetics, drug-likeness, basicity predictions, virtual screening and molecular dynamics analysis (200 ns) were carried out to predict the inhibition potential of the analogous compounds on the spike protein. This work identifies the six potential analogues, out of which two compounds, namely 1-[1-(6-Chloroquinolin-4-yl) piperidin-4-yl]piperidin-3-ol and (1R,2R)-2-N-(7-Chloroquinolin-4-yl)cyclohexane-1,2-diamine interact with the active site of the spike protein similar to HCQ and CQ respectively with augmented safety profile.