mRNA Expression Levels In Tissues Research Articles

Welding Fume Instillation in Isolated Perfused Mouse Lungs-Effects of Zinc- and Copper-Containing Welding Fumes.

Zinc- and copper-containing welding fumes can cause systemic inflammation after exposure in humans. Recent ex vivo studies have shown that the observed inflammation originates from exposed immune cells. In vitro studies identified the soluble fraction of metal particles as the main effectors. Isolated perfused mouse lungs (IPLs) were perfused and ventilated for 270 min. Lungs were instilled with saline solution (control), welding fume particle suspension (WFs) or the soluble fraction of the welding fumes (SF-WFs). Bronchoalveolar lavage fluid (BALF) and perfusate samples were analyzed for cytokine levels and lung tissue mRNA expression levels were analyzed via RT-PCR. All lungs instilled with WFs did not complete the experiments due to a fatal reduction in tidal volume. Accordingly, IL-6 and MPO levels were significantly higher in BALF of WF lungs compared to the control. IL-6 and MPO mRNA expression levels were also increased for WFs. Lungs instilled with SF-WFs only showed mild reactions in tidal volume, with BALF and mRNA expression levels not significantly differing from the control. Zinc- and copper-containing welding fume particles adversely affect IPLs when instilled, as evidenced by the fatal loss in tidal volume and increased cytokine expression and secretion. The effects are mainly caused by the particles, not by the soluble fraction.

Selective VEGFR-2 inhibitors: Synthesis of pyridine derivatives, cytotoxicity and apoptosis induction profiling

VEGFR-2 is a key regulator in cancer angiogenesis. This research displays the design and synthesis of novel 3-cyano-6-naphthylpyridine scaffold-based derivatives as selective VEGFR-2 inhibitors and cytotoxic agents. In vitro percent kinase activity inhibition screening against a panel of 23 kinases at a single high dose (30 nM) affirmed that VEGFR-2 was selectively the most responsive to inhibition by the investigated chemotypes. IC50 values determination demonstrated kinase inhibitory activities of the test compounds at the sub-nanomolar level. In vitro testing of the new compounds against two prostate cancer cell lines namely PC3 and DU145 and two breast cancer cell lines namely MCF-7 and MDA-MB435 confirmed their potent cytotoxic activity with IC50s at the nanomolar level. The most active compound against MCF-7 viz.11d was subjected to an in vivo examination against a xenograft mouse model and was found effective. Studying the tissue mRNA expression levels of various cell cycle controlling biomolecules in 11d-treated MCF-7 cells demonstrated (i) upregulation of p53, p21 and p27, (ii) cleavage of PARP protein, (iii) activation of caspase-3, −8 and −9, (iv) downregulation of the anti-apoptotic protein Bcl, (v) upregulation of the pro-apoptotic protein Bax, and (vi) decreased expression of Cdks 2, 4, 6 and cyclin D1. Additionally, 11d affected a cell cycle arrest at the G1 phase in treated MCF-7 cells and an S phase arrest in MCF-7 p53 knockdown cells. Additionally, molecular docking was performed to predict how 11d might bind to its biological target VEGFR-2. Finally, in-silico ADME and drug-likeness profiling of these derivatives demonstrated favorable properties thereof.

Subchronic Noise and Metabolism: Rodent Model Identifies Potential Mechanistic Links.

Vol. 128, No. 3 Science SelectionOpen AccessSubchronic Noise and Metabolism: Rodent Model Identifies Potential Mechanistic Linksis accompanied byImpacts of Subchronic, High-Level Noise Exposure on Sleep and Metabolic Parameters: A Juvenile Rodent Model Silke Schmidt Silke Schmidt Search for more papers by this author Published:13 March 2020CID: 034004https://doi.org/10.1289/EHP6304View Article in:中文版AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractMultiple epidemiologic studies1,2,3 have linked chronic exposure to environmental noise with an increased risk of metabolic disorders, including obesity and diabetes. An experimental study published recently in Environmental Health Perspectives found consistent results in rodents, with gene expression and tissue analyses suggesting potential underlying mechanisms.4Epidemiological evidence has associated chronic noise exposure with metabolic changes, potentially stemming from sleep disturbances. A rat study of noise exposures during regular sleeping hours sought to tease out a mechanism that might explain these observations. Image: © iStockphoto/unoL.Researchers at Université de Picardie Jules Verne in Amiens, France, exposed 12 male Wistar rats to subchronic noise, meaning intermittent noisy periods of relatively long duration. For 37 days, starting at 3 weeks of age, the rats were exposed during their 12-hour sleep period to different sounds at levels comparable to the threshold commonly used in human studies of urban traffic noise. The noise exposure was followed by a noise-free 12-hour active period. Another 12 rats made up the control group.In the two groups of rats, the researchers compared physiologic parameters, sleep patterns, sleep apnea symptoms, post-sacrifice organ weights, and tissue mRNA expression levels of genes that regulate food intake and appetite. The noise-exposed rats consumed more food and water and gained 6% more body weight than the control animals. They also had heavier thymus and adrenal glands. Forebrain (hypothalamus) mRNA expression levels were higher for one gene (the cocaine- and amphetamine-regulated transcript gene, or CART) and lower for another (leptin receptor). Differences in sleep parameters between exposed and nonexposed animals, such as the duration of REM and non-REM sleep, were subtle, and there was no evidence of an effect on apnea during sleep.“Our study’s main finding is that noise exposure during the rest period increased food and water consumption, resulting in body and organ weight gain,” says senior author Amandine Pelletier, a lecturer in physiology. “Similar effects have been observed in human studies.”1,2,3An association between chronic noise exposure and greater thymus weight was previously reported in female adult rats.5 That finding suggested that noise-induced stress may influence metabolic function.6 Another study found that noise exposure increased the adrenal gland production of corticosterone in rats,7 which may lead to hypertrophy of the organ.8 Measuring blood levels of corticosterone and other hormones under a similar exposure protocol would be an interesting next step, says Pelletier.The noise-associated changes in mRNA expression levels may partially explain the observed body weight gain. Leptin is a hormone that inhibits hunger.9,10 The CART gene codes for neuropeptides involved in stress and reward pathways, whose expression is partially regulated by leptin.11 It is plausible that the leptin receptor and CART mRNAs may jointly influence circulating levels of leptin and other hormones, contributing to an increased appetite.Jennifer Teske, an associate professor of nutritional sciences at the University of Arizona in Tucson, who was not involved in the project, notes that an earlier study of rodents12 reported similar associations between noise exposure and weight gain. Recent work in Teske’s lab using female rats also found similar results.13 The current study’s gene expression analysis adds to the body of evidence by suggesting potential underlying mechanisms, she says.Zorana Jovanovic Andersen, a professor of environmental epidemiology at the University of Copenhagen, notes that, given the rat’s life span, the 36-day exposure period mimics long-term exposures that are relevant for human chronic disease development. For Andersen, who also was not involved in the study, the rodent findings are consistent with associations between chronic noise, sleep disturbances, and human diabetes. “The researchers also identified interesting and plausible explanations for these associations,” she says, “pointing at noise-related increased appetite and weight gain as the most relevant pathway.”Silke Schmidt, PhD, writes about science, health, and the environment from Madison, Wisconsin.

Goat CMTM2: mRNA expression profiles of different alternative spliced variants and associations analyses with growth traits.

CKLF like MARVEL transmembrane domain containing 2 (CMTM2) plays crucial roles in spermiogenesis, skeletogenous, growth, and development through PI3K/Akt and other pathways.The purpose of this study was to explore the expression profile and variation of different spliced CMTM2 gene in Shaanbei white cashmere goats, as well as to find the relationships between a CMTM2 promoter region 14bp genetic variant and growth traits in 1366 Shaanbei white cashmere goats. In this study, we identified alternative CMTM2 splicing and detected the effects of the spliced variants on mRNA expression levels in tissues. Meanwhile, an unreported spliced variant of CMTM2 in goat was identified using in CDS cloning and RT-PCR, namely, CMTM2-AS2. Compared with the normal transcript (CMTM2-AS1), the novel variant had the higher expression level in muscle and liver tissues, indicating that it plays an effective role in growth traits. Furthermore, a 14bp deletion was detected within CMTM2 promoter region, and the different genotypes were significantly associated with growth traits (e.g., body length, circumferenceofcannonbone) in the large group of 1366 individuals in Shaanbei white cashmere goats. We found that the body length of the individuals with II (n = 571) genotype had better phenotypes than those with DD (n = 118) and ID (n = 650) genotypes. These results have direct guiding significance for goat breeding in the future and provide a new idea for studying the characteristics and functions of CMTM2 gene in goats.

MircoRNA-129-5p suppresses the development of glioma by targeting HOXC10

BackgroundmiR-129-5p has been reported to be abnormally expressed and plays an important role in the progression of various malignancies. However, its role in gliomas and its exact molecular mechanism need further research. Methods and materialsRT-qPCR was performed to evaluate miR-129-5p and HOXC10 mRNA expression levels in tissues and cell lines. Cell proliferation was detected via Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) and clone formation assays. Luciferase assays were used to validate the binding of seeds between miR-129-5p and HOXC10. A tumor xenograft model was developed to study the effect of miR‐129-5p on glioma growth in vivo. ResultsmiR-129-5p was expressed at low levels in glioma tissues and cell lines. miR-129-5p overexpression inhibited glioma proliferation, migration and invasion. miR-129-5p negatively and directly targeted HOXC10. At the same time, HOXC10 was upregulated in glioma cancer, and HOXC10 knockdown inhibited cell proliferation, migration and invasion. ConclusionmiR-129-5p inhibits glioma development by altering HOXC10 expression and may therefore serve as a new diagnostic marker and therapeutic target for glioma in the future.

Abstract 863: Functions of FRAS1 as a driver gene of liver metastasis from gastric cancer analyzed by the genome editing technology

Abstract Background: The prognosis of patients with liver metastasis from gastric cancer (GC) is dismal and it remains a serious problem in the management of GC. To elucidate complex molecular mechanisms underlying formation of liver metastasis is necessary for development of novel therapeutic modality. Methods: Transcriptome analysis of 57749 molecules with 4 groups (no recurrence, peritoneal recurrence, liver recurrence and nodal recurrence) was conducted to screen out the candidate molecules specific for liver metastasis. Subsequently, the stable knockout (KO) cells were established using the CRISPR/Cas9 system and cell functions, apoptosis, cell cycle, oxidative stress and cancer stemness were analyzed. PCR and antibody array analyses were conducted to identify the co-regulated molecules. Mouse subcutaneous xenograft models and liver metastasis models were generated to evaluate tumorigenicity of the KO cells in vivo. Tissue mRNA expression levels were determined by qRT-PCR in patients with stage II/III GC and correlated with recurrence patterns. Results: Fraser extracellular matrix complex subunit 1 (FRAS1) was identified as a candidate molecule overexpressed specifically in the liver metastasis group. Proliferation, adhesion, invasion and migration abilities were suppressed in FRAS1-KO cells compared to the control cells. Cell cycle alternation, increased proportions of apoptotic cells, increased oxidative stress and decreased cancer stemness were also observed in FRAS1-KO cells. PCR array analysis revealed that FRAS1 mRNA expression was positively correlated with TGFB1, MAP1B, AHNKA and negatively correlated with BMP2. Pathway analysis demonstrated that expression of MUC1, Survivin, Ki67 and Met proteins were decreased in FRAS1-KO cells. The tumorigenicity of FRAS1-KO cells was reduced in a mouse subcutaneous model and more clearly in liver metastasis models. Patients with high FRAS1 mRNA expression had a significantly higher incidence of liver recurrences after curative gastrectomy. Conclusion: Our results indicated that FRAS1 contributes to formation of liver metastasis from GC and is a potential therapeutic target. Citation Format: Shinichi Umeda, Mitsuro Kanda, Haruyoshi Tanaka, Fuminori Sonohara, Hideki Takami, Norifumi Hattori, Masamichi Hayashi, Chie Tanaka, Daisuke Kobayashi, Suguru Yamada, Goro Nakayama, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera. Functions of FRAS1 as a driver gene of liver metastasis from gastric cancer analyzed by the genome editing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 863.

Characterization of kallikrein-related peptidase 4 (KLK4) mRNA expression in tumor tissue of advanced high-grade serous ovarian cancer patients.

Overexpression of several members of the kallikrein-related peptidase (KLK) family, including KLK4, has been reported in ovarian cancer tissue, consistent with the fact that elevated levels of KLK protein are often also found in serum and in effusion fluids of ovarian cancer patients. In the present study, we quantitatively analyzed KLK4 tumor tissue mRNA expression levels in a homogeneous cohort including 138 patients of advanced high-grade serous ovarian cancer (FIGO stage III/IV). Age as well as ascites fluid volume were found to be significantly associated with KLK4 mRNA expression levels. In univariate Cox regression analysis, the clinical factors residual tumor mass and ascites fluid volume represented univariate predictors for both overall survival (OS) and progression-free survival (PFS). Furthermore, elevated KLK4 mRNA expression levels were significantly linked with reduced OS (p = 0.001), but not with PFS. The results concerning the association of KLK4 mRNA expression with OS were validated in a publicly available Affymetrix-based mRNA data set from The Cancer Genome Atlas (n = 252) applying the Kaplan-Meier Plotter tool (p = 0.047). In multivariable analyses, elevated KLK4 mRNA values turned out as an additional, independent predictive marker for shortened OS (p = 0.006), whereas residual tumor mass, but not ascites fluid volume, remained an independent indicator for both OS and PFS (p < 0.001 and p = 0.002, respectively). The results of the present study, obtained in a well-defined, homogenous cohort of patients afflicted with advanced high-grade serous ovarian cancer, are in line with previous reports describing high KLK4 levels as an unfavorable marker in ovarian cancer patients.

Bovine pituitary homeobox 2 (PITX2): mRNA expression profiles of different alternatively spliced variants and association analyses with growth traits

Pituitary homeobox 2 (PITX2) plays crucial roles in embryogenesis, ontogenesis, growth, and development via the Wnt/beta-catenin and POU1F1 pathways. To better understand the characteristics and genetic effects of the cattle PITX2 gene, we identified alternative PITX2 splicings, examined the effects of the spliced variants on mRNA expression levels in tissues, and then used association analyses to explore the relationships between a PITX2 deletion genetic variant and growth traits in 750 native Chinese cattle. An unreported spliced variant of PITX2, designated here as PITX2-V1, was identified in cattle using in silico cloning and RT-PCR. The entire coding sequence of PITX2 is 978 bp, encoding 325 amino acids, whereas that of PITX2-V1 is 357 bp encoding 118 amino acids. Cattle PITX2 exhibited both a perfect homeodomain and an OAR domain, but PITX2-V1 lacked the homeodomain. Analyses with qRT-PCR showed that the expression level of PITX2 in cattle testis was very low, and PITX2-V1 was only very slightly expressed in the brain and testis. Furthermore, a 24 bp deletion was detected within PITX2 intron, and the different genotypes were significantly associated with growth traits (e.g., body height, body length, heart girth) in four cattle breeds (P < 0.05). These results are of direct benefit to future cattle breeding, and provide new insights into the characteristics and functions of cattle PITX2 gene.

A novel dual-marker expression panel for easy and accurate risk stratification of patients with gastric cancer.

Development of specific biomarkers is necessary for individualized management of patients with gastric cancer. The aim of this study was to design a simple expression panel comprising novel molecular markers for precise risk stratification. Patients (n = 200) who underwent gastrectomy for gastric cancer were randomly assigned into learning and validation sets. Tissue mRNA expression levels of 15 candidate molecular markers were determined using quantitative PCR analysis. A dual‐marker expression panel was created according to concordance index (C‐index) values of overall survival for all 105 combinations of two markers in the learning set. The reproducibility and clinical significance of the dual‐marker expression panel were evaluated in the validation set. The patient characteristics of the learning and validation sets were well balanced. The C‐index values of combinations were significantly higher compared with those of single markers. The panel with the highest C‐index (0.718) of the learning set comprised SYT8 and MAGED2, which clearly stratified patients into low‐, intermediate‐, and high‐risk groups. The reproducibility of the panel was demonstrated in the validation set. High expression scores were significantly associated with larger tumor size, vascular invasion, lymph node metastasis, peritoneal metastasis, and advanced disease. The dual‐marker expression panel provides a simple tool that clearly stratifies patients with gastric cancer into low‐, intermediate‐, and high risk after gastrectomy.

Abstract P2-09-34: An mRNA-based method to measure PI3K activity in cancer tissue using a computational pathway model to assess FOXO transcriptional activity

Abstract Introduction The PI3K signaling pathway is frequently active in breast cancer, and therapeutic inhibitors have been developed. However, it has proven difficult to correctly predict treatment response. We developed a method that measures functional activity of the PI3K pathway using a computational model that infers transcriptional FOXO activity (downstream of PI3K) from expression levels of its target genes. In principle, PI3K pathway activity inhibits transcriptional FOXO activity, hence inactive FOXO is indicative of active PI3K. Method We developed a knowledge-based computational model to infer transcriptional FOXO activity from cancer tissue mRNA expression levels, using a Bayesian network approach (Verhaegh et al., Cancer Res 2014). Model calibration was done on microarray data from HUVEC cells with inducible FOXO3.A3-ER (GSE16573). Results The FOXO model was biologically validated with in-house microarray data from independent breast cancer cell lines. ER positive, PIK3CAE545K mutant MCF7 and triple negative MDA-MB-231 cells were stably transduced with a doxycycline inducible FOXO3.A3 expression vector, allowing controlled induction of FOXO3 protein activity. FOXO activity was determined to be low in untreated and 20% FBS treated MCF7 cells, and high after doxycycline, LY294002, and combination treatment. Next, we tested our FOXO model on independent MCF7, BT-20 and MDA-MB-453 cell line data treated with EGFR inhibitor erlotinib (GSE30516), showing an increase of FOXO activity upon treatment, due to reduced PI3K pathway activity (combined Wilcox rank sum test p = 7.8x10−5). We further analyzed independent publicly available data from breast cancer patients. FOXO was generally active in healthy breast tissue. Compared to healthy breast tissue, FOXO activity was higher in normal-like and luminal A breast cancer samples (p = 1.9x10−6 and 0.025, resp.), and lower in luminal B samples (p = 4.2x10−7). In addition to the above mechanism for regulating FOXO activity, literature suggests that FOXO can also be activated by cellular oxidative stress, which is often associated with PI3K signaling. This may be assessed using expression levels of the FOXO target gene SOD2, which is differentially expressed between the two FOXO activity modes, and whose function is to reduce oxidative stress. Public data shows an increasing percentage of elevated SOD2 levels among FOXO-active samples with increasing breast cancer aggressiveness: 7% in normal-like, 5% in luminal A, 18% in luminal B, 31% in HER2-enriched and 74% in basal like breast cancer. Conclusion Our computational model to measure PI3K activity using FOXO target gene mRNA levels was able to measure increased FOXO activity in multiple cancer cell lines after PI3K inhibition. FOXO activity was measured high in healthy breast tissue and in normal-like and luminal A breast cancer, and lower in luminal B, indicating PI3K activity in the latter group. In more aggressive subtypes, FOXO activity was increasingly accompanied by high SOD2 expression, suggesting oxidative stress with associated PI3K activity as the FOXO activating mechanism. Clinical utility for improved response prediction and monitoring of PI3K pathway inhibitors is being investigated with clinical partners. Citation Format: Verhaegh W, van Ooijen H, Hornsveld M, Dam C, Eijkelenboom A, Dou M, Velter R, Burgering B, van de Stolpe A. An mRNA-based method to measure PI3K activity in cancer tissue using a computational pathway model to assess FOXO transcriptional activity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-34.

Correlation of TLR4 and KLF7 in Inflammation Induced by Obesity

Objective Recent studies have revealed a link between toll-like receptors (TLRs), Kruppel-like factors (KLFs), and the adipose tissue inflammation associated with obesity. TLR4 is associated with chronic inflammation in obesity. KLF7 is known to play an important role in the differentiation of adipocytes, but its role in visceral adipose tissue inflammation has not yet been investigated. Thus, the objective of this study was to determine the correlation of TLR4 and KLF7 in inflammation induced by obesity. Methods A total of 32 Wistar male rat subjects were fed in the center for experimental animals of Shihezi University. The rats were divided into normal control (NC) and high-fat diet (HFD) group. Surgical instruments were used to collect rats' visceral adipose tissue samples in the 10th week after HFD feeding. Ninety-five Uygur subjects between 20 and 90years old were enrolled in the present study. The subjects were divided into two groups: the normal control group (NC, 18.0kg/m2 ≤ BMI ≤ 23.9kg/m2, n = 50) and the obesity group (OB, BMI ≥ 28kg/m2, n = 45), and visceral adipose tissue was collected from the subjects. Anthropometric and clinical parameters were measured using standard procedures; biochemical indices were detected using the glucose oxidase-peroxidase method and a standardized automatic biochemistry analyzer; the plasma levels of inflammatory factors and adipocytokines were measured by enzyme-linked immunosorbent assay (ELISA); the mRNA and protein expression levels of key genes involved in the inflammatory signaling pathway were measured by real-time PCR and Western blot. Results In rats, compared with the NC group, the weight, Lee's index, waist circumference, visceral fat mass, and the plasma level of Glu, TG, FFA, and TNF-α were higher in the HFD group, while the plasma levels of LPT and APN were significantly lower in the HFD group in the 10th week. Furthermore, compared with the NC group, visceral adipose tissue's mRNA expression levels of TLR4, KLF7, and SRC were higher in the HFD group, and KLF7 was significantly positively correlated with LDL, TLR4, SRC, and IL-6 (P < 0.05). Meanwhile, in the Uygur population, the plasma levels of TG, LDL, and TNF-α in the OB group were significantly higher than those in the NC group (P < 0.05). Moreover, compared with the NC group, visceral adipose tissue's mRNA expression levels of TLR4, KLF7, and SRC were significantly higher in the OB group (P < 0.05), and KLF7 was significantly positively correlated with TC, TLR4, MYD88, SRC, and IL-6 (P < 0.05); the protein expression levels of TLR4 and KLF7 were significantly higher than those in the NC group (P < 0.05). Conclusion Higher expression of TLR4 and KLF7 may play a vital role in the process of inflammation induced by obesity in visceral adipose tissue.

An integrated cell line-based discovery strategy identified follistatin and kallikrein 6 as serum biomarker candidates of breast carcinoma

Secreted proteins constitute a relevant source of putative cancer biomarkers. Here, we compared the secretome of a series of four genetically-related breast cancer cell lines as a model of aggressiveness using quantitative mass spectrometry. 537 proteins (59.5% of the total identified proteins) predicted to be released or shed from cells were identified. Using a scoring system based on i) iTRAQ value, ii) breast cancer tissue mRNA expression levels, and iii) immunohistochemical staining (public database), a short list of 10 candidate proteins was selected. Using specific ELISA assays, the expression level of the top five proteins was measured in a verification set of 56 patients. The four significantly differentially expressed proteins were then validated in a second independent set of 353 patients. Finally, follistatin (FST) and kallikrein 6 (KLK6) in serum were significantly higher (p-value < 0.0001) in invasive breast cancer patients compared with non-cancerous controls. Using specific cut-off values, FST distinguished breast cancer samples from healthy controls with a sensitivity of 65% and an accuracy of 68%, whereas KLK6 achieved a sensitivity of 55% and an accuracy of 61%. Therefore, we concluded that FST and KLK6 may have significance in breast cancer detection. Biological significanceDiscovery of new serum biomarkers that exhibit increased sensitivity and specificity compared to current biomarkers appears to be an essential field of research in cancer. Most biological markers show insufficient diagnostic sensitivity for early breast cancer detection and, for the majority of them, their concentrations are elevated only in metastatic forms of the disease. It is therefore essential to identify clinically reliable biomarkers and develop effective approaches for cancer diagnosis. One promising approach in this field is the study of secreted proteins through proteomic analysis of in vitro progression breast cancer models. Here we have shown that FST and KLK6 are elevated in breast cancer patient serum compared to healthy controls. We expect that our discovery strategy will help to identify cancer-specific and body-fluid-accessible biomarkers.

Molecular and functional characterization of autophagy marker LC3 in orange-spotted grouper (Epinephelus coioides)

Autophagy is a conserved cellular process in response to stress, sustain normal cell growth by degrade and recycle unnecessary cytosolic components in autophagosome and autolysosome. Microtubuleassociated protein light chain 3 known as LC3 is a major constituent of the autophagosome and used as a reliable marker for autophagy. In this study, a full length of orange-spotted grouper LC3 (osgLC3) mRNA had been cloned with a size of 1,440 bp including the sequence encoded for a 126 a.a. protein. The phylogenetic relationship and relative tissue mRNA expression levels for osgLC3 has been evaluated. The punctate structures increased in the GF-1 cells with over-expressed osgLC3 under nutrient deprivation revealed that starvation induced autophagy formation and the colocalization of punctate structures and lysosome indicated autolysosome formation during autophagy induction. Additionally, it has found that autophagy formation in GF-1 cells induced by nervous necrosis virus (NNV) infection through quantitation of endogenous osgLC3-positive punctate structures. In grouper larvae, the mRNA expression of osgLC3 were increased under NNV infection and the osgLC3 was able to convert to lipidated form of LC3 (osgLC3-II) for synthesis of autophagosome. Moreover, it has been verified that Hsp90 plays a role in autophagy and here the osgLC3 and Hsp90AB had higher FRET efficiency that implied the interaction between osgLC3 and Hsp90AB. Based on these results, osgLC3 contributed to the autophagy formation in response to NNV infection in grouper and the working mechanisms of osgLC3 and Hsp90AB during virus infection need further studies.

Molecular and biochemical evidence on the protective effects of embelin and carnosic acid in isoproterenol-induced acute myocardial injury in rats

AimsAcute myocardial infarction is a serious acute cardiac disorder and heart disease is still a major public health problem in adults. We investigated the effects of embelin (EMB) and carnosic acid (CA) in animals with isoproterenol (ISO)-induced myocardial injury. Main methodsAdult male Wistar–Albino rats were divided into four groups: control, ISO, ISO with EMB, and ISO with CA. Before myocardial injury was induced, drugs were administered by oral gavage. Myocardial injury was induced by subcutaneous injection of ISO hydrochloride for 2 consecutive days. Serum cardiac troponin I (cTnI), ischemia modified albumin (IMA), heart fatty acid binding protein (HFABP) levels and paraoxonase-1 (PON-1) activity, tissue total oxidant status (TOS), total antioxidant status (TAS), total thiol (TT), tumor necrosis factor-α (TNF-α) levels, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity were measured. Tissue mRNA expression levels of nuclear factor-kappa B (NF-κB), P38 mitogen-activated protein kinase (p38 MAPK), and nuclear factor erythroid 2-related factor 2 (Nrf2) were analyzed. In addition, cardiac tissues were evaluated histopathologically and immunohistochemically. Key findingsAll tested compounds reduced myocardial damage, apoptosis, cTnI, IMA, HFABP, TOS, and TNF-α levels, NF-κB, p38 MAPK, and phosphorylated c-Jun N-terminal protein kinase (pJNK 1/2) expressions. All tested compounds increased SOD activity, GSH-Px activity, TAS levels, TT levels, phosphorylated extracellular signal-regulated kinase (pERK 1/2), and Nrf2 expressions. SignificanceOur results suggest that EMB and CA pretreatment could reduce myocardial injury via antiinflammatory, antioxidant, and antiapoptotic effects.

Clinical significance of ECT2 expression in tissue and serum of gastric cancer patients.

The ECT2 (epithelial cell transforming sequence 2) oncogene acted as a guanine nucleotide exchange factor for RhoGTPases, and regulates cytokinesis; thus, it may play a role in the pathogenesis of gastric cancer. In this study, we investigated the expression ECT2 gene in tissues and serum of gastric cancer patients to explore its clinical significance. ECT2 mRNA expression levels in tissues and serum were examined by RT-PCR, and ECT2 protein expression in tissue was evaluated by Western blot, and was further validated by immunohistochemistry and enzyme-linked immunosorbent assay at serum level. ECT2 level was significantly increased in the GC tissues and serum compared to normal control. ECT2 expression was positively correlated with the histologic differentiation, stages of TNM, and lymph node metastasis in GC (P<0.05). Our results suggest that ECT2 plays an important role during GC progression and it may become a new diagnostic marker and therapeutic molecular target for management of GC.

Association of Ketone Body Levels With Hyperglycemia and Type 2 Diabetes in 9,398 Finnish Men

We investigated the association of the levels of ketone bodies (KBs) with hyperglycemia and with 62 genetic risk variants regulating glucose levels or type 2 diabetes in the population-based Metabolic Syndrome in Men (METSIM) study, including 9,398 Finnish men without diabetes or newly diagnosed type 2 diabetes. Increasing fasting and 2-h plasma glucose levels were associated with elevated levels of acetoacetate (AcAc) and β-hydroxybutyrate (BHB). AcAc and BHB predicted an increase in the glucose area under the curve in an oral glucose tolerance test, and AcAc predicted the conversion to type 2 diabetes in a 5-year follow-up of the METSIM cohort. Impaired insulin secretion, but not insulin resistance, explained these findings. Of the 62 single nucleotide polymorphisms associated with the risk of type 2 diabetes or hyperglycemia, the glucose-increasing C allele of GCKR significantly associated with elevated levels of fasting BHB levels. Adipose tissue mRNA expression levels of genes involved in ketolysis were significantly associated with insulin sensitivity (Matsuda index). In conclusion, high levels of KBs predicted subsequent worsening of hyperglycemia, and a common variant of GCKR was significantly associated with BHB levels.

Adipose tissue and liver expression of SIRT1, 3, and 6 increase after extensive weight loss in morbid obesity

Severe obesity is associated with a state of chronic inflammation. Sirtuins (SIRT) are a family of conserved enzymes which are able to affect many metabolic and inflammatory pathways thereby potentially improving health and increasing lifespan. We investigated the effect of weight loss on subcutaneous adipose tissue and liver mRNA and immunohistochemical expression of SIRT1, SIRT3, and SIRT6. Twenty-nine severely obese patients undergoing laparoscopic adjustable gastric banding (LAGB) were studied. Tissue samples were collected before and 6months after LAGB surgery. Tissue mRNA expression levels of SIRT1, SIRT3, and SIRT6 were correlated with clinical, biochemical, and histological parameters. In vitro, we studied sirtuin expression in native and stimulated monocytes, adipocytes, and hepatocytes. SIRT1, SIRT3, and SIRT6 mRNA expression was higher in the subcutaneous adipose tissue than in the liver. Weight loss resulted in a significant induction of SIRT1, SIRT3, and SIRT6 expression in the subcutaneous adipose tissue. In the liver, a significant increase after weight loss was observed, particularly for SIRT3 and SIRT6 mRNA expression; immunohistochemically, SIRT1 and SIRT3 expression was upregulated. Endotoxin and tumor necrosis factor-alpha suppressed SIRT1, SIRT3, and SIRT6 expression in human monocytes. The same stimuli suppressed total sirtuin deacetylase activity again, mainly in monocytes and less in adipocytes and hepatocytes. The relative abundance of adipose tissue mRNA expression of certain sirtuins exceeds its expression in the liver. Extensive weight loss increases sirtuin expression significantly both in adipose tissue and liver, probably as a consequence of reduced inflammation.

The impact of reference gene selection in quantification of gene expression levels in guinea pig cervical tissues and cells

BackgroundAccurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix.ResultsGene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes.ConclusionsWe recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells from guinea pig.

Nature of fatty acids in high fat diets differentially delineates obesity-linked metabolic syndrome components in male and female C57BL/6J mice

BackgroundAdverse effects of high-fat diets (HFD) on metabolic homeostasis are linked to adipose tissue dysfunction. The goal of this study was to examine the effect of the HFD nature on adipose tissue activity, metabolic disturbances and glucose homeostasis alterations in male mice compared with female mice.MethodsC57BL/6J mice were fed either a chow diet or HFD including vegetal (VD) or animal (AD) fat. Body weight, plasmatic parameters and adipose tissue mRNA expression levels of key genes were evaluated after 20 weeks of HFD feeding.ResultsHFD-fed mice were significantly heavier than control at the end of the protocol. Greater abdominal visceral fat accumulation was observed in mice fed with AD compared to those fed a chow diet or VD. Correlated with weight gain, leptin levels in systemic circulation were increased in HFD-fed mice in both sexes with a significant higher level in AD group compared to VD group. Circulating adiponectin levels as well as adipose tissue mRNA expression levels were significantly decreased in HFD-fed male mice. Although its plasma levels remained unchanged in females, adiponectin mRNA levels were significantly reduced in adipose tissue of both HFD-fed groups with a more marked decrease in AD group compared to VD group. Only HFD-fed male mice were diabetic with increased fasting glycaemia. On the other hand, insulin levels were only increased in AD-fed group in both sexes associated with increased resistin levels. VD did not induce any apparent metabolic alteration in females despite the increased weight gain. Peroxisome Proliferator-Activated Receptors gamma-2 (PPARγ2) and estrogen receptor alpha (ERα) mRNA expression levels in adipose tissue were decreased up to 70% in HFD-fed mice but were more markedly reduced in male mice as compared with female mice.ConclusionsThe nature of dietary fat determines the extent of metabolic alterations reflected in adipocytes through modifications in the pattern of adipokines secretion and modulation of key genes mRNA expression. Compared with males, female mice demonstrate higher capacity in controlling glucose homeostasis in response to 20 weeks HFD feeding. Our data suggest gender specific interactions between the diet's fatty acid source, the adipocyte-secreted proteins and metabolic disorders.

Chitinase 3-like 1 promotes macrophage recruitment and angiogenesis in colorectal cancer

Chitinase 3-like 1 (CHI3L1), one of mammalian members of the chitinase family, is expressed in several types of human cancer, and elevated serum level of CHI3L1 is suggested to be a biomarker of poor prognosis in advanced cancer patients. However, the overall biological function of CHI3L1 in human cancers still remains unknown. Studies were performed to characterize the role of CHI3L1 in cancer pathophysiology utilizing human colorectal cancer samples and human cell lines. Plasma protein and tissue mRNA expression levels of CHI3L1 in colorectal cancer were strongly upregulated. Immunohistochemical analysis showed that CHI3L1 was expressed in cancer cells and CHI3L1 expression had a significant association with the number of infiltrated macrophages and microvessel density. By utilizing trans-well migration and tube formation assays, overexpression of CHI3L1 in SW480 cells (human colon cancer cells) enhanced the migration of THP-1 cells (human macrophage cells) and HUVECs (human endothelial cells), and the tube formation of HUVECs. The knockdown of CHI3L1 by RNA interference or the neutralization of CHI3L1 by anti-CHI3L1 antibody displayed strong suppression of CHI3L1-induced migration and tube formation. Cell proliferation assay showed that CHI3L1 overexpression significantly enhanced the proliferation of SW480 cells. ELISA analysis showed that CHI3L1 increased the secretion of inflammatory chemokines, IL-8 and MCP-1, from SW480 cells through mitogen-activated protein kinase (MAPK) signaling pathway. Both neutralization of IL-8 or MCP-1 and inhibition or knockdown of MAPK in SW480 cells significantly inhibited CHI3L1-induced migration and tube formation. In a xenograft mouse model, overexpression of CHI3L1 in HCT116 cells (human colon cancer cells) enhanced the tumor growth as well as macrophage infiltration and microvessel density. In conclusion, CHI3L1 expressed in colon cancer cells promotes cancer cell proliferation, macrophage recruitment and angiogenesis. Thus, the inhibition of CHI3L1 activity may be a novel therapeutic strategy for human colorectal cancer.