Overexpression of human DNA polymerase theta is a biomarker of aggressive and DNA repair-deficient papillary thyroid cancers.

BACKGROUND: DNA polymerase theta (POLQ) is an enzyme that repairs double-strand DNA breaks. POLQ is overexpressed in several cancer types, and increased expression is associated with a poor prognosis. Ablating POLQ function in vitro increases drug sensitivity to agents that cause double-strand DNA breaks, including chemotherapies and ionizing radiation. POLQ's role in thyroid cancer remains poorly understood. METHODS: Expression of POLQ and other genes of interest were analyzed in 513 papillary thyroid cancers (505 primary tumors and 8 metastatic lesions) and 59 normal thyroid samples available in the Cancer Genome Atlas. The Cancer Genome Atlas RNA and DNA sequencing data were queried with the Xena platform. The Recombination Proficiency Score was calculated to assess DNA repair efficiency. Other signaling events associated with thyroid tumorigenesis and clinical outcomes were analyzed. Univariate and multivariate analyses were performed. Treatment with the POLQ inhibitors ART558 and Novobiocin tested the effect of POLQ inhibition on in vitro thyroid cancer growth. RESULTS: POLQ expression was increased in papillary thyroid cancers compared to normal thyroid tissue (P < .05). POLQ expression levels were inversely correlated with Recombination Proficiency Score levels (P < .05). POLQ expression was highest in tall cell papillary thyroid cancers and in metastases. Higher POLQ expression was also associated with dedifferentiation, BRAF signaling, and shorter progression-free intervals (P < .05). Treatment with POLQ inhibitors decreased in vitro thyroid cancer growth (P < .05). CONCLUSION: These findings suggest that increased POLQ expression could serve as a valuable clinical marker and a potential therapeutic target in the treatment of thyroid cancer.

Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2.

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

Association of IL-22 and IL-22RA1 gene variants in Iranian patients with colorectal cancer.

AIM: In the current study, it was hypothesized that single nucleotide polymorphisms (SNPs) in the regulatory region of the IL-22 signaling pathway genes, including IL-22 and IL-22RA1 variants, may be associated with CRC susceptibility. BACKGROUND: The important role of pro-inflammatory cytokines during tumorigenesis is well-established. In recent years, IL-22 has been linked with colorectal cancer (CRC) through a number of mechanistic and observational studies. METHODS: The association of four polymorphisms in the IL-22 (rs1179251 and rs1179246) and IL-22RA1 (rs4648936 and rs10794665) genes with CRC risk were studied using a case-control design with 304 cases and 345 controls from the Iranian population. All 649 subjects were evaluated by PCR-RFLP method. RESULTS: No significant difference was found in genotype and allele frequencies between the cases and controls for either IL-22 and IL-22RA1 gene variants or CRC risk before or after adjusting for confounders. CONCLUSION: The current findings do not present any significant evidence for associations between variants in IL-22 signaling pathway genes and CRC. Complementary studies with greater sample sizes may be necessary to fully elucidate the nature of these associations.

Association of Interleukin-17 gene polymorphisms with susceptibility to chronic hepatitis B virus infection and clearance in Iranian population.

Hepatitis B virus (HBV) approximately infects 350 million people. Interleukin-17 (IL-17) as a pro-inflammatory cytokine, have been found to modulate the immune system in infectious and inflammatory diseases. Recently, the influence of genetic changes like single nucleotide polymorphisms (SNP) on expression rate and function of cytokine has been widely investigated. This study was performed to determine any possible association between four IL-17 SNPs (rs2397084, rs763780, rs2275913 and rs10484879) and chronic HBV infection. A total of 466 samples were recruited and studied including 199 chronic patients, 172 healthy controls and 95 spontaneous clearance individuals between genotype and allele frequencies. Genomic DNA was extracted from peripheral blood cells and Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) was used to determine the SNPs genotypes. Genotypes frequencies for rs10484879 were 63.8% CC, 31.7% AC, 4.5% AA in chronic group, 54.7% CC, 36.6% AC, 8.7% AA in control and 63.2% CC, 33.7% AC, 5.8% AA in cleared samples. The AC genotype for rs10484879 was significantly associated with a decreased risk of HBV chronicity (Pvalue = 0.031, OR = 2.699, 95%CI: 1.097-6.639). The genotype and allele frequencies of rs2397084, rs763780 and rs2275913 did not show significant difference between chronic HBV patients and healthy controls. Indeed, there is no significant difference between clearance and chronic patient's genotypes in four SNPs. Our results suggest that IL-17A rs10484879 single nucleotide polymorphism genotype is probably associated with susceptibility to HBV chronic infection, while no significant differences in IL-17 rs2397084, rs763780 and rs227591 distribution were found between HBV patients and spontaneous clearance individuals and control participants.

Spatial regulation of GPR64/ADGRG2 signaling by ß-arrestins and GPCR kinases.

Mechanisms of activation, signaling, and trafficking of adhesion G protein-coupled receptors (aGPCRs) have remained largely unknown. Several aGPCRs, including GPR56/ADGRG1 and GPR64/ADGRG2, show increased activity in the absence of their N-terminal fragment (NTF). This constitutive signaling is plausibly caused by the binding of extracellular N-terminal 15-25 amino acid-long tethered agonist to extracellular domains of the cognate aGPCRs. To test the role of NTF and tethered agonist in GPR64 signaling and endocytosis, we generated mutants that lack either NTF alone (ΔNTF) or NTF and tethered agonist (P622). We discover that unlike full-length GPR64, ΔNTF and P622 mutants interact with ß-arrestin1 and ß-arrestins2 and are constitutively internalized in steady states. However, only ΔNTF shows exaggerated basal activation of the Gαs -cAMP-CRE signaling cascade. Neither ΔNTF nor P622 shows constitutive activation of the Gα13 -SRE pathway, but both mutants respond to exogenously added agonistic peptide via CRE and SRE. GPCR kinases and dynamin mediate the constitutive internalization of ΔNTF and P622 to early endosomes, where ΔNTF constantly induces CRE. These data suggest that NTF not only shields the tethered agonist to prevent G protein signaling but also confers a conformation that inhibits the interaction with ß-arrestins and the consequent endocytosis and sustained signaling from endosomes.

V617F-independent upregulation of JAK2 gene expression in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is one of the most important immune-mediated disorders of the gastrointestinal tract. Besides, IBD is associated with numerous extraintestinal complications such as venous thromboembolism (VTE), an important risk factor for vascular complications, which results in the increased morbidity and mortality. The JAK2 (Janus kinase 2) V617F mutation is a well-known point mutation which is involved in the pathogenesis of IBD, and VTE. Therefore, the aims of this study were to evaluate expression of JAK2 and association of V617F mutation in JAK2 of Iranian patients with IBD. METHODS: Two hundred and forty-six patients with IBD (209 UC and 37 CD) and 206 healthy controls were enrolled in this study. The genomic DNA and total RNA were extracted from peripheral blood mononuclear cells (PBMCs). Then, the JAK2 V617F mutation detection was performed using the restriction fragment length polymorphism (RFLP) method. In addition, the JAK2 mRNA expression was evaluated using a quantitative polymerase chain reaction (q-PCR) using the SYBR Green assay. RESULTS: There was no association of V61F mutation in patients with IBD with or without thrombosis compared with healthy control. However, the relative mRNA expression of JAK2 was significantly upregulated in patients with IBD in comparison with healthy control (P < 0.0001). In addition, the JAK2 mRNA expression was significantly decreased in patients with IBD having thrombosis compared with those without thrombosis ( P < 0.0001). CONCLUSIONS: Taken together our findings suggested that JAK2 V61F-independent upregulation of JAK2 mRNA expression in patients with IBD. Moreover, despite the absence of JAK2 V617F mutation in patients with IBD, the increased gene expression of JAK2 can be explained by another molecular mechanism such as regulation of gene expression at the transcriptional level which may play crucial roles in the pathogenesis of IBD.

Parathyroid-Targeted Overexpression of Regulator of G-Protein Signaling 5 (RGS5) Causes Hyperparathyroidism in Transgenic Mice.

The relationship between impaired calcium sensing, dysregulated parathyroid hormone (PTH) secretion, and parathyroid cell proliferation in parathyroid neoplasia is not understood. We previously reported that a GTPase activating protein, regulator of G-protein signaling 5 (RGS5) is overexpressed in a subset of parathyroid tumors associated with primary hyperparathyroidism (PHPT) and that RGS5 can inhibit signaling from the calcium-sensing receptor (CASR). In vivo, we found that RGS5-null mice have abnormally low PTH levels. To gain a better understanding of the potential role of RGS5 overexpression in parathyroid neoplasia and PHPT and to investigate whether inhibition of CASR signaling can lead to parathyroid neoplasia, we created and characterized a transgenic mouse strain overexpressing RGS5 specifically in the parathyroid gland. These mice develop hyperparathyroidism, bone changes reflective of elevated PTH, and parathyroid neoplasia. Further, expression of exogenous RGS5 in normal human parathyroid cells results in impaired signaling from CASR and negative feedback on PTH secretion. These results provide evidence that RGS5 can modulate signaling from CASR and support a role for RGS5 in the pathogenesis of PHPT through inhibition of CASR signaling. © 2019 American Society for Bone and Mineral Research.

Interleukin 12B mRNA level and rs3212227 genotyping in peripheral blood mononuclear cells of inflammatory bowel disease patients

Background/aim: Inflammatory bowel disease (IBD) is a multifactorial disorder. Single nucleotide polymorphisms (SNPs) in the IL-12 gene, which are the main factors to regulate the immune reaction, play an important role in the production of IL-12 molecules. The aim of this study was to evaluate the correlation between the SNP on position +1188 of the 3 ' UTR region of the IL-12 p40 subunit gene and expression of the IL-12 p40 gene. Materials and methods: This case-control study was performed with 102 patients with IBD and 107 healthy people. PCR-RFLP and comparative real-time PCR were performed to assess the association between genotype and IL-12 gene expression. Results: The frequency of AA, CA, and CC genotypes of this gene at position +1188 was calculated to be 58.8%, 32.4%, and 8.8% in patients and 61.7%, 26.2%, and 12.1% in the control group, respectively, with no significant difference between the two groups (IL- 12B rs3212227: AA (Reference 1), CA (P = 0.407); OR (95% CI) 0.771 (0.418­1.424), CC (P = 0.561); OR (95% CI) 1.313 (0.524­3.292)). Also, the IL-12B mRNA expression level was compared between IBD patients and healthy controls and demonstrated a significant association (R 2 0.136, 95% CI 1.892­3.872, P < 0.0001). Conclusion: Our results show that IL-12B expression in IBD may be associated with altered immune and inflammatory responses.

The Association between Angiotensin II Type 1 Receptor Gene A1166C Polymorphism and Non-alcoholic Fatty Liver Disease and Its Severity.

BACKGROUND Genetic predisposition may have important role in pathogenesis of non-alcoholic fatty liver disease (NAFLD). Angiotensin II type I receptor (AGTR1) has been known to involve in the process of liver steatosis and fibrosis. This study aimed to investigate the association between AGTR1 A1166C polymorphism and NAFLD. METHODS A cross-sectional study was conducted during May 2014-May 2015 among healthy adults referring to our radiology clinic for abdominal sonography. AGTR1 A1166C polymorphism was evaluated in subjects with NAFLD and healthy individuals using allelic discrimination method. RESULTS 58 subjects with NAFLD were compared with 88 healthy individuals without NAFLD. The frequency of AA and CC genotypes of AGTR1 was significantly higher in patients with NAFLD compared with controls (p = 0.029 and 0.042, respectively). C allele was more detected in subjects with NAFLD compared with the healthy controls (OR: 2.1; 95% CI: 1.23-3.61, p = 0.006). CC genotype (OR: 10.62; 95% CI: 1.05-106.57, p = 0.045) and C allele (OR: 6.81; 95% CI: 1.42- 32.48, p = 0.016) were also predictors of severe fatty liver disease in our study population. CONCLUSION Our results provide the first evidence that AGTR1 gene A1166C polymorphism not only is associated with NAFLD and but also may predict its severity.

Detection ofParvovirus4 in Iranian patients with HBV, HCV, HIV mono-infection, HIV and HCV co-infection.

AIM: In this study, we investigated the prevalence of PARV4 virus among the healthy population and four other groups of HBV infected, HCV infected, HIV infected and HIV/HCV co-infected individuals in Iran. BACKGROUND: Parvovirus 4 (PARV4) was first discovered in 2005, in a hepatitis B virus-infected injecting drug user (IDU). To date, the best evidence about PARV4 transmission is parenteral roots which comes from IDU individuals. It seems that the prevalence of the virus in the normal population is very low. METHODS: A total of 613 patients, including chronic HCV (n=103), HBV (n=193), HIV (n=180) infected individuals, HIV/HCV (n=34) co-infected patients and 103 healthy controls, were studied by using nested-PCR and also real-time PCR techniques. RESULTS: Of those 180 samples were positive for HIV RNA, co-infection of PARV4 was detected in 3 cases (1.66%). All these three patients were male with the age of 28, 32 and 36 years (mean: 32). No statistical differences were found between HIV positive group and the healthy individuals. (P>0.05) The result of PARV4 PCR was negative in all other samples and healthy controls as well. CONCLUSION: This study is the first to investigate the occurrence of PARV4 among these groups in Iran. The results show that the virus is not significant in Iranian population, even in patients with blood born infections such as HCV, HBV or even HIV patients. Further studies in other areas and various groups are required.

Genetic association between a single nucleotide polymorphism in Interleukin-16 (rs4072111) and susceptibility to chronic HCV infection in an Iranian population.

AIM: Our goal was to identify the putative association of rs4072111 variant in IL-16 gene and HCV susceptibility in an Iranian population. BACKGROUND: Interleukin 16 (IL-16), a multifunctional cytokine, plays a vital role in modulation of immune system. METHODS: In present case control and cross sectional study, IL-16 gene variant in 300 patients with hepatitis C (HCV) infection and 300 healthy individuals were analyzed. To evaluate this possible association, genomic DNA from venous blood was extracted and genotypes of IL-16 rs4072111 variant were determined by polymerase chain reaction- Fragments Length Polymorphism Technique (PCR-RFLP). Then, rs4072111 C/T genotypes frequency and allelic distribution were evaluated in each group. RESULTS: The results of genotyping showed 82% CC, 17.3% CT, 0.7% TT in the control group and 78% CC, 20% CT and 2% TT in the case group. The distribution of rs4072111 C allele was 90.7% in controls and 88% in case group respectively.However, no correlation between IL-16 rs4072111 C/T variants and susceptibility to chronic HCV infection was found in the present study. CONCLUSION: We concluded the rs4072111 C/T cannot be considered as a proper biomarker to identify susceptibility to chronic hepatitis C virus infection.

Polyclonal origin of parathyroid tumors is common and is associated with multiple gland disease in primary hyperparathyroidism.

BACKGROUND: Parathyroid tumors are mostly considered monoclonal neoplasms, the rationale for focused parathyroidectomy in primary hyperparathyroidism. We reported that flow sorting parathyroid tumor cells and methylation-sensitive polymerase chain reaction (me-PCR) of polymorphic human androgen receptor gene and phosphoglycerate kinase gene alleles in deoxyribonucleic acid reveals that ≤35% of parathyroid tumors are polyclonal. We sought to confirm these findings and assess for clinical relevance. METHODS: Parathyroid tumors from 286 female primary hyperparathyroidism patients were analyzed for clonal status. Tumor clonal status was compared with clinical variables and operative findings. Statistical analysis was performed and significance was established at P < .05. RESULTS: In the study, 176 (62%) patients were informative for human androgen receptor gene and/or phosphoglycerate kinase gene. Assignment of clonal status was made in 119 (68%) tumors, of which 64 (54%) were monoclonal and 55 (46%) were polyclonal. Comparison of tumor clonal status to clinical variables in patients with complete operative data (N = 82) showed that while clinical features were the same between tumor types, patients with polyclonal tumors more often had multiple gland disease (risk ratio 4.066, confidence interval, 1.016-16.26; P = .039) potentially missed at unilateral neck exploration. CONCLUSION: This work confirms that primary hyperparathyroidism is often the result of polyclonal tumors and that parathyroid tumor clonal status may be associated with multiple gland disease.

Variants in two gene members of the TNF ligand superfamily and hepatitis C virus chronic disease.

AIM: To assess the possible correlation between single nucleotide polymorphisms (SNPs) of two members of TNF ligand superfamily genes, tumor necrosis factor-α (TNF-α) and lymphotoxin-α (LTA), and HCV chronic disease. BACKGROUND: The causes of disease progression from hepatitis C virus (HCV) infection to chronic liver disease still remains unclear. Abnormal production of the cytokines alleged to be contributed to progression of the disease or viral persistence. Regulatory mechanisms that control the production of cytokines including genetic polymorphisms, especially at coding/regulatory regions of genes, may affect expression and secretion of the cytokines. METHODS: In this case-control investigation, 258 individuals with serologically proven chronic HCV infection and 277 healthy controls were studied. Genotyping of rs1799964 variant of TNF-α and rs909253 intronic variant in LTA gene were performed. To confirm the results of genotyping, 10% of the specimens analyzed again by sequencing approach. RESULTS: In this investigation, a significant association was observed between the TNF-α TC genotype and chronic HCV infection (P = 0.035). Moreover, the frequency of C allele was significantly different between control subjects in comparison with chronic HCV patients (P=0.02). On the other hand, no association was found between LTA gene polymorphism and susceptibility to chronic HCV infection. CONCLUSION: These findings indicate that genetic variants like single nucleotide polymorphism in TNF-α rs1799964, could be a host factor associated with susceptibility to HCV chronic infection. However, further large scale investigations are needed to confirm this finding.

Reporter gene assays for investigating GPCR signaling.

Luciferase-based assays are applied to evaluate various cellular processes due to their sensitivity and feasibility. The field of GPCR research has also benefited from this enzymatic reaction both in deorphanization campaigns and in delineation of the signaling pathways. Here, we describe the details of this assay in GPCR studies in 96-well format and will provide examples where the assay can show constitutive activity of an orphan GPCR and demonstrate the impact of cell type on the efficacy and potency of ligands.

Evaluation of tumor necrosis factor (TNF)-α mRNA expression level and the rs1799964 polymorphism of the TNF-α gene in peripheral mononuclear cells of patients with inflammatory bowel diseases.

Crohn's disease (CD) and ulcerative colitis (UC) are types of chronic inflammatory bowel disease (IBD) of which the actual causes remain unknown. Emerging data indicate that alterations in cytokine synthesis may be involved in IBD pathogenesis. The aim of the present study was to determine whether the tumor necrosis factor (TNF)-α mRNA expression level and rs1799964 polymorphism are the genetic susceptibility component of IBD development. The TNF-α mRNA expression level of peripheral blood mononuclear cells (PBMCs) was measured using comparative reverse-transcription quantitative polymerase chain reaction (PCR). Genomic DNA from 201 individuals (CD: n=15; UC: n=86; control subjects: n=100) was analyzed for the presence of the TNF-α-1031 polymorphism by PCR-restriction fragment length polymorphism. An increased TNF-α mRNA expression level was additionally observed in the CC genotype of the -1031 TNF-α gene polymorphism compared with the TC and TT genotypes (P<0.05). Furthermore, the present results revealed that there was no significant difference in the genotype/allele frequencies of the -1031 TNF-α gene polymorphism in Iranian IBD patients. By comparison, the TNF-α mRNA expression level was evaluated in patients with a history of taking medications and demonstrated a significant association in the group that received the 5-ASA + Pred + AZA,5. 5-ASA + Pred + AZA + IFX when compared with the other groups (P<0.05). Thus, these results support the hypothesis that overexpression of the TNF-α gene, which correlated with the CC genotype, may represent a genetic risk factor for Iranian IBD.

TGF-ß1 polymorphisms -509 C>T and +915 G>C and risk of pancreatic cancer.

BACKGROUND: Ninety percent of pancreatic cancer patients have less than 5-year overall survival and approximately 50% of cases were diagnosed with metastasis in the time of admission. Previous evidences have demonstrated the strong association between TGF-ß1 variations and cancer susceptibility so far. METHODS: A total of 78 patients with pancreatic cancer and 94 healthy controls were enrolled in this case- control study between 2007 and 2012. Genomic DNA was isolated from peripheral blood samples according to phenol chloroform extraction. The genotypes of TGF-ß1 rs rs1800469 and rs1800471 were determined using the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The mean age of cases and the control group were 64.50 ± 13.718 and 40.12 ± 16.001, respectively. For polymorphism -509 C>T, the frequency of TT genotype were 31 (33.0), CT, 47(50) and CC, 16 (17) in control and 19 (24.4), 45 (57.7) and 14 (17.9) in cases respectively. In position +915 G>C, the frequency of GG genotype was 84 (89.4) and GC, 10 (10.6) in control and 71 (91.0) and 7 (9) in cases, respectively. We did not observe any significant differences in the genotype and allele frequencies of the TGF-ß1-509 C>T (rs1800469) and codon +915 G>C (rs1800471) between the two study groups (P>0.05). CONCLUSION: we found that TGF-ß1 gene polymorphisms rs1800469 and rs1800471 might not play a role in pancreatic cancer susceptibility in Iranian population.

Acute Myeloid Leukemia as the Main Cause of Pancytopenia in Iranian Population.

BACKGROUND & OBJECTIVE: Pancytopenia is the reduction in the number of all 3 major cellular elements of blood and leads to anemia, leukopenia, and thrombocytopenia. A wide variety of etiologies result in pancytopenia including leukemia, aplastic anemia, and megaloblastic anemia. The current study identified the different etiologies of pancytopenia based on bone marrow examination in Iranian patients with pancytopenia. METHODS: A total of 683 cases of pancytopenia with various etiologies were selected for this retrospective study. Bone marrow biopsy was performed with the standard technique using Jamshidi needle. The inclusion criteria for patients with pancytopenia were hemoglobin (Hb) <10 g/dL, total leukocyte count (TLC) <4 x 109/L, and platelet count <140 x 109/L. RESULTS: In the present study acute leukemia was the first most common etiology detected in 235 (35.4%) patients in which acute myeloid leukemia (AML) comprised the majority of cases 142 (21.4%), followed by myelodysplastic syndrome (MDS) 100 (15%). In patients less than 20 years old, acute leukemia was also the commonest cause identified in 56 (57.7%) cases in which acute lymphoblastic leukemia (ALL) with 38.7% was the most common etiology; however in adults (>45 year old), AML accounted for the majority of cases 76 (53.5%). CONCLUSION: Since acute leukemia was the commonest etiology in both young and adults in which AML accounted for the majority of cases with pancytopenia in Iranian population, there was an urgent need to identify the underlying molecular or genetic mechanism of this malignancy for better further medical management and patients` survival.

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium-Sensing Receptor-Mediated Signaling.

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and ß-catenin), the molecular basis of abnormal calcium sensing by calcium-sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein-coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N-terminally truncated human GPR64 is constitutively active and a 15-amino acid-long peptide C-terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full-length GPR64 physically interacts with CaSR and attenuates the CaSR-mediated intracellular Ca2+ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.

Neuroprotective Effects of Herbal Extract (Rosa canina, Tanacetum vulgare and Urtica dioica) on Rat Model of Sporadic Alzheimer's Disease.

BACKGROUND: Sporadic Alzheimer's Disease (SAD) is caused by genetic risk factors, aging and oxidative stresses. The herbal extract of Rosa canina (R. canina), Tanacetum vulgare (T. vulgare) and Urtica dioica (U. dioica) has a beneficial role in aging, as an anti-inflammatory and anti-oxidative agent. In this study, the neuroprotective effects of this herbal extract in the rat model of SAD was investigated. METHODS: The rats were divided into control, sham, model, herbal extract -treated and ethanol-treated groups. Drug interventions were started on the 21(st) day after modeling and each treatment group was given the drugs by intraperitoneal (I.P.) route for 21 days. The expression levels of the five important genes for pathogenesis of SAD including Syp, Psen1, Mapk3, Map2 and Tnf-α were measured by qPCR between the hippocampi of SAD model which were treated by this herbal extract and control groups. The Morris Water Maze was adapted to test spatial learning and memory ability of the rats. RESULTS: Treatment of the rat model of SAD with herbal extract induced a significant change in expression of Syp (p=0.001) and Psen1 (p=0.029). In Morris Water Maze, significant changes in spatial learning seen in the rat model group were improved in herbal-treated group. CONCLUSION: This herbal extract could have anti-dementia properties and improve spatial learning and memory in SAD rat model.

Low Level of Microsatellite Instability Correlates with Poor Clinical Prognosis in Stage II Colorectal Cancer Patients.

The influence of microsatellite instability (MSI) on the prognosis of colorectal cancer (CRC) requires more investigation. We assessed the role of MSI status in survival of individuals diagnosed with primary colorectal cancer. In this retrospective cross-sectional study the MSI status was determined in 158 formalin-fixed paraffin-embedded tumors and their matched normal tissues from patients who underwent curative surgery. Cox proportional hazard modeling was performed to assess the clinical prognostic significance. In this study we found that MSI-H tumors were predominantly located in the colon versus rectum (p = 0.03), associated with poorer differentiation (p = 0.003) and TNM stage II/III of tumors (p = 0.02). In CRC patients with stage II, MSI-L cases showed significantly poorer survival compared with patients who had MSI-H or MSS tumors (p = 0.04). This study indicates that MSI-L tumors correlate with poorer clinical outcome in patients with stage II tumors (p = 0.04) or in tumors located in the colon (p = 0.02). MSI-L characterizes a distinct subgroup of CRC patients who have a poorer outcome. This study suggests that MSI status in CRC, as a clinical prognostic marker, is dependent on other factors, such as tumor stage and location.