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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.

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.

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.

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]

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]

[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]

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]

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]

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.

Research dealing with early diagnosis and efficient treatment in colon cancer to improve patient's survival is still under investigation. Chemotherapeutic agent result in high systemic toxicity due to their non‐specific actions on DNA repair and/or cell replication. Traditional medicine such as Lycopodium clavatum (LC) has been claimed to have therapeutic potentials against cancer. The present study focuses on targeted drug delivery of cationic liposomal nanoformulated LC (CL‐LC) in colon cancer cells (HCT15) and comparing the efficacy with an anti‐colon cancer drug, 7‐ethyl‐10‐hydroxy‐camptothecin (SN38) along with its nanoformulated form (CL‐SN38). The colloidal suspension of LC was made using thin film hydration method. The drugs were characterised using ultraviolet, dynamic light scattering, scanning electron microscopy, energy, dispersive X‐ray spectroscopy. Invitro drug release showed kinetics of 49 and 89% of SN38 and LC, whereas CL‐SN38 and CL‐LC showed 73 and 74% of sustained drug release, respectively. Studies on morphological changes, cell viability, cytotoxicity, apoptosis, cancer‐associated gene expression analysis of Bcl‐2, Bax, p53 by real‐time polymerase chain reaction and western blot analysis of Bad and p53 protein were performed. Nanoformulated LC significantly inhibited growth and increased the apoptosis of colon cancer cells indicating its potential anti‐cancer activity against colon cancer cells. [ABSTRACT FROM AUTHOR]

Silencing of tumor suppressor genes by hypermethylation in gene promoter regions is a crucial mechanism in carcinogenesis. Gene methylation may be reversible and evaluated easily, thus providing a promising substrate for the development of biomarkers for the detection and prevention of cancer, including colorectal cancer (CRC). In the present study, the protein expression and methylation status of smooth muscle protein 22α (SM22α) was investigated in 78 cases of CRC and adjacent normal tissue. The aim of the study was to investigate the function of SM22α in the pathogenesis of CRC and to identify a candidate biomarker for the early detection of CRC. The methylation status of promoter of SM22α gene was detected using methylation-specific polymerase chain reaction. The protein expression of SM22α was evaluated using western blot analysis. The results demonstrated a significant decrease of SM22α protein expression in 50 (68.5%) cases of CRC compared with that in adjacent normal tissues (P<0.001). The methylation status of SM22α promoter in CRC was significantly increased compared with that in adjacent normal tissues (P<0.001). Additionally, there was a negative correlation between the expression of SM22α protein and methylation levels of SM22α gene in CRC (P<0.001). Kaplan-Meier curves revealed that patients with CRC with an unmethylated promoter of SM22α gene exhibited an increased survival time (34.8±0.6 vs. 30.9±1.3 months; P=0.025) compared with that in patients with a methylated promoter of SM22α gene. The present study demonstrated that the protein expression of SM22α is downregulated in CRC tissues by hypermethylation of its promoter, and that the methylation of SM22 α promoter may be used as a biomarker for early detection, prognosis and prediction of CRC. [ABSTRACT FROM AUTHOR]

Epithelial cell transforming 2 (Ect2) protein is a member of the human diffuse B-cell lymphoma family of guanine nucleotide exchange factors, which activate the Ras homolog gene family of small GTPases; however, the clinical implications of Ect2 in colorectal carcinoma (CRC) are unclear. The present study aimed to determine the relationship between Ect2 expression and prognosis in patients with CRC. Western blot analysis and immunohistochemistry assays were used to determine the expression of Ect2 in CRC and paired non-cancerous tissues from 66 patients. The correlation between Ect2 expression and clinicopathological parameters was assessed using χ² tests. Patient survival was determined using the Kaplan-Meier method and log-rank test. Cox regression was used for multivariate analysis of prognostic factors. Results demonstrated that Ect2 protein was highly expressed in human CRC samples [29/45 (64.45%)] and significantly correlated with a poor prognosis (P<0.05). Compared with normal tissues, CRC tissues demonstrated higher expression levels of Ect2 mRNA [44/66 (66.67%)]. In addition, highly-expressed Ect2 was significantly associated with recurrence (P=0.023) and invasion (P=0.008) of CRC. High Ect2 expression levels in patients were associated with poorer overall survival (OS) and disease-free survival (DFS) compared with lower expression levels of Ect2. Based on multivariate analysis, Ect2 overexpression was significantly correlated with OS and DFS (P=0.015 and 0.020, respectively). In conclusion, Ect2 overexpression is an independent and important prognostic factor for OS and DFS in patients with CRC. [ABSTRACT FROM AUTHOR]

Compounds were isolated from Ecklonia stolonifera Okamura, a marine brown alga widely consumed as food. Among the isolated compounds, 974-A was demonstrated for the first time to be a potent competitive inhibitor of mushroom tyrosinase activity towards l-tyrosine and l-DOPA (IC50 values = 1.57 ± 0.08 and 3.56 ± 0.22 µM, respectively). Molecular docking simulations clarified that the hydroxyl residues of the isolated compounds formed hydrogen bonds with residues at the catalytic and allosteric sites of tyrosinase, while other residues participated in hydrophobic interactions. Moreover, 974-A, phlorofucofuroeckol-A and eckol reduced the cellular melanin content and tyrosinase activity, and downregulated the expression of melanogenesis enzymes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 in B16F10 melanoma cells. These compounds also effectively scavenged radicals at the cellular level. Thus, our results revealed that compounds isolated from E. stolonifera are potent tyrosinase inhibitors with potential applications in the cosmetic industry for treatment of hyperpigmentation and for the anti-browning effect in the agricultural field.

Citrus tristeza virus (CTV) is among the most destructive pathogens of citrus and causes substantial economic losses in citrus-growing industry worldwide. Considering recent distribution of this pathogen and its capability of transmission by existing aphid vectors in Iran, detection of this virus is enforceable for controlling the damage caused by this pathogen in Iran, as one of the major citrus producing countries. Toward this aim, developing a reliable and sensitive detection method such as enzyme- linked immunosorbant assay (ELISA) would be the first step to detect CTV in large scale screenings of field samples. As the serological method requires great amounts of specific antibody, the consequent preparation of a large scale antigen source for immunization process is necessary. In this study the coat protein gene of CTV (CP25) was amplified by polymerase chain reaction from a cloned CP25 gene in pTZ57R/T and subcloned in pET26b expression vector and named pET-CP25. Two Escherichia coli strains of BL21 and Rosetta Gami (DE3) were transformed by pET-CP25. Expression of recombinant protein was induced by IPTG. The authenticity of recombinant protein was confirmed by western immunoblot analysis using a polyclonal antiserum against CTV particles. The results indicated that CTV coat protein gene was expressed in E.coli. This recombinant protein could be used as a source of antigen for immunization process.

In Leishmania (L.) major-infected BALB/c mice Th2-type cells results in disease progression, whereas C57BL/6-infected mice mount a Th1-type response, which leads to control of the infection. Th2 response correlates with IgG1 whereas, Th1 response supports switching to IgG2a subclasses. Since IgG isotype-dominated response depends on different CD4 + T cell subsets, we studied the antigenic profile of L. major promastigotes that induce IgG1, IgG2a and IgG2b isotypes in BALB/c and C57BL/6 mice to provide insight into Th1 and Th2 inducing antigens. Humoral immune responses were studied in sera from L. major-infected BALB/c and C57BL/6 mice by ELISA and immunoblot analysis. These techniques were used to detect IgG, IgG1, IgG2a, and IgG2b antibodies against crude promastigote antigens. BALB/c mice showed higher IgG, IgG1 and IgG2a antibody levels compared to C57BL/6 mice (p

High expression of B-cell specific Moloney leukemia virus insert site 1 (Bmi-1) and peptidyl arginine deiminase IV (PADI4) is associated with esophageal carcinoma. However, few studies have investigated the association between the Bmi-1 and PADI4 genes. The aim of the present study was to evaluate the expression of Bmi-1 and PADI4 and identify the association between the Bmi-1 and PADI4 genes in esophageal squamous cell carcinoma (ESCC) tissues. Bmi-1 and PADI4 gene expression levels were measured using immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction in ESCC tissues from 86 patients who had not received pre-operative chemoradiation. The results revealed that the Bmi-1 and PADI4 genes had increased expression in carcinoma tissues compared with pericarcinous tissue (P<0.05). Bmi-1 gene expression was revealed to be associated with differentiation degree, clinical stage and lymph node metastasis (P<0.05), but had no association with gender, age or depth of invasion (P>0.05). The expression of PADI4 was associated with clinical stage, depth of invasion and lymph node metastasis (P<0.05), but was not associated with gender, age or differentiation degree (P>0.05). In addition, there was a positive association between Bmi-1 and PADI4 gene expression in ESCC (P<0.05). These results indicated that Bmi-1 and PADI4 positively regulate carcinogenesis and progression of ESCC. [ABSTRACT FROM AUTHOR]

To quantitatively evaluate the production of staphylococcal enterotoxin A (SEA) in cooked rice, we inoculated 11 strains of SEA-producing Staphylococcus aureus into cooked rice, incubated the samples at 15 - 37°C, and quantified SEA. The maximal concentrations of SEA were 0.0 (not detected)-4.0 ng/g at an incubation temperature of 15°C, 0.4 - 8.4 ng/g at 20°C, and 2.3-53.8 ng/g at 37°C with an inoculum size of 106CFU/g. There were few correlations among the maximal concentrations of SEA at incubation temperatures of 15, 20, and 37°C or between the maximal concentrations of SEA produced in a liquid medium and in cooked rice. Although the predicted amounts of SEA produced in a rice ball under conditions of 16 h of incubation at 20°C or 4 h at 37°C appeared to reach levels capable of causing staphylococcal food poisoning, S. aureus growth under these conditions was not organoleptically detected. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the possible mechanism of Janus kinase (JAK)-signal transduction and activator of transcription (STAT)/PSMβ3 signaling in the occurrence of asthenozoospermia. We examined seminal fluid samples from 30 cases of asthenozoospermia and 30 healthy controls. Sperm was collected using the Percoll density gradient centrifugation method. The expression of JAK, STAT and PSMβ3 mRNA was assessed by reverse-transcription quantitative PCR and the protein levels of p-JAK, p-STAT and PSMβ3 were measured by western blot analysis. The PSMβ3 mRNA and protein expression levels were also measured after application of a JAK inhibitor, AG-490, to the control group, with a FITC-labeled monoclonal rabbit anti-human PSMβ3 primary antibody. The cells were observed under a laser confocal microscope. The mRNA levels of JAK, STAT and PSMβ3 in asthenozoospermia were decreased significantly (P<0.05). The protein levels of p-JAK, p-STAT and PSMβ3 in asthenozoospermia were also reduced and the differences were statistically significant (P<0.05). The PSMβ3 mRNA and protein expression levels were decreased in the control group after treatment with the JAK inhibitor, and levels were approximately equal to those of the asthenozoospermia group. PSMβ3 was mainly expressed in round-headed sperm, and less in asthenozoospermia. In conclusion, the JAK-STAT/PSMβ3 signaling transduction pathway may be involved in the pathogenic mechanism of asthenozoospermia. [ABSTRACT FROM AUTHOR]

Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc.

Outer membrane proteins (OMPs) of three strains of B. abortus i.e. S19, RB51 and a local field isolate of biotype 1 were isolated through disrupting cells to generate membranes by centrifugation and sodium lauryl sarcosinate solubilisation of inner membrane proteins. Distinct OMP profiles of each strain were seen on SDS-PAGE. SDS-PAGE analysis of S19 and field isolate revealed eight protein bands in each strain. The OMPs of S19 had molecular masses 89.0, 73.0, 53.7, 49.0, 38.0, 27.0, 22.3, and 17.7 kDa, while field isolate had OMPs of 151.3, 89.0, 75.8, 67.6, 37.0, 27.0, 24.0 and 19.0 kDa. B. abortus RB51 yielded 11 OMP bands ranging from 12.5 to 107.1 kDa, with 34.2, 15.8 and 12.5 kDa as additional OMPs. Western immunoblot analysis using antisera raised against all three strains in buffaloes indicated an almost similar pattern of immuno-reactive OMPs in S19 and field strain. Two OMPs of molecular weight 37-38 and 19 kDa were immuno-reactive in all strains in buffaloes. There is possibility of use of these OMPs in a recombinant vaccine for B. abortus. A distinct protein of molecular weight of 151.3 kDa was identified in field strain but not in both vaccine strains of B. abortus. Use of this OMP in a diagnostic assay may differentiate between vaccinated and infected animals.