RNA Expression Patterns Research Articles

Diverse repetitive element RNA expression defines epigenetic and immunologic features of colon cancer.

There is tremendous excitement for the potential of epigenetic therapies in cancer, but the ability to predict and monitor response to these drugs remains elusive. This is in part due to the inability to differentiate the direct cytotoxic and the immunomodulatory effects of these drugs. The DNA-hypomethylating agent 5-azacitidine (AZA) has shown these distinct effects in colon cancer and appears to be linked to the derepression of repeat RNAs. LINE and HERV are two of the largest classes of repeats in the genome, and despite many commonalities, we found that there is heterogeneity in behavior among repeat subtypes. Specifically, the LINE-1 and HERV-H subtypes detected by RNA sequencing and RNA in situ hybridization in colon cancers had distinct expression patterns, which suggested that these repeats are correlated to transcriptional programs marking different biological states. We found that low LINE-1 expression correlates with global DNA hypermethylation, wild-type TP53 status, and responsiveness to AZA. HERV-H repeats were not concordant with LINE-1 expression but were found to be linked with differences in FOXP3+ Treg tumor infiltrates. Together, distinct repeat RNA expression patterns define new molecular classifications of colon cancer and provide biomarkers that better distinguish cytotoxic from immunomodulatory effects by epigenetic drugs.

Global expression profile of tumor stem-like cells isolated from MMQ rat prolactinoma cell

BackgroundCancer stem cells (CSCs), which have been isolated from various malignancies, were closely correlated with the occurrence, progression, metastasis and recurrence of the malignant cancer. Little is known about the tumor stem-like cells (TSLCs) isolated from benign tumors. Here we want to explore the global expression profile of RNA of tumor stem-like cells isolated from MMQ rat prolactinoma cells.MethodsIn this study, total RNA was extracted from MMQ cells and MMQ tumor stem-like cells. RNA expression profiles were determined by Agilent Rat 8 × 60 K Microarray. Then we used the qRT-PCR to test the result of Microarray, and found VEGFA had a distinct pattern of expression in MMQ tumor stem-like cells. Then WB and ELISA were used to confirm the VEGFA protein level of tumor sphere cultured from both MMQ cell and human prolactinoma cell. Finally, CCK-8 was used to evaluate the reaction of MMQ tumor stem-like cells to small interfering RNAs intervention and bevacizumab treatment.ResultThe results of Microarray showed that 566 known RNA were over-expressed and 532 known RNA were low-expressed in the MMQ tumor stem-like cells. These genes were mainly involved in 15 different signaling pathways. In pathway in cancer and cell cycle, Bcl2, VEGFA, PTEN, Jun, Fos, APC2 were up-regulated and Ccna2, Cdc25a, Mcm3, Mcm6, Ccnb2, Mcm5, Cdk1, Gadd45a, Myc were down-regulated in the MMQ tumor stem-like cells. The expression of VEGFA were high in tumor spheres cultured from both MMQ cell and human prolactinomas. Down-regulation of VEGFA by small interfering RNAs partially decreased cell viability of MMQ tumor stem-like cells in vitro. Bevacizumab partially suppressed the proliferation of MMQ tumor stem-like cells.ConclusionsOur findings characterize the pattern of RNA expression of tumor stem-like cells isolated from MMQ cells. VEGFA may act as a potential therapeutic target for tumor stem-like cells of prolactinomas.

Mucosal Expression of Type 2 and Type 17 Immune Response Genes Distinguishes Ulcerative Colitis From Colon-Only Crohn’s Disease in Treatment-Naive Pediatric Patients

There is controversy regarding the role of the type 2 immune response in the pathogenesis of ulcerative colitis (UC)-few data are available from treatment-naive patients. We investigated whether genes associated with a type 2 immune response in the intestinal mucosa are up-regulated in treatment-naive pediatric patients with UC compared with patients with Crohn's disease (CD)-associated colitis or without inflammatory bowel disease (IBD), and whether expression levels are associated with clinical outcomes. We used a real-time reverse-transcription quantitative polymerase chain reaction array to analyze messenger RNA (mRNA) expression patterns in rectal mucosal samples from 138 treatment-naive pediatric patients with IBD and macroscopic rectal disease, as well as those from 49 children without IBD (controls), enrolled in a multicenter prospective observational study from 2008 to 2012. Results were validated in real-time reverse-transcription quantitative polymerase chain reaction analyses of rectal RNA from an independent cohort of 34 pediatric patients with IBD and macroscopic rectal disease and 17 controls from Cincinnati Children's Hospital Medical Center. We measured significant increases in mRNAs associated with a type 2 immune response (interleukin [IL]5 gene, IL13, and IL13RA2) and a type 17 immune response (IL17A and IL23) in mucosal samples from patients with UC compared with patients with colon-only CD. In a regression model, increased expression of IL5 and IL17A mRNAs distinguished patients with UC from patients with colon-only CD (P= .001; area under the receiver operating characteristic curve, 0.72). We identified a gene expression pattern in rectal tissues of patients with UC, characterized by detection of IL13 mRNA, that predicted clinical response to therapy after 6 months (odds ratio [OR], 6.469; 95% confidence interval [CI], 1.553-26.94), clinical response after 12 months (OR, 6.125; 95% CI, 1.330-28.22), and remission after 12 months (OR, 5.333; 95% CI, 1.132-25.12). In an analysis of rectal tissues from treatment-naive pediatric patients with IBD, we observed activation of a type 2 immune response during the early course of UC. We were able to distinguish patients with UC from those with colon-only CD based on increased mucosal expression of genes that mediate type 2 and type 17 immune responses. Increased expression at diagnosis of genes that mediate a type 2 immune response is associated with response to therapy and remission in pediatric patients withUC.

Transcriptomic signatures differentiate survival from fatal outcomes in humans infected with Ebola virus

BackgroundIn 2014, Western Africa experienced an unanticipated explosion of Ebola virus infections. What distinguishes fatal from non-fatal outcomes remains largely unknown, yet is key to optimising personalised treatment strategies. We used transcriptome data for peripheral blood taken from infected and convalescent recovering patients to identify early stage host factors that are associated with acute illness and those that differentiate patient survival from fatality.ResultsThe data demonstrate that individuals who succumbed to the disease show stronger upregulation of interferon signalling and acute phase responses compared to survivors during the acute phase of infection. Particularly notable is the strong upregulation of albumin and fibrinogen genes, which suggest significant liver pathology. Cell subtype prediction using messenger RNA expression patterns indicated that NK-cell populations increase in patients who survive infection. By selecting genes whose expression properties discriminated between fatal cases and survivors, we identify a small panel of responding genes that act as strong predictors of patient outcome, independent of viral load.ConclusionsTranscriptomic analysis of the host response to pathogen infection using blood samples taken during an outbreak situation can provide multiple levels of information on both disease state and mechanisms of pathogenesis. Host biomarkers were identified that provide high predictive value under conditions where other predictors, such as viral load, are poor prognostic indicators. The data suggested that rapid analysis of the host response to infection in an outbreak situation can provide valuable information to guide an understanding of disease outcome and mechanisms of disease.

Microarray analysis of circular RNA expression patterns in polarized macrophages

Circular RNAs (circRNAs) are generated from diverse genomic locations and are a new player in the regulation of post-transcriptional gene expression. Recent studies have revealed that circRNAs play a crucial role in fine-tuning the level of microRNA (miRNA)-mediated regulation of gene expression by sequestering miRNAs. The interaction of circRNAs with disease-associated miRNAs suggests that circRNAs are important in the pathology of disease. However, the effects and roles of circRNAs in macrophage polarization have yet to be explored. In the present study, we performed a circRNA microarray to compare the circRNA expression profiles of bone marrow-derived macrophages (BMDMs) under two distinct polarizing conditions (M1 macrophages induced by interferon-γ and LPS stimulation, and M2 macrophages induced by interleukin-4 stimulation). Our results showed that a total of 189 circRNAs were differentially expressed between M1 and M2 macrophages. Differentially expressed circRNAs with a high fold-change were selected for validation by RT-qPCR: circRNA-003780, circRNA-010056, and circRNA-010231 were upregulated and circRNA-003424, circRNA-013630, circRNA-001489 and circRNA-018127 were downregulated (fold-change >4, P<0.05) in M1 compared to M2, which was found to correlate with the microarray data. Furthermore, the most differentially expressed circRNAs within all the comparisons were annotated in detail with circRNA/miRNA interaction information using miRNA target prediction software. In conclusion, the present study provides novel insight into the role of circRNAs in macrophage differentiation and polarization.

Defining the human copper proteome and analysis of its expression variation in cancers.

Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is associated with increased cellular Cu concentrations, whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer versus normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-molecular studies of Cu-dependent processes in cancer.

Hypoxia Stress Modifies Na+/K+-ATPase, H+/K+-ATPase, [Formula: see text], and nkaα1 Isoform Expression in the Brain of Immune-Challenged Air-Breathing Fish.

Fishes are equipped to sense stressful stimuli and are able to respond to environmental stressor such as hypoxia with varying pattern of stress response. The functional attributes of brain to hypoxia stress in relation to ion transport and its interaction during immune challenge have not yet delineated in fish. We, therefore, explored the pattern of ion transporter functions and messenger RNA (mRNA) expression of α1-subunit isoforms of Na+/K+-ATPase (NKA) in the brain segments, namely, prosencephalon (PC), mesencephalon (MC), and metencephalon (MeC) in an obligate air-breathing fish exposed either to hypoxia stress (30 minutes forced immersion in water) or challenged with zymosan treatment (25-200 ng g−1 for 24 hours) or both. Zymosan that produced nonspecific immune responses evoked differential regulation of NKA, H+/K+-ATPase (HKA), and (NNA) in the varied brain segments. On the contrary, hypoxia stress that demanded activation of NKA in PC and MeC showed a reversed NKA activity pattern in MeC of immune-challenged fish. A compromised HKA and NNA regulation during hypoxia stress was found in immune-challenged fish, indicating the role of these brain ion transporters to hypoxia stress and immune challenges. The differential mRNA expression of α1-subunit isoforms of NKA, nkaα1a, nkaα1b, and nkaα1c, in hypoxia-stressed brain showed a shift in its expression pattern during hypoxia stress-immune interaction in PC and MC. Evidence is thus presented for the first time that ion transporters such as HKA and NNA along with NKA act as functional brain markers which respond differentially to both hypoxia stress and immune challenges. Taken together, the data further provide evidence for a differential Na+, K+, H+, and ion signaling that exists in brain neuronal clusters during hypoxia stress-immune interaction as a result of modified regulations of NKA, HKA, and NNA transporter functions and nkaα1 isoform regulation.

Identifying Novel Glioma-Associated Noncoding RNAs by Their Expression Profiles.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play a significant role in cancer development as regulators of protein-coding genes. Their dysregulation was in some extent already associated with glioma, the most aggressive primary brain tumours in adults. The correct diagnosis and treatment selection due to high tumour heterogeneity might be difficult and inadequate, resulting in poor prognosis. Studies of expression patterns of noncoding RNAs (ncRNAs) could provide useful insight in glioma molecular development. We used the qPCR approach to screen and investigate the expression of lncRNAs that were previously deregulated in other cancer types. The study showed altered expression levels for numerous lncRNAs across histologically different glioma samples. Validation of few lncRNAs showed association of expression levels with histological subtype and/or malignancy grade. We also observed deregulated and subtype-distinctive expression for four lncRNA-associated miRNAs. Expression of few lncRNAs and miRNA was also associated with patients' survival, showing potential prognostic value. Several ncRNAs, some already related to glioma and some, to the best of our knowledge, investigated for the first time, might be of greater importance in glioma molecular development and progression. Finding the subtype-specific lncRNA and/or miRNA expression patterns may contribute additional information for a more objective classification.

Logic programming to infer complex RNA expression patterns from RNA-seq data.

To meet the increasing demand in the field, numerous long noncoding RNA (lncRNA) databases are available. Given many lncRNAs are specifically expressed in certain cell types and/or time-dependent manners, most lncRNA databases fall short of providing such profiles. We developed a strategy using logic programming to handle the complex organization of organs, their tissues and cell types as well as gender and developmental time points. To showcase this strategy, we introduce 'RenalDB' (http://renaldb.uni-frankfurt.de), a database providing expression profiles of RNAs in major organs focusing on kidney tissues and cells. RenalDB uses logic programming to describe complex anatomy, sample metadata and logical relationships defining expression, enrichment or specificity. We validated the content of RenalDB with biological experiments and functionally characterized two long intergenic noncoding RNAs: LOC440173 is important for cell growth or cell survival, whereas PAXIP1-AS1 is a regulator of cell death. We anticipate RenalDB will be used as a first step toward functional studies of lncRNAs in the kidney.

Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle

Six male Tharparkar cattle aged 2-3years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15days and then exposed to 42°C temperature up to 23days followed by 12days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P<0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P<0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P<0.05) after 10days of heat exposure (corresponds to chronic heat stress) as compared to the first 5days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

Microarray analysis of long noncoding RNA expression patterns in diabetic nephropathy

AimsLong noncoding RNAs (lncRNAs) are implicated in various biological processes and human diseases. Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). We explored the potential functions of lncRNAs in DN. MethodsWe established a mouse model of DN and compared lncRNA expression patterns between DN model and db/m control mouse kidney tissues using microarray analysis. lncRNA function was predicted by gene ontology enrichment and KEGG pathway analyses of lncRNAs–coexpressed mRNAs. Quantitative reverse-transcription PCR was used for validation. Cis- and trans-regulation analyses were conducted to reveal potential relationships between lncRNAs and their target genes. ResultsIn DN, 311 lncRNAs were dysregulated. LncRNA–coexpressed mRNAs were mainly targeted to golgi apparatus (ontology: cellular component), catalytic activity (ontology: molecular function), and mitotic nuclear division (ontology: biological process), and were mostly enriched in glutathione metabolism signaling. One hundred forty-seven lncRNAs were regarded as cis-regulatory. Several groups of lncRNAs may participate in biological pathways related to DN via trans-regulation of protein-coding genes. ConclusionHundreds of lncRNAs are dysregulated in DN. These lncRNAs might be involved in the pathogenesis of DN by modulating multiple molecular pathways. Our findings provide potential candidate biomarkers for predicting or diagnosing DN.

Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1.

Using RNA sequencing (RNA-Seq), we compared the expression patterns of circular RNAs in proliferating (early-passage) and senescent (late-passage) human diploid WI-38 fibroblasts. Among the differentially expressed senescence-associated circRNAs (which we termed ‘SAC-RNAs’), we identified CircPVT1, generated by circularization of an exon of the PVT1 gene, as a circular RNA showing markedly reduced levels in senescent fibroblasts. Reducing CircPVT1 levels in proliferating fibroblasts triggered senescence, as determined by a rise in senescence-associated β-galactosidase activity, higher abundance of CDKN1A/P21 and TP53, and reduced cell proliferation. Although several microRNAs were predicted to bind CircPVT1, only let-7 was found enriched after pulldown of endogenous CircPVT1, suggesting that CircPVT1 might selectively modulate let-7 activity and hence expression of let-7-regulated mRNAs. Reporter analysis revealed that CircPVT1 decreased the cellular pool of available let-7, and antagonizing endogenous let-7 triggered cell proliferation. Importantly, silencing CircPVT1 promoted cell senescence and reversed the proliferative phenotype observed after let-7 function was impaired. Consequently, the levels of several proliferative proteins that prevent senescence, such as IGF2BP1, KRAS and HMGA2, encoded by let-7 target mRNAs, were reduced by silencing CircPVT1. Our findings indicate that the SAC-RNA CircPVT1, elevated in dividing cells and reduced in senescent cells, sequesters let-7 to enable a proliferative phenotype.

Upregulation of MAPK/MCL-1 Maintaining Mitochondrial Oxidative Phosphorylation Confers Acquired Resistance to BCL-2 Inhibitor Venetoclax in AML

ABT-199 (venetoclax), a selective small-molecule antagonist of the anti-apoptotic protein BCL-2, enables the activation of pro-apoptotic proteins and the induction of cancer cell death. Our previous studies found that AML is a BCL-2 dependent disease and responds robustly to venetoclax by induction of apoptotic cell death (Pan et al., Cancer Discovery 2014). Despite initial responses to single agent venetoclax in a Phase II trial of relapsed AML, patients ultimately developed resistance and progressed (Konopleva et al., Cancer Discovery 2016). In this study we investigated mechanisms of acquired resistance to venetoclax in preclinical AML models.First, we generated 5 AML cell lines resistant to ³1µM venetoclax. No BAX (exon5 and 6) or BCL2 (exon2) mutations were found in resistant cells. Immunoblotting analysis demonstrated increased expression of anti-apoptotic proteins MCL-1, BCL-2 A1, and BCL-XL, and a decrease of pro-apoptotic PUMA protein in selected resistant cell lines. To probe the functional interactions between the pro- and anti-apoptotic proteins, we next performed co-immunoprecipitation (co-IP) studies. The anti-BIM and anti-MCL-1 co-IPs revealed reduced levels of BIM:BCL-2 complexes and increased BIM:MCL-1 complexes in resistant cells compared to their parental counterparts (Fig 1B). The BH3 profiling technique examines mitochondrial sensitivity to different BH3 mimetic peptides, and has proven to be a useful tool to determine cell dependence on anti-apoptotic BCL-2 family proteins. BH3 profiling demonstrated that resistant cells had increased responses to NOXA, MS1 and HRK peptides, indicating increased dependence on MCL-1 and/or BCL-XL (Fig 1C). To characterize the functional role of MCL-1 in resistance to venetoclax, we co-treated parental and resistant cells with selective BCL-XL or MCL-1 inhibitors A-1155463 (Leverson et al. Science Transl Med 2015) and A-1210477 (Leverson et al., Cell Death Dis 2015). The combination of venetoclax with either A-1155463 or A-1210477 showed synergistic growth inhibition in all 5 parental cell lines. Notably, 4 of the 5 resistant cell lines (OCI-AML2, Kasumi, MV4-11, MOLM13) became more sensitive to an MCL-1 inhibitor but not to a BCL-XL inhibitor (Fig 1E). However, no further sensitization was seen in combination with venetoclax in resistant cells.To characterize additional mechanisms of resistance to venetoclax in AML cells, we conducted RNA sequencing of single cell clones (2 clones/cell line) isolated from paired isogenic cells (OCI-AML2, MV4-11, MOLM13). Analysis of RNA expression patterns by gene set enrichment analysis (GSEA) revealed elevated expression of genes in the RAS/MAPK pathway (Fig 1F), consistent with increased p-ERK and p-p90-RSK protein levels (Fig 1G). Inhibition of MAPK with MEK inhibitor GDC-0973 reduced MCL-1 expression in parental but not in resistant cells, indicating that MAPK activation partially contributed to high MCL-1 levels (Fig 1G).GSEA of RNAseq data further uncovered altered expression of genes involved in mitochondrial oxidative phosphorylation (OxPhos) in 3 resistant cell lines with high MCL-1 expression (OCI-AML2, MV4-11 and MOLM-13). Notably, BCL-2 was reported to sustain AML stem cell survival through maintenance of the mitochondrial activity of OxPhos (Lagadinou etal., Cell Stem Cell, 2013). Analysis of mitochondrial respiration using a Seahorse Bioanalyzer demonstrated similar levels of oxygen consumption rate (OCR) in parental and resistant cells. Inhibition of BCL-2 with 100nM venetoclax for only 2 hrs. fully blocked baseline and maximal respiratory activity in parental but not in resistant cells. In turn, inhibition of MCL-1 with A-1210477 inhibited respiration in both parental and resistant cells, indicating a role for MCL-1 in sustaining mitochondrial activity in venetoclax-resistant AML cells, which can maintain unperturbed mitochondrial function.In summary, we identified a novel mechanism of resistance to targeted BCL-2 inhibition through upregulation of MAPK leading to increased levels of anti-apoptotic MCL-1 that binds and neutralizes BIM and maintains the mitochondrial OxPhos pathway in AML cells. Concomitant inhibition of BCL-2 and MCL-1, or of BCL-2 and OxPhos could induce synergistic cell death in AML and conceivably prevent the emergence of venetoclax resistance. [Display omitted] DisclosuresTyner:Constellation Pharmaceuticals: Research Funding; Janssen Research & Development: Research Funding; Agios Pharmaceuticals: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding; Inctye: Research Funding; Seattle Genetics: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Leverson:AbbVie: Employment, Other: Shareholder in AbbVie. Letai:Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Differentially Expressed Long Non-Coding RNAs Were Predicted to Be Involved in the Control of Signaling Pathways in Pediatric Astrocytoma.

Expression changes for long non-coding RNAs (lncRNAs) have been identified in adult glioblastoma multiforme (GBM) and in a mixture of adult and pediatric astrocytoma. Since adult and pediatric astrocytomas are molecularly different, the mixture of both could mask specific features in each. We determined the global expression patterns of lncRNAs and messenger RNA (mRNAs) in pediatric astrocytoma of different histological grades. Transcript expression changes were determined with an HTA 2.0 array. lncRNA interactions with microRNAs and mRNAs were predicted by using an algorithm and the LncTar tool, respectively. Interactomes were constructed with the HIPPIE database and visualized with the Cytoscape platform. The array showed expression changes in 156 and 207 lncRNAs in tumors (versus the control) and in pediatric GBM (versus low-grade astrocytoma), respectively. Predictions identified lncRNAs that have putative microRNA binding sites, which might suggest that they function as sponges in these tumors. Also, lncRNAs were shown to interact with many mRNAs, such as Pleckstrin homology-like domain, family A, member 1 (PHLDA1) and sulfatase 2 (SULF2). For example, qPCR found long intergenic non-coding RNA regulator of reprogramming (linc-RoR) expression levels upregulated in pediatric GBM when they were compared with control tissues or with low-grade tumors. Meanwhile, PHLDA1 and ELAV-like RNA binding protein 1 (ELAV1) showed expression changes in tumors relative to the control. Our data showed many lncRNAs with expression changes in pediatric astrocytoma, which might be involved in the regulation of different signaling pathways.

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells.

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality.

Differential protein abundance of a basolateral MCT1 transporter in the human gastrointestinal tract.

Bacterially derived short chain fatty acids (SCFAs), such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the transport of SCFAs across colonic epithelial cells via MCT1 transporters. This study investigated MCT1 protein abundance in various human intestinal tissues. Initial RT-PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, immunoblot analysis detected higher abundance of a 45 kDa MCT1 protein in colonic tissue compared to ileum tissue (P < 0.001, N = 4, unpaired t-test). Importantly, MCT1 abundance was found to be significantly lower in sigmoid colon compared to ascending colon (P < 0.01, N = 8-11, ANOVA). Finally, immunolocalization studies confirmed MCT1 to be abundant in the basolateral membranes of surface epithelial cells of the ascending, transverse, and descending colon, but significantly less prevalent in the sigmoid colon (P < 0.05, N = 5-21, ANOVA). In conclusion, these data confirm that basolateral MCT1 protein abundance is correlated to levels of bacterially derived SCFAs along the human gastrointestinal tract. These findings highlight the importance of precise tissue location in studies comparing colonic MCT1 abundance between normal and diseased states.

Sensitive whole mount insitu localization of small RNAs in plants.

Small RNAs, such as microRNAs (miRNAs), regulate gene expression and play important roles in many plant processes. Although our knowledge of their biogenesis and mode of action has significantly progressed, we still have comparatively little information about their biological functions. In particular, knowledge about their spatio-temporal expression patterns rely on either indirect detection by use of reporter constructs or labor-intensive direct detection by insitu hybridization on sectioned material. None of the current approaches allows a systematic investigation of small RNA expression patterns. Here, we present a sensitive method for insitu detection of miRNAs and siRNAs in intact plant tissues that utilizes both double-labeled probes and a specific cross-linker. We determined the expression patterns of several small RNAs in diverse plant tissues.

Cytogenetic or molecular analysis for prognosis?

SummaryPrognostication in uveal melanoma can have several functions: to advice the patient, to council patients with regard to monitoring, and to stratify patients in trials. Scientifically, determination of the parameters that define the development of metastases is of interest to understand tumour behaviour and develop treatments.The behaviour of tumor cells is related to changes at the DNA level, which lead to differences in mRNA, lncRNA, miRNA, and protein expression. A variety of tests using different technical platforms identify these differences, and can, after validation, be used for prognostication. Commonly‐used tests use the chromosome status (chrom 3, 8q), or the RNA expression pattern (Class 1 and 2). Expression of BAP1 can also be used. Some patients with low risk uveal melanoma also develop metastases, and these have been associated with the presence of extra copies of chromosome 8q, and with a specific marker, PRAME.The biological pathways that lead to metastases are still under investigation, although it is clear that a combination of loss of one chromosome 3 and loss of expression of BAP1 on the other chromosome 3 plays an important role in this pathway.

The Expression of B-Type Natriuretic Peptide After CaCl2-Induced Arrhythmias in Rats

To investigate the patterns of B-type natriuretic peptide (BNP) expression after arrhythmia, BNP was assessed at different time points (0 minute, 10 minutes, 30 minutes, 1 hour, 3 hours, and 6 hours) in CaCl2-induced arrhythmia in rats through various methods such as immunohistochemistry, Western blotting, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Immunohistochemistry results showed that the expression of BNP in the endocardium was higher than that in the epicardium in rats undergoing sustained arrhythmias. The BNP-to-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) ratios determined by Western blotting analysis revealed no change at 0 minute but increased at 10 minutes and reached the first peak (0.48 [0.03]) at 30 minutes. After a brief decline, the second peak was observed at 6 hours (0.54 [0.03]). Similar patterns of BNP messenger RNA expression were also observed by quantitative real-time polymerase chain reaction. The plasma BNP concentrations did not change after initial bouts of cardiac arrhythmias but significantly increased 30 minutes after CaCl2 injections. The results demonstrate that arrhythmia causes an elevation of BNP in the myocardium and blood, and BNP messenger RNA increases in initial arrhythmia while its protein in myocardium and plasma does not; however, both of them were elevated after sustained arrhythmia. Such an elevated BNP expression, which is directly related to the severity and duration of the arrhythmias, may suggest the existence of fatal arrhythmia in sudden cardiac death.

Hypoxia-Inducible Factor 2α Mutation-Related Paragangliomas Classify as Discrete Pseudohypoxic Subcluster

Recently, activating mutations of the hypoxia-inducible factor 2α gene (HIF2A/EPAS1) have been recognized to predispose to multiple paragangliomas (PGLs) and duodenal somatostatinomas associated with polycythemia, and ocular abnormalities. Previously, mutations in the SDHA/B/C/D, SDHAF2, VHL, FH, PHD1, and PHD2 genes have been associated with HIF activation and the development of pseudohypoxic (cluster-1) PGLs. These tumors overlap in terms of tumor location, syndromic presentation, and noradrenergic phenotype to a certain extent. However, they also differ especially by clinical outcome and by presence of other tumors or abnormalities. In the present study, we aimed to establish additional molecular differences between HIF2A and non-HIF2A pseudohypoxic PGLs. RNA expression patterns of HIF2A PGLs (n=6) from 2 patients were compared with normal adrenal medullas (n=8) and other hereditary pseudohypoxic PGLs (VHL: n=13, SDHB: n=15, and SDHD: n=14). Unsupervised hierarchical clustering showed that HIF2A PGLs made up a separate cluster from other pseudohypoxic PGLs. Significance analysis of microarray yielded 875 differentially expressed genes between HIF2A and other pseudohypoxic PGLs after normalization to adrenal medulla (false discovery rate 0.01). Prediction analysis of microarray allowed correct classification of all HIF2A samples based on as little as three genes (TRHDE, LRRC63, IGSF10; error rate: 0.02). Genes with the highest expression difference between normal medulla and HIF2A PGLs were selected for confirmatory quantitative reverse transcriptase polymerase chain reaction. In conclusion, HIF2A PGLs show a characteristic expression signature that separates them from non-HIF2A pseudohypoxic PGLs. Unexpectedly, the most significantly differentially expressed genes have not been previously described as HIF target genes.