The Influence of Race/Ethnicity on the Transcriptomic Landscape of Uterine Fibroids.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2, SOX4, TNFRSF19, ACP7, GRIP1, IRS4, PLEKHG4B, PGR, COL24A1, KRT17, MMP17, SLN, CCDC177, FUT2, MYO5B, MYOG, ZNF703, CDC25A, and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Differential Expression of MED12-Associated Coding RNA Transcripts in Uterine Leiomyomas.

Recent studies have demonstrated that somatic MED12 mutations in exon 2 occur at a frequency of up to 80% and have a functional role in leiomyoma pathogenesis. The objective of this study was to elucidate the expression profile of coding RNA transcripts in leiomyomas, with and without these mutations, and their paired myometrium. Next-generation RNA sequencing (NGS) was used to systematically profile the differentially expressed RNA transcripts from paired leiomyomas (n = 19). The differential analysis indicated there are 394 genes differentially and aberrantly expressed only in the mutated tumors. These genes were predominantly involved in the regulation of extracellular constituents. Of the differentially expressed genes that overlapped in the two comparison groups, the magnitude of change in gene expression was greater for many genes in tumors bearing MED12 mutations. Although the myometrium did not express MED12 mutations, there were marked differences in the transcriptome landscape of the myometrium from mutated and non-mutated specimens, with genes regulating the response to oxygen-containing compounds being most altered. In conclusion, MED12 mutations have profound effects on the expression of genes pivotal to leiomyoma pathogenesis in the tumor and the myometrium which could alter tumor characteristics and growth potential.

Characterization of eosinophilic esophagitis variants by clinical, histological, and molecular analyses: A cross-sectional multi-center study.

OBJECTIVE: Physicians are increasingly confronted with patients presenting with symptoms of esophageal dysfunction resembling eosinophilic esophagitis (EoE), but absence of significant esophageal eosinophilia. The purpose of this study was to characterize and classify this group of EoE variants. DESIGN: Patients from six EoE-centers with symptoms of esophageal dysfunction, but peak eosinophil counts of <60/mm2 (<15/hpf) in esophageal biopsies and absence of gastro-esophageal reflux disease (GERD) were included. Clinical, endoscopic, (immuno)-histological, and molecular features were determined and compared with EoE, GERD, and healthy controls. RESULTS: We included 69 patients with EoE variants. Endoscopic abnormalities were found in 53.6%. We identified three histological subtypes: EoE-like esophagitis (36/69, 52.2%), lymphocytic esophagitis (14/69, 20.3%), and non-specific esophagitis (19/69, 27.5%). Immunohistochemistry revealed-in contrast to EoE-no significant increase in inflammatory cell infiltrates compared with GERD and healthy controls, except for lymphocytes in lymphocytic esophagitis. EoE-typical Th2-response was absent in all EoE variants. However, considerable structural changes were detected based on histology and protein expression. Using next generation mRNA sequencing, we found the three EoE variants to have distinct molecular fingerprints partially sharing pronounced traits of EoE. Hierarchical sample clustering of RNA sequencing data confirmed the presence of an EoE-like (characterized by eotaxin-3 expression), non-specific, and lymphocytic variant cluster (characterized by CD3 cells and TSLP expression). CONCLUSION: All EoE variants are clinically and histologically active conditions despite the absence of esophageal eosinophilia. EoE variants appear to be part of a disease spectrum, where classical EoE represents the most common and apparent phenotype.

Expression of cartilage oligomeric matrix protein (COMP) associates with capsular invasion in salivary gland pleomorphic adenoma.

BACKGROUND: Accumulating evidence shows that pleomorphic adenoma (PA) exhibits a unique capsular invasion and with a crucial role in recurrence. This study was designed to explore RNA expression profiles in salivary gland PA in an attempt to further analyse genes associate with capsule invasion. METHODS: We evaluated the expression profiles of 4 salivary gland PA patients by RNA-sequencing. The principal functions of the differentially expressed mRNAs (DEGs) were explored using GO and KEGG analysis. Then, RT-qPCR and correlation analyses were performed to verify the candidate DEGs in 59 PA patients, and immunohistochemical examinations were conducted to validate candidate DEGs. Finally, the COMP-related genes were screened using correlation and biological pathway enrichment analysis, and further validated by RT-qPCR. RESULTS: A total of 974 DEGs were significantly upregulated, and 1464 were downregulated (fold change ≥2.0; p < 0.05). Based on GO and KEGG analyses, extracellular matrix organization and the PI3K-Akt signalling pathway might play pivotal roles in the tumorigenesis of PA. 40 DEGs were screened and validated by RT-qPCR, 11 upregulated and 5 downregulated DEGs were consistent with the sequencing results. Cartilage oligomeric matrix protein (COMP) was identified to have a significant correlation with the capsular invasion of PA and expression of COMP in patients with invasive capsular PA was significantly stronger than PA. Finally, further results could reveal that 5 highest scoring genes were screened as hub genes for COMP. CONCLUSIONS: These findings suggested that COMP may be a prognostic target for PA and might contribute to its capsular invasion.

Single-Cell RNA Sequencing of Ovarian Cancer: Promises and Challenges.

Ovarian cancer remains the leading cause of death from gynecologic malignancy in the Western world. Tumors are comprised of heterogeneous populations of various cancer, immune, and stromal cells; it is hypothesized that rare cancer stem cells within these subpopulations lead to disease recurrence and treatment resistance. Technological advances now allow for the analysis of tumor genomes and transcriptomes at the single-cell level, which provides the resolution to potentially identify these rare cancer stem cells within the larger tumor.In this chapter, we review the evolution of next-generation RNA sequencing techniques, the methodology of single-cell isolation and sequencing, sequencing data analysis, and the potential applications in ovarian cancer. We also summarize the current published work using single-cell sequencing in ovarian cancer.By utilizing this novel technique to characterize the gene expression of rare subpopulations, new targets and treatment pathways may be identified in ovarian cancer to change treatment paradigms.

RNA-seq analysis of galaninergic neurons from ventrolateral preoptic nucleus identifies expression changes between sleep and wake.

BACKGROUND: Previous studies show that galanin neurons in ventrolateral preoptic nucleus (VLPO-Gal) are essential for sleep regulation. Here, we explored the transcriptional regulation of the VLPO-Gal neurons in sleep by comparing their transcriptional responses between sleeping mice and those kept awake, sacrificed at the same diurnal time. RESULTS: RNA-sequencing (RNA-seq) analysis was performed on eGFP(+) galanin neurons isolated using laser captured microdissection (LCM) from VLPO. Expression of Gal was assessed in our LCM eGFP(+) neurons via real time qPCR and showed marked enrichment when compared to LCM eGFP(-) cells and to bulk VLPO samples. Gene set enrichment analysis utilizing data from a recent single-cell RNA-seq study of the preoptic area demonstrated that our VLPO-Gal samples were highly enriched with galanin-expressing inhibitory neurons, but not galanin-expressing excitatory neurons. A total of 263 genes were differentially expressed between sleep and wake in VLPO-Gal neurons. When comparing differentially expressed genes in VLPO-Gal neurons to differentially expressed genes in a wake-active neuronal region (the medial prefrontal cortex), evidence indicates that both systemic and cell-specific mechanisms contribute to the transcriptional regulation in VLPO-Gal neurons. In both wake-active and sleep-active neurons, ER stress pathways are activated by wake and cold-inducible RNA-binding proteins are activated by sleep. In contrast, expression of DNA repair genes is increased in VLPO-Gal during wakefulness, but increased in wake-active cells during sleep. CONCLUSION: Our study identified transcriptomic responses of the galanin neurons in the ventrolateral preoptic nucleus during sleep and sleep deprivation. Data indicate that VLPO contains mainly sleep-active inhibitory galaninergic neurons. The VLPO galanin neurons show responses to sleep and wake similar to wake-active regions, indicating these responses, such as ER stress and cold-inducible RNA-binding proteins, are systemic affecting all neuronal populations. Region-specific differences in sleep/wake responses were also identified, in particular DNA repair. Our study expands knowledge about the transcriptional response of a distinct group of neurons essential for sleep.

Decrypting the PAK4 transcriptome profile in mammary tumor forming cells using Next Generation Sequencing.

The p-21 Activated Kinase 4 (PAK4) protein kinase is implicated in many cancers, including breast cancer. Overexpression of PAK4 is sufficient to cause mouse mammary epithelial cells (iMMECs) to become tumorigenic. To gain insight into the long-term gene expression changes that occur downstream to PAK4, we performed Next Generation Sequencing of RNA collected from PAK4 overexpressing iMMECs and wild-type iMMECs. We identified a list of genes whose expression levels were altered in response to PAK4 overexpression in iMMECs. Some of these genes, including FoxC2 and ParvB, are consistent with a role for PAK4 in cancer. In addition, PAK4 regulates many genes that are frequently associated with the inflammatory response, raising the possibility that there is a connection between PAK4, inflammation, and the tumor microenvironment. This study delineates the PAK4 transcriptome profile in transformed mammary cells and can provide translational utility in other types of cancers as well.

Identification of core gene networks and hub genes associated with progression of non-alcoholic fatty liver disease by RNA sequencing.

AIM: Non-alcoholic fatty liver disease (NAFLD) progresses because of the interaction between numerous genes. Thus, we carried out a weighted gene coexpression network analysis to identify core gene networks and key genes associated with NAFLD progression. METHODS: We enrolled 39 patients with mild NAFLD (fibrosis stages 0-2) and 21 with advanced NAFLD (fibrosis stages 3-4). Total RNA was extracted from frozen liver biopsies, and sequenced to capture a large dynamic range of expression levels. RESULTS: A total of 1777 genes differentially expressed between mild and advanced NAFLD (q-value <0.05) clustered into four modules. One module was enriched for genes that encode cell surface or extracellular matrix proteins, and are involved in cell adhesion, proliferation, and signaling. This module formed a scale-free network containing four hub genes (PAPLN, LBH, DPYSL3, and JAG1) overexpressed in advanced NAFLD. PAPLN is a component of the extracellular matrix, LBH and DPYSL3 are reported to be tumor suppressors, and JAG1 is tumorigenic. Another module formed a random network, and was enriched for genes that accumulate in the mitochondria. These genes were downregulated in advanced NAFLD, reflecting impaired mitochondrial function. However, the other two modules did not form unambiguous networks. KEGG analysis indicated that 71 differentially expressed genes were involved in "pathways in cancer". Strikingly, expression of half of all differentially expressed genes was inversely correlated with methylation of CpG sites (q-value <0.05). Among clinical parameters, serum type IV collagen 7 s was most strongly associated with the epigenetic status in NAFLD. CONCLUSIONS: Newly identified core gene networks suggest that the NAFLD liver undergoes mitochondrial dysfunction and fibrosis, and acquires tumorigenic potential epigenetically. Our data provide novel insights into the pathology and etiology of NAFLD progression, and identify potential targets for diagnosis and treatment.

Circular RNA_0025843 Alleviated Cigarette Smoke Extract Induced Bronchoalveolar Epithelial Cells Ferroptosis.

Purpose: Circular RNA (circRNA) plays an important role in various biological processes. However, their functions in cigarette smoke extract (CSE) induced human normal lung epithelial cells (BEAS-2B) injury remain vague. The study aimed to explore circRNA expression profiles and reveal their potential roles in CSE-treated BEAS-2B cells. Methods: 5% CSE exposure for 24 hours were used to build the BEAS-2B cells ferroptosis model. Differentially expressed circRNAs (DECs) were identified by next-generation RNA sequencing. Six randomly selected DECs were validated via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis were conducted to clarify the potential functions of the DECs. Furthermore, the role of hsa_circ_0025843 in CSE-related BEAS-2B cells ferroptosis was confirmed. Results: 5% CSE exposure induced BEAS-2B cells ferroptosis. Fifty-one up-regulated cirRNAs and 80 down-regulated circRNAs were revealed in CSE-treated BEAS-2B cells. Hsa_circ_0003461, hsa_circ_0007548, hsa_circ_0025843, hsa_circ_0068896, hsa_circ_0005832, and hsa_circ_0053378 were selected randomly to validate the reliability of next-generation RNA sequencing by qRT-PCR. After KEGG pathway analysis, DECs were found to participate in the process of EGFR tyrosine kinase inhibitor resistance and glycerophospholipid metabolism. The knockdown of hsa_circ_0025843 significantly alleviated CSE-induced BEAS-2B cells ferroptosis. Conclusion: The study indicated the circRNA expression profiles in CSE-treated BEAS-2B cells. Hsa_circ_0025843 alleviated CSE induced BEAS-2B cells ferroptosis, which might be a potential therapeutic target of CSE related lung injury.

Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing.

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS.

Association of epilepsy, anti-epileptic drugs (AEDs), and type 2 diabetes mellitus (T2DM): a population-based cohort retrospective study, impact of AEDs on T2DM-related molecular pathway, and via peroxisome proliferator-activated receptor γ transactivation.

Introduction: A potential association between epilepsy and subsequent type 2 diabetes mellitus (T2DM) has emerged in recent studies. However, the association between epilepsy, anti-epileptic drugs (AEDs), and the risk of T2DM development remains controversial. We aimed to conduct a nationwide, population-based, retrospective, cohort study to evaluate this relationship. Methods: We extracted data from the Taiwan Longitudinal Generation Tracking Database of patients with new-onset epilepsy and compared it with that of a comparison cohort of patients without epilepsy. A Cox proportional hazards regression model was used to analyze the difference in the risk of developing T2DM between the two cohorts. Next-generation RNA sequencing was used to characterize T2DM-related molecularchanges induced by AEDs and the T2DM-associated pathways they alter. The potential of AEDs to induce peroxisome proliferator-activated receptor γ (PPARγ) transactivation was also evaluated. Results: After adjusting for comorbidities and confounding factors, the case group (N = 14,089) had a higher risk for T2DM than the control group (N = 14,089) [adjusted hazards ratio (aHR), 1.27]. Patients with epilepsy not treated with AEDs exhibited a significantly higher risk of T2DM (aHR, 1.70) than non-epileptic controls. In those treated with AEDs, the risk of developing T2DM was significantly lower than in those not treated (all aHR ≤ 0.60). However, an increase in the defined daily dose of phenytoin (PHE), but not of valproate (VPA), increased the risk of T2DM development (aHR, 2.28). Functional enrichment analysis of differentially expressed genes showed that compared to PHE, VPA induced multiple beneficial genes associated with glucose homeostasis. Among AEDs, VPA induced the specific transactivation of PPARγ. Discussion: Our study shows epilepsy increases the risk of T2DM development, however, some AEDs such as VPA might yield a protective effect against it. Thus, screening blood glucose levels in patients with epilepsy is required to explore the specific role and impact of AEDs in the development of T2DM. Future in depth research on the possibility to repurpose VPA for the treatment of T2DM, will offer valuable insight regarding the relationship between epilepsy and T2DM.

Integrated genomic, transcriptomic and metabolomic analysis reveals MDH2 mutation-induced metabolic disorder in recurrent focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) has an over 30% risk of recurrence after kidney transplantation (Ktx) and is associated with an extremely high risk of graft loss. However, mechanisms remain largely unclear. Thus, this study identifies novel genes related to the recurrence of FSGS (rFSGS). Whole genome-wide sequencing and next-generation RNA sequencing were used to identify the candidate mutant genes associated with rFSGS in peripheral blood mononuclear cells (PBMCs) from patients with biopsy-confirmed rFSGS after KTx. To confirm the functional role of the identified gene with the MDH2 c.26C >T mutation, a homozygous MDH2 c.26C >T mutation in HMy2.CIR cell line was induced by CRISPR/Cas9 and co-cultured with podocytes, mesangial cells, or HK2 cells, respectively, to detect the potential pathogenicity of the c.26C >T variant in MDH2. A total of 32 nonsynonymous single nucleotide polymorphisms (SNPs) and 610 differentially expressed genes (DEGs) related to rFSGS were identified. DEGs are mainly enriched in the immune and metabolomic-related pathways. A variant in MDH2, c.26C >T, was found in all patients with rFSGS, which was also accompanied by lower levels of mRNA expression in PBMCs from relapsed patients compared with patients with remission after KTx. Functionally, co-cultures of HMy2.CIR cells overexpressing the mutant MDH2 significantly inhibited the expression of synaptopodin, podocin, and F-actin by podocytes compared with those co-cultured with WT HMy2.CIR cells or podocytes alone. We identified that MDH2 is a novel rFSGS susceptibility gene in patients with recurrence of FSGS after KTx. Mutation of the MDH2 c.26C >T variant may contribute to progressive podocyte injury in rFSGS patients.

Comparative Analysis of Differentially Expressed Circular RNAs in Polarized Macrophages.

Macrophage polarization is a process that macrophages exert different functions according to surrounding micro-environment. Macrophages commonly exist in two distinct subsets: classically activated M1 macrophages and alternatively activated M2 macrophages. Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated by back-splicing. Thousands of circRNAs were identified in different cells and tissues. Recent studies have revealed that circRNAs play a crucial role in regulating transcriptional and post-transcriptional gene expression. However, the effects and roles of circRNAs in macrophage polarization have not been well elucidated. Here, circRNAs expression profiles were determined in human THP-1 macrophages incubated in conditions causing activation toward M1 (interferon-γ + LPS) or M2 (interleukin-4) phenotypes. Overall, 9,720 circular RNA were detected from RNA sequencing data. Compared with M2 macrophages, a total of 140 circRNAs were aberrantly expressed in M1 macrophages, including 71 up-regulated circRNAs and 69 down-regulated circRNAs. Quantitative real-time PCR (qRT-PCR) results were generally consistent with the selected differentially expressed circRNAs. Gene Ontology (GO) and KEGG pathway analyses were used to predict biological functions and potential mechanisms of the host linear transcripts of these up-regulated and down-regulated circRNAs. Furthermore, we found that the expression level of circRNA-RNF19B (circRNF19B) in M1 macrophages was significantly higher than that in THP-1 macrophages and M2 macrophages. circRNF19B expression was increased when M2 converted to M1 whereas decreased when M1 converted to M2. Knockdown of circRNF19B following the activation of THP-1 cells using interferon-γ + LPS diminished the expression of M1 macrophages markers and elevated the expression of M2 macrophages markers. In conclusion, these data suggest the involvement of altered circRNAs expression patterns in macrophages exposure to different activating conditions. Circular RNAs may play important roles in regulating macrophage polarization.

Analysis of Annotated and Unannotated Long Noncoding RNAs from Exosome Subtypes Using Next-Generation RNA Sequencing.

Long noncoding RNAs (lncRNAs) contain >200 nucleotides and act as regulatory molecules in transcription and translation processes in both normal and pathological conditions. LncRNAs have been reported to localize in nuclei, cytoplasm, and, more recently, extracellular vesicles such as exosomes. Exosomal lncRNAs have gained much attention as exosomes secreted from one cell type can transfer their cargo (e.g., protein, RNA species, and lipids) to recipient cells and mediate phenotypic changes in the recipient cell. In recent years, many exosomal lncRNAs have been discovered and annotated and are attracting much attention as potential markers for disease diagnosis and prognosis. It is expected that many exosomal lncRNAs are yet to be identified. However, characterization of unannotated exosomal RNAs with non-protein-coding sequences from massive RNA sequencing data is technically challenging. Here, we describe a method for the discovery of annotated and unannotated exosomal lncRNA. This method includes a large-scale isolation and purification strategy for exosome subtypes, using the human colorectal cancer cell line (LIM1863) as a model. The method inputs RNA sequencing clean reads and performs transcript assembly to identify annotated and unannotated exosomal lncRNAs. Cutoffs (length, number of exon, classification code, and human protein-coding probability) are used to identify potentially novel exosomal lncRNAs. Raw read count calculation and differential expression analysis are also introduced for downstream analysis and candidate selection. Exosomal lncRNA candidates are validated using RT-qPCR. This method provides a template for exosomal lncRNA discovery and analysis from next-generation RNA sequencing.

Pathophysiological Significance of Neutrophilic Transfer RNA-Derived Small RNAs in Asymptomatic Moyamoya Disease.

Understanding asymptomatic moyamoya disease (aMMD), for which treatment options are currently limited, is key to the development of therapeutic strategies that will slow down the progression of this disease, as well as facilitate the discovery of therapeutic targets for symptomatic MMD. Newly found transfer RNA-derived small RNAs (tsRNAs) perform potential regulatory functions in neovascularization, which is a well-known pathological manifestation of MMD. In this study, the neutrophilic tsRNA transcriptome in aMMD was profiled using next-generation RNA sequencing in five patients and five matched healthy subjects. A negative binominal generalized log-linear regression was used to identify differentially expressed (DE)-tsRNAs in aMMD. Gene Ontology and functional pathway analyses were used to identify biological pathways involved with the targeted genes of the DE-tsRNAs. Four tsRNAs were selected and validated using quantitative reverse transcription polymerase chain reaction. In total, 186 tsRNAs were DE between the two groups. Pathophysiological events, including immune response, angiogenesis, axon guidance, and metabolism adjustment, were enriched for the DE-tsRNAs. The expression levels of the four DE-tsRNAs were consistent with those in the neutrophilic transcriptome. These aberrantly expressed tsRNAs and their targeted pathophysiological processes provide a basis for potential future interventions for aMMD.

Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice.

BACKGROUND: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. METHODS: The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. RESULTS: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol-HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs' generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. CONCLUSION: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

Age attenuates the transcriptional changes that occur with sleep in the medial prefrontal cortex.

Sleep abnormalities are common with aging. Studies show that sleep plays important roles in brain functions, and loss of sleep is associated with increased risks for neurological diseases. Here, we used RNA sequencing to explore effects of age on transcriptome changes between sleep and sleep deprivation (SD) in medial prefrontal cortex and found that transcriptional changes with sleep are attenuated in old. In particular, old mice showed a 30% reduction in the number of genes significantly altered between sleep/wake and, in general, had smaller magnitudes of changes in differentially expressed genes compared to young mice. Gene ontology analysis revealed differential age effects on certain pathways. Compared to young mice, many of the wake-active functions were similarly induced by SD in old mice, whereas many of the sleep-active pathways were attenuated in old mice. We found similar magnitude of changes in synaptic homeostasis genes (Fos, Arc, and Bdnf) induced by SD, suggesting intact synaptic upscaling on the transcript level during extended wakefulness with aging. However, sleep-activated processes, such as DNA repair, synaptogenesis, and axon guidance, were sensitive to the effect of aging. Old mice expressed elevated levels of immune response genes when compared to young mice, and enrichment analysis using cell-type-specific markers indicated upregulation of microglia and oligodendrocyte genes in old mice. Moreover, gene sets of the two cell types showed age-specific sleep/wake regulation. Ultimately, this study enhances understanding of the transcriptional changes with sleep and aging, providing potential molecular targets for future studies of age-related sleep abnormalities and neurological disorders.

Unique RNA signature of different lesion types in the brain white matter in progressive multiple sclerosis.

The heterogeneity of multiple sclerosis is reflected by dynamic changes of different lesion types in the brain white matter (WM). To identify potential drivers of this process, we RNA-sequenced 73 WM areas from patients with progressive MS (PMS) and 25 control WM. Lesion endophenotypes were described by a computational systems medicine analysis combined with RNAscope, immunohistochemistry, and immunofluorescence. The signature of the normal-appearing WM (NAWM) was more similar to control WM than to lesions: one of the six upregulated genes in NAWM was CD26/DPP4 expressed by microglia. Chronic active lesions that become prominent in PMS had a signature that were different from all other lesion types, and were differentiated from them by two clusters of 62 differentially expressed genes (DEGs). An upcoming MS biomarker, CHI3L1 was among the top ten upregulated genes in chronic active lesions expressed by astrocytes in the rim. TGFß-R2 was the central hub in a remyelination-related protein interaction network, and was expressed there by astrocytes. We used de novo networks enriched by unique DEGs to determine lesion-specific pathway regulation, i.e. cellular trafficking and activation in active lesions; healing and immune responses in remyelinating lesions characterized by the most heterogeneous immunoglobulin gene expression; coagulation and ion balance in inactive lesions; and metabolic changes in chronic active lesions. Because we found inverse differential regulation of particular genes among different lesion types, our data emphasize that omics related to MS lesions should be interpreted in the context of lesion pathology. Our data indicate that the impact of molecular pathways is substantially changing as different lesions develop. This was also reflected by the high number of unique DEGs that were more common than shared signatures. A special microglia subset characterized by CD26 may play a role in early lesion development, while astrocyte-derived TGFß-R2 and TGFß pathways may be drivers of repair in contrast to chronic tissue damage. The highly specific mechanistic signature of chronic active lesions indicates that as these lesions develop in PMS, the molecular changes are substantially skewed: the unique mitochondrial/metabolic changes and specific downregulation of molecules involved in tissue repair may reflect a stage of exhaustion.

Molecular signature of different lesion types in the brain white matter of patients with progressive multiple sclerosis.

To identify pathogenetic markers and potential drivers of different lesion types in the white matter (WM) of patients with progressive multiple sclerosis (PMS), we sequenced RNA from 73 different WM areas. Compared to 25 WM controls, 6713 out of 18,609 genes were significantly differentially expressed in MS tissues (FDR < 0.05). A computational systems medicine analysis was performed to describe the MS lesion endophenotypes. The cellular source of specific molecules was examined by RNAscope, immunohistochemistry, and immunofluorescence. To examine common lesion specific mechanisms, we performed de novo network enrichment based on shared differentially expressed genes (DEGs), and found TGFß-R2 as a central hub. RNAscope revealed astrocytes as the cellular source of TGFß-R2 in remyelinating lesions. Since lesion-specific unique DEGs were more common than shared signatures, we examined lesion-specific pathways and de novo networks enriched with unique DEGs. Such network analysis indicated classic inflammatory responses in active lesions; catabolic and heat shock protein responses in inactive lesions; neuronal/axonal specific processes in chronic active lesions. In remyelinating lesions, de novo analyses identified axonal transport responses and adaptive immune markers, which was also supported by the most heterogeneous immunoglobulin gene expression. The signature of the normal-appearing white matter (NAWM) was more similar to control WM than to lesions: only 465 DEGs differentiated NAWM from controls, and 16 were unique. The upregulated marker CD26/DPP4 was expressed by microglia in the NAWM but by mononuclear cells in active lesions, which may indicate a special subset of microglia before the lesion develops, but also emphasizes that omics related to MS lesions should be interpreted in the context of different lesions types. While chronic active lesions were the most distinct from control WM based on the highest number of unique DEGs (n = 2213), remyelinating lesions had the highest gene expression levels, and the most different molecular map from chronic active lesions. This may suggest that these two lesion types represent two ends of the spectrum of lesion evolution in PMS. The profound changes in chronic active lesions, the predominance of synaptic/neural/axonal signatures coupled with minor inflammation may indicate end-stage irreversible molecular events responsible for this less treatable phase.

Expression Profiling of lncRNAs, miRNAs, and mRNAs and Their Differential Expression in Leiomyoma Using Next-Generation RNA Sequencing.

The objective of this study was to identify the expression profile of long noncoding RNAs (lncRNAs) with concurrent assessment of microRNA (miRNA) and messenger RNA (mRNA) profiles in leiomyomas and paired myometrium using next-generation RNA sequencing and assembly of RNA transcripts. Total RNA was isolated from leiomyoma and paired myometrium (N = 8) and samples from 3 pairs were subjected to RNA sequencing. Normalized assembly of over 48 000 lncRNAs resulted in identification of 45 936 lncRNAs. Of these lncRNAs, 22 148 representing overlapping, intergenic, intronic, and antisense subtypes were expressed in all paired tissues, with 5941 (2813 up- and 3128 downregulated at ≥1.5 fold) differentially expressed in leiomyomas. Concurrent RNA sequencing revealed the expression of 2588 miRNAs and 21 814 mRNAs, of which 392 miRNAs and 16 559 mRNAs were expressed in all paired tissues. Of these transcripts, 56 and 92 miRNAs and 2030 and 1825 mRNAs were up- or downregulated at ≥1.5 fold, respectively, in leiomyoma as compared to myometrium. Using quantitative reverse transcription-polymerase chain reaction (QRT-PCR), we confirmed the expression of hepatocellular carcinoma upregulated (HULC), lnc-maternally expressed 3 (MEG3), long intergenic ncRNA 890 (LINC00890), TSIX, long intergenic ncRNA 473 (LINC00473), lnc-KLF9-1, and lnc-POTEM-3 (lncRNA-ATB) in leiomyoma and matched myometrium (N = 8). Collectively, the results presented here provide a comprehensive expression profile of lncRNAs in leiomyomas with concurrent integrated expression of miRNAs and mRNAs and implicate potential regulatory functions of lncRNAs through interactions with specific miRNAs and mRNAs which are known to be critical in the pathogenesis of leiomyoma.