Follicular oocyte as a potential target for severe acute respiratory syndrome coronavirus 2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in December 2019 and rapidly became a pandemic as coronavirus disease 2019 (COVID-19). Apart from other organs, presence of specific receptor angiotensin-converting enzyme (ACE2) and corresponding proteases such as transmembrane serine protease 2, basigin and cysteine protease cathepsin L make follicular somatic cells as well as oocyte as potential targets for SARS-CoV-2 infection. The SARS-CoV-2 causes inflammation and hypoxia that generate reactive oxygen species (ROS) in critically ill patients. In addition, a large number of casualties and insecurity of life due to repeated waves of SARS-CoV-2 infection generate psychological stress and cortisol resulting in the further generation of ROS. The excess levels of ROS under physiological range cause meiotic instability, while high levels result in oxidative stress that trigger various death pathways and affect number as well as quality of follicular oocytes. Although, emerging evidence suggests that the SARS-CoV-2 utilises cellular machinery of ovarian follicular cells, generates ROS and impairs quality of follicular oocytes, the underlying mechanism of viral entry into host cell and its negative impact on the follicular oocyte remains poorly understood. Therefore, this review summarises emerging evidence on the presence of cellular machinery for SARS-CoV-2 in ovarian follicles and the potential negative impact of viral infection on the follicular oocytes that affect ovarian functions in critically ill and stressed women.

In silico prediction of COVID-19 cytokine storm in lung cancer types.

Lung cancer is one of the most frequently diagnosed malignant tumors and the leading cause of cancer-related death worldwide. Mainly, Non-small-cell lung cancer (NSCLC), which accounts for more than eighty-five percent of all lung cancers, consists of two major subtypes: lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Novel coronavirus disease (COVID-19) affected millions of people caused by acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) around the globe. Lung cancer patients and COVID-19 present unique and unfortunate lethal combinations because the lungs are the primary target organ of SARS-CoV-2 infection. Clinical studies have demonstrated that an over-activated inflammatory response associated with severe COVID-19 cases is characterized by excessive auto-amplifying cytokine release, which is defined as a "cytokine storm." ACE2 and TMPRSS2 receptors play an essential role in SARS-CoV-2 infection; therefore, using in silico analysis, we did correlation analysis with immune infiltration markers in LUAD and LUSC patient groups. Our study identified a promising correlation between immune-modulators and receptor proteins (ACE-2 and TMPRSS2), creating a domain that requires further laboratory studies for clinical authentication.

Meiotic Instability Generates a Pathological Condition in Mammalian Ovum.

Maintenance of metaphase-II (M-II) arrest in ovum is required to present itself as a right gamete for successful fertilization in mammals. Surprisingly, instability of meiotic cell cycle results in spontaneous exit from M-II arrest, chromosomal scattering and incomplete extrusion of second polar body (PB-II) without forming pronuclei so called abortive spontaneous ovum activation (SOA). It remains unclear what causes meiotic instability in freshly ovulated ovum that results in abortive SOA. We propose the involvement of various signal molecules such as reactive oxygen species (ROS), cyclic 3',5' adenosine monophosphate (cAMP) and calcium (Ca2+) in the induction of meiotic instability and thereby abortive SOA. These signal molecules through their downstream pathways modulate phosphorylation status and activity of cyclin dependent kinase (cdk1) as well as cyclin B1 level. Changes in phosphorylation status of cdk1 and its activity, dissociation and degradation of cyclin B1 destabilize maturation promoting factor (MPF). The premature MPF destabilization and defects in other cell cycle regulators possibly cause meiotic instability in ovum soon after ovulation. The meiotic instability results in a pathological condition of abortive SOA and deteriorates ovum quality. These ova are unfit for fertilization and limit reproductive outcome in several mammalian species including human. Therefore, global attention is required to identify the underlying causes in greater details in order to address the problem of meiotic instability in ova of several mammalian species icluding human. Moreover, these activated ova may be used to create parthenogenetic embryonic stem cell lines in vitro for the use in regenerative medicine.Graphical abstract.

Cyclic nucleotide phosphodiesterase inhibitors: possible therapeutic drugs for female fertility regulation.

Cyclic nucleotide phosphodiesterases (PDEs) are group of enzymes responsible for the hydrolysis of cyclic adenosine 3', 5' monophosphate (cAMP) and cyclic guanosine 3', 5' monophosphate (cGMP) levels in wide variety of cell types. These PDEs are detected in encircling granulosa cells or in oocyte with in follicular microenvironment and responsible for the decrease of cAMP and cGMP levels in mammalian oocytes. A transient decrease of cAMP level initiates downstream pathways to cause spontaneous meiotic resumption from diplotene arrest and induces oocyte maturation. The nonspecific PDE inhibitors (caffeine, pentoxifylline, theophylline, IBMX etc.) as well as specific PDE inhibitors (cilostamide, milrinone, org 9935, cilostazol etc.) have been used to elevate cAMP level and inhibit meiotic resumption from diplotene arrest and oocyte maturation, ovulation, fertilization and pregnancy rates both in vivo as well as under in vitro culture conditions. The PDEs inhibitors are used as powerful experimental tools to demonstrate cyclic nucleotide mediated changes in ovarian functions and thereby fertility. Indeed, non-hormonal nature and reversible effects of nonspecific as well as specific PDE inhibitors hold promise for the development of novel therapeutic drugs for female fertility regulation.

A Catalytic Trisulfide in Human Sulfide Quinone Oxidoreductase Catalyzes Coenzyme A Persulfide Synthesis and Inhibits Butyrate Oxidation.

Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H2S to glutathione persulfide with concomitant reduction of CoQ10. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ1-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates 201Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and 379Cys-SSH, which transfers sulfur to an external acceptor.

S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase-dependent hydrogen sulfide synthesis.

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 µm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine ß-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

Necrosis and necroptosis in germ cell depletion from mammalian ovary.

The maximum number of germ cells is present during the fetal life in mammals. Follicular atresia results in rapid depletion of germ cells from the cohort of the ovary. At the time of puberty, only a few hundred (<1%) germ cells are either culminated into oocytes or further get eliminated during the reproductive life. Although apoptosis plays a major role, necrosis as well as necroptosis, might also be involved in germ cell elimination from the mammalian ovary. Both necrosis and necroptosis show similar morphological features and are characterized by an increase in cell volume, cell membrane permeabilization, and rupture that lead to cellular demise. Necroptosis is initiated by tumor necrosis factor and operated through receptor interacting protein kinase as well as mixed lineage kinase domain-like protein. The acetylcholinesterase, cytokines, starvation, and oxidative stress play important roles in necroptosis-mediated granulosa cell death. The granulosa cell necroptosis directly or indirectly induces susceptibility toward necroptotic or apoptotic cell death in oocytes. Indeed, prevention of necrosis and necroptosis pathways using their specific inhibitors could enhance growth/differentiation factor-9 expression, improve survivability as well as the meiotic competency of oocytes, and prevent decline of reproductive potential in several mammalian species and early onset of menopause in women. This study updates the information and focuses on the possible involvement of necrosis and necroptosis in germ cell depletion from the mammalian ovary.

Cytochrome c Reduction by H2S Potentiates Sulfide Signaling.

Hydrogen sulfide (H2S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by H2S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H2S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H2S oxidized. H2S increases O2 consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H2S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.

Role of granulosa cell mitogen-activated protein kinase 3/1 in gonadotropin-mediated meiotic resumption from diplotene arrest of mammalian oocytes.

In mammals, preovulatory oocytes are encircled by several layers of granulosa cells (GCs) in follicular microenvironment. These follicular oocytes are arrested at diplotene arrest due to high level of cyclic nucleotides from encircling GCs. Pituitary gonadotropin acts at the level of encircling GCs and increases adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) and activates mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. The MAPK3/1 disrupts the gap junctions between encircling GCs and oocyte. The disruption of gap junctions interrupts the transfer of cyclic nucleotides to the oocyte that results a drop in intraoocyte cAMP level. A transient decrease in oocyte cAMP level triggers maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggers meiotic resumption from diplotene arrest in follicular oocyte. Thus, MAPK3/1 from GCs origin plays important role in gonadotropin-mediated meiotic resumption from diplotene arrest in follicular oocyte of mammals.

Journey of oocyte from metaphase-I to metaphase-II stage in mammals.

In mammals, journey from metaphase-I (M-I) to metaphase-II (M-II) is important since oocyte extrude first polar body (PB-I) and gets converted into haploid gamete. The molecular and cellular changes associated with meiotic cell cycle progression from M-I to M-II stage and extrusion of PB-I remain ill understood. Several factors drive oocyte meiosis from M-I to M-II stage. The mitogen-activated protein kinase3/1 (MAPK3/1), signal molecules and Rho family GTPases act through various pathways to drive cell cycle progression from M-I to M-II stage. The down regulation of MOS/MEK/MAPK3/1 pathway results in the activation of anaphase-promoting complex/cyclosome (APC/C). The active APC/C destabilizes maturation promoting factor (MPF) and induces meiotic resumption. Several signal molecules such as, c-Jun N-terminal kinase (JNK2), SENP3, mitotic kinesin-like protein 2 (MKlp2), regulator of G-protein signaling (RGS2), Epsin2, polo-like kinase 1 (Plk1) are directly or indirectly involved in chromosomal segregation. Rho family GTPase is another enzyme that along with cell division cycle (Cdc42) to form actomyosin contractile ring required for chromosomal segregation. In the presence of origin recognition complex (ORC4), eccentrically localized haploid set of chromosomes trigger cortex differentiation and determine the division site for polar body formation. The actomyosin contractile activity at the site of division plane helps to form cytokinetic furrow that results in the formation and extrusion of PB-I. Indeed, oocyte journey from M-I to M-II stage is coordinated by several factors and pathways that enable oocyte to extrude PB-I. Quality of oocyte directly impact fertilization rate, early embryonic development, and reproductive outcome in mammals.

Hydrogen Sulfide Oxidation by Myoglobin.

Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal muscle cytochrome c oxidase to sulfide poisoning in ethylmalonic encephalopathy, resulting from the deficiency in a mitochondrial sulfide oxidation enzyme, might be due to the concentration of H2S by myoglobin in this tissue.

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine ß-Synthase.

Cystathionine ß-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO(•)), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2to Fe(III)-CBS, forming superoxide radical anion (O2 (̇̄)). In this study, we describe the kinetics of nitrite (NO2 (-)) reduction by Fe(II)-CBS to form Fe(II)NO(•)-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO(•)-CBS by O2showed complex kinetic behavior and led to peroxynitrite (ONOO(-)) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO(•)and peroxynitrite.

Biosynthesis and Reactivity of Cysteine Persulfides in Signaling.

Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.

1-Triacontanol cerotate; isolated from Marsilea quadrifolia Linn. ameliorates reactive oxidative damage in the frontal cortex and hippocampus of chronic epileptic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsilea quadrifolia Linn. (MQ) has been used for insomnia and epileptic disorders in traditional Indian medicine. The present study is to isolate the active component responsible for antiepileptic property of MQ by evaluating its ability to minimize the reactive oxidative damage in brain due to chronic epilepsy in rat. MATERIALS AND METHODS: 1-Triacontanol cerotate (1TAC) was isolated after chromatography on a silica gel from dried petroleum ether fraction of methanolic extract of MQ. Acute oral toxicity studies of 1TAC were carried out and efficacy of 1TAC on malondialdehyde (MDA) and reduced glutathione (GSH) production in different brain areas of chronic pentylenetetrazole (PTZ) induced epileptic rats were evaluated. RESULTS: Our results showed that PTZ-kindled chronic epileptic rats had an increase MDA and decreased GSH concentration in the frontal cortex as well as hippocampus, compared to the normal control. MDA and GSH concentrations in those brain areas were normalized after treatment with sodium valproate (SV) in 200 mg kg(-1)bw; as well as 1TAC in 40 and 80 mg kg(-1)bw doses. CONCLUSION: Production of reactive oxygen species (ROS) is known to worsen epileptogenesis. The isolated component 1TAC which reduced the reactive oxidative damage in hippocampus and frontal cortex of PTZ kindled rats could be responsible for antiepileptic property of MQ. Its action is found to be dose dependent, with 80 mg kg(-1)bw showing even better efficacy than 200 mg kg(-1)bw of SV.

Assay methods for H2S biogenesis and catabolism enzymes.

H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine ß-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter.

Nitrite reductase activity and inhibition of H2S biogenesis by human cystathionine ß-synthase.

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H2S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II)-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of Fe(II)-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H2S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H2S biology.