Proximate Analysis of Moringa oleifera Leaves and the Antimicrobial Activities of Successive Leaf Ethanolic and Aqueous Extracts Compared with Green Chemically Synthesized Ag-NPs and Crude Aqueous Extract against Some Pathogens.

Research on the use of different parts of the Moringa oleifera plant as a nutritional and pharmaceutical resource for human and animals has increased in recent years. This study aimed to investigate the chemical composition and the TPCs and TFCs of Moringa leaves, the antimicrobial activities of Moringa successive ethanolic, aqueous, crude aqueous extracts, and green-chemically synthesized characterized Ag-NPs. The results indicated that the ethanolic extract recorded the highest activity against E. coli. On the other side, the aqueous extract showed higher activity, and its effects ranged from 0.03 to 0.33 mg/mL against different strains. The MIC values of Moringa Ag-NPs against different pathogenic bacteria ranged from 0.05 mg/mL to 0.13 mg/mL, and the activity of the crude aqueous extract ranged from 0.15 to 0.83 mg/mL. For the antifungal activity, the ethanolic extract recorded the highest activity at 0.04 mg/mL, and the lowest activity was recorded at 0.42 mg/mL. However, the aqueous extract showed effects ranging from 0.42 to 1.17 mg/mL. Moringa Ag-NPs showed higher activity against the different fungal strains than the crude aqueous extract, and they ranged from 0.25 to 0.83 mg/mL. The MIC values of the Moringa crude aqueous extract ranged from 0.74 to 3.33 mg/mL. Moringa Ag-NPs and their crude aqueous extract may be utilized to boost antimicrobial attributes.

Short term ciprofloxacin and clindamycin combination antibiotic therapy before and after transrectal ultrasound scan and prostate biopsy: Its impact on major components of gut microbiome.

The perturbation of gut microbiome is a risk factor for a number of adverse conditions. Among other factors antibiotic therapy is a common culprit. We characterized the short-term alteration of gut microbiome after antibiotic therapy. Nine patients (age (median [range]): 67 [57-75 years]) were subjected to prostate biopsy. Ciprofloxacin and clindamycin, 500 mg and 150 mg, respectively, were administered twice a day; this combination therapy was started the day before and continued until 5th and 8th day, respectively, following biopsy. 16s RNA sequencing data from fecal swabs taken before antibiotic therapy and 14 days after biopsy were analysed. At phylum level, the abundance of Actinobacteria and Firmicutes decreased, while that of Bacteroides and Proteobacteria increased after antibiotic therapy. The ratio of Firmicutes:Bacteroides inversed (from 2.81 to 0.74, p = 0.035). At order level, the abundance of Bacteroidales and Veillonellales increased, while that of Clostridiales and Coriobacteriales decreased. At genus level the abundance of Bacteroides increased, while those of Roseburia, Faecalibacterium and Collinsella decreased. These findings indicate that short-term antibiotic exposure skews gut microbiome composition. The current level of knowledge does not allow to decide whether this skewness is detrimental and has any long-term effect on disease including prostate pathology.

Reproductive Healthcare in Women with Rheumatoid Arthritis and Psoriatic Diseases in Routine Clinical Practice: Survey Results of Rheumatologists and Dermatologists.

INTRODUCTION: The proportion of women being treated with biologics is growing. However, data on treatment recommendation awareness among treating physicians and women who are considering pregnancy and family planning are limited. In this study, we used a questionnaire survey to learn how rheumatologists and dermatologists address women's needs for family planning, pregnancy, and breastfeeding, as well as their possible concerns with concurrent inflammatory rheumatic disease or psoriasis. METHODS: A 55-question (in English) survey aimed at identifying surveyed physicians' current practices regarding the reproductive health needs of women with rheumatoid arthritis, psoriasis, or psoriatic arthritis. This survey included 82 rheumatologists and 38 dermatologists from the Czech Republic, Hungary, and Slovakia. RESULTS: The proportion of female patients of reproductive age with the moderate-to-severe disease was 10-30% of all patients treated by the respondents. At the time of diagnosis, approximately two-thirds of the respondents discussed family planning with their patients. Rheumatologists collaborated with other specialists more frequently than dermatologists and gynecologist-obstetricians. Half of the rheumatologists revised systemic treatment 6 months before the patient planned to become pregnant, whereas dermatologists appear to act much sooner. Rheumatologists chose systemic glucocorticoids as the first-line treatment for pregnancy flares, whereas dermatologists chose topical corticosteroids. Congresses and interdisciplinary forums were rated the most valuable sources of information by physicians. CONCLUSIONS: There is a need for more holistic, multidisciplinary, collaborative, and integrated communication between clinicians and women of childbearing age. Physicians should consider the implications of these conditions and medical treatment for women of childbearing age and family planning for those with rheumatoid arthritis and psoriatic disease. Patient-centered care that includes patients' reproductive choices should be a routine clinical practice.

Physiological and transcriptional profiling of surfactin exerted antifungal effect against Candida albicans.

Given the risk of Candida albicans overgrowth in the gut, novel complementary therapies should be developed to reduce fungal dominancy. This study highlights the antifungal characteristics of a Bacillus subtilis-derived secondary metabolite, surfactin with high potential against C. albicans. Surfactin inhibited the growth of C. albicans following a 1-hour exposure, in addition to reduced adhesion and morphogenesis. Specifically, surfactin did not affect the level of reactive oxygen species but increased the level of reduced glutathione. Surprisingly, ethanol production was increased following 2 h of surfactin exposure. Surfactin treatment caused a significant reduction in intracellular iron, manganese and zinc content compared to control cells, whereas the level of copper was not affected. Alongside these physiological properties, surfactin also enhanced fluconazole efficacy. To gain detailed insights into the surfactin-related effects on C. albicans, genome-wide gene transcription analysis was performed. Surfactin treatment resulted in 1390 differentially expressed genes according to total transcriptome sequencing (RNA-Seq). Of these, 773 and 617 genes with at least a 1.5-fold increase or decrease in transcription, respectively, were selected for detailed investigation. Several genes involved in morphogenesis or related to metabolism (e.g., glycolysis, ethanol and fatty acid biosynthesis) were down-regulated. Moreover, surfactin decreased the expression of ERG1, ERG3, ERG9, ERG10 and ERG11 involved in ergosterol synthesis, whereas genes associated with ribosome biogenesis and iron metabolism and drug transport-related genes were up-regulated. Our data demonstrate that surfactin significantly influences the physiology and gene transcription of C. albicans, and could contribute to the development of a novel innovative complementary therapy.

Isolation and molecular characterization of a tomato brown rugose fruit virus mutant breaking the tobamovirus resistance found in wild Solanum species.

A new tobamovirus named tomato brown rugose fruit virus (ToBRFV) overcomes the effect of the Tm-1, Tm-2, and Tm-22 resistance genes introgressed from wild Solanum species into cultivated tomato (Solanum lycopersicum). Here, we report the isolation and molecular characterization of a spontaneous mutant of ToBRFV that breaks resistance in an unknown genetic background, demonstrated recently in Solanum habrochaites and Solanum peruvianum. The wild isolate ToBRFV-Tom2-Jo and the mutant ToBRFV-Tom2M-Jo were fully sequenced and compared to each other and to other ToBRFV sequences available in the NCBI GenBank database. Sequence analysis revealed five nucleotide substitutions in the ToBRFV-Tom2M-Jo genome compared to ToBRFV-Tom2-Jo. Two substitutions were located in the movement protein (MP) gene and resulted in amino acid changes in the 30-kDa MP (Phe22 → Asn and Tyr82 → Lys). These substitutions were not present in any of the previously described ToBRFV isolates. No amino acid changes were found in the 126-kDa and 183-kDa replicase proteins or the 17.5-kDa coat protein. Our data strongly suggest that breaking the newly discovered resistance in wild tomatoes is associated with one or two mutations on the MP gene of ToBRFV.

Topical Dosage Formulation of Lyophilized Philadelphus coronarius L. Leaf and Flower: Antimicrobial, Antioxidant and Anti-Inflammatory Assessment of the Plant.

Philadelphus coronarius is a versatile plant and its use in folk medicine has a long tradition; however, scientifically, the medical utilization of the herb is a less explored research field. The aim of our study was to identify and determine the quantity of the bioactive compounds of both the leaf and the flower and prepare a lyophilized product of them, from which medical ointments were formulated, since the topical application of P. coronarius has also not been studied. In vitro drug release, texture analysis and biocompatibility experiments were carried out, as well as the investigation of microbiological, antioxidant and anti-inflammatory properties. According to our results the composition and the selected excipients of the ointments have a great impact on the drug release, texture and bioavailability of the preparation. During the microbiological testing, the P. coronarius leaf was effective against Escherichia coli and Staphylococcus aureus, but it did not significantly decrease IL-4 production when it was tested on HaCaT cells. P. coronarius is a promising herb, and its topical application in antimicrobial therapy can be a useful addition to modern medical therapy.

Transcriptional Profiling of the Candida auris Response to Exogenous Farnesol Exposure.

The antifungal resistance threat posed by Candida auris necessitates bold and innovative therapeutic options. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment regimens. To gain further insights into the farnesol-related effect on C. auris, genome-wide gene transcription analysis was performed using transcriptome sequencing (RNA-Seq). Farnesol exposure resulted in 1,766 differentially expressed genes. Of these genes, 447 and 304 genes with at least 1.5-fold increase or decrease in transcription, respectively, were selected for further investigation. Genes involved in morphogenesis, biofilm events (maturation and dispersion), gluconeogenesis, iron metabolism, and regulation of RNA biosynthesis showed downregulation, whereas those related to antioxidative defense, transmembrane transport, glyoxylate cycle, fatty acid ß-oxidation, and peroxisome processes were upregulated. In addition, farnesol treatment increased the transcription of certain efflux pump genes, including MDR1, CDR1, and CDR2. Growth, measured by the change in the number of CFU, was significantly inhibited within 2 h of the addition of farnesol (5.8 × 107 ± 1.1 × 107 and 1.1 × 107 ± 0.3 × 107 CFU/ml for untreated control and farnesol-exposed cells, respectively) (P < 0.001). In addition, farnesol treatment caused a significant reduction in intracellular iron (152.2 ± 21.1 versus 116.0 ± 10.0 mg/kg), manganese (67.9 ± 5.1 versus 18.6 ± 1.8 mg/kg), and zinc (787.8 ± 22.2 versus 245.8 ± 34.4 mg/kg) (P < 0.05 to 0.001) compared to untreated control cells, whereas the level of cooper was significantly increased (274.6 ± 15.7 versus 828.8 ± 106.4 mg/kg) (P < 0.001). Our data demonstrate that farnesol significantly influences the growth, intracellular metal ion contents, and gene transcription related to fatty acid metabolism, which could open new directions in developing alternative therapies against C. auris. IMPORTANCE Candida auris is a dangerous fungal pathogen that causes outbreaks in health care facilities, with infections associated with a high mortality rate. As conventional antifungal drugs have limited effects against the majority of clinical isolates, new and innovative therapies are urgently needed. Farnesol is a key regulator molecule of fungal morphogenesis, inducing phenotypic adaptations and influencing biofilm formation as well as virulence. Alongside these physiological modulations, it has a potent antifungal effect alone or in combination with traditional antifungals, especially at supraphysiological concentrations. However, our knowledge about the mechanisms underlying this antifungal effect against C. auris is limited. This study has demonstrated that farnesol enhances the oxidative stress and reduces the fungal survival strategies. Furthermore, it inhibits manganese, zinc transport, and iron metabolism as well as increases fungal intracellular copper content. In addition, metabolism was modulated toward ß-oxidation. These results provide definitive explanations for the observed antifungal effects.

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312-0.5, 0.155-0.5, 0.037-0.375, 0.064-0.375, and 0.064-0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 µM2%, 2.16 µM2%, 33.31 µM2%, 10.72 µM2%, and 111.19 µM2% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.

Poor in vivo efficacy of caspofungin, micafungin and amphotericin B against wild-type Candida krusei clinical isolates does not correlate with in vitro susceptibility results.

We determined micafungin, caspofungin and amphotericin B (AMB) minimum inhibitory concentration (MICs) and killing rates in RPMI-1640 and in RPMI-1640 with 50% serum against three Candida krusei bloodstream isolates. MIC ranges in RPMI-1640 were 0.125-0.25, 0.25 and 0.125-0.5 mg/L, in RPMI-1640 with 50% serum, MICs were 64-128-, 8- and 4-16-fold higher, respectively. In RPMI-1640 micafungin and caspofungin at 1, 4, 16 and 32 mg/L as well as AMB at 2 mg/L were fungicidal against all isolates in ≤3.96, ≤4.42 and 14.96 h, respectively. In RPMI-1640 with 50% serum, caspofungin was fungicidal for all isolates only at 32 mg/L, micafungin and AMB were fungistatic. In neutropenic mice, 5 mg/kg caspofungin and 1 mg/kg AMB were ineffective against two of the three isolates. Thus, in vivo efficacy of echinocandins and AMB is weak or absent against C. krusei. Prescribers treating C. krusei infections with echinocandins should watch out for clinical resistance and therapeutic failure.

Identification of agents that reduce renal hypoxia-reoxygenation injury using cell-based screening: purine nucleosides are alternative energy sources in LLC-PK1 cells during hypoxia.

Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay.

Myocyte injury due to myocardial reperfusion injury plays a crucial role in the pathogenesis of acute myocardial infarction even after successful coronary revascularization. Identification of compounds that reduce reperfusion-associated myocyte death is important. Therefore, we developed an in vitro model of myocardial reperfusion injury in H9c2 rat cardiomyocytes and applied a cell-based high-throughput approach to screen a standard library of pharmacologically active compounds (LOPAC) in order to identify drugs with cardioprotective effects. Oxidative stress was induced with hydrogen peroxide (H2O2) treatment, which resulted in approximately 50% reduction in cell viability. Test compounds were added at a 3-microM final concentration as a pretreatment or in a delayed fashion (30 min after the peroxide challenge in order to imitate pharmacological treatment following angioplasty). Cells were cultured for 3 or 24 h. Viability was quantitated with the methylthiazolyldiphenyl-tetrazolium bromide method. Cytotoxicity and cytoprotection were also evaluated by measuring the lactate dehydrogenase activity in the cell culture supernatant. The screening identified a number of compounds with cytoprotective action, including molecules that are known to interfere with components of DNA repair and cell cycle progression, e.g. poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, and cyclin dependent kinase inhibitors, or reduce energy consumption by interfering with cardiac myofilament function. A number of dopamine D1 receptor agonists also provided significant cytoprotection at 3 h, but only three of them showed a similar effect at 24 h: chloro- and bromo-APB and chloro-PB hydrobromide. Chloro-APB hydrobromide significantly reduced peroxide-induced PARP activation in the myocytes independently of its action on dopamine D1 receptors, but lacked PARP inhibitor capacity in a cell-free PARP assay system. In conclusion, the pattern of cytoprotective drugs identified in the current assay supports the overall validity of our model system. The findings demonstrate that cytoprotective agents, including novel indirect inhibitors of cellular PARP activation can be identified with the method, chloro-APB hydrobromide being one such compound. The current experimental setting can be employed for cell-based high-throughput screening of various compound libraries.