Seyit Ali Kayis

Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy

The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), late diestrus (LD, n=4), early luteolysis (EL, n=4) and after luteolysis (AL, n=4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily and ultrasound examinations. Biopsies were also taken on days 14 (P14; n=4), 15 (P15, n=4), 18 (P18, n=4) and 22 (P22; n=4) of pregnancy. Relative mRNA expressions were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Expression of PTGS1 mRNA was low throughout the estrous cycle and early days of pregnancy, but upregulated on P18 and P22. PTGS2 expression was increased on EL, but it was suppressed by pregnancy on P15, P18, and P22. PTGFS expression was upregulated in both cyclic and pregnant mares compared to d0 and its level was the highest on LD. PTGFR expression was transiently increased on LD and EL and was suppressed during early pregnancy. Both PTGES and PTGER2 expressions were increased on LD, EL, and early pregnancy, but were decreased after the luteolysis in cyclic mares as they remained high on P18 and P22. PTGER4 expression did not change throughout the cycle and early pregnancy. Levels of HPGD and SLCO2A1 were significantly increased only on P22. In conclusion, PTGS2 expression increases around the time of luteolysis and concurrent upregulation of PTGFS and PTGES indicates that equine endometrium has increased capability of PTG production around the time of luteolysis. However, pregnancy reduces PTGS2 expression, but maintains the high levels of PTGES during early pregnancy along with PTGER2 while PTGFR expression was suppressed. These findings suggest that possible luteotrophic action of PGE₂ is required in early equine pregnancy. PTGS1 is only upregulated later in the early pregnancy suggesting that it is not involved in luteolysis, but could be the main PTGS enzyme at this time during early pregnancy. An increase in HPGD and SLCO2A1 levels on P22 indicates a tight regulation of PTG action by pregnancy.

Bone morphogenetic protein‐2, ‐6, and ‐7 differently regulate osteogenic differentiation of human periodontal ligament stem cells

The utility of adult stem cells for bone regeneration may be an attractive alternative in the treatment of extensive injury, congenital malformations, or diseases causing large bone defects. To create an environment that is supportive of bone formation, signals from molecules such as the bone morphogenetic proteins (BMPs) are required to engineer fully viable and functional bone. We therefore determined whether BMP-2, -6, and -7 differentially regulate the (1) proliferation, (2) mineralization, and (3) mRNA expression of bone/mineralized tissue associated genes of human periodontal ligament stem cells (hPDLSCs), which were obtained from periodontal ligament tissue of human impacted third molars. hPDLSCs from six participants were isolated and characterized using histochemical and immunohistochemical methods. A real-time cell analyzer was used to evaluate the effects of BMP-2, -6, and -7 on the proliferation of hPDLSCs. hPDLSCs were treated with Dulbeccos modified Eagles medium containing different concentrations of BMP-2, -6, and -7 (10, 25, 50, 100 ng/mL) and monitored for 264 hours. After dose-response experiments, 50 and 100 ng/mL concentrations of BMPs were used to measure bone/mineralized tissue-associated gene expression. Type I collagen, bone sialoprotein, osteocalcin, osteopontin, and osteoblastic transcription factor Runx2 mRNA expression of hPDLSCs treated with BMP-2, -6, and -7, were evaluated using quantitative RT-PCR. Biomineralization of hPDLSCs was assessed using von Kossa staining. This study demonstrated that BMPs at various concentrations differently regulate the proliferation, mineralization, and mRNA expression of bone/mineralized tissue associated genes in hPDLSCs. BMPs regulate hPDLSC proliferation in a time and dose-dependent manner when compared to an untreated control group. BMPs induced bone/mineralized tissue-associated gene mRNA expression and biomineralization of hPDLSCs. The most pronounced induction occurred in the BMP-6 group in the biomineralization of the hPDLSCs. Our data suggest that BMP-2, -6, and -7 are potent regulators of hPDLSC gene expression and biomineralization. Employing BMPs with hPDLSCs isolated from periodontal ligament tissues provides a promising strategy for bone tissue engineering.

Comparison of Mesenchymal Stem Cells Isolated From Pulp and Periodontal Ligament

BACKGROUND Cell-based therapy using mesenchymal stem cells (MSCs) seems promising to obtain regeneration of dental tissues. A comparison of tissue sources, including periodontal ligament (PDL) versus pulp (P), could provide critical information to select an appropriate MSC population for designing predictable regenerative therapies. The purpose of this study is to compare the proliferation and stemness and the MSC-specific and mineralized tissue-specific gene expression of P-MSCs and PDL-MSCs. METHODS MSCs were obtained from PDL and P tissue of premolars (n = 3) extracted for orthodontic reasons. MSC proliferation was evaluated using a real-time cell analyzer for 160 hours. Telomerase activity was evaluated by a telomeric repeat amplification protocol assay based on enzyme-linked immunosorbent assay. Total RNA was isolated from the MSCs on day 3. A polymerase chain reaction (PCR) array was used to compare the expression of MSC-specific genes. The expression of mineralized tissue-associated genes, including Type I collagen (COL I), runt-related transcription factor 2 (RunX2), bone sialoprotein (BSP), and osteocalcin (OCN) messenger RNA (mRNA), was evaluated using quantitative real-time PCR. RESULTS Higher proliferation potential and telomerase activity were observed in the P-MSCs compared to PDL-MSCs of premolar teeth. Fourteen of 84 genes related to MSCs were expressed differently in the PDL-MSCs versus the P-MSCs. The expressions of bone morphogenetic protein 2 (BMP2) and BMP6; sex-determining region Y-box 9 (SOX9); integrin, alpha 6 (ITGA6); melanoma cell adhesion molecule (MCAM); phosphatidylinositol glycan anchor biosynthesis, class S (PIGS); prominin 1 (PROM1); ribosomal protein L13A (RPL13A); and microphthalmia-associated transcription factor (MITF) were higher in the P-MSCs compared to the PDL-MSCs, and higher expression of matrix metalloproteinase 2 (MMP2), interleukin (IL)-6, insulin (INS), alanyl (membrane) aminopeptidase (ANPEP), and IL-10 were observed in the PDL-MSCs. However, there was no statistically significant difference in the expression of mineralized tissue-associated genes, including BSP and RunX2, between the P-MSCs and the PDL-MSCs. Higher expression of COL I and lower expression of OCN mRNA transcripts were noted in the PDL-MSCs compared to the P-MSCs. CONCLUSIONS The results of this study suggest that MSCs isolated from P and PDL tissues show different cellular behavior. To increase the predictability of MSC-based regenerative treatment, differences in dental tissue-derived MSCs and favorable aspects of cell sources should be further clarified.

Rating of putative housekeeping genes for quantitative gene expression analysis in cyclic and early pregnant equine endometrium

The aim was an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in the equine endometrium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine ribosyl transferase 1 (HPRT1), ubiquitin B (UBB), tubulin alpha 1 (TUBA1), ribosomal protein L32 (RPL32), beta-2-microglobulin (B2M), 18S rRNA (18S), and 28S rRNA (28S) HKGs were evaluated using real-time PCR and were compared in different physiological stages of the endometrium. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), at late diestrus (LD, n=4), after luteolyis (AL, n=4) of the cycle and on days 14 (P14; n=3), 18 (P18, n=3) and 22 (P22; n=3) of pregnancy. A model based on REML with support of descriptive statistics was proposed in accordance with experimental design and was further confirmed with principal component analysis (PCA). Results were compared with widely used software including geNorm, BestKeeper, and NormFinder. Results indicated that GAPDH was the most stable HKG and RPL32 was ranked as the second best. 18S and 28S were found to be the least stable. The proposed model, PCA, geNorm, and BestKeeper were in agreement in detecting the most stable and the least stable HKGs in the equine endometrium during the estrous cycle and early pregnancy.

The Adiponectin variants contribute to the genetic background of type 2 diabetes in Turkish population

Adiponectin, an adipose tissue specific protein encoded by the Adiponectin gene, modulates insulin sensitivity and plays an important role in regulating energy homeostasis. Many studies have shown that single nucleotide polymorphisms (SNPs) in the Adiponectin gene are associated with low plasma adiponectin levels, insulin resistance and an increased risk of type 2 diabetes mellitus. The aim of the present study was to evaluate the contribution of the Adiponectin gene polymorphisms in genetic background of type 2 diabetes in a Turkish population. In total, 169 unrelated and non-obese diabetic patients and 119 age- and BMI-matched non-diabetic individuals with no family history of diabetes were enrolled in this study. We detected a significant association between type 2 diabetes and two SNPs: SNP -11391G>A, which is located in the promoter region of the Adiponectin gene, and SNP +276G>T, which is found in intron 2 of the gene (P<0.05). The silence SNP G15G (+45T>G) in exon 1 and SNP +349A>G in intron 2 also showed a weak association with type 2 diabetes (P=0.06 and P=0.07, respectively), while SNPs -3971A>G in intron 1 and Y111H, R112C and H241P in exon 3 showed no association (P>0.05). In conclusion, these findings suggest that Adiponectin gene polymorphisms might be effective on susceptibility for type 2 diabetes development which emerged from the interactions between multiple genes, variants and environmental factors.

Boron enhances strength and alters mineral composition of bone in rabbits fed a high energy diet

An experiment was performed to determine whether boron had a beneficial effect on bone strength and composition in rabbits with apparent adiposity induced by a high energy diet. Sixty female New Zealand rabbits, aged 8 months, were randomly divided into five groups with the following treatments for seven months: control 1, fed alfalfa hay only (5.91 MJ/kg); control 2, high energy diet (11.76 MJ and 3.88 mg boron/kg); B10, high energy diet+10 mg/kg body weight boron gavage/96 h; B30, high energy diet+30 mg/kg body weight boron gavage/96 h; B50, high energy diet+50mg/kg body weight boron gavage/96 h. Bone boron concentrations were lowest in rabbits fed the high energy diet without boron supplementation, which suggested an inferior boron status. Femur maximum breaking force was highest in the B50 rabbits. Tibia compression strength was highest in B30 and B50 rabbits. All boron treatments significantly increased calcium and magnesium concentrations, and the B30 and B50 treatments increased the phosphorus concentration in tibia of rabbits fed the high energy diet. The B30 treatment significantly increased calcium, phosphorus and magnesium concentrations in femur of rabbits fed the high energy diet. Principal component analysis of the tibia minerals showed that the three boron treatments formed a separate cluster from controls. Discriminant analysis suggested that the concentrations of the minerals in femur could predict boron treatment. The findings indicate boron has beneficial effects on bone strength and mineral composition in rabbits fed a high energy diet.

Red cell distribution width and neurological scoring systems in acute stroke patients.

Objectives The purpose of the present study was to evaluate the association between the red blood cell distribution width (RDW) and the Glasgow Coma Scale (GCS), Canadian Neurological Scale (CNS), and National Institutes of Health Stroke Scale (NIHSS) scores in patients who had acute ischemic stroke. Methods This prospective observational cohort study included 88 patients who have had acute ischemic stroke and a control group of 40 patients who were evaluated in the Emergency Department for disorders other than acute ischemic stroke. All subjects had RDW determined, and stroke patients had scoring with the GCS, CNS, and NIHSS scores. The GCS, CNS, and NIHSS scores of the patients were rated as mild, moderate, or severe and compared with RDW. Results Stroke patients had significantly higher median RDW than control subjects. The median RDW values were significantly elevated in patients who had more severe rather than milder strokes rated with all three scoring systems (GCS, CNS, and NIHSS). The median RDW values were significantly elevated for patients who had moderate rather than mild strokes rated by GCS and CNS and for patients who had severe rather than mild strokes rated by NIHSS. The area under the receiver operating characteristic curve was 0.760 (95% confidence interval, 0.676–0.844). Separation of stroke patients and control groups was optimal with RDW 14% (sensitivity, 71.6%; specificity, 67.5%; accuracy, 70.3%). Conclusion In stroke patients who have symptoms <24 hours, the RDW may be useful in predicting the severity and functional outcomes of the stroke.

Inter simple sequence repeats separate efficiently hemp from marijuana (Cannabis sativa L.)

Cannabis sativa L. is a multiple-use plant that provides raw material for the production of seed oil, natural fiber for textiles, automotive and pulp industries. It has also been used in insulating boards, ropes, varnishes, animal feed, and as medicinal agents. Cannabis has potential to be used for phytoremediation: however, its cultivation is strictly controlled due to its psychoactive nature and usage in producing drugs such as marijuana, and hashish. In this study, psychoactive type Cannabis samples, which were seized from 23 different locations of Turkey, and nine hemp type Cannabis accessions, as well as an unknown accession were used. Our interest was to identify the genetic relatedness of the seized samples and to separate drug and hemp type plants. Inter Simple Sequence Repeats (ISSRs) were employed for analysis based on single plant material (SET1) and bulked samples of them (SET2). Data was analysed via cluster analysis and principal coordinate analysis (PCoA). PCoA analyses, by using SET1 and SET2, were able to efficiently discriminate the seized samples from the fiber type accessions. However, separation of the plants was not clear via unweighted pair-group method using arithmetic average (UPGMA) dendogram in SET1, while they were clearly separated in SET2. Hemp type accessions showed high levels of variation compared to drug type Cannabis both in SET1 and SET2.

Effects of Single Nucleotide Polymorphisms in KATP Channel Genes on Type 2 Diabetes in a Turkish Population

BACKGROUND AND AIMS ATP-sensitive potassium (K(ATP)) channels of pancreatic β-cells play a key role in glucose-stimulated insulin secretion mechanism. The Kir6.2 protein, forming the K(ATP) channel pore inwardly, and the SUR1 protein that surrounds it forming the outside part of the channel were encoded by ABCC8 and KCNJ11 genes, respectively. Recent studies reported that the single nucleotide polymorphisms (SNPs) established in these genes are associated with defects in insulin secretion and type 2 diabetes mellitus (T2DM). We aimed to investigate the allele profiles and the risk alleles of the ABCC8 and KCNJ11 genes and to highlight the associations with the disease in patients in Konya region of Turkey where T2DM is common. METHODS In this study, 169 patients with T2DM and 119 healthy controls were included. A total of 29 SNPs in ABCC8 and KCNJ11 genes were screened by PCR-SSCP technique and sequenced. Biochemical parameters and genotype-phenotype relationships were analyzed using variance analysis. RESULTS R1273R silent substitution in exon 31 and 16/-3t→c substitution in noncoding region of exon 16 of ABCC8 gene showed a significant association (OR 4.8 [95% CI 2.41-9.77], p <0.001 and OR 3.5 [95% CI 1.64-7.40], p <0.001 under dominant and recessive models, respectively). We detected a significant association between E/K heterozygote genotype and reduced plasma insulin level in patients with T2DM (p <0.05). CONCLUSIONS ABCC8 exons 16 and 31 variants increase susceptibility to T2DM and KCNJ11 E23K decreases insulin secretion in a Turkish population.

Expression profile of interferon tau–stimulated genes in ovine peripheral blood leukocytes during embryonic death

Early and efficient detection of embryonic death (ED) has a valuable impact as important as early pregnancy diagnosis in ruminants. Among early pregnancy diagnosis methods, detection of the expression of interferon tau-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) is well documented in cows and ewes. Therefore, we hypothesized that the expression profile of ISGs in PBLs might also be useful for detecting ED in these animals. For this purpose, pregnant ewes were used as an experimental model. Pregnancy was detected on Day 18 after mating by transrectal ultrasonography. Pregnant ewes were divided into a control group (sham injection on Day 18, n = 10) and ED group (treated with 75 μg synthetic PGF2α on Day 18, n = 12). PBLs and plasma were collected on Days 0 (mating day), 15, 18, 19, 20, 21, 23, and 25 by jugular venipuncture. Total RNA was isolated from PBLs. ISGs expression levels were determined by real-time polymerase chain reaction in triplicate. Electrochemiluminescence immunoassay was used to measure progesterone (P4) levels in plasma. In the ED group, the P4 level declined to less than 1 ng/mL on Day 19 and remained at a low level until the end of the study. Compared with that on Day 0, receptor transporter protein 4 (RTP4) and ISG15 expression was upregulated on Day 15 and remained high until Day 21 in both groups, and RTP4 and ISG15 mRNA levels were attenuated on Days 23 and 25 only in the ED group (P < 0.001). Myxovirus resistance 1 expression was upregulated on Day 15 and remained high until Day 23 in both groups, but was attenuated on Day 25 in the ED group (P < 0.05). The B2-microglobulin mRNA level did not change significantly during the study in either group. These results indicate that the decline in P4 concentration was an immediate response to PGF2α and that the embryo may have survived longer than the CL on the basis of the extended period of ISGs expression. This suggests that the absence of P4 could be the reason for ED rather than a direct effect of PGF2α. In conclusion, the expression of ISGs, including ISG15, RTP4, and myxovirus resistance 1, but not B2-microglobulin, in PBLs may serve as a marker of ED.