Muscle Transcriptome Sequencing Revealed Thermal Stress-Responsive Regulatory Genes in Farmed Rohu, Labeo rohita (Hamilton, 1822).

Rohu, Labeo rohita, is one of the most important aquaculture species in the Indian subcontinent. Understanding the molecular-level physiological responses to thermal stress or climate change is essential. In the present work, transcriptome sequencing was carried out in the muscle tissue of the rohu in response to heat stress (35 °C) in comparison with the control (28 °C). A total of 125 Gb of sequence data was generated, and the raw-reads were filtered and trimmed, which resulted in 484 million quality reads. Reference-based assembly of reads was performed using L. rohita genome, and a total of 90.17% of reads were successfully mapped. A total of 37,462 contigs were assembled with an N50 value of 1854. The differential expression analysis revealed a total of 107 differentially expressed genes (DEGs) (15 up-, 37 down-, and 55 neutrally regulated) as compared to the control group (Log2FC > 2, P < 0.05). Gene enrichment analysis of DEGs indicates that transcripts were associated with molecular, biological, and cellular activities. The randomly selected differentially expressed transcripts were validated by RT-qPCR and found consistent expression patterns in line with the RNA-seq data. Several transcripts such as SERPINE1(HSP47), HSP70, HSP90alpha, Rano class II histocompatibility A beta, PGC-1 and ERR-induced regulator, proto-oncogene c-Fos, myozenin2, alpha-crystallin B chain-like protein, angiopoietin-like protein 8, and acetyl-CoA carboxylases have been identified in muscle tissue of rohu that are associated with stress/immunity. This study identified the key biomarker SERPINE1 (HSP47), which showed significant upregulation (~ 2- to threefold) in muscle tissue of rohu exposed to high temperature. This study can pave a path for the identification of stress-responsive biomarkers linked with thermal adaptations in the farmed carps.

DNA Methylation Profiling of Ovarian Tissue of Climbing Perch (Anabas testudienus) in Response to Monocrotophos Exposure.

Epigenetic modifications like DNA methylation can alter an organism's phenotype without changing its DNA sequence. Exposure to environmental toxicants has the potential to change the resilience of aquatic species. However, little information is available on the dynamics of DNA methylation in fish gonadal tissues in response to organophosphates. In the present work, reduced-representation bisulfite sequencing was performed to identify DNA methylation patterns in the ovarian tissues of Anabas testudienus exposed to organophosphates, specifically monocrotophos (MCP). Through sequencing, an average of 41,087 methylated cytosine sites were identified and distributed in different parts of genes, i.e., in transcription start sites (TSS), promoters, exons, etc. A total of 1058 and 1329 differentially methylated regions (DMRs) were detected as hyper-methylated and hypo-methylated in ovarian tissues, respectively. Utilizing whole-genome data of the climbing perch, the DMRs, and their associated overlapping genes revealed a total of 22 genes within exons, 45 genes at transcription start sites (TSS), and 218 genes in intergenic regions. Through gene ontology analysis, a total of 16 GO terms particularly involved in ovarian follicular development, response to oxidative stress, oocyte maturation, and multicellular organismal response to stress associated with reproductive biology were identified. After functional enrichment analysis, relevant DMGs such as steroid hormone biosynthesis (Cyp19a, 11-beta-HSD, 17-beta-HSD), hormone receptors (ar, esrrga), steroid metabolism (StAR), progesterone-mediated oocyte maturation (igf1ar, pgr), associated with ovarian development in climbing perch showed significant differential methylation patterns. The differentially methylated genes (DMGs) were subjected to analysis using real-time PCR, which demonstrated altered gene expression levels. This study revealed a molecular-level alteration in genes associated with ovarian development in response to chemical exposure. This work provides evidence for understanding the relationship between DNA methylation and gene regulation in response to chemicals that affect the reproductive fitness of aquatic animals.

Phytogenic silver nanoparticles from tissue-cultured Kaempferia angustifolia - an underutilized medicinal herb: a comparative antibacterial study on urinary pathogens.

BACKGROUND: Ethnomedicinally important Kaempferia angustifolia is a rhizomatous aromatic herb belonging to the family Zingiberaceae. The present manuscript deals with the green synthesis of silver nanoparticles through a rapid reduction process mediated by the rhizome extract of tissue culture-raised plants. The present study was conducted to evaluate the antimicrobial activity of the bio-nanoparticles, and the plant extracts themselves against seven multidrug-resistant urinary tract infecting (MDR-UTI) pathogens. RESULT: The ethanolic extracts of the rhizomes of the plant executed a very rapid synthesis of silver bio-nanoparticles, and the generation of the nanoparticles was confirmed through UV-vis spectrophotometry, dynamic light scattering (DLS), and electron dispersion spectroscopic (EDS) analysis. Finally, the precise shapes and dimensions of these nanoparticles were confirmed under the transmission electron microscope (TEM). The shapes of the nanoparticles obtained were diverse in nature and varied from rod, triangular, spherical, to oval shaped, with the size, ranging from 10-60 nm. Silver nanoparticles exhibited a maximum zone of inhibition (ZI) of 16.93 ± 0.04 mm against isolate no. 42332. The ex vitro and in vivo extracts exhibited ZI 14.03 ± 0.04 mm and 11.56 ± 0.04 mm, respectively, against the same strain, which are comparatively lower than the nanoparticles but unignorable. CONCLUSION: Although the pathogens used in the present study are resistant to at least three or more types of pharmacologically important antibiotics, nanoparticles, as well as the plant extracts, exhibited significant inhibition to all the seven MDR-UTI pathogens, which confirms that they are highly antimicrobic. Hence, this underutilized medicinal plant extracts of K. angustifolia and the bio-nanoparticles synthesized from these can be explored in pharmaceutical industries to treat multidrug-resistant human pathogenic bacteria. Furthermore, their broad-spectrum activity leads to the opportunity for the synthesis of future generation drugs.

Silver nanoparticle synthesis and their potency against multidrug-resistant bacteria: a green approach from tissue-cultured Coleus forskohlii.

Drug resistance is a major concern nowadays, and finding alternatives of the well-known antibiotic is necessary. Green nanoparticles are emerging as a tenable alternative to this with a large spectrum of activity. The present manuscript describes an eco-friendly approach for green synthesis of silver nanoparticles from both in vitro and in vivo leaf extract of Coleus forskohlii. Leaf extracts were used in synthesis of nanoparticles which were further analyzed through UV-Vis, dynamic light scattering, energy-dispersive spectroscopy, and transmission electron microscopy. Antimicrobial activity of silver nanoparticles alone, as well as crude extract of the plant itself, was carried out against eight multidrug-resistant respiratory tract infecting pathogenic strains. Satisfactory antimicrobial activities were found with nanoparticles, in vitro and in vivo leaf extracts. However, gradually higher to lower inhibition potential against pathogenic bacterial strains was found in silver nanoparticles, in vitro and in vivo leaf extracts. Seven bioactive compounds were detected in the crude extract through gas chromatography-mass spectroscopy analysis. Results revealed that nanoparticle formation occurred in a wide range of sizes (10-50 nm) and shapes (trigonal, hexagonal, spherical, rod). The diversity in size and shape of the nanoparticles makes them biologically active. Silver nanoparticle exhibits significantly better antimicrobial activities as compared to the plant extract in case of nearly all pathogens with a maximum zone of inhibition of 15.33 ± 0.94 mm where more than 12 well-known antibiotics failed to respond. Because of this broad-spectrum activity of nanoparticles as well as the leaf extracts against life-threatening microbes, it can be used as future generation drugs.

Exposure to sub-inhibitory concentrations of gentamicin, ciprofloxacin and cefotaxime induces multidrug resistance and reactive oxygen species generation in meticillin-sensitive Staphylococcus aureus.

PURPOSE: The role of antibiotics below their MIC in the development of bacterial drug resistance is becoming increasingly important. We investigated the effect of sub-MICs of bactericidal antibiotics on the susceptibility pattern of Staphylococcus aureus and evaluated the role of free radicals. METHODOLOGY: A total of 12 S. aureus strains were recovered from pus samples and their antibiograms determined. The test isolates were treated with sub-MIC levels of tetracycline, gentamicin, ciprofloxacin and cefotaxime. Alterations in their respective breakpoints were observed along with measurements of free radical generation by nitro blue tetrazolium test.Results/Key findings. Gentamicin, ciprofloxacin and cefotaxime exposure significantly altered the breakpoints of exposed isolates against several tested antibiotics and higher levels of free radicals were generated after antibiotic exposure. CONCLUSIONS: Our study demonstrates that sub-MIC levels of antimicrobials can lead to resistance and cross-resistance across several classes of antibiotics in wild strains of S. aureus, possibly by free radical production. The molecular mechanisms behind the acquisition of drug resistance at low antibiotic concentrations and the specific target genes of reactive oxygen speciesneed to be explored further.

Polyphenolic Secondary Metabolites Synergize the Activity of Commercial Antibiotics against Clinical Isolates of ß-Lactamase-producing Klebsiella pneumoniae.

Emergence of worldwide antimicrobial resistance prompted us to study the resistance modifying potential of plant-derived dietary polyphenols, mainly caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin. These compounds were studied in logical combination with clinically significant antibiotics (ciprofloxacin/gentamicin/tetracycline) against Klebsiella pneumoniae, after conducting phenotypic screening of a large number of clinical isolates and selecting the relevant strains possessing extended-spectrum ß-lactamase (ESBL) and K. pneumoniae carbapenemase (KPC)-type carbapenemase enzymes only. The study demonstrated that EGCG and caffeic acid could synergize the activity of tested antibiotics within a major population of ß-lactamase-producing K. pneumoniae. In spectrofluorimetric assay, ~17-fold greater ciprofloxacin accumulation was observed within K. pneumoniae cells pre-treated with EGCG in comparison with the untreated control, indicating its ability to synergize ciprofloxacin to restrain active drug-efflux. Further, electron micrograph of ESBL-producing K. pneumoniae clearly demonstrated the prospective efficacy of EGCG towards biofilm degradation.

Antimicrobial activity of pomegranate fruit constituents against drug-resistant Mycobacterium tuberculosis and ß-lactamase producing Klebsiella pneumoniae.

CONTEXT: The global surge in multi-drug resistant bacteria and the imminence of tuberculosis pandemic necessitate alternative therapeutic approaches to augment the existing medications. Pomegranate, the fruit of Punica granatum Linn. (Punicaceae), widely recognized for potency against a broad spectrum of bacterial pathogens, deserves further investigation in this respect. OBJECTIVE: This study determines the therapeutic potential of pomegranate juice, extracts of non-edible peel prepared with methanol/water, and its four polyphenolic constituents, namely caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin, against drug-resistant clinical isolates. MATERIALS AND METHODS: Phenotypic characterisation of Mycobacterium tuberculosis, extended-spectrum ß-lactamase (ESBL) and KPC-type carbapenemase producing Klebsiella pneumoniae was performed by biochemical and molecular methods. Resistance profiles of M. tuberculosis and K. pneumoniae were determined using LJ proportion and Kirby-Bauer methods, respectively. Pomegranate fruit extracts, and the compounds, were evaluated at a dose range of 1024-0.5 µg/mL, and 512-0.25 µg/mL, respectively, to determine minimum inhibitory (MIC) and bactericidal concentrations (MBC) against the drug-resistant isolates by the broth micro-dilution method. RESULTS: The peel extracts exhibited greater antimycobacterial activity (MIC 64-1024 µg/mL) than the potable juice (MIC 256 - > 1024 µg/mL). EGCG and quercetin exhibited higher antitubercular (MIC 32-256 µg/mL) and antibacterial (MIC 64-56 µg/mL) potencies than caffeic acid and ellagic acid (MIC 64-512 µg/mL). DISCUSSION AND CONCLUSION: The pomegranate fruit peel and pure constituents were active against a broad panel of M. tuberculosis and ß-lactamase producing K. pneumoniae isolates. EGCG and quercetin need further investigation for prospective application against respiratory infections.

Antitubercular and antibacterial activity of quinonoid natural products against multi-drug resistant clinical isolates.

Multi-drug resistant Mycobacterium tuberculosis and other bacterial pathogens represent a major threat to human health. In view of the critical need to augment the current drug regime, we have investigated therapeutic potential of five quinonoids, viz. emodin, diospyrin, plumbagin, menadione and thymoquinone, derived from natural products. The antimicrobial activity of quinonoids was evaluated against a broad panel of multi-drug and extensively drug-resistant tuberculosis (M/XDR-TB) strains, rapid growing mycobacteria and other bacterial isolates, some of which were producers of ß-lactamase, Extended-spectrum ß-lactamase (ESBL), AmpC ß-lactamase, metallo-beta-lactamase (MBL) enzymes, as well as their drug-sensitive ATCC counterparts. All the tested quinones exhibited antimycobacterial and broad spectrum antibacterial activity, particularly against M. tuberculosis (lowest MIC 0.25 µg/mL) and Gram-positive bacteria (lowest MIC <4 µg/mL) of clinical origin. The order of antitubercular activity of the tested quinonoids was plumbagin > emodin ~ menadione ~ thymoquinone > diospyrin, whereas their antibacterial efficacy was plumbagin > menadione ~ thymoquinone > diospyrin > emodin. Furthermore, this is the first evaluation performed on these quinonoids against a broad panel of drug-resistant and drug-sensitive clinical isolates, to the best of our knowledge.

Pomegranate pericarp extract enhances the antibacterial activity of ciprofloxacin against extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL) producing Gram-negative bacilli.

A methanolic extract of Punica granatum (pomegranate) fruit pericarp (PGME) was tested in combination with ciprofloxacin against extended-spectrum ß-lactamase (ESBL) producing Escherichia coli, Klebsiella pneumoniae, and metallo-ß-lactamase (MBL) producing Pseudomonas aeruginosa, which were screened for their resistance profile against fluoroquinolone antibiotics. The minimum inhibitory concentrations (MIC) of ciprofloxacin and PGME, alone, were determined, and synergy of ciprofloxacin-PGME combinations evaluated by checkerboard assay and fractional inhibitory concentration (FIC). Nineteen out of forty-nine strains exhibited synergy with ciprofloxacin (FIC of 0.125-0.5 for ciprofloxacin) further verified by agar-well assay. This could be due to the bacterial efflux pump inhibitor (EPI) activity of the polyphenolic constituents of PGME. However, the isolates exhibiting a high level of ciprofloxacin resistance did not respond to ciprofloxacin-PGME combinations, which could be due to target site modification not influenced further by EPI activity of PGME. Again, some strains were sensitive or weakly resistant to ciprofloxacin, which exhibited 'indifference' to the combination, probably due to a lack of over-expressed efflux mechanism. Thus, a synergy of a ciprofloxacin-PGME combination was demonstrated for the first time against ESBL- and MBL-producing Gram-negative bacilli, and the efficacy of an existing drug improved with the help of an inexpensive alternative therapy.