Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn's Disease.

BACKGROUND & AIMS: The incidence of Crohn's disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. We aimed to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. METHODS: We performed single-cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. RESULTS: We identified 5 distinct FOXP3+ regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to tumor necrosis factor-α signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. CONCLUSIONS: We identified a novel, proinflammatory FOXP3+ T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration-approved drug vorinostat.

Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice.

BACKGROUND & AIMS: Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. METHODS: Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing. RESULTS: HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. CONCLUSIONS: Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.

G9a Modulates Lipid Metabolism in CD4 T Cells to Regulate Intestinal Inflammation.

BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Assessment of factors influencing the olfactory bulb volume in patients with post-viral olfactory dysfunction.

PURPOSE: To investigate the factors influencing the volume of the olfactory bulb (OB) in patients with post-viral olfactory dysfunction (PVOD). METHODS: We collected 92 olfactory bulb volumes from patients with PVOD who underwent a sinus computed tomographic and magnetic resonance imaging (MRI) scan of the head and collected clinical information including gender, age, disease course, minimal cross-sectional area, nasal airway resistance, and olfactory function. OB volume was measured in MRI and the scans were evaluated according to the Lund-Mackay (LM) scoring system. RESULTS: Male patients with PVOD had a larger OB volume (ß = 0.284, P < 0.05). OB volume was smaller in patients with a longer course of olfactory dysfunction (ß = - 0.254, P < 0.05). According to the LM scoring system, patients with a higher anterior ethmoidal sinus score had smaller OB volume (ß = - 0.476, P < 0.05). CONCLUSIONS: The study revealed that gender, disease course, and the score of anterior ethmoidal sinusitis can affect the OB volume in patients with PVOD.

MCAM abnormal expression and clinical outcome associations are highly cancer dependent as revealed through pan-cancer analysis.

MCAM (CD146) is a cell surface adhesion molecule that has been reported to promote cancer development, progression and metastasis and is considered as a potential tumor biomarker and therapeutic target. However, inconsistent reports exist, and its clinical value is yet to be confirmed. Here we took advantage of several large genomic data collections (Genotype-Tissue Expression, The Cancer Genome Atlas and Cancer Cell Line Encyclopedia) and comprehensively analyzed MCAM expression in thousands of normal and cancer samples and cell lines along with their clinical phenotypes and drug response information. Our results show that MCAM is very highly expressed in large vessel tissues while majority of tissues have low or minimal expression. Its expression is dramatically increased in a few tumors but significantly decreased in most other tumors relative to their pairing normal tissues. Increased MCAM expression is associated with a higher tumor stage and worse patient survival for some less common tumors but not for major ones. Higher MCAM expression in primary tumors may be complicated by tumor-associated or normal stromal blood vessels yet its significance may differ from the one from cancer cells. MCAM expression is weakly associated with the response to a few small molecular drugs and the association with targeted anti-BRAF agents suggests its involvement in that pathway which warrants further investigation.

A novel ferroptosis phenotype-related clinical-molecular prognostic signature for hepatocellular carcinoma.

Ferroptosis is a newly identified cell death mechanism and potential biomarker for hepatocellular carcinoma (HCC) therapy; however, its clinical relevance and underlying mechanism remain unclear. In this study, transcriptome and methylome data from 374 HCC cases were investigated for 41 ferroptosis-related genes to identify ferroptosis activity-associated subtypes. These subtypes were further investigated for associations with clinical and pathological variables, gene mutation landscapes, deregulated pathways and tumour microenvironmental immunity. A gene expression signature and predictive model were developed and validated using an additional 232 HCC cases from another independent cohort. Two distinct ferroptosis phenotypes (Ferroptosis-H and Ferroptosis-L) were identified according to ferroptosis gene expression and methylation in the patients with HCC. Patients with the Ferroptosis-H had worse overall and disease-specific survival, and the molecular subtypes were significantly associated with different clinical characteristics, mRNA expression patterns, tumour mutation profiles and microenvironmental immune status. Furthermore, a 15-gene ferroptosis-related prognostic model (FPM) for HCC was developed and validated which demonstrated accurate risk stratification ability. A nomogram included the FPM risk score, ECOG PS and hepatitis B status was developed for eventual clinical translation. Our results suggest that HCC subtypes defined by ferroptosis gene expression and methylation may be used to stratify patients for clinical decision-making.

Evaluation of circulating cell-free DNA in cholestatic liver disease using liver-specific methylation markers.

BACKGROUND: Quantification of circulating organ-specific cell-free DNA (cfDNA) provides a sensitive measure of ongoing cell death that could benefit evaluation of the cholestatic liver diseases primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), which lack reliable non-invasive biomarkers. Our goal in this pilot study was to determine whether liver-specific cfDNA levels are increased in PBC and PSC patients relative to controls and in advanced versus early disease, to evaluate their potential as novel disease biomarkers. METHODS: Peripheral blood derived bisulfite-treated DNA was PCR amplified from patients with PBC (n = 48), PSC (n = 48) and controls (n = 96) to evaluate methylation status at 16 CpG sites reported to be specifically unmethylated in liver tissue near the genes IGF2R, ITIH4 and VTN. Amplicons were used to prepare paired end libraries which were sequenced on a MiSeq sequencer. Trimmed reads were aligned and used to determine unmethylation ratios and to calculate concentration of liver-specific cfDNA. Comparisons between groups were performed using the two-tailed Mann-Whitney Test and relationships between variables were evaluated using Pearson's Correlation. RESULTS: Levels of liver-specific cfDNA, as measured at the 3 genetic loci, were increased in PBC and PSC patients relative to controls and in late-stage relative to early-stage patients. As well, cfDNA levels were correlated with levels of alkaline phosphatase, a commonly used biochemical test to evaluate disease severity in liver disease, in patients, but not in controls. CONCLUSIONS: cfDNA offers promise as a non-invasive liquid-biopsy to evaluate liver-specific cell-death in patients with cholestatic liver diseases.

Prognostic value of olfactory evoked potentials in patients with post-infectious olfactory dysfunction.

PURPOSE: Prognostic assessment of patients with post-infectious olfactory dysfunction (PIOD) poses a challenge for clinicians. While there have been some studies on the prognostic factors of PIOD focusing on demographic factors, the aim of this study was to investigate whether event-related potentials (ERPs) could be used as a new predictor of olfactory recovery in PIOD. METHODS: This was a retrospective study involving patients who underwent olfactory examinations using Sniffin' Sticks test before treatment and after 1 year of follow-up. The responder group was defined by an increase of threshold-discrimination-identification (TDI) score of ≥ 6 points. All patients underwent ERP examination and the amplitude and latency of each wave of ERPs were recorded before treatment. RESULTS: A total of 61 patients (age 47.50 ± 11.04 years, 27 males) were analyzed. The presence of olfactory ERPs (oERPs) was greater in the responder group than in the non-responder group (P = 0.007), while that of trigeminal ERPs (tERPs) did not differ between the two groups (P = 0.346). Logistic-regression analyses showed that factors associated with improvement of subjective olfactory function were duration (OR, 1.604; 95% CI, 1.062-2.423; P = 0.025), initial threshold (odds ratio [OR], 0.043; 95% confidence interval [CI], 0.004-0.439; P = 0.008), and latency of N1 in oERPs (OR, 1.007; 95% CI, 1.001-1.013; P = 0.021). CONCLUSION: Our study shows that duration of OD, initial threshold, and latency of N1 in oERPs were associated with olfactory improvement in PIOD patients, which may provide guidance for clinicians.

Altered glucose metabolism of the olfactory-related cortices in anosmia patients with traumatic brain injury.

PURPOSE: Impaired brain cortices contribute significantly to the pathophysiological mechanisms of post-traumatic olfactory dysfunction (PTOD). This study aimed to use 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) to measure cerebral cortices' metabolism activity and then to explore their associations with olfaction in patients with PTOD. METHODS: Ethics committee-approved prospective studies included 15 patients with post-traumatic anosmia and 11 healthy volunteers. Olfactory function was assessed using the Sniffin' Sticks. Participants underwent 18F-FDG PET/CT scan and the image data were collected for the voxel-based whole brain analysis. Furthermore, the standardized uptake value ratio (SUVR) of the whole brain regions was measured and correlated with olfactory function. RESULTS: Patients with post-traumatic anosmia showed significantly reduced glucose metabolism in bilateral rectus, bilateral superior and medial orbitofrontal cortex (OFC), bilateral thalamus, left hippocampus and parahippocampus and left superior temporal pole (all p < 0.001). In contrast, patients with post-traumatic anosmia had significantly increased glucose metabolism in the bilateral insula (all p < 0.001). SUVR values among a total of 17 cerebral cortices including frontal, limbic, and temporal regions were significantly and positively correlated with olfactory function. The cerebral cortices with the top three correlations were the right middle frontal OFC (r = 0.765, p = 0.001), right caudate (r = 0.652, p = 0.010) and right putamen (r = 0.623, p = 0.002). CONCLUSION: Patients with post-traumatic anosmia presented with distinct patterns of brain metabolism and key cortices that highly associated with the retained olfactory function were identified. The preliminary results further support the potential use of PET imaging for precisely assessing brain metabolism in patients with PTOD.

Statistical method evaluation for differentially methylated CpGs in base resolution next-generation DNA sequencing data.

High-throughput bisulfite methylation sequencing such as reduced representation bisulfite sequencing (RRBS), Agilent SureSelect Human Methyl-Seq (Methyl-seq) or whole-genome bisulfite sequencing is commonly used for base resolution methylome research. These data are represented either by the ratio of methylated cytosine versus total coverage at a CpG site or numbers of methylated and unmethylated cytosines. Multiple statistical methods can be used to detect differentially methylated CpGs (DMCs) between conditions, and these methods are often the base for the next step of differentially methylated region identification. The ratio data have a flexibility of fitting to many linear models, but the raw count data take consideration of coverage information. There is an array of options in each datatype for DMC detection; however, it is not clear which is an optimal statistical method. In this study, we systematically evaluated four statistic methods on methylation ratio data and four methods on count-based data and compared their performances with regard to type I error control, sensitivity and specificity of DMC detection and computational resource demands using real RRBS data along with simulation. Our results show that the ratio-based tests are generally more conservative (less sensitive) than the count-based tests. However, some count-based methods have high false-positive rates and should be avoided. The beta-binomial model gives a good balance between sensitivity and specificity and is preferred method. Selection of methods in different settings, signal versus noise and sample size estimation are also discussed.

Molecular profiling reveals immunogenic cues in anaplastic large cell lymphomas with DUSP22 rearrangements.

Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell non-Hodgkin lymphomas broadly segregated into ALK-positive and ALK-negative types. Although ALK-positive ALCLs consistently bear rearrangements of the ALK tyrosine kinase gene, ALK-negative ALCLs are clinically and genetically heterogeneous. About 30% of ALK-negative ALCLs have rearrangements of DUSP22 and have excellent long-term outcomes with standard therapy. To better understand this group of tumors, we evaluated their molecular signature using gene expression profiling. DUSP22-rearranged ALCLs belonged to a distinct subset of ALCLs that lacked expression of genes associated with JAK-STAT3 signaling, a pathway contributing to growth in the majority of ALCLs. Reverse-phase protein array and immunohistochemical studies confirmed the lack of activated STAT3 in DUSP22-rearranged ALCLs. DUSP22-rearranged ALCLs also overexpressed immunogenic cancer-testis antigen (CTA) genes and showed marked DNA hypomethylation by reduced representation bisulfate sequencing and DNA methylation arrays. Pharmacologic DNA demethylation in ALCL cells recapitulated the overexpression of CTAs and other DUSP22 signature genes. In addition, DUSP22-rearranged ALCLs minimally expressed PD-L1 compared with other ALCLs, but showed high expression of the costimulatory gene CD58 and HLA class II. Taken together, these findings indicate that DUSP22 rearrangements define a molecularly distinct subgroup of ALCLs, and that immunogenic cues related to antigenicity, costimulatory molecule expression, and inactivity of the PD-1/PD-L1 immune checkpoint likely contribute to their favorable prognosis. More aggressive ALCLs might be pharmacologically reprogrammed to a DUSP22-like immunogenic molecular signature through the use of demethylating agents and/or immune checkpoint inhibitors.

Effects of a high-fat diet on intracellular calcium (Ca2+) handling and cardiac remodeling in Wistar rats without hyperlipidemia.

A high-fat diet is often associated with cardiovascular diseases. Research has suggested that consumption of a high-fat diet for 10 weeks is associated with cardiac dysfunction, including arrhythmias, through alterations in cardiac remodeling and myocardial intracellular calcium (Ca2+) handling. In this study, rats were randomly divided into two groups: the standard diet (N = 5) and high-fat diet (N = 5) groups. To evaluate the effects of a high-fat diet on cardiac remodeling, we investigated the myocardium obtained from male Wistar rats fed a high-fat diet or standard diet for ten weeks via scanning electron microscopy, polarization microscopy, and RT-PCR. We found that compared with the standard diet cohort, the high-fat diet cohort exhibited increased levels of SERCA2a and SERCA2b mRNA and a decreased level of PLB mRNA (P < .05). These findings showed that a high-fat diet may lead to cardiac upregulation of Ca2+ transport-related genes in the sarcoplasmic reticulum. Additionally, we observed endocardial injury accompanied by focal dense layered collagen, increased spacing between endocardial cells that was often filled with collagen debris, and increased amounts of collagen fibers among enlarged cardiomyocytes in the high-fat diet cohort. The abnormal intracellular calcium (Ca2+) handling and cardiac remodeling may be contributing factors in arrhythmias and sudden cardiac death in high-fat diet-fed rats.

Type 1/type 2 inflammatory cytokines correlate with olfactory function in patients with chronic rhinosinusitis.

BACKGROUND: Olfactory dysfunction secondary to chronic rhinosinusitis (CRS) has been highly associated with impaired quality of life. Asian CRS patients showed a distinct inflammatory profile, with less type 2 endotype compared with European and North American. This study aimed to explore the pattern of the inflammatory cytokines in CRS patients from China and their association with olfactory function. METHODS: Institutional review board-approved prospective study in which the olfactory function of 71 CRS patients was assessed with Sniffin' Sticks before the nasal endoscopic surgery. A set of cytokines and inflammatory mediators including type 1 and type 2 inflammatory cytokines were measured in nasal mucus by using a multiplex flow cytometric bead assay (CBA). Baseline characteristics in CRS patients were collected and the Spearman r statistic was performed to assess the association of olfactory function with cytokines and inflammatory mediators. RESULTS: A total of 71 nasal mucus samples of CRS patients, including 25 chronic rhinosinusitis without nasal polyposis (CRSsNP) patients and 46 chronic rhinosinusitis with nasal polyposis (CRSwNP) patients, were evaluated in this study. The nasal mucus levels of type 1 inflammatory cytokine IFN-γ (interferon-γ), type 2 inflammatory cytokines including IL-4, IL-5 and GM-CSF (granulocyte-macrophage colony-stimulating factor) and anti-inflammatory cytokine IL-10 were significantly and inversely correlated with olfactory function in total patients with CRS (r = -0.308, p = 0.009; r = -0.250, p = 0.036; r = -0.399, p = 0.001; r = -0.269, p = 0.023; r = -0.273, p = 0.021, respectively). In CRSsNP, the olfactory function was inversely correlated with levels of type 1 inflammatory cytokine TNF-α (tumor necrosis factor-α) (r = -0.637, p = 0.001) and IL-10 (r = -0.468, p = 0.018). Nevertheless, the olfactory function in CRSwNP was inversely correlated with type 2 inflammatory cytokines including IL-4 (r = -0.303, p = 0.041) and IL-5 (r = -0.383, p = 0.009). CONCLUSION: Both type 1 and type 2 inflammatory cytokines may contribute to the pathogenesis of CRS-associated olfactory dysfunction in the Chinese population.

Association between ALS and retroviruses: evidence from bioinformatics analysis.

BACKGROUND: Emerging evidence suggests retroviruses play a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). Specifically, activation of ancient viral genes embedded in the human genome is theorized to lead to motor neuron degeneration. We explore whether connections exist between ALS and retroviruses through protein interaction networks (PIN) and pathway analysis, and consider the potential roles in drug target discovery. Protein database and pathway/network analytical software including Ingenuity Pathway BioProfiler, STRING, and CytoScape were utilized to identify overlapping protein interaction networks and extract core cluster (s) of retroviruses and ALS. RESULTS: Topological and statistical analysis of the ALS-PIN and retrovirus-PIN identified a shared, essential protein network and a core cluster with significant connections with both networks. The identified core cluster has three interleukin molecules IL10, Il-6 and IL-1B, a central apoptosis regulator TP53, and several major transcription regulators including MAPK1, ANXA5, SQSTM1, SREBF2, and FADD. Pathway enrichment analysis showed that this core cluster is associated with the glucocorticoid receptor singling and neuroinflammation signaling pathways. For confirmation purposes, we applied the same methodology to the West Nile and Polio virus, which demonstrated trivial connectivity with ALS, supporting the unique connection between ALS and retroviruses. CONCLUSIONS: Bioinformatics analysis provides evidence to support pathological links between ALS and retroviral activation. The neuroinflammation and apoptotic regulation pathways are specifically implicated. The continuation and further analysis of large scale genome studies may prove useful in exploring genes important in retroviral activation and ALS, which may help discover new drug targets.

NUCLIZE for quantifying epigenome: generating histone modification data at single-nucleosome resolution using genuine nucleosome positions.

BACKGROUND: Defining histone modification at single-nucleosome resolution provides accurate epigenomic information in individual nucleosomes. However, most of histone modification data deposited in current databases, such as ENCODE and Roadmap, have low resolution with peaks of several kilo-base pairs (kb), which due to the technical defects of regular ChIP-Seq technology. RESULTS: To generate histone modification data at single-nucleosome resolution, we developed a novel approach, NUCLIZE, using synergistic analyses of histone modification data from ChIP-Seq and high-resolution nucleosome mapping data from native MNase-Seq. With this approach, we generated quantitative epigenomics data of single and multivalent histone modification marks in each nucleosome. We found that the dominant trivalent histone mark (H3K4me3/H3K9ac/H3K27ac) and others showed defined and specific patterns near each TSS, indicating potential epigenetic codes regulating gene transcription. CONCLUSIONS: Single-nucleosome histone modification data render epigenomic data become quantitative, which is essential for investigating dynamic changes of epigenetic regulation in the biological process or for functional epigenomics studies. Thus, NUCLIZE turns current epigenomic mapping studies into genuine functional epigenomics studies with quantitative epigenomic data.

Indel detection from RNA-seq data: tool evaluation and strategies for accurate detection of actionable mutations.

Driver somatic mutations are a hallmark of a tumor that can be used for diagnosis and targeted therapy. Mutations are primarily detected from tumor DNA. As dynamic molecules of gene activities, transcriptome profiling by RNA sequence (RNA-seq) is becoming increasingly popular, which not only measures gene expression but also structural variations such as mutations and fusion transcripts. Although single-nucleotide variants (SNVs) can be easily identified from RNA-seq, intermediate long insertions/deletions (indels > 2 bases and less than sequence reads) cause significant challenges and are ignored by most RNA-seq analysis tools. This study evaluates commonly used RNA-seq analysis programs along with variant and somatic mutation callers in a series of data sets with simulated and known indels. The aim is to develop strategies for accurate indel detection. Our results show that the RNA-seq alignment is the most important step for indel identification and the evaluated programs have a wide range of sensitivity to map sequence reads with indels, from not at all to decently sensitive. The sensitivity is impacted by sequence read lengths. Most variant calling programs rely on hard evidence indels marked in the alignment and the programs with realignment may use soft-clipped reads for indel inferencing. Based on the observations, we have provided practical recommendations for indel detection when different RNA-seq aligners are used and demonstrated the best option with highly reliable results. With careful customization of bioinformatics algorithms, RNA-seq can be reliably used for both SNV and indel mutation detection that can be used for clinical decision-making.

Apatinib preferentially inhibits PC9 gefitinib-resistant cancer cells by inducing cell cycle arrest and inhibiting VEGFR signaling pathway.

BACKGROUND: Lung cancer is one of the most common and deadly tumors around the world. Targeted therapy for patients with certain mutations, especially by use of tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR), has provided significant benefit to patients. However, gradually developed resistance to the therapy becomes a major challenge in clinical practice and an alternative to treat such patients is needed. Herein, we report that apatinib, a novel anti-angiogenic drug, effectively inhibits obtained gefitinib-resistant cancer cells but has no much effect on their parental sensitive cells. METHODS: Gefitinib-resistant lung cancer cell line (PC9GR) was established from its parental sensitive line (PC9) with a traditional EGFR mutation after long time exposure to gefitinib. Different concentrations of apatinib were used to treat PC9, PC9GR, and other two lung cancer cell lines for its anti-growth effects. RNA sequencing was performed on PC9, PC9GR, and both after apatinib treatment to detect differentially expressed genes and involved pathways. Protein expression of key cycle regulators p57, p27, CDK2, cyclin E2, and pRb was detected using Western blot. Xenograft mouse model was used to assess the anti-tumor activity of apatinib in vivo. RESULTS: The established PC9GR cells had over 250-fold increased resistance to gefitinib than its sensitive parental PC9 cells (IC50 5.311 ± 0.455 µM vs. 0.020 ± 0.003 µM). The PC9GR resistance cells obtained the well-known T790M mutation. Apatinib demonstrated much stronger ( ~ fivefold) growth inhibition on PC9GR cells than on PC9 and other two lung cancer cell lines, A549 and H460. This inhibition was mostly achieved through cell cycle arrest of PC9GR cells in G1 phase. RNA-seq revealed multiple changed pathways in PC9GR cells compared to the PC9 cells and after apatinib treatment the most changed pathways were cell cycle and DNA replication where most of gene activities were repressed. Consistently, protein expression of p57, CDK2, cyclin E2, and pRb was significantly impacted by apatinib in PC9GR cells. Oral intake of apatinib in mouse model significantly inhibited establishment and growth of PC9GR implanted tumors compared to PC9 established tumors. VEGFR2 phosphorylation in PC9GR tumors after apatinib treatment was significantly reduced along with micro-vessel formation. CONCLUSIONS: Apatinib demonstrated strong anti-proliferation and anti-growth effects on gefitinib resistant lung cancer cells but not its parental sensitive cells. The anti-tumor effect was mostly due to apatinib induced cell cycle arrest and VEGFR signaling pathway inhibition. These data suggested that apatinib may provide a benefit to patients with acquired resistance to EGFR-TKI treatment.

Correlation of tissue eosinophil count and chemosensory functions in patients with chronic rhinosinusitis with nasal polyps after endoscopic sinus surgery.

PURPOSE: To investigate the correlation of tissue eosinophil count and chemosensory functions in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) after endoscopic sinus surgery (ESS). METHODS: This was a cross-sectional study including 40 patients with a history of ESS for CRSwNP recruited consecutively. Visual analog scale score and the Lund-Kennedy endoscopic score were recorded. Biopsies of the ethmoidal sinus mucosal were performed and evaluated. Chemosensory functions were measured by Sniffin' Sticks and chemosensory event-related potentials (CSERP). The associations between chemosensory functions and tissue eosinophil count were analyzed using Spearman correlation and partial correlation after adjusting the confounding factors. Kendall's tau-b correlation was performed between sneezing score and CSERP with ethyl alcohol (EAL) stimulation. RESULTS: Olfactory and trigeminal nerve function was successfully evaluated using CSERP. Postoperative tissue eosinophil count was correlated with threshold (T) score (partial correlation coefficient r = - 0.460, p = 0.012) and CSERP peak latency for olfactory (N1: partial r = 0.471, p = 0.010; P2: partial r = 0.487, p = 0.007) and mixed olfactory-trigeminal (N1: partial r = - 0.516, p = 0.008; P2: partial r = - 0.590, p = 0.002). There were also correlations between T score and N1 latency with phenyl ethyl alcohol (PEA) (partial r = - 0.560, p < 0.001), between sneezing score and N1 latency with EAL (Kendall's tau-b = - 0.40, p = 0.005). CONCLUSIONS: Postoperative tissue eosinophilia is significantly associated with postoperative olfactory disorders as assessed by Sniffin' Sticks and CSERP peak latency. Furthermore, olfaction as measured by T score correlates with olfactory ERP latency in inflammation-associated olfactory dysfunction. Trigeminal sensitivity also appears to relate to tissue eosinophilia, indicating mucosal inflammation can affect both sensory systems in the nose.