Lower mRNA Expression Research Articles

Correlation of Lipin gene expression with hepatic fat content in rats with intrauterine growth retardation

To study the correlation of the expression of Lipin1 in visceral adipose tissue and Lipin2 in liver tissue with hepatic fat content in rats with intrauterine growth retardation (IUGR). Pregnant rats were given a low-protein (10% protein) diet during pregnancy to establish a model of IUGR in neonatal rats. The pregnant rats in the control group were given a normal-protein (21% protein) diet during pregnancy. The neonatal rats were weighed and liver tissue was collected on day 1 and at weeks 3, 8, and 12 after birth, and visceral adipose tissue was collected at weeks 3, 8, and 12 after birth. The 3.0T 1H-magnetic resonance spectroscopy was used to measure hepatic fat content at weeks 3, 8, and 12 after birth. Real-time PCR was used to measure mRNA expression levels of Lipin2 in liver tissue and Lipin1 in visceral adipose tissue. Western blot was used to measure protein levels of Lipin2 in liver tissue and Lipin1 in visceral adipose tissue. A Pearson correlation analysis was performed to investigate the correlation of mRNA and protein expression of Lipin with hepatic fat content. The IUGR group had significantly higher mRNA and protein expression levels of Lipin1 in visceral adipose tissue than the control group at weeks 3, 8, and 12 after birth (P<0.05). Compared with the control group, the IUGR group had significantly lower mRNA and protein expression levels of Lipin2 in liver tissue on day 1 after birth and significantly higher mRNA and protein expression levels of Lipin2 at weeks 1, 3, 8, and 12 after birth (P<0.05). At week 3 after birth, there was no significant difference in hepatic fat content between the IUGR and control groups (P>0.05), while at weeks 8 and 12 after birth, the IUGR group had a significantly higher hepatic fat content than the control group (P<0.05). The protein and mRNA expression levels of Lipin1 were positively correlated with hepatic fat content (r=0.628 and 0.521 respectively; P<0.05), and the protein and mRNA expression levels of Lipin2 were also positively correlated with hepatic fat content (r=0.601 and 0.524 respectively; P<0.05). Upregulation of the mRNA and protein expression levels of Lipin1 in visceral adipose tissue and Lipin2 in liver tissue can increase hepatic fat content in rats with IUGR and may be associated with obesity in adulthood.

DNA methylation regulates TIGIT expression within the melanoma microenvironment, is prognostic for overall survival, and predicts progression-free survival in patients treated with anti-PD-1 immunotherapy

BackgroundTIGIT is an immune checkpoint under investigation as therapeutic target. Understanding the regulation of TIGIT on an epigenetic level might support the development of companion biomarkers.MethodsWe correlated TIGIT DNA methylation of single CpG sites with gene expression, signatures of immune infiltrates and interferon-γ, and survival in melanoma. We further analyzed methylation levels in immune cell subsets, melanocyte and melanoma cell lines. TIGIT expression patterns within components of the melanoma microenvironment were analyzed by single cell sequencing. We used quantitative methylation-specific PCR, flow cytometry, and immunohistochemistry for correlations between expression and methylation and to assess the effect of pharmacological demethylation of melanoma cells treated with 5‐aza‐2‐deoxycytidine (decitabine). Finally, we investigated the association of patients’ survival with TIGIT mRNA and methylation.ResultsDepending on the sequence context of the analyzed CpG site, we found a cell type-specific TIGIT gene locus methylation pattern and significant correlations of TIGIT methylation with mRNA expression, an interferon γ signature, and distinct immune cell infiltrates, including TIGIT+ lymphocytes. We detected a melanoma cell-intrinsic TIGIT protein expression. Pharmacological demethylation of the A375 melanoma cell line led to a constitutive TIGIT expression. Low promoter flank methylation and high mRNA expression was associated with patients’ prognosis and predicted progression-free survival in patients treated with anti-PD-1 immunotherapy. A high TIGIT+ lymphocyte score was associated with better progression-free survival under anti-PD-1 immunotherapy.ConclusionsOur data demonstrate an epigenetic regulation of TIGIT expression via DNA methylation within the melanoma microenvironment. TIGIT DNA methylation and expression may serve as predictive biomarkers in the context of immunotherapies in melanoma.

Aldh1a1 and Scl25a30 in diaphragmatic dysfunction.

New methods to prevent ventilator-induced diaphragmatic dysfunction (VIDD) are urgently needed, and the cellular basis of VIDD is poorly understood. This study evaluated whether transvenous phrenic nerve stimulation (PNS) could prevent VIDD in rabbits undergoing mechanical ventilation (MV) and explored whether oxidative stress-related genes might be candidate molecular markers for VIDD. Twenty-four adult male New Zealand white rabbits were allocated to control, MV, and PNS groups (n = 8 in each group). Rabbits in the MV and PNS groups underwent MV for 24 h. Intermittent bilateral transvenous PNS was performed in rabbits in the PNS group. Transdiaphragmatic pressure was recorded using balloon catheters. The diameters and cross-sectional areas (CSAs) of types I and II diaphragmatic fibers were measured using immunohistochemistry (IHC) techniques. Genes associated with VIDD were identified by RNA sequencing (RNA-seq), differentially expressed gene (DEG) analysis, and weighted gene co-expression network analysis (WGCNA). Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and IHC analyses were carried out to verify the transcriptome profile. Pdi60Hz, Pdi80Hz, and Pdi100Hz were significantly higher in the PNS group than in the MV group at 12 and 24 h (P < 0.05 at both time points). The diameters and CSAs of types I (slow-twitch) and II (fast-twitch) fibers were significantly larger in the PNS group than in the MV group (P < 0.05). RNA-seq, RT-PCR, Western blotting, and IHC experiments identified two candidate genes associated with VIDD: Aldh1a1 and Scl25a30. The MV group had significantly higher mRNA and protein expressions of Aldh1a1/ALDH1A1 and significantly lower mRNA and protein expressions of Scl25a30/SCL25A30 than the control or PNS groups (P < 0.05). We have identified two candidate genes involved in the prevention of VIDD by transvenous PNS. These two key genes may provide a theoretical basis for targeted therapy against VIDD.

Obesogenic and diabetic effects of CD44 in mice are sexually dimorphic and dependent on genetic background

IntroductionCD44 is a candidate gene for obesity and diabetes development and may be a critical mediator of a systemic inflammation associated with obesity and diabetes.MethodsWe investigated the relationship of CD44 with obesity in CD44-deficient mice challenged with a high-fat diet.ResultsIn mice fed a diet high in fat, cholesterol, and sucrose for 12 weeks fat mass accumulation was reduced in CD44-deficient mice bred onto both a C57BL/6J and the naturally TLR deficient C3H/HeJ background. Reduced fat mass could not be attributed to lower food intake or an increase in energy expenditure as measured by indirect calorimetry. However, we observed a 40–60% lower mRNA expression of the inflammation markers, F4/80, CD11b, TNF-α, and CD14, in adipose tissue of CD44-deficient mice on the C57BL/6J background but not the C3H/HeJ background, perhaps indicating that alternative factors may be affecting adiposity in this model. Measures of hepatic steatosis and insulin sensitivity were improved in CD44-deficient mice on a C57BL/6J but not in the C3H/HeJ mice. These results were highly sexually dimorphic as there were no detectable effects of CD44 inactivation in female mice on a C57BL/6 J or C3H/HeJ background.ConclusionCD44 was associated with adiposity, liver fat, and glucose in male mice. However, the effects of CD44 on obesity may be independent of TLR4 signaling.

Exploring sex differences in fetal programming for childhood emotional disorders

In examining maternal depression, placental 11β-HSD2 mRNA expression and offspring cortisol regulation as a potential fetal programming pathway in relation to later child emotional disorders, it has become clear that sex differences may be important to consider. This study reports on data obtained from 209 participants in the Mercy Pregnancy and Emotional Wellbeing Study (MPEWS) recruited before 20 weeks of pregnancy. Maternal depressive disorders were diagnosed using the SCID-IV and maternal childhood trauma using the Childhood Trauma Questionnaire. Placental 11β-HSD2 mRNA was measured using qRT-PCR. For assessment of stress-induced cortisol reactivity, salivary cortisol samples were taken at 12 months of age. At 4 years of age, measurement of Childhood Emotional Disorders (depression and anxiety) was based on maternal report using the Preschool Age Psychiatric Assessment (PAPA) and internalizing symptoms using the Child Behavior Checklist (CBCL). Maternal depression in pregnancy and postpartum, and infant cortisol reactivity, was associated with internalizing symptoms for females only. For female offspring only, increased 12-month cortisol reactivity was also associated with increased emotional disorders at 4 years of age; however, there was no association with placental 11β-HSD2 mRNA expression. In females only, the combination of lower placental 11β-HSD2 mRNA expression and higher cortisol reactivity at 12 months of age predicted increased internalising problems. These findings suggest there may be sex differences in prenatal predictors and pathways for early childhood depression and anxiety symptoms and disorder.

Effects of dietary supplementation of trans-anethole on the intestinal antioxidant status, immune function, and liver lipid metabolism in broilers

This study was aimed to investigate the effects of trans-anethole (TA) on intestinal antioxidant status, immune function and liver lipid metabolism in broilers. A total of 256 1-day-old Arbour Acres broilers were allocated to 4 treatment groups with 8 birds per replicate. TA was added to diets at a concentration of 0 (control), 400, 600, and 800 mg/kg for 42 days. Dietary supplementation of TA increased the average daily feed intake (ADFI) of birds throughout the entire period. TA supplemented at 400 mg/kg of diet contributed to the lowest jejunal and ileal malondialdehyde (MDA) concentration on d 42, and the highest ileal total superoxide dismutase (T-SOD) activity on d 21 and d 42. TA supplementation at the concentration of 400 and 600 mg/kg of diet increased mRNA expression of ileal interleukin (IL)-4 on d 21, and intestinal secretory immunoglobulin A (sIgA) concentration on d 42. Supplementation of TA at the concentration up to 800 mg/kg of diet had adverse effects on intestinal antioxidant status, immune function and liver lipid metabolism compared with control group. Differently, TA supplemented at 400 mg/kg led to lower mRNA expression of liver ACC1, FASN, sterol regulatory element-binding protein 1c (SREBP-1), and higher CPT1 on d 42. In conclusion, TA inclusion improved intestinal antioxidant status and immune function, and enhanced liver lipid metabolism of broilers. HIGHLIGHTS Supplementation of 400 mg/kg of TA increased intestinal antioxidant status and immune function, and promoted liver lipid metabolism of broilers compared with non-supplemented group.

The prognostic role of combining Krüppel-like factor 4 score and grade of inflammation in a Norwegian cohort of oral tongue squamous cell carcinomas.

Krüppel‐like factor 4 (KLF4) is a zinc‐finger transcription factor involved in inflammation, cancer development, and progression. However, the relationship between KLF4, inflammation, and prognosis in oral cancer is not fully understood. KLF4 expression levels were examined in a multicenter cohort of 128 oral squamous cell carcinoma (OSCC) specimens from the tongue (OTSCC) using immunohistochemistry. In two external KLF4 mRNA datasets (The Cancer Genome Atlas/The Genotype‐Tissue Expression Portal), lower KLF4 mRNA expression was found in OSCC and head and neck squamous cell carcinomas (HNSCC) than in control oral epithelium. These data indicate that down‐regulation of KLF4 mRNA is linked to OSCC/HNSCC progression. Using Cox‐multivariate analysis, a significantly favorable 5‐year disease‐specific survival rate was observed for a subgroup of patients with a combination of high levels of KLF4 expression and inflammation. OSCC cell lines exposed to IFN‐γ showed a significant upregulation of nuclear KLF4 expression, indicating a link between inflammation and KLF4 expression in OSCC. Overall, the current data suggest a functional link between KLF4 and inflammation. The combination of high KLF4 nuclear expression and marked/moderate stromal inflammation might be useful as a favorable prognostic marker for a subgroup of OTSCC patients.

Low placental angiotensin-converting enzyme expression is related to fetal small for gestational age but not to metabolic control in type 1 diabetic pregnancies.

The maternal renin-angiotensin system is involved in blood pressure control and plays a crucial role in fetoplacental nutrition. Pre-gestational type 1 diabetes (PGDM) leads to serious pregnancy complications. We thus performed a longitudinal study to analyse the association of maternal angiotensin-converting enzyme (ACE) serum levels and placental mRNA expression with fetal newborns gestational weight in type 1 diabetes mellitus (T1DM) women. We recruited 65 singleton pregnant women with T1DM. Placental mRNA ACE gene expression was examined using quantitative real-time PCR. Serum ACE levels were measured in the first, second and third trimesters of pregnancy by ELISA commercial kits. Placental expression of ACE mRNA was significantly lower in small for gestational age (SGA) than appropriate for gestational age (AGA) and large for gestational age (LGA) mothers (0.55±0.06 vs 0.78±0.06 and 0.85±0.07 respectively, p=0.003). In the SGA group, the mRNA expression of ACE positively correlated with maternal body mass index (BMI) in the third trimester (r=0.49; p=0.04). In all study groups maternal ACE level was significantly higher in the third trimester (mean 139.91±SD 69.64) compared to the first and second trimesters of pregnancy (13.57±4.32 and 15.69±15.92 respectively). Our data suggest that lower placental ACE gene mRNA expression may have a vital role in the etiology of SGA babies.

Clinical Importance of FNDC-5 and Selectin-E mRNA Expression Among Type 2 Diabetics with and without Obesity.

BackgroundType 2 diabetes mellitus (T2DM) is growing illnesses associated with metabolic dysregulation such as obesity affecting a large population become leading causes of death worldwide. Fibronectin type III domain-containing protein 5 (FNDC-5) and selectin-E were suggested to have effects on metabolism and diabetes, therefore present study aimed to evaluate the clinical importance of FNDC-5 and selectin-E among the T2DM patients with and without obesity.MethodsStudy included cohort of 200 T2DM patients with and without obesity. We evaluated FNDC-5, selectin-E mRNA expression as well as vitamin-D, and vitamin-B12 levels in among the T2DM patients with and without obesity.ResultsStudy observed significant difference in biochemical parameters included in study. T2DM patients with obesity had significantly higher fasting blood glucose levels (p<0.0001) and HbA1c (glycated hemoglobin) (p<0.0001) compared to those T2DM patients without obesity. T2DM patients with obesity also had higher systolic blood pressure (p=0.001), LDL (low density lipoprotein) (p=0.02), TG (triglycerides) (p=0.02) and cholesterol (p=0.01) compared to T2DM patients without obesity. The mRNA expression of FNDC-5 (p<0.0001) was lower in T2DM patients with obesity compared to T2DM patients without obesity. It was observed that the T2DM patients with vitamin-D deficiency had significantly lower FNDC-5 mRNA expression (p=0.03) when compared with those with sufficient vitamin-D level. T2DM patients with clinically normal vitamin-B12 level expressed 0.60 fold FNDC-5 mRNA expression while B12 deficient T2DM patients had 0.28 fold FNDC-5 mRNA expression (p=0.005). No as such significant association was was observed with selectin-E. A negative correlation of FNDC-5 mRNA expression with Post prandial glucose (mg/dl) (p=0.04) and TG (mg/dl) (p=0.02) was observed.ConclusionFNDC-5 down regulation was observed with T2DM with obesity, vitamin-D and vitamin-B12 deficiency suggesting obesity, vitamin-D and vitamin-B12 deficiency could be the factor for FNDC-5 down-regulation leading to worseness or progression of disease. We suggest that FNDC-5 down-regulation could be used as an indicator for T2DM worseness and development of other associated complications.

Sodium-glucose co-transporter 1 (SGLT1) differentially regulates gluconeogenesis and GLP-1 receptor (GLP-1R) expression in different diabetic rats: a preliminary validation of the hypothesis of "SGLT1 bridge" as an indication for "surgical diabetes".

BackgroundSodium-glucose co-transporter 1 (SGLT1) may play a synergistic role in gluconeogenesis (GNG) and glucagon-like peptide-1 (GLP-1) expression. We proposed the hypothesis of a “SGLT1 bridge” as an indication for “surgical diabetes” that was preliminary validated in the present study.MethodsWe selected nonobese diabetic Goto-Kakizaki (GK) rats and Zuker diabetic fat (ZDF) rats to represent advanced and early diabetes, respectively. Based on glucose gavage with or without SGLT1 inhibitor phlorizin, the rats were divided into 4 groups: Gk-Glu, GK-P, ZDF-Glu, and ZDF-P. The expressions of SGLT1, GLP-1 receptor (GLP-1R), glucose-6 phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase-1 (Pck1) were determined by immunohistochemistry (IHC) or quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the effects of phlorizin were analyzed.ResultsGlucose tolerance was worse in GK rats and the homeostasis model assessment-insulin resistance (HOMA-IR) was higher in ZDF rats, indicating different pathophysiological conditions between the different diabetic rats. GK rats showed higher activity of duodenal SGLT1 (P=0.022) and jejunal SGLT1 mRNA expression (P=0.000) and lower SGLT1 mRNA expression in the liver (P=0.000) and pancreas (P=0.000). Phlorizin effectively inhibited the activity of duodenal SGLT1 in both GK rats (P=0.000) and ZDF rats (P=0.000). In ZDF rats, the expression of GLP-1R mRNA was downregulated in the jejunum (P=0.001) and upregulated in the pancreas (P=0.021) by phlorizin, but there were no regulatory effects on GLP-1R mRNA in the jejunum and pancreas of GK rats. As for the regulatory effects on GNG, phlorizin upregulated Pck1 mRNA in the duodenum (P=0.000) and the jejunum (P=0.038), whereas it downregulated hepatic G6Pase mRNA in ZDF rats (P=0.005) and Pck1 mRNA expression in GK rats (P=0.001), suggesting that SGLT1 inhibitor may have upregulated intestinal GNG in ZDF rats and downregulated hepatic GNG in both ZDF and GK rats.ConclusionsSGLT1 showed synergistic regulatory effects on the entero-insular axis (EIA) and the gut-brain-liver axis (GBLA), preliminarily validating the hypothesis of a “SGLT1 bridge”. The distinct expression of SGLT1 and its differentially regulatory effects on diabetic rats with different pathophysiological conditions may provide probable potential indications involved in the “Surgical Diabetes” that is supposed as the inclusion for diabetic surgery.

Associations between the C3orf20 rs12496846 Polymorphism and Both Postoperative Analgesia after Orthognathic and Abdominal Surgeries and C3orf20 Gene Expression in the Brain.

Considerable individual differences are widely observed in the sensitivity to opioid analgesics. We focused on rs12496846, rs698705, and rs10052295 single-nucleotide polymorphisms (SNPs) in the C3orf20, SLC8A2, and CTNND2 gene regions that we previously identified as possibly associated with postoperative analgesia after orthognathic surgery. We investigated associations between these SNPs and postoperative analgesia in 112 patients who underwent major open abdominal surgery in hospitals and were treated with analgesics, including opioids, after surgery. Total genomic DNA was extracted from peripheral blood or oral mucosa samples for genotyping each SNP. Effects of these potent SNPs on gene expression in the brain were also investigated in samples that were provided by the Stanley Foundation Brain Bank. In the association studies, carriers of the G allele of the rs12496846 SNP in the C3orf20 gene region were significantly associated with greater 24 h postoperative analgesic requirements among the three SNPs that were investigated (p = 0.0015), which corroborated a previous study of orthognathic patients (p < 0.0001). In the gene expression analysis, carriers of the G allele of the rs12496846 SNP were significantly associated with lower mRNA expression of the C3orf20 gene (p < 0.0001). These results indicate that this SNP could serve as a marker that predicts analgesic requirements.

Loss of ARID1A expression is associated with systemic inflammation markers and has important prognostic significance in gastric cancer.

The tumor suppressor gene AT-rich interactive domain 1A (ARID1A) and systemic inflammatory response (SIR) have been reported to be related to the sensitivity to immunotherapy. This study intended to explore the relationship between ARID1A expression and SIR, and to further elucidate the prognostic value of ARID1A expression in gastric cancer (GC). The mRNA and protein expression of ARID1A were detected in 272 formalin-fixed paraffin-embedded (FFPE) tumor tissues. The data of nine systemic inflammation markers were collected 1 week before gastrectomy. Univariate and multivariate COX analysis were used to screen out independent predictors of GC. Negative expression of ARID1A protein was related to GC with deficient mismatch repair (dMMR) (p = 0.033), positive programmed cell death-ligand 1 (PD-L1) (p = 0.005) and lower albumin level (p = 0.0064). Low expression of ARID1A mRNA was common in GC with abnormal E-cadherin (p = 0.020) and a higher platelet/lymphocyte ratio (PLR) (p = 0.0391). Multivariate COX analysis showed that the expression of ARID1A protein (p = 0.023), age (p = 0.004), T stage (p = 0.009) and N stage (p = 0.009) were independent predictors of GC. The nomogram established by independent predictors can accurately evaluate the survival risk of patients with GC. The loss of ARID1A protein expression was associated with the dMMR subtype and high expression of PD-L1 in GC. Negative ARID1A protein and low expression of mRNA were associated with aberrant systemic inflammatory markers. The expression of ARID1A protein had important prognostic significance in GC.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Genetic association of IL17 and the importance of ABO blood group antigens in saliva to COVID-19

The outbreak of COVID-19 caused by infection with SARS-CoV-2 virus has become a worldwide pandemic, and the number of patients presenting with respiratory failure is rapidly increasing in Japan. An international meta-analysis has been conducted to identify genetic factors associated with the onset and severity of COVID-19, but these factors have yet to be fully clarified. Here, we carried out genomic analysis based on a genome-wide association study (GWAS) in Japanese COVID-19 patients to determine whether genetic factors reported to be associated with the onset or severity of COVID-19 in the international meta-GWAS are replicated in the Japanese population, and whether new genetic factors exist. Although no significant genome-wide association was detected in the Japanese GWAS, an integrated analysis with the international meta-GWAS identified for the first time the involvement of the IL17A/IL17F gene in the severity of COVID-19. Among nine genes reported in the international meta-GWAS as genes involved in the onset of COVID-19, the association of FOXP4-AS1, ABO, and IFNAR2 genes was replicated in the Japanese population. Moreover, combined analysis of ABO and FUT2 genotypes revealed that the presence of oral AB antigens was significantly associated with the onset of COVID-19. FOXP4-AS1 and IFNAR2 were also significantly associated in the integrated analysis of the Japanese GWAS and international meta-GWAS when compared with severe COVID-19 cases and the general population. This made it clear that these two genes were also involved in not only the onset but also the severity of COVID-19. In particular, FOXP4-AS1 was not found to be associated with the severity of COVID-19 in the international meta-GWAS, but an integrated analysis with the Japanese GWAS revealed an association with severity. Individuals with the SNP risk allele found between IL17A and IL17F had significantly lower mRNA expression levels of IL17F, suggesting that activation of the innate immune response by IL17F may play an important role in the severity of SARS-CoV-2 infection.

Expression levels of sonic hedgehog pathway genes and their targets are upregulated in early clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU-SKI43, cyclopamine and GLI-antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786-O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription-quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma-associated zinc finger protein (GLI)1-3, BCL2 apoptosis regulator (BCL2), MYC proto-oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786-O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU-SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU-SKI43 appear to be potential anti-renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.

Effects of dietary Glycyrrhiza polysaccharide on growth performance, blood parameters and immunity in weaned piglets.

The purpose of this study was to evaluate the effects of dietary Glycyrrhiza polysaccharide (GCP) on growth performance, blood parameters and immunity in weaned piglets. A total of 240 (10.33 ± 0.62 kg body weight) 35-day-old (Duroc × Landrace × White) weaned piglets were randomly assigned to four dietary treatments, with six replicate pens per treatment and 10 piglets per pen (five males and five females). The dietary treatments continued for 21 days and comprised a basal diet supplemented with 0 (control group), 500, 1000 and 2000 mg/kg GCP. The results showed that the inclusion of 1000 and 2000 mg/kg GCP increased the average daily gain and decreased the feed conversion rate compared with the control group (p < 0.05). The piglets treated with 500 and 1000 mg/kg GCP had a lower diarrhoeal incidence than the control group (p < 0.05). Moreover, supplementation with 1000 mg/kg GCP increased the counts of white blood cells, neutrophils, red blood cells, and platelets, and elevated alkaline phosphatase, total protein, globulin, glucose, triglyceride, immunoglobulin A, immunoglobulin G, and total antioxidant capacity levels (p < 0.05), and decreased malondialdehyde content compare with the control group (p < 0.05). In addition, relative to the control group, piglets fed 500 and 1000 mg/kg GCP had significantly lower expression of interleukin-6 mRNA in spleen (p < 0.05). Our results indicate that dietary supplementation with GCP can improve growth performance, blood parameters and immunity in weaned piglets. Our study suggests that adding 1000 mg/kg GCP to the diet had the most beneficial effect.

Cytokine and LL-37 gene expression levels in Bartonella spp. seropositive and seronegative patients of a rheumatology clinic

PurposeThe variation in the immune response to Bartonella spp. infection in humans remains unclear. The present study compares the expression of selected interleukins, cytokines and cathelicidin (LL-37) in rheumatology clinic patients suffering from musculoskeletal symptoms with healthy blood donors. The patients had previously been tested for the presence of Bartonella henselae antibodies. MethodsGene expression of LL-37, interleukin (IL)-2, IL-4, IL-6, IL-12, interferon-(IFN)-γ, and tumor necrosis factor (TNF-α)-α was determined in blood samples using quantitative Polymerase Chain Reaction (qPCR). Statistical analysis was prepared with STATISTICA. ResultsStatistically significant differences in the mRNA levels of the tested cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-12; p<0.0001) were observed between the healthy controls and patients; however, no difference was observed for LL37 mRNA (p ​= ​0.1974). No significant differences in mRNA expression were observed between IgG in anti-Bartonella seropositive and seronegative individuals (p>0.05). The only significant differences between the Bartonella spp. DNA positive and negative patients, indicated by PCR, were observed for TNF-α and IL-12 mRNA (p ​= ​0.0045 and p ​= ​0.0255, respectively). ConclusionA broadly similar immune response to the tested cytokines was observed among the participants irrespective of anti-Bartonella spp. IgG seropositivity. However, the Bartonella DNA-positive participants demonstrated significantly lower expression of IL-12 and TNF-α mRNA; this may indicate that these bacteria have a suppressive influence on the immune system.

Local injection of botulinum toxin type A (BTX-A) prevents scarring esophageal stricture caused by electrocautery in rabbit models.

BackgroundBenign esophageal strictures are common in clinical practice. The commonly used methods for preventing benign esophageal strictures still have many shortcomings. In this study, we investigated the preventive effect and possible mechanism of endoscopic local injection of botulinum toxin type A (BTX-A) on scarring esophageal stricture caused by electrocautery in rabbit models, with an attempt to provide a theoretical basis for the clinical application of BTX-A in the prevention of benign esophageal stricture.MethodsAdult male New Zealand rabbits were randomly divided into 4 groups: cautery group (cauterized with 30 W electrocoagulation power without other intervention), saline group (injected with normal saline at 4 spots in the local esophagus after modeling), BTX-A I group (injected with 10 U of BTX-A after modeling), and BTX-A II group (injected with 20 U of BTX-A after modeling). Body weight was measured at postoperative weeks 1, 2, and 4. Esophagography was performed, and the internal diameter of the esophagus was measured. The esophageal tissues were harvested for hematoxylin and eosin (HE) staining and Masson staining. Type I, type III collagen levels and the mRNA expression of transforming growth factor β1 (TGF-β1) in esophageal tissues were detected.ResultsCompared with the cautery and saline groups, the BTX-A I and BTX-A II groups had significantly higher body weight, larger esophageal internal diameter, lower type I and type III collagen levels, and lower TGF-β1 mRNA expression levels in esophageal tissues at postoperative week 4. Comparisons between the BTX-A I and BTX-A II groups showed no significant differences in terms of body weight, esophageal internal diameter, and type I collagen level at postoperative week 4. However, the BTX-A II group had a significantly lower type III collagen level and TGF-β1 mRNA expression level than the BTX-A I group.ConclusionsLocal injection of BTX-A can alleviate esophageal stricture after electrocautery and has a preventive effect on benign esophageal stricture caused by electrocautery in rabbits. The mechanism may be that BTX-A down-regulates the expression of TGF-β1 in the esophageal tissue at the burn site and reduces the deposition of collagen.

PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas

The similarities between sporadic basal-like breast cancer (BLBC) and BRCA1-mutated breast tumours raise the possibility that deregulation of the same pathway may underlie these tumour types. The aim of this study was to determine if PTEN aberrations are characteristic of both BRCA1 tumours and sporadic TN breast carcinomas with low BRCA1 expression, and can thus be used to identify sporadic tumours potentially sensitive to PARP inhibitors. Twelve BRCA1 tumours, 19 non-BRCA familial breast tumours and 71 unselected TN breast carcinomas were screened for PTEN mutations and assessed for PTEN expression and BRCA1 mRNA expression. Loss of PTEN expression was observed in 67% of BRCA1 tumours and more specifically in 89% of TN BRCA1 tumours highlighting the link between PTEN loss and BLBC in the context of germline BRCA1 mutations. Regarding unselected TN tumours, 56% showed PTEN expression loss and 35% displayed low BRCA1 mRNA expression. Unlike familial breast cancers with low BRCA1 mRNA expression, no significant correlation was observed between the loss of PTEN expression and low BRCA1 mRNA expression in this unselected TN tumours panel. Our data suggest that, unlike the germinal context, PTEN and BRCA1 alterations in sporadic TN breast tumours are independent events.

Genomic analysis of paired IDHwt glioblastomas reveals recurrent alterations of MPDZ at relapse after radiotherapy and chemotherapy

PurposeWe aimed to identify genomic drivers of glioblastoma inevitable recurrence. MethodsTen pairs of initial and recurrent frozen IDHwt glioblastoma samples were screened by CGH Array. Next Generation Sequencing (NGS) was then performed on an enriched cohort of 19 pairs. MPDZ alterations were analyzed using TCGA dataset. ResultsNineteen IDHwt glioblastoma patients were included. Median age was 54.5 y/o (37.2–72.8). Using CGH array, unsupervised analysis aggregated the cohort by paired initial and recurrent tumors. Only 44% of CGH Array alterations were conserved at recurrence (amplifications: 55%; deletions: 30%). Two regions (including FPR1, 2 and 3) were lost at relapse: 19q13.33 and 19q13.41. MPDZ and 25 other genes were altered in ≥20% of recurrent tumors. NGS analysis of 29 candidate genes revealed 4 genes with pathogenic mutations: (FPR2, REL, TYRP1 and MPDZ). MPDZ (Multiple PDZ Domain Crumbs Cell Polarity Complex Component) was altered by two pathogenic mutations occurring at relapse. Using TCGA dataset we observed that a lower MPDZ mRNA expression was associated with IDHwt (p < 0.001) and grade IV (p < 0.001) gliomas. Finally, a low mRNA MPDZ expression was significantly correlated to poor overall survival in both IDHwt and IDH mutated gliomas, reinforcing the potential pejorative impact of MPDZ loss. ConclusionOur results suggest that MPDZ is more frequently altered at relapse after radio-chemotherapy in glioblastoma IDHwt patients, suggesting that MPDZ impairment could contribute to the systematic resistance of these tumors opening new therapeutic perspectives.