Nigella sativa, Anthemis hyaline and Citrus sinensis extracts reduce SARS-CoV-2 replication by fluctuating Rho GTPase, PI3K-AKT, and MAPK/ERK pathways in HeLa-CEACAM1a cells.

Traditional remedies have long utilized Anthemis hyaline, Nigella sativa, and Citrus sinensis peel extracts as treatments for microbial infections. This study aimed to investigate the influence of Anthemis hyaline, Nigella sativa, and Citrus sinensis extracts on coronavirus replication and apoptosis-related pathways. HeLa-CEACAM1a cells were exposed to mouse hepatitis virus-A59. After viral inoculation, the mRNA levels of 36 genes were quantified using a Fluidigm Dynamic Array nanofluidic chip. IL-8 level and intracellular Ca2+ concentration was measured, and viral titer was assessed by the TCID50/ml assay to detect the extent of infection. Treatment with Nigella sativa extract surged the inflammatory cytokine IL-8 level at both 24 and 48-hour. Changes in gene expression were notable for RHOA, VAV3, ROCK2, CFL1, RASA1, and MPRIP genes following treatment with any of the extracts. The addition of Anthemis hyaline, Nigella sativa, or Citrus sinensis extracts to coronavirus-infected cells reduced viral presence, with Anthemis hyaline extract leading to a virtually undetectable viral load at 6- and 8-hours after infection. While all treatments influenced IL-8 production and viral levels, Anthemis hyaline extract displayed the most pronounced reduction in viral load. Consequently, Anthemis hyaline extract emerges as the most promising agent, harboring potential therapeutic compounds.

High-throughput screening and clinical importance of autophagy-associated genes in basal cell carcinoma.

Autophagy is a type II programmed cell death mechanism that plays a critical role in preserving cellular homeostasis through the regulation of protein, lipid, and organelle quality control. It has become gradually evident that autophagy plays a fundamental role in the initiation and progression of various types of human cancers. Nevertheless, its significance in non-melanoma skin cancers, particularly in basal cell carcinoma, has not been well documented and remains largely elusive. In this study, we aimed to illuminate the role of autophagy-associated signaling signatures during development and progression of basal cell carcinoma. For the study, a total of 72 autophagy-related genes were screened using a high-throughput qPCR approach integrating Fluidigm 96.96 Dynamic Array™ integrated fluidic circuits (IFC) and BioMark™ HD Real-Time PCR system, which enabled efficient and precise analysis of gene expression patterns. Results were analyzed using Fluidigm's Real-Time PCR Analysis software and 2-ΔΔCt formula was used for the calculation of expression changes. Notably, expression levels of INS, TMEM74 and IFNA2 genes were identified to be prominently altered in BCC comparted to adjacent healthy tissues. However, only IFNA2 expression showed statistically significant change in BCC. Consequently, these findings suggest that IFNA2 might play significant role in the regulation of autophagy in BCC development and progression and can be therapeutically targeted.

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation, migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-ß/Src signaling in breast cancer cells.

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin ß1 and ß3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

Investigation of Cytotoxic Effects of Oxymetazoline on Lungs in a Rat Model of Rhinitis Medicamentosa.

BACKGROUND: Rhinitis medicamentosa, also known as 'rebound congestion,' is inflammation of the nasal mucosa caused by the overuse of topical nasal decongestants. Although local decongestants resolve the initial nasal obstruction, the overuse causes rebound obstruction. However, how the overuse of the decongestant causes rhinitis medicamentosa is not known. OBJECTIVES: Here, we show the intracellular effects of oxymetazoline, commonly used a local decongestant, on the cell death pathways. We also investigated the antioxidative effects of erdosteine suspension (175 mg/5mL), an antioxidative agent. METHODS: Thirty Wistar-albino rats were used to form the rhinitis medicamentosa model. After rhinitis medicamentosa was clinically detected, we removed the whole lungs of animals to perform the molecular analyses of cell death pathways. RESULTS: We found a statistically significant decrease in the expression levels of Atg5 (p=0.021), Atg7 (p=0.013) and Ulk1 (p=0.036) in the oxymetazoline group compared to the control group (p<0.05); however, Caspase 3 expression level was recorded to be significantly increased in the oxymetazoline group, and the expression level of Beclin1 recorded to be substantially increased in the erdosteine group (p=0.001). CONCLUSION: Based on these grounds, we suggest that vasoconstriction in capillary vessels caused by oxymetazoline could lead to a decrease in the blood supply, which triggers autophagy to ensure cellular homeostasis.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of defined molecular targets, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and is associated with high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC represents an unmet therapeutic challenge. Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical calcium/calmodulin-dependent serine/threonine kinase that promotes TNBC tumorigenesis, progression, and drug resistance, representing a potential novel molecular target. However, the mechanisms regulating eEF2K expression are unknown. Here, we report that eEF2K protein expression is highly up-regulated in TNBC cells and patient tumors and it is associated with poor patient survival and clinical outcome. We found that loss/reduced expression of miR-603 leads to eEF2K overexpression in TNBC cell lines. Its expression results in inhibition of eEF2K by directly targeting the 3-UTR and the inhibition of tumor cell growth, migration and invasion in TNBC. In vivo therapeutic gene delivery of miR-603 into TNBC xenograft mouse models by systemic administration of miR-603-nanoparticles led to a significant inhibition of eEF2K expression and tumor growth, which was associated with decreased activity of the downstream targets of eEF2K, including Src, Akt, cyclin D1 and c-myc. Our findings suggest that miR-603 functions as a tumor suppressor and loss of miR-603 expression leads to increase in eEF2K expression and contributes to the growth, invasion, and progression of TNBC. Taken together, our data suggest that miR-603-based gene therapy is a potential strategy against TNBC.

Polymorphisms in the MTHFR gene are associated with recurrence risk in lymph node-positive breast cancer patients.

PURPOSE: The aim of this study is to clarify the relationship between recurrence risk of breast cancer and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms. PATIENTS AND METHODS: Breast cancer patients who had undergone surgery in Gaziantep University Oncology Hospital between June 2005 and June 2012 were followed-up and retrospectively enrolled in this study. Blood samples were collected from all patients to assess MTHFR C677T polymorphisms. Stage according to tumor-node-metastasis system, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 status, grade of disease, menopausal status, and administered chemotherapy or hormonal therapy were recorded. Effects of these parameters on recurrence risk were evaluated using univariate analysis and multivariate binary logistic regression model. RESULTS: Association of MTHFR C677T polymorphisms with recurrence risk was evaluated in 298 patients whose median age was 47 years (range: 21-79 years). In all patients, age (odds ratio [OR] =0.953, P=0.005) and N3 lymph node status (OR =6.293, P=0.001) were found to affect the recurrence risk. While MTHFR homozygote genotype did not have an effect on recurrence risk in all patients, increased risk was observed in lymph node-positive subgroup (OR =4.271; 95% CI 1.515-12.023; P=0.006). Adjusting for age, tumor size (T), and node status (N), MTHFR homozygote genotype had more statistically significant risk for recurrence (OR =3.255; 95% CI 1.047-10.125; P=0.041). CONCLUSION: MTHFR TT genotype was found to be associated with increased recurrence risk in patients with lymph node-positive breast cancer.

Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel.

BACKGROUND: Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. METHODS: From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m(2) every 3 weeks for four cycles, or IV 80 mg/m(2) weekly for 12 cycles, and IV 100 mg/m(2) docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. RESULTS: Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172-6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033-4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001-3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. CONCLUSION: ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy.

Gene expression profiles of autophagy-related genes in multiple sclerosis.

Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate<0.05). Thus, altered expression levels of several autophagy related genes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis.

Expression Levels of miR-30a-5p in Papillary Thyroid Carcinoma: A Comparison Between Serum and Fine Needle Aspiration Biopsy Samples.

BACKGROUND: Fine needle aspiration biopsy (FNAB) is a useful tool in the diagnosis of thyroid nodules. However, some limitations exist as approximately 25% of the cases cannot be distinguished with this method. Therefore, identification of novel diagnostic markers is very important in improving the papillary thyroid carcinoma (PTC) diagnosis. microRNAs (miRNAs) are small regulatory RNA molecules that have been involved in a variety of biological processes, including tumorigenesis. Moreover, determination of miRNAs with prognostic, diagnostic, and therapeutic potential is of a great interest today. AIMS: In the present study, we evaluated the expression level of miR-30a-5p in serum and FNAB samples of PTC patients. METHODS: A total of 60 cases were included in the study, with the patients subdivided into four groups; benign, atypical cells of undetermined significance (ACUS), malignant group, including Hurthle cell PTC (HC-PTC), and malignant without Hurthle cell PTC (non-HC-PTC). Peripheral blood and FNAB samples of the cases were collected. The serum and FNAB expression levels of miR-30a-5p among the groups were compared. The miR-30a-5p expression level was determined using real-time polymerase chain reaction (RT-PCR). RESULTS: According to both pre- and postoperative pathological diagnosis, miR-30a-5p levels were significantly increased in both serum and FNAB samples of HC-PTC and non-HC-PTC groups compared to other groups. This increase was more evident in the non-HC-PTC group (p=0.0245 for FNAB, p=0.0166 for serum). CONCLUSIONS: The results of this study suggest that miR-30a-5p might be a novel diagnostic marker candidate in PTC. Further studies are required to investigate this possibility.

Novel Βeta (ß)-Thalassemia Mutation in Turkish Children.

Beta (ß)-thalassemia is the most frequently observed hereditary blood disorder in the world. It is characterized by deficiency of hemoglobin ß-globin gene and is also a profoundly heterogeneous both at the molecular and clinical level. In the case of ß-thalassemia, there is reduced (ß(+) type) or absent (ß(o) type) synthesis of the beta chains of hemoglobin. ß-Thalassemia clinically occurs in three main forms: major, intermedia and minor according to requirement of transfusion. The objective of this study was to evaluate ß-thalassemia mutations in 89 patients ranging from 2 months to 16 years of age, who enrolled to Medical School Research and Training Hospital, Gaziantep University. The direct DNA sequence analysis was performed for mutation scanning of ß-globin gene. 89 children with ß-Thalassemia including all types were analyzed, 16 different ß-thalassemia mutations were detected. We have also identified a novel mutation (HBB.c.-80delT, rs397509430) in the promoter region (-30 TATA box) of ß-globin gene, and clinical data of patient having novel mutation was given. The ß-Thalassemia mutations were determined as ß-Thalassemia major type in 42 patients (47.19 %), ß-Thalassemia intermedia in 4 (4.49 %), ß-Thalassemia minor in 43, (48.31 %) patients. The most frequent mutation was IVS I-110 G>A, followed by IVS I-1 G>A, IVS I-6 T>C, IVS II-1 G>A, respectively.

The association of the expression of miR-122-5p and its target ADAM10 with human breast cancer.

MicroRNAs can regulate many biological functions. miR-122-5p has a tumor suppressor function through different molecular pathways. Also, our second hit, ADAM10, targeted by miR-122-5p, is a major determinant of HER2 shedding causing that trastuzumab cannot bind to HER2 receptors. Therefore, our analysis upon ADAM10 expression and miR-122-5p was a good point to understand molecular mechanism of breast cancer. In our study, we investigated the expression profiles of miR-122-5p and its target ADAM10 in 71 breast cancer patients. Immunohistochemical analysis of ER, PR and HER2 gene products was used to categorize tumors in patients. Expression data and immunohistochemical findings were evaluated to comment on the relationship between miR-122-5p and ADAM10. ADAM10 expression was higher in tumor than that of normal tissue but miR-122-5p expression was lower in tumor than that of normal tissue. The expression pattern in HER2+ patients was reverse of the overall result. It can be explained like that miR-122-5p expression increases especially in HER2+ cancer cell to suppress ADAM10 shedding activity on HER2 receptor. However, increase in expression of tumor suppressor miR-122-5p is not enough to inhibit ADAM10. All in all, we can think miR-122-5p as potential regulator of ADAM10 and trastuzumab resistance. Since if we increase miR-122-5p activity together with trastuzumab administration, then HER2+ breast cancer cells may overcome trastuzumab resistance by inhibiting ADAM10 shedding activity on HER2 receptors and increase the efficiency of trastuzumab.

Synthesis, molecular modeling, and biological evaluation of novel chiral thiosemicarbazone derivatives as potent anticancer agents.

A series of new chiral thiosemicarbazones derived from homochiral amines in both enantiomeric forms were synthesized and evaluated for their in vitro antiproliferative activity against A549 (human alveolar adenocarcinoma), MCF-7 (human breast adenocarcinoma), HeLa (human cervical adenocarcinoma), and HGC-27 (human stomach carcinoma) cell lines. Some of compounds showed inhibitory activities on the growth of cancer cell lines. Especially, compound exhibited the most potent activity (IC50 4.6 µM) against HGC-27 as compared with the reference compound, sindaxel (IC50 10.3 µM), and could be used as a lead compound to search new chiral thiosemicarbazone derivatives as antiproliferative agents.

Do fasudil and Y-27632 affect the level of transient receptor potential (TRP) gene expressions in breast cancer cell lines?

Breast cancer (BC) is the most frequent cancer type in women, and the mortality rate is high especially in metastatic disease. Ion channels such as the transient receptor potential (TRP) channels correlate with malignant growth and cancer progression. Hence, some authors have suggested that the expression levels of TRP channels may be used as a marker in the diagnosis and predicting the prognosis of BC. Also, in some recent studies, targeting TRP channels are suggested as a novel treatment strategy in BC. The aim of this study was to investigate the effect of two Rho-kinase (ROCK) inhibitors, fasudil and Y-27632, on the expression levels of TRP channel genes in breast cancer cell lines (ZR-75-1, MCF7, and MDA-MB-231) and breast epithelial cell line (hTERT-HME1). The expression levels of TRP genes were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We found that fasudil had reduced the TRPC1, TRPV2 expression levels in the ZR-75-1, MCF7, and MDA-MB-231 cell lines. On the other hand, fasudil and Y-27632 had reduced TRPM6 expression levels in all cell lines. Y-27632 increased the expression levels of TRPC7 in all cell lines. In conclusion, this is the first study demonstrating that the inhibition of ROCK pathway changes the expression levels of some TRP genes. Also, our study has firstly shown that the expression levels of the TRP genes which are suggested as a diagnostic and prognostic biomarker in BC, were changed with the treatment of fasudil and Y-27632.

Evaluation of Factor V G1691A, prothrombin G20210A, Factor XIII V34L, MTHFR A1298C, MTHFR C677T and PAI-1 4G/5G genotype frequencies of patients subjected to cardiovascular disease (CVD) panel in south-east region of Turkey.

Cardiovascular disease (CVD) risk factors, such as arterial hypertension, obesity, dyslipidemia or diabetes mellitus, as well as CVDs, including myocardial infarction, coronary artery disease or stroke, are the most prevalent diseases and account for the major causes of death worldwide. In the present study, 4,709 unrelated patients subjected to CVD panel in south-east part of Turkey between the years 2010 and 2013 were enrolled and DNA was isolated from the blood samples of these patients. Mutation analyses were conducted using the real-time polymerase chain reaction method to screen six common mutations (Factor V G1691A, PT G20210A, Factor XIII V34L, MTHFR A1298C and C677T and PAI-1 -675 4G/5G) found in CVD panel. The prevalence of these mutations were 0.57, 0.25, 2.61, 13.78, 9.34 and 24.27 % in homozygous form, respectively. Similarly, the mutation percent of them in heterozygous form were 7.43, 3.44, 24.91, 44.94, 41.09 and 45.66%, respectively. No mutation was detected in 92 (1.95%) patients in total. Because of the fact that this is the first study to screen six common mutations in CVD panel in south-east region of Turkey, it has a considerable value on the diagnosis and treatment of these diseases. Upon the results of the present and previous studied a careful examination for these genetic variants should be carried out in thrombophilia screening programs, particularly in Turkish population.

Screening of common and novel familial mediterranean fever mutations in south-east part of Turkey.

Familial mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder (MIM# 249100), particularly common in populations of Mediterranean extraction. MEFV gene, responsible for FMF, encoding pyrin has recently been mapped to chromosome 16p13.3. In the present study, 3,341 unrelated patients with the suspicion of FMF in south-east part of Turkey between the years 2009 and 2013 were enrolled and genomic sequences of exon 2 and exon 10 of the MEFV gene were scanned for mutations by direct sequencing. We identified 43 different type of mutations and 9 of them were novel. DNA was amplified by PCR and subjected to direct sequencing for the detection of MEFV gene mutations. Among the 3,341 patients, 1,598 (47.8 %) were males and 1,743 (52.1 %) were females. The mutations were heterozygous in 806 (62.3 %), compound heterozygous in 188 (14.5 %), homozygous in 281 (21.8 %) and mutations had complex genotype in 17 (1.32 %) patients. No mutation was detected in 2,051 (61.4 %) patients. The most frequent mutations were M694V, E148Q, M680I(G/C) and V726A. We could not find any significant differences between the two common mutations according to the gender. Molecular diagnosis of MEFV is a useful tool in clinical practice, thus a future study relating to genotype/phenotype correlation of FMF in more and larger group in Turkish population involving the whole MEFV gene mutations is necessary.

The effects of Nigella sativa (Ns), Anthemis hyalina (Ah) and Citrus sinensis (Cs) extracts on the replication of coronavirus and the expression of TRP genes family.

Extracts of Anthemis hyalina (Ah), Nigella sativa (Ns) and peels of Citrus sinensis (Cs) have been used as folk medicine to fight antimicrobial diseases. To evaluate the effect of extracts of Ah, Ns and Cs on the replication of coronavirus (CoV) and on the expression of TRP genes during coronavirus infection, HeLa-CEACAM1a (HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a) cells were inoculated with MHV-A59 (mouse hepatitis virus-A59) at moi of 30. 1/50 dilution of the extracts was found to be the safe active dose. ELISA kits were used to detect the human IL-8 levels. Total RNA was isolated from the infected cells and cDNA was synthesized. Fluidigm Dynamic Array nanofluidic chip 96.96 was used to analyze the mRNA expression of 21 TRP genes and two control genes. Data was analyzed using the BioMark digital array software. Determinations of relative gene expression values were carried out by using the 2(-∆∆Ct) method (normalized threshold cycle (Ct) value of sample minus normalized Ct value of control). TCID50/ml (tissue culture infectious dose that will produce cytopathic effect in 50% of the inoculated tissue culture cells) was found for treatments to determine the viral loads. The inflammatory cytokine IL-8 level was found to increase for both 24 and 48 h time points following Ns extract treatment. TRPA1, TRPC4, TRPM6, TRPM7, TRPM8 and TRPV4 were the genes which expression levels changed significantly after Ah, Ns or Cs extract treatments. The virus load decreased when any of the Ah, Ns or Cs extracts was added to the CoV infected cells with Ah extract treatment leading to undetectable virus load for both 6 and 8 hpi. Although all the extract treatments had an effect on IL-8 secretion, TRP gene expression and virus load after CoV infection, it was the Ah extract treatment that showed the biggest difference in virus load. Therefore Ah extract is the best candidate in our hands that contains potential treatment molecule(s).

Thr21Met (T21M) but not Ser89Asn (S89N) polymorphisms of the urotensin-II (UTS-II) gene are associated with Behcet's disease (BD).

Behcet's disease (BD) is multisytemic vasculitis or chronic inflammation that may lead to various autoimmune and autoinflammatory syndromes. Exact etiopathogenesis of BD has not been clarified yet. Urotensin II (UTS-II) is predominantly a vasoactive peptide and Thr21Met polymorphism in UTS-II gene was proved to increasing in some autoimmune diseases. Considering these, our objective was to evaluate whether two UTS-II gene polymorphisms (Thr21Met and Ser89Asn) were responsible in genetic susceptibility to BD in a Turkish population. A total of 198 patients with BD and 275 healthy controls were enrolled. We analyzed the genotype and allele frequencies of two UTS-II gene polymorphisms, Thr21Met and Ser89Asn, in BD patients and in controls. We found that Thr21Met but not Ser89Asn polymorphisms of the UTS-II gene were markedly associated with the risk of developing BD (p<0.0001), The Met21Met genotype was less common among BD patients (6.1% in patients vs. 17.1% in controls; p<0.0001). There was also an increase in the 21Thr allele (54.8% in BD patients vs. 43.8% in controls) and a decrease in 21Met allele frequencies (45.2% in controls vs. 56.2% in patients) in the BD groups (p<0.0044). To the best of our knowledge, for the first time in the literature, our study claims that there is an association between Thr21Met, and not between Ser89Asn polymorphisms in the UTS-II gene and BD. These results put a new player to the field of undiscovered pathogenesis of BD and hopefully provide new insights to the treatment options.

Correlation between Rho-kinase pathway gene expressions and development and progression of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and the most aggressive primary malignant tumor of the brain. Prognostic factors in GBM can be sorted as age, tumor localization, tumor diameter, symptom period and type, the extent of surgery, postoperative tumor volume, and adjuvant radiotherapy and/or chemotherapy status. Besides the interactions between actin microfilaments, microtubules, and intermediate filaments, environmental factors and intracellular signals which regulate them affect the cell invasion. Rho proteins and therefore Rho-kinase activation play important role at these changes. The aim of this study is to evaluate the relationship between the Rho-kinase pathway gene expressions and prognosis in GBM. Ninety-eight patients diagnosed as GBM between 2001 and 2010 were enrolled into the study. RNA was obtained from the paraffinized tumor tissue of the patients with formalin-fixed, paraffin-embedded RNA isolation kit and the mRNA expressions of 26 genes were investigated. There was a statistically significant negative correlation between the ages at the diagnosis and survival. There was a significant relationship between the overexpression of Rho-kinase pathway-related genes LIMK1, CFL1, CFL2, and BCL2 and low expression of MAPK1 gene and the survival of the patients. These results demonstrate for the first time that there is a marked contribution of Rho-kinase pathway-related genes to the progression and survival of the GBM. The expression of these genes may be related to response of multimodal therapy or these parameters could be used to determine possible unresponsive patients before treatment.