High-throughput Expression Profiling Research Articles

Paving the way to a neural fate – RNA signatures in naive and trans-differentiating mesenchymal stem cells

Mesenchymal Stem Cells (MSCs) derived from the embryonic mesoderm persist as a viable source of multipotent cells in adults and have a crucial role in tissue repair. One of the most promising aspects of MSCs is their ability to trans-differentiate into cell types outside of the mesodermal lineage, such as neurons. This characteristic positions MSCs as potential therapeutic tools for neurological disorders. However, the definition of a clear MSC signature is an ongoing topic of debate. Likewise, there is still a significant knowledge gap about functional alterations of MSCs during their transition to a neural fate. In this study, our focus is on the dynamic expression of RNA in MSCs as they undergo trans-differentiation compared to undifferentiated MSCs. To track and correlate changes in cellular signaling, we conducted high-throughput RNA expression profiling during the early time-course of human MSC neurogenic trans-differentiation. The expression of synapse maturation markers, including NLGN2 and NPTX1, increased during the first 24 h. The expression of neuron differentiation markers, such as GAP43 strongly increased during 48 h of trans-differentiation. Neural stem cell marker NES and neuron differentiation marker, including TUBB3 and ENO1, were highly expressed in mesenchymal stem cells and remained so during trans-differentiation. Pathways analyses revealed early changes in MSCs signaling that can be linked to the acquisition of neuronal features. Furthermore, we identified microRNAs (miRNAs) as potential drivers of the cellular trans-differentiation process. We also determined potential risk factors related to the neural trans-differentiation process. These factors include the persistence of stemness features and the expression of factors involved in neurofunctional abnormalities and tumorigenic processes. In conclusion, our findings contribute valuable insights into the intricate landscape of MSCs during neural trans-differentiation. These insights can pave the way for the development of safer treatments of neurological disorders.

Novel melatonin-producing Bacillus safensis EH143 mitigates salt and cadmium stress in soybean.

Recently, microorganism and exogenous melatonin application has been recognized as an efficient biological tool for enhancing salt tolerance and heavy metal detoxification in agriculture crops. Thus, the goal of this study was to isolate and evaluate a novel melatonin-producing plant growth promoting bacterium. With high-throughput whole genome sequencing, phytohormone measurements, expression profiling, and biochemical analysis, we can identify a novel PGPB that produces melatonin and unravel how it promotes soybean growth and development and protects against salt and Cd stress. We identify the melatonin synthesis pathway (tryptophan→tryptamine→serotonin melatonin) of the halotolerant (NaCl > 800 mM) and heavy metal-resistant (Cd >3 mM) rhizobacterium Bacillus safensis EH143 and use it to treat soybean plants subjected to Cd and NaCl stresses. Results show that EH143 will highly bioaccumulate heavy metals and significantly improve P and Ca2+ uptake and the K+/Na+ (93%↑under salt stress) ratio while reducing Cd uptake (49% under Cd stress) in shoots. This activity was supported by the expression of the ion regulator HKT1, MYPB67, and the calcium sensors CDPK5 and CaMK1 which ultimately led to increased plant growth. EH143 significantly decreased ABA content in shoots by 13%, 20%, and 34% and increased SA biosynthesis in shoots by 14.8%, 31%, and 48.2% in control, salt, and Cd-treated plants, upregulating CYP707A1 and CYP707A2 and PAL1 and ICS, respectively. The melatonin content significantly decreased along with a reduced expression of ASMT3 following treatment with EH143; moreover, reduced expression of peroxidase (POD) and superoxide dismutase (SOD) by 134.5% and 39% under salt+Cd stress, respectively and increased level of total amino acids were observed. Whole-genome sequencing and annotation of EH143 revealed the presence of the melatonin precursor tryptophan synthase (trpA, trpB, trpS), metal and other ion regulators (Cd: cadA, potassium: KtrA and KtrB, phosphate: glpT, calcium: yloB, the sodium/glucose cotransporter: sgIT, and the magnesium transporter: mgtE), and enzyme activators (including the siderophore transport proteins yfiZ and yfhA, the SOD sodA, the catalase katA1, and the glutathione regulator KefG) that may be involved in programming the plant metabolic system. As a consequence, EH143 treatment significantly reduced the contents of lipid peroxidation (O2-, MDA, and H2O2) up to 69%, 46%, and 29% in plants under salt+Cd stress, respectively. These findings suggest that EH143 could be a potent biofertilizer to alleviate NaCl and Cd toxicity in crops and serve as an alternative substitute for exogenous melatonin application.

Individualized detection of TMPRSS2-ERG fusion status in prostate cancer: a rank-based qualitative transcriptome signature

BackgroundTMPRSS2-ERG (T2E) fusion is highly related to aggressive clinical features in prostate cancer (PC), which guides individual therapy. However, current fusion prediction tools lacked enough accuracy and biomarkers were unable to be applied to individuals across different platforms due to their quantitative nature. This study aims to identify a transcriptome signature to detect the T2E fusion status of PC at the individual level.MethodsBased on 272 high-throughput mRNA expression profiles from the Sboner dataset, we developed a rank-based algorithm to identify a qualitative signature to detect T2E fusion in PC. The signature was validated in 1223 samples from three external datasets (Setlur, Clarissa, and TCGA).ResultsA signature, composed of five mRNAs coupled to ERG (five ERG-mRNA pairs, 5-ERG-mRPs), was developed to distinguish T2E fusion status in PC. 5-ERG-mRPs reached 84.56% accuracy in Sboner dataset, which was verified in Setlur dataset (n = 455, accuracy = 82.20%) and Clarissa dataset (n = 118, accuracy = 81.36%). Besides, for 495 samples from TCGA, two subtypes classified by 5-ERG-mRPs showed a higher level of significance in various T2E fusion features than subtypes obtained through current fusion prediction tools, such as STAR-Fusion.ConclusionsOverall, 5-ERG-mRPs can robustly detect T2E fusion in PC at the individual level, which can be used on any gene measurement platform without specific normalization procedures. Hence, 5-ERG-mRPs may serve as an auxiliary tool for PC patient management.

Location matters: spatial dynamics of tumor-infiltrating T cell subsets is prognostic in colon cancer.

Colon cancer is a heterogeneous disease and consists of various molecular subtypes. Despite advances in high-throughput expression profiling, limitations remain in predicting clinical outcome and assigning specific treatment to individual cases. Tumor-immune interactions play a critical role, with tumors that activate the immune system having better outcome for the patient. The localization of T cells within tumor epithelium, to enable direct contact, is essential for antitumor function, but bulk DNA/RNA sequencing data lacks spatial distribution information. In this study, we provide spatial T cell tumor distribution and connect these data with previously determined genomic data in the AC-ICAM colon cancer patient cohort. Colon cancer patients (n=90) with transcriptome data available were selected. We used a custom multiplex immunofluorescence assay on colon tumor tissue sections for quantifying T cell subsets spatial distribution in the tumor microenvironment, in terms of cell number, location, mutual distance, and distance to tumor cells. Statistical analyses included the previously determined Immunologic Constant of Rejection (ICR) transcriptome correlation and patient survival, revealing potential prognostic value in T cell spatial distribution. T cell phenotypes were characterized and CD3+CD8-FoxP3- T cells were found to be the predominant tumor-infiltrating subtype while CD3+FoxP3+ T cells and CD3+CD8+ T cells showed similar densities. Spatial distribution analysis elucidated that proliferative T cells, characterized by Ki67 expression, and Granzyme B-expressing T cells were predominantly located within the tumor epithelium. We demonstrated an increase in immune cell density and a decrease in the distance of CD3+CD8+ T cells to the nearest tumor cell, in the immune active, ICR High, immune subtypes. Higher densities of stromal CD3+FoxP3+ T cells showed enhanced survival outcomes, and patients exhibited superior clinical benefits when greater spatial distances were observed between CD3+CD8-FoxP3- or CD3+CD8+ T cells and CD3+FoxP3+ T cells. Our study's in-depth analysis of the spatial distribution and densities of major T cell subtypes within the tumor microenvironment has provided valuable information that paves the way for further research into the intricate relationships between immune cells and colon cancer development.

Subtype-selective prenylated isoflavonoids disrupt regulatory drivers of MYCN-amplified cancers

Transcription factors have proven difficult to target with small molecules because they lack pockets necessary for potent binding. Disruption of protein expression can suppress targets and enable therapeutic intervention. To this end, we developed a drug discovery workflow that incorporates cell-line-selective screening and high-throughput expression profiling followed by regulatory network analysis to identify compounds that suppress regulatory drivers of disease. Applying this approach to neuroblastoma (NBL), we screened bioactive molecules in cell lines representing its MYC-dependent (MYCNA) and mesenchymal (MES) subtypes to identify selective compounds, followed by PLATESeq profiling of treated cells. This revealed compounds that disrupt a sub-network of MYCNA-specific regulatory proteins, resulting in MYCN degradation invivo. The top hit was isopomiferin, a prenylated isoflavonoid that inhibited casein kinase 2 (CK2) in cells. Isopomiferin and its structural analogs inhibited MYC and MYCN in NBL and lung cancer cells, highlighting the general MYC-inhibiting potential of this unique scaffold.

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling

BackgroundHypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200–500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and resultsUsing high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. ConclusionsAltogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

A high-throughput single-cell RNA expression profiling method identifies human pericyte markers.

We sought to identify and optimise a universally available histological marker for pericytes in the human brain. Such a marker could be a useful tool for researchers. Further, identifying a gene expressed relatively specifically in human pericytes could provide new insights into the biological functions of this fascinating cell type. We analysed single-cell RNA expression profiles derived from different human and mouse brain regions using a high-throughput and low-cost single-cell transcriptome sequencing method called EasySci. Through this analysis, we were able to identify specific gene markers for pericytes, some of which had not been previously characterised. We then used commercially (and therefore universally) available antibodies to immunolabel the pericyte-specific gene products in formalin-fixed paraffin-embedded (FFPE) human brains and also performed immunoblots to determine whether appropriately sized proteins were recognised. In the EasySci data sets, highly pericyte-enriched expression was notable for SLC6A12 and SLC19A1. Antibodies against these proteins recognised bands of approximately the correct size in immunoblots of human brain extracts. Following optimisation of the immunohistochemical technique, staining for both antibodies was strongly positive in small blood vessels and was far more effective than a PDGFRB antibody at staining pericyte-like cells in FFPE human brain sections. In an exploratory sample of other human organs (kidney, lung, liver, muscle), immunohistochemistry did not show the same pericyte-like pattern of staining. The SLC6A12 antibody was well suited for labelling pericytes in human FFPE brain sections, based on the combined results of single-cell RNA-seq analyses, immunoblots and immunohistochemical studies.

Molecular mechanisms of NMDA excitotoxicity in the retina

NMDA excitotoxicity, as a part of glutamate excitotoxicity, has been proposed to contribute significantly to many retinal diseases. Therefore, understanding mechanisms of NMDA excitotoxicity will provide further insight into the mechanisms of many retinal diseases. To study mechanisms of NMDA excitotoxicity in vivo, we used an animal model in which NMDA (20 mM, 2 µL) was injected into the vitreous of mice. We also used high-throughput expression profiling, various animals with reduced expression of target genes, and animals treated with the oral iron chelator deferiprone. We found that the expression of many genes involved in inflammation, programmed cell death, free radical production, oxidative stress, and iron and calcium signaling was significantly increased 24 h after NMDA treatment. Meanwhile, decreased activity of the pro-inflammatory TNF signaling cascade and decreased levels of ferrous iron (Fe2+, required for free radical production) led to significant neuroprotection in NMDA-treated retinas. Since increased TNF signaling activity and high Fe2+ levels trigger regulated necrosis, which, in turn, lead to inflammation, we proposed an important role in NMDA excitotoxicity of a positive feedback loop in which regulated necrosis promotes inflammation, which subsequently triggers regulated necrosis.

Characterization of human umbilical cord blood-derived mast cells using high-throughput expression profiling and next-generation knowledge discovery platforms

Mast cells (MCs) are tissue-resident innate immune cells that express the high-affinity receptor for immunoglobulin E and are responsible for host defense and an array of diseases related to immune system. We aimed in this study to characterize the pathways and gene signatures of human cord blood-derived MCs (hCBMCs) in comparison to cells originating from CD34− progenitors using next-generation knowledge discovery methods. CD34+ cells were isolated from human umbilical cord blood using magnetic activated cell sorting and differentiated into MCs with rhIL-6 and rhSCF supplementation for 6–8 weeks. The purity of hCBMCs was analyzed by flow cytometry exhibiting the surface markers CD117+CD34−CD45−CD23−FcεR1αdim. Total RNA from hCBMCs and CD34− cells were isolated and hybridized using microarray. Differentially expressed genes were analyzed using iPathway Guide and Pre-Ranked Gene Set Enrichment Analysis. Next-generation knowledge discovery platforms revealed MC-specific gene signatures and molecular pathways enriched in hCBMCs and pertain the immunological response repertoire.

Small RNA transcriptome analysis using parallel single-cell small RNA sequencing

miRNA and other forms of small RNAs are known to regulate many biological processes. Single-cell small RNA sequencing can be used to profile small RNAs of individual cells; however, limitations of efficiency and scale prevent its widespread application. Here, we developed parallel single-cell small RNA sequencing (PSCSR-seq), which can overcome the limitations of existing methods and enable high-throughput small RNA expression profiling of individual cells. Analysis of PSCSR-seq data indicated that diverse cell types could be identified based on patterns of miRNA expression, and showed that miRNA content in nuclei is informative (for example, cell type marker miRNAs can be detected in isolated nuclei). PSCSR-seq is very sensitive: analysis of only 732 peripheral blood mononuclear cells (PBMCs) detected 774 miRNAs, whereas bulk small RNA analysis would require input RNA from approximately 106 cells to detect as many miRNAs. We identified 42 miRNAs as markers for PBMC subpopulations. Moreover, we analyzed the miRNA profiles of 9,533 cells from lung cancer biopsies, and by dissecting cell subpopulations, we identified potentially diagnostic and therapeutic miRNAs for lung cancers. Our study demonstrates that PSCSR-seq is highly sensitive and reproducible, thus making it an advanced tool for miRNA analysis in cancer and life science research.

MicroRNA Expression Signatures in Clear Cell Renal Cell Carcinoma: High-Throughput Searching for Key miRNA Markers in Patients from the Volga-Ural Region of Eurasian Continent.

Clear cell renal cell carcinoma (ccRCC) is characterized by high molecular genetic heterogeneity, metastatic activity and unfavorable prognosis. MicroRNAs (miRNA) are 22-nucleotide noncoding RNAs that are aberrantly expressed in cancer cells and have gained serious consideration as non-invasive cancer biomarkers. We investigated possible differential miRNA signatures that may differentiate high-grade ccRCC from primary disease stages. High-throughput miRNAs expression profiling, using TaqMan OpenArray Human MicroRNA panel, was performed in a group of 21 ccRCC patients. The obtained data was validated in 47 ccRCC patients. We identified nine dysregulated miRNAs (miRNA-210, -642, -18a, -483-5p, -455-3p, -487b, -582-3p, -199b and -200c) in tumor ccRCC tissue compared to normal renal parenchyma. Our results show that the combination of miRNA-210, miRNA-483-5p, miRNA-455 and miRNA-200c is able to distinguish low and high TNM ccRCC stages. Additionally, miRNA-18a, -210, -483-5p and -642 showed statistically significant differences between the low stage tumor ccRCC tissue and normal renal tissue. Contrariwise, the high stages of the tumor process were accompanied by alteration in the expression levels of miRNA-200c, -455-3p and -582-3p. Although the biological roles of these miRNAs in ccRCC are not totally clear, our findings need additional investigations into their involvement in the pathogenesis of ccRCC. Prospective studies with large study cohorts of ccRCC patients are important to further establish the clinical validity of our miRNA markers to predict ccRCC.

High-throughput time series expression profiling of Plasmopara halstedii infecting Helianthus annuus reveals conserved sequence motifs upstream of co-expressed genes

Downy mildew disease of sunflower, caused by the obligate biotrophic oomycete Plasmopara halstedii, can have significant economic impact on sunflower cultivation. Using high-throughput whole transcriptome sequencing, four developmental phases in 16 time-points of Pl. halstedii infecting Helianthus annuus were investigated. With the aim of identifying potential functional and regulatory motifs upstream of co-expressed genes, time-series derived gene expression profiles were clustered based on their time-course similarity, and their upstream regulatory gene sequences were analyzed here. Several conserved motifs were found upstream of co-expressed genes, which might be involved in binding specific transcription factors. Such motifs were also found associated with virulence related genes, and could be studied on a genetically tractable model to clarify, if these are involved in regulating different stages of pathogenesis.

Effect and Related Mechanism of Platelet-Rich Plasma on the Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

Objective Human adipose-derived stem cells (hADSCs) are ideal seed cells for the regeneration of alveolar bone defects. Platelet-rich plasma (PRP), which is rich in growth factors, promotes tissue repair. The purpose of the present study was to investigate whether PRP promotes the osteogenic differentiation of hADSCs and to perform high-throughput sequencing to explore the possible mechanism. Methods hADSCs were divided into the three following groups: CON group, OM group, and PRP group. Osteogenesis was detected by Alizarin Red staining on day 14. Total RNA was extracted from the OM and PRP groups for high-throughput sequencing. The target genes of the differentially expressed osteogenic-related miRNAs were predicted, and combined miRNA/mRNA analysis was then performed. The mRNA and protein expression levels of hsa-miR-212-5p, type 1 cannabinoid receptor (CNR1), alkaline phosphatase (ALP), Runx2, osteocalcin (OCN), and collagen 1 A1 (COL1A1) in the OM and PRP groups were detected by qRT–PCR and Western blot analyses. The binding between hsa-miR-212-5p and CNR1 was detected by a dual-luciferase reporter assay. Results Both the OM and PRP groups exhibited enhanced proliferation of hADSCs, and the differences at 48 h and 72 h were statistically significant (P < 0.05). The PRP group had significantly more calcium nodules than the CON group (P < 0.05). Through high-throughput sequencing analysis, differential miRNA and mRNA expression profiles were obtained. During hADSC osteogenesis, the expression of hsa-miR-212-5p was downregulated, and the expression of CNR1 was upregulated. hsa-miR-212-5p was found to bind directly to the 3′ UTR of CNR1. Conclusions The present findings indicated that downregulation of hsa-miR-212-5p and upregulation of CNR1 may be involved in the process by which PRP promotes the osteogenic differentiation of hADSCs.

Comprehensive analysis of ncRNA involvement in brain microglia immunology

Microglia is a major class of brain-resident myeloid cells and non-coding RNAs (ncRNAs) serves as key regulators in microglia homeostasis and inflammatory process. Here, we constructed the systematical association between microglia and ncRNAs including miRNAs, lncRNAs and circRNAs from two aspects, manual retrieval and computational detection. A total of 648 experimental verified ncRNA-microglia associations were obtained from published studies, including ncRNA regulatory patterns within different experimental models. Furthermore, we extracted 9 miRNA and 1 lncRNA expression profiles from the GEO database. Also, we obtained 31 sample-match miRNA and mRNA expression profiles, containing a total of 2335 normal or disordered brain samples. Finally, we developed a platform named MG-ncRexplorer (http://bio-bigdata.hrbmu.edu.cn/MG-ncRexplorer/), exploring the associations between ncRNAs and microglia among experimental validated and computational detection. To demonstrate the usage of MG-ncRexplorer, we constructed regulatory target networks based on manual retrieval associations and identified risk glioma miRNAs among multiple high-throughput expression profiles.

Gene Expression Transcriptome Profile Validation: A Critical Appraisal of Methods and Signature Analyses

<h3>Purpose</h3> The aim of our study is to assess the reliability of transcriptome profile platform outcomes between different tissue preservation conditions, RNA extraction methods and heart-failure-related genes, minimizing intra- and inter-experimental variability. <h3>Methods</h3> Our cohort of study is composed by 6 FFPE endomyocardial biopsies (EMBs) and 6 fresh frozen (FF) heart tissues samples belonging to the same adult end-stage heart failure patient, limiting inter-patient variability bias. Two major RNA extraction procedure were tested for FF and FFPE tissues. Then, we performed a high-throughput microarray expression profiling without a bias of transcript selection. We analyzed mRNAs expression through R programming and transcriptome analysis software, exploring >20000 transcripts. We refined the transcriptome analysis selecting, from a literature systematic review appraisal 30 heart-failure related genes, coupling the overlapping genes in the differential expression and network analysis. <h3>Results</h3> We compared the analysis of up- and down-regulated genes between the groups, addressing both the different RNA extraction procedure and tissue preservation, while sharing the same transcriptome platform. The differential expressed genes (DEG) analyzed had fold change set at 2 and -2, and a cut-off threshold of FDR adjusted p-value < 0,05. Heatmap showed a great samples' clusterization according to preservation and extraction methods. Only 284 genes passed filter criteria in FFPE inter-groups comparison of extraction methods, while 52 differed in FFPE vs. FF, showing a great overlap of transcriptome signature, corroborated by the heart-failure related set of genes. The network analysis highlighted that most of the DEG are strongly associated (p-value adjusted <0.004) to the dysregulation of reticulum and collagen related cell component, in accordance with heart failure stage. <h3>Conclusion</h3> To the best of our knowledge, this is the first study in heart transplantation fields comparing genetic extraction and preservation methods for microarray expression profile evaluation correlated with pathophysiological mechanism. A proof of concept that RNAs from FFPE-EMBs, and not only FF, are feasible for microarray expression profile evaluation, and derived transcriptome profiles have a strong potential for research and diagnostic clinical application.

Altered Circulating microRNAs in Patients with Diabetic Neuropathy and Corneal Nerve Loss: A Pilot Study.

An alteration in circulating miRNAs may have important diagnostic and therapeutic relevance in diabetic neuropathy. Patients with type 2 diabetes mellitus (T2DM) underwent an assessment of neuropathic symptoms using Douleur Neuropathique 4 (DN4), the vibration perception threshold (VPT) using a Neurothesiometer, sudomotor function using the Sudoscan, corneal nerve morphology using corneal confocal microscopy (CCM) and circulating miRNAs using high-throughput miRNA expression profiling. Patients with T2DM, with (n = 9) and without (n = 7) significant corneal nerve loss were comparable in age, gender, diabetes duration, BMI, HbA1c, eGFR, blood pressure, and lipid profile. The VPT was significantly higher (p < 0.05), and electrochemical skin conductance (p < 0.05), corneal nerve fiber density (p = 0.001), corneal nerve branch density (p = 0.013), and corneal nerve fiber length (p < 0.001) were significantly lower in T2DM patients with corneal nerve loss compared to those without corneal nerve loss. Following a q-PCR-based analysis of total plasma microRNAs, we found that miR-92b-3p (p = 0.008) was significantly downregulated, while miR-22-3p (p = 0.0001) was significantly upregulated in T2DM patients with corneal nerve loss. A network analysis revealed that these miRNAs regulate axonal guidance and neuroinflammation genes. These data support the need for more extensive studies to better understand the role of dysregulated miRNAs’ in diabetic neuropathy.

Better Agreement of Human Transcriptomic and Proteomic Cancer Expression Data at the Molecular Pathway Activation Level.

Previously, we have shown that the aggregation of RNA-level gene expression profiles into quantitative molecular pathway activation metrics results in lesser batch effects and better agreement between different experimental platforms. Here, we investigate whether pathway level of data analysis provides any advantage when comparing transcriptomic and proteomic data. We compare the paired proteomic and transcriptomic gene expression and pathway activation profiles obtained for the same human cancer biosamples in The Cancer Genome Atlas (TCGA) and the NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) projects, for a total of 755 samples of glioblastoma, breast, liver, lung, ovarian, pancreatic, and uterine cancers. In a CPTAC assay, expression levels of 15,112 protein-coding genes were profiled using the Thermo QE series of mass spectrometers. In TCGA, RNA expression levels of the same genes were obtained using the Illumina HiSeq 4000 engine for the same biosamples. At the gene level, absolute gene expression values are compared, whereas pathway-grade comparisons are made between the pathway activation levels (PALs) calculated using average sample-normalized transcriptomic and proteomic profiles. We observed remarkably different average correlations between the primary RNA- and protein expression data for different cancer types: Spearman Rho between 0.017 (p = 1.7 × 10−13) and 0.27 (p < 2.2 × 10−16). However, at the pathway level we detected overall statistically significantly higher correlations: averaged Rho between 0.022 (p < 2.2 × 10−16) and 0.56 (p < 2.2 × 10−16). Thus, we conclude that data analysis at the PAL-level yields results of a greater similarity when comparing high-throughput RNA and protein expression profiles.

Persistence of Lgr5+ colonic epithelial stem cells in mouse models of inflammatory bowel disease.

Intestinal mucosal healing is the primary therapeutic goal of medical treatments for inflammatory bowel disease (IBD). Epithelial stem cells are key players in the healing process. Lgr5+ stem cells maintain cellular turnover during homeostasis in the colonic crypt. However, they are lost and dispensable for repair in a wide variety of injury models, including dextran sulfate sodium (DSS) colitis, radiation, helminth infection, and T-cell activation. The direct loss of Lgr5+ cells activates a plasticity response in the epithelium in which other cell types can serve as stem cells. Whether this paradigm applies to mouse models of IBD remains unknown. In contrast to previously tested models, IBD models involve an inflammatory response rooted in the loss of immunologic tolerance to intestinal luminal contents including the microbiome. Here, we show the persistence of Lgr5+ cells in oxazolone, 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Il10-/-, and Il10-/- Tnfr1-/- IBD models. This contrasts with results obtained from DSS-induced injury. Through high-throughput expression profiling, we find that these colitis models were associated with distinct patterns of cytokine expression. Direct exposure of colonic epithelial organoids to DSS, oxazolone, or TNBS resulted in increased apoptosis and loss of Lgr5+ cells. Targeted ablation of Lgr5+ cells resulted in severe exacerbation of chronic, antibody-induced IL-10-deficient colitis, but had only modest effects in TNBS-induced colitis. These results show that distinct mouse models of IBD-like colitis induce different patterns of Lgr5+ stem cell retention and function.NEW & NOTEWORTHY Acute intestinal injury and epithelial repair are associated with the loss of fast-cycling Lgr5+ stem cells and plasticity in the activation of formerly quiescent cell populations. In contrast, here we show in murine inflammatory bowel disease the persistence of the Lgr5+ stem cell population and its essential role in restricting the severity of chronic colitis. This demonstrates a diversity of stem cell responses to colitis.

Identification of a Novel Prognostic Signature for Gastric Cancer Based on Multiple Level Integration and Global Network Optimization.

Gastric Cancer (GC) is a common cancer worldwide with a high morbidity and mortality rate in Asia. Many prognostic signatures from genes and non-coding RNA (ncRNA) levels have been identified by high-throughput expression profiling for GC. To date, there have been no reports on integrated optimization analysis based on the GC global lncRNA-miRNA-mRNA network and the prognostic mechanism has not been studied. In the present work, a Gastric Cancer specific lncRNA-miRNA-mRNA regulatory network (GCsLMM) was constructed based on the ceRNA hypothesis by combining miRNA-target interactions and data on the expression of GC. To mine for novel prognostic signatures associated with GC, we performed topological analysis, a random walk with restart algorithm, in the GCsLMM from three levels, miRNA-, mRNA-, and lncRNA-levels. We further obtained candidate prognostic signatures by calculating the integrated score and analyzed the robustness of these signatures by combination strategy. The biological roles of key candidate signatures were also explored. Finally, we targeted the PHF10 gene and analyzed the expression patterns of PHF10 in independent datasets. The findings of this study will improve our understanding of the competing endogenous RNA (ceRNA) regulatory mechanisms and further facilitate the discovery of novel prognostic biomarkers for GC clinical guidelines.

Protein Microarrays for Ocular Diseases.

The eye is a multifaceted organ organized in several compartments with particular properties that reflect their diverse functions. The prevalence of ocular diseases is increasing, mainly because of its relationship with aging and of generalized lifestyle changes. However, the pathogenic molecular mechanisms of many common eye pathologies remain poorly understood. Considering the unquestionable importance of proteins in cellular processes and disease progression, proteomic techniques, such as protein microarrays, represent a valuable approach to analyze pathophysiological protein changes in the ocular environment. This technology enables to perform multiplex high-throughput protein expression profiling with minimal sample volume requirements broadening our knowledge of ocular proteome network in eye diseases.In this review, we present a brief summary of the main types of protein microarrays (antibody microarrays, reverse-phase protein microarrays, and protein microarrays) and their application for protein change detection in chronic ocular diseases such as dry eye, age-related macular degeneration, diabetic retinopathy, and glaucoma. The validation of these specific protein changes in eye pathologies may lead to the identification of new biomarkers, depiction of ocular disease pathways, and assistance in the diagnosis, prognosis, and development of new therapeutic options for eye pathologies.