A Continuous Flow PCR Array Microfluidic Chip Applied for Simultaneous Amplification of Target Genes of Periodontal Pathogens

The concept of time to place conversion makes continuous flow polymerase chain reaction (CF-PCR) microfluidic chip an ideal way to reduce the time required for amplification of target genes, however,...

Detection of invasive Trichosporon asahii in patient blood by a fungal PCR array

Rare invasive fungal infections are increasingly emerging in hosts with predisposing factors such as immunodeficiency. Their timely diagnosis remains difficult, as their clinical picture may initially mimic infections with more common fungal species and species identification may be difficult with routine methods or may require time-consuming subcultures. This often results in ineffective drug administration and fatal outcomes. We report on a patient in their early twenties with mixed cellularity classical Hodgkin lymphoma with a disseminated Trichosporon asahii (T. asahii) infection. Even though pathogen detection and identification was possible via the standard procedure consisting of culture followed by matrix-assisted laser desorption ionisation–time of flight (MALDI-TOF) mass spectrometry, the patient passed away in the course of multi organ failure. Herein, we report on a retrospectively applied experimental diagnostic fungal PCR-analysis used on an EDTA blood sample and consisting of two pan-fungal reactions and seven branch-specific reactions. Regarding invasive T. asahii infection, this PCR array could considerably shorten time to diagnosis and switch to a targeted therapy with triazoles.

Peritoneal Modulators of Endometriosis-Associated Ovarian Cancer

Ovarian cancer is the 4th largest cause of cancer death in women. Approximately 10–15% of women of childbearing age suffer from endometriosis. Endometriosis is defined by the growth and presence of endometrial tissue (lesions) outside of the uterus. The women with endometriosis also have an increased presence of peritoneal fluid (PF) that comprises of inflammatory cells, growth factors, cytokines/chemokines, etc. Epidemiological studies have shown that >3% of women with endometriosis develop ovarian cancer (low-grade serous or endometrioid types). Our hypothesis is that the PF from women with endometriosis induces transformative changes in the ovarian cells, leading to ovarian cancer development. PF from women with and without endometriosis was collected after IRB approval and patient consent. IOSE (human normal ovarian epithelial cells) and TOV-21G cells (human ovarian clear cell carcinoma cell line) were treated with various volumes of PF (no endometriosis or endometriosis) for 48 or 96 h and proliferation measured. Expression levels of epigenetic regulators and FoxP3, an inflammatory tumor suppressor, were determined. A Human Cancer Inflammation and Immunity Crosstalk RT2 Profiler PCR array was used to measure changes in cancer related genes in treated cells. Results showed increased growth of TOV-21G cells treated with PF from women with endometriosis versus without endometriosis and compared to IOSE cells. Endo PF treatment induced EZH2, H3K27me3, and FoxP3. The RT2 PCR array of TOV-21G cells treated with endo PF showed upregulation of various inflammatory genes (TLRs, Myd88, etc.). These studies indicate that PF from women with endometriosis can both proliferate and transform ovarian cells and hence this microenvironment plays a major mechanistic role in the progression of endometriosis to ovarian cancer.

Glucocorticoid-induced Changes in the Transcriptional Activity of Genes of the Innate and Adaptive Immune System in the Blood of Patients with Acute Urticaria

Background: A number of the main effects of glucocorticoids (GCs) are their direct action on T cells, mainly through the transcriptional regulation: elevated expression of immune-regulatory proteins, inhibitory receptors, and reduced expression of pro-inflammatory cytokines, co-stimulatory molecules, and cell cycle mediators. But controversies arise due to the clinical effectiveness of GCs in the treatment of acute urticaria. Methods: In our research, we applied a pathway-specific PCR array (Human Innate & Adaptive Immune Responses RT2 Profiler PCR Array, QIAGEN, Germany) to detect and verify innate & adaptive immune responses pathway-focused genes expression in the blood of patients with acute urticaria who received treatment with glucocorticoids in addition to standard therapy. Results: Adding glucocorticoids to standard therapy did not notably affect the nature of the clinical presentation of acute urticaria, which was assessed according to the UAS scale (urticaria activity score). Analysis of the transcriptional profile of peripheral blood mononuclear cells in patients with acute urticaria against the background of glucocorticoid therapy showed the induction expression of the FOXP3 and IL10 genes against the background of repression of the transcriptional activity of the genes for chemokines and cytokines CCL5, CXCL8, IFNG, IL2, IL5, IL17A, IL1B, and TNF. Glucocorticoid-induced changes in the transcriptome also manifested by pronounced repression in genes of CD40 and CD80 (B7-1) co-stimulatory molecules, transcriptional regulators of Th1-cells differentiation - TBX21 and STAT1, Th17 cells - RORC, NLRP3-inflammasome genes, and the transcription factor NFKB1 compared with the control group. Conclusions: Adding glucocorticoids to the standard therapy of acute urticaria has a pronounced immunosuppressive potential at the transcriptome level of immune response genes in the blood; however, it does not have any noticeable clinical effect.

Comparative Expression Analysis of Genes Encoding Neurotransmitter Receptors Between Entrepreneurs and Non-Entrepreneurs

Introduction and Purpose: Recent studies revealed that different behavioral and physiological processes are closely related to different receptor levels in humans. On the other hand, there weren’t any studies that investigated neurotransmitter activity and entrepreneurship relationships. Little is known about how genes expressed in the blood are associated with entrepreneurship. In this study, we measured the expression of 84 genes encoding neurotransmitter receptors between the entrepreneurs and non-entrepreneurs (n=25) using RT-PCR arrays to monitor differentially expressed genes for exploring molecular mechanism behind the entrepreneurship mindset. Materials and Methods: After determining whether participants are entrepreneurs or not, blood samples were collected. Blood samples were collected in Vacutainer® EDTA tubes as 10 ml and RNA isolation was performed at the Erzurum Technical University, Cell Culture Laboratory. Trizol® solution for RNA isolation (Thermo®, USA) was applied according to the manufacturer's instructions. Concentrations of RNA samples were measured at 260 nm using a spectrophotometer (Epoch®, Biotek). Then, total RNA was reverse transcribed into cDNA by using High-Capacity cDNA Reverse Transcription Kit (Thermo®). Total 84 genes were analyzed via RT² Profiler™ Human Neurotransmitter Receptors PCR Array (Qiagen®, USA) in Rotor-Gene Q real-time PCR cycler (Qiagen®, USA). Findings: The gene expression results obtained from RT-PCR were compared between entrepreneurs and non-entrepreneurs, and presented as Fold Changes (FC). According to our results, positive FC values indicated an increase in the expression of the genes and negative FC values indicated decrease in gene expression levels in entrepreneurs. Results: These 84 different genes regulate neurotransmitter biosynthesis, uptake, transport, and signaling via neurotransmitter receptors. According to gene expression analyses, gene expressions that could be related to the entrepreneur behavior might be connected to not only undesired psychological outcomes like various addictions and also neurological cases such as schizophrenia and depressive disorder. Our results firstly indicated that entrepreneurship was not only associated with neurotransmitter release but also with receptor levels. Keywords: Entrepreneurship, Entrepreneurs, Genes, Neurotransmitter Receptors, RT-PCR Array

Synaptotagmin 13 Is Highly Expressed in Estrogen Receptor-Positive Breast Cancer

Background: Accumulating evidence indicates tumor-promoting roles of synaptotagmin 13 (SYT13) in several cancers; however, no studies have investigated its expression in breast cancer (BC). This study aimed to clarify the significance of SYT13 in BC. Methods: SYT13 mRNA expression levels were evaluated in BC cell lines. Polymerase chain reaction (PCR) array analysis was conducted to determine the correlation between expression levels of SYT13 and other tumor-associated genes. Then, the association of SYT13 expression levels in the clinical BC specimens with patients’ clinicopathological factors was evaluated. These findings were subsequently validated using The Cancer Genome Atlas (TCGA) database. Results: Among 13 BC cell lines, estrogen receptor (ER)-positive cells showed higher SYT13 mRNA levels than ER-negative cells. PCR array analysis revealed positive correlations between SYT13 and several oncogenes predominantly expressed in ER-positive BC, such as estrogen receptor 1, AKT serine/threonine kinase 1, and cyclin-dependent kinases 4. In 165 patients, ER-positive specimens exhibited higher SYT13 mRNA expression levels than ER-negative specimens. The TCGA database analysis confirmed that patients with ER-positive BC expressed higher SYT13 levels than ER-negative patients. Conclusion: This study suggests that SYT13 is highly expressed in ER-positive BC cells and clinical specimens, and there is a positive association of SYT13 with the ER signaling pathways.

Osteocrin, a bone-derived humoral factor, exerts a renoprotective role in ischemia-reperfusion injury in mice

Background Osteocrin (OSTN), a bone-derived humoral factor, was reported to act on heart and bone by potentiating the natriuretic peptide (NP) system. Ostn gene polymorphisms have been associated with renal function decline, but its pathophysiological role in the kidney remains unclear. Methods The role of endogenous OSTN was investigated using systemic Ostn-knockout mice (KO). As a model for OSTN administration, liver-specific Ostn-overexpressing mice crossed with KO (KO-Tg) were generated. These mice were subjected to the unilateral ischemia-reperfusion injury, and renal lesions after 21 days of insult were evaluated. A comprehensive analysis of the Wnt/β-catenin pathway was performed using a PCR array. Reporter plasmid-transfected proximal tubular cells (NRK52E) were used to investigate the mechanism by which OSTN affects the pathway. Results After injury, KO showed marginal worsening of renal fibrosis compared to wild-type mice, with comparable renal atrophy. KO-Tg showed significantly ameliorated renal atrophy, fibrosis and tubular injury, together with reduced expressions of fibrosis- and inflammation-related genes. PCR array showed that the activation of the Wnt/β-catenin pathway was attenuated in KO-Tg. The downstream targets, Mmp7, Myc, and Axin2 showed similar results. MMP7 and Wnt2 were induced in corticomedullary proximal tubules after injury, but not in KO-Tg. In NRK52E, OSTN significantly potentiated the inhibitory effects of NP on TGFβ1-induced activation of the Wnt/β-catenin pathway, which was reproduced by a cGMP analog. Conclusions Ectopic Ostn overexpression ameliorated subsequent renal injury following ischemia-reperfusion. OSTN could represent possible renoprotection in acute to chronic kidney disease transition, thus serving as a potential therapeutic strategy.

Comparative Expression Analysis of Genes Encoding Neurotransmitter Receptors Between Entrepreneurs and Non-Entrepreneurs

Introduction and Purpose: Recent studies revealed that different behavioral and physiological processes are closely related to different receptor levels in humans. On the other hand, there weren’t any studies that investigated neurotransmitter activity and entrepreneurship relationships. Little is known about how genes expressed in the blood are associated with entrepreneurship. In this study, we measured the expression of 84 genes encoding neurotransmitter receptors between the entrepreneurs and non-entrepreneurs (n=25) using RT-PCR arrays to monitor differentially expressed genes for exploring molecular mechanism behind the entrepreneurship mindset. Materials and Methods: After determining whether participants are entrepreneurs or not, blood samples were collected. Blood samples were collected in Vacutainer® EDTA tubes as 10 ml and RNA isolation was performed at the Erzurum Technical University, Cell Culture Laboratory. Trizol® solution for RNA isolation (Thermo®, USA) was applied according to the manufacturer's instructions. Concentrations of RNA samples were measured at 260 nm using a spectrophotometer (Epoch®, Biotek). Then, total RNA was reverse transcribed into cDNA by using High-Capacity cDNA Reverse Transcription Kit (Thermo®). Total 84 genes were analyzed via RT² Profiler™ Human Neurotransmitter Receptors PCR Array (Qiagen®, USA) in Rotor-Gene Q real-time PCR cycler (Qiagen®, USA). Findings: The gene expression results obtained from RT-PCR were compared between entrepreneurs and non-entrepreneurs, and presented as Fold Changes (FC). According to our results, positive FC values indicated an increase in the expression of the genes and negative FC values indicated decrease in gene expression levels in entrepreneurs. Results: These 84 different genes regulate neurotransmitter biosynthesis, uptake, transport, and signaling via neurotransmitter receptors. According to gene expression analyses, gene expressions that could be related to the entrepreneur behavior might be connected to not only undesired psychological outcomes like various addictions and also neurological cases such as schizophrenia and depressive disorder. Our results firstly indicated that entrepreneurship was not only associated with neurotransmitter release but also with receptor levels. Keywords: Entrepreneurship, Entrepreneurs, Genes, Neurotransmitter Receptors, RT-PCR Array

Ranibizumab Inhibits Human Tenon’s Fibroblast Proliferation via p21 Dependent p53 Mechanisms

Trabeculectomy is the gold standard procedure performed in glaucoma when topical medication and laser intervention failed. In a trabeculectomy, number of clinical trials have shown the efficacy of ranibizumab in minimizing extracellular matrix accumulation at the filtering site. Ranibizumab (LucentisTM) is a drug that targets vascular endothelial growth factor (VEGF). However, to date the actual mechanisms of this anti-VEGF in trabeculectomy is still not well understood. Hence, in here we aimed to elucidate the effects of ranibizumab on human Tenon’s fibroblast (HTF) isolated from patients undergoing trabeculectomy. In our previous study, we had reported that ranibizumab reduces the level of spermidine metabolite whereby spermidine is an important polyamine for cell proliferation. For this current study, cultured HTFs were divided into untreated, control IgG, ranibizumab only, difluoromethylornithine (DFMO; inhibitor of spermidine) only and ranibizumab with DFMO. All cells were extracted for PCR array (expression of CDKN1A, CDK2, and CDK4) and protein expression of p53, p21, CDK2, and CDK4 by Western Blot. In here, our result demonstrated that cells treated with ranibizumab or DFMO and cells treated with ranibizumab-DFMO have similar effects as both show increased in p53 and p21. Meanwhile, no significant differences in expression of CDKN1A, CDK2 and CDK4 were observed in all groups. In essence, our findings suggest that ranibizumab action is mediated by p21 and p53.