Repurposing of dibucaine and niflumic acid as antimicrobial agents in combination with antibiotics against Staphylococcus aureus.

The versatile human commensal bacteria and pathogen Staphylococcus aureus cause several community and hospital-acquired illnesses associated with significant morbidity and death. Antibiotic therapy for S. aureus infections has grown increasingly difficult as the organism has developed a wide spectrum of antibiotic resistance mechanisms. This situation emphasizes the significance of developing and advocating new antimicrobials for preventative and therapeutic measures. Our study aimed to identify and evaluate new therapeutic options against S. aureus. We investigated the efficacy of two drugs, dibucaine, and niflumic acid, as potential adjuvant for anti-staphylococcal therapeutics. Dibucaine and niflumic acid found to have bactericidal activity against S. aureus. These drugs acted synergistically with antibiotics reducing the required dose of antibiotics up to 4 times. In combination with antibiotics, they were effectively and synergistically inhibited the formation of biofilms of S. aureus. The best synergistic partner of dibucaine was with kanamycin and tetracycline, whereas niflumic acid was with streptomycin and ampicillin. Both the drugs showed significant efflux inhibition in the bacteria. Moreover, the drugs are found to be safe at synergistic doses. Our findings suggest that dibucaine and niflumic acid could be potential adjuvant with antibiotics for the treatment of S. aureus infections. Their ability to significantly enhance the efficacy of antibiotics highlights their potential clinical significance as adjunct therapies.

Insights into the identification and evolutionary conservation of key genes in the transcriptional circuits of meiosis initiation and commitment in budding yeast.

Initiation of meiosis in budding yeast does not commit the cells for meiosis. Thus, two distinct signaling cascades may differentially regulate meiosis initiation and commitment in budding yeast. To distinguish between the role of these signaling cascades, we reconstructed protein-protein interaction networks and gene regulatory networks with upregulated genes in meiosis initiation and commitment. Analyzing the integrated networks, we identified four master regulators (MRs) [Ume6p, Msn2p, Met31p, Ino2p], three transcription factors (TFs), and 279 target genes (TGs) unique for meiosis initiation, and three MRs [Ndt80p, Aro80p, Rds2p], 11 TFs, and 948 TGs unique for meiosis commitment. Functional enrichment analysis of these distinct members from the transcriptional cascades for meiosis initiation and commitment revealed that nutritional cues rewire gene expression for initiating meiosis and chromosomal recombination commits cells to meiosis. As meiotic chromosomal recombination is highly conserved in eukaryotes, we compared the evolutionary rate of unique members in the transcriptional cascade of two meiotic phases of Saccharomyces cerevisiae with members of the phylum Ascomycota, revealing that the transcriptional cascade governing chromosomal recombination during meiosis commitment has experienced greater purifying selection pressure (P value = 0.0013, 0.0382, 0.0448, 0.0369, 0.02967, 0.04937, 0.03046, 0.03357 and < 0.00001 for Ashbya gossypii, Yarrowia lipolytica, Debaryomyces hansenii, Aspergillus fumigatus, Neurospora crassa, Kluyveromyces lactis, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, and Schizosaccharomyces octosporus, respectively). This study demarcates crucial players driving meiosis initiation and commitment and demonstrates their differential rate of evolution in budding yeast.

A systematic drug repurposing approach to identify promising inhibitors from FDA-approved drugs against Nsp4 protein of SARS-CoV-2.

COVID-19 is caused by SARS-CoV-2 and responsible for the ongoing global pandemic in the world. After more than a year, we are still in lurch to combat and control the situation. Therefore, new therapeutic options to control the ongoing COVID-19 are urgently in need. In our study, we found that nonstructural protein 4 (Nsp4) of SARS-CoV-2 could be a potential target for drug repurposing. Due to availability of only the crystal structure of C-terminal domain of Nsp4 (Ct-Nsp4) and its crucial participation in viral RNA synthesis, we have chosen Ct-Nsp4 as a target for screening the 1600 FDA-approved drugs using molecular docking. Top 102 drugs were found to have the binding energy equal or less than -7.0 kcal/mol. Eribulin and Suvorexant were identified as the two most promising drug molecules based on the docking score. The dynamics of Ct-Nsp4-drug binding was monitored using 100 ns molecular dynamics simulations. From binding free energy calculation over the simulation, both the drugs were found to have considerable binding energy. The present study has identified Eribulin and Suvorexant as promising drug candidates. This finding will be helpful to accelerate the drug discovery process against COVID-19 disease.Communicated by Ramaswamy H. Sarma.

CRISPR/Cas-Based Biosensor As a New Age Detection Method for Pathogenic Bacteria.

Methods enabling rapid and on-site detection of pathogenic bacteria are a prerequisite for public health assurance, medical diagnostics, ensuring food safety and security, and research. Many current bacteria detection technologies are inconvenient and time-consuming, making them unsuitable for field detection. New technology based on the CRISPR/Cas system has the potential to fill the existing gaps in detection. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a part of the bacterial adaptive immune system to protect them from intruding bacteriophages. The immunological memory is saved by the CRISPR array of bacteria in the form of short DNA sequences (spacers) from invading viruses and incorporated with the CRISPR DNA repeats. Cas proteins are responsible for triggering and initiating the adaptive immune function of CRISPR/Cas systems. In advanced biological research, the CRISPR/Cas system has emerged as a significant tool from genome editing to pathogen detection. By considering its sensitivity and specificity, this system can become one of the leading detection methods for targeting DNA/RNA. This technique is well applied in virus detection like Dengue, ZIKA, SARS-CoV-2, etc., but for bacterial detection, this CRISPR/Cas system is limited to only a few organisms to date. In this review, we have discussed the different techniques based on the CRISPR/Cas system that have been developed for the detection of various pathogenic bacteria like L. monocytogenes, M. tuberculosis, Methicillin-resistant S. aureus, Salmonella, E. coli, P. aeruginosa, and A. baumannii.

FSH mediated cAMP signalling upregulates the expression of Gα subunits in pubertal rat Sertoli cells.

Follicle Stimulating Hormone (FSH) acts via FSH-Receptor (FSH-R) by employing cAMP as the dominant secondary messenger in testicular Sertoli cells (Sc) to support spermatogenesis. Binding of FSH to FSH-R, results the recruitment of the intracellular GTP binding proteins, either stimulatory Gαs or inhibitory Gαi that in turn regulate cAMP production in Sc. The cytosolic concentration of cAMP being generated by FSH-R thereafter critically determines the downstream fate of the FSH signalling. The pleiotropic action of FSH due to differential cAMP output during functional maturation of Sc has been well studied. However, the developmental and cellular regulation of the Gα proteins associated with FSH-R is poorly understood in Sc. In the present study, we report the differential transcriptional modulation of the Gα subunit genes by FSH mediated cAMP signalling in neonatal and pubertal rat Sc. Our data suggested that unlike in neonatal Sc, both the basal and FSH/forskolin induced expression of Gαs, Gαi-1, Gαi-2 and Gαi-3 transcripts was significantly (p < 0.05) up-regulated in pubertal Sc. Further investigations involving treatment of Sc with selective Gαi inhibitor pertussis toxin, confirmed the elevated expression of Gi subunits in pubertal Sc. Collectively our results indicated that the high level of Gαi subunits serves as a negative regulator to optimize cAMP production in pubertal Sc.

In silico identification of key regulators instigating the pre-meiotic phase during respiration in Saccharomyces cerevisiae.

Like higher eukaryotes, diploid MATa/MATα budding yeasts can undergo both mitosis and meiosis. Although the potential reason for their phase switching is elucidated by two consecutive processes, i.e. transition from fermentation (mitotic growth) to respiration in glucose-deficient media and then complete shift to meiotic phase in combined nitrogen- and glucose-starved media, the genomic interactions and regulatory cascade operating this drive remain elusive. Here, we aim to explore the regulatory cross-talk that mediates the phase transition. We have hypothesized that pre-growth in glucose-starved condition (yeast extract-peptone-acetate media) not only causes switch from fermentation to respiration but also prepares them for meiosis via a myriad of signaling events regulated by transcription factors (TFs). We have identified 23 putative TFs from integrated protein-protein interaction and gene regulatory network that were reconstructed from predicted and experimentally validated data. A total of six TFs (Xbp1p, Abf1p, Cbf1p, Ste12p, Reb1p and Gcn4p) are found to be highly connected in the network and involved in the cross-talk between respiration and cellular preparation for meiosis. We have identified Abf1p and Adr1p as the master regulators of the integrated network. This study in yeast will help to decipher the pre-meiotic initiation that occurs in higher eukaryotes.

Testosterone augments FSH signaling by upregulating the expression and activity of FSH-Receptor in Pubertal Primate Sertoli cells.

The synergistic actions of Testosterone (T) and FSH via testicular Sertoli cells (Sc) regulate male fertility. We have previously reported that the actions of these hormones (T and FSH) in infant monkey testes are restricted only to the expansion of Sc and spermatogonial cells. The robust differentiation of male Germ cells (Gc) occurs after pubertal maturation of testis. The present study was aimed to investigate the molecular basis of the synergy between T and FSH action in pubertal primate (Macaca mulatta) Sc. Using primary Sc culture, we here have demonstrated that T (but not FSH) downregulated AMH and Inhibin-ß-B (INHBB) mRNAs in pubertal Sc. We also found that, prolonged stimulation of T in pubertal Sc significantly elevated the expression of genes involved in FSH signaling pathway like FSH-Receptor (FSHR), GNAS and RIC8B, and this was associated with a rise in cAMP production. T also augmented FSH induced expression of genes like SCF, GDNF, ABP and Transferrin (TF) in pubertal Sc. We therefore conclude that T acts in synergy with FSH signaling in pubertal Sc. Such a coordinated network of hormonal signaling in Sc may facilitate the timely onset of the first spermatogenic wave in pubertal primates and is responsible for quantitatively and qualitatively normal spermatogenesis.

Advantages of pulsatile hormone treatment for assessing hormone-induced gene expression by cultured rat Sertoli cells.

In response to various hormonal (follicle-stimulating hormone [FSH] and testosterone [T]) and biochemical inputs, testicular Sertoli cells (Sc) produce factors that regulate spermatogenesis. A number of FSH- and T-responsive Sc-specific genes, necessary for spermatogenesis, have been identified to date. However, the hormone-induced in vitro expression pattern of most of these genes is reported to be inconsistent at various time points in primary rat Sc cultures. As a matter of convenience, cultured Sc are constantly exposed to hormones for a few hours to days in the reported literature, although Sc are exposed to pulsatile FSH and T in vivo. The major aim of the present study is to evaluate the advantage, if any, of the in vitro administration of pulsatile hormone (FSH and T in combination) treatment on gene expression of cultured Sc as compared with that of constant hormone treatment. Pulsatile treatment (a 30-min hormonal exposure every 3 h) mimicking the in vivo condition reveals a more prominent effect of hormones in augmenting gene expression as compared with constant treatment. Our results indicate that the expressions of Stem cell factor (Scf, only responsive to FSH), Claudin11 (only responsive to T) and Transferrin (both FSH- and T-responsive) mRNAs are significantly higher at 12 h upon pulsatile treatment than upon constant hormonal treatment. Maximal expression of relevant genes because of pulsatile treatment with hormones suggests that this protocol provides a more suitable premise for assessing hormone-induced gene expression in isolated Sc than one involving constant exposure to hormones.

Robust generation of transgenic mice by simple hypotonic solution mediated delivery of transgene in testicular germ cells.

Our ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools, but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious, and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

A Study of Differential Expression of Testicular Genes in Various Reproductive Phases of Hemidactylus flaviviridis (Wall Lizard) to Derive Their Association with Onset of Spermatogenesis and Its Relevance to Mammals.

Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.

Low levels of Gαs and Ric8b in testicular sertoli cells may underlie restricted FSH action during infancy in primates.

FSH acts via testicular Sertoli cells (Sc) bearing FSH receptor (FSH-R) for regulating male fertility. Despite an adult-like FSH milieu in infant boys and monkeys, spermatogenesis is not initiated until the onset of puberty. We used infant and pubertal monkey Sc to reveal the molecular basis underlying developmental differences of FSH-R signaling in them. Unlike pubertal Sc, increasing doses of FSH failed to augment cAMP production by infant Sc. The expression of Gαs subunit and Ric8b, which collectively activate adenylyl cyclase (AC) for augmenting cAMP production and gene transcription, were significantly low in infant Sc. However, forskolin, which acts directly on AC bypassing FSH-R, augmented cAMP production and gene transcription uniformly in both infant and pubertal Sc. FSH-induced Gαs mRNA expression was higher in pubertal Sc. However, Gαi-2 expression was down-regulated by FSH in pubertal Sc, unlike infant Sc. FSH failed, but forskolin or 8-Bromoadenosine 3',5'-cyclic monophosphate treatment to infant Sc significantly augmented the expression of transferrin, androgen binding protein, inhibin-ß-B, stem cell factor, and glial-derived neurotropic factor, which are usually up-regulated by FSH in pubertal Sc during spermatogenic onset. This suggested that lack of FSH mediated down-regulation of Gαi-2 expression and limited expression of Gαs subunit as well as Ric8b may underlie limited FSH responsiveness of Sc during infancy. This study also divulged that intracellular signaling events downstream of FSH-R are in place and can be activated exogenously in infant Sc. Additionally, this information may help in the proper diagnosis and treatment of infertile individuals having abnormal G protein-coupled FSH-R.

A non-surgical approach for male germ cell mediated gene transmission through transgenesis.

Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.