Quantitative Analysis Of mRNA Expression Research Articles

Gastric Cancer Mesenchymal Stem Cells Trigger Endothelial Cell Functional Changes to Promote Cancer Progression.

Our previous studies have highlighted the pivotal role of gastric cancer mesenchymal stem cells (GCMSCs) in tumor initiation, progression, and metastasis. In parallel, it is well-documented that endothelial cells (ECs) undergo functional alterations in response to challenging tumor microenvironment. This study aims to elucidate whether functional changes in ECs might be induced by GCMSCs and thus influence cancer progression. Cell proliferation was assessed through CCK-8 and colony formation assays, while cell migration and invasion capabilities were evaluated by wound-healing and Transwell assays. Immunohistochemistry was employed to examine protein distribution and expression levels. Additionally, quantitative analysis of protein and mRNA expression was carried out through Western blotting and qRT-PCR respectively, with gene knockdown achieved using siRNA. Our findings revealed that GCMSCs effectively stimulate cell proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs), both in vitro and in vivo. GCMSCs promote the migration and invasion of gastric cancer cells by inducing the expression of Slit2 in HUVECs. Notably, the inhibition of phosphorylated AKT partially mitigates the aforementioned effects. In conclusion, GCMSCs may exert regulatory control over Slit2 expression in ECs via the AKT signaling pathway, thereby inducing functional changes in ECs that promote tumor progression.

Intra-articular injection of ascorbic acid enhances microfracture-mediated cartilage repair

Previous studies have confirmed that ascorbic acid (AA) can promote cartilage repair and improve cartilage differentiation in bone marrow mesenchymal stem cells. However, the use of microfracture (MFX) combined with AA to repair cartilage damage has not been studied. This study established a rabbit animal model and treated cartilage injury with different concentrations of AA combined with MFX. Macroscopic observations, histological analysis, immunohistochemical analysis and reverse transcription quantitative polymerase chain reaction analysis of TGF-β, AKT/Nrf2, and VEGF mRNA expression were performed. The results showed that intra-articular injection of AA had a positive effect on cartilage repair mediated by microfractures. Moreover, 10 mg/ml AA was the most effective at promoting cartilage repair mediated by microfractures. Intra-articular injection of AA promoted the synthesis of type II collagen and the formation of glycosaminoglycans by downregulating the mRNA expression of TGF-β and VEGF. In summary, this study confirmed that AA could promote cartilage repair after MFX surgery.

Acute exercise in a hot environment increases heat shock protein 70 and peroxisome proliferator-activated receptor γ coactivator 1α mRNA in Thoroughbred horse skeletal muscle.

Heat acclimatization or acclimation training in horses is practiced to reduce physiological strain and improve exercise performance in the heat, which can involve metabolic improvement in skeletal muscle. However, there is limited information concerning the acute signaling responses of equine skeletal muscle after exercise in a hot environment. The purpose of this study was to investigate the hypothesis that exercise in hot conditions induces greater changes in heat shock proteins and mitochondrial-related signaling in equine skeletal muscle compared with exercise in cool conditions. Fifteen trained Thoroughbred horses [4.6 ± 0.4 (mean ± SE) years old; 503 ± 14 kg] were assigned to perform a treadmill exercise test in cool conditions [COOL; Wet Bulb Globe Temperature (WBGT), 12.5°C; n = 8] or hot conditions (HOT; WBGT, 29.5°C; n = 7) consisting of walking at 1.7 m/s for 1 min, trotting at 4 m/s for 5 min, and cantering at 7 m/s for 2 min and at 90% of VO2max for 2 min, followed by walking at 1.7 m/s for 20 min. Heart rate during exercise and plasma lactate concentration immediately after exercise were measured. Biopsy samples were obtained from the middle gluteal muscle before and at 4 h after exercise, and relative quantitative analysis of mRNA expression using real-time RT-PCR was performed. Data were analyzed with using mixed models. There were no significant differences between the two groups in peak heart rate (COOL, 213 ± 3 bpm; HOT, 214 ± 4 bpm; p = 0.782) and plasma lactate concentration (COOL, 13.1 ± 1.4 mmoL/L; HOT, 17.5 ± 1.7 mmoL/L; p = 0.060), while HSP-70 (COOL, 1.9-fold, p = 0.207; HOT, 2.4-fold, p = 0.045), PGC-1α (COOL, 3.8-fold, p = 0.424; HOT, 8.4-fold, p = 0.010), HIF-1α (COOL, 1.6-fold, p = 0.315; HOT, 2.2-fold, p = 0.018) and PDK4 (COOL, 7.6-fold, p = 0.412; HOT, 14.1-fold, p = 0.047) mRNA increased significantly only in HOT at 4 h after exercise. These data indicate that acute exercise in a hot environment facilitates protective response to heat stress (HSP-70), mitochondrial biogenesis (PGC-1α and HIF-1α) and fatty acid oxidation (PDK4).

Quantitative Spatial Analysis of Neuroligin-3 mRNA Expression in the Enteric Nervous System Reveals a Potential Role in Neuronal-Glial Synapses and Reduced Expression in Nlgn3R451C Mice.

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00–07, S-00–11, S-00–16 and S-00–23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

Estrogen Receptor-Beta2 (ERβ2)-Mutant p53-FOXM1 Axis: A Novel Driver of Proliferation, Chemoresistance, and Disease Progression in High Grade Serous Ovarian Cancer (HGSOC).

Simple SummaryHigh grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer without effective therapeutic options. The high prevalence of mutations (~96%) in tumor suppressor p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERβ) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unclear. The aim of this study was to analyze the crosstalk between ERβ and mutant p53 and its impact on the pro-tumorigenic processes in HGSOC. Using the HGSOC cell line models and patient tumor tissue specimens, we demonstrated functional interaction between the ERβ2 isoform and mutant p53 and their ability to co-dependently increase FOXM1 gene transcription, decrease cell death, increase cell proliferation, and mediate resistance to carboplatin treatment. Furthermore, high levels of ERβ2 as well as FOXM1 correlated with worse patient survival. Collectively, our data suggest that the ERβ2-mutant p53-FOXM1 axis could be a novel therapeutic target for HGSOC.High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of epithelial ovarian cancer. Prevalence (~96%) of mutant p53 is a hallmark of HGSOC. Estrogen receptor-beta (ERβ) has been reported to be another important player in HGSOC, although the pro-versus anti-tumorigenic role of its different isoforms remains unsettled. However, whether there is functional interaction between ERβ and mutant p53 in HGSOC is unknown. ERβ1 and ERβ2 mRNA and protein analysis in HGSOC cell lines demonstrated that ERβ2 is the predominant isoform in HGSOC. Specificity of ERβ2 antibody was ascertained using cells depleted of ERβ2 and ERβ1 separately with isoform-specific siRNAs. ERβ2-mutant p53 interaction in cell lines was confirmed by co-immunoprecipitation and in situ proximity ligation assay (PLA). Expression levels of ERβ2, ERα, p53, and FOXM1 proteins and ERβ2-mutant p53 interaction in patient tumors were determined by immunohistochemistry (IHC) and PLA, respectively. ERβ2 levels correlate positively with FOXM1 levels and negatively with progression-free survival (PFS) and overall survival (OS). Quantitative chromatin immunoprecipitation (qChIP) and mRNA expression analysis revealed that ERβ2 and mutant p53 co-dependently regulated FOXM1 gene transcription. The combination of ERβ2-specific siRNA and PRIMA-1MET that converts mutant p53 to wild type conformation increased apoptosis. Our work provides the first evidence for a novel ERβ2-mutant p53-FOXM1 axis that can be exploited for new therapeutic strategies against HGSOC.

The therapeutic effect of Jiawei Danshen Decoction on myocardial ischemia-reperfusion injury by inhibiting H2S-mediated autophagy signaling pathway

ObjectiveTo investigate the protective effects of Jiawei Danshen Decoction (加味丹参饮, JWDSD) on myocardial ischemia-reperfusion injury (MIRI) via the regulation of serum Hydrogen sulfide (H2S) and cardiac Beclin1, light Chain 3A/B (LC3A/B), p62, and autophagy protein5 (ATG5). MethodsSeventy specific pathogen free (SPF) Sprague-Dawley (SD) rats were randomly assigned to seven groups (n = 10 in each group), including normal control, sham operation, MIRI model (model), ischemic preconditioning, NaHS, JWDSD, and JWDSD +CSE inhibitor (JWDSD + PPG) groups, and orally administered the indicated drugs for 14 d. Two hours after the last administration, the left anterior decreased branch of the coronary artery of each rat in model, NaHS, JWDSD, and JWDSD + PPG groups was ligated for 30 min and subsequently reperfused for 90 min to establish the MIRI model, and the rats in the sham operation group were only exposed to the thorax after surgery without coronary ligation. Blood samples were collected to detect H2S levels using an enzyme-linked immunosorbent assay (ELISA). Heart tissues were harvested for histopathological and immunohistochemical examination and quantitative reverse transcription polymerase chain reaction analysis of Beclin1 and ATG5 mRNA expression and Western blot analysis of Beclin1, LC3A/B, and p62 protein expression. Results(1) The serum H2S content in model group rats was significantly reduced (P < 0.01), JWDSD significantly increased the serum H2S content of model group rats (P < 0.01), and the CSE inhibitor (PPG) significantly reduced H2S levels in the JWDSD group rats (P < 0.01). (2) Compared with the normal control group, the myocardial tissue necrosis and cell destruction occurred in the MIRI model group, and JWDSD could alleviate the myocardial tissue necrosis of model rats, but the ameliorative effect of JWDSD could be reversed by PPG. (3) Beclin1, LC3A/B, and p62 expression levels in the heart tissues of the model group were significantly increased (P < 0.001), whereas decreased by JWDSD (P < 0.05, P < 0.01, and P < 0.001, respectively), and the inhibitory effects of JWDSD on Beclin1, LC3A/B, and p62 expression were partially reversed by PPG (P < 0.01, P < 0.05, and P < 0.01, respectively). (4) The expression levels of autophagy-related genes Beclin1 and ATG5 were significantly increased in the model group (P < 0.001). JWDSD clearly downregulated the expression levels of Beclin1 and ATG5 (P < 0.05 and P < 0.001, respectively), which were reversed by PPG (P < 0.001). ConclusionOur experimental data show that JWDSD can exhibit an anti-MIRI role by increasing endogenous H2S generation, and downregulating the expression of Beclin1, LC3A/B, p62 and ATG5, which are related to inhibiting autophagy signaling.

Calcineurin Gamma Catalytic Subunit PPP3CC Inhibition by miR-200c-3p Affects Apoptosis in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) outpaces all the other forms of the female reproductive system malignancies. MicroRNAs have emerged as promising predictive biomarkers to therapeutic treatments as their expression might characterize the tumor stage or grade. In EOC, miR-200c is considered a master regulator of oncogenes or tumor suppressors. To investigate novel miR-200c-3p target genes involved in EOC tumorigenesis, we evaluated the association between this miRNA and the mRNA expression of several potential target genes by RNA-seq data of both 46 EOC cell lines from Cancer Cell line Encyclopedia (CCLE) and 456 EOC patient bio-specimens from The Cancer Genome Atlas (TCGA). Both analyses showed a significant anticorrelation between miR-200c-3p and the protein phosphatase 3 catalytic subunit γ of calcineurin (PPP3CC) levels involved in the apoptosis pathway. Quantitative mRNA expression analysis in patient biopsies confirmed the inverse correlation between miR-200c-3p and PPP3CC levels. In vitro regulation of PPP3CC expression through miR-200c-3p and RNA interference technology led to a concomitant modulation of BCL2- and p-AKT-related pathways, suggesting the tumor suppressive role of PPP3CC in EOC. Our results suggest that inhibition of high expression of miR-200c-3p in EOC might lead to overexpression of the tumor suppressor PPP3CC and subsequent induction of apoptosis in EOC patients.

Empty spiracles homeobox genes EMX1 and EMX2 regulate WNT pathway activation in sarcomagenesis

BackgroundSarcomas are a very heterogeneous group of tumors with intrinsic developmental programs derived from the cell of origin. This implies a functional hierarchy inside tumors governed by sarcoma stem cells. Therefore, genetic and/or epigenetic changes profoundly affect the biology of sarcoma tumor stem cells. EMX genes are proposed to be transcription factors that are involved in the sarcomagenesis process, regardless of the neural or mesodermal embryological sarcoma origin. It has been shown that EMX1 or EMX2 overexpression reduces tumorigenic properties, while reducing the levels of these genes enhances these properties. Furthermore, it has been shown that EMX genes decrease the expression of stem cell regulatory genes and the stem cell phenotype. Taken together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-remodeling populations or sarcoma stem cells, acting as tumor suppressors in sarcoma.MethodsBioinformatic analysis, quantitative mRNA and protein expression analysis, cell models of sarcoma by ectopic expression of EMX genes. By cell biology methods we measured tumorigenesis and populations enriched on stem cell phenotypes, either in vitro or in vivo.ResultsIn this work, we showed that the canonical Wnt pathway is one of the mechanisms that explains the relationships of EMX1/EMX2 and stem cell genes in sarcoma. The Wnt-EMX1/EMX2 relationship was validated in silico with sarcoma patient datasets, in vitro in primary derived sarcoma cell lines, and in vivo. EMX expression was found to negatively regulate the Wnt pathway. In addition, the constitutive activation of the Wnt pathway revers to a more aggressive phenotype with stem cell properties, and stemness gene transcription increased even in the presence of EMX1 and/or EMX2 overexpression, establishing the relationship among the Wnt pathway, stem cell genes and the EMX transcription factors.ConclusionsOur data showed that Empty Spiracles Homeobox Genes EMX1 and EMX2 represses WNT signalling and activation of WNT pathway bypass EMX-dependent stemness repression and induces sarcomagenesis. These results also suggest the relevance of the Wnt/b-catenin/stemness axis as a therapeutic target in sarcoma.

Mechanistic Actions between Garcinia atroviridis Essential Oil and 2 Deoxy-d-glucose in Cultured PANC-1 Human Pancreatic Cancer Cells.

Pancreatic cancer is an aggressive disease that progresses in a relatively symptom-free manner; thus, is difficult to detect and treat. Essential oil is reported to exhibit pharmacological properties, besides its common and well-known function as aromatherapy. Therefore, this study herein aimed to investigate the anti-proliferative effect of essential oil extracted from leaves of Garcinia atroviridis (EO-L) against PANC-1 human pancreatic cancer cell line. The cell growth inhibitory concentration at 50% (IC50) and selective index (SI) values of EO-L analyses were determined as 78 µg/mL and 1.23, respectively. Combination index (CI) analysis revealed moderate synergism (CI values of 0.36 to 0.75) between EO-L and 2 deoxy-d-glucose (2-DG) treatments. The treatments of PANC-1 cells with EO-L, 2-DG and EOL+2DG showed evidence of depolarization of mitochondrial membrane potential, cell growth arrest and apoptosis. The molecular mechanism causing the anti-proliferative effect between EO-L and 2-DG is potentially through pronounced up-regulation of P53 (4.40-fold), HIF1α (1.92-fold), HK2 (2.88-fold) and down-regulation of CYP3A5 (0.11-fold), as supported by quantitative mRNA expression analysis. Collectively, the current data suggest that the combination of two anti-proliferative agents, EO-L and 2-DG, can potentially be explored as therapeutic treatments and as potentiating agents to conventional therapy against human pancreatic cancer.

Altered Expression of Glucocorticoid Receptor and Neuron-Specific Enolase mRNA in Peripheral Blood in First-Episode Schizophrenia and Chronic Schizophrenia.

IntroductionIt is well-known that altered hypothalamus–pituitary–adrenal (HPA) axis process has an important role in the neurodegenerative process in schizophrenia (SZ). However, this neurodegenerative mechanism has not been clarified in SZ. Therefore, the main purpose of this study was to determine HPA axis damage in the first-episode, unmedicated schizophrenia (FES) patients and chronic schizophrenia (CSZ) patients in comparison with healthy controls (HC) by means of quantitative analysis of the peripheral blood mRNA expression of glucocorticoid receptor (GR), GR transcripts containing exons 1B (GR-1B), and neuron specific enolase (NSE) genes and serum cortisol and NSE, a specific serum marker for neuronal damage.MethodsIn the present study, 43 FES patients, 39 CSZ, and 47 HC were included. The peripheral blood mRNA expressions for GR, GR-1B, and NSE genes were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Serum cortisol and NSE were analyzed by electrochemiluminescence immunoassay technique.ResultsLevels of GR mRNA were significantly lower in FES and CSZ than that in HC. The expression of GR-1B mRNA was significantly decreased in CSZ when compared with that in FES. Levels of NSE mRNA were significantly lower in CSZ than that in FES patients or HC patients. CSZ patients showed significantly lower cortisol concentrations than FES and HC patients. FES patients showed significantly higher NSE concentrations than CSZ and HC.ConclusionOur findings support that there is disrupted HPA axis system in the SZ and suggest that CSZ patients suffer a greater HPA axis damage than FES patients. Our research implicated underlying GR mRNA dysregulation in SZ and the potential importance of the functional GR-1B transcription in CSZ.

A rare genomic duplication in 2p14 underlies autosomal dominant hearing loss DFNA58.

Here we define a ~200Kb genomic duplication in 2p14 as the genetic signature that segregates with postlingual progressive sensorineural autosomal dominant hearing loss (HL) in 20 affected individuals from the DFNA58 family, first reported in 2009. The duplication includes two entire genes, PLEK and CNRIP1, and the first exon of PPP3R1 (protein coding), in addition to four uncharacterized long non-coding (lnc) RNA genes and part of a novel protein-coding gene. Quantitative analysis of mRNA expression in blood samples revealed selective overexpression of CNRIP1 and of two lncRNA genes (LOC107985892 and LOC102724389) in all affected members tested, but not in unaffected ones. Qualitative analysis of mRNA expression identified also fusion transcripts involving parts of PPP3R1, CNRIP1 and an intergenic region between PLEK and CNRIP1, in the blood of all carriers of the duplication, but were heterogeneous in nature. By in situ hybridization and immunofluorescence, we showed that Cnrip1, Plek and Ppp3r1 genes are all expressed in the adult mouse cochlea including the spiral ganglion neurons, suggesting changes in expression levels of these genes in the hearing organ could underlie the DFNA58 form of deafness. Our study highlights the value of studying rare genomic events leading to HL, such as copy number variations. Further studies will be required to determine which of these genes, either coding proteins or non-coding RNAs, is or are responsible for DFNA58 HL.

Quantitative analysis of HER2 mRNA expression by RNA in situ hybridization in canine mammary gland tumors: Comparison with immunohistochemistry analysis.

Spontaneously occurring canine mammary gland tumors share many features with human breast cancer, including biological behavior and histologic features. Compared to transgenic murine model, canine models have advantages including naturally occurring models of human diseases and cancer. In humans, breast cancer is divided into molecular subtypes based on ER, PR, and HER2 expression. In contrast with humans, few studies have evaluated these subtypes in canine mammary gland tumors, including expression of HER2. HER2 expression in canine mammary tissues has been further complicated by controversy regarding the antibody’s specificity. This study aimed to investigate c-erbB2 mRNA expression in retrospective formalin-fixed paraffin embedded samples, using RNA in situ hybridization with a novel quantitative assay and to compare this method with immunohistochemistry. Using 48 canine mammary tumor samples and 14 non-neoplastic canine mammary tissues, RNA in situ hybridization was performed with RNAscope® using a canine-specific target gene probe (ERBB2), and quantitative measurement was performed using the housekeeping gene (POLR2A) to calculate the target gene/housekeeping gene ratio. The ratio of ERBB2/POLR2A was quantified using open-source image analysis programs and compared with the immunohistochemistry results. A significant correlation was observed between the HER2 immunohistochemistry score and ERBB2/POLR2A RNA in situ hybridization (P < 0.001). When the HER2 immunohistochemistry score was 3+, significantly higher expression of HER2 mRNA was observed by RNA in situ hybridization. Interestingly, HER2 mRNA was also observed in non-neoplastic mammary tissues by RNA in situ hybridization. This assay potentially facilitates the reliable quantification of mRNA expression levels in retrospective formalin-fixed paraffin-embedded samples. Further studies are required to elucidate the role of HER2 in canine mammary gland tumors and to implement clinical trials in dogs.

Neurally adjusted ventilatory assist mitigates ventilator-induced diaphragm injury in rabbits

BackgroundVentilator-induced diaphragmatic dysfunction is a serious complication associated with higher ICU mortality, prolonged mechanical ventilation, and unsuccessful withdrawal from mechanical ventilation. Although neurally adjusted ventilatory assist (NAVA) could be associated with lower patient-ventilator asynchrony compared with conventional ventilation, its effects on diaphragmatic dysfunction have not yet been well elucidated.MethodsTwenty Japanese white rabbits were randomly divided into four groups, (1) no ventilation, (2) controlled mechanical ventilation (CMV) with continuous neuromuscular blockade, (3) NAVA, and (4) pressure support ventilation (PSV). Ventilated rabbits had lung injury induced, and mechanical ventilation was continued for 12 h. Respiratory waveforms were continuously recorded, and the asynchronous events measured. Subsequently, the animals were euthanized, and diaphragm and lung tissue were removed, and stained with Hematoxylin-Eosin to evaluate the extent of lung injury. The myofiber cross-sectional area of the diaphragm was evaluated under the adenosine triphosphatase staining, sarcomere disruptions by electron microscopy, apoptotic cell numbers by the TUNEL method, and quantitative analysis of Caspase-3 mRNA expression by real-time polymerase chain reaction.ResultsPhysiological index, respiratory parameters, and histologic lung injury were not significantly different among the CMV, NAVA, and PSV. NAVA had lower asynchronous events than PSV (median [interquartile range], NAVA, 1.1 [0–2.2], PSV, 6.8 [3.8–10.0], p = 0.023). No differences were seen in the cross-sectional areas of myofibers between NAVA and PSV, but those of Type 1, 2A, and 2B fibers were lower in CMV compared with NAVA. The area fraction of sarcomere disruptions was lower in NAVA than PSV (NAVA vs PSV; 1.6 [1.5–2.8] vs 3.6 [2.7–4.3], p < 0.001). The proportion of apoptotic cells was lower in NAVA group than in PSV (NAVA vs PSV; 3.5 [2.5–6.4] vs 12.1 [8.9–18.1], p < 0.001). There was a tendency in the decreased expression levels of Caspase-3 mRNA in NAVA groups. Asynchrony Index was a mediator in the relationship between NAVA and sarcomere disruptions.ConclusionsPreservation of spontaneous breathing using either PSV or NAVA can preserve the cross sectional area of the diaphragm to prevent atrophy. However, NAVA may be superior to PSV in preventing sarcomere injury and apoptosis of myofibrotic cells of the diaphragm, and this effect may be mediated by patient-ventilator asynchrony.

Cancer-Specific Loss of Urocortin3 in Human Renal Cancer.

The corticotropin-releasing hormone (CRH) system, its receptors corticotropin-releasing hormone receptor 1 (CRHR1) and 2 (CRHR2), and its corresponding binding protein corticotropin-releasing hormone-binding protein (CRHBP) as well as the urocortin proteins-structural homologues to CRH, which are included in this peptide family-have become interesting oncological targets recently. Carcinogenesis of various human tumors has been reported with an altered presence of members of this system. The aim of the present study was to examine the role of urocortin3 (UCN3) in renal cell carcinoma (RCC). Therefore, tumoral tissues of 106 patients with RCC and available corresponding normal tissues were analyzed using qPCR for quantitative mRNA expression analysis. Tissue localization and protein signals of UCN3 in normal and tumoral renal specimens were evaluated using western blot and immunohistochemistry. In addition, correlation studies of UCN3 mRNA expression with clinicopathological parameters of patients with RCC and different histological subtypes were evaluated. UCN3 mRNA was significantly downregulated in nearly all tumoral tissues (p = 7.92 × 10-13). The same effect was observed at protein level using immunohistochemistry. Level of UCN3 mRNA expression was not directly correlated with clinicopathological parameters. We report for the first time the significant downregulation of UCN3 in RCC. These results demonstrate a possible involvement of the CRH system and its significance in carcinogenesis of RCC.

Evaluation of ERAP1 gene single nucleotide polymorphisms in immunomodulation of pro-inflammatory and anti-inflammatory cytokines profile in ankylosing spondylitis

BackgroundAnkylosing spondylitis (AS) is a prototype of chronic inflammatory arthritis termed seronegative spondyloarthropathies that typically affects the joints. Among the non-Human leukocyte antigen (HLA) loci, the strongest association has been observed with Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene single nucleotide polymorphisms (SNPs). Moreover, the effect of ERAP1 gene SNPs on the pro-inflammatory and anti-inflammatory cytokines in AS disease has still been poorly elucidated. In this study, we aimed to determine the association of ERAP1 gene SNPs (rs30187 and rs2287987) with AS risk as well as their effect on the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, with emphasis on the immunoregulation of the IL-17/IL-23 pathway, in an Iranian population. MethodsWe performed Single specific primer (SSP)-PCR for genotyping of 160 AS patients and 160 healthy controls. After isolation of peripheral blood mononuclear cells (PBMCs), total RNA of PBMCs was isolated, complementary DNA (cDNA) was synthesized, and quantitative analyses of mRNA expression of cytokines were performed by Real-time PCR for 40 HLA-B27 positive AS patients and 40 healthy individuals as controls. ResultsIt was seen that T allele of rs30187 (OR = 1.54, 95% CI = 1.07–2.22, P = 0.017) and C allele of rs2287987 (OR 1.50, 95% CI 1.05–2.14, P = 0.024) were associated with the risk of AS. Both of these alleles were associated more strongly in the HLA-B27 positive AS patients. There was a significant overexpression of mRNAs of pro-inflammatory (IL-17A, IL-17F, IL-23, TNF-α and IFN-γ), while downregulation of anti-inflammatory cytokines (IL-10 and TGF-β) in PBMCs from 40 HLA-B27 positive AS patients in comparison to controls. AS patients with rs30187 SNP TT genotype expressed mRNA of IL-17A, IL-17F, and IL-23 significantly higher than patents with CT and CC genotypes for this SNP. ConclusionsThis study represented the association of ERAP1 gene rs30187 and rs2287987 polymorphism with the risk of AS. Additionally, it appears that rs30187 polymorphism may be involved in the immunomodulation of the IL-17/IL-23 pathway in the AS disease.

Metformin ameliorates stress-induced depression-like behaviors via enhancing the expression of BDNF by activating AMPK/CREB-mediated histone acetylation

BackgroundMetformin, a first-line antiglycemic drug, has been reported to have anti-depressant effects in patients with type 2 diabetes; however, its exact role and underlying mechanism still need to be investigated. MethodC57BL/6J mice were subjected to the Chronic social defeat stress (SDS) and drug administration (Control + Vehicle, SDS + Vehicle, SDS + MET (200 mg kg−1), SDS + FLUOX (3 mg kg−1), SDS + MET + FLUOX). And the depression phenotypes were evaluated by the sucrose preference test, social interaction, tail suspension test and forced swimming test. The potential mechanisms underlying the effects of metformin on depression was discussed by using Chromatin immunoprecipitation, Quantitative real-time PCR mRNA expression analysis and Western blot in vivo and in primary cultured hippocampal neurons. ResultThe metformin treatment counteracted the development of depression-like behaviors in mice suffering SDS when administered alone and enhanced the anti-depressant effect of fluoxetine when combined with fluoxetine. Further RNA sequencing analysis revealed that metformin treatment prevented the transcriptional changes in the medial prefrontal cortex (mPFC) of the animals and Golgi staining indicated favorable morphological changes in the neurite plasticity of CA1 pyramidal neurons, which approximated to those found in unstressed mice. At a molecular level, metformin significantly upregulated the expression of the brain-derived neurotrophic factor (BDNF) by increasing the histone acetylation along with the BDNF promoter, which was attributed to the activation of AMP-activated protein kinase (AMPK) and cAMP-response element binding protein (CREB). ConclusionOur findings suggest that metformin can produce antidepressant effects, which provides empirical insights into the clinical value of metformin in the prevention and therapy of depression.

Dysregulation of the histone demethylase KDM6B in alcohol dependence is associated with epigenetic regulation of inflammatory signaling pathways.

Epigenetic enzymes oversee long‐term changes in gene expression by integrating genetic and environmental cues. While there are hundreds of enzymes that control histone and DNA modifications, their potential roles in substance abuse and alcohol dependence remain underexplored. A few recent studies have suggested that epigenetic processes could underlie transcriptomic and behavioral hallmarks of alcohol addiction. In the present study, we sought to identify epigenetic enzymes in the brain that are dysregulated during protracted abstinence as a consequence of chronic and intermittent alcohol exposure. Through quantitative mRNA expression analysis of over 100 epigenetic enzymes, we identified 11 that are significantly altered in alcohol‐dependent rats compared with controls. Follow‐up studies of one of these enzymes, the histone demethylase KDM6B, showed that this enzyme exhibits region‐specific dysregulation in the prefrontal cortex and nucleus accumbens of alcohol‐dependent rats. KDM6B was also upregulated in the human alcoholic brain. Upregulation of KDM6B protein in alcohol‐dependent rats was accompanied by a decrease of trimethylation levels at histone H3, lysine 27 (H3K27me3), consistent with the known demethylase specificity of KDM6B. Subsequent epigenetic (chromatin immunoprecipitation [ChIP]–sequencing) analysis showed that alcohol‐induced changes in H3K27me3 were significantly enriched at genes in the IL‐6 signaling pathway, consistent with the well‐characterized role of KDM6B in modulation of inflammatory responses. Knockdown of KDM6B in cultured microglial cells diminished IL‐6 induction in response to an inflammatory stimulus. Our findings implicate a novel KDM6B‐mediated epigenetic signaling pathway integrated with inflammatory signaling pathways that are known to underlie the development of alcohol addiction.

Physical, histological, endocrinological and steroidogenical evaluation of male cats postnatally exposed to sexual steroids

To test the hypothesis that postnatal sexual steroids induce an impairment of domestic male cat reproductive function, this study describes the physical, endocrine, steroidogenical and histological effects of a single, high dose of a postnatal sexual steroid in this species. Twenty male kittens were randomly assigned within the first 24 h of birth to: Testosterone enanthate 12.5 mg sc (TE; n = 8), medroxyprogesterone acetate 10 mg sc (MA; n = 6), or Placebo sc (PL; n = 6). The cats were followed until puberty when they were castrated. Kittens achieved puberty without age differences among groups (P > 0.05). Two MA cats presented abnormal testicular descent. Histological evaluation of the MA (P < 0.01), but not of TE testes revealed decreased diameter (P < 0.01) and epithelial height (P < 0.01) of the seminiferous tubules. Leydig cell nuclear area was also reduced in this group. Conversely, tubular/intertubular ratio was increased in TE animals (P < 0.01). Quantitative real-time PCR analysis of mRNA expression of testicular tissue revealed no significant differences among groups for StAR, CYP17A1 and androgen receptors. TE animals showed decreased CYP19A1 mRNA expression (P < 0.05). In the first 4 postnatal weeks, fecal testosterone (T) values were high, basal and intermediate in TE, MA and PL (P < 0.05), respectively. These differences progressively diminished and the three groups presented basal T concentrations from the 7th week on (P > 0.05). It was concluded that the postnatal progestagen initially suppressed the gonadal axis and caused an impairment of spermatogenesis and testicular descent at puberty. Androgen treatment caused downregulation of the final steroidogenic cascade.