Discordant Phenotypes of Nephritis in Patients with X-linked Agammaglobulinemia.

PURPOSE: To define the clinical and histological characteristics of nephritis in patients with X-linked agammaglobulinemia (XLA) and their immunological profiles. METHODS: The clinical, immunological, and histological findings of nine patients with XLA and nephritis were retrospectively analyzed. RESULTS: Based on kidney histological findings, patients with XLA and nephritis could be divided into two groups, viz., chronic glomerulonephritis (CGN) and tubulointerstitial nephritis (TIN). The two groups showed different immunological profiles. Patients in the CGN group exhibited an atypical immunological profile of XLA, with pathogenic leaky B cells producing immunoglobulins that may play a role in forming immune complexes and causing immune-mediated glomerulonephritis. In contrast, patients in the TIN group exhibited a typical immunological profile of XLA, suggesting that antibody-independent/other BTK-dependent mechanisms, or immunoglobulin replacement therapy (IgRT)-related immune/nonimmune-mediated nephrotoxicity causes TIN. CONCLUSION: Nephritis occurring in patients with XLA could have links between their renal pathology and immunological status. Careful observation is recommended to detect kidney pathology in patients with XLA on IgRT.

CXCR7 activation evokes the anti-PD-L1 antibody against glioblastoma by remodeling CXCL12-mediated immunity.

The interaction between glioblastoma cells and glioblastoma-associated macrophages (GAMs) influences the immunosuppressive tumor microenvironment, leading to ineffective immunotherapies. We hypothesized that disrupting the communication between tumors and macrophages would enhance the efficacy of immunotherapies. Transcriptomic analysis of recurrent glioblastoma specimens indicated an enhanced neuroinflammatory pathway, with CXCL12 emerging as the top-ranked gene in secretory molecules. Single-cell transcriptome profiling of naïve glioblastoma specimens revealed CXCL12 expression in tumor and myeloid clusters. An analysis of public glioblastoma datasets has confirmed the association of CXCL12 with disease and PD-L1 expression. In vitro studies have demonstrated that exogenous CXCL12 induces pro-tumorigenic characteristics in macrophage-like cells and upregulated PD-L1 expression through NF-κB signaling. We identified CXCR7, an atypical receptor for CXCL12 predominantly present in tumor cells, as a negative regulator of CXCL12 expression by interfering with extracellular signal-regulated kinase activation. CXCR7 knockdown in a glioblastoma mouse model resulted in worse survival outcomes, increased PD-L1 expression in GAMs, and reduced CD8+ T-cell infiltration compared with the control group. Ex vivo T-cell experiments demonstrated enhanced cytotoxicity against tumor cells with a selective CXCR7 agonist, VUF11207, reversing GAM-induced immunosuppression in a glioblastoma cell-macrophage-T-cell co-culture system. Notably, VUF11207 prolonged survival and potentiated the anti-tumor effect of the anti-PD-L1 antibody in glioblastoma-bearing mice. This effect was mitigated by an anti-CD8ß antibody, indicating the synergistic effect of VUF11207. In conclusion, CXCL12 conferred immunosuppression mediated by pro-tumorigenic and PD-L1-expressing GAMs in glioblastoma. Targeted activation of glioblastoma-derived CXCR7 inhibits CXCL12, thereby eliciting anti-tumor immunity and enhancing the efficacy of anti-PD-L1 antibodies.

Kidney Cysts in Children With Alport Syndrome: A Report of 3 Cases.

Alport syndrome (AS) is a progressive hereditary kidney disease characterized by hematuria, proteinuria, and progressive kidney dysfunction accompanied by sensorineural hearing loss and ocular abnormalities. Pathogenic COL4A3-5 variants can result in different AS spectra. Further, kidney cysts have been reported in adults with AS. However, the relationship between kidney cysts and AS remains unclear. Here, we report 3 cases of AS in children that occurred with kidney cysts. The patient in case 1 was initially diagnosed with IgA nephropathy at the age of 8 years but later developed bilateral multiple kidney cysts at the age of 17 years, suggesting autosomal-dominant polycystic kidney disease. Whole-exome sequencing identified a pathogenic COL4A5 variant and confirmed the AS diagnosis. The patients in cases 2 and 3 had already been diagnosed with X-linked AS using kidney biopsy and genetic analysis. Initial kidney ultrasonography showed nephromegaly; however, kidney cyst formation was observed during their annual follow-up. Our study supports the association between AS and kidney cysts. Kidney cysts in adolescents with suspected AS should not discourage clinicians from testing for pathogenic COL4A3-COL4A5 variants. Early detection of kidney cysts is critical because it may indicate kidney disease progression.

NTRK3 exhibits a pro-oncogenic function in upper tract urothelial carcinomas.

Neurotrophic receptor tyrosine kinase 3 (NTRK3) has pleiotropic functions: it acts not only as an oncogene in breast and gastric cancers but also as a dependence receptor in tumor suppressor genes in colon cancer and neuroblastomas. However, the role of NTRK3 in upper tract urothelial carcinoma (UTUC) is not well documented. This study investigated the association between NTRK3 expression and outcomes in UTUC patients and validated the results in tests on UTUC cell lines. A total of 118 UTUC cancer tissue samples were examined to evaluate the expression of NTRK3. Survival curves were generated using Kaplan-Meier estimates, and Cox regression models were used for investigating survival outcomes. Higher NTRK3 expression was correlated with worse progression-free survival, cancer-specific survival, and overall survival. Moreover, the results of an Ingenuity Pathway Analysis suggested that NTRK3 may interact with the PI3K-AKT-mTOR signaling pathway to promote cancer. NTRK3 downregulation in BFTC909 cells through shRNA reduced cellular migration, invasion, and activity in the AKT-mTOR pathway. Furthermore, the overexpression of NTRK3 in UM-UC-14 cells promoted AKT-mTOR pathway activity, cellular migration, and cell invasion. From these observations, we concluded that NTRK3 may contribute to aggressive behaviors in UTUC by facilitating cell migration and invasion through its interaction with the AKT-mTOR pathway and the expression of NTRK3 is a potential predictor of clinical outcomes in cases of UTUC.

Dominant dystrophic epidermolysis bullosa is associated with glycolytically active GATA3+ T helper 2 cells which may contribute to pruritus in lesional skin.

BACKGROUND: Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by trauma-induced blisters and, in some individuals, intense pruritus. Precisely what causes itch in DDEB and optimal ways to reduce it have not been fully determined. OBJECTIVES: To characterize DDEB skin transcriptomes to identify therapeutic targets to reduce pruritus in patients. METHODS: Using bulk RNA sequencing, we evaluated affected and unaffected skin biopsy samples from six patients with DDEB (all with the very itchy pruriginosa subtype) and four healthy individuals. Single-cell transcriptomes of affected (n = 2) and unaffected (n = 1) DDEB skin and healthy skin (n = 2) were obtained. Dupilumab treatment was provided for three patients. RESULTS: The skin bulk transcriptome showed significant enrichment of T helper (Th)1/2 and Th17 pathways in affected DDEB skin compared with nonlesional DDEB skin and healthy skin. Single-cell transcriptomics showed an association of glycolytically active GATA3+ Th2 cells in affected DDEB skin. Treatment with dupilumab in three people with DDEB led to significantly reduced visual analogue scale (VAS) itch scores after 12 weeks (mean VAS 3.83) compared with pretreatment (mean VAS 7.83). Bulk RNAseq and quantitative polymerase chain reaction showed that healthy skin and dupilumab-treated epidermolysis bullosa (EB) pruriginosa skin have similar transcriptomic profiles and reduced Th1/Th2 and Th17 pathway enrichment. CONCLUSIONS: Single-cell RNAseq helps define an enhanced DDEB-associated Th2 profile and rationalizes drug repurposing of anti-Th2 drugs in treating DDEB pruritus.

Dominant dystrophic epidermolysis bullosa (DDEB) is a rare inherited skin disease that causes fragile skin that blisters easily, often triggered by minor injuries. These blisters are accompanied by intense itching, which can be distressing. The underlying cause of DDEB lies in genetic mutations in a gene called COL7A1. This gene encodes 'type VII collagen', a protein crucial for attaching the outer skin layer (epidermis) to the layer beneath (dermis). Although the genetic basis of DDEB is understood, the causes of itch are not known. As well as this, effective treatments for DDEB are lacking, which has driven scientists to explore innovative approaches like repurposing existing drugs. Drug repurposing involves using medications that have already been approved for other health conditions. One such drug is dupilumab, which is used for severe atopic dermatitis (eczema). Dupilumab targets immune cells called Th2 cells, which play a role in inflammation and allergies. While dupilumab has shown promise in relieving DDEB itching, the way it works in this condition is unclear. This study, carried out by a group of researchers in Taiwan, looked at gene expression in DDEB-affected and unaffected skin, and compared it to gene expression in healthy skin samples. We found heightened activity in Th2 immune cells and abnormal gene signals related to itching, similar to atopic dermatitis. These findings support using dupilumab and other anti-inflammatory drugs to alleviate itching in DDEB. Clinical trials will be crucial to evaluate the effectiveness of these drugs for managing DDEB symptoms. This research opens doors for enhanced treatment options and improving the quality of life of people living with DDEB.

Germline mutations of homologous recombination genes and clinical outcomes in pancreatic cancer: a multicenter study in Taiwan.

BACKGROUND: Cancer susceptibility germline mutations are associated with pancreatic ductal adenocarcinoma (PDAC). However, the hereditary status of PDAC and its impact on survival is largely unknown in the Asian population. METHODS: Exome sequencing was performed on 527 blood samples from PDAC individuals and analyzed for mutations in 80 oncogenic genes. Pathogenic and likely pathogenic (P/LP) germline variants were diagnosed according to the ACMG variant classification categories. The association between germline homologous recombination gene mutations (gHRmut, including BAP1, BRCA1, BRCA2, PALB2, ATM, BLM, BRIP1, CHEK2, NBN, MUTYH, FANCA and FANCC) and the treatment outcomes was explored in patients with stage III/IV diseases treated with first-line (1L) platinum-based versus platinum-free chemotherapy. RESULTS: Overall, 104 of 527 (19.7%) patients carried germline P/LP variants. The most common mutated genes were BRCA2 (3.60%), followed by ATR (2.66%) and ATM (1.9%). After a median follow-up duration of 38.3-months (95% confidence interval, 95% CI 35.0-43.7), the median overall survival (OS) was not significantly different among patients with gHRmut, non-HR germline mutations, or no mutation (P = 0.43). Among the 320 patients with stage III/IV disease who received 1L combination chemotherapy, 32 (10%) had gHRmut. Of them, patients receiving 1L platinum-based chemotherapy exhibited a significantly longer median OS compared to those with platinum-free chemotherapy, 26.1 months (95% CI 12.7-33.7) versus 9.6 months (95% CI 5.9-17.6), P = 0.001. However, the median OS of patients without gHRmut was 14.5 months (95% CI 13.2-16.9) and 12.6 months (95% CI 10.8-14.7) for patients receiving 1L platinum-based and platinum-free chemotherapy, respectively (P = 0.22). These results were consistent after adjusting for potential confounding factors including age, tumor stage, performance status, and baseline CA 19.9 in the multivariate Cox regression analysis. CONCLUSIONS: Our study showed that nearly 20% of Taiwanese PDAC patients carried germline P/LP variants. The longer survival observed in gHRmut patients treated with 1L platinum-based chemotherapy highlights the importance of germline testing for all patients with advanced PDAC at diagnosis.

Profibrotic Subsets of SPP1+ Macrophages and POSTN+ Fibroblasts Contribute to Fibrotic Scarring in Acne Keloidalis.

Acne keloidalis is a primary scarring alopecia characterized by longstanding inflammation in the scalp causing keloid-like scar formation and hair loss. Histologically, acne keloidalis is characterized by mixed leukocytic infiltrates in the acute stage followed by a granulomatous reaction and extensive fibrosis in the later stages. To further explore its pathogenesis, bulk RNA sequencing, single-cell RNA sequencing, and spatial transcriptomics were applied to occipital scalp biopsy specimens of lesional and adjacent no-lesional skin in patients with clinically active disease. Unbiased clustering revealed 19 distinct cell populations, including 2 notable populations: POSTN+ fibroblasts with enriched extracellular matrix signatures and SPP1+ myeloid cells with an M2 macrophage phenotype. Cell communication analyses indicated that fibroblasts and myeloid cells communicated by SPP1 signaling networks in lesional skin. A reverse transcriptomics in silico approach identified corticosteroids as possessing the capability to reverse the gene expression signatures of SPP1+ myeloid cells and POSTN+ fibroblasts. Intralesional corticosteroid injection greatly reduced SPP1 and POSTN gene expression as well as acne keloidalis disease activity. Spatial transcriptomics and immunofluorescence staining verified microanatomic specificity of SPP1+ myeloid cells and POSTN+ fibroblasts with disease activity. In summary, the communication between POSTN+ fibroblasts and SPP1+ myeloid cells by SPP1 axis may contribute to the pathogenesis of acne keloidalis.

Polycythemia Secondary to Renal Hemangioblastoma: A Case Report and Literature Review.

Secondary polycythemia is a paraneoplastic syndrome observed in tumors with excessive erythropoietin (EPO) production. Renal cell carcinoma (RCC) and cerebellar hemangioblastoma are the 2 most well-known tumors to induce secondary polycythemia. Hemangioblastomas occurring in the kidney are rare. In this work we present a case of renal hemangioblastoma that caused erythrocytosis in a 19-year-old man. We demonstrated intratumoural EPO production by immunohistochemistry, and conducted whole-exome sequencing to evaluate possible genetic alterations that reported to induce tumor-related polycythemia. In spite of an indolent clinical behavior, renal hemangioblastoma is difficult to differentiate from RCC not only clinically, but also histopathologically. Given that RCC is the most well-known renal tumor to induce erythrocytosis, the uncommon manifestation of polycythemia in renal hemangioblastoma, as shown in our case, can cause further diagnostic challenges. Renal hemangioblastoma should be listed in the differential diagnoses of renal tumors presenting with erythrocytosis, apart from the most common RCC.

TRPM1 promotes tumor progression in acral melanoma by activating the Ca2+/CaMKIIδ/AKT pathway.

INTRODUCTION: Acral melanoma is a predominant and aggressive subtype of melanoma in non-Caucasian populations. There is a lack of genotype-driven therapies for over 50% of patients. TRPM1 (transient receptor potential melastatin 1), a nonspecific cation channel, is mainly expressed in retinal bipolar neurons and skin. Nonetheless, the function of TRPM1 in melanoma progression is poorly understood. OBJECTIVES: We investigated the association between TRPM1 and acral melanoma progression and revealed the molecular mechanisms by which TRPM1 promotes tumor progression and malignancy. METHODS: TRPM1 expression and CaMKII phosphorylation in tumor specimens were tested by immunohistochemistry analysis and scored by two independent investigators. The functions of TRPM1 and CaMKII were assessed using loss-of-function and gain-of-function approaches and examined by western blotting, colony formation, cell migration and invasion, and xenograft tumor growth assays. The effects of a CaMKII inhibitor, KN93, were evaluated using both in vitro cell and in vivo xenograft mouse models. RESULTS: We revealed that TRPM1 protein expression was positively associated with tumor progression and shorter survival in patients with acral melanoma. TRPM1 promoted AKT activation and the colony formation, cell mobility, and xenograft tumor growth of melanoma cells. TRPM1 elevated cytosolic Ca2+ levels and activated CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) to promote the CaMKIIδ/AKT interaction and AKT activation. The functions of TRPM1 in melanoma cells were suppressed by a CaMKII inhibitor, KN93. Significant upregulation of phospho-CaMKII levels in acral melanomas was related to increased expression of TRPM1. An acral melanoma cell line with high expression of TRPM1, CA11, was isolated from a patient to show the anti-tumor activity of KN93 in vitro and in vivo. CONCLUSIONS: TRPM1 promotes tumor progression and malignancy in acral melanoma by activating the Ca2+/CaMKIIδ/AKT pathway. CaMKII inhibition may be a potential therapeutic strategy for treating acral melanomas with high expression of TRPM1.

Methylation of TET2 Promoter Is Associated with Global Hypomethylation and Hypohydroxymethylation in Peripheral Blood Mononuclear Cells of Systemic Lupus Erythematosus Patients.

(1) Background: It is widely accepted that aberrant methylation patterns contribute to the development of systemic lupus erythematosus (SLE). Ten-eleven translocation (TET) methylcytosine dioxygenase is an essential enzyme of which there are three members, TET1, 2, and 3, involved in hydroxymethylation, a newly uncovered mechanism of active DNA methylation. The epigenomes of gene transcription are regulated by 5-hydroxymethylcytocine (5-hmC) and TETs, leading to dysregulation of the immune system in SLE. The purpose of this study was to investigate the global hydroxymethylation status in SLE peripheral blood mononuclear cells (PBMCs) and to explore the role of TETs in changing the patterns of methylation. (2) Methods: We collected PBMCs from 101 SLE patients and 100 healthy donors. TaqMan real-time polymerase chain-reaction assay was performed for the detection of 5-methylcytosine (5-mC), 5-hmC, and TET2 mRNA expression and single-nucleotide polymorphism genotyping. The methylation rates in different CpG sites of TET2 promoters were examined using next-generation sequencing-based deep bisulfite sequencing. Putative transcription factors were investigated using the UCSC Genome Browser on the Human Dec. 2013 (GRCh38/hg38) Assembly. (3) Results: 5-mC and 5-hmC were both decreased in SLE. The mRNA expression level of TET2 was notably high and found to be correlated with the levels of immunologic biomarkers that are indicative of SLE disease activity. The analysis of methylation rates in the TET2 promoter revealed that SLE patients had significantly higher and lower rates of methylation in TET2 105146072-154 and TET2 105146218-331, respectively. (4) Conclusions: TET2 may play an important role in 5-mC/5-hmC dynamics in the PBMCs of SLE patients. The epigenetic modification of TET2 promoters could contribute to the pathogenesis of SLE and the intensity of the immunologic reaction.

UMOD Mutations in Chronic Kidney Disease in Taiwan.

UMOD is the first identified and the most commonly mutated gene that causes autosomal dominant tubulointerstitial kidney disease (ADTKD). Recent studies have shown that ADTKD-UMOD is a relatively common cause of chronic kidney disease (CKD). However, the status of ADTKD-UMOD in Taiwan remains unknown. In this study, we identified three heterozygous UMOD missense variants, c.121T > C (p.Cys41Arg), c.179G > A (p.Gly60Asp), and c.817G > T (p.Val273Phe), in a total of 221 selected CKD families (1.36%). Two of these missense variants, p.Cys41Arg and p.Gly60Asp, have not been reported previously. In vitro studies showed that both uromodulin variants have defects in cell membrane trafficking and excretion to the culture medium. The structure model predicted altered disulfide bond formation in both variants, but only p.Gly60Asp was predicted to cause protein destabilization. Our findings extend the mutation spectrum and indicate that the ADTKD-UMOD contributed to a small but significant cause of CKD in the Taiwanese population.

PKD2 founder mutation is the most common mutation of polycystic kidney disease in Taiwan.

Autosomal Dominant polycystic kidney disease (ADPKD) is the most common inherited adult kidney disease. Although ADPKD is primarily caused by PKD1 and PKD2, the identification of several novel causative genes in recent years has revealed more complex genetic heterogeneity than previously thought. To study the disease-causing mutations of ADPKD, a total of 920 families were collected and their diagnoses were established via clinical and image studies by Taiwan PKD Consortium investigators. Amplicon-based library preparation with next-generation sequencing, variant calling, and bioinformatic analysis was used to identify disease-causing mutations in the cohort. Microsatellite analysis along with genotyping and haplotype analysis was performed in the PKD2 p.Arg803* family members. The age of mutation was calculated to estimate the time at which the mutation occurred or the founder arrived in Taiwan. Disease-causing mutations were identified in 634 families (68.9%) by detection of 364 PKD1, 239 PKD2, 18 PKHD1, 7 GANAB, and 6 ALG8 pathogenic variants. 162 families (17.6%) had likely causative but non-diagnostic variants of unknown significance (VUS). A single PKD2 p.Arg803* mutation was found in 17.8% (164/920) of the cohort in Taiwan. Microsatellite and array analysis showed that 80% of the PKD2 p.Arg803* families shared the same haplotype in a 250 kb region, indicating those families may originate from a common ancestor 300 years ago. Our findings provide a mutation landscape as well as evidence that a founder effect exists and has contributed to a major percentage of the ADPKD population in Taiwan.

SH3GLB1-related autophagy mediates mitochondrial metabolism to acquire resistance against temozolomide in glioblastoma.

BACKGROUND: The mechanism by which glioblastoma evades temozolomide (TMZ)-induced cytotoxicity is largely unknown. We hypothesized that mitochondria plays a role in this process. METHODS: RNA transcriptomes were obtained from tumor samples and online databases. Expression of different proteins was manipulated using RNA interference or gene amplification. Autophagic activity and mitochondrial metabolism was assessed in vitro using the respective cellular and molecular assays. In vivo analysis were also carried out in this study. RESULTS: High SH3GLB1 gene expression was found to be associated with higher disease grading and worse survival profiles. Single-cell transcriptome analysis of clinical samples suggested that SH3GLB1 and the altered gene levels of oxidative phosphorylation (OXPHOS) were related to subsets expressing a tumor-initiating cell signature. The SH3GLB1 protein was regulated by promoter binding with Sp1, a factor associated with TMZ resistance. Downregulation of SH3GLB1 resulted in retention of TMZ susceptibility, upregulated p62, and reduced LC3B-II. Autophagy inhibition by SH3GLB1 deficiency and chloroquine resulted in attenuated OXPHOS expression. Inhibition of SH3GLB1 in resistant cells resulted in alleviation of TMZ-enhanced mitochondrial metabolic function, such as mitochondrial membrane potential, mitochondrial respiration, and ATP production. SH3GLB1 modulation could determine tumor susceptibility to TMZ. Finally, in animal models, resistant tumor cells with SH3GLB1 knockdown became resensitized to the anti-tumor effect of TMZ, including the suppression of TMZ-induced autophagy and OXPHOS. CONCLUSIONS: SH3GLB1 promotes TMZ resistance via autophagy to alter mitochondrial function. Characterizing SH3GLB1 in glioblastoma may help develop new therapeutic strategies against this disease in the future.

Whole-exome sequencing identified mutational profiles of urothelial carcinoma post kidney transplantation.

Kidney transplantation is a lifesaving option for patients with end-stage kidney disease. In Taiwan, urothelial carcinoma (UC) is the most common de novo cancer after kidney transplantation (KT). UC has a greater degree of molecular heterogeneity than do other solid tumors. Few studies have explored genomic alterations in UC after KT. We performed whole-exome sequencing to compare the genetic alterations in UC developed after kidney transplantation (UCKT) and in UC in patients on hemodialysis (UCHD). After mapping and variant calling, 18,733 and 11,093 variants were identified in patients with UCKT and UCHD, respectively. We excluded known single-nucleotide polymorphisms (SNPs) and retained genes that were annotated in the Catalogue of Somatic Mutations in Cancer (COSMIC), in the Integrative Onco Genomic cancer mutations browser (IntOGen), and in the Cancer Genome Atlas (TCGA) database of genes associated with bladder cancer. A total of 14 UCKT-specific genes with SNPs identified in more than two patients were included in further analyses. The single-base substitution (SBS) profile and signatures showed a relative high T > A pattern compared to COMSIC UC mutations. Ingenuity pathway analysis was used to explore the connections among these genes. GNAQ, IKZF1, and NTRK3 were identified as potentially involved in the signaling network of UCKT. The genetic analysis of posttransplant malignancies may elucidate a fundamental aspect of the molecular pathogenesis of UCKT.

Single-Cell RNA Sequencing Analysis for Oncogenic Mechanisms Underlying Oral Squamous Cell Carcinoma Carcinogenesis with Candida albicans Infection.

Oral squamous cell carcinoma (OSCC) carcinogenesis involves heterogeneous tumor cells, and the tumor microenvironment (TME) is highly complex with many different cell types. Cancer cell-TME interactions are crucial in OSCC progression. Candida albicans (C. albicans)-frequently pre-sent in the oral potentially malignant disorder (OPMD) lesions and OSCC tissues-promotes malignant transformation. The aim of the study is to verify the mechanisms underlying OSCC car-cinogenesis with C. albicans infection and identify the biomarker for the early detection of OSCC and as the treatment target. The single-cell RNA sequencing analysis (scRNA-seq) was performed to explore the cell subtypes in normal oral mucosa, OPMD, and OSCC tissues. The cell composi-tion changes and oncogenic mechanisms underlying OSCC carcinogenesis with C. albicans infec-tion were investigated. Gene Set Variation Analysis (GSVA) was used to survey the mechanisms underlying OSCC carcinogenesis with and without C. albicans infection. The results revealed spe-cific cell clusters contributing to OSCC carcinogenesis with and without C. albicans infection. The major mechanisms involved in OSCC carcinogenesis without C. albicans infection are the IL2/STAT5, TNFα/NFκB, and TGFß signaling pathways, whereas those involved in OSCC carcinogenesis with C. albicans infection are the KRAS signaling pathway and E2F target down-stream genes. Finally, stratifin (SFN) was validated to be a specific biomarker of OSCC with C. albicans infection. Thus, the detailed mechanism underlying OSCC carcinogenesis with C. albicans infection was determined and identified the treatment biomarker with potential precision medicine applications.

HDAC6 involves in regulating the lncRNA-microRNA-mRNA network to promote the proliferation of glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is the most aggressive and lethal brain tumor. Although the histone deacetylase (HDAC)/transcription factor axis promotes growth in GBM, whether HDACs including HDAC6 are involved in modulating long non-coding RNAs (lncRNAs) to affect GBM malignancy remains obscure. METHODS: Integrative analysis of microarray and RNA-seq was performed to identify lncRNAs governed by HDAC6. Half-life measurement and RNA-protein pull-down assay combined with isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis were conducted to identify RNA modulators. The effect of LINC00461 on GBM malignancy was evaluated using animal models and cell proliferation-related assays. Functional analysis of the LINC00461 downstream networks was performed comprehensively using ingenuity pathway analysis and public databases. RESULTS: We identified a lncRNA, LINC00461, which was substantially increased in stem-like/treatment-resistant GBM cells. LINC00461 was inversely correlated with the survival of mice-bearing GBM and it was stabilized by the interaction between HDAC6 and RNA-binding proteins (RBPs) such as carbon catabolite repression-negative on TATA-less (CCR4-NOT) core exoribonuclease subunit 6 and fused in sarcoma. Targeting LINC00461 using azaindolylsulfonamide, an HDAC6 inhibitor, decreased cell-division-related proteins via the lncRNA-microRNA (miRNA)-mRNA networks and caused cell-cycle arrest, thereby suppressing proliferation in parental and drug-resistant GBM cells and prolonging the survival of mice-bearing GBM. CONCLUSIONS: This study sheds light on the role of LINC00461 in GBM malignancy and provides a novel therapeutic strategy for targeting the HDAC6/RBP/LINC00461 axis and its downstream effectors in patients with GBM.

Genetic and epigenetic alterations of cyclic AMP response element modulator in rheumatoid arthritis.

BACKGROUND: Genetic and epigenetic factors are strongly associated with the autoimmune disease rheumatoid arthritis (RA). Cyclic AMP response element modulator (CREM), a gene related to immune system regulation, has been implicated in various immune-mediated inflammatory processes, although it remains unknown whether CREM is involved in RA. METHODS: This study enrolled 278 RA patients and 262 controls. Three variants [rs12765063, rs17499247, rs1213386] were identified through linkage disequilibrium and expression quantitative trait locus analysis, and CREM transcript abundance was determined by quantitative real-time polymerase chain reaction. The identified variants were genotyped using the TaqMan Allelic Discrimination assay, and CREM promoter methylation was assessed by bisulphite sequencing. Differences between groups and correlations between variables were assessed with Student's t-tests and Pearson's correlation coefficients. Associations between phenotypes and genotypes were evaluated with logistic regression. RESULTS: Rheumatoid arthritis patients exhibited increased CREM expression (p < .0001), which was decreased by methotrexate (p = .0223) and biologics (p = .0001), but could not be attributed to CREM variants. Interestingly, rs17499247 displayed a significant association with serositis (p = .0377), and rs1213386 increased the risk of lymphadenopathy (p = .0398). Furthermore, seven CpG sites showed decreased methylation in RA (p = .0477~ p < .0001). CONCLUSIONS: Collectively, our results indicate that CREM hypomethylation and CREM upregulation occur in RA and that CREM variants are involved in the development of serositis and lymphadenopathy in RA. This study highlights the novel roles of CREM in RA pathophysiology.

Novel Mutations Detection with Next-Generation Sequencing and Its Association with Clinical Outcome in Unilateral Primary Aldosteronism.

Somatic mutations have been identified in adrenal tissues of unilateral primary aldosteronism (uPA). The spectrum of somatic mutations in uPAs was investigated using a customized and targeted next-generation sequencing (cNGS) approach. We also assessed whether cNGS or Sanger sequencing-identified mutations have an association with clinical outcomes in uPA. Adrenal tumoral tissues of uPA patients who underwent adrenalectomy were obtained. Conventional somatic mutation hotspots in 240 extracted DNA samples were initially screened using Sanger sequencing. A total of 75 Sanger-negative samples were further investigated by sequencing the entire coding regions of the known aldosterone-driver genes by our cNGS gene panel. Somatic mutations in aldosterone-driver genes were detected in 21 (28%) of these samples (8.8% of all samples), with 9 samples, including mutations in CACNA1D gene (12%), 5 in CACNA1H (6.6%), 3 in ATP2B3 (4%), 2 in CLCN2 (2.6%), 1 in ATP1A1 (1.3%), and 1 in CTNNB1 (1.3%). Via combined cNGS and Sanger sequencing aldosterone-driver gene mutations were detected in altogether 186 of our 240 (77.5%) uPA samples. The complete clinical success rate of patients containing cNGS-identified mutations was higher than those without mutations (odds ratio (OR) = 10.9; p = 0.012). Identification of somatic mutations with cNGS or Sanger sequencing may facilitate the prediction of complete clinical success after adrenalectomy in uPA patients.

AUY922 induces retinal toxicity through attenuating TRPM1.

BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)­based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Pyuria, urinary tract infection and renal outcome in patients with chronic kidney disease stage 3-5.

Pyuria is common in chronic kidney disease (CKD), which could be due to either urinary tract infection (UTI) or renal parenchymal inflammation. Only little is known regarding the association of pyuria or UTI with renal outcomes. We investigated 3226 patients with stage 3-5 CKD. Pyuria was defined as ≥ 50 WBC per high-power field (hpf) and was correlated to old age, female, diabetes, hypoalbuminemia, lower eGFR, and higher inflammation status. In Cox regression, patients with more than one episode of pyuria in the first year (11.8%) had increased risks for end-stage renal disease (ESRD) [hazard ratio (95% CI): 1.90 (1.58-2.28); p < 0.001], rapid renal function progression [odds ratio (95% CI): 1.49 (1.13-1.95); p = 0.001], and all-cause mortality [hazard ratio: 1.63 (1.29-2.05); p < 0.001], compared to those without pyuria. In a subgroup analysis, the risk of pyuria for ESRD was modified by CKD stages. We investigated the effects of UTI (urinary symptoms and treated by antibiotics) and pyuria without UTI (urine WBC < 50 to ≥ 10/hpf without any episodes of ≥ 50 WBC/hpf or UTI), while both groups were associated with clinical outcomes. In conclusion, CKD stage 3-5 patients with frequent pyuria or UTI episodes have increased risks of renal outcomes.