Catecholamines modulate differentially nonapeptide precursor mRNA expression in the preoptic area and ovary of the catfish Heteropneustes fossilis: An in vitro study.

In the catfish Heteropneustes fossilis, three nonapeptide hormone genes were identified in the brain preoptic area (POA) and ovary: a pro-vasotocin (pro-vt) and two isotocin gene paralogs viz., a novel pro-ita and conventional pro-itb. In the present study, the regulatory role of catecholamines [CA: dopamine (DA), noradrenaline (NA), adrenaline (AD)] on the expression of these genes were investigated in vitro. DA (1, 10, and 100 ng/mL) inhibited significantly the mRNA expression in both the POA and ovary. NA upregulated the POA mRNA expression in a biphasic manner, the lower concentrations (1 ng and 10 ng) scaled up and the higher concentration (100 ng) scaled down the expression of pro-vt and pro-itb, while only the 1 ng NA scaled up the pro-ita expression. In the ovary, NA upregulated the mRNA expressions at all concentrations; the pro-vt expression was stimulated only at 10 and 100 ng. AD stimulated pro-vt and pro-ita expression in the POA at all concentrations but the pro-itb expression was inhibited at 1 and 10 ng, and stimulated at 100 ng concentrations. In the ovary, AD elicited varied effects; no significant change in pro-vt, a stimulation of pro-ita, and an inhibition of pro-itb at 1 ng, and stimulation of pro-itb at the 10 and 100 ng. The incubation of the POA and ovary with α-methylparatyrosine (MPT, 250 µg/mL, a tyrosine hydroxylase inhibitor) for 8 h downregulated the mRNA expression in the POA but unaltered the expression in the ovary. Pre-incubation with MPT for 4 h, followed by co-incubation with DA, NA or AD for 4 h elicited varied effects. In the POA, the co-incubations with the CAs rescued the inhibition due to MPT. The MPT + DA and MPT + AD treatments reduced the magnitude of the inhibition of pro-vt and pro-itb by MPT. But the pro-ita expression was modestly stimulated in the MPT + AD group. On the other hand, the MPT + NA treatment rescued the MPT effect and elicited 10-folds increase in the expression levels. In the ovary, the changes were: an inhibition in the MPT + DA group, no significant alteration in the MPT + NA group, and a mild stimulation in the MPT + AD group. The results suggest that CAs modulate brain and ovarian nonapeptide gene expression differentially, which is important in the neuroendocrine/endocrine integration of reproduction in the catfish.

Kisspeptin modulation of nonapeptide and cytochrome P450 aromatase mRNA expression in the brain and ovary of the catfish Heteropneustes fossilis: in vivo and in vitro studies.

In Heteropneustes fossilis, kisspeptins (Kiss) and nonapeptides (NPs; vasotocin, Vt; isotocin, Itb; Val8-isotocin, Ita) stimulate the hypothalamus-pituitary-gonadal (HPG) axis, and estrogen feedback modulates the expression of these systems. In this study, functional interactions among these regulatory systems were demonstrated in the brain and ovary at the mRNA expression level. Human KISS1 (hKISS1) and H. fossilis Kiss2 (HfKiss2) produced biphasic effects on brain and ovarian vt, itb and ita expression at 24 h post injection: low and median doses produced inhibition, no change or mild stimulation, and the highest dose consistently stimulated the mRNA levels. The Kiss peptides produced an upregulation of NP mRNA expression at 24 h incubation of brain and ovarian slices by increasing the concentration of hKISS1 and HfKiss2. The kiss peptides stimulated brain cyp19a1b and ovary cyp19a1a expression, both in vivo and in vitro. Peptide234, a Kiss1 receptor antagonist, inhibited basal mRNA expression of the NPs, cyp19a1b and cyp19a1a, which was prevented by the Kiss peptides, both in vivo and in vitro. In all the experiments, HfKiss2 was more effective than hKISS1 in modulating mRNA expression. The results suggest that the NP and E2 systems are functional targets of Kiss peptides and interact with each other.

Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

Molecular cloning and characterization of secretogranin II in the catfish Heteropneustes fossilis: Sex and seasonal brain regional variations and its gonadotropin regulation.

Secretoneurin (SN) is a novel functional peptide derived from secretogranin II, a protein belonging to the class of chromogranins. Catfish Heteropneustes fossilis is an economically important species of the Asian subcontinent and holds an important place in reproductive physiology study. In the present study, the full-length cDNA encoding secretogranin IIb (SgIIb) was cloned from the brain of catfish H. fossilis. Sequence analysis showed that a 33 amino acid SN peptide (SNb) is present in SgIIb proprotein. The full-length sequence of SgIIb is 2912 bp with open-reading frame 1761 bp long. The 5'UTR is 681 bp long upstream and the 3'UTRis 470 bp long downstream. The ORF encodes 586 amino acid residues comprising signal peptide (24 amino acids) and SNb (33 amino acids), EM66 (66 amino acids). Catfish SgIIb showed 88% nucleotide homology and 90% protein identity with Ictalurus punctatus. Tissue expression showed that it is highly expressed in the brain among all tissues studied. In situ hybridization and quantitative real-time PCR showed significant brain regional distribution with highest transcript abundance in the pituitary. In ovary tissue SgIIb transcripts were localized in the granulosa layer. In the brain, hCG administration stimulated expression highly at 16 h. In ovary, hCG treatment under in vivo and in vitro condition decreased SgIIb expression at all doses. Thus, in the present study in situ localization of SgIIb mRNAs in different regions of brain and pituitary, and ovary suggest its putative role in regulating hypothalamic-pituitary-gonadal (HPG) axis and also suggest that the SgIIb expression pattern in the brain and ovary could be related to reproductive activity and may be involved in the neuroendocrine role of SgIIb in the catfish H. fossilis.

In situ localization of vasotocin receptor gene transcripts in the brain-pituitary-gonadal axis of the catfish Heteropneustes fossilis: a morpho-functional study.

In the catfish Heteropneustes fossilis, three vasotocin (VT) receptor subtype genes, v1a1, v1a2, and v2a, were cloned and characterized previously. In the present study, using RNA probes, we localized the distribution of the gene transcripts in the brain-pituitary-gonadal (BPG) axis. The V1a-type receptor, v1a1 and v1a2, genes showed similar and overlapping distribution in the brain. The gene paralogs are distributed in the radial glial cells (RGCs) of the telencephalic ventricle and around the third ventricle in the hypothalamus and thalamus, olfactory tract, nucleus preopticus, nucleus lateralis tuberis, nucleus recessus lateralis and posterioris, nucleus saccus vasculosi, thalamic nuclei, habenular nucleus, habenular commissure, basal part of pineal stalk, accessory pretectal nucleus, optic tectum, corpus and valvula of the cerebellum, and facial and vagal lobes. The V2a receptor gene (v2a) has restricted distribution and is largely confined to the anterior subependymal region of the telencephalon. The localization pattern shows that the V1a-type receptors are distributed in major sensorimotor processing centers and the neuroendocrine/reproductive centers of the brain. In the pituitary, the receptor genes were localized differentially in the three divisions with the V1a-type receptor genes strongly expressed in the rostral pars distalis compared to the v2a paralog. In the ovary, the V1a-type receptor genes were localized in the follicular layer while v2a was localized in the oocyte membrane. In the testis, v1a2 and v2a are densely distributed in the interstitial tissue and seminiferous epithelium but the v1a1 is lowly expressed. The results suggest that the VT receptor genes have an extensive but differential distribution in the BPG axis. Future experimental studies are required to correlate the cellular localizations with specific functions of VT in the BPG axis.

Distribution and localization of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the brain and its regions of the catfish Heteropneustes fossilis.

In vertebrates, steroids are synthesized de novo in the central and peripheral nervous system, independent of peripheral steroidogenic glands, such as the adrenal, gonads and placenta. 3ß-Hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3ß-HSD) is a key steroidogenic enzyme in vertebrate gonads, placenta and adrenal. It mediates the oxidation and isomerization reactions of progesterone from pregnenolone, 17-hydroxyprogesterone from 17-hydroxypregnenolone and androstenedione from dehydroepiandrosterone. In the present study, we examined the expression of 3ß-HSD cDNA by real time-PCR and localization of the mRNA by in situ hybridization in the brain and its regions during the different phases of the reproductive cycle of the catfish Heteropneustes fossilis. Further, 3ß-HSD activity was assayed biochemically to show seasonal variations. We showed significant seasonal and sexual dimorphic changes in the levels of transcript abundance in the whole brain and its regions. In whole brain, level was the highest in post-spawning phase and lowest in spawning phase in males. In females, there was a progressive increase through resting phase to pre-spawning phase, a decline in the spawning phase and increase in the post-spawning phase. In the preparatory phase, the highest transcript level was seen in medulla oblongata and the lowest in pituitary in males. In females, the level was the highest in the hypothalamus and lowest in olfactory bulb and pituitary. However, in the pre-spawning phase, in males it was the highest in telencephalon and hypothalamus and lowest in pituitary. In females, the highest transcript level was in olfactory bulb and lowest in pituitary. 3ß-HSD enzyme activity showed significant seasonal variation in the brain, the highest in the resting phase and lowest in the preparatory and spawning phases. In situ hybridization showed the presence of 3ß-HSD transcript was especially high in the cerebellum region. The presence of 3ß-HSD in the brain may indicate steroidogenesis in the catfish brain.

Molecular cloning and characterization of brain and ovarian cytochrome P450 aromatase genes in the catfish Heteropneustes fossilis: Sex, tissue and seasonal variation in, and effects of gonadotropin on gene expression.

Cytochrome P450 aromatase (Cyp19arom) is the rate-limiting enzyme controlling estrogen biosynthesis, coded by Cyp19a1 in most gnathostomes. Most teleosts have two forms expressed differentially in ovary (cyp19a1a) and neural tissue (cyp19a1b). In this study, full length cDNAs of 2006 bp and 1913 bp with ORFs of 1575 bp and 1488 bp were isolated from the brain and ovary, respectively, of the catfish Heteropneustes fossilis, an air-breathing species with high aquaculture potential. The ORFs encode predicted proteins of 495 and 524 amino acid residues, respectively. The proteins show 62% identity with each other and cluster in two distinct clades (the brain type and ovary type) in the teleost taxon, separated from the tetrapod type. In the in situ localization study, both cyp19a1a and cyp19a1b transcripts were localized in the brain but the signal intensity was higher for the brain type paralog. The transcript signals were observed in the radial glial cells and in neuronal populations of the dorso-lateral region of the telencephalon, pre-tectum, hypothalamus and medulla oblongata. In the ovary, both paralogs were expressed in the follicular layer with a high signal intensity of the ovarian type (cyp19a1a). The differential expression of the gene paralogs was evident from qPCR analysis. Cyp19a1b has relatively a high abundance in the female brain, followed by other peripheral tissues (gonads, liver, gill, kidney and muscle). On the other hand, cyp19a1a has relatively a high transcript abundance in the ovary and female brain, followed by the testis and male brain, and female liver and muscle. The expression was low in male liver and muscle, and the lowest in the gill and kidney. The expression of the two paralogs exhibit brain regional differences; both types have relatively a high transcript abundance in telencephalon-preoptic area with the cyp19a1b expression higher in females than males. In hypothalamus, the expression of both types is higher in males than females. In medulla, the expression of the cyp19a1b is higher than cyp19a1a, and the transcript abundance of the ovarian type is higher in females than males. The expression of the gene paralogs elicits significant seasonal variations in the ovary and brain. In both tissues, the expression increases from the resting to preparatory phases, and decreases through the prespawning phase to low levels in spawning phase. In vivo and/or in vitro treatments with human chorionic gonadotropin (hCG) stimulated the expression of the gene paralogs in the brain and ovary, time-dependently. In conclusion, both paralogs have an overlapping distribution at different levels of the brain-pituitary-gonad axis and may function as a single functional unit as far as the estrogen synthesis is concerned.

Changes in vasotocin levels in relation to ovarian development in the catfish Heteropneustes fossilis exposed to altered photoperiod and temperature.

Photoperiod and temperature are the major proximate factors that activate the brain-pituitary-gonadal-endocrine axis stimulating gonadal recrudescence. Vasotocin (VT), the basic nonapeptide hormone, is secreted by the nucleus preopticus in the hypothalamus and released from the pituitary into circulation as a neurohormone for physiological actions. Additionally, VT is secreted de novo in the ovary of the catfish and has been implicated in ovarian functions. In the present study, we evaluated the changes in VT secretion during altered photoperiod and temperature exposure. The ovarian changes were monitored over gonadosomatic index (GSI) and plasma steroid hormone levels. Exposure of the catfish to long photoperiod (LP, 16L:08D) daily, alone or in combination with high temperature (HT, 28 ± 2 °C), for 14 or 28 days resulted in a decrease in brain-pituitary VT level with a concomitant increase in plasma and ovarian VT levels. The changes were greater in the LP + HT group on day 28. Concurrently, the treatments stimulated the GSI and plasma estradiol-17ß (E2), testosterone (T) and progesterone (P4) levels with higher more responses in the LP + HT group. Exposure of the catfish to short photoperiod (SP, 08L:16D) daily or total darkness (TD, 24L:00D) daily, with or without changing the ambient temperature, for 14 or 28 days produced a depressing effect on VT, GSI and steroid hormone levels, the range of the response varied with the temperature. The brain VT level was low except in the TD + NT group. Plasma and ovarian VT levels decreased more in the SP and TD groups under ambient temperature than in the groups at the raised temperature. The GSI and plasma steroid hormones (E2, T and P4) responded in a similar manner. Plasma cortisol level registered a significant increase in all the groups compared to the initial control groups, and the increase was significantly higher on day 28. The simultaneous activation of VT secretion and ovarian recrudescence by photoperiod and temperature suggests the peptide's involvement in the hormonal control of gametogenesis.

Molecular cloning, sequencing and phylogeny of vasotocin receptor genes in the air-breathing catfish Heteropneustes fossilis with sex dimorphic and seasonal variations in tissue expression.

Vasotocin (VT) is the ortholog of vasopressin (VP) in non-mammalian vertebrates and is known for multiple functions. Teleost fishes have a complete repertoire of known VP/VT receptor subtypes (vasopressin type, VR): two V1A subtypes (V1Aa and V1Ab or V1a1 and V1a2) and five V2 subtypes (V2A1, V1A2, V2B1, V2B2 and V2C). Full-length cDNAs of v1a1, v1a2 and v2 (v2a1) with ORFs of 1,308, 1,137 and 1,527 bp, respectively, were cloned and characterized in the catfish Heteropneustes fossilis (Siluriformes, Ostariophysi). BLAST analysis revealed that the genes encoded three VT receptors, V1a1, V1a2 and V2 of 436, 379 and 509 amino acid residues, respectively. The predicted proteins showed typical features of the seven-transmembrane domain receptor core structure with hallmark triplets Asp-Arg-Tyr/Asp-Arg-His (DRY/DRH) and the variable intracellular loop III of vertebrate neurohypophysial hormone receptors. Phylogenetic analysis of the deduced protein sequences revealed that they clustered with the V1Aa, V1Ab and V2A1, respectively, of other teleosts. The V2R has a sequence identity of 70-76% with V2A1 than with the V2B type (sequence identity 43-49%). Semiquantitative PCR analysis showed that the receptor gene transcripts were expressed ubiquitously in the tissues examined (brain, pituitary, gonads, liver, muscle, kidney and gills) and displayed sex and seasonal fluctuations in a tissue-specific manner. The results form a basis for functional studies on the VT receptors in the catfish.

Differential and reproductive stage-dependent regulation of vasotocin secretion by catecholamines in the catfish Heteropneustes fossilis.

Vasotocin (VT) is the basic nonapeptide hormone secreted by the neurohypophysis of non-mammalian vertebrates and is involved in the regulation of osmoregulation, metabolism, cardiovascular function, reproduction and behaviour. Among the reproductive function, VT is specifically implicated in final oocyte maturation, ovulation, oviposition/parturition in teleosts, amphibians, reptiles, and birds. The central catecholaminergic system is involved in the regulation of pituitary hormone secretion including gonadotropin, and mediates also changes in environmental photoperiod and temperature. The close apposition of the VT and catecholaminergic systems in the preoptic area of the hypothalamus signifies a strong possibility of their functional interaction. The aim of the present study was to investigate the effects of exogenously administered catecholamines on VT secretion in two different reproductive phases of female catfish Heteropneustes fossilis. For this, the catecholamine precursor L-dihydroxyphenylalanine (L-DOPA) and catecholamines (dopamine-DA, norepinephrine-NE, and epinephrine-E) were intraperitoneally injected in normal catfish and/or along with α-methylparatyrosine (α-MPT, a tyrosine hydroxylase inhibitor). Brain and plasma VT levels were measured by specific ELISA, 24h post injection. Both L-DOPA and DA inhibited brain and plasma VT levels in a concentration-dependent manner in preparatory and prespawning phases. In contrast, NE elicited dose-dependent effects: the lowest dose (0.5 ng/g body mass, BM) was ineffective, the median dose (1 ng/g BM) stimulated, and the high doses (10 and 100 ng/g BM) inhibited VT levels. E stimulated VT levels dose-dependently. A single injection of α-MPT (250 µg/g BM) strongly inhibited VT when given alone and enhanced the inhibitory effects of L-DOPA and DA in the combination groups. The α-MPT inhibition of VT was significantly reduced by the injection of NE (5 ng/g BM) and was restored or elevated by E. When the adrenergic neurotransmitters were given together with α-MPT, the inhibitory effect of the latter was abolished and VT levels were significantly elevated. Thus, the present data indicate that the physiological changes in VT is differentially regulated by the catecholamines (DA inhibits and NE/E stimulates VT).

Teratogenic effects of 4-nonylphenol on early embryonic and larval development of the catfish Heteropneustes fossilis.

Alkylphenol polyethoxylates (APEs), which are widely used in detergents, paints, herbicides, insecticides, and in many other formulations, have been widely detected in aquatic environments. 4-Nonylphenol (NP) is an important APE detected at microgram levels per litre (0.1-336 µg/L) in water. The objective of the present study was to evaluate NP's toxic effects at low and high sublethal concentrations (0.1 and 1 µg/L) on embryonic development of the catfish Heteropneustes fossilis at different time intervals. The data show that fertilization rate was decreased and cleavage and blastula were severely affected leading to complete mortality of embryos. NP exposure resulted in various body malformations in larvae, such as vertebral deformations, e.g., fin blistering/necrosis, axial deformities (lordosis, kyphosis, and scoliosis) of the spine in the abdominal and caudal region, tail curved completely backward, shortened body, severe spinal and yolk sac malformations, C-shaped severe spinal curvature, cranial malformation with undeveloped head, and failure of eye development. The level of body malformations increased with the concentration and exposure time. After 72 h of exposure, all larvae were dead at both concentrations. Scanning electron microscope study showed that epidermal cells (keratinocytes) were severely damaged in both low- and high-dose treatments throughout development, leading to development of numerous depressions representing sinking holes on the skin. Mucous glands increased significantly in treatment groups compared with control groups. The present study highlights the severe teratogenic effects of NP. The prevalence of the contaminant, if not checked, can lead to decreased population and ultimate disappearance of the species.

Bioinspired fabrication of zinc hydroxide-based nanostructure from lignocellulosic biomass Litchi chinensis leaves and its efficacy evaluation on antibacterial, antioxidant, and anticancer activity.

Zinc-based nanostructures are known for their numerous potential biomedical applications. In this context, the biosynthesis of nanostructures using plant extracts has become a more sustainable and promising alternative to effectively replace conventional chemical methods while avoiding their toxic impact. In this study, following a low-temperature calcination process, a green synthesis of Zn-hydroxide-based nanostructure has been performed using an aqueous extract derived from the leaves of Litchi chinensis, which is employed as a lignocellulose waste biomass known to possess a variety of phytocompounds. The biogenic preparation of Zn-hydroxide based nanostructures is enabled by bioactive compounds present in the leaf extract, which act as reducing and capping agents. In order to evaluate its physicochemical characteristics, the produced Zn-hydroxide-based nanostructure has been subjected to several characterization techniques. Further, the multifunctional properties of the prepared Zn-hydroxide-based nanostructure have been evaluated for antioxidant, antimicrobial, and anticancer activity. The prepared nanostructure showed antibacterial efficacy against Bacillus subtilis and demonstrated its anti-biofilm activity as evaluated through the Congo red method. In addition, the antioxidant activity of the prepared nanostructure has been found to be dose-dependent, wherein 91.52 % scavenging activity could be recorded at 200 µg/ml, with an IC50 value of 45.22 µg/ml, indicating the prepared nanostructure has a high radical scavenging activity. Besides, the in vitro cytotoxicity investigation against HepG2 cell lines explored that the as-prepared nanostructure exhibited a higher cytotoxic effect and 73.21 % cell inhibition could be noticed at 25.6 µg/ml with an IC50 of 2.58 µg/ml. On the contrary, it was found to be significantly lower in the case of HEK-293 cell lines, wherein ~47.64 % inhibition could be noticed at the same concentration. These findings might be further extended to develop unique biologically derived nanostructures that can be extensively evaluated for various biomedical purposes.

Estrogen regulation of brain vasotocin secretion in the catfish Heteropneustes fossilis: an interaction with catecholaminergic system.

Vasotocin (VT) is a basic neurohypophysial nonapeptide in non-mammalian vertebrates and is involved in diverse functions like osmoregulation, reproduction, metabolism and behavior. In this study, we report that estradiol-17ß (E(2)) regulates brain and plasma VT secretion through the involvement of the catecholaminergic (CA) system. To demonstrate this, E(2) level was altered through ovariectomy (OVX, 3 weeks) and replacement study with low and high E(2) doses (0.1 and 0.5 µg/g body weight). CA activity was inhibited by treatment with α-methylparatyrosine (α-MPT; 250 µg/g body weight), a competitive inhibitor of tyrosine hydroxylase. VT was assayed by an enzyme immunoassay method. In the sham group, the low E(2) dose produced 82% and 104% increase, respectively, in brain and plasma VT levels. The high E(2) dose decreased the VT levels significantly. The low E(2) dose decreased brain E(2) but elevated plasma E(2). In the high E(2) group, the E(2) level increased further in both brain and plasma. OVX resulted in a significant inhibition (69% and 25%, respectively) of both brain and plasma VT, which was correlated with low E(2) levels. The low E(2) dose not only reversed the inhibition, but increased the VT level in both brain and plasma in comparison to the sham groups. The high E(2) replacement inhibited VT levels further low in both brain and plasma. The α-MPT treatment inhibited VT levels significantly in both sham and OVX groups. The drug treatment abolished partially the restorative effect of the low E(2) dose in the ovariectomized fish. In the high E(2) dose group, α-MPT decreased brain and plasma VT levels further low compared to the sham + 0. 5 µg E(2) group or OVX + 0.5 µg E(2) group except the brain VT level, which increased in the OVX+0.5 µg E(2) group. It is inferred that E(2) may exert biphasic effects on VT through the mediation of the CA system.

Targeting COVID-19 (SARS-CoV-2) main protease through active phytochemicals of ayurvedic medicinal plants - Withania somnifera (Ashwagandha), Tinospora cordifolia (Giloy) and Ocimum sanctum (Tulsi) - a molecular docking study.

COVID-19 (Coronavirus disease 2019) is a transmissible disease initiated and propagated through a new virus strain SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) since 31st December 2019 in Wuhan city of China and the infection has outspread globally influencing millions of people. Here, an attempt was made to recognize natural phytochemicals from medicinal plants, in order to reutilize them against COVID-19 by the virtue of molecular docking and molecular dynamics (MD) simulation study. Molecular docking study showed six probable inhibitors against SARS-CoV-2 Mpro (Main protease), two from Withania somnifera (Ashwagandha) (Withanoside V [10.32 kcal/mol] and Somniferine [9.62 kcal/mol]), one from Tinospora cordifolia (Giloy) (Tinocordiside [8.10 kcal/mol]) and three from Ocimum sanctum (Tulsi) (Vicenin [8.97 kcal/mol], Isorientin 4'-O-glucoside 2″-O-p-hydroxybenzoagte [8.55 kcal/mol] and Ursolic acid [8.52 kcal/mol]). ADMET profile prediction showed that the best docked phytochemicals from present work were safe and possesses drug-like properties. Further MD simulation study was performed to assess the constancy of docked complexes and found stable. Hence from present study it could be suggested that active phytochemicals from medicinal plants could potentially inhibit Mpro of SARS-CoV-2 and further equip the management strategy against COVID-19-a global contagion. HighlightsHolistic approach of Ayurvedic medicinal plants to avenge against COVID-19 pandemic.Active phytoconstituents of Ayurvedic medicinal plants Withania somnifera (Ashwagandha), Tinospora cordifolia (Giloy) and Ocimum sanctum (Tulsi) predicted to significantly hinder main protease (Mpro or 3Clpro) of SARS-CoV-2.Through molecular docking and molecular dynamic simulation study, Withanoside V, Somniferine, Tinocordiside, Vicenin, Ursolic acid and Isorientin 4'-O-glucoside 2″-O-p-hydroxybenzoagte were anticipated to impede the activity of SARS-CoV-2 Mpro.Drug-likeness and ADMET profile prediction of best docked compounds from present study were predicted to be safe, drug-like compounds with no toxicity.Communicated by Ramaswamy H. Sarma.

Reproductive roles of the vasopressin/oxytocin neuropeptide family in teleost fishes.

The vertebrate nonapeptide families arginine vasopressin (AVP) and oxytocin (OXT) are considered to have evolved from a single vasopressin-like peptide present in invertebrates and termed arginine vasotocin in early vertebrate evolution. Unprecedented genome sequence availability has more recently allowed new insight into the evolution of nonapeptides and especially their receptor families in the context of whole genome duplications. In bony fish, nonapeptide homologues of AVP termed arginine vasotocin (Avp) and an OXT family peptide (Oxt) originally termed isotocin have been characterized. While reproductive roles of both nonapeptide families have historically been studied in several vertebrates, their roles in teleost reproduction remain much less understood. Taking advantage of novel genome resources and associated technological advances such as genetic modifications in fish models, we here critically review the current state of knowledge regarding the roles of nonapeptide systems in teleost reproduction. We further discuss sources of plasticity of the conserved nonapeptide systems in the context of diverse reproductive phenotypes observed in teleost fishes. Given the dual roles of preoptic area (POA) synthesized Avp and Oxt as neuromodulators and endocrine/paracrine factors, we focus on known roles of both peptides on reproductive behaviour and the regulation of the hypothalamic-pituitary-gonadal axis. Emphasis is placed on the identification of a gonadal nonapeptide system that plays critical roles in both steroidogenesis and gamete maturation. We conclude by highlighting key research gaps including a call for translational studies linking new mechanistic understanding of nonapeptide regulated physiology in the context of aquaculture, conservation biology and ecotoxicology.

Elevated Circulatory Proline to Glutamine Ratio (PQR) in Endometriosis and Its Potential as a Diagnostic Biomarker.

Endometriosis (EM) is a hormone-dependent gynecological disease associated with chronic pelvic pain and altered immuno-inflammatory processes. It shares some cancer-like characteristics such as increased proline biosynthesis and activated glutaminolysis. Both proline and glutamine are interconvertible metabolically, and studies have shown their roles in cancer cell metabolic reprogramming, redox homeostasis, occurrence/development of endometrial carcinoma, and its further progression toward the malignant state. So based on this, we hypothesized that the circulatory proline to glutamine ratio (PQR) would be altered in EM and may serve as an indicative biomarker to improve the clinical diagnosis of EM. In present study, the circulatory-PQR levels were estimated for 39 EM patients and 48 age matched healthy female subjects using 800 MHz NMR spectroscopy. Among 39 EM patients, 15 were in the clinical stages I to II and referred to here as moderate EM (MEM) patients and 24 were in the clinical stages III to IV and referred here as severe EM (SEM) patients. The circulatory-PQR levels were significantly increased in EM patients (0.99 ± 0.13 µM in MEM; 1.39 ± 0.22 µM in SEM) compared to normal control (NC) subjects (0.52 ± 0.05 µM in NC). Further, the circulatory PQR levels exhibit the highest diagnostic potential with area under receiver operating characteristic (AUROC) curve values equal to 0.87 ± 0.04 [95%CI = 0.79-0.96] for MEM and 0.89 ± 0.04 [95% CI = 0.82-0.96] for SEM. These results suggested that circulatory-PQR has significant potential to serve as a noninvasive biomarker for diagnostic/prognostic screening of EM and further underscored the importance of these two nonessential amino acids (proline and glutamine) in cancer metabolism.

In silico screening of Pueraria tuberosa (PTY-2) for targeting COVID-19 by countering dual targets Mpro and TMPRSS2.

COVID-19 pandemic was started in Wuhan city of China in December 2019; immensely affected global population. Herein, an effort was made to identify potential inhibitors from active phytochemicals of Pueraria tuberosa (PTY-2) via molecular docking study. Our study showed five potential inhibitors (Robinin, Genistin, Daidzin, Hydroxytuberosone, Tuberostan) against Mpro and five inhibitors (Robinin, Anhydrotuberosin, Daidzin, Hydroxytuberosone, Stigmasterol) against TMPRSS2. Out of these, Robinin, Daidzin and Hydroxytuberosone were common inhibitors for Mpro and TMPRSS2. Among these, Robinin showed the highest binding affinity, therefore, tested for MD simulation runs and found stable. ADMET analysis revealed the best-docked compounds are safe and follow the Lipinski Rule of Five. Thus, it could be suggested that phytochemicals of PTY-2 could serve as potential inhibitors for COVID-19 targets.Communicated by Ramaswamy H. Sarma.

Molecular and functional characterization of catfish (Heteropneustes fossilis) aquaporin-1b: changes in expression during ovarian development and hormone-induced follicular maturation.

The oocytes of the freshwater catfish Heteropneustes fossilis hydrate during hormone-induced meiotic maturation. To investigate if this process may be mediated by aquaporins (AQPs), as it occurs in marine fish producing highly hydrated eggs, the cloning of ovarian AQPs in catfish was carried out. Using degenerate primers for conserved domains of the major intrinsic protein (MIP) family, and 5' and 3'end amplification procedures, a full-length cDNA encoding for an AQP1-like protein was isolated. The predicted protein showed the typical six transmembrane domains and two Asn-Pro-Ala (NPA) motifs conserved among the members of the AQP superfamily. Phylogenetic analysis indicated that the catfish AQP clustered with the teleost-specific aquaporin-1b subfamily, and accordingly it was termed HfAqp1b. Heterologous expression in Xenopus laevis oocytes indicated that HfAqp1b encoded for a functional AQP, water permeability being enhanced by cAMP. Site-directed mutagenesis revealed that cAMP induced the translocation of HfAqp1b into the oocyte plasma membrane most likely through the phosphorylation of HfAqp1b Ser(227). In adult catfish, hfaqp1b transcripts were detected exclusively in ovary and brain and showed significant seasonal variations; in the ovary, hfaqp1b was maximally expressed during the pre-spawning period, whereas in the brain the highest expression was detected during spawning. In vitro stimulation of isolated catfish ovarian follicles with vasotocin (VT) or human chorionic gonadotropin (hCG), which induce oocyte maturation and hydration, elevated the hfaqp1b transcript levels after 6 or 16 h of incubation, respectively. These results suggest that HfAqp1b may play a role during VT- and hCG-induced oocyte hydration in catfish, and that VT may regulate HfAqp1b at the transcriptional and post-translational level in a manner similar to the vasopressin-dependent mammalian AQP2.

Vasotocin stimulates maturation-inducing hormone, oocyte maturation and ovulation in the catfish Heteropneustes fossilis: Evidence for a preferential calcium involvement.

Arginine vasotocin (VT) is the basic neurohypophysial nonapeptide hormone in teleosts. VT is also distributed in the ovary of the catfish Heteropneustes fossilis and induces final oocyte maturation (FOM) and ovulation by stimulating the maturation-inducing hormone (MIH). The present study reports the effects of cAMP (0.5 mM), phosphodiesterase inhibitors (IBMX -0.5 mM and theophylline- 0.5 mM), the inositol triphosphate (IP3) receptor inhibitor heparin (10 µg/mL) and the Ca2+ chelator BAPTA-AM (25 µM) on VT (100 nM) - induced progestin stimulation, FOM and ovulation. Incubation of post-vitellogenic follicles with cAMP, IBMX and theophylline for 0, 8, 16 and 24 h stimulated basal secretion of progesterone (P4), 17-hydroxyprogesterone (17-P) and 17, 20ß-dihydroxy-4-pregnen-3-one (MIH) in a duration-dependent manner. The incubation of the follicles with heparin stimulated P4 modestly, and 17-P and MIH levels in a duration-dependent manner. The incubation of the follicles with BAPTA-AM stimulated P4 and MIH levels marginally and 17-P robustly. The stimulation was in the order cAMP > IBMX > theophylline > heparin > BAPTA-AM. The incubation of the follicles with VT stimulated P4, 17-P, MIH, GVBD and ovulation in a duration-dependent manner. The co-incubations with VT and the test compounds inhibited the VT-induced stimulation of P4, 17-P and MIH levels in a time-dependent manner in the order heparin > BAPTA-AM > cAMP > IBMX > theophylline. Concurrently, the VT-induced stimulation of GVBD and ovulation were also inhibited by the test compounds in the same order. The results show that VT induces FOM and ovulation preferentially acting through Ca2+ pathway and a crosstalk between Ca2+ and cAMP signaling pathways seems to integrate the processes.