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.

Hypomethylation and Overexpression of Th17-Associated Genes is a Hallmark of Intestinal CD4+ Lymphocytes in Crohn's Disease.

BACKGROUND: The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated. MATERIALS AND METHODS: Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells. RESULTS: CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites. CONCLUSIONS: The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells.

Host immunity and KLF 11 deficiency together promote fibrosis in a mouse model of endometriosis.

BACKGROUND: Endometriosis is a debilitating disease typically characterized by prolific fibrotic scarring. Earlier we reported downregulation of two transcription factors belonging TGF-ßR signaling pathway Sp/Krüppel-like factor 11 (KLF11) and 10 (KLF10) in human endometriosis lesions. Here we investigated the role of these nuclear factors and immunity in the scaring fibrosis associated with endometriosis. METHODS: We used a well characterized experimental mouse model of endometriosis. WT, KLF10 or KLF11 deficient mice were compared. The lesions were evaluated histologically, fibrosis was quantified with Masons' Trichome staining, immune-infiltrates were quantified by immunohistochemistry, peritoneal adhesions were score, gene expression was evaluated by bulk RNA sequencing. RESULTS: Intense fibrotic reactions and large changes in gene expression were detected in KLF11 deficient implants associated with squamous metaplasia of the ectopic endometrium, as compared to KLF10 deficient or WT implants. Fibrosis was mitigated with pharmacologic agents that blocked histone acetylation or TGF-ßR signaling or with genetic deficiency for SMAD3. The lesions were richly infiltrated with T-cells, regulatory T-cells, and innate immune cells. Fibrosis was exacerbated when implants expressed ectopic genes implicating autoimmunity as a major factor contributing to the scaring fibrosis. CONCLUSIONS: Our findings identify KLF11 and TGF-ßR signaling as cell intrinsic mechanisms and autoimmune responses as cell extrinsic mechanisms of scaring fibrosis in ectopic endometrium lesions. GENERAL SIGNIFICANCE: Immunological factors associated with inflammation and tissue repair drive scaring fibrosis in experimental endometriosis, providing the rationale for immune therapy of endometriosis.

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.

Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer.

Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases, yet is responsible for a disproportionately high percentage of breast cancer mortalities. Thus, there is an urgent need to identify novel biomarkers and therapeutic targets based on the molecular events driving TNBC pathobiology. Estrogen receptor beta (ERß) is known to elicit anti-cancer effects in TNBC, however its mechanisms of action remain elusive. Here, we report the expression profiles of ERß and its association with clinicopathological features and patient outcomes in the largest cohort of TNBC to date. In this cohort, ERß was expressed in approximately 18% of TNBCs, and expression of ERß was associated with favorable clinicopathological features, but correlated with different overall survival outcomes according to menopausal status. Mechanistically, ERß formed a co-repressor complex involving enhancer of zeste homologue 2/polycomb repressive complex 2 (EZH2/PRC2) that functioned to suppress oncogenic NFκB/RELA (p65) activity. Importantly, p65 was shown to be required for formation of this complex and for ERß-mediated suppression of TNBC. Our findings indicate that ERß+ tumors exhibit different characteristics compared to ERß- tumors and demonstrate that ERß functions as a molecular switch for EZH2, repurposing it for tumor suppressive activities and repression of oncogenic p65 signaling.

[Prognostic value of olfactory bulb volume in patients with post-viral olfactory dysfunction].

Objective:The purpose of this study was to compare the olfactory function examination results of patients with post-viral olfactory dysfunction（PVOD） in different prognostic groups and analyze prognostic factors, especially the influence of olfactory bulb volume（OBV） on prognosis, so as to provide objective basis for clinical diagnosis and treatment. Methods:After approval by the hospital ethics committee, the patients with PVOD admitted to Beijing Anzhen Hospital's outpatient department from January 2019 to December 2019 were followed up for at least 1 year. These patients completed the Sniffin' Sticks test and MRI examination of the olfactory pathway before treatment. According to the results of the Sniffin' Sticks test after 1 year follow-up（threshold-discrimination-identification（TDI） score of the patients was increased at least 6 points）, the patients were divided into two groups as the improvement group and the non-improvement group. The prognostic factors of PVOD patients were preliminarily determined by comparing the differences of various factors and the results of olfactory function examination between the two groups. Results:In this study, 47 patients with PVOD were included, with the smell improvement rate was 53.2%. Compared with the improvement group, the patients in the non-improvement group had longer duration, poorer initial olfactory function, higher olfactory threshold, and poorer olfactory discrimination and recognition ability（All P<0.01）. There was no statistical difference in terms of gender, age, allergic rhinitis and smoking between the two groups（All P>0.05）.The OBV of the non-improvement group was （59.48±23.92） mm³, which was significantly lower than that in the improvement group（[92.77±14.35]mm³, P<0.001）. Multiple logistic regression analysis showed that prognostic factors included course of disease（OR 0.677, 95%CI 0.461-0.993, P=0.046）, initial T value（OR 263.806, 95%CI 1.028-67 675.884, P=0.049） and OBV（OR 1.160, 95%CI 1.002-1.343, P=0.047）. The area under the receiver operating characteristic curve（ROC curve） of OBV was 0.888（0.797-0.979, P<0.001）. The correct diagnostic index of OBV≥78.50 mm³was used to determine the prognosis of olfactory function, with a specificity of 0.818 and a sensitivity of 0.840. The ROC curve analysis showed that the area under the ROC curve of duration was 0.822（0.703-0.940, P<0.001）. The correct diagnostic index of the duration ≤6 months was used to determine the prognosis of olfactory function, with a specificity of 0.727 and a sensitivity of 0.800. The area of T score was 0.793（0.662-0.924, P=0.001）. T score ≥1.25 was used as the correct diagnostic index to determine the prognosis of olfactory function. The specificity and sensitivity were 0.818 and 0.680, respectively. Conclusion:The prognosis of olfactory function in PVOD patients is related to the course of disease, the degree of olfactory loss and OBV. Those with no improvement in olfactory function have a longer disease course, aggravated olfactory damage and reduced OBV than those with improved olfactory function. The factors of Duration ≤6 months, T value ≥1.25 and OBV≥78.50 mm³suggested better prognosis, and the results of objective olfactory examination have greater value in evaluating the prognosis of olfactory function.

[Predictors of posttreatment olfactory improvement in patients with postviral olfactory dysfunction].

Objective:To analyzed the results of olfactory function test in patients with post-viral olfactory dysfunction（PVOD）, and evaluated the prognostic factors, so as to provide a basis for clinical diagnosis and treatment. Methods:This study included patients who were diagnosed with PVOD at least one year ago in Beijing Anzhen Hospital and whose telephone interviews of subjective olfactory function were available. The general condition of the patients, the results Sniffin' Sticks olfactory test and the event-related potentials（ERPs） were analyzed in different improvement groups. This study retrospectively analyzed PVOD patients treated in the outpatient department of Beijing Anzhen Hospital. They were given olfactory training for 4 months. The Sniffin' Sticks test was performed on the patients before and after the treatment. The Sniffin' Sticks test and event-related potentials（ERPs） results were used to evaluate the prognostic factors. Results:In this study, the olfactory improvement rate of 63 PVOD patients was 52.38%（33/63）. Compared to the non-improvement group, the course of disease in the group with improved subjective olfactory function was significantly shorter（P<0.001）, the initial olfactory function was significantly better（P<0.001）, and the olfactory threshold was much lower（P<0.001）. The presence of olfactory event-related potentials and trigeminal ERPs（tERPs） were 52.38%（33/63） and 87.30%（55/63）, respectively. The presence of oERPs in the olfactory function improvement group was significantly higher than that in the non-improvement group（P<0.05）, but there was no difference in the presence of tERPs（P>0.05）. Latency of N1 and P2 waves in oERPs with improvement group（ON1L, OP2L） were longer than those in the non-improvement group（P<0.05）, N1 and P2 wave amplitudes（ON1A, OP2A） had no difference（P>0.05）. The N1 and P2 amplitudes and latency of tERPs showed no difference between the two groups. Multivariate Logistic regression analysis showed that threshold value before treatment（OR=21.376, 95%CI: 2.172-210.377, P=0.009）; ON1L（OR=0.994, 95%CI: 0.988-0.999, P=0.029） and course of disease（OR=0.607, 95%CI: 0.405-0.920, P=0.016） was significantly associated with olfactory prognosis. Conclusion:The course of olfactory dysfunction, the severity of olfactory dysfunction, the threshold of olfactory function, and the latency of N1 wave of oERPs can be used to evaluate the prognosis of PVOD patients. However, age, olfactory discrimination, recognition ability, oERPs amplitude and tERPs wave value had less prognostic value.

BMI1 maintains the Treg epigenomic landscape to prevent inflammatory bowel disease.

FOXP3+ Tregs are expanded within the inflamed intestine of human Crohn's disease, yet FOXP3-mediated gene repression within these cells is lost. The polycomb repressive complexes play a role in FOXP3 target gene regulation, but deeper mechanistic insight is incomplete. We have now specifically identified the polycomb-repressive complex 1 (PRC1) family member, BMI1 in the regulation of a proinflammatory enhancer network in both human and murine Tregs. Using human Tregs and lamina propria T cells, we inferred PRC1 to regulate Crohn's associated gene networks through assays of chromatin accessibility. Conditional deletion of BMI1 in murine FOXP3+ cells led to systemic inflammation. BMI1-deficient Tregs beared a TH1/TH17-like phenotype as assessed by assays of genome wide transcription, chromatin accessibility and proteomic techniques. Finally, BMI1 mutant FOXP3+ cells did not suppress colitis in the adoptive transfer model of human inflammatory bowel disease. We propose that BMI1 plays an important role in enforcing Treg identity in vitro and in vivo. Loss of Treg identity via genetic or transient BMI1 depletion perturbs the epigenome and converts Tregs into Th1/Th17-like proinflammatory cells, a transition relevant to human Crohn's disease associated CD4+ T cells.

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.

RNF20 and RNF40 regulate vitamin D receptor-dependent signaling in inflammatory bowel disease.

Despite the identification of several genetic factors linked to increased susceptibility to inflammatory bowel disease (IBD), underlying molecular mechanisms remain to be elucidated in detail. The ubiquitin ligases RNF20 and RNF40 mediate the monoubiquitination of histone H2B at lysine 120 (H2Bub1) and were shown to play context-dependent roles in the development of inflammation. Here, we aimed to examine the function of the RNF20/RNF40/H2Bub1 axis in intestinal inflammation in IBD patients and mouse models. For this purpose, intestinal sections from IBD patients were immunohistochemically stained for H2Bub1. Rnf20 or Rnf40 were conditionally deleted in the mouse intestine and mice were monitored for inflammation-associated symptoms. Using mRNA-seq and chromatin immunoprecipitation (ChIP)-seq, we analyzed underlying molecular pathways in primary intestinal epithelial cells (IECs) isolated from these animals and confirmed these findings in IBD resection specimens using ChIP-seq.The majority (80%) of IBD patients displayed a loss of H2Bub1 levels in inflamed areas and the intestine-specific deletion of Rnf20 or Rnf40 resulted in spontaneous colorectal inflammation in mice. Consistently, deletion of Rnf20 or Rnf40 promoted IBD-associated gene expression programs, including deregulation of various IBD risk genes in these animals. Further analysis of murine IECs revealed that H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes is RNF20/40-dependent. Finally, these effects were confirmed in a subgroup of Crohn's disease patients which displayed epigenetic and expression changes in RNF20/40-dependent gene signatures. Our findings reveal that loss of H2B monoubiquitination promotes intestinal inflammation via decreased VDR activity thereby identifying RNF20 and RNF40 as critical regulators of IBD.

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.

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.

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.

Identification of Viruses in Patients With Postviral Olfactory Dysfunction by Multiplex Reverse-Transcription Polymerase Chain Reaction.

OBJECTIVES/HYPOTHESIS: To investigate causative viruses in patients with postviral olfactory disorders (PVOD). STUDY DESIGN: Case-control study. METHODS: One hundred fifty-one consecutive patients diagnosed with PVOD were enrolled, and samples from 38 patients who visited the doctor within 3 months of symptom onset were collected and analyzed. Thirty-two individuals who underwent surgery for nasal septal deviation during the same time period were collected as the control group. The Sniffin' Sticks psychophysical olfactory test was used to evaluate olfactory function. Olfactory cleft specimens were collected using nasopharyngeal flocked swabs (COPAN FLOQSwabs). Eighteen viruses were tested for with the Luminex xTAG RVP FAST v2 Assay Kit. RESULTS: Out of the 38 patients with PVOD, rhinoviruses were detected in 13 patients, and coronavirus OC43 was detected in one patient. The frequency of positive virus detection in the patients with anosmia was higher than in those with hyposmia (58.8% vs. 19.0%, P = 0.018). In control group, rhinovirus was identified in one patient (3.1%). Nasal obstruction was the most common symptom and was experienced by 71.0% of patients. CONCLUSIONS: Rhinovirus and coronavirus are more commonly identified in PVOD. Our methods represent an approach to screen for viruses that may be involved in PVOD. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:158-164, 2021.

Transcriptome Profiling of Acquired Gefitinib Resistant Lung Cancer Cells Reveals Dramatically Changed Transcription Programs and New Treatment Targets.

Background: Targeted therapy for lung cancer with epidermal growth factor receptor (EGFR) mutations with tyrosine kinase inhibitors (TKIs) represents one of the major breakthroughs in lung cancer management. However, gradually developed resistance to these drugs prevents sustained clinical benefits and calls for resistant mechanism research and identification of new therapeutic targets. Acquired T790M mutation accounts for the majority of resistance cases, yet transcriptome changes in these cells are less characterized, and it is not known if new treatment targets exist by available drugs. Methods: Transcriptome profiling was performed for lung cancer cell line PC9 and its resistant line PC9GR after long-term exposure to gefitinib through RNA sequencing. Differentially expressed genes and changed pathways were identified along with existing drugs targeting these upregulated genes. Using 144 lung cancer cell lines with both gene expression and drug response data from the cancer cell line encyclopedia (CCLE) and Cancer Therapeutics Response Portal (CTRP), we screened 549 drugs whose response was correlated with these upregulated genes in PC9GR cells, and top drugs were evaluated for their response in both PC9 and PC9GR cells. Results: In addition to the acquired T790M mutation, the resistant PC9GR cells had very different transcription programs from the sensitive PC9 cells. Multiple pathways were changed with the top ones including TNFA signaling, androgen/estrogen response, P53 pathway, MTORC1 signaling, hypoxia, and epithelial mesenchymal transition. Thirty-two upregulated genes had available drugs that can potentially be effective in treating the resistant cells. From the response profiles of CCLE, we found 17 drugs whose responses were associated with at least four of these upregulated genes. Among the four drugs evaluated (dasatinib, KPT-185, trametinib, and pluripotin), all except trametinib demonstrated strong inhibitory effects on the resistant PC9GR cells, among which KPT185 was the most potent. KPT-185 suppressed growth, caused apoptosis, and inhibited migration of the PC9GR cells at similar (or better) rates as the sensitive PC9 cells in a dose-dependent manner. Conclusions: Acquired TKI-resistant lung cancer cells (PC9GR) have dramatically changed transcription and pathway regulation, which expose new treatment targets. Existing drugs may be repurposed to treat those patients with developed resistance to TKIs.

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.

Protein expression shift and potential diagnostic markers through proteomics profiling of tuberculous pleurisy biopsy tissues.

OBJECTIVES: Tuberculous pleurisy is a common type of tuberculosis (TB), but its diagnosis is challenging. This study aimed to profile the protein expression of this disease and identify new diagnostic makers. METHODS: Biopsy tissues from patients with tuberculous pleurisy and controls were taken through thoracoscopy, and proteins were extracted for Tandem Mass Tag Mass Spectrometry. Differential protein expression was performed between patients and controls, and the identified proteins were analyzed for pathway enrichment. Selected proteins were further validated in another set of samples using a more quantitative method. RESULTS: A total of 5101 proteins were detected and quantified in a discovery set of patients and controls. Overall protein expression was quite different between patients and controls. Most proteins were down-expressed, while a minority were overly expressed in the patient samples. At p value < 0.05 and absolute fold change >2, 295 proteins were found to be up-expressed and 608 down-expressed. The top enriched pathways included ECM-receptor interaction, complement and coagulation cascades and focal adhesion. All 19 selected candidates were validated in an independent set of patient and control samples. CONCLUSION: This unbiased proteomics approach not only provided unique insights into protein expression and pathways, but also discovered potential diagnostic markers for tuberculous pleurisy.