Bioactive Compounds, Antioxidant, and Antibacterial Activity Against MDR and Food-Borne Pathogenic Bacteria of Psidium guajava. L Fruit During Ripening.

Psidium guajava fruits are highly appreciated for their nutrients and bioactive compounds content, which contribute to their antioxidant and antimicrobial capacities. The purpose of this study was to determine bioactive compound (phenolic, flavonoids, and carotenoid contents), antioxidant activity (DPPH, ABTS, ORAC, and FRAP), and antibacterial potential against MDR and food-borne pathogenic strains of Escherichia coli, and Staphylococcus aureus during different stages of fruit ripening.The results elucidated that ripe fruits (methanolic extract) contain the highest total phenolic, flavonoids, and carotenoid contents (417.36 ± 2.63 µg GAE/gm of FW, 711.78 ± 0.70 µg QE/gm of FW and 0.683 ± 0.06 µg/gm of FW) followed by hexane, ethyl acetate, and aqueous. Methanolic extract of the ripe fruits showed the highest antioxidant activity when measured by DPPH (61.55 ± 0.91%), FRAP (31.83 ± 0.98 mM Fe(II)/gm of FW), ORAC (17.19 ± 0.47 mM TE/ gm of FW), and ABTS (41.31 ± 0.99 µmol Trolox/gm of FW) assays. In the antibacterial assay, the ripe stage had the highest antibacterial activity against MDR and food-borne pathogenic strains of Escherichia coli, and Staphylococcus aureus. The methanolic ripe extract was found to possess maximum antibacterial activity ZOI, MIC, and IC50 18.00 ± 1.00 mm, 95.95 ± 0.05%, and 0.58 µg/ml; 15.66 ± 0.57 mm, 94.66 ± 0.19%, and 0.50 µg/ml, respectively, against pathogenic and MDR strains of E. coli and 22.33 ± 0.57 mm, 98.97 ± 0.02%, and 0.26 µg/ml; 20.33 ± 1.15 mm, 96.82 ± 0.14%, and 0.39 µg/ml, respectively, against pathogenic and MDR strains of S. aureus. Considering the bioactive compounds and beneficial effects, these fruit extracts could be promising antibiotic alternatives, avoiding antibiotic overuse and its negative effects on human health and the environment, and can be recommended as a novel functional food.

ACC Deaminase Produced by PGPR Mitigates the Adverse Effect of Osmotic and Salinity Stresses in Pisum sativum through Modulating the Antioxidants Activities.

Salinity-induced ethylene production and reactive oxygen species (ROS) inhibit agricultural productivity. The plant synthesizes ethylene directly from aminocyclopropane-1-carboxylic acid (ACC). By using ACC as a nitrogen source, bacteria with ACC deaminase (ACCD) inhibit the overproduction of ethylene, thereby maintaining the ROS. The present study investigated the ACCD activity of previously identified rhizobacterial strains in Dworkin and Foster (DF) minimal salt media supplemented with 5 mM ACC (as N-source). Bacterial isolates GKP KS2_7 (Pseudomonas aeruginosa) and MBD 133 (Bacillus subtilis) could degrade ACC into α-ketobutyrate, exhibiting ACCD activity producing more than ~257 nmol of α-ketobutyrate mg protein−1 h−1, and were evaluated for other plant growth-promoting (PGP) traits including indole acetic acid production (>63 µg/mL), phosphate solubilization (>86 µg mL−1), siderophore (>20%) ammonia and exopolysaccharide production. Furthermore, Fourier Transform Infrared analysis also demonstrated α-ketobutyrate liberation from ACC deamination in DF minimal salt media, thereby confirming the ACCD activity. These isolates also showed enhanced tolerance to salinity stress of 3% w/v NaCl in vitro, in addition to facilitating multifarious PGP activities. Seed bacterization by these ACCD-producing bacterial isolates (GKP KS2_7 and MBD 133) revealed a significant decline in stress-stimulated ethylene levels and its associated growth inhibition during seedling germination. They also mitigated the negative effects of salt stress and increased the root-shoot length, fresh and dry weight of root and shoot, root-shoot biomass, total sugar, protein, reducing sugar, chlorophyll content, and antioxidants enzymes in Pisum sativum. As a result, these strains (GKP KS2_7 and MBD 133) might be applied as biofertilizers to counteract the negative effects of soil salinity.

Mechanistic Insights of Plant Growth Promoting Bacteria Mediated Drought and Salt Stress Tolerance in Plants for Sustainable Agriculture.

Climate change has devastating effects on plant growth and yield. During ontogenesis, plants are subjected to a variety of abiotic stresses, including drought and salinity, affecting the crop loss (20-50%) and making them vulnerable in terms of survival. These stresses lead to the excessive production of reactive oxygen species (ROS) that damage nucleic acid, proteins, and lipids. Plant growth-promoting bacteria (PGPB) have remarkable capabilities in combating drought and salinity stress and improving plant growth, which enhances the crop productivity and contributes to food security. PGPB inoculation under abiotic stresses promotes plant growth through several modes of actions, such as the production of phytohormones, 1-aminocyclopropane-1-carboxylic acid deaminase, exopolysaccharide, siderophore, hydrogen cyanide, extracellular polymeric substances, volatile organic compounds, modulate antioxidants defense machinery, and abscisic acid, thereby preventing oxidative stress. These bacteria also provide osmotic balance; maintain ion homeostasis; and induce drought and salt-responsive genes, metabolic reprogramming, provide transcriptional changes in ion transporter genes, etc. Therefore, in this review, we summarize the effects of PGPB on drought and salinity stress to mitigate its detrimental effects. Furthermore, we also discuss the mechanistic insights of PGPB towards drought and salinity stress tolerance for sustainable agriculture.

ACC deaminase producing plant growth promoting rhizobacteria enhance salinity stress tolerance in Pisum sativum.

Salinity stress is one of the most serious environmental stresses which limit plant growth, development and productivity. In this study, we screened 25 bacterial isolates based on the biochemical activity of ACC deaminase. Two potent PGPR namely Bacillus marisflavi (CHR JH 203) and Bacillus cereus (BST YS1_42) having the highest ACC deaminase (ACCD) activity were selected for further analyses such as polymerase chain reaction (PCR), salt tolerance assay, expression analysis, antioxidant assay, etc. The structural gene for ACCD activity was further confirmed by PCR showing the amplicon size ~ 800 bp. The acdS positive isolates exhibited optimum growth at 3% w/v (NaCl), indicating its ability to survive and thrive in induced saline soil. Inoculation of acdS + strain on pea plants was found to be efficient and ameliorated the induced NaCl-stress by enhancing the various parameters like plant-biomass, carbohydrates, reducing sugars, protein, chlorophylls, phenol, flavonoids content and increasing antioxidants enzymes levels in plants. Moreover, the expression of ROS scavenging genes (PsSOD, PsCAT, PsPOX, PsNOS, PsAPX, PsChla/bBP), defense genes and cell rescue genes (PsPRP, PsMAPK, PsFDH) were analyzed. Inoculated plants exhibited a higher gene expression level and salt tolerance under 1%NaCl concentration. Thus, our results indicate that CHR JH 203 and BST YS1_42 strain showed the highest plant growth-promoting attributes could be used as bio-inoculants for crops under saline stress in the field towards sustainable crop development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03047-5.

Targeting progesterone metabolism in breast cancer with l-proline derived new 14-azasteroids.

Breast cancer cell proliferation is promoted by a variety of mitogenic signals. Classically estrogen is considered as most predominant mitogenic signal in hormone-dependent breast cancer and progesterone is primarily considered to have protective effect. However, it is suggested that some progesterone metabolite may promote breast cancer and progesterone metabolites like 5α-pregnane and 4-pregnene could serve as regulators of estrogen-responsiveness of breast cancer cells. Here, we estimated the potential of alternate targeting of breast cancer via progesterone signalling. l-Proline derived novel 14-azasteroid compounds were screened against MCF-7 and MDA-MB-231 cell lines using MTT assay. In silico studies, cell cycle, Annexin-V-FITC/PI, JC-1 mitochondrial assay, ROS analysis were performed to analyse the impact of hit compound 3b on breast cancer cells. Further, we analysed the impact of hit 3b on the progesterone, its metabolites and enzymes responsible for the conversion of progesterone and its metabolites using ELISA. Data suggests that compound 3b binds and down regulates of 5α-reductase by specifically inhibiting production of progesterone metabolites that are capable of promoting breast cancer proliferation, epithelial mesenchymal transition and migration. This study establishes the proof of concept and generation of new leads for additional targeting of breast cancer.

Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

BACKGROUND: In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates. CONCLUSIONS/SIGNIFICANCE: Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.

Leishmania donovani trypanothione reductase: role of urea and guanidine hydrochloride in modulation of functional and structural properties.

Trypanothione reductase [TR], an NADPH-dependent disulfide oxidoreductase, unique to kinetoplastid parasites including Trypanosoma and Leishmania, is a validated target for the design of improved drugs. TR is a stable homodimer with a FAD molecule tightly bound to each subunit. In this paper, structure, function, stability properties and cofactor protein interactions of recombinant TR from Leishmania donovani were investigated under equilibrium unfolding/denaturing conditions. Urea induced unfolding was non-reductive in nature and led to the formation of partially folded intermediate. This intermediate species lacks catalytic activity and characteristic conformation of native LdTR but has significant secondary structure and could be partially reactivated. Guanidine hydrochloride-induced irreversible denaturation was marked by the presence of molten globule intermediate. Reactivation and cross-linking experiments clearly demonstrated that the loss of activity at lower denaturant concentrations was not coincided by dimer dissociation or structural unfolding. The studies demonstrate that functional conformation and stability are largely governed by ionic interactions and active site disulfide plays a vital role in maintaining functional conformation. The results obtained from this study provide intriguing insight into the possible mechanism/s of modulation of structure, function and stability of LdTR induced by the cationic, guanidine hydrochloride and the neutral denaturant, urea.

Synthesis of covalently linked unsymmetrical porphyrin pentads containing three different porphyrin subunits.

The tetrafunctionalized AB3-type porphyrin building blocks containing two different types of functional groups with N4, N3O, N3S, and N2S2 porphyrin cores were synthesized by following various synthetic routes. The AB3-type tetrafunctionalized N4 porphyrin building block was synthesized by a mixed condensation approach, the N3S and N3O porphyrin building blocks by a mono-ol method, and N2S2 porphyrin building block by an unsymmetrical diol method. The tetrafunctionalized porphyrin building blocks were used to synthesize monofunctionalized porphyrin tetrads containing two different types of porphyrin subunits by coupling of 1 equiv of tetrafunctionalized N4, N3O, N3S, and N2S2 porphyrin building block with 3 equiv of monofunctionalized ZnN4 porphyrin building block under mild copper-free Pd(0) coupling conditions. The monofunctionalized porphyrin tetrads were used further to synthesize unsymmetrical porphyrin pentads containing three different types of porphyrin subunits by coupling 1 equiv of monofunctionalized porphyrin tetrad with 1 equiv of monofunctionalized N2S2 porphyrin building blocks under the same mild Pd(0) coupling conditions. The NMR, absorption, and electrochemical studies on porphyrin tetrads and porphyrin pentads indicated that the monomeric porphyrin subunits in tetrads and pentads retain their individual characteristic features and exhibit weak interaction among the porphyrin subunits. The steady state and time-resolved fluorescence studies support an efficient energy transfer from donor porphyrin subunit to acceptor porphyrin subunit in unsymmetrical porphyrin tetrads and porphyrin pentads.

Characterization of natural antimony resistance in Leishmania donovani isolates.

Clinical resistance to pentavalent antimonial compounds has long been recognized as a major problem in the treatment of visceral leishmaniasis in India. However, mechanisms of natural resistance are unclear. In this study, we observed that Leishmania donovani clinical isolates not responsive to sodium stibogluconate showed resistance to antimony treatment in both in vitro and in vivo laboratory conditions. The resistant isolates have increased levels of intracellular thiols. This increase in thiol levels was not mediated by the amplification of gamma-glutamylcysteine synthetase, but was accompanied by amplification of trypanothione reductase and an intracellular ATP-binding cassette transporter gene MRPA. The resistance of parasites to antimony could be reversed by the glutathione biosynthesis-specific inhibitor, buthionine sulfoximine, which resulted in increased drug susceptibility. These results suggest the possible role of thiols and MRPA in antimony resistance in field isolates.