Unraveling the Nephroprotective Potential of Papaverine against Cisplatin Toxicity through Mitigating Oxidative Stress and Inflammation: Insights from In Silico, In Vitro, and In Vivo Investigations.

Cisplatin is a potent compound in anti-tumor chemotherapy; however, its clinical utility is hampered by dose-limiting nephrotoxicity. This study investigated whether papaverine could mitigate cisplatin-induced kidney damage while preserving its chemotherapeutic efficacy. Integrative bioinformatics analysis predicted papaverine modulation of the mechanistic pathways related to cisplatin renal toxicity; notably, mitogen-activated protein kinase 1 (MAPK1) signaling. We validated protective effects in normal kidney cells without interfering with cisplatin cytotoxicity on a cancer cell line. Concurrent in vivo administration of papaverine alongside cisplatin in rats prevented elevations in nephrotoxicity markers, including serum creatinine, blood urea nitrogen, and renal oxidative stress markers (malondialdehyde, inducible nitric oxide synthase (iNOS), and pro-inflammatory cytokines), as tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). Papaverine also reduced apoptosis markers such as Bcl2 and Bcl-2-associated X protein (Bax) and kidney injury molecule-1 (KIM-1), and histological damage. In addition, it upregulates antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) while boosting anti-inflammatory signaling interleukin-10 (IL-10). These effects were underlined by the ability of Papaverine to downregulate MAPK-1 expression. Overall, these findings show papaverine could protect against cisplatin kidney damage without reducing its cytotoxic activity. Further research would allow the transition of these results to clinical practice.

Walnut Kernel Oil and Defatted Extracts Enhance Mesenchymal Stem Cell Stemness and Delay Senescence.

Decreased stemness and increased cellular senescence impair the ability of mesenchymal stem cells (MSCs) to renew themselves, change into different cell types, and contribute to regenerative medicine. There is an urgent need to discover new compounds that can boost MSCs' stemness and delay senescence. Therefore, this study aimed to investigate the impact of walnut kernel oil (WKO) and defatted (WKD) extracts on bone marrow (BM)-MSC stemness and senescence. Premature senescence and inflammation were induced in BM-MSCs using H2O2 and LPS, respectively. Phytochemical constituents of WKO and WKD extracts were detected by HPLC. The stemness (proliferation and migration), senescence-related markers (p53, p21, SIRT1, and AMPK), oxidative stress/antioxidant markers, inflammatory cytokines, and cell cycle of BM-MSCs were measured by MTT assay, qPCR, ELISA, and flow cytometry. WKO and WKD extracts improved rat BM-MSC stemness, as evidenced by (1) increased cell viability, (2) decreased apoptosis (low levels of Bax and caspase3 and high levels of Bcl2), (3) upregulated MMP9 and downregulated TIMP1 expression, and (4) cell cycle arrest in the G0/G1 phase and declined cell number in the S and G2/M phases. Additionally, WKO and WKD extracts reduced rat BM-MSC senescence, as indicated by (1) decreased p53 and p21 expression, (2) upregulated expression and levels of SIRT1 and AMPK, (3) reduced levels of ROS and improved antioxidant activity (higher activity of CAT, SOD, and GPx and upregulated expression of NrF2 and HO-1), and (4) declined levels of TNFα, IL1ß, and NF-κB. When compared to the WKO extract, the WKD extract had a greater impact on the induction of stemness and reduction of senescence of BM-MSCs due to its stronger antioxidant activity, which could be attributed to its higher levels of flavonoids and phenolic compounds, as detected by HPLC analysis. WKO and WKD extracts enhance rat BM-MSC stemness and protect them from senescence, suggesting their potential use as enhancers to increase MSCs' therapeutic efficacy.

Association of LEPR polymorphisms with egg production and growth performance in female Japanese quails.

This study aimed to screen intron 8 of the leptin receptor (LEPR) gene for polymorphisms in female Japanese quails. Two adjacent novel SNPs (A277G and A304G) were detected using PCR-SSCP and sequencing. These SNPs produced three haplotypes (AA/AA, AG/AG, and GG/GG) that were significantly (p ≤ 0.05) associated with growth and egg production traits. GG/GG haplotype-quails had significantly (p ≤ 0.05) lower egg production, feed intake, growth performance, lipid profile, serum levels of sex hormones (estradiol, progesterone, FSH, LH), and ovarian expressions of survivin, FSHR, and IGF1 than other quails. However, GG/GG quails had significantly (p ≤ 0.05) higher serum levels of LEP and mRNA levels of LEPR, LEP, and caspase 3 in the hypothalamus and ovaries. These higher levels of LEP/LEPR could not only reduce feed intake and body weight gain but also could induce apoptosis of ovarian cells (as indicated by lower survivin and IGF1 and higher caspase3 expression) which could inhibit the development of the follicles and the release of sex hormones with a subsequent decrease in egg production in GG/GG quails. Therefore, with these results, we suggest selecting Japanese quails with AA/AA and AG/AG haplotypes to improve the reproduction and growth performance of this flock.

Quercetin-Ameliorated, Multi-Walled Carbon Nanotubes-Induced Immunotoxic, Inflammatory, and Oxidative Effects in Mice.

The expanding uses of carbon nanotubes (CNTs) in industry and medicine have raised concerns about their toxicity on human and animal health. CNTs, including multi-walled nanotubes (MWCNTs), have been reported to induce immunotoxic, inflammatory, and oxidative effects. Quercetin is a natural flavonoid present in many vegetables and fruits and has immunomodulatory, anti-inflammatory, and antioxidant properties. Herein, we investigated the protective effects of quercetin on pristine MWCNTs-induced immunotoxicity in mice. In comparison with two doses of MWCNTs, high doses [0.5 mg/kg body weight (BW), once intraperitoneally (IP)] caused higher immunotoxic, inflammatory, and oxidative effects than low doses (0.25 mg/kg BW, once IP). Administration of quercetin (30 mg/kg BW, IP for 2 weeks) relieved these deleterious effects as evidenced by (1) reduced spleen weight, (2) increased number of total leukocytes, lymphocytes, and neutrophils, (3) elevated serum levels of IgM, IgG, and IgA, (4) decreased lipid peroxide malondialdehyde levels and increased levels of antioxidant markers reduced glutathione, superoxide dismutase, and catalase in the spleen, (5) decreased concentrations and mRNA levels of inflammatory markers tumor necrosis factor-alpha (TNFα), interleukin 1 beta (IL1ß), and IL6 in the spleen, (6) downregulated expression of immunomodulatory genes transforming growth factor-beta (TGFß), cyclooxygenase2 (COX2), and IL10, and (7) regenerative histological changes as indicated by decreased mononuclear cell infiltration, minimized degenerative changes and restored lymphocytes depletion in the spleen. These results infer that quercetin can ameliorate MWCNTs-induced immunotoxic, inflammatory, and oxidative effects.

Avocado Seeds-Mediated Alleviation of Cyclosporine A-Induced Hepatotoxicity Involves the Inhibition of Oxidative Stress and Proapoptotic Endoplasmic Reticulum Stress.

Previous studies reported disrupted hepatic function and structure following the administration of cyclosporine A (CsA) in humans and animals. Recently, we found that avocado seeds (AvS) ameliorated CsA-induced nephrotoxicity in rats. As a continuation, herein we checked whether AvS could also attenuate CsA-induced hepatotoxicity in rats. Subcutaneous injection of CsA (5 mg/kg) for 7 days triggered hepatotoxicity in rats, as indicated by liver dysfunction, redox imbalance, and histopathological changes. Oral administration of 5% AvS powder for 4 weeks ameliorated CsA-induced hepatotoxicity, as evidenced by (1) decreased levels of liver damage parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin), (2) resumed redox balance in the liver (reduced malondialdehyde (MDA) and increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), (3) downregulated hepatic expression of endoplasmic reticulum (ER) stress-related genes (X-box binding protein 1 (XBP1), binding immunoglobulin protein (BIP), C/EBP homologous protein (CHOP)), and apoptosis-related genes (Bax and Casp3), (4) upregulated expression of the anti-apoptotic gene Bcl2, (5) reduced DNA damage, and (6) improved liver histology. These results highlight the ability of AvS to ameliorate CsA-induced hepatotoxicity via the inhibition of oxidative stress and proapoptotic ER stress.

Bee venom and its active component Melittin synergistically potentiate the anticancer effect of Sorafenib against HepG2 cells.

There are current attempts to find a safe substitute or adjuvant for Sorafenib (Sorf), the standard treatment for advanced hepatocellular carcinoma (HCC), as it triggers very harsh side effects and drug-resistance. The therapeutic properties of Bee Venom (BV) and its active component, Melittin (Mel), make them suitable candidates as potential anti-cancer agents per-se or as adjuvants for cancer chemotherapy. Hence, this study aimed to evaluate the combining effect of BV and Mel with Sorf on HepG2 cells and to investigate their molecular mechanisms of action. Docking between Mel and different tumor-markers was performed. The cytotoxicity of BV, Mel and Sorf on HepG2 and THLE-2 cells was conducted. Combinations of BV/Sorf and Mel/Sorf were performed in non-constant ratios on HepG2. Expression of major cancer-related genes and oxidative stress status was evaluated and the cell cycle was analyzed. The computational analysis showed that Mel can bind to and inhibit XIAP, Bcl2, MDM2, CDK2 and MMP12. Single treatments of BV, Mel and Sorf on HepG2 showed lower IC50than on THLE-2. All combinations revealed a synergistic effect at a combination index (CI) < 1. Significant upregulation (p < 0.05) of p53, Bax, Cas3, Cas7 and PTEN and significant downregulation (p < 0.05) of Bcl-2, Cyclin-D1, Rac1, Nf-κB, HIF-1a, VEGF and MMP9 were observed. The oxidative stress markers including MDA, SOD, CAT and GPx showed insignificant changes, while the cell cycle was arrested at G2/M phase. In conclusion, BV and Mel have a synergistic anticancer effect with Sorf on HepG2 that may represent a new enhancing strategy for HCC treatment.

Differential molecular and hormonal changes in oocytes, granulosa cells and follicular fluid of pregnant and non-pregnant camels.

This study aimed to compare the expression of genes regulating follicles development, survival and steroid hormones secretion in oocytes and granulosa cells (GCs) and study the correlation between their expression and follicular fluid (FF) levels of progesterone (P4) in pregnant and non-pregnant camels. In total, 138 ovarian pairs from slaughtered camels were used. Gene expression and hormonal assay were determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The obtained results revealed that the number of follicles (3-8 mm) was significantly (P < 0.05) lower in pregnant, compared with non-pregnant, camels. P4 level in the FF was significantly (P < 0.05) higher in pregnant, compared with non-pregnant, camels. However, no significant (P > 0.05) difference was noticed in the oestradiol (E2) level. STAR, PTEN, IGF1 and BCL2 mRNA levels were significantly higher in GCs and significantly lower in oocytes of pregnant, compared with non-pregnant, camels. However, follicle-stimulating hormone receptor (FSHR) mRNA level was significantly lower in GCs and oocytes, and the BMP15 mRNA level was significantly lower in oocytes of pregnant, compared with non-pregnant, camels. P4 level in FF was positively correlated with STAR, PTEN, IGF1 and BCL2 mRNA levels in GCs and negatively correlated with BMP15 mRNA levels in oocytes and FSHR mRNA levels in GCs and oocytes of pregnant camels. It could be concluded that pregnancy-induced variations in oocytes and GC expression of BMP15, IGF1, FSHR, STAR, BCL2, and PTEN genes might be associated with a decrease in the number of follicles and an increase in the FF level of P4.

Panax ginseng modulates oxidative stress, DNA damage, apoptosis, and inflammations induced by silicon dioxide nanoparticles in rats.

Silicon dioxide nanoparticles (SiO2 NPs) are extensively used in cosmetics, food, and drug delivery. The main mechanism of SiO2 NPs toxicities depends on oxidative stress. Ginseng (Panax ginseng Meyer) is used in various medicinal applications because of its antioxidant efficiency. Therefore, the present study was carried out to investigate the possible combated role of ginseng against SiO2 NPs toxicity in rat liver. Thirty-five male rats (160-180 g) were allocated into five groups of seven rats each, randomly. The first group was used as a control while groups 2, 3, 4, and 5 were treated orally with ginseng (Gin; 75 mg/kg, 1/10 LD50 ), SiO2 NPs, (200 mg/kg, 1/10 LD50 ), Gin + SiO2 NPs (protection group), and SiO2 NPs + Gin (therapeutic group) for 5 weeks, respectively. Treatment with SiO2 NPs increased lipid peroxidation, liver function enzymes, and decreased antioxidant enzymes (SOD, CAT, GPx, GST) activity and non-enzymatic antioxidant (GSH) level. SiO2 NPs administration motivated liver apoptosis as revealed by the upregulation of the apoptotic genes, Bcl2-associated x protein (Bax), and Beclin 1 and downregulation of the anti-apoptotic gene, B-cell lymphoma 2 (Bcl2) as well as increase in DNA damage. Also, SiO2 NPs administration caused inflammation as indicated by upregulation of the inflammation-related genes (interleukin 1 beta [IL1ß], tumor necrosis factor-alpha [TNFα], nuclear factor kappa B [NFκB], cyclooxygenase 2 [Cox2], transforming growth factor-beta 1 [TGFß1]) as well as cell cycle arrest in the G0/G1 phase of liver cells. Moreover, histopathological examination proved the biochemical and molecular perturbations occurred due to SiO2 NPs toxicity. On the other hand, ginseng caused a significant modulation on the deleterious effects induced by SiO2 NPs in rat liver. In conclusion, ginseng has a potent preventive effect than the therapeutic one and might be used in the treatment of SiO2 NPs hepatotoxicity.

Untargeted Metabolic Profiling of Extracellular Vesicles of SARS-CoV-2-Infected Patients Shows Presence of Potent Anti-Inflammatory Metabolites.

Extracellular vesicles (EVs) carry important biomolecules, including metabolites, and contribute to the spread and pathogenesis of some viruses. However, to date, limited data are available on EV metabolite content that might play a crucial role during infection with the SARS-CoV-2 virus. Therefore, this study aimed to perform untargeted metabolomics to identify key metabolites and associated pathways that are present in EVs, isolated from the serum of COVID-19 patients. The results showed the presence of antivirals and antibiotics such as Foscarnet, Indinavir, and lymecycline in EVs from patients treated with these drugs. Moreover, increased levels of anti-inflammatory metabolites such as LysoPS, 7-α,25-Dihydroxycholesterol, and 15-d-PGJ2 were detected in EVs from COVID-19 patients when compared with controls. Further, we found decreased levels of metabolites associated with coagulation, such as thromboxane and elaidic acid, in EVs from COVID-19 patients. These findings suggest that EVs not only carry active drug molecules but also anti-inflammatory metabolites, clearly suggesting that exosomes might play a crucial role in negotiating with heightened inflammation during COVID-19 infection. These preliminary results could also pave the way for the identification of novel metabolites that might act as critical regulators of inflammatory pathways during viral infections.

Oriental Hornet (Vespa orientalis) Larval Extracts Induce Antiproliferative, Antioxidant, Anti-Inflammatory, and Anti-Migratory Effects on MCF7 Cells.

The use of insects as a feasible and useful natural product resource is a novel and promising option in alternative medicine. Several components from insects and their larvae have been found to inhibit molecular pathways in different stages of cancer. This study aimed to analyze the effect of aqueous and alcoholic extracts of Vespa orientalis larvae on breast cancer MCF7 cells and investigate the underlying mechanisms. Our results showed that individual treatment with 5% aqueous or alcoholic larval extract inhibited MCF7 proliferation but had no cytotoxic effect on normal Vero cells. The anticancer effect was mediated through (1) induction of apoptosis, as indicated by increased expression of apoptotic genes (Bax, caspase3, and p53) and decreased expression of the anti-apoptotic gene Bcl2; (2) suppression of intracellular reactive oxygen species; (3) elevation of antioxidant enzymes (CAT, SOD, and GPx) and upregulation of the antioxidant regulator Nrf2 and its downstream target HO-1; (4) inhibition of migration as revealed by in vitro wound healing assay and downregulation of the migration-related gene MMP9 and upregulation of the anti-migratory gene TIMP1; and (5) downregulation of inflammation-related genes (NFκB and IL8). The aqueous extract exhibited the best anticancer effect with higher antioxidant activities but lower anti-inflammatory properties than the alcoholic extract. HPLC analysis revealed the presence of several flavonoids and phenolic compounds with highest concentrations for resveratrol and naringenin in aqueous extract and rosmarinic acid in alcoholic extract. This is the first report to explain the intracellular pathway by which flavonoids and phenolic compounds-rich extracts of Vespa orientalis larvae could induce MCF7 cell viability loss through the initiation of apoptosis, activation of antioxidants, and inhibition of migration and inflammation. Therefore, these extracts could be used as adjuvants for anticancer drugs and as antioxidant and anti-inflammatory agents.

The role of chick Ebf genes in the mediolateral patterning of the somites.

This study was conducted to check whether the three chick Early B-cell Factor (Ebf) genes, particularly cEbf1, would be targets for Shh and Bmp signals during somites mediolateral (ML) patterning. Tissue manipulations and gain and loss of function experiments for Shh and Bmp4 were performed and the results revealed that cEbf1 expression was initiated in the cranial presomitic mesoderm by low dose of Bmp4 from the lateral mesoderm and maintained in the ventromedial part of the epithelial somite and the medial sclerotome by Shh from the notochord; while cEbf2/3 expression was induced and maintained by Bmp4 and inhibited by high dose of Shh. To determine whether Ebf1 plays a role in somite patterning, transfection of a dominant-negative construct was carried out; this showed suppression of cPax1 expression in the medial sclerotome and upregulation and medial expansion of cEbf3 and cPax3 expression in sclerotome and dermomyotome, respectively, suggesting that Ebf1 is important for ML patterning. Thus, it is possible that low doses of Bmp4 set up Ebf1 expression which, together with Shh from the notochord, leads to establishment of the medial sclerotome and suppression of lateral identities. These data also conclude that Bmp4 is required in both the medial and lateral domain of the somitic mesoderm to keep the ML identity of the sclerotome through maintenance of cEbf gene expression. These striking findings are novel and give a new insight on the role of Bmp4 on mediolateral patterning of somites.

Design, synthesis, modeling studies and biological evaluation of thiazolidine derivatives containing pyrazole core as potential anti-diabetic PPAR-γ agonists and anti-inflammatory COX-2 selective inhibitors.

Nowadays, diabetes and its associated inflammatory complications are important public health problems worldwide. Market limitations of drugs with dual actions as anti-inflammatory (AI) and anti-diabetic have been led to a temptation for focusing on the discovery and development of new compounds with potential AI and anti-diabetic activities. Herein, we synthesized two new series containing pyrazole ring with vicinal diaryl rings as selective COX-2 moiety and thiazolidindione (series 12a-f) or thiazolidinone (series 13a-f) as anti-diabetic moiety and the two moieties were linked together with methylene or methylenehydrazone functionality. The two series were evaluated for their COX inhibition, AI activity and ulcerogenic liability and for the anti-diabetic activity; 12a-f and 13a-f were assessed in vitro against α-glucosidase, ß- glucosidase, in vivo hypoglycemic activity (one day and 15 days studies) in addition to PPARγ activation study. Four compounds (12c, 12f, 13b and 13f) had higher COX-2 S.I. (8.69-9.26) than the COX-2 selective drug celecoxib (COX-2 S.I. = 8.60) and showed the highest AI activities and the lowest ulcerogenicity than other derivatives. Also, two thiazolidindione derivatives 12e and 12f and two thiazolidinone derivatives 13b and 13c showed higher inhibitory activities against α- and ß-glucosidase (% inhibitory activity = 62.15, 55.30, 65.37, 59.08 for α-glucosidase and 57.42, 60.07, 58.19, 66.90 for ß-glucosidase respectively) than reference compounds (acarbose with % inhibitory activity = 49.50 for α-glucosidase and d-saccharic acid 1,4-lactone monohydrate with % inhibitory activity = 53.42 for ß-glucosidase) and also showed good PPAR-γ activation and good hypoglycemic effect in comparison to pioglitazone and rosiglitazone. Moreover, Shape comparison and docking studies were carried out to understand their interaction and similarity with standard drugs.

Soya bean rich diet is associated with adult male rat aggressive behavior: relation to RF amide-related peptide 3-aromatase-neuroestrogen pathway in the brain.

Relation between soya bean (SB) consumption and aggressive behavior has not been elucidated yet. Thus, this study was conducted to investigate the effect of large amount of SB consumption on adult male rats' aggressive behavior through investigating changes in the expression of gonadotropin-inhibitory hormone/ RF amide-related peptide 3 (GnIH/RFRP3), neuropeptide FF receptor, cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19A1), estrogen receptors α and ß and the levels of neuroestrogen, dopamine, glutamate and testosterone as well as aromatase activity in the brain. Adult male rats were divided into three equal groups: group I, control group, received standard diet; group II and group III received 25% and 50% SB of their standard diet contents, respectively, for 12 weeks. The obtained results showed that feeding male rats with large amount of SB could induce aggressive behavior in a dose dependant manner possibly through inhibition of brain GnIH/RFRP-aromatase-neuroestrogen pathway. These effects may be through decreasing aromatase activity, neuroestrogen concentration, Cyp19A1 and ER ß mRNA levels and increasing ER α mRNA levels and immunostaining as well as testosterone, dopamine and glutamate levels in the brain. These findings also provide further support for the inhibitory role of RFRP3 on aggressive behavior of male rats. These data may open new avenues for the potential harmful effects of consumption large amounts of SB rich food on humans.

Association between a novel G94A single nucleotide polymorphism in ATP1A1 gene and type 2 diabetes mellitus among Egyptian patients.

BACKGROUND: Na+/K+ ATPase enzyme is essential for nerve cell membrane integrity, and reduction in its activity, probably due to ATP1A1 gene polymorphisms, is related to diabetic neuropathy progression. Therefore, the goal of the existent study is to evaluate the Na+/K+ ATPase activity in type 2 diabetes mellitus (T2DM) Egyptian patients with or without neuropathy, search for polymorphism(s) in the highly polymorphic region of ATP1A1 gene, exon 2, and study its (their) associations with T2DM with and without neuropathy. MATERIALS AND METHODS: A total number of 150 individuals were subclassified into healthy controls (n = 30), T2DM without complications (n = 60), and T2DM with neuropathy (n = 60). RESULTS: The biochemical results exhibited a significant reduction in fasting C-Peptide and activity of Na+/K+ ATPase in T2DM patients with neuropathy followed by T2DM without complication in comparison with healthy controls. ATP1A1 exon2 was amplified by polymerase chain reaction (PCR) then digested by the PstI restriction enzyme, and the obtained data from restriction fragment length polymorphism-PCR and sequencing revealed the existence of a novel synonymous G94A single nucleotide polymorphism (SNP) at nucleotide 27 in exon 2 of ATP1A1 gene (rs1060366). Diabetic groups had only allele A, while the control group had G allele. Interestingly, individuals carrying AA genotype had a significantly lower Na+/K+ ATPase, C-peptide, and higher glycosylated hemoglobin (HBA1c %) than those having GG genotype, suggesting a possible association for this SNP, and this developed phenomenon of not only T2DM but also diabetic neuropathy. CONCLUSION: Thus, allele A of G94A SNP (rs1060366) could be a risk allele for diabetes susceptibility among Egyptian patients.

Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes.

Cytochrome P450 aromatase (encoded by the CYP19A1 gene) regulates oestrogen biosynthesis and so plays an essential role in female fertility. We investigated the genetic association of CYP19A1 with the risk of anoestrus in Egyptian water buffaloes. A total of 651 animals (326 anoestrous and 325 cycling) were used in this case-control study. Using single-strand conformation polymorphisms and sequencing, four single nucleotide polymorphisms (SNPs) were detected; c.-135T>C SNP in the 5'UTR and three non-synonymous SNPs: c.559G>A (p. V187M) in Exon 5, c.1285C>T (p. P429S) and c.1394A>G (p. D465G) in Exon 10. Individual SNP-anoestrus association analyses revealed that genotypes (CC, AA and GG) and alleles (C, A and G) of the -135T>C, c.559G>A and c.1394A>G SNPs respectively were high risk for anoestrus. A further analysis confirmed that these three SNPs were in linkage disequilibrium. Additionally, haplotypes with two (TAG/122 and CAA/221) or three (CAG/222) risk alleles were significantly associated with susceptibility to anoestrus, lower blood levels of both oestradiol and antioxidant enzymes (superoxide dismutase, glutathione peroxidase (GPX) and catalase) and downregulated expression levels of CYP19A1, oestrogen receptor α and Gpx3 in the ovary, as well as increased serum level of malondialdehyde. This suggests the occurrence of a high incidence of oxidative ovarian damage and subsequently ovarian inactivity in buffaloes carrying risk alleles. Therefore, with this study we suggest the selection of buffaloes with protective alleles at these SNPs to improve the reproductive efficiency of the herd.

Isolates from Alpinia officinarum Hance attenuate LPS-induced inflammation in HepG2: Evidence from in silico and in vitro studies.

In an attempt to connect the legacy of centuries of invaluable knowledge from traditional medicine and the current understanding to the molecular mechanism of diseases, we took the advantage of the emergence of in silico screening as a promising tool for identification of potential leads from libraries of natural products. Traditional Chinese Medicine database was subjected to structure based virtual screening for identification of anti-inflammatory compounds using the 3D crystal structure of p38 alpha mitogen activated protein kinase. The molecular docking studies revealed the potential activity of several classes of compounds known to be the constituents of the rhizomes of Alpinia officinarum Hance (Lesser galangal). Five compounds, galangin, kaempferide, isorhamnetin, and two diarylheptanoids, were isolated from the rhizomes of the plant using vacuum liquid chromatography and flash chromatography techniques. The anti-inflammatory activity of these compounds was investigated on HepG2 cells stimulated by lipopolysaccharide. The latter induced the gene expression of proinflammatory cytokines; interleukin-1ß, interleukin-6, tumor necrosis factor alpha. Addition of the 5 isolated compounds downregulated this increased gene expression in a dose dependent manner. Thus, these results indicate that the isolated compounds from A. officinarum could be used as a beneficial source for preventing and treating inflammatory diseases.

Bmp4 regulates chick Ebf2 and Ebf3 gene expression in somite development.

The chick Early B-cell Factor-2 and 3 (cEbf2 and cEbf3) genes are members of EBF family of helix loop helix transcription factors. The expression, regulation and importance of these genes have been extensively studied in lymphatic, nervous and muscular tissues. Recently, a new role for some members of EBF in bone development has been investigated. However, the expression profile and regulation in the axial skeleton precursor, the somite, have yet to be elucidated. Therefore, this study was aimed to investigate the expression and regulation of cEbf2 and cEbf3 genes in the developing chick embryo somite from HH4 to HH28. The spatiotemporal expression study revealed predominant localization of cEbf2 and cEbf3 in the lateral sclerotomal domains and later around vertebral cartilage anlagen of the arch and the proximal rib. Subsequently, microsurgeries, ectopic gene expression experiments were performed to analyze which tissues and factors regulate cEbf2 and cEbf3 expression. Lateral barriers experiments indicated the necessity for lateral signal(s) in the regulation of cEbf2 and cEbf3 genes. Results from tissue manipulations and ectopic gene expression experiments indicate that lateral plate-derived Bmp4 signals are necessary for the initiation and maintenance of cEbf2 and cEbf3 genes in somites. In conclusion, cEbf2 and cEbf3 genes are considered as lateral sclerotome markers which their expression is regulated by Bmp4 signals from the lateral plate mesoderm.

Effect of c.1431C > T mutation, a causative mutation of Glanzmann's thrombasthenia, on ITGB3 splicing, gene and protein expression.

BACKGROUND/AIM: Recently, it was reported that the non-synonymous c.1431C > T (p. G477=) mutation of the integrin subunit ß3 (ITGB3) gene is the cause of Glanzmann's thrombasthenia (GT). However, the functional consequences of this mutation on the ITGB3 gene and protein expression remain to be elucidated. Therefore, this study was conducted to cover this scientific shortage. METHODS: Peripheral blood samples were collected from Chinese family members (parents and proband and his sister), and DNA was extracted and sequenced using whole-exome and Sanger sequencing. The effect of c.1431C > T mutation on the splicing of mRNA was verified by the in vitro minigene assay and the three variants that resulted from the mutation were cloned into a phage vector and pEGFP-C1 vector, and ITGB3 gene and protein expression was detected in the transfected 293 T cells using qPCR and Western blotting. RESULTS: Minigene splicing assay showed that c.1431C > T mutation causes three kinds of alternative splicing; (1) a 95 bp deletion in the middle of exon10, (2) a 155 bp deletion (95 bp deletion in the middle of exon10 plus a 60 bp deletion in the right side of exon10), and (3) a 261 bp deletion in the right side of exon10. The in vitro expression assay showed that the c.1431C > T variant did not affect the ITGB3 mRNA levels, but directly led to protein truncation and declined expression. CONCLUSION: Due to its significant impact on protein expression, c.1431C > T mutation in ITGB3 could be considered a pathogenic variant of GT. This could enrich the ITGB3 mutation spectrum and provide a base for the genetic diagnosis of GT.

25 (S)-Hydroxycholesterol acts as a possible dual enzymatic inhibitor of SARS-CoV-2 Mpro and RdRp-: an insight from molecular docking and dynamics simulation approaches.

The coronavirus disease (COVID-19) pandemic has rapidly extended globally and killed approximately 5.83 million people all over the world. But, to date, no effective therapeutic against the disease has been developed. The disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enters the host cell through the spike glycoprotein (S protein) of the virus. Subsequently, RNA-dependent RNA polymerase (RdRp) and main protease (Mpro) of the virus mediate viral transcription and replication. Mechanistically inhibition of these proteins can hinder the transcription as well as replication of the virus. Recently oxysterols and its derivative, such as 25 (S)-hydroxycholesterol (25-HC) has shown antiviral activity against SARS-CoV-2. But the exact mechanisms and their impact on RdRp and Mpro have not been explored yet. Therefore, the study aimed to identify the inhibitory activity of 25-HC against the viral enzymes RdRp and Mpro simultaneously. Initially, a molecular docking simulation was carried out to evaluate the binding activity of the compound against the two proteins. The pharmacokinetics (PK) and toxicity parameters were analyzed to observe the 'drug-likeness' properties of the compound. Additionally, molecular dynamics (MD) simulation was performed to confirm the binding stability of the compound to the targeted protein. Furthermore, molecular mechanics generalized Born surface area (MM-GBSA) was used to predict the binding free energies of the compound to the targeted protein. Molecular docking simulation identified low glide energy -51.0 kcal/mol and -35.0 kcal/mol score against the RdRp and Mpro, respectively, where MD simulation found good binding stability of the compound to the targeted proteins. In addition, the MM/GBSA approach identified a good value of binding free energies (ΔG bind) of the compound to the targeted proteins. Therefore, the study concludes that the compound 25-HC could be developed as a treatment and/or prevention option for SARS-CoV-2 disease-related complications. Although, experimental validation is suggested for further evaluation of the work.Communicated by Ramaswamy H. Sarma.

Antibacterial, Antifungal, and Anticancer Effects of Camel Milk Exosomes: An In Vitro Study.

Camel milk (CM) has potent antibacterial and antifungal effects and camel milk exosomes (CM-EXO) have been shown to inhibit the proliferation of a large variety of cancer cells including HepaRG, MCF7, Hl60, and PANC1. However, little is known regarding the effects of CM-EXO on bacteria, fungi, HepG2, CaCo2, and Vero cells. Therefore, this study aimed to evaluate the antibacterial, antifungal, and anticancer effects of CM-EXO. EXOs were isolated from CM by ultracentrifugation and characterized by transmission electron microscope and flow cytometry. Unlike CM, CM-EXO (6 mg/mL) had no bactericidal effects on Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus, and Enterococcus feacalis) but they had bacteriostatic effects, especially against Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis), and fungistatic effects on Candida albicans. HepG2, CaCo2, and Vero cells were respectively treated with CM-EXOs at low (6.17, 3.60, 75.35 µg/mL), moderate (12.34, 7.20, 150.70 µg/mL), and high (24.68, 14.40, 301.40 µg/mL) doses and the results revealed that CM-EXOs triggered apoptosis in HepG2 and CaCo2 cells, but not in normal Vero cells, as revealed by high Bax expression and caspase 3 activities and lower expression of Bcl2. Interestingly, CM-EXOs also induced the elevation of intracellular reactive oxygen species and downregulated the expression of antioxidant-related genes (NrF2 and HO-1) in cancer cells but not in normal cells. CM-EXOs have antibacterial and antifungal effects as well as a selective anticancer effect against HepG2 and CaCo2 cells with a higher safety margin on normal cells.