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Feline morbillivirus (FeMV) has been associated with feline health, although its exact role in pathogenesis is still debated. In this study, an indirect enzyme-linked immunosorbent assay (i-ELISA) targeting a recombinant matrix protein of FeMV (rFeMV-M) was developed and assessed in comparison to a Western blotting (WB) assay. The i-ELISA was evaluated using blood samples from 136 cats that were additionally tested with real-time reverse-transcription PCR (RT-qPCR). The i-ELISA exhibited a sensitivity of 90.1%, specificity of 75.6%, positive predictive value of 88.2%, and negative predictive value of 79.1%. The agreement between i-ELISA and WB analyses was substantial (a κ coefficient of 0.664 with a 95% confidence interval of 0.529 to 0.799). Within the study group, 68.4% (93/136) of the cats were serologically positive in the i-ELISA and 66.9% (91/136) in the WB assay, with 11.8% (11/93) of false positivity with the i-ELISA. However, only 8.1% (11/136) of the cats tested positive for FeMV using RT-qPCR (p < 0.001). The developed i-ELISA proved effective in identifying FeMV-infected cats and indicated the prevalence of FeMV exposure. Combining FeMV antibody detection through i-ELISA with FeMV RT-qPCR could offer a comprehensive method to determine and monitor FeMV infection status. Nevertheless, this assay still requires refinement due to a significant number of false positive results, which can lead to the misdiagnosis of cats without antibodies as having antibodies. This study also provided the first evidence of seroprevalence against FeMV among cat populations in Thailand, contributing valuable insights into the geographic distribution and prevalence of this virus. [ABSTRACT FROM AUTHOR]

Silibinin is a flavonoid compound extracted from the seeds of Silybum marianum (L.) Gaertn. It has the functions of liver protection, blood-lipid reduction and anti-tumor effects. However, the potential molecular mechanism of silibinin against tumors is still unknown. This study aimed to assess the anti-tumor effects of silibinin in adenoid cystic carcinoma (ACC2) cells and Balb/c nude mice, and explore its potential mechanism based on network pharmacology prediction and experimental verification. A total of 347 targets interacting with silibinin were collected, and 75 targets related to the tumor growth process for silibinin were filtrated. Based on the PPI analysis, CASP3, SRC, ESR1, JAK2, PRKACA, HSPA8 and CAT showed stronger interactions with other factors and may be the key targets of silibinin for treating tumors. The predicted target proteins according to network pharmacology were verified using Western blot analysis in ACC2 cells and Balb/c nude mice. In the pharmacological experiment, silibinin was revealed to significantly inhibit viability, proliferation, migration and induce the apoptosis of ACC2 cells in vitro, as well as inhibit the growth and development of tumor tissue in vivo. Western blot analysis showed that silibinin affected the expression of proteins associated with cell proliferation, migration and apoptosis, such as MMP3, JNK, PPARα and JAK. The possible molecular mechanism involved in cancer pathways, PI3K-Akt signaling pathway and viral carcinogenesis pathway via the inhibition of CASP3, MMP3, SRC, MAPK10 and CDK6 and the activation of PPARα and JAK. Overall, our results provided insight into the pharmacological mechanisms of silibinin in the treatment of tumors. These results offer a support for the anti-tumor uses of silibinin. [ABSTRACT FROM AUTHOR]

The present study was conducted to scrutinize the pharmacological effect of Estragole (ESG) against CFA-induced arthritis in rats. The rats underwent induction of arthritis using the administration of CFA and after that, the rats were randomly divided into five different groups, where three groups correspond to diverse dosages of ESG, and the other two were control and CFA-arthritic control. Results of the study suggested that ESG in a dose-dependent manner, improves body weight and arthritis score of rats as evidenced by reduction of hind-paw volume. ESG also improved the antioxidant status of rats by reducing MDA levels and enhancing the concentration of endogenous antioxidants SOD and GPx. The level of pro-inflammatory cytokines was also found to be reduced in the case of ESG treated group as compared to CFA-group. In a western blot analysis, ESH showed downregulation of p-JAK-2/STAT-3. The study provided concrete evidence for the protective effect of ESG against rheumatoid arthritis in rats. [ABSTRACT FROM AUTHOR]

Feline morbillivirus (FeMV) has been associated with feline health, although its exact role in pathogenesis is still debated. In this study, an indirect enzyme-linked immunosorbent assay (i-ELISA) targeting a recombinant matrix protein of FeMV (rFeMV-M) was developed and assessed in comparison to a Western blotting (WB) assay. The i-ELISA was evaluated using blood samples from 136 cats that were additionally tested with real-time reverse-transcription PCR (RT-qPCR). The i-ELISA exhibited a sensitivity of 90.1%, specificity of 75.6%, positive predictive value of 88.2%, and negative predictive value of 79.1%. The agreement between i-ELISA and WB analyses was substantial (a κ coefficient of 0.664 with a 95% confidence interval of 0.529 to 0.799). Within the study group, 68.4% (93/136) of the cats were serologically positive in the i-ELISA and 66.9% (91/136) in the WB assay, with 11.8% (11/93) of false positivity with the i-ELISA. However, only 8.1% (11/136) of the cats tested positive for FeMV using RT-qPCR (p < 0.001). The developed i-ELISA proved effective in identifying FeMV-infected cats and indicated the prevalence of FeMV exposure. Combining FeMV antibody detection through i-ELISA with FeMV RT-qPCR could offer a comprehensive method to determine and monitor FeMV infection status. Nevertheless, this assay still requires refinement due to a significant number of false positive results, which can lead to the misdiagnosis of cats without antibodies as having antibodies. This study also provided the first evidence of seroprevalence against FeMV among cat populations in Thailand, contributing valuable insights into the geographic distribution and prevalence of this virus.

Epilobium hirsutum L., commonly known as hairy willowherb, is a perennial herbaceous plant native to Europe and Asia. In Romania, the Epilobium genus includes 17 species that are used in folk medicine for various purposes. This study aimed to investigate the anti-inflammatory and antitumor potential of the optimized extract of Epilobium hirsutum (EH) in animal models. The first study investigated the anti-inflammatory properties of EH optimized extract and the model used was carrageenan-induced paw inflammation. Wistar rats were divided into three groups: negative control, positive control treated with indomethacin, and a group treated with the extract. Oxidative stress markers, cytokine levels, and protein expressions were assessed. The extract demonstrated anti-inflammatory properties comparable to those of the control group. In the second study, the antitumor effects of the extract were assessed using the tumor model of Ehrlich ascites carcinoma. Swiss albino mice with Ehrlich ascites were divided into four groups: negative, positive treated with cyclophosphamide (Cph), Group 3 treated with Cph and EH optimized extract, and Group 4 treated with extract alone. Samples from the ascites fluid, liver, and heart were analyzed to evaluate oxidative stress, inflammation, and cancer markers. The extract showed a reduction in tumor-associated inflammation and oxidative stress. Overall, the EH optimized extract exhibited promising anti-inflammatory and antitumor effects in the animal models studied. These findings suggest its potential as a natural adjuvant therapeutic agent for addressing inflammation and oxidative stress induced by different pathologies. [ABSTRACT FROM AUTHOR]

Silibinin is a flavonoid compound extracted from the seeds of Silybum marianum (L.) Gaertn. It has the functions of liver protection, blood-lipid reduction and anti-tumor effects. However, the potential molecular mechanism of silibinin against tumors is still unknown. This study aimed to assess the anti-tumor effects of silibinin in adenoid cystic carcinoma (ACC2) cells and Balb/c nude mice, and explore its potential mechanism based on network pharmacology prediction and experimental verification. A total of 347 targets interacting with silibinin were collected, and 75 targets related to the tumor growth process for silibinin were filtrated. Based on the PPI analysis, CASP3, SRC, ESR1, JAK2, PRKACA, HSPA8 and CAT showed stronger interactions with other factors and may be the key targets of silibinin for treating tumors. The predicted target proteins according to network pharmacology were verified using Western blot analysis in ACC2 cells and Balb/c nude mice. In the pharmacological experiment, silibinin was revealed to significantly inhibit viability, proliferation, migration and induce the apoptosis of ACC2 cells in vitro, as well as inhibit the growth and development of tumor tissue in vivo. Western blot analysis showed that silibinin affected the expression of proteins associated with cell proliferation, migration and apoptosis, such as MMP3, JNK, PPARα and JAK. The possible molecular mechanism involved in cancer pathways, PI3K-Akt signaling pathway and viral carcinogenesis pathway via the inhibition of CASP3, MMP3, SRC, MAPK10 and CDK6 and the activation of PPARα and JAK. Overall, our results provided insight into the pharmacological mechanisms of silibinin in the treatment of tumors. These results offer a support for the anti-tumor uses of silibinin.

A new ligand DFIP (2-(dibenzo[b,d]furan-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its two complexes iridium(III) [Ir(ppy)2(DFIP)](PF6) (ppy = 2-phenylpyridine, Ir1) and ruthenium(II) [Ru(bpy)2(DFIP)](PF6)2 (bpy = 2,2′-bipyridine, Ru1) were synthesized and characterized. The anticancer effects of the two complexes on A549, BEL-7402, HepG2, SGC-7901, HCT116 and normal LO2 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complex Ir1 shows high cytotoxic activity on A549, BEL-7402, SGC-7901 and HepG2, Ru1 exhibits moderate anticancer activity toward A549, BEL-7402 and SGC-7901 cells. The IC50 values of Ir1 and Ru1 toward A549 are 7.2 ± 0.1 and 22.6 ± 1.4 μM, respectively. The localization of complexes Ir1 and Ru1 in the mitochondrial, intracellular accumulation of reactive oxygen species (ROS) levels, and the changes of mitochondrial membrane potential (MMP) and cytochrome c (cyto-c) were investigated. Apoptosis and cell cycle were detected by flow cytometry. Immunogenic cell death (ICD) was used to detect the effects of Ir1 and Ru1 on the A549 using a confocal laser scanning microscope. The expression of apoptosis-related proteins was detected by western blotting. Ir1 and Ru1 can increase the intracellular ROS levels and release cyto-c, reduce the MMP, leading to the apoptosis of A549 cells and blocking the A549 cells at the G0/G1 phase. Additionally, the complexes caused a decrease of the expression of polyADP-ribose polymerase (PARP), caspase 3, Bcl-2 (B-cell lymphoma-2), PI3K (phosphoinositide-3 kinase) and upregulated the expression of Bax. All these findings indicated that the complexes exert anticancer efficacy to induce cell death through immunogenic cell death, apoptosis, and autophagy. [ABSTRACT FROM AUTHOR]

Epilobium hirsutum L., commonly known as hairy willowherb, is a perennial herbaceous plant native to Europe and Asia. In Romania, the Epilobium genus includes 17 species that are used in folk medicine for various purposes. This study aimed to investigate the anti-inflammatory and antitumor potential of the optimized extract of Epilobium hirsutum (EH) in animal models. The first study investigated the anti-inflammatory properties of EH optimized extract and the model used was carrageenan-induced paw inflammation. Wistar rats were divided into three groups: negative control, positive control treated with indomethacin, and a group treated with the extract. Oxidative stress markers, cytokine levels, and protein expressions were assessed. The extract demonstrated anti-inflammatory properties comparable to those of the control group. In the second study, the antitumor effects of the extract were assessed using the tumor model of Ehrlich ascites carcinoma. Swiss albino mice with Ehrlich ascites were divided into four groups: negative, positive treated with cyclophosphamide (Cph), Group 3 treated with Cph and EH optimized extract, and Group 4 treated with extract alone. Samples from the ascites fluid, liver, and heart were analyzed to evaluate oxidative stress, inflammation, and cancer markers. The extract showed a reduction in tumor-associated inflammation and oxidative stress. Overall, the EH optimized extract exhibited promising anti-inflammatory and antitumor effects in the animal models studied. These findings suggest its potential as a natural adjuvant therapeutic agent for addressing inflammation and oxidative stress induced by different pathologies.

Java Ginger or Curcuma zanthorrhiza Roxb. has long gained focus among tribal people of Java, for its medicinal properties mainly against gynaecological challenges. The present study aims to identify the most potent phytocompound present in the extract and determine primary mode of action accountable for cytotoxic activity of Curcuma zanthorrhiza rhizome extract against HPV16-positive SiHa cervical cancer cells. The phytochemically-rich extract of rhizome (CZM) was capable to inhibit proliferation of target cells in a dose-dependent manner with an IC50 of 150 μg/ml. Dysregulation of intercellular antioxidant defence system resulted to surges in ROS and RNS level, increased calcium concentration and compromised mitochondrial membrane potential. Nucleus got affected, cell cycle dynamics got impaired while clonogenicity and migration ability diminished. Expression of viral oncogenes E7 and E6 decreased significantly. Accumulation of toxic cell metabolite and decrease in level of essential ones continued. Finally, alteration in PI3K/AKT/mTOR signalling route was followed by onset of autophagic cell death concomitant with the upregulated expression of Beclin1, Atg5-12 and LC3II. Curcumin and a novel crystal as well as few phyto-fractions were isolated by column chromatography. Of these, curcumin was found to be most potent in inducing cytotoxicity in SiHa while two other fractions also showed significant activity. Thus, CZM acted against SiHa cells by inducing autophagy that commences in compliance to the changes in PI3K/AKT/mTOR pathway mainly in response to oxidative stress. To the best of our knowledge this is the first report of Curcuma zanthorrhiza Roxb. inducing autophagy. [ABSTRACT FROM AUTHOR]

Transcription factor 4 (TCF4) belongs to the class I basic helix–loop–helix family of transcription factors (also known as E-proteins) and is vital for the development of the nervous system. Aberrations in the TCF4 gene are associated with several neurocognitive disorders such as schizophrenia, intellectual disability, post-traumatic stress disorder, depression, and Pitt-Hopkins Syndrome, a rare but severe autism spectrum disorder. Expression of the human TCF4 gene can produce at least 18 N-terminally distinct protein isoforms, which activate transcription with different activities and thus may vary in their function during development. We used long-read RNA-sequencing and western blot analysis combined with the analysis of publicly available short-read RNA-sequencing data to describe both the mRNA and protein expression of the many distinct TCF4 isoforms in rodent and human neural and nonneural tissues. We show that TCF4 mRNA and protein expression is much higher in the rodent brain compared to nonneural tissues. TCF4 protein expression is highest in the rodent cerebral cortex and hippocampus, where expression peaks around birth, and in the rodent cerebellum, where expression peaks about a week after birth. In human, highest TCF4 expression levels were seen in the developing brain, although some nonneural tissues displayed comparable expression levels to adult brain. In addition, we show for the first time that out of the many possible TCF4 isoforms, the main TCF4 isoforms expressed in the rodent and human brain and other tissues are TCF4-B, -C, -D, -A, and-I. Taken together, our isoform specific analysis of TCF4 expression in different tissues could be used for the generation of gene therapy applications for patients with TCF4-associated diseases. [ABSTRACT FROM AUTHOR]

Transcription factor 4 (TCF4) belongs to the class I basic helix–loop–helix family of transcription factors (also known as E-proteins) and is vital for the development of the nervous system. Aberrations in the TCF4 gene are associated with several neurocognitive disorders such as schizophrenia, intellectual disability, post-traumatic stress disorder, depression, and Pitt-Hopkins Syndrome, a rare but severe autism spectrum disorder. Expression of the human TCF4 gene can produce at least 18 N-terminally distinct protein isoforms, which activate transcription with different activities and thus may vary in their function during development. We used long-read RNA-sequencing and western blot analysis combined with the analysis of publicly available short-read RNA-sequencing data to describe both the mRNA and protein expression of the many distinct TCF4 isoforms in rodent and human neural and nonneural tissues. We show that TCF4 mRNA and protein expression is much higher in the rodent brain compared to nonneural tissues. TCF4 protein expression is highest in the rodent cerebral cortex and hippocampus, where expression peaks around birth, and in the rodent cerebellum, where expression peaks about a week after birth. In human, highest TCF4 expression levels were seen in the developing brain, although some nonneural tissues displayed comparable expression levels to adult brain. In addition, we show for the first time that out of the many possible TCF4 isoforms, the main TCF4 isoforms expressed in the rodent and human brain and other tissues are TCF4-B, -C, -D, -A, and-I. Taken together, our isoform specific analysis of TCF4 expression in different tissues could be used for the generation of gene therapy applications for patients with TCF4-associated diseases.

The potential for a donor‐derived transmission of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) to the recipient has been recognized since pigs were considered candidate donors for xenotransplantation. This review gives a short description of the viral properties and summarizes the current evidence of the effects of PCMV/PRV transmission in preclinical xenotransplantation. Despite evidence that PCMV/PRV does not infect human and non‐human primate cells, activation in the transplanted organ and detrimental systemic complications have been described. As PCMV/PRV is a herpesvirus able to establish latency, the importance of adequate screening of donor pigs is emphasized, as no efficient treatment is available. Furthermore, easy and successful ways of elimination of PCMV/PRV from pig herds are indicated. [ABSTRACT FROM AUTHOR]

Emodin, a natural anthraquinone derivative, is an active ingredient in many Chinese traditional herbs. Interestingly, although it is generally considered to possess hepatoprotective activity, some studies have also reported that it has a certain degree of hepatotoxicity. Additionally, the underlying metabolic regulation of emodin remains uncertain. Therefore, we conducted a nontargeted metabolomic study based on UHPLC/Q-Orbitrap-MS and NMR. Data are available via ProteomeXchange with the identifier PXD055000. The results indicated a close association between the short-term administration of emodin and lipid metabolism. Moreover, a lipidomics investigation utilizing QTRAP 6500 + UHPLC-MS/MS was conducted, with a focus on determining the position of C═C double bonds in unsaturated lipids based on Paternò-Büchi (PB) reaction to discover the metabolic disturbance more precisely. Specifically, lipidomics revealed elevated levels of free fatty acids (FFA) alongside notable reductions in sphingomyelin (SM) and triacylglycerol (TAG) levels. Furthermore, the combination of PB reaction and molecular biology results indicated that short-term administration of emodin may lead to the accumulation of n-6 polyunsaturated fatty acids by up-regulating the expression of FASN, stearyl CoA desaturase 1 (SCD1), and cytosolic phospholipase A 2 (cPLA2). Simultaneously, up-regulation of cyclooxygenase-2 (Cox-2) expression was observed, potentially fostering the production of prostaglandin E2 (PGE2) and subsequent inflammation.

Background: Cyrtopodion scabrum is a kind of lizard, widely distributed in southwestern, central, and eastern Iran. In our previous studies, we showed the selective anticancer properties of Cyrtopodion scabrum extract (CSE) against certain gastrointestinal cancer cell lines with no significant harmful effects on the normal cells. Method: This in vitro study was conducted to investigate the mechanism of action of CSE in SW742 (colon cancer cell line) using western blotting, real-time polymerase chain reaction (PCR), and PI staining/flow cytometry analysis. Results: Western blot analysis showed that CSE upregulates P53 protein expression significantly in SW742 cancer cell line, while QRT-PCR results revealed that P53 mRNA did not increase accordingly. It is proposed that TP53 protein play its role as a tumor suppressor through the protein stability mechanism and not through the increase in the gene expression. Considering this fact, for TP53 stabilization, two of its target genes, p21 and mdm2, were regulated. We evaluated the mRNA expression of these two genes as well. The obtained data showed a significant increase in both genes by CSE (1000 μg/ml), suggesting that the induction of G2 cell cycle arrest, which we previously reported, may happen through P53 and P21over-expression. No significant decrease was observed in apoptosis when SW742 cells were co-treated with CSE and the inhibitor of P53 transcriptional activity, (PFT)-α, indicating that the induction of apoptosis with CSE did not occur through P53-dependent transcriptional activity. Conclusion: The obtained results herein revealed that the observed anticancer effect of CSE may occur through TP53 upregulation, yet with P53-independent transcriptional activity.

Aims: Phytonematodes are a constraint on crop production and have been controlled using nematicides; these are highly toxic and legislation in Europe and elsewhere is prohibiting their use and alternatives are being sought. Pasteuria penetrans is a hyperparasitic bacterium that form endospores and have potential to control root‐knot nematodes (Meloidogyne spp.), but their attachment to the nematode cuticle is host‐specific. Understanding host specificity has relied upon endospore inhibition bioassays using immunological and biochemical approaches. Phylogenetic analysis of survey sequences has shown P. penetrans to be closely related to Bacillus and to have a diverse range of collagen‐like fibres which we hypothesise to be involved in the endospore adhesion. However, due to the obligately hyperparasitic nature of Pasteuria species, identifying and characterizing these collagenous‐like proteins through gain of function has proved difficult and new approaches are required. Methods and Results: Using antibodies raised to synthetic peptides based on Pasteuria collagen‐like genes we show similarities between P. penetrans and the more easily cultured bacterium Bacillus thuringiensis and suggest it be used as a gain of function platform/model. Using immunological approaches similar proteins between P. penetrans and B. thuringiensis are identified and characterized, one >250 kDa and another ~72 kDa are glycosylated with N‐acetylglucosamine and both of which are digested if treated with collagenase. These treatments also affected endospore attachment and suggest these proteins are involved in adhesion of endospores to nematode cuticle. Conclusion: There are conserved similarities in the collagen‐like proteins present on the surface of endospores of both P. penetrans and B. thuringiensis. Significance and Impact of Study: As B. thuringiensis is relatively easy to culture and can be transformed, it could be developed as a platform for studying the role of the collagen‐like adhesins from Pasteuria in endospore adhesion. [ABSTRACT FROM AUTHOR]

Objective Liver cancer is the third rank amongst the common malignancies, causing maximum death in the patients diagnosed with cancers. Currently available biomarkers are not enough sensitive for early diagnosis of hepatocellular carcinoma (HCC). This makes difficult management of HCC. With the aim of finding new generation of proteomic-based biomarkers, the represented study was designed to characterize the differentially expressed proteins at different stages of HCC initiation and at progression. This could lead to find potential biomarkers for early detection of HCC. Materials And Methods In this experimental study, we report induction of HCC by administrating chemical carcinogens in male Wistar rats. Disease progression was monitored by histological evaluation. Serum proteomic analyses such as 2 dimensional (2D)-electrophoresis, MALDI-TOF-MS/MS and Western blot have been used to analyze and characterize the differentially expressed proteins during HCC development. Results HCC initiation and tumorigenesis were observed at one and four months post carcinogen treatment, respectively. One of the differentially-expressed proteins, namely, cytosolic phospholipase A2delta was significantly up-regulated at very early stage of HCC development. Its expression continued to increase during cancer progression and hepatotumorigenesis stages. Its elevated expression has been confirmed by Western blot analysis. Consistent to this, analyses of the sera in the clinically confirmed liver cancer patients showed elevated expression of this protein, further validating our experimental results. Conclusion This study suggests that elevation in the expression of cytosolic phospholipase A2delta is associated with progression of HCC.

Abstract Background This study is intended to clarify the influence of exogenous bovine lactoferrin (bLf) treatment on glycemic homeostasis, gene expression, and production of lactoferrin (Lf) in rats with experimentally induced diabetes. Methodology Fifty adult male rats were used; 15 rats were used as the normal control rats (group A) and were injected an intraperitoneal (I/P) with 1 ml of isotonic saline daily for 3 months. Thirty-five rats were administered I/P injection of 60 mg of streptozotocin per kilogram body weight to induce diabetes. Thirty streptozotocin-treated rats were selected and used as diabetic rats, and they were subdivided into 2 equal groups (15 rats each): the untreated diabetic group (group B) and treated diabetic group (group C). The rats in the diabetic group were administered an I/P injection of 1 ml of isotonic saline daily for 3 months, while rats in the diabetic treatment group were administered an I/P injection of 300 mg of bLf/kg body weight/day/3 months. At the end of the experiment, blood and renal tissue samples were collected from all rats. The levels of insulin, glucose, and lactoferrin in plasma were measured. Homeostatic model assessment for insulin resistance (HOMA-IR) was performed. The numbers of Lf-mRNA copies in renal tissue were assessed using quantitative RT-PCR. To measure Lf production in renal tissue, Western blot analysis was applied. Results The obtained data demonstrated that the treatment of diabetic rats with bLf maintained glycemic homeostasis at normal levels but increased the mRNA expression of renal Lf. Conclusion In diabetic rats, bovine lactoferrin treatment offers the potential for protection against incidences of insulin resistance (IR) by stimulating the suppressed expression and production of the LTF gene.

Despite its prevalence, preeclampsia (PE) remains unclear as to its etiology. Here, we aimed to investigate the mechanisms regulating differences in the gene expression of zinc-finger protein 516 (ZNF516) in the placenta. The expression of the placental ZNF516 gene and its association with critical clinical markers were verified, and a rigorous correlation analysis was conducted. With a dual-luciferase reporter gene assay, microRNA targeting the ZNF516 gene was predicted and confirmed. Finally, the molecular processes associated with ZNF516 were explored via microarray and bioinformatic analyses. In hypoxic conditions, miR-371-5p expression was reduced, resulting in ZNF516 expression being induced. Moreover, ZNF516 was shown to hinder trophoblast cell migration and invasion while enhancing trophoblast cell death in various in vitro cellular assays, such as cell counting kit-8, colony formation, wound healing, and Transwell assays. Our findings reveal a new regulatory network facilitated by ZNF516. ZNF516 overexpression inhibits trophoblast growth, movement, and penetration, potentially causing problems with placenta formation with the help of miR-371-5p suppression.

The aim of the study was to produce polyclonal antibodies against human lactoferrin in order to apply them in evaluation of lactoferrin levels in tear fluid collected from healthy eye and after corneal injury. Materials and methods. Affine chromatography on Protein A-sepharose was applied in order to isolate immunoglobulin G (IgG) fraction from the blood serum of lactoferrin-immunized rabbits. Each step of protein purification was monitored by denaturing gel electrophoresis (SDS-PAGE). Target antigen recognition by produced antibodies was established by western blot analysis with the use of diluted IgG fraction. Lactoferrin levels in the tear fluids collected from healthy individuals (n = 4) and patients with non-penetrating corneal injures (n = 6) were determined immunochemically with the use of purified antibodies. The results of western blot of lactoferrin levels in the tear fluids of healthy individuals and patients with corneal wounds were analysed using Mann-Whitney U-test. The difference between group mean values was considered significant at P<0.05. Results. Using affine chromatography on Protein A-sepharose, antibodies against human lactoferrin were purified as IgG fraction from blood serum of lactoferrin-immunized rabbits. Western blot analysis showed that obtained antibodies recognize the antigen as a 75-kDa band, which corresponds to the intact human lactoferrin polypeptide. The same major polypeptide band was visualized by western blot with enhanced chemiluminescence detection in the tear fluid samples. Densitometry analysis of 75-kDa lactoferrin band showed 3.2-fold decrease in lactoferrin level in the tear fluid samples obtained from patients with non-penetrating corneal traumas as compared with samples collected from healthy persons ( P<0.05). Besides, tear fluid of patients with injured corneas contained large amounts of truncated lactoferrin immunoreactive polypeptides as well as high molecular weight bands, which could correspond to lactoferrin complexes with other proteins occurring during inflammation. Conclusions. According to our data, obtained anti-lactoferrin antibodies can be used as a valuable tool for development of advanced tests and procedures for diagnostics of eye diseases associated with the corneal lesions. Reduced lactoferrin concentration might represent a potential prognostic biomarker for diagnosis of ocular diseases including non-penetrating corneal injuries in a simple and non-invasive way. [ABSTRACT FROM AUTHOR]

Background: Cyrtopodion scabrum is a kind of lizard, widely distributed in southwestern, central, and eastern Iran. In our previous studies, we showed the selective anticancer properties of Cyrtopodion scabrum extract (CSE) against certain gastrointestinal cancer cell lines with no significant harmful effects on the normal cells. Method: This in vitro study was conducted to investigate the mechanism of action of CSE in SW742 (colon cancer cell line) using western blotting, real-time polymerase chain reaction (PCR), and PI staining/flow cytometry analysis. Results: Western blot analysis showed that CSE upregulates P53 protein expression significantly in SW742 cancer cell line, while QRT-PCR results revealed that P53 mRNA did not increase accordingly. It is proposed that TP53 protein play its role as a tumor suppressor through the protein stability mechanism and not through the increase in the gene expression. Considering this fact, for TP53 stabilization, two of its target genes, p21 and mdm2, were regulated. We evaluated the mRNA expression of these two genes as well. The obtained data showed a significant increase in both genes by CSE (1000 μg/ml), suggesting that the induction of G2 cell cycle arrest, which we previously reported, may happen through P53 and P21over-expression. No significant decrease was observed in apoptosis when SW742 cells were co-treated with CSE and the inhibitor of P53 transcriptional activity, (PFT)-α, indicating that the induction of apoptosis with CSE did not occur through P53-dependent transcriptional activity. Conclusion: The obtained results herein revealed that the observed anticancer effect of CSE may occur through TP53 upregulation, yet with P53-independent transcriptional activity. [ABSTRACT FROM AUTHOR]

The study was intended to determine the antineoplastic effects of two new iridium(III) complexes [Ir(ppy)2(PTTP)](PF6) (1) (ppy = 2-phenylpyridine) and [Ir(piq)2(PTTP)](PF6) (2) (piq = 1-phenylisoquinoline, PTTP = 2-phenoxy-1,4,8,9-tetraazatriphenylene). In MTT assay, the ligand PTTP displayed ineffective inhibition on cell growth in SGC-7901, BEL-7402, HepG2 as well as NIH3T3 cell lines, while complexes 1 and 2 showed high cytotoxic activity on SGC-7901 cells with an IC50 value of 0.5 ± 0.1 µM and 4.4 ± 0.6 µM, respectively. Cellular uptake, cell cloning experiments, wound healing assay and cell cycle arrest indicated that the two complexes can inhibit the cell proliferation in SGC-7901 and induce cell cycle arrest at G0/G1 phase. Additionally, reactive oxygen species (ROS) and mitochondrial membrane potential suggested that the two complexes induced cell apoptosis through disrupting mitochondrial functions. Further, western blot analysis illustrated that the two complexes caused apoptosis via regulating expression levels of Bcl-2 family proteins. Moreover, complex 1 could suppress tumor growth in vivo with an inhibitory rate of 49.41%. Altogether, these results demonstrated that complexes 1 and 2 exert a potent anticancer effect against SGC-7901 cells via mitochondrial apoptotic pathway and have a potential to be developed as antineoplastic drug candidates for human gastric cancer. [ABSTRACT FROM AUTHOR]

Purpose: TILRR (Toll-like interleukin-1 receptor regulator), a variant of FREM1 (Fras-related extracellular matrix 1), is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and inflammatory responses. It enhanced the expression of multiple genes in the NF-κB signaling pathway and promoted the production of multiple pro-inflammatory cytokines/chemokines. TILRR is an extracellular matrix protein and expressed in cells and tissues, and has never been considered to exist in the blood. The study aimed to identify circulating TILRR protein in human plasma as a biomarker of systemic inflammation. Methods and Results: We developed a multiplex bead array method (Bio-Plex) using 4 monoclonal antibodies targeting different protein domains of FREM1/TILRR to investigate whether TILRR can be detected in blood plasma. The results of the multiplex bead array method were validated by Western blot analysis of affinity-purified TILRR from patient plasma samples. We subsequently analyzed 640 plasma samples from women enrolled in the Pumwani Sex Worker cohort (PSWC) (Nairobi, Kenya). Our study showed that TILRR exists in all patient plasma samples, but its quantities vary greatly among the patients, ranging from 2.38 ng/mL to 5196.79 ng/mL. The plasma TILRR below 2.38 ng/mL can only be detected by affinity purification and Western blot analysis. Conclusion: Our in-house developed multiplex bead array method can successfully quantify TILRR protein in plasma samples. Because TILRR is an important modulator of many inflammation-responsive genes, it may be an inflammation biomarker in blood and play a role in modulating systemic inflammation. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the impact of the herbicide monuron on various aspects of photosynthesis and diazotrophy in the cyanobacterium Nostoc muscorum Meg 1 isolated from a rice field in Cherrapunji, Meghalaya, India. The consequences of monuron exposure on various photosynthetic pigments, functioning of PSII, RuBisCO, nitrogenase and glutamine synthetase enzyme activities, heterocyst frequency, and on production of proteins and carbohydrates in the cyanobacterium were studied in a range of monuron doses (20–100 ppm) to gauge the herbicide's effect on CO2 and N2 fixation and on net biomass production. The total amount of D1 protein (the host for PSII complex), RuBisCO (the key enzyme for CO2 fixation), nitrogenase (the enzyme responsible for the reduction of atmospheric nitrogen to ammonia), and ammonia assimilating enzyme glutamine synthetase (GS) contents under western blot analysis indicated interruption of new protein synthesis and breakdown of their existing enzyme molecules when exposed to higher monuron concentrations. All parameters studied showed enhanced expression under low dose monuron treatment (20 ppm) indicating a hormetic effect in the exposed organism. The expression of monuron toxicity on various parameters of CO2 and N2 fixation in a dose-dependent manner was immediately visible when cultures were treated with higher doses (40–100 ppm). Scanning and transmission electron microscopic studies further uncovered several undesirable changes in the morphology and ultrastructure of the organism due to herbicide treatment that could be correlated to compromised CO2 and N2 fixation. [ABSTRACT FROM AUTHOR]

Dehydrins are well-known components of plant responses to different stresses that cause dehydration, including drought, freezing, salinity, etc. In conifers, the dehydrin gene family is very large, implying that the members of this family have important physiological functions in conifer stress tolerance. However, dehydrin gene expression displays a wide range of responses to stress, from thousand-fold increased expression to decreased expression, and it is generally unknown how regulatory systems are connected at the mRNA and protein levels. Therefore, we studied these aspects of dehydrin regulation in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) seedlings under polyethylene glycol 6000-induced osmotic stress ranging from relatively low (culture medium water potential of −0.15 MPa) to very high (−1.0 MPa) intensities. In pine, the major dehydrin protein was Dhn1 in both the roots and needles, and in spruce, two isoforms of the Dhn4 protein were the major dehydrins; both of these proteins are AESK-type dehydrins. The genes encoding these major proteins were highly expressed even under control conditions; surprisingly, we also observed several highly expressed dehydrin genes that were not abundantly translated. Under osmotic stress, the most prominent expression changes were observed for the dehydrin genes with low basal expression levels, whereas highly expressed genes generally demonstrated rather modest changes in expression. We report proposed constitutive physiological functions of the AESK-type dehydrins in Pinaceae plants. • In pine and spruce seedlings, AESK-type proteins are the major DHNs. • DHNs are more responsive to stress at the mRNA level than at the protein level. • AESK-type DHNs probably function constitutively in both pine and spruce seedlings. [ABSTRACT FROM AUTHOR]

[Display omitted] • Chemistry: A series of novel pyrimidine-5-carbonitriles was synthesised. • Biological screening: Compound 7c was the most potent member in our study. The most sensitive cell lines were SNB-75, and OVAR-4. • Compound 7c have dual inhibitory activity against EGFRWT/T790M/PI3K. • Compound 7c block cell cycle at G0-G1 phase. • Compound 7c decrease the expression level of p-AKT protein but increase caspase 3 & 9 & BAX proteins. • Compounds 7c demonstrated less cytotoxicity against normal epithelial colon cells compared to Staurosporine. • Compound 7c showed decrease in tumor weight, and the number of tumor nodules in Mice in which cancer was induced. A new series of 6-(4-fluorophenyl)-2-(methylthio) pyrimidine-5-carbonitrile derivatives were designed and synthesized as EGFR/PI3K dual inhibitors, and potential antiproliferative agents. The new 22 compounds were screened by DTP-NCI against all NCI60 cell lines. Almost all compounds showed cytotoxic activity. Compound 7c showed a promising antitumour activity on CNS cancer (SNB-75), and ovarian cancer (OVAR-4) with IC 50 < 0.01, and 0.64 µM, respectively. Fortunately, 7c exhibited a better safety profile on normal cells (WI-38) than doxorubicin by 2.2-fold. Compound 7c displayed selective inhibitory activity on EGFRt790m over EGFRWT with IC 50 = 0.08, and 0.13 µM, respectively, wherefore it might overcome EGFR-TKIs resistance. In addition to its remarkable inhibitory activity on all PI3K isoforms, specifically PI3K-δ with IC 50 = 0.64 µM Compared with LY294002 IC 50 = 7.6 µM. Compound 7c arrested the cell cycle of SNB-75 & OVAR-4 at the G0-G1 phase coupled with apoptosis induction. The western blotting analysis approved decreasing the expression level of p-AKT coupled with an increase in Casp3, Casp9, and BAX proteins in the SNB-75 & OVAR-4 after being treated with 7c which may support the suggested mechanism of action of 7c as EGFR/PI3K dual inhibitor. Physicochemical parameters were forecasted using SwissADME online tool. MD showed the interaction of 7c with the crucial amino acids of the active domain of both EGFR/PI3K which may explain its potent inhibitory activities. In vivo study disclosed a significant decrease in tumor weight and the number of nodules in the group of mice treated with 7c compared with the control group. [ABSTRACT FROM AUTHOR]

There are limited and equivocal data regarding potential fiber type-specific differences in the human skeletal muscle response to sprint interval training (SIT), including how this compares with moderate-intensity continuous training (MICT). We examined mixed-muscle and fiber type-specific responses to a single session (study 1) and to 12 wk (study 2) of MICT and SIT using Western blot analysis. MICT consisted of 45 min of cycling at ∼70% of maximal heart rate, and SIT involved 3 × 20-s "all-out" sprints interspersed with 2 min of recovery. Changes in signaling proteins involved in mitochondrial biogenesis in mixed-muscle and pooled fiber samples were similar after acute MICT and SIT. This included increases in the ratios of phosphorylated to total acetyl-CoA carboxylase and p38 mitogen-activated protein kinase protein content (main effects, P < 0.05). Following training, mitochondrial content markers including the protein content of cytochrome c oxidase subunit IV and NADH:ubiquinone oxidoreductase subunit A9 were increased similarly in mixed-muscle and type IIa fibers (main effects, P < 0.05). In contrast, only MICT increased these markers of mitochondrial content in type I fibers (interactions, P < 0.05). MICT and SIT also similarly increased the content of mitochondrial fusion proteins optic atrophy 1 (OPA1) and mitofusin 2 in mixed-muscle, and OPA1 in pooled fiber samples (main effects, P < 0.02). In summary, acute MICT and SIT elicited similar fiber type-specific responses of signaling proteins involved in mitochondrial biogenesis, whereas 12 wk of training revealed differential responses of mitochondrial content markers in type I but not type IIa fibers.NEW & NOTEWORTHY We examined mixed-muscle and fiber type-specific responses to a single session and to 12 wk of moderate-intensity continuous training (MICT) and sprint interval training (SIT) in humans. Both interventions elicited generally similar responses, although the training-induced increases in type I fiber-specific markers of mitochondrial content were greater in MICT than in SIT. These findings advance our understanding of the potential role of fiber type-specific changes in determining the human skeletal muscle response to intermittent and continuous exercise. [ABSTRACT FROM AUTHOR]

Background and Objective: Fractional radiofrequency microneedling (FRM) is widely used as an option for skin rejuvenation, however there is a lack of histological evidence for the various energy delivery systems available. The objective was to assess thermal denaturation of tissue and the wound healing response in monopolar mode versus bipolar mode. Histological analysis was performed to demonstrate the efficacy of automatic impedance feedback system in monopolar mode., Study Design and Methods: In this study, the acute thermal effects caused by monopolar FRM treatment to the dorsal skin of pigs were assessed histologically by hematoxylin & eosin (H&E) staining. Then, one session of either monopolar or bipolar FRM was used to treat one or the other side of the pig using varying power levels and pulse widths. The acute and chronic tissue reactions were assessed using H&E, immunofluorescence, and western blot analysis at 0, 14, 30, and 90 days after treatment. The efficacy of the impedance feedback system was also monitored histologically., Results: High-energy FRM treatment produced tissue loss and necrosis. The power level and pulse duration significantly affected the coagulation amount. Histopathology at 0, 14, 30, and 90 days showed that the skin tissue reaction was more pronounced for bipolar compared to monopolar FRM. Immunofluorescence showed the expression of TGF-β, Ki67, MMP3, and elastin increased dramatically with both modes, but were higher in the bipolar FRM treated side. The automatic impedance feedback system could effectively adjust the output energy., Conclusions: We found that bipolar FRM produced greater thermal effects, more collagen coagulation, and more pronounced molecular changes compared with monopolar mode in a porcine animal model., (© 2024 Wiley Periodicals LLC.)

The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813-829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC-mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F-L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity "GNAL" domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to antibody affinity for the GNAL domain of the D2 protein and has no bearing on the identity or function of the TLA1 protein.

Background/Aims: Approximately 10%-20% of patients with acute cardiovascular disease who have received coronary intervention suffer restenosis and high inflammation. The stent compound paclitaxel+hirudin was prepared for the treatment of post-intervention restenosis. This study aimed to explore the anti-inflammatory and anti-restenosis mechanisms of paclitaxel+hirudin with regard to the TLR4/MyD88/NF-κB pathway. Methods: Human coronary artery smooth muscle cells (HCASMCs) at 4-6 generations after in vitro culture were used as a model. Lipopolysaccharide (LPS) was used as an inducer to maximally activate the TLR4/MyD88/NF-κB inflammation pathway. After MyD88 knockdown and selective blocking of MyD88 degradation with epoxomicin, the effects of paclitaxel+hirudin stenting on key sites of the TLR4/MyD88/NF-κB pathway were detected using ELISA, Q-PCR, and western blot analysis. Results: LPS at 1 μg/mL for 48 h was the optimal modeling condition for inflammatory activation of HCASMCs. Paclitaxel+hirudin inhibited the levels of key proteins and the gene expression, except for that of the MyD88 gene, of the TLR4-MyD88 pathway. The trend of the effect of paclitaxel+hirudin on the pathway proteins was similar to that of MyD88 knockdown. After epoxomicin intervention, the inhibitory effects of paclitaxel+hirudin on the key genes and proteins of the TLR4-MyD88 pathway were significantly weakened, which even reached pre-intervention levels. Paclitaxel+hirudin affected the MyD88 protein in a dosage-dependent manner. Conclusion: The paclitaxel+hirudin compound promotes MyD88 degradation in the TLR4/MyD88/NF-κB pathway to reduce the activity of TLR4 and NF-κB p65 and to weaken the LPS-initiated inflammatory reactions of IL-1β, IL-6, and TNF-α.

Abstract Objective PKD is a genetic disease that is characterized by abnormally proliferative epithelial cells in the kidney and liver. Urinary exosomes have been previously examined as a source of unique proteins that may be used to diagnose and monitor the progression of PKD. Previous studies by our group have shown that AGS3, which is a receptor-independent regulator G-proteins, was markedly upregulated in RTECs during kidney injury including PKD. In this study, our goal was to determine whether AGS3 could be measured in exosomes using animals and humans with PKD. Results In our study, urinary exosomes were isolated from PCK rats and the control Sprague–Dawley (SD) rats. AGS3 expression was significantly increased (P

Background. Previous studies have evaluated the biomechanical, histological, and clinical effects of rotator cuff repair with different suture materials. However, a histological and western blot analysis after repair comparing nonabsorbable polyester and polyethylene suture materials in a chronic rotator cuff tear model has not been conducted in any previous studies. The aim of this study is to evaluate the effect the two different suture materials, specifically Ethibond and Ultrabraid, have on repair based on a histological evaluation and protein expression by means of a western blot analysis. Methods. Twenty-six rabbits were included in the study. Two rabbits served as normal controls (control group). A bilateral tenotomy of the subscapularis tendon was performed in the remaining 24 rabbits. Six weeks after the tenotomy, the animals were randomly divided into four repair groups (five rabbits in each group) according to the suture material used and the sacrifice time (Ultrabraid two weeks after repair, Ethibond two weeks after repair, Ultrabraid six weeks after repair, and Ethibond six weeks after repair). Two sham-operated groups with two rabbits each were sacrificed at eight and 12 weeks after the tenotomy without repair. Both shoulders of all the animals were used for a western blot (collagen type 1-3, MMP-2, TGF-beta-1, IGF-1, COX-2) analysis and a histological evaluation (fibrocartilage formation, inflammation, vascularisation, collagen density). Results. All groups showed minimal-to-mild histological changes irrespective of the type of suture material used. There was no statistically significant difference in histological scores (p > 0.05). Fibrocartilage formation was greater at six weeks in the polyester suture repair groups without statistical significance (p > 0.05). Collagen type 1-3, MMP-2, TGF-beta-1, IGF-1, and COX-2 were significantly upregulated at two weeks compared to six weeks after repair (p < 0.05). Protein expression levels were statistically significantly higher in the Ultrabraid groups compared to the Ethibond groups (p < 0.05). Conclusions. Changes in the expression of the markers related to rotator cuff healing were demonstrated when repairing with different suture materials, with these being in favour of polyester sutures; however, the histological findings are similar. This finding could influence the design of new suture materials. [ABSTRACT FROM AUTHOR]

Background: This study is intended to clarify the influence of exogenous bovine lactoferrin (bLf) treatment on glycemic homeostasis, gene expression, and production of lactoferrin (Lf) in rats with experimentally induced diabetes. Methodology: Fifty adult male rats were used; 15 rats were used as the normal control rats (group A) and were injected an intraperitoneal (I/P) with 1 ml of isotonic saline daily for 3 months. Thirty-five rats were administered I/P injection of 60 mg of streptozotocin per kilogram body weight to induce diabetes. Thirty streptozotocin-treated rats were selected and used as diabetic rats, and they were subdivided into 2 equal groups (15 rats each): the untreated diabetic group (group B) and treated diabetic group (group C). The rats in the diabetic group were administered an I/P injection of 1 ml of isotonic saline daily for 3 months, while rats in the diabetic treatment group were administered an I/P injection of 300 mg of bLf/kg body weight/day/3 months. At the end of the experiment, blood and renal tissue samples were collected from all rats. The levels of insulin, glucose, and lactoferrin in plasma were measured. Homeostatic model assessment for insulin resistance (HOMA-IR) was performed. The numbers of Lf-mRNA copies in renal tissue were assessed using quantitative RT-PCR. To measure Lf production in renal tissue, Western blot analysis was applied. Results: The obtained data demonstrated that the treatment of diabetic rats with bLf maintained glycemic homeostasis at normal levels but increased the mRNA expression of renal Lf. Conclusion: In diabetic rats, bovine lactoferrin treatment offers the potential for protection against incidences of insulin resistance (IR) by stimulating the suppressed expression and production of the LTF gene. [ABSTRACT FROM AUTHOR]

Objective: Liver cancer is the third rank amongst the common malignancies, causing maximum death in the patients diagnosed with cancers. Currently available biomarkers are not enough sensitive for early diagnosis of hepatocellular carcinoma (HCC). This makes difficult management of HCC. With the aim of finding new generation of proteomic-based biomarkers, the represented study was designed to characterize the differentially expressed proteins at different stages of HCC initiation and at progression. This could lead to find potential biomarkers for early detection of HCC. Materials and Methods: In this experimental study, we report induction of HCC by administrating chemical carcinogens in male Wistar rats. Disease progression was monitored by histological evaluation. Serum proteomic analyses such as 2 dimensional (2D)-electrophoresis, MALDI-TOF-MS/MS and Western blot have been used to analyze and characterize the differentially expressed proteins during HCC development. Results: HCC initiation and tumorigenesis were observed at one and four months post carcinogen treatment, respectively. One of the differentially-expressed proteins, namely, cytosolic phospholipase A2 delta was significantly up-regulated at very early stage of HCC development. Its expression continued to increase during cancer progression and hepatotumorigenesis stages. Its elevated expression has been confirmed by Western blot analysis. Consistent to this, analyses of the sera in the clinically confirmed liver cancer patients showed elevated expression of this protein, further validating our experimental results. Conclusion: This study suggests that elevation in the expression of cytosolic phospholipase A2 delta is associated with progression of HCC. [ABSTRACT FROM AUTHOR]

The production of streptococcus pyogenes serum opacity factor is associated with GAS strains that are tropic for the skin, and as such are correlated with diseases such as impetigo and acute glomerulonephritis. In this study Aboriginal sera and saliva samples were used to study the immune response to streptococcus pyogenes serum opacity factor resulting from infection by streptococcus pyogenes. [ABSTRACT FROM AUTHOR]

Serovars of Salmonella enterica encounter various microenvironments in the process of cycling through their numerous host and non-host niches. Many of these environs are less than optimal for growth and can even threaten viability. [ABSTRACT FROM AUTHOR]

This study was conducted to evaluate the influence of DNA methylation of metastasis suppressor 1 (MTSS1) on prostate cancer (PCa) progression. Forty‐nine paired PCa tissue samples and normal tissue samples from The Cancer Genome Atlas were analyzed. Methylome analysis, CpG island arrays and Hierarchical clustering were used to analyze methylation profiles of PCa tissues. MTSS1 methylation level was detected by methylation‐specific PCR. Relative messenger RNA and the expression level of MTSS1 protein were identified by quantitative real‐time PCR (qRT‐PCR) and western blot analysis. The migration, invasion, proliferation, and cell cycle were detected separately by wound‐healing assay, transwell chamber assay, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and flow cytometry. The roles of MTSS1 in PCa progression were demonstrated in vivo by tumor formation assays in nude mice. MTSS1 expression was decreased in PCa tissues in comparison with paired adjacent normal prostate tissues. Compared to the methylation of MTSS1 in normal prostate tissues based on the MethHC website, the MTSS1 in PCa tissues was hypermethylated. The expression of MTSS1 detected by qRT‐PCR and western blot analysis was found to be downregulated in PCa cells and tissues. The reduced expression of MTSS1 by small interfering RNA‐MTSS1 was recovered by 5‐aza‐2′‐deoxycytidine treatment. Besides, MTSS1 demethylation inhibited migration, invasion, and proliferation of PCa cells, and induced cell cycle to be arrested at G0/G1 phase. Furthermore, it was shown by tumor xenograft assay that MTSS1 inhibited the growth of tumor in vivo. Hypermethylated MTSS1 promoted PCa cells migration, invasion, and proliferation, and suppressed cell cycle arrest at the G0/G1 phase. [ABSTRACT FROM AUTHOR]

This study aimed to characterize the morphological changes in the ovary of the female crab Neptunus pelagicus and to identify specific fractions of vitelloginin and vitelline molecules during primary and secondary vitellogenesis. Samples of the blue crab were collected from the Mediterranean Sea of Alexandria monthly during 2017. Ovaries and oocytes in primary and secondary vitellogenesis were detached and treated for histological test. Native polyacrylamide gel electrophoresis (PAGE) Bis-Tris Gels was applied to identify vitelloginin (VN) and vitelline (VL) molecules. Protein Analyses were done by PAGE-SDS. The initial degenerate primers were built regarding the conserved amino acid domains of the yolk proteins. Primary and secondary vitellogeneses consisted of 8 phases. Lipoprotein fraction with molecular weight 550 kDa was identified in the hemolymph in secondary vitellogenesis. Two protein fractions (VLI & VLII) were identified in secondary vitellogenic oocytes. The electrophoresis performed with extract of stage I oocyte showed two protein fractions with molecular weights 550 kDa and 460 kDa. In stage II and III oocyte, 4 subunits were presented of 180, 195, 140 and 120 kDa in VLI and 2 subunits with molecular weight of 110 kDa and 95 kDa in VLII. Another two fractions in stage V oocyte presented with molecular weights of 380 kDa and 360 kDa. Western blot analysis proved that both fractions were of four major polypeptide subunits with molecular weight of 180, 125, 90 and 85 kDa in each of the two VLs. The hybridization signal obtained by the Northern blot was detected in the hepatopancreas during ovarian cycle and in the ovary during secondary vitellogenesis. The result of the reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNA that encodes the C-terminal region of the VN cDNA was found in the ovary in secondary vitellogenesis and in the hepatopancreas. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that the expression of CARD10 is closely associated with the occurrence of tumors, and its role is mainly to promote tumor progression by activating the transcription factor NF-κB. However, the signaling pathway in renal cancer remains unclear. The objective of the present study was to investigate the ability of caspase recruitment domain 10 (CARD10) to regulate the NF-κB signaling pathway and promote the progression of renal cell carcinoma (RCC). Expression of CARD10 in ACHN, 786-O and HK-2 cells was evaluated via western blot analysis, as was the epidermal growth factor (EGF)-induced activation of NF-κB signaling pathway-related proteins in cells. The expression of CARD10 was inhibited by CARD10 short hairpin RNA transfection. Cell cycle analysis and MTT assays were used to evaluate cell proliferation. Cell apoptosis was analyzed via flow cytometry. The invasion of renal cell lines was detected via Transwell cell migration and invasion assays in vitro. The results showed that CARD10 expression was significantly higher in RCC cells than in normal renal tubular epithelial cells. CARD10 silencing inhibited the proliferation, invasion and migration of RCC cells. EGF stimulation upregulated the activation of the NF-κB pathway in RCC cells. Inhibition of CARD10 expression inhibited NF-κB activation in RCC cells. Taken together, these data suggested that CARD10 promotes the progression of renal cell carcinoma by regulating the NF-κB signaling pathway. Thus, this indicated that CARD10 may be a novel therapeutic target in RCC. [ABSTRACT FROM AUTHOR]

Continuous optimization of in vitro analytical techniques is ever more important, especially given the development of new materials for tissue engineering studies. In particular, isolation of cellular components for downstream applications is often hindered by the presence of biomaterials, presenting a major obstacle in understanding how cell–matrix interactions influence cell behavior. Here, we describe an approach for western blot analysis of cells that have been encapsulated in self-assembling peptide hydrogels (SAPHs), which highlights the need for complete solubilization of the hydrogel construct. We demonstrate that both the choice of buffer and multiple cycles of sonication are vital in obtaining complete solubilization, thereby enabling the detection of proteins otherwise lost to SAP aggregation. Moreover, we show that the presence of self-assembling peptides (SAPs) does not interfere with the standard immunoblotting technique, offering the potential for use in more full-scale proteomic studies.

Clear cell renal cell carcinoma (ccRCC) is the most common and the most aggressive histopathological subtype of kidney cancer, with patients exhibiting high mortality rates for metastatic tumors. The Sonic Hedgehog (SHH) pathway serves a crucial role in embryonic development. The abnormal activity of SHH signaling is observed in a broad range of malignancies. However, its role in ccRCC is still undetermined. The aim of the present study was to assess the expression of the SHH pathway genes in ccRCC. Neoplastic and morphologically unchanged kidney tissues were obtained during radical nephrectomy from 37 patients with ccRCC. The SHH, PTCH1, SMO and GLI1 mRNA levels were assessed using the reverse transcription-quantitative PCR. Western blot analysis was used to assess the full-length and C-terminal SHH protein level. The mRNA levels of SHH, SMO and GLI1 were approximately 2-, 2,5- and 7-fold higher in ccRCC tissue compared with control kidney tissue, respectively. Correlational analysis between the mRNA levels of SHH pathway genes and patients' clinicopathological factors revealed decreased and increased mRNA levels of PTCH1 and SMO respectively, in tumor samples derived from older patients (age >62). Furthermore, the level of C-terminal SHH protein in ccRCC samples was significantly lower in a group of males compared with females. No correlation was exhibited between molecular data and patient survival. Western blot analysis indicated a ~3-fold higher level of SHH full-length protein, and a 4-fold lower level of the C-terminal SHH protein domain, in ccRCC tumor tissues compared with normal kidney samples. The current study indicated an involvement of the SHH pathway in ccRCC development. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Novel pyrrolopyrimidine derivatives were synthesized and evaluated for anticancer activity against A549, PC3 and MCF-7 cells. • Compounds 6b , 8a , 9a and 7a , 8b displayed the significant cytotoxic activities against A549 and PC3 cells with IC 50 values ranging from 0.35 to 1.89 µM. • Compounds 6b , 8a and 9a induced apoptosis in A549 cells by intrinsic pathway. • Also, compounds 6b , 8a and 9a induced apoptosis in HCT116 wt cells. Bax/Bak KO cells were found resistance to 6b , 8a and 9a treatment. Abstract In this work we described the synthesis and evaluation of cytotoxic and apoptotic activity of novel pyrrolopyrimidine derivatives against A549, PC3 and MCF-7 cells. Among the synthesized compounds, 6b , 8a , 9a and 7a , 8b displayed the significant cytotoxic activities against A549 and PC3 cells with IC 50 value of 0.35, 1.48, 1.56 and 1.04, 1.89 µM, respectively. It was found that A549 cells were more sensitive to synthesized compounds than PC3 and MCF-7 cells. In order to evaluate the mechanism of cytotoxic activity in A549, compounds 6b , 8a and 9a were selected for further studies. Annexin V binding assay and western blot analysis results revealed that 6b , 8a and 9a induced apoptosis in A549 cells by intrinsic apoptotic pathway through the activation pro-apoptotic proteins such as Bim, Bax, Bak, Puma and deactivation of anti-apoptotic proteins including Bcl-2, Mcl-1 and Bcl-XL accompanied by the activation of caspase-3, caspase-9 and cleavage of PARP. Also, compounds 6b , 8a and 9a triggered apoptosis in HCT116 wt cells via activation of caspase-3 and caspase-9, but not in HCT116 Bax/Bak KO cells, indicating resistance to 6b , 8a and 9a treatment. [ABSTRACT FROM AUTHOR]

Due to the observed increase of consumption of lupine-fortified food products and the high homology of lupine protein to other legumes, occurrence of new lupine allergy cases can increase significantly. Therefore, the aim of this study was to compare the seeds of 18 new Polish lupine breeding lines for immunoreactivity in relation to the sera of the patients sensitized to cross-reacting allergens, i.e. to soybeans and peanuts, using the western blot method. Antibodies present in the sera obtained from two adult outpatients cross-reacted with the studied extracts and made it possible to indicate six lines with decreased expression of Lup an 1 (high molecular weight subunit of β-conglutin): Mandelup, Mirela, Puławski Różowy Wczesny, Ignis, 25-65-M-4-1 and Stadoliszczenskij L-610 lines. Two more lines (Schmalblaettrige Schwerzplatzen and Rammiespielyj) are probably also less immunoreactive, due to the decreased content of alkaline subunit of α-conglutin—Lup an 2. The highest variability in immunoreactivity of proteins in question was noted for peptides with molecular weights ~ 20, 23, 28, 33, 38, 39, 43, 49, 50 and 63 kDa. Unfortunately, different patterns of immunoreactivity of the seeds obtained from various cultivation sites was noted for some lines. The correlation between the total protein content and reduced immunoreactivity of the tested lines was not confirmed. Thus, the lines with decreased immunoreactivity could be considered material for future crossing the narrow-leafed lupine lines to obtain varieties intended for food production. [ABSTRACT FROM AUTHOR]

Background/Aims: Approximately 10%-20% of patients with acute cardiovascular disease who have received coronary intervention suffer restenosis and high inflammation. The stent compound paclitaxel+hirudin was prepared for the treatment of post-intervention restenosis. This study aimed to explore the anti-inflammatory and anti-restenosis mechanisms of paclitaxel+hirudin with regard to the TLR4/MyD88/NF-κB pathway. Methods: Human coronary artery smooth muscle cells (HCASMCs) at 4-6 generations after in vitro culture were used as a model. Lipopolysaccharide (LPS) was used as an inducer to maximally activate the TLR4/MyD88/NF-κB inflammation pathway. After MyD88 knockdown and selective blocking of MyD88 degradation with epoxomicin, the effects of paclitaxel+hirudin stenting on key sites of the TLR4/MyD88/NF-κB pathway were detected using ELISA, Q-PCR, and western blot analysis. Results: LPS at 1 μg/mL for 48 h was the optimal modeling condition for inflammatory activation of HCASMCs. Paclitaxel+hirudin inhibited the levels of key proteins and the gene expression, except for that of the MyD88 gene, of the TLR4-MyD88 pathway. The trend of the effect of paclitaxel+hirudin on the pathway proteins was similar to that of MyD88 knockdown. After epoxomicin intervention, the inhibitory effects of paclitaxel+hirudin on the key genes and proteins of the TLR4-MyD88 pathway were significantly weakened, which even reached pre-intervention levels. Paclitaxel+hirudin affected the MyD88 protein in a dosage-dependent manner. Conclusion: The paclitaxel+hirudin compound promotes MyD88 degradation in the TLR4/MyD88/NF-κB pathway to reduce the activity of TLR4 and NF-κB p65 and to weaken the LPS-initiated inflammatory reactions of IL-1β, IL-6, and TNF-α. [ABSTRACT FROM AUTHOR]

Two polypyridyl ruthenium(II) complexes [Ru(DIP) 2 (BIP)](PF 6) 2 (DIP = 4,7-diphenyl-1,10-phenanthrolie, BIP = 2-(1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f][1,10]phenanthroline, Ru1) and [Ru(DIP) 2 (CBIP)](PF 6) 2 (CBIP = 2-(4′-chloro-1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f][1,10]phenanthroline, Ru2) were synthesized. The cytotoxic activities in vitro of Ru1, Ru2 toward B16, A549, HepG2, SGC-7901, HeLa, BEL-7402, non-cancer LO2 were investigated using MTT method (3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide). Unexpectedly, Ru1, Ru2 can't prevent these cancer cells proliferation. To improve the anti-cancer effect, we used liposomes to entrap the complexes Ru1, Ru2 to form Ru1lipo, Ru2lipo. As expectation, Ru1lipo and Ru2lipo exhibit high anti-cancer efficacy, especially, Ru1lipo (IC 50 3.4 ± 0.1 μM), Ru2lipo (IC 50 3.5 ± 0.1 μM) display strong ability to block the cell proliferation in SGC-7901. The cell colony, wound healing, and cell cycle distribution show that the complexes can validly inhibit the cell growth at G2/M phase. Apoptotic studied with Annex V/PI doubling method showed that Ru1lipo and Ru2lipo can effectively induce apoptosis. Reactive oxygen species (ROS), malondialdehyde, glutathione and GPX4 demonstrate that Ru1lipo and Ru2lipo improve ROS and malondialdehyde levels, inhibit generation of glutathione, and finally result in a ferroptosis. Ru1lipo and Ru2lipo interact on the lysosomes and mitochondria and damage mitochondrial dysfunction. Additionally, Ru1lipo and Ru2lipo increase intracellular Ca2+ concentration and induce autophagy. The RNA-sequence and molecular docking were performed, the expression of Bcl-2 family was investigated by Western blot analysis. Antitumor in vivo experiments confirm that 1.23 mg/kg, 2.46 mg/kg of Ru1lipo possesses a high inhibitory rate of 53.53% and 72.90% to prevent tumor growth, hematoxylin-eosin (H&E) results show that Ru1lipo doesn't cause chronic organ damage and strongly promotes the necrosis of solid tumor. Taken together, we conclude that Ru1lipo and Ru2lipo cause cell death through the following pathways: autophagy, ferroptosis, ROS-regulated mitochondrial dysfunction, and blocking the PI3K/AKT/mTOR. Two new Ru(II) polypyridyl complexes [Ru(dip) 2 (BIP)](PF 6) 2 (dip = 4,7-diphenyl-1,10-phenanthrolie, BIP = 2-(1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f] [1,10]phenanthroline, Ru1) and [Ru(dip) 2 (CBIP)](PF 6) 2 (CBIP = 2-(4′-chloro-1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f] [1,10]phenanthroline, Ru2) and complexes-loaded liposomes Ru1lipo and Ru2lipo were synthesized and characterized. The cytotoxicity of the complexes Ru1, Ru2, Ru1lipo and Ru2lipo against B16, BEL-7402, A549, HepG2, SGC-7901 cancer cells and non-cancer cell LO2 was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. The high inhibitory rate in vivo of Ru1lipo inhibiting SGC-7901 tumor growth reaches 72.90%. [Display omitted] • Two new Ru(II) polypyridyl complexes were synthesized and characterized. • The cytotoxicity in vitro and in vivo of the complexes and liposomes was investigated. • Apoptosis, reactive oxygen species and cell cycle arrest were performed. • RNA-sequence assay was investigated. • The antitumor activity in vivo was investigated. [ABSTRACT FROM AUTHOR]

In this work, four phenolic compounds, N1,N5-di-[(E)-p-coumaroyl]-spermidine (1), p-coumaric acid (2), ferulic acid (3), and rutin (4) were isolated from n-butanol fraction of acetone extract of defatted adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) seed meal. Compound 1 was identified from adlay for the first time. The structures of these compounds were characterised by ESI-MS, 1H NMR, 13C NMR and UV–vis techniques. These compounds were evaluated for their anti-tumour activities against HepG-2, MCF-7 and CaCo-2 cancer cell lines by MTT assay. Of all purified compounds, compound 1 possessed the highest anti-tumour activity than other compounds with IC50 values between 46.34 ± 3.99 and 92.69 ± 5.63 µg/ml. Furthermore, activation of the caspase cascade in HepG-2 cells for intrinsic pathways caused by compound 1 were observed using western blot analysis. The result also showed that the expression of caspase-3, caspase-9, cytochrome c and Bax protein increased and the protein level of Bcl-2 decreased.

Cross-reactive immunological material (CRIM) status is an important prognostic factor in patients with infantile Pompe disease (IPD) being treated with enzyme replacement therapy. Western blot analysis of cultured skin fibroblast lysates has been the gold standard for determining CRIM status. Here, we evaluated CRIM status using peripheral blood mononuclear cell (PBMC) protein. For 6 of 33 patients (18%) CRIM status determination using PBMC was either indeterminate or discordant with GAA genotype or fibroblast CRIM analysis results. While the use of PBMCs for CRIM determination has the advantage of a faster turnaround time, further evaluation is needed to ensure the accuracy of CRIM results.