The first implementation of allele-specific primers for detecting the knockdown and acetylcholinesterase target site mutations in malaria vector, Anopheles sacharovi.

Anopheles sacharovi, a primer malaria vector species of Turkey, have a significant public health importance. It is aimed to determine the insecticide resistance status in Anopheles sacharovi populations in the Aegean and Mediterranean regions of Turkey. A total of 1638 individuals were analysed from 15 populations. Bioassay results indicated all An. sacharovi populations were resistant to DDT, malathion, fenitrothion, bendiocarb, propoxur. Many populations have begun to have resistance against permethrin and deltamethrin. Biochemical analyses results revealed that glutathione-S-transferases and P450 monooxygenases might be responsible from the mechanisms of DDT resistance; esterases and acetylcholinesterase might be responsible for organophosphate and carbamate resistance; P450 monooxygenases and esterases might be responsible for pyrethroid resistance into populations sampled from the study area. Allele-specific primers detected L1014F and L1014S mutations that provide kdr resistance against pyrethroids and DDT. Increased acetylcholinesterase insensitivity was detected while Ace-1 G119S mutations were not detected in An. sacharovi populations by using allele-specific primers. Overall results indicate the presence of multiple resistance mechanisms in Turkish An. sacharovi field populations suggesting that populations might gain resistance against all possible insecticide in the future. Therefore, insecticide resistance management strategies are urgently needed for effective vector control implementation.

The effect of baicalein on Wnt/ß-catenin pathway and miR-25 expression in Saos-2 osteosarcoma cell line

Background/aim: Osteosarcoma is the most common primary bone malignancy that occurs frequently in children and adolescents. Baicalein, a flavonoid that has attracted great attention in recent years with its strong antitumor activity, shows a wide range of biological and pharmaceutical effects.MicroRNAs have been found to be involved in many critical processes in cancers. This study aimed to investigate the effect of baicalein and miR-25 on Wnt/ß-catenin signaling pathway of osteosarcoma cell line Saos-2. Materials and methods: Cell viability was assessed, and qRT-PCR and Western blot were performed to study the effects of baicalein on expression of Wnt/ß-catenin signaling pathway-realted genes (ß-catenin, GSK-3ß, and Axin2) of Saos-2 cells. Results: Our results indicated that baicalein can inhibit the proliferation (IC50 value 35 µM), regulate Wnt/ß-catenin pathway and also increase miR-25 expression of Saos-2. Baicalein and also miR-25 decreased the expression of ß-catenin and Axin2, while increasing the expression of GSK-3ß. Down regulation of miR-25 decreased the expression of GSK-3ß, while ß-catenin and Axin2 expression increased. Conclusion: These findings demonstrate that baicalein may target genes related to the Wnt/ß-catenin pathway by regulating miR-25 expression and may be a potential Wnt/ß-catenin pathway inhibitor for osteosarcoma therapy.

Current insecticide resistance status in Anopheles sacharovi and Anopheles superpictus populations in former malaria endemic areas of Turkey.

Anopheles sacharovi and Anopheles superpictus have a significant public health importance since they are primer and seconder malaria vectors of Turkey, respectively. As a result of intensive insecticide usage in historically malaria endemic regions of Turkey for long years, insecticide resistance problem has occurred inevitably. In this study, we aimed to investigate the involvement of the detoxification enzymes in insecticide resistance in Turkish An. sacharovi and An. superpictus populations in the Mediterranean and South-eastern Anatolia region where have a malaria history in the past. Bioassay results indicated that both An. sacharovi and An. superpictus populations are resistant to DDT, resistant or possible resistant to organophosphates and carbamates and finally mostly susceptible to pyrethroids. Although bioassays results indicated high DDT resistance in all mosquito populations, biochemical assays did not show significantly high GST levels in all strains. Almost all An. sacharovi and An. superpictus populations had an increased α and ß esterase activity levels while nearly half of the overall populations had an increased p-NPA esterase than the control group. Elevated levels of MFO frequency have been shown in the majority of the populations. Consequently, our results reveal that biochemical resistance mechanisms may play an important role in insecticide resistance in Turkish An. sacharovi and An. superpictus populations. These results give useful cues to monitor the insecticide resistance before it spreads throughout an entire population, enabling early intervention.

Distribution pattern and genetic structure of Aedes zammitii (Diptera: Culicidae) along the Mediterranean and Aegean coasts of Turkey.

The Mariae species complex, consisting of Aedes mariae, Aedes phoeniciae, and Aedes zammitii, has a limited distribution worldwide. All three species are found in rocky habitats on the coastal areas of Mediterranean countries. Aedes phoeniciae and Ae. zammitii are two members of the Mariae complex that exist in Turkey. The aim of this study was to determine the distribution pattern and genetic structure of Ae. zammitii along the Mediterranean and Aegean regions. For this purpose, larval and adult samples of Ae. zammitii were collected from 19 different rocky habitats along the coastal regions of Antalya, Mugla, Aydin, Izmir, Balikesir, and Çanakkale provinces. DNA isolation was performed primarily from collected samples, and mitochondrial NADH dehydrogenase 4 (ND4) gene was amplified by polymerase chain reaction. Based on ND4 sequence analyses, 21 haplotypes were detected along the distribution range of the species. Analyses of molecular variance (AMOVA) and spatial analyses of molecular variance (SAMOVA) indicated six groups, and most of the variation was among groups, demonstrating the population structuring at group level. Isolation by distance analyses (IBD) showed a correlation between geographic and genetic distances.

Genetic variation of the Nile soft-shelled turtle (Trionyx triunguis).

We studied the genetic structure of Trionyx triunguis populations from the Mediterranean and African continent based on mtDNA D-loop (776 bp) and nine microsatellite loci. A total of 102 polymorphic sites and 13 mtDNA haplotypes were described. Nucleotide diversity and haplotypes diversity were 0.047 and 0.974 respectively. Both mtDNA and nDNA supported the existence of two main management units as the Mediterranean and Africa. Based on the mtDNA results, the Mediterranean can be divided into two subunits; western Turkey and the eastern Mediterranean.

Expression profiling reveals MSX1 and EphB2 expression correlates with the invasion capacity of Wilms tumors.

BACKGROUND: Wilms tumor is the most common pediatric renal malignancy, but the parameters that are important to its invasion capacity are poorly understood. The aim of this study was to identify new proteins associated with the invasion capacity of Wilms tumor. PROCEDURE: Gene expression profiles for 15 primary Wilms tumor samples were determined by Affymetrix Genechip® Human Genome Ul33A microarray analysis. The gene expression profiles for selected genes was further confirmed by quantitative RT-PCR analysis. Immunohistochemical analysis was performed on 25 Wilms tumor cases to confirm expression for Bcl2A1, EphB2, MSX1, and RIN1. RESULTS: Using microarray analysis 14 genes showed differential expression (P < 0.05) comparing stage 1 non-invasive Wilms tumor to stages 2-4 invasive Wilms tumor. The differential expression for Bcl2A1, EphB2, MSX1, and RIN1 was confirmed by quantitative RT-PCR. MSX1 protein was statistically significantly lower in stages 2-4 invasive Wilms tumor cases compared to stage 1 non-invasive cases (P = 0.013). EphB2 protein was higher in stages 2-4 Wilms tumor cases compared to stage 1 cases (P = 0.006). There was no statistically significant difference between stages 1 and 2-4 Wilms tumor for Bcl2A1 (P = 0.230) or RIN1 (P = 0.969) at the protein level. CONCLUSION: Our results indicate that MSX1 may be associated with the invasion capacity of Wilms tumors. RIN1 is a downstream effector of RAS and Bcl2A1 functions as an anti-apoptotic protein. EphB2 is an ephrin receptor and is up-regulated in invasive tumors but its role needs to be confirmed in further cases of Wilms tumors.

The zymographic evaluation of gelatinase (MMP-2 and -9) levels in acute and chronic periapical abscesses.

OBJECTIVE: This study investigated the presence and levels of matrix metalloproteinases (MMP)-2 and -9 in periapical abscesses. STUDY DESIGN: Eighteen samples of intracanal exudates containing pus were collected from teeth with clinically and radiographically verified primary or secondary acute and chronic apical abscesses. Pro- and active forms of MMP-2 and MMP-9 levels were analyzed by using substrate gel zymography followed by an image analysis system. Statistical analysis was performed using the Kruskall-Wallis and Mann-Whitney U tests with Bonferroni adjustment. RESULTS: Both forms of MMP-9 were detected in all pus samples and demonstrated marked differences among the experimental groups (P < .05). Primary or secondary acute apical abscess samples demonstrated significantly higher MMP-9 levels compared with MMP-2 levels (P < .01). However, MMP-2 could not be detected in chronic apical abscesses. CONCLUSION: According to the results of this study, gelatinases might affect the pathogenesis of acute and chronic periapical abscesses.

Genome profiling of chondrosarcoma using oligonucleotide array-based comparative genomic hybridization.

Chondrosarcomas of the bone are malignant hyaline cartilage-forming tumors with an annual incidence rate of 3.6% of all primary bone malignancies in the United States. Specimens of 25 chondrosarcomas (10 grade I, 9 grade II, 1 grade III, and 5 dedifferentiated) from 23 patients were collected from the Department of Pathology at the University Hospital at UMDNJ-New Jersey Medical School from 1996 to 2007. Array-based comparative genomic hybridization (array-CGH) studies were performed on frozen tumor specimens. Recurrent deletions observed in at least in six tumors were 5q13.2, 5q14.2 approximately q21.3, 6q12 approximately q13, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There was a statistically significant association between high-grade tumor (grade III and dedifferentiated) and the recurrent genetic deletions at 5q14.2 approximately q21.3, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There is consistency between increased levels of aneuploidy and the progression of chondrosarcoma from lower to higher grades.

Association between gene expression profile and tumor invasion in oral squamous cell carcinoma.

There are limited studies attempting to correlate the expression changes in oral squamous cell carcinoma with clinically relevant variables. We determined the gene expression profile of 16 tumor and 4 normal tissues from 16 patients by means of Affymetrix Hu133A GeneChips. The hybridized RNA was isolated from cells obtained with laser capture microdissection, then was amplified and labeled using T7 polymerase-based in vitro transcription. The expression of 53 genes was found to differ significantly (33 upregulated, 20 downregulated) in normal versus tumor tissues under two independent statistical methods. The expression changes in four selected genes (LGALS1, MMP1, LAGY, and KRT4) were confirmed with reverse transcriptase polymerase chain reaction. Two-dimensional hierarchical clustering of the 53 genes resulted in the samples clustering according to the extent of tumor infiltration: normal epithelial tissue, tumors less than or equal to 4 cm in dimension, and tumors more than 4 cm in dimension (P = 0.0014). The same pattern of clustering was also observed for the 20 downregulated genes. We did not observe any associations with lymph node metastasis (P = 0.097).

Dopamine receptor 4 (DRD4) 7-repeat allele predicts methylphenidate dose response in children with attention deficit hyperactivity disorder: a pharmacogenetic study.

OBJECTIVE: Genetic polymorphisms of the dopamine neurotransmitter system have been identified in attention deficit hyperactivity disorder (ADHD). Since stimulant medications act through this system, we sought to determine if the 48 base pair VNTR polymorphism (7- repeat allele) of dopamine receptor gene DRD4 predicts methylphenidate responsiveness. METHODS: Forty-five children, aged 7-15 years, with ADHD, confirmed by NIMH-DISC-IV, participated in this prospective pharmacogenetic study. Subjects received increasing methylphenidate doses based on serial Conners' Global Index-Parent assessments. Doses to obtain a 10-point improvement and normalization (T-score, 60) were determined. Blood and buccal screening for DRD4 7R was correlated with outcomes. RESULTS: Mean dose for a 10-point CGI-P improvement with DRD4 7R (n=20) was 30 mg (1.00 mg/kg) versus 20 mg (0.49 mg/kg) without 7R (n=25) (log rank=13.69; df=1; p=0.0002). Mean dose for CGI-P normalization for children with 7R was 47 mg (1.70 mg/kg) of methylphenidate versus 31 mg (0.79 mg/kg) of methylphenidate without 7R (log rank=14.17; df=1; p=0.0002). ADHD symptom normalization at < or =50 mg methylphenidate was achieved in 58% with 7R versus 95% without (log rank=9.45; df=1; p=0.002). CONCLUSIONS: Children with ADHD possessing the DRD4 7R allele require higher doses of methylphenidate for symptom improvement and symptom normalization. This pharmacogenetic study demonstrates that the 7-repeat allele of the DRD4 gene VNTR polymorphism correlates with treatment outcomes.

Comprehensive genome-wide comparison of DNA and RNA level scan using microarray technology for identification of candidate cancer-related genes in the HL-60 cell line.

Genome-wide scans for DNA and RNA changes in the HL-60 cell line relative to normal leukocytes were conducted. Microarray-based comparative genome hybridization (CGH) studies were performed with the Spectral Genomics Human Bacterial Artificial Chromosome (BAC) 3MB system. Transcriptional measurements of approximately 12,500 human genes were monitored using Affymetrix U95A GeneChips. In HL-60, genomic DNA amplification of the 8q24 locus, trisomy 18, and deletions at loci 5q11.2 approximately q31, 6q12, 9p21.3 approximately p22, 10p12 approximately p15, 14q22 approximately q31, 17p12 approximately p13.3, and monosomy X were detected. After obtaining locus information about the RNA transcripts from the Affymetrix database, 4368 genes were stratified both according to status of RNA expression and the DNA copy number of their designated loci. The expression level of 2326 (53.25%) of 4368 transcripts is concordant with DNA copy number. Examples of specific, highly expressed, cancer-associated genes in amplified loci include SERPINB10, MYC, TYMS, HEC, and EPB41L3, while CD14, GZMK, TCF7, FOS, MLH3, CTNNA1, IRF1, VIM, CRK, MAP3K1, STAM, MAX, SFRG5, ENC1, PURA, MNT, RASA1, GLRX, UBE2B, NR3C1, PTENP1, BS69, COPEB, SKIP, PIM2, and MIC2 represent cancer-associated genes in deleted loci with decreased expression. The complementary usage of genome-wide DNA and RNA scans should enhance the identification of candidate genes in the neoplastic process.