Largest Fold Change Research Articles

Implications of Lead (Pb)-Induced Transcriptomic and Phenotypic Alterations in the Aged Zebrafish (Danio rerio).

Lead (Pb) is a well-known neurotoxin with established adverse effects on the neurological functions of children and younger adults, including motor, learning, and memory abilities. However, its potential impact on older adults has received less attention. Using the zebrafish model, our study aims to characterize the dose-response relationship between environmentally relevant Pb exposure levels and their effects on changes in behavior and transcriptomics during the geriatric periods. We exposed two-year-old zebrafish to waterborne lead acetate (1, 10, 100, 1000, or 10,000 µg/L) or a vehicle (DMSO) for 5 days. While lower concentrations (1-100 µg/L) reflect environmentally relevant Pb levels, higher concentrations (1000-10,000 µg/L) were included to assess acute toxicity under extreme exposure scenarios. We conducted adult behavior assessment to evaluate the locomotor activity following exposure. The same individual fish were subsequently sacrificed for brain dissection after a day of recovery in the aquatic system. RNA extraction and sequencing were then performed to evaluate the Pb-induced transcriptomic changes. Higher (1000-10,000 ug/L) Pb levels induced hyperactive locomotor patterns in aged zebrafish, while lower (10-100 ug/L) Pb levels resulted in the lowest locomotor activity compared to the control group. Exposure to 100 µg/L led to the highest number of differentially expressed genes (DEGs), while 10,000 µg/L induced larger fold changes in both directions. The neurological pathways impacted by Pb exposure include functions related to neurotransmission, such as cytoskeletal regulation and synaptogenesis, and oxidative stress response, such as mitochondrial dysfunction and downregulation of heat shock protein genes. These findings emphasize a U-shape dose-response relationship with Pb concentrations in locomotor activity and transcriptomic changes in the aging brain.

Integrative mRNA and miRNA Expression Profiles from Developing Zebrafish Head Highlight Brain-Preference Genes and Regulatory Networks.

Zebrafish is an emerging animal model for studying molecular mechanism underlying neurodevelopmental disorder due to its advantage characters. miRNAs are small non-coding RNAs that play a key role in brain development. Understanding of dynamic transcriptional and post-transcriptional molecules and their regulation during the head development is important for the study of neurodevelopmental disorder. In this study, we performed the high-throughput sequencing of mRNAs and miRNAs in developing zebrafish head from pharyngula to early larval stages and carried out bioinformatic analysis including differential expression and functional enrichment as well as joint analysis of miRNAs and mRNAs, and also compared with other related public sequencing datasets to aid our interpretation. A large number of differential expression genes with a large fold change were detected during the head development. Further clustering and functional enrichment analyses indicated that genes in late stage were most related with synaptic signaling. Overlap test analysis showed a significant enrichment of brain-preference and synapse-associated gene set in the head transcriptome compared with the whole embryo transcriptome. We also constructed miRNA-mRNA network for those brain-preference genes and focused on those densely connected network components. CRISPR-Cas9-mediated snap25b mutants led to embryonic development defects and decreases locomotor activity. Altogether, the present study provides developmental profiles of head-enriched mRNAs and miRNAs at three critical windows for nervous system development, which may contribute to the study of neurodevelopmental disorder.

Modeling of new markers for the diagnosis and prognosis of pancreatic cancer based on the transition from inflammation to cancer.

Pancreatic adenocarcinoma (PAAD) is a lethal disease with a poor prognosis. Genes involved in acute pancreatitis (AP) or chronic pancreatitis (CP) might be important for PAAD development. This study sought to identify potential PAAD diagnosis markers and to establish a PAAD prognosis prediction model based on AP- and CP-related genes. The significantly differentially expressed genes in both AP or CP and PAAD were obtained by a bioinformatics analysis. A risk-score model for predicting survival was constructed based on The Cancer Genome Atlas (TCGA) data and validated using an International Cancer Genome Consortium (ICGC) cohort. Protein expression and the effects of the genes in the risk models were validated by immunohistochemistry, or Cell Counting Kit-8 (CCK-8) and transwell assays. The study sample data included six AP tissue samples and five normal pancreatic tissue samples, six CP tissue samples and six normal pancreatic tissue samples from the Gene Expression Omnibus (GEO) expression profiling microarrays GSE109227 and GSE41418 data sets, respectively, and fragments per kilobase per million mapped fragments (FPKM) data from four normal controls and 150 PAAD cases from TCGA database, and 182 cancer patient samples with complete survival prognostic data from the ICGC database. In total, 508 significantly differentially expressed genes were found in both AP or CP and PAAD. Trefoil factor 2 (TFF2), tubulointerstitial nephritis antigen (TINAG), trefoil factor 1 (TFF1), aquaporin 5 (AQP5), SAM pointed domain containing ETS transcription factor (SPDEF), anterior gradient protein 2 (AGR2), apolipoprotein B messenger RNA editing enzyme catalytic subunit 1 (APOBEC1), kallikrein-related peptidase 6 (KLK6), dopa decarboxylase (DDC), mucin 13 (MUC13), claudin 18 (CLDN18), annexin A10 (ANXA10), and tetraspanin 1 (TSPAN1) were found to be present in PAAD and had the largest fold change. A risk-score model, comprising 19 genes, was constructed for prognostic prediction. A high-risk score indicated a poor prognosis. TINAG, DDC, SPDEF, and APOBEC1 proteins were increased in PAAD, while TINAG and DDC were correlated with the pathologic grade. Decreased TINAG, APOBEC1, transmembrane protein 94 (TMEM94), and kelch like family member 36 (KLHL36) expression inhibited PAAD cell proliferation, while decreased SPDEF, TMEM94, and KLHL36 expression significantly inhibited PAAD cell migration. The AP and CP co-related genes were significantly correlated with PAAD. TINAG, DDC, SPDEF, and APOBEC1 could serve as new PAAD predictors. The risk model developed in this study could be used to predict the prognosis of PAAD patients.

Targeting EGFR/PI3K/AKT/mTOR signaling in lung and colon cancers: synthesis, antitumor evaluation of new 1,2,4-oxdiazoles tethered 1,2,3-triazoles.

The EGFR/PI3K/Akt/mTOR pathway is important for metastasis, medication resistance, apoptosis prevention, and malignant transformation. Mutations in lung and colon cancer typically change this pathway's expression. As a result, a novel class of 1,2,4-oxdiazoles that are attached to 1,2,3-triazoles, 5-11, were created as possible anticancer drugs. The produced compounds are all examined by spectroscopic and micro-analytical techniques. MTT assay results on lung (A549) colon (Caco-2) and normal lung fibroblast (WI38) revealed that compounds 6a, 6b, 8a, and 11b demonstrated strong and selective antiproliferative activities against lung (A549) and colon (Caco-2) cancer cell lines while the remaining derivatives showed moderate to low activity. qPCR data revealed that the potential hits had large fold changes in the downregulation of EGFR, mTOR, and PI3K; they upregulate the amount of p53 to support their mode of action even more. Interestingly, docking investigations validated the biological outcomes by demonstrating a strong affinity of our compounds against EGFR active regions. Computational predictions of all the synthesized compounds' pharmacokinetic profiles, physicochemical characteristics, and drug-likeness data indicated that the promising hits might be taken into consideration as drug-like prospects.

Aronia melanocarpa extract extends the lifespan and health-span of Caenorhabditis elegans via mitogen-activated protein kinase 1.

Aging is a highly complex process and one of the largest risk factors for many chronic diseases. Aronia melanocarpa (AM) is rich in bioactive phytochemicals with antioxidant, anti-inflammatory, and anticancer properties. However, little is known about its effects on aging. The objective of this study was to evaluate the effects of AM extract on lifespan and health-span using Caenorhabditis elegans as a representative model. The mechanisms of its effects were explored using transcriptomics and untargeted metabolomics. Results showed that the lifespan of C. elegans was significantly extended by 22.2% after high-dose AM treatment. AM improved the behavior and physiological functions of C. elegans by increasing the pharyngeal pumping rate, decreasing lipofuscin accumulation and the reactive oxygen species level, enhancing resistance to oxidative stress, and increasing the activities of superoxide dismutase and catalase. Transcriptome analysis showed that the pmk-1 gene (mitogen-activated protein kinase 1), which is involved in the MAPK signaling pathway, was the gene with the largest fold change after AM intervention. However, in the C. elegans pmk-1(km25) mutant, the beneficial effect of AM in improving nematode senescence disappeared. An untargeted metabolomics study showed that the levels of 4-hydroxyproline, rhamnose, and cysteine were increased after AM supplementation, and their extending effect on the lifespan and health-span of C. elegans were partly dependent on the pmk-1 gene. In conclusion, our results revealed that AM can promote the lifespan and health-span of C. elegans via the PMK-1 pathway, highlighting the potential of AM as a dietary supplement to delay aging.

DiffN Selection of Tandem Mass Spectrometry Precursors.

Current data-dependent acquisition (DDA) approaches select precursor ions for tandem mass spectrometry (MS/MS) characterization based on their absolute intensity, known as a TopN approach. Low-abundance species may not be identified as biomarkers in a TopN approach. Herein, a new DDA approach is proposed, DiffN, which uses the relative differential intensity of ions between two samples to selectively target species undergoing the largest fold changes for MS/MS. Using a dual nano-electrospray (nESI) ionization source which allows samples contained in separate capillaries to be analyzed in parallel, the DiffN approach was developed and validated with well-defined lipid extracts. A dual nESI source and DiffN DDA approach was applied to quantify the differences in lipid abundance between two colorectal cancer cell lines. The SW480 and SW620 lines represent a matched pair from the same patient: the SW480 cells from a primary tumor and the SW620 cells from a metastatic lesion. A comparison of TopN and DiffN DDA approaches on these cancer cell samples highlights the ability of DiffN to increase the likelihood of biomarker discovery and the decreased probability of TopN to efficiently select lipid species that undergo large fold changes. The ability of the DiffN approach to efficiently select precursor ions of interest makes it a strong candidate for lipidomic analyses. This DiffN DDA approach may also apply to other molecule classes (e.g., other metabolites or proteins) that are amenable to shotgun analyses.

OA16 Identification of surrogate serum proteomic biomarkers associated with ultrasound power Doppler in psoriatic arthritis

Abstract Background/Aims An area of key unmet need in psoriatic arthritis (PsA) is the identification of biomarker(s) which predict those likely to respond to a specific therapy; in order to achieve that, an objective measure of disease activity would be ideal. Ultrasound power Doppler (PD) is a semi-quantitative measure of inflammation. However, ultrasound is time-consuming and requires specific expertise, whereas a blood-based surrogate biomarker of PD would be easier to apply clinically, and could serve as an objective outcome measure of response. Our aim is to identify proteomic biomarkers associated with PD in PsA patients. Methods Twenty-six patients with PsA (fulfilling CASPAR criteria) were recruited prospectively. All underwent 66-joint ultrasound examination and presence of PD was recorded. There were 7 patients without PD(-) and 19 with PD(+) detectable in at least 1 joint. Proteomics data for 1,073 proteins were generated using sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Proteins with high levels of missing data were filtered (>30%) followed by random forest analysis. Differentially expressed proteins (DEPs) based on PD status were identified. Protein-protein interaction network analysis was performed using the search tool for retrieval of interacting genes (STRING), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO). Results Following pre-processing, 553 proteins were available for analysis. Seventeen DEPs were identified (p-values <0.05) (Table 1). Among these, ERAP2, an enzyme involved in MHC-I ligand processing and a known risk factor for inflammatory diseases, had the largest fold change (-0.64 95%CI -1.20 to -0.07, p = 0.03). Network analysis revealed that the 17 DEPs were significantly enriched (p < 0.001) in the PD+ group. KEGG pathway analysis identified the association of the DEPs in leukocyte transendothelial migration (strength 1.5 FDR=0.04). GO analysis of the DEPs revealed involvement in immunoglobulin receptor binding (strength 2.28 FDR=0.03). Conclusion We identified several potential serum proteomic biomarkers correlated with disease activity, as assessed by the presence of PD, in PsA; these provide insights into the biological mechanisms underlying synovitis in PsA. Validation in a larger cohort and functional studies are required to determine the role of these proteins in mediating inflammation in PsA. Disclosure R.M. Hum: None. N. Nair: None. S.F. Ling: None. A. Barton: None. P. Ho: None. D. Plant: None.

Transcriptome profile in Drosophila Kc and S2 embryonic cell lines.

Drosophila melanogaster cell lines are an important resource for a range of studies spanning genomics, molecular genetics, and cell biology. Amongst these valuable lines are Kc167 (Kc) and Schneider 2 (S2) cells, which were originally isolated in the late 1960s from embryonic sources and have been used extensively to investigate a broad spectrum of biological activities including cell-cell signaling and immune system function. Whole-genome tiling microarray analysis of total RNA from these two cell types was performed as part of the modENCODE project over a decade ago and revealed that they share a number of gene expression features. Here, we expand on these earlier studies by using deep-coverage RNA-sequencing approaches to investigate the transcriptional profile in Kc and S2 cells in detail. Comparison of the transcriptomes reveals that ∼75% of the 13,919 annotated genes are expressed at a detectable level in at least one of the cell lines, with the majority of these genes expressed at high levels in both cell lines. Despite the overall similarity of the transcriptional landscape in the two cell types, 2,588 differentially expressed genes are identified. Many of the genes with the largest fold change are known only by their "CG" designations, indicating that the molecular control of Kc and S2 cell identity may be regulated in part by a cohort of relatively uncharacterized genes. Our data also indicate that both cell lines have distinct hemocyte-like identities, but share active signaling pathways and express a number of genes in the network responsible for dorsal-ventral patterning of the early embryo.

Independent Evolution Has Led to Distinct Genomic Signatures in Dutch Elm Disease-Causing Fungi and Other Vascular Wilts-Causing Fungal Pathogens.

Vascular wilts are important diseases caused by plant pathogenic fungi that result in the rapid death of their plant hosts. This is due to a systemic defense mechanism whereby the plant induces the compartmentalization of the infected vascular system in order to reduce the propagation of the fungus. The ascomycete class Sordariomycetes contains several species that cause vascular wilts in diverse plant hosts, and they can be classified into four taxonomic orders. The genetic mechanisms of pathogenesis have already been investigated in Fusarium and Verticillium species, but they have not yet been compared with other well-known wilt-causing species, especially fungi causing oak wilt or Dutch elm disease (DED). Here we analyzed 20 whole genome assemblies of wilt-causing fungi together with 56 other species using phylogenetic approaches to trace expansions and contractions of orthologous gene families and gene classes related to pathogenicity. We found that the wilt-causing pathogens evolved seven times, experiencing the largest fold changes in different classes of genes almost every time. However, some similarities exist across groups of wilt pathogens, particularly in Microascales and Ophiostomatales, and these include the common gains and losses of genes that make up secondary metabolite clusters (SMC). DED pathogens do not experience large-scale gene expansions, with most of the gene classes, except for some SMC families, reducing in number. We also found that gene family expansions in the most recent common ancestors of wilt pathogen groups are enriched for carbohydrate metabolic processes. Our study shows that wilt-causing species evolve primarily through distinct changes in their repertoires of pathogenicity-related genes and that there is the potential importance of carbohydrate metabolism genes for regulating osmosis in those pathogens that penetrate the plant vascular system.

Plasma Olink Proteomics Identifies CCL20 as a Novel Predictive and Diagnostic Inflammatory Marker for Preeclampsia.

Inflammation is generally thought to be involved in the occurrence and development of preeclampsia (PE), but its specific effect on PE remains unclear. In the present study, the expression levels of 92 inflammation-related proteins were measured in the late pregnancy maternal plasma from patients with PE (n = 15) and normal pregnant controls (n = 15) using the Olink inflammation panel based on the highly sensitive and specific proximity extension assay technology. A total of 28 inflammation-related markers differed between the PE and control groups. Among them, fibroblast growth factor 21 (FGF-21) and cysteine-cysteine motif chemokine ligand 20 (CCL20) had the largest fold changes. We further validated the levels of CCL20 in the late (43 with PE and 44 controls) and early (37 with PE and 37 controls) pregnancy maternal plasma using enzyme-linked immunosorbent assay (ELISA). To the best of our knowledge, for the first time, CCL20 was found to be upregulated in the late and early pregnancy plasma of patients with PE and had an area under the curve (AUC) of 0.753 and 0.668, respectively. In conclusion, patients with PE had increased levels of most inflammatory markers, and CCL20 might be a novel potential predictive and diagnostic biomarker for PE.

Chronic intermittent hypoxia impaired collagen synthesis in mouse genioglossus via ROS accumulation: A transcriptomic analysis

Obstructive sleep apnea (OSA) is a sleep-related breathing disorder characterized by intermittent and recurrent upper airway collapse during sleep that leads to chronic intermittent hypoxia (CIH). The genioglossus (GG) is the largest dilator muscle, which controls the upper airway and plays an important role in OSA pathology. Elucidating its genetic alterations may help identify potential targets for OSA. However, the genetic aspects of the GG in CIH mice remain unclear. Here, we have conducted an RNA sequencing (RNA-Seq) analysis to assess the differentially expressed genes (DEGs) in the GG between CIH mice and normoxia (NOR) mice. A total of 637 DEGs were identified to be dysregulated in CIH mice compared with control mice. Bioinformatics analysis showed that the DEGs were related to various physiological processes, such as the endogenous stimulus responses, cellular component organization and metabolic processes. Extracellular matrix (ECM)–receptor interaction was the top KEGG pathway in the environmental information processing category with high significance and large fold changes. From the gene weight distributions of collagen (Col)-related biological processes (BPs), we found several significant DEGs, such as Col1a1, Col1a2, Mmp2, Col3a1, Col5a1, Fmod, and Col5a2. A PPI network showed that Col1a1 was linked to ECM–receptor interactions, responses to reactive oxygen species (ROS) and Col-related BPs. It was verified in vivo and in vitro that hypoxia can induce excess ROS and reduce Col expression levels. Moreover, we found NAC can effectively scavenge ROS and restore collagen synthesis. These findings contribute to a better understanding of the mechanisms linking OSA and upper airway muscle injury and may help identify potential therapeutic targets.

Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice.

Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

Screening seven hub genes associated with prognosis and immune infiltration in glioblastoma.

Glioblastoma (GBM) is the most common and deadly primary brain tumor in adults. Diagnostic and therapeutic challenges have been raised because of poor prognosis. Gene expression profiles of GBM and normal brain tissue samples from GSE68848, GSE16011, GSE7696, and The Cancer Genome Atlas (TCGA) were downloaded. We identified differentially expressed genes (DEGs) by differential expression analysis and obtained 3,800 intersected DEGs from all datasets. Enrichment analysis revealed that the intersected DEGs were involved in the MAPK and cAMP signaling pathways. We identified seven different modules and 2,856 module genes based on the co-expression analysis. Module genes were used to perform Cox and Kaplan-Meier analysis in TCGA to obtain 91 prognosis-related genes. Subsequently, we constructed a random survival forest model and a multivariate Cox model to identify seven hub genes (KDELR2, DLEU1, PTPRN, SRBD1, CRNDE, HPCAL1, and POLR1E). The seven hub genes were subjected to the risk score and survival analyses. Among these, CRNDE may be a key gene in GBM. A network of prognosis-related genes and the top three differentially expressed microRNAs with the largest fold-change was constructed. Moreover, we found a high infiltration of plasmacytoid dendritic cells and T helper 17 cells in GBM. In conclusion, the seven hub genes were speculated to be potential prognostic biomarkers for guiding immunotherapy and may have significant implications for the diagnosis and treatment of GBM.

Circulating MicroRNAs as Potential Biomarkers for Ischemic Stroke in Patients with Asymptomatic Intracranial Artery Stenosis.

Circulating microRNAs have been shown to be biomarkers of various diseases. We aimed to investigate whether circulating microRNA can serve as a biomarker to predict ischemic stroke risk in asymptomatic intracranial artery stenosis. A total of 716 participants from the Asymptomatic Polyvascular Abnormalities Community study who had asymptomatic intracranial artery stenosis at baseline were enrolled (2010-11). Patients who suffered incident ischemic stroke were classified into the case group, and age- and sex-matched individuals without stroke were used as controls. MicroRNA microarrays were used to distinguish baseline circulating serum microRNA levels between the case and the control groups (GEO accession number GSE201860). The differentially expressed microRNAs were validated by real-time PCR. MicroRNA microarrays were performed in baseline serum samples from12 subjects who developed ischemic stroke and 12 age- and sex-matched subjects without stroke during the 2014-15 follow-up period. Twenty microRNAs were differentially expressed between the two groups (fold change > 1.3 and p < 0.05 for all). Hsa-miR-486-5p, hsa-miR-92a-3p, hsa-miR-6089 from them were selected and validated in the baseline serum samples of ten subjects with incident ischemic stroke and another ten age- and sex-matched subjects without stroke during the 2016-17 follow-up period. Hsa-miR-1225-5p, with a large fold change value and a reported relationship with cardiovascular or cerebrovascular diseases, was also validated. Ultimately, only hsa-miR-6089 was differentially downregulated among patients with intracranial artery stenosis who developed ischemic stroke (p < 0.05). In patients with asymptomatic intracranial artery stenosis, downregulated serum hsa-miR-6089 may be associated with the risk of ischemic stroke.

Identification and verification of differentially expressed key genes in peripheral blood-derived T cells between chronic immune thrombocytopenia patients and healthy controls

ABSTRACT Immune thrombocytopenia (ITP), characterized by decreased platelet counts, is a complex immune-mediated disorder with unelucidated pathogenesis. Accumulating evidence shows that T cell-mediated platelet destruction is one crucial process during the progression of ITP. Here, we attempted to identify core genes in peripheral blood-derived T-cells of chronic ITP through the analysis of microarray data (GSE43179) and clinical verification, with the aim to further understand the pathogenesis and progression of ITP. Compared with healthy controls, 97 differentially expressed genes (DEGs), including 63 up-regulated and 34 down-regulated were identified in ITP patients. Functional enrichment analysis showed that the DEGs were mainly enriched in innate immune response, inflammatory response, and IL-17 signaling pathway. Among the DEGs, top 15 hub genes ranked by degree score were identified via protein-protein interaction (PPI) network and were further confirmed by quantitative reverse transcription PCR (qRT-PCR). Among top 15 hub genes, the expression levels of 14 DEGs like TLR4, S100A8, S100A9, and S100A12 were significantly up-regulated, while one DEG IFNG was down-regulated in ITP patients. Noticeably, TLR4 exhibited the highest degree score, and S100A8 had the largest fold change in qRT-PCR analysis. Altogether, our results suggested that the pathogenesis and progression of ITP are related with multiple immune-related pathways, and that TLR4 and S100A8 are likely to play crucial roles.

Urinary Protein Array Analysis to Identify Key Inflammatory Markers in Children with IgA Vasculitis Nephritis.

Chronic kidney disease is a recognised complication of immunoglobulin A vasculitis, (IgAV; formerly Henoch–Schonlein purpura—HSP). The pathophysiology of IgAV and why some patients develop significant renal involvement remains largely unknown. Identifying urinary inflammatory markers could direct targets for earlier intervention. The aim of this cross-sectional exploratory study was to perform a large protein array analysis to identify urinary markers to provide insight into the mechanisms of kidney inflammation in children with established IgAV nephritis (IgAVN). Determination of the relative levels of 124 key proteins was performed using commercially available proteome profiler array kits. Twelve children were recruited: IgAVN, n = 4; IgAV without nephritis (IgAVwoN), n = 4; healthy controls (HCs), n = 4. The urinary concentrations of twenty proteins were significantly different in IgAVN compared to IgAVwoN. The largest fold changes were reported for B-cell activating factor (BAFF), Cripto-1, sex-hormone-binding globulin and angiotensinogen. The urinary levels of complement components C5/C5a and factor D were also significantly elevated in patients with IgAVN. A total of 69 urinary proteins significantly raised levels in comparisons made between IgAVN vs. HCs and nine proteins in IgAVwoN vs. HCs, respectively. This study identified key urinary proteins potentially involved in IgAVN providing new insight into the pathophysiology. Further longitudinal studies with larger cohorts are needed to quantitatively analyse these biomarkers.

Interpretation of exercise-induced changes in human skeletal muscle mRNA expression depends on the timing of the post-exercise biopsies.

BackgroundExercise elicits a range of adaptive responses in skeletal muscle, which include changes in mRNA expression. To better understand the health benefits of exercise training, it is important to investigate the underlying molecular mechanisms of skeletal muscle adaptation to exercise. However, most studies have assessed the molecular events at only a few time-points within a short time frame post-exercise, and the variations of gene expression kinetics have not been addressed systematically.MethodsWe assessed the mRNA expression of 23 gene isoforms implicated in the adaptive response to exercise at six time-points (0, 3, 9, 24, 48, and 72 h post exercise) over a 3-day period following a single session of high-intensity interval exercise.ResultsThe temporal patterns of target gene expression were highly variable and the expression of mRNA transcripts detected was largely dependent on the timing of muscle sampling. The largest fold change in mRNA expression of each tested target gene was observed between 3 and 72 h post-exercise.Discussion and ConclusionsOur findings highlight an important gap in knowledge regarding the molecular response to exercise, where the use of limited time-points within a short period post-exercise has led to an incomplete understanding of the molecular response to exercise. Muscle sampling timing for individual studies needs to be carefully chosen based on existing literature and preliminary analysis of the molecular targets of interest. We propose that a comprehensive time-course analysis on the exercise-induced transcriptional response in humans will significantly benefit the field of exercise molecular biology.

Cranial manipulation affects cholinergic pathway gene expression in aged rats.

Age-dependent dementia is a devastating disorder afflicting a growing older population. Although pharmacological agents improve symptoms of dementia, age-related comorbidities combined with adverse effects often outweigh their clinical benefits. Therefore, nonpharmacological therapies are being investigated as an alternative. In a previous pilot study, aged rats demonstrated improved spatial memory after osteopathic cranial manipulative medicine (OCMM) treatment. In this continuation of the pilot study, we examine the effect of OCMM on gene expression to elicit possible explanations for the improvement in spatial memory. OCMM was performed on six of 12 elderly rats every day for 7days. Rats were then euthanized to obtain the brain tissue, from which RNA samples were extracted. RNA from three treated and three controls were of sufficient quality for sequencing. These samples were sequenced utilizing next-generation sequencing from Illumina NextSeq. The Cufflinks software suite was utilized to assemble transcriptomes and quantify the RNA expression level for each sample. Transcriptome analysis revealed that OCMM significantly affected the expression of 36 genes in the neuronal pathway (false discovery rate [FDR] <0.004). The top five neuronal genes with the largest-fold change were part of the cholinergic neurotransmission mechanism, which is known to affect cognitive function. In addition, 39.9% of 426 significant differentially expressed (SDE) genes (FDR<0.004) have been previously implicated in neurological disorders. Overall, changes in SDE genes combined with their role in central nervous system signaling pathways suggest a connection to previously reported OCMM-induced behavioral and biochemical changes in aged rats. Results from this pilot study provide sufficient evidence to support a more extensive study with a larger sample size. Further investigation in this direction will provide a better understanding of the molecular mechanisms of OCMM and its potential in clinical applications. With clinical validation, OCMM could represent a much-needed low-risk adjunct treatment for age-related dementia including Alzheimer's disease.

Identification of miRNA Regulatory Networks and Candidate Markers for Fracture Healing in Mice.

Background It is important to improve the understanding of the fracture healing process at the molecular levels, then to discover potential miRNA regulatory mechanisms and candidate markers. Methods Expression profiles of mRNA and miRNA were obtained from the Gene Expression Omnibus database. We performed differential analysis, enrichment analysis, protein-protein interaction (PPI) network analysis. The miRNA-mRNA network analysis was also performed. Results We identified 499 differentially expressed mRNAs (DEmRs) that were upregulated and 534 downregulated DEmRs during fracture healing. They were mainly enriched in collagen fibril organization and immune response. Using the PPI network, we screened 10 hub genes that were upregulated and 10 hub genes downregulated with the largest connectivity. We further constructed the miRNA regulatory network for hub genes and identified 13 differentially expressed miRNAs (DEmiRs) regulators. Cd19 and Col6a1 were identified as key candidate mRNAs with the largest fold change, and their DEmiR regulators were key candidate regulators. Conclusion Cd19 and Col6a1 might serve as candidate markers for fracture healing in subsequent studies. Their expression is regulated by miRNAs and is involved in collagen fibril organization and immune responses.

Integrative analysis of long non-coding RNA and messenger RNA expression in toll-like receptor 4-primed mesenchymal stem cells of ankylosing spondylitis.

BackgroundThe precise pathogenesis of ankylosing spondylitis (AS) is still largely unknown at present. Our previous study found that toll-like receptor 4 (TLR4) downregulated and performed immunoregulatory dysfunction in mesenchymal stem cells from AS patients (AS-MSCs). The aim of this study was to explore the expression profiles of long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) in TLR4-primed AS-MSCs, and to clarify the potential mechanisms.MethodsThe immunoregulatory effects of MSCs were determined after TLR4 activation. Next, the differentially-expressed (DE) lncRNAs and mRNAs between AS-MSCs and TLR4-primed AS-MSCs [stimulated by lipopolysaccharide (LPS)] were identified via high-throughput sequencing followed by quantitative real-time PCR (qRT-PCR) confirmation. Finally, bioinformatics analyses were performed to identify the critical biological functions, signaling pathways, and associated functional networks involved in the TLR4-primed immunoregulatory function of AS-MSCs.ResultsA total of 147 DE lncRNAs and 698 DE mRNAs were identified between TLR4-primed AS-MSCs and unstimulated AS-MSCs. Of these, 107 lncRNAs were upregulated and 40 were downregulated (fold change ≥2, P<0.05), while 504 mRNAs were upregulated and 194 were downregulated (fold change ≥2, P<0.05). Five lncRNAs and five mRNAs with the largest fold changes were respectively verified by qRT-PCR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that the DE mRNAs and lncRNAs were highly associated with the inflammatory response, such as NOD-like receptor (NLR) signaling pathway, the TNF signaling pathway and the NF-κB signaling pathway. Cis-regulation prediction revealed eight novel lncRNAs, while trans-regulation prediction revealed 15 lncRNAs, respectively. Eight core pairs of lncRNA and target mRNA in the lncRNA-transcription factor (TF)-mRNA network were as follows: PACERR-PTGS2, LOC105378085-SOD2, LOC107986655-HIVEP2, MICB-DT-MICB, LOC105373925-SP140L, LOC107984251-IFIT5, LOC112268267-GBP2, and LOC101926887-IFIT3, respectively.ConclusionsTLR4 activation in AS can enhance the immunoregulatory ability of MSCs. Eight core pairs of lncRNA and target mRNA were observed in TLR4-primed AS-MSCs, which could contribute to understanding the potential mechanism of AS-MSC immunoregulatory dysfunction.