Epitope-based vaccine design against the membrane and nucleocapsid proteins of SARS-CoV-2.

BACKGROUND: The high prevalence and mortality rate of coronavirus disease 2019 (COVID-19) is a major global concern. Bioinformatics approaches have helped to develop new strategies to combat infectious agents, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Indeed, the structural proteins of microorganisms provide suitable epitopes for the development of vaccines to prevent infectious diseases. OBJECTIVES: The present study aimed to use bioinformatics tools to find peptides from the membrane (M) and nucleocapsid (N) proteins with effective cellular and humoral immunogenicity. MATERIAL AND METHODS: Sequences of the M and N proteins were sourced from the National Center for Biotechnology Information (NCBI). The conserved regions of the proteins with the highest immunogenicity were identified and assessed using different servers, and the physicochemical and biochemical properties of the epitopes were evaluated. Finally, allergenicity, antigenicity and docking to human leukocyte antigen (HLA) were investigated. RESULTS: The data indicated that the best epitopes were LVIGFLFLT and LFLTWICLL (as membrane epitopes), and KLDDKDPNFKDQ (as a nucleocapsid epitope), with significant immunogenicity and no evidence of allergenicity. The 3 epitopes are stable peptides that can interact with HLA to induce strong immune responses. CONCLUSIONS: The findings indicate that 3 common epitopes could effectively elicit an immune response against the disease. Hence, in vitro and in vivo studies are recommended to confirm the theoretical information.

Antibacterial, Antioxidant and Nutraceutical Potential of New Culinary-Medicinal Mushroom Russula lakhanpalii (Agaricomycetes) from India.

Russula lakhanpalii is a wild edible mushroom, collected from Pedkhal block of Pauri Garhwal, India. The nutritional composition, antioxidant activity (AOA), and antibacterial activity (ABA) of R. lakhanpalii were analyzed for the first time in this study. Dried fruiting bodies of R. lakhanpalii were reported to contain 17.7% ash, 10% crude fiber, 13.4% protein, 30.9% carbohydrate, and 5% unsaturated lipids. In addition, 10.22-72.56% DPPH scavenging activity also confirmed the good antioxidant nature of R. lakhanpalii. The methanolic extract of R. lakhanpalii fruiting bodies inhibited the growth of five pathogenic bacteria in vitro; Klebsiella pneumoniae (MTCC 4030), Micrococcus luteus (MTCC 1809), Staphylococcus aureus (MTCC 1144), Escherichia coli (MTCC 68), and Streptococcus pneumoniae (MTCC 655). The maximum and minimum zone of inhibitions (ZOIs) reported were 17.8 ± 1.04 mm (K. pneumoniae) and 11.16 ± 0.76 mm, (E. coli), respectively. The noticeable feature of the extract was the inhibition of erythromycin-resistant E. coli and M. luteus by it, which were resistant to 15 µg/disc concentration of erythromycin. Dietary components, antibacterial and antioxidant potentials of R. lakhanpalii suggested its nutraceutical and medicinal applications.

Prevalence of enteric adenovirus and co-infection with rotavirus in children under 15 years of age with gastroenteritis in Qom, Iran.

Aim: The current study is the first performed in Qom to determine the prevalence of adenovirus and co-infections with rotavirus in children aged <15 years with gastroenteritis symptoms. Background: Gastroenteritis-associated viral infections are a cause of death among young children worldwide, especially in developing countries. The Adenovirus species F (40 and 41) are responsible for a range of acute diarrhea cases among infants and children. Methods: Over a period of 9 months, a total of 130 children suffering from intestinal problems who referred to the infectious ward of Children's Hospital were enrolled in the current study. After clinical examination and collection of demographic information, fecal samples were obtained from the patients. Viral genomes were extracted with a commercial kit and amplified and typed by adenovirus-specific PCR assay. Adenovirus-positive samples were also evaluated for co-infection with rotavirus. Results: Patients had a mean±SD age of 2.66±2.72 years; 63.1% of patients were male and 36.9% were female. Adenovirus infection was identified in 23 cases (17.7%), 21 (91.0%) and 2 (9.0%) of which were type 41 and type 40, respectively. Fever was the most common clinical manifestation among adenovirus-positive patients. No significant difference was observed between adenovirus infection and clinical symptoms, seasonal pattern, or serum laboratory results. Co-infection was found in only 5 cases (21.7%). Conclusion: This study was the first to demonstrate adenovirus infection with a relatively high prevalence among children, especially infants, in Qom. The findings further revealed co-infection with rotavirus, indicating a health problem in this region.

miR-149-PARP-2 Signaling Regulates E-cadherin and N-cadherin Expression in the Murine Model of Endometrium Receptivity.

Cadherins play an essential role in the attachment of the blastocyst to the endometrium, a process known as endometrial receptivity. Loss of E-cadherin expression is essential during the process, while the expression level of the other cadherin, N-cadherin, has been reported to be altered in cases of infertility. Both E-cadherin and N-cadherin can be regulated by members of the PARP family. Specifically, PARP-2, which is under the epigenetic control of miR-149, has been observed to promote E-cadherin expression in other human cells. We investigated the roles of E-cadherin and N-cadherin in endometrial receptivity using mouse models for normal endometrial receptivity, pseudopregnancy, and LPS-induced endometrial receptivity failure. E-cadherin and phosphorylated E-cadherin were predominantly expressed during pre-receptive stages as well as in the implantation site of the receptive stage, which were observed reduced during the later stages of implantation in both implantation and non-implantation regions, while N-cadherin was detected only at pre-receptive stages. E-cadherin and N-cadherin were also seen in the uterus during pseudopregnancy, showing a downregulation trend during receptive and post-receptive stages. LPS-induced failed endometrial receptivity showed upregulation of E-cadherin and downregulation of N-cadherin. The E-cadherin expression promoter, GSK-3, was lost and its suppressor, SLUG was upregulated during normal course of endometrial receptivity in mouse model, while GSK-3 was increased during LPS-induced failed embryo implantation. In an in vitro model of embryo implantation, E-cadherin expression is promoted by PARP-2 and regulated by miR-149 epigenetically in human endometrium epithelial cells. In conclusion, E-cadherin is predominantly expressed during pre-receptive stage and promoted by PARP-2, which is regulated by miR-149 in the endometrial epithelial cells.

Ethnomedicinal Plants Used in the Health Care System: Survey of the Mid Hills of Solan District, Himachal Pradesh, India.

The study was performed in the mid hills of the Dharampur region in Solan district of Himachal Pradesh, India. At the study site, a total of 115 medicinal plants were documented (38 trees, 37 herbs, 34 shrubs, 5 climbers, 1 fern, and 1 grass). In the study region, extensive field surveys were performed between March 2020 and August 2021. Indigenous knowledge of wild medicinal plants was collected through questionnaires, discussions, and personal interviews during field trips. Plants with their correct nomenclature were arranged by botanical name, family, common name, habitat, parts used, routes used, and diseases treated. In the present study, the predominant family was Rosaceae, which represented the maximum number of plant species, 10, followed by Asteraceae and Lamiaceae, which represented 8 plant species. The rural inhabitants of the Dharampur region in the Solan district have been using local plants for primary health care and the treatment of various diseases for a longer time. However, information related to the traditional knowledge of medicinal plants was not documented. The rural inhabitants of the Dharampur region reported that the new generation is not so interested in traditional knowledge of medicinal plants due to modernization in society, so there is an urgent need to document ethnomedicinal plants before such knowledge becomes inaccessible and extinct.

Poly(ADP-ribose) polymerase-2 is essential for endometrial receptivity and blastocyst implantation, and regulated by caspase-8.

Embryo implantation is a very complex process and several factors play important roles. Using a mouse model, we investigated the functions of PARP-2 and caspase-8 during endometrial receptivity for blastocyst implantation. We found that PARP-2 was upregulated at the receptive stage's implantation region and predominantly expressed in the endometrial stromal region, but downregulated during pregnancy failure and pseudopregnancy. To reinforce the necessity of PARP-2 for embryo implantation, we pharmacologically inhibited PARP-2 'before' & 'after' embryo arrival and observed a reduction in blastocyst implantation. Conversely, elevated caspase-8 expression and activity during pseudopregnancy, delayed implantation, and embryo implantation failure conditions and decreased levels in the decidualization exhibited an inverse pattern with PARP-2, suggesting caspase-8 as a negative regulator for embryo implantation. In vitro caspase-8 downregulates the PARP-2 activity in the mouse endometrial epithelial and stromal cells. These data suggest that PARP-2 and its negative regulation by caspase-8 constitute a crucial step in embryo implantation.

Antibacterial potential of Saussurea obvallata petroleum ether extract: A spiritually revered medicinal plant.

Uttarakhand Himalayan region holds Asteraceae or Compositae as the largest family of flowering, medicinal and aromatic plants. Species belonging to this family rises from low altitude to the alpine region. Among Asteraceae, Saussurea obvallata (DC.) Edgew. is widely used in several indigenous systems of medicine. Flowers, leaves and rhizomes of S. obvallata are used for several traditional, religious, therapeutic and ornamental purposes. Aim of this study was to determine the chemical composition and antibacterial efficacy of petroleum ether extract (PEE) of S. obvallata. Gas chromatography-mass spectrometry (GC-MS) analysis was used for identifying phytochemicals present in the plant extract. Furthermore, the PEE was assessed for in-vitro antibacterial activity against selected Gram positive and negative strains. Structure of squalene and α-linolenic acid methyl ester were identified in PEE by GC-MS analysis, by comparing the results obtained with NIST library and literature reports. PEE exhibited significant activity against Staphylococcus aureus, Escherichia coli, Bacillus cereus, Bacillus subtilis with IC50 value of 87.2 ± 1.6, 98.4 ± 1.1 and 90.2 ± 1.8 µg/ml, respectively. These results showed that squalene and α-linolenic acid derivative identified in S. obvallata may be responsible for the observed antibacterial activity. To the best of our knowledge, this is the first report focused on the chemical composition and antibacterial activity of S. obvallata.

Integrin beta8 (ITGB8) activates VAV-RAC1 signaling via FAK in the acquisition of endometrial epithelial cell receptivity for blastocyst implantation.

Integrin beta8 (ITGB8) is involved in the endometrial receptivity. The blastocyst first interacts with the luminal endometrial epithelial cells during its implantation; therefore, we have investigated the signaling of ITGB8 via FAK and VAV-RAC1 in the endometrial epithelial cells. Integrin beta8 was found elevated in epithelial cells at late-pre-receptive (day4, 1600 h) and receptive (day5, 0500 h) stages of endometrial receptivity period in the mouse. Integrins downstream molecule FAK has demonstrated an increased expression and phosphorylation (Y397) in the endometrium as well as in the isolated endometrial epithelial cells during receptive and post-receptive stages. Integrin beta8 can functionally interact with FAK, VAV and RAC1 as the levels of phosphorylated-FAK, and VAV along with the RAC-GTP form was reduced after ITGB8 knockdown in the endometrial epithelial cells and uterus. Further, VAV and RAC1 were seen poorly active in the absence of FAK activity, suggesting a crosstalk of ITGB8 and FAK for VAV and RAC1 activation in the endometrial epithelial cells. Silencing of ITGB8 expression and inhibition of FAK activity in the Ishikawa cells rendered poor attachment of JAr spheroids. In conclusion, ITGB8 activates VAV-RAC1 signaling axis via FAK to facilitate the endometrial epithelial cell receptivity for the attachment of blastocyst.

S-Enantiomer of the Antitubercular Compound S006-830 Complements Activity of Frontline TB Drugs and Targets Biogenesis of Mycobacterium tuberculosis Cell Envelope.

A synthetic molecule S006-830, belonging to the class of thiophene-containing trisubstituted methanes, had shown good in vitro and in vivo bactericidal activity against drug-sensitive and drug-resistant Mycobacterium tuberculosis (Mtb). The molecule had also shown good druglike pharmacokinetic properties. However, S006-830 is a racemic mixture of two enantiomers, one of which could possess a better pharmacological profile than the other. We purified both the enantiomers on a chiral column and observed that S-enantiomer has a significantly higher inhibitory and cidal activity against Mtb than the R-enantiomer. Action of S-S006-830 was "synergistic" for rifampicin and "additive" for isoniazid and ethambutol. The combination of S-S006-830 and rifampicin produced 100% kill of Mtb within 8 days. In a chemical proteomics approach using matrix-bound compound to pull down its target protein(s) from Mtb membrane, FabG4 (ß-ketoacyl CoA reductase, EC 1.1.1.100) emerged as the most likely target for S-S006-830. In target validation assays, the compound exhibited 2-fold higher inhibitory concentration for an Mtb construct overexpressing FabG4. In addition, it inhibited mycolic acid biosynthesis and formation of biofilms by Mtb. Molecular docking of S-S006-830 with FabG4 was consistent with the experimental data. These results support the development of S-S006-830 as a novel lead against tuberculosis.

Correction to "S-Enantiomer of the Antitubercular Compound S006-830 Complements Activity of Frontline TB Drugs and Targets Biogenesis of Mycobacterium tuberculosis Cell Envelope".

[This corrects the article DOI: 10.1021/acsomega.7b01281.].

RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model.

The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCK1, and RAC1 in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCK1, VAV, and RAC1 expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.

Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation.

Endoglin is a TGF-ß receptor that is expressed in uterine endothelial and stromal cells in addition to trophoblast expression. However, the functional importance of endoglin in the embryo implantation process is not clear. We observed endoglin expression in the endometrium throughout the stages of its receptivity; however, its expression was enhanced during the receptive stage. Endoglin expression was predominant in epithelial cells of the lumen and glands, but showed a milder expression in stromal cells. Endoglin expression was initially observed in the primary decidual zone and later extended to the secondary decidua zone. Knockdown of endoglin via siRNA reduced the implantation sites along with the blastocyst numbers. Mouse blastocyst with endoglin-silenced endometrial epithelial cells (human and mouse origin) showed poor trophoblast outgrowth, which suggests an essential role for endoglin during endometrial receptivity. In conclusion, our findings reveal the association of endoglin with endometrial receptivity, which is important for embryo attachment.

Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.

Expression and activity of Rac1 is negatively affected in the dehydroepiandrosterone induced polycystic ovary of mouse.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is characterized by the presence of multiple follicular cysts, giving rise to infertility due to anovulation. This syndrome affects about 10% of women, worldwide. The exact molecular mechanism leading to PCOS remains obscure. RhoGTPase has been associated with oogenesis, but its role in PCOS remains unexplored. Therefore, we attempted to elucidate the Vav-Rac1 signaling in PCOS mice model. METHODS: We generated a PCOS mice model by injecting dehydroepiandrosterone (DHEA) for a period of 20 days. The expression levels of Rac1, pRac1, Vav, pVav and Caveolin1 were analyzed by employing immuno-blotting and densitometry. The association between Vav and Rac1 proteins were studied by immuno-precipitation. Furthermore, we analyzed the activity of Rac1 and levels of inhibin B and 17ß-estradiol in ovary using biochemical assays. RESULTS: The presence of multiple follicular cysts in ovary were confirmed by histology. The activity of Rac1 (GTP bound state) was significantly reduced in the PCOS ovary. Similarly, the expression levels of Rac1 and its phosphorylated form (pRac1) were decreased in PCOS in comparison to the sham ovary. The expression level and activity (phosphorylated form) of guanine nucleotide exchanger of Rac1, Vav, was moderately down-regulated. We observed comparatively increased expressions of Caveolin1, 17ß-estradiol, and inhibin B in the polycystic ovary. CONCLUSION: We conclude that hyperandrogenization (PCOS) by DHEA diminishes ovarian Rac1 and Vav expression and activity along with an increase in expression of Caveolin1. This is accompanied by an increase in the intra-ovarian level of '17 ß-estradiol and inhibin B.

PARP1 during embryo implantation and its upregulation by oestradiol in mice.

Pregnancy requires successful implantation of an embryo, which occurs during a restricted period defined as 'receptivity of the endometrium' and is influenced by the ovarian steroids progesterone and oestradiol. The role of poly(ADP-ribose)polymerase-1 (PARP1) in apoptosis is well established. However, it is also involved in cell differentiation, proliferation and tissue remodelling. Previous studies have described the presence of PARP in the uterus, but its exact role in embryo implantation is not yet elucidated. Hence, in this study, we studied the expression of PARP1 in the uterus during embryo implantation and decidualisation, and its regulation by ovarian steroids. Our results show upregulation of the native form of PARP1 (∼116 kDa) in the cytosolic and nuclear compartments of implantation and non-implantation sites at day 5 (0500 h), followed by downregulation at day 5 (1000 h), during the embryo implantation period. The transcript level of Parp1 was also augmented during day 5 (0500 h). Inhibition of PARP1 activity by the drug EB-47 decreased the number of embryo implantation sites and blastocysts at day 5 (1000 h). Further, cleavage of native PARP1 was due to the activity of caspase-3 during the peri-implantation stage (day 5 (0500 h)), and is also required for embryo implantation, as inhibition of its activity compromised blastocyst implantation. The native (∼116 kDa) and cleaved (∼89 kDa) forms of PARP1 were both elevated during decidualisation of the uterus. Furthermore, the expression level of PARP1 in the uterus was found to be under the control of the hormone oestrogen. Our results clearly demonstrate that PARP1 participates in the process of embryo implantation.

Transforming growth factor-beta 1 (TGF-B1) liberation from its latent complex during embryo implantation and its regulation by estradiol in mouse.

Transforming growth factor-beta (TGF-B) plays an important role in embryo implantation; however, TGF-B requires liberation from its inactive latent forms (i.e., large latent TGF-B complex [LLC] and small latent TGF-B complex [SLC]) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors (TGF-BRs), which in turn activate SMAD2/3. Activation of TGF-B1 from its latent complexes in the uterus is not yet deciphered. We investigated uterine latent TGF-B1 complex and its biologically active form during implantation, decidualization, and delayed implantation. Our study, utilizing nonreducing SDS-PAGE followed by Western blotting and immunoblotting with TGF-B1, LTBP1, and latency-associated peptide, showed the presence of LLC and SLC in the uterine extracellular matrix and plasma membranous protein fraction during stages of the implantation period. A biologically active form of TGF-B1 (~17-kDa monomer) was highly elevated in the uterine plasma membranous compartment at the peri-implantation stage (implantation and nonimplantation sites). Administration of hydroxychloroquine (an inhibitor of pro-TGF-B processing) at the preimplantation stage was able to block the liberation of biologically active TGF-B1 from its latent complex at the postimplantation stage; as a consequence, the number of implantation sites was reduced at Day 5 (1000 h), as was the number of fetuses at Day 13. The inhibition of TGF-B1 showed reduced levels of phosphorylated SMAD3. Further, the delayed-implantation mouse model showed progesterone and estradiol coordination to release the active TGF-B1 form from its latent complex in the receptive endometrium. This study demonstrates the importance of liberation of biologically active TGF-B1 during the implantation period and its regulation by estradiol.