In-silico and in-vitro evaluation of antifungal bioactive compounds from Streptomyces sp. strain 130 against Aspergillus flavus.

Streptomyces spp. are considered excellent reservoirs of natural bioactive compounds. The study evaluated the bioactive potential of secondary metabolites from Streptomyces sp. strain 130 through PKS-I and NRPS gene-clusters screening. GC-MS analysis was done for metabolic profiling of bioactive compounds from strain 130 in the next set of experiments. Identified antifungal compounds underwent ADMET analyses to screen their toxicity. All compounds' molecular docking was done with the structural gene products of the aflatoxin biosynthetic pathway of Aspergillus flavus. MD simulations were utilized to evaluate the stability of protein-ligand complexes under physiological conditions. Based on the in-silico studies, compound 2,4-di-tert butyl-phenol (DTBP) was selected for in-vitro studies against Aspergillus flavus. Simultaneously, bioactive compounds were extracted from strain 130 in two different solvents (ethyl-acetate and methanol) and used for similar assays. The MIC value of DTBP was found to be 314 µg/mL, whereas in ethyl-acetate extract and methanol-extract, it was 250 and 350 µg/mL, respectively. A mycelium growth assay was done to analyze the effect of compounds/extracts on the mycelium formation of Aspergillus flavus. In agar diffusion assay, zone of inhibitions in DTBP, ethyl-acetate extract, and methanol extract were observed with diameters of 11.3, 13.3, and 7.6 mm, respectively. In the growth curve assay, treated samples have delayed the growth of fungi, which signified that the compounds have a fungistatic nature. Spot assay has determined the fungal sensitivity to a sub-minimum inhibitory concentration of antifungal compounds. The study's results suggested that DTBP can be exploited for antifungal-drug development.Communicated by Ramaswamy H. Sarma.

Natural products from Streptomyces spp. as potential inhibitors of the major factors (holoRdRp and nsp13) for SARS-CoV-2 replication: an in silico approach.

The COVID-19 pandemic caused unprecedented damage to humanity, and while vaccines have been developed, they are not fully effective against the SARS-CoV-2 virus. Limited targeted drugs, such as Remdesivir and Paxlovid, are available against the virus. Hence, there is an urgent need to explore and develop new drugs to combat COVID-19. This study focuses on exploring microbial natural products from soil-isolated bacteria Streptomyces sp. strain 196 and RI.24 as a potential source of new targeted drugs against SARS-CoV-2. Molecular docking studies were performed on holoRdRp and nsp13, two key factors responsible for virus replication factor. Our in silico studies, K-252-C aglycone indolocarbazole alkaloid (K252C) and daunorubicin were found to have better binding affinities than the respective control drugs, with K252C exhibiting binding energy of - 9.1 kcal/mol with holoRdRp and - 9.2 kcal/mol with nsp13, and daunorubicin showing binding energy at - 8.1 kcal/mol with holoRdRp and - 9.3 kcal/mol with nsp13. ADMET analysis, MD simulation, and MM/GBSA studies indicated that K252C and daunorubicin have the potential to be developed as targeted drugs against SARS-CoV-2. The study concludes that K252C and daunorubicin are potential lead compounds that might suppress the inhibition of SARS-CoV-2 replication among the tested microbial compounds and could be developed as targeted drugs against COVID-19. In the future, further in vitro studies are required to validate these findings.

Harnessing the Biocontrol Potential of Bradyrhizobium japonicum FCBP-SB-406 to Manage Charcoal Rot of Soybean with Increased Yield Response for the Development of Sustainable Agriculture.

Plant growth-promoting bacteria (PGPRs) have the potential to act as biofertilizers and biopesticides. This study was planned to explore indigenously isolated PGPRs as a potential candidate to control charcoal rot that affects various crops including soybean. Among the four different tested species of PGPRs, Bradyrhizobium japonicum (FCBP-SB-406) showed significant potential to enhance growth and control soil borne pathogens such as Macrophomina phaseolina. Bacillus subtilis (FCBP-SB-324) followed next. Bradyrhizobium japonicum (FCBP-SB-406) reduced disease severity up to 81.25% in comparison to the control. The strain showed a strong fertilizing effect as a highly significant increase in biomass and other agronomic parameters was recorded in plants grown in its presence. The same was supported by the Pearson's correlation and principal component analysis. A decrease in disease incidence and severity may be due to the induced resistance imparted by the bacterium. This resulted in significant increments in quantities of defense enzymes, including catalase, peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD). A significant production of proteases, catalases and hydrogen cyanide by B. japonicum (FCBP-SB-406) can also be associated to mycoparasitism. The establishment of PGPRs in treated soils also showed positive effects on soil health. Total metabolite profiling of treated plants in comparison to the control showed the upregulation of many flavonoids, isoflavonoids and amino acids. Many of these compounds have been well reported with antimicrobial activities. Bradyrhizobium japonicum (FCBP-SB-406) can be employed for the production of a potential formulation to support sustainable agriculture by reducing the input of synthetic pesticides and fertilizers.

Monoterpenes as potential antifungal molecules against Candida cell membranes: in-vitro and in-silico studies.

Azoles are the frequently used antifungal drugs that target the enzyme lanosterol 14 α-demethylase (erg11p). This enzyme plays a vital role in ergosterol biosynthesis and hence maintainenance of cell membrane fluidity and integrity. The emergence of resistance to azoles and their fungistatic nature against several fungal pathogens is the major challenge to combat invasive candidiasis. Therefore, there is an urgent need to discover new antifungals with better efficacy. This study targets erg11 protein using in silico approach and identifies the monoterpene compounds (α-terpineol, carveol, and terpinene-4-ol) based on docking score and ligand interaction analysis. Further dynamic behavior of best-docked compounds with erg11p was analyzed by various parameters of MD simulation. The binding free energy of selected compounds towards the definitive pocket was also calculated. To further investigate the antifungal activity of selected compounds, in vitro studies were conducted on C. albicans. Studies thus suggest that the proposed the mechanism of antifungal action of test compounds involves targeting the ergosterol biosynthetic pathway. The compounds were explored for their effect on the disruption of membrane integrity by studying ERG11gene expression analysis, scanning electron microscopy, PI uptake (fluorescence microscopy,) and H+-extrusion. The results suggest that the selected monoterpenes are safer natural antifungals that disrupt membrane integrity by inhibiting ergosterol biosynthesis and other membrane associated structures.Communicated by Ramaswamy H. Sarma.

Racial and Ethnic Disparities in Peripheral Vascular Disease Admissions Using a Nationally Representative Sample.

Our study aimed to identify clinical outcomes and resource utilization associated with race and ethnicity in patients admitted with peripheral vascular disease (PVD) across the United States. We queried the National Inpatient Sample database from 2015 to 2019 and identified 622,820 patients admitted with PVD. Patients across 3 major race and ethnic categories were compared in terms of baseline characteristics, inpatient outcomes, and resource utilization. Black and Hispanic patients were more likely to be younger and of the lowest median income but incur higher total hospital costs. Black race predicted higher rates of acute kidney injury, need for blood transfusion, and need for vasopressor but lower rates of circulatory shock, and mortality. Black and Hispanic patients were less likely to undergo limb-salvaging procedures and more likely to undergo amputation than White patients. In conclusion, our findings indicate that Black and Hispanic patients experience health disparities in resource utilization and inpatient outcomes for PVD admissions.

Comparison of Efficacy and Safety of Angiotensin Receptor-Neprilysin Inhibitors in Patients With Heart Failure With Reduced Ejection Fraction: A Meta-Analysis.

The present meta-analysis was conducted to compare the safety and efficacy of angiontensin receptor neprilysin inhibitor (ARNI) with angiotensin receptor blockers (ARBs) and angiotensin-converting-enzyme inhibitors (ACEi) in patients with heart failure with reduced ejection fraction (HFrEF). This meta-analysis was conducted and reported in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. Two authors carried out a scientific literature search on online databases, including EMBASE, PubMed, and the Cochrane Library. The following keywords or corresponding Medical Subject Headings (MeSH) were used for the search of relevant articles: "heart failure with reduced ejection fraction," "angiotensin receptor-neprilysin inhibitor," "Angiotensin receptor blockers," and "clinical outcomes." Outcomes assessed in the present meta-analysis included changes in ejection fraction (EF) from baseline in percentage. Other outcomes assessed in the present meta-analysis included all-cause mortality, cardiovascular death, and hospitalization due to heart failure. Adverse events assessed in the present meta-analysis included hypokalemia, acute kidney injury, and hypotension. Total 10 studies were included. This meta-analysis showed that treatment with ARNI was associated with a significantly lower risk of all-cause mortality and cardiovascular death compared to control groups. There was no significant difference between the two groups in terms of change of EF from baseline or hospitalization related to heart failure. However, the risk of hypotension was significantly higher in patients receiving ARNI. The study findings support the use of ARNI as first-line therapy for heart failure with reduced ejection fraction. Further studies are required to determine the optimal use of ARNI in heart failure management and to investigate the mechanisms underlying the increased risk of hypotension.

Incorporation of engineered nanoparticles of biochar and fly ash against bacterial leaf spot of pepper.

In agriculture, the search for higher net profit is the main challenge in the economy of the producers and nano biochar attracts increasing interest in recent years due to its unique environmental behavior and increasing the productivity of plants by inducing resistance against phytopathogens. The effect of rice straw biochar and fly ash nanoparticles (RSBNPs and FNPs, respectively) in combination with compost soil on bacterial leaf spot of pepper caused by Xanthomonas campestris pv. vesicatoria was investigated both in vitro and in vivo. The application of nanoparticles as soil amendment significantly improved the chili pepper plant growth. However, RSBNPs were more effective in enhancing the above and belowground plant biomass production. Moreover, both RSBNPs and FNPs, significantly reduced (30.5 and 22.5%, respectively), while RSBNPs had shown in vitro growth inhibition of X. campestris pv. vesicatoria by more than 50%. The X-ray diffractometry of RSBNPs and FNPs highlighted the unique composition of nano forms which possibly contributed in enhancing the plant defence against invading X. campestris pv. vesicatoria. Based on our findings, it is suggested that biochar and fly ash nanoparticles can be used for reclaiming the problem soil and enhance crop productivity depending upon the nature of the soil and the pathosystem under investigation.

A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis.

Chromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

Diosmin in combination with naringenin enhances apoptosis in colon cancer cells.

Colon cancer is one of the most commonly diagnosed malignancies, which begins as a polyp and grows to become cancer. Diosmin (DS) and naringenin (NR) are naturally occurring flavonoids that exhibit various pharmacological activities. Although several studies have illustrated the effectiveness of these flavonoids as anti­cancerous agents individually, the combinatorial impact of these compounds has not been explored. In the present study, the combined effect of DS and NR (DiNar) in colon cancer cell lines HCT116 and SW480 were assessed by targeting apoptosis and inflammatory pathways. The MTT assay was used to evaluate the effect of DiNar on cell proliferation, while Chou­Talalay analysis was employed to determine the combination index of DS and NR. Moreover, flow cytometry was used to monitor cell cycle arrest and population study. The onset of apoptosis was assessed by DAPI staining, DNA fragmentation, and Annexin V­fluorescein isothiocyanate/propidium iodide (Annexin V­FITC/PI). The expression levels of apoptotic pathway markers, Bcl­2, Bax, caspase3, caspase8, caspase9 and p53, and inflammatory markers, NF­κß, IKK­α and IKK­ß, were assessed using western blotting and reverse transcription­quantitative PCR. These results suggested that DiNar treatment acts synergistically and induces cytotoxicity with a concomitant increase in chromatin condensation, DNA fragmentation and cell cycle arrest in the G0/G1 phase. Annexin V­FITC/PI apoptosis assay also showed increased number of cells undergoing apoptosis in the DiNar treatment group. Furthermore, the expression of apoptosis and inflammatory markers was also more effectively regulated under the DiNar treatment. Thereby, these findings demonstrated that DiNar treatment could be a potential novel chemotherapeutic alternative in colon cancer.

Compatibility of a novel filter paper-based bio-safe sputum transport kit with line probe assay for diagnosing drug-resistant tuberculosis: a single-site evaluation study.

BACKGROUND: Near-patient access to appropriate tests is a major obstacle for the efficient diagnosis of tuberculosis (TB) and associated drug resistance. METHODS: We recently developed the "TB Concentration & Transport" kit for bio-safe, ambient-temperature transportation of dried sputum on Trans-Filter, and the "TB DNA Extraction" kit for DNA extraction from Trans-Filter for determining drug resistance by DNA sequencing. In the present study, we evaluated the compatibility of Kit-extracted DNA with Hain's line probe assays (LPAs), which are endorsed by National TB programmes for the detection of drug resistance in sputum collected from presumptive multidrug-resistant TB patients (n=207). RESULTS: Trans-Filter-extracted DNA was seamlessly integrated with the LPA protocol (Kit-LPA). The sensitivity of Kit-LPA for determining drug resistance was 83.3% for rifampicin (95% CI 52-98%), 77.7% for isoniazid (95% CI 52-94%), 85.7% for fluoroquinolones (95% CI 42-100%) and 66.6% for aminoglycosides (95% CI 9-99%), with a specificity range of 93.7% (95% CI 87-97) to 99.1% (95% CI 95-100) using phenotypic drug susceptibility testing (DST) as a reference standard. A high degree of concordance was noted between results obtained from Kit-LPA and LPA (99% to 100% (κ value: 0.83-1.0)). CONCLUSIONS: This study demonstrates successful integration of our developed kits with LPA. The adoption of these kits across Designated Microscopy Centres in India can potentially overcome the existing challenge of transporting infectious sputum at controlled temperature to centralised testing laboratories and can provide rapid near-patient cost-effective "Universal DST" services to TB subjects residing in remote areas.

Carbon ion radiotherapy eradicates medulloblastomas with chromothripsis in an orthotopic Li-Fraumeni patient-derived mouse model.

BACKGROUND: Medulloblastomas with chromothripsis developing in children with Li-Fraumeni Syndrome (germline TP53 mutations) are highly aggressive brain tumors with dismal prognosis. Conventional photon radiotherapy and DNA-damaging chemotherapy are not successful for these patients and raise the risk of secondary malignancies. We hypothesized that the pronounced homologous recombination deficiency in these tumors might offer vulnerabilities that can be therapeutically utilized in combination with high linear energy transfer carbon ion radiotherapy. METHODS: We tested high-precision particle therapy with carbon ions and protons as well as topotecan with or without PARP inhibitor in orthotopic primary and matched relapsed patient-derived xenograft models. Tumor and normal tissue underwent longitudinal morphological MRI, cellular (markers of neurogenesis and DNA damage-repair), and molecular characterization (whole-genome sequencing). RESULTS: In the primary medulloblastoma model, carbon ions led to complete response in 79% of animals irrespective of PARP inhibitor within a follow-up period of 300 days postirradiation, as detected by MRI and histology. No sign of neurologic symptoms, impairment of neurogenesis or in-field carcinogenesis was detected in repair-deficient host mice. PARP inhibitors further enhanced the effect of proton irradiation. In the postradiotherapy relapsed tumor model, median survival was significantly increased after carbon ions (96 days) versus control (43 days, P < .0001). No major change in the clonal composition was detected in the relapsed model. CONCLUSION: The high efficacy and favorable toxicity profile of carbon ions warrants further investigation in primary medulloblastomas with chromothripsis. Postradiotherapy relapsed medulloblastomas exhibit relative resistance compared to treatment-naïve tumors, calling for exploration of multimodal strategies.

Evaluation of 'TBDetect' sputum microscopy kit for improved detection of Mycobacterium tuberculosis: a multi-centric validation study.

OBJECTIVES: The present study aimed to evaluate the performance of the 'TBDetect' kit-based bio-safe fluorescent microscopy filter (BioFM-Filter) microscopy in comparison with direct smear microscopy and culture for the detection of pulmonary tuberculosis (TB) in a multi-centric setting in India. METHODS: The TBDetect kit enables sputum concentration through filtration using the BioFM-Filter for improved and bio-safe smear microscopy. We evaluated the performance of the TBDetect kit in a six-site multi-centric validation study on sputum collected from 2086 presumptive TB patients. RESULTS: The combined positivity of TBDetect microscopy performed on these sputum samples was 20% (n = 417/2086) vs 16.1% of light-emitting diode fluorescence microscopy (LED-FM, n = 337/2086) and 16% of Ziehl Neelsen (ZN) smear microscopy (n = 333/2086). The increment in positivity of TBDetect over both LED-FM and ZN smears was significant (p < 0.001). The overall sensitivity of TBDetect for six sites was ~55% (202/367, 95% confidence interval (CI): 50, 60%) vs 52% (191/367, 95% CI: 47, 57%) for LED-FM (p 0.14) and 50.9% (187/367, 95% CI: 46, 56%) for ZN smear (p < 0.05), using Mycobacterium Growth Indicator Tube culture (MGIT, n = 1949, culture positive, n = 367) as the reference standard. A bio-safety evaluation at six sites confirmed efficient sputum disinfection by TBDetect; 99.95% samples (1873/1874) were sterile after 42 days of incubation. Scientists and technicians at the study sites indicated the ease of use and convenience of TBDetect microscopy during feedback. CONCLUSIONS: TBDetect added value to the smear microscopy test due to its improved performance, convenience and user safety. These findings indicate that equipment-free TBDetect technology has the potential to improve TB diagnosis in basic laboratory settings by leveraging on the existing nationwide network of designated microscopy centres and primary healthcare centres.

A versatile system to introduce clusters of genomic double-strand breaks in large cell populations.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5-bromo-2-deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

Phytochemical based nanomedicines against cancer: current status and future prospects.

Cancer continues to be one in all the leading reasons for death worldwide. The mean cancer survival through standard therapeutic strategies has not been significantly improved over the past few decades. Hence, alternate remedies are needed to treat this terrible disease. Recently, natural compounds present in the plants, i.e. phytochemicals have been widely exploited for their anticancer potential. Phytochemicals may exhibit their anticancer activity through targeting different cancer cell signalling pathways, promoting cell cycle arrest and apoptosis, regulating antioxidant status and detoxification. Despite their excellent anticancer activity, the phytochemicals are limited by their low aqueous solubility, poor bioavailability, and poor penetration into cells, hepatic disposition, narrow therapeutic index and rapid uptake by normal tissues. Therefore, to address these challenges, the scientific community has shifted its significant interests towards nanocarriers-based delivery of phytochemicals due to their ability to enhance aqueous solubility, and bioavailability, specific tumour cell/tissue targeting, improved cellular uptake, reducing doses of phytochemicals and achieving steady-state therapeutic levels of the phytochemicals over an extended period of time. Additional advantages include excellent blood stability, multifunctional design of nanocarriers and improvement in anticancer activities. This review aims to summarise recent progress in phytochemical based nanomedicines for effective treatment of cancer.

Sputum smear positivity at two months in previously untreated pulmonary tuberculosis patients.

BACKGROUND AND OBJECTIVES: In pulmonary tuberculosis, bacteriological status at two months affects subsequent treatment and prognosis. The effect on treatment outcome and risk factors for sputum conversion at two months treatment in previously untreated pulmonary tuberculosis (PTB) patients was studied in the following report. METHODS: A 1:1 case-control study was performed from June 2006 to February 2008 on patients in the Revised National Tuberculosis Control Program in a tertiary level institute in Delhi, India. Patients with previously untreated PTB with sputum smear positive at 2months of treatment (cases) were compared with those who achieved conversion (controls). RESULTS: In 74 cases and 74 controls, independent risk factors for sputum smear positive at two months were: illness for >2months, presence of cavity or extensive disease on chest X-ray, and interruption in intensive phase of treatment. Patients with smear positive at 2 or 3months of treatment were more likely to fail or default from treatment. Aforesaid factors were also associated with sputum culture positive status at 2months in univariate analysis. Patients who interrupted treatment ⩾3 times in the first two months were more likely to be culture positive at two months and had a higher rate of default and failure. CONCLUSIONS: Illness for more than 2months, presence of cavity or extensive disease on chest X-ray, and interruption in intensive phase of treatment are independent risk factors for sputum smear positivity at two months, which in turn is associated with poor treatment outcomes. Patients with these factors merit special attention under the national program.