Analysis of somatic mutations in whole blood from 200,618 individuals identifies pervasive positive selection and novel drivers of clonal hematopoiesis.

Human aging is marked by the emergence of a tapestry of clonal expansions in dividing tissues, particularly evident in blood as clonal hematopoiesis (CH). CH, linked to cancer risk and aging-related phenotypes, often stems from somatic mutations in a set of established genes. However, the majority of clones lack known drivers. Here we infer gene-level positive selection in whole blood exomes from 200,618 individuals in UK Biobank. We identify 17 additional genes, ZBTB33, ZNF318, ZNF234, SPRED2, SH2B3, SRCAP, SIK3, SRSF1, CHEK2, CCDC115, CCL22, BAX, YLPM1, MYD88, MTA2, MAGEC3 and IGLL5, under positive selection at a population level, and validate this selection pattern in 10,837 whole genomes from single-cell-derived hematopoietic colonies. Clones with mutations in these genes grow in frequency and size with age, comparable to classical CH drivers. They correlate with heightened risk of infection, death and hematological malignancy, highlighting the significance of these additional genes in the aging process.

Metabolic signature and response to glutamine deprivation are independent of p53 status in B cell malignancies.

The tumor suppressor p53 has been described to control various aspects of metabolic reprogramming in solid tumors, but in B cell malignancies that role is as yet unknown. We generated pairs of p53 functional and knockout (KO) clones from distinct B cell malignancies (acute lymphoblastic leukemia, chronic lymphocytic leukemia, diffuse large B cell lymphoma, and multiple myeloma). Metabolomics and isotope tracing showed that p53 loss did not drive a common metabolic signature. Instead, cell lines segregated according to cell of origin. Next, we focused on glutamine as a crucial energy source in the B cell tumor microenvironment. In both TP53 wild-type and KO cells, glutamine deprivation induced cell death through the integrated stress response, via CHOP/ATF4. Lastly, combining BH3 mimetic drugs with glutamine starvation emerged as a possibility to target resistant clones. In conclusion, our analyses do not support a common metabolic signature of p53 deficiency in B cell malignancies and suggest therapeutic options for exploration based on glutamine dependency.

Machine learning algorithms being an auxiliary tool to predict the overall survival of patients with renal cell carcinoma using the SEER database.

Background: The clinical prognosis assessment of renal cell carcinoma (RCC) still relies on nuclear grading and nuclear score by naked eye with microscope, which has defects long time, low efficiency, and uneven evaluation level criteria. There are few machine learning (ML) studies investigating the prognosis in the RCC literature which could also quantify the risk of postoperative recurrence of RCC patients and guide cancer patients to conduct individualized postoperative clinical management. This study evaluated the suitability of ML algorithms for survival prediction in patients with RCC. Methods: A total of 192,912 RCC patients from the Surveillance, Epidemiology, and End Results (SEER) were obtained from 2004 to 2015. Six ML algorithms including support vector machine (SVM), Bayesian method, decision tree, random forest, neural network, and Extreme Gradient Boosting (XGBoost) were applied to predict overall survival (OS) of RCC. Results: Patients from the SEER with a median age of 62 years and the pathological types were clear cell RCC (47.6%), papillary RCC (9.5%), chromophobe RCC (4.0%) and others (4.1%) were collected. In the deleting patients with missing data, the highest accurate model was XGBoost [area under the curve (AUC) 67.0%]. In the deleting patients with missing data and survival time <5 years, the accuracy of random forest, neural network and XGBoost were high, with AUC of 80.8%, 81.5% and 81.8%, respectively. In the only deleting the missing tumor diameter and filling the missing dataset with missForest, the highest accurate model was random forest (AUC: 71.9%). In this study, the overall accuracy of the SVM model was not high, apart from in the population of patients with deleting the missing tumor diameter and survival time <5 years, and filling the missing data with missForest. Random forest, neural network and XGBoost had high accuracy, with AUC of 84.1%, 84.7% and 84.8%, respectively. Conclusions: ML algorithms could be used to predict the prognosis of RCC. It could quantify the recurrence possibility of patients and help more individualized postoperative clinical management. Given the limitations and complexity of datasets, ML may be used as an auxiliary tool to analyze and process larger datasets and complex data.

Electron transport chain and mTOR inhibition synergistically decrease CD40 signaling and counteract venetoclax resistance in chronic lymphocytic leukemia.

CD40 signaling upregulates BCL-XL and MCL-1 expression in the chronic lymphocytic leukemia (CLL) lymph node microenvironment, affording resistance to the BCL-2 inhibitor, venetoclax. Venetoclax resistance in the therapeutic setting and after long-term laboratory selection has been linked to metabolic alterations, but the underlying mechanism(s) are unknown. We aimed here to discover how CD40 stimulation as a model for tumor microenvironment-mediated metabolic changes, affects venetoclax sensitivity/resistance. CD40 stimulation increased oxidative phosphorylation and glycolysis, but only inhibition of oxidative phosphorylation countered venetoclax resistance. Furthermore, blocking mitochondrial import of pyruvate, glutamine or fatty acids affected CLL metabolism, but did not prevent CD40-mediated resistance to venetoclax. In contrast, inhibition of the electron transport chain (ETC) at complex I, III or V attenuated CLL activation and ATP production, and downregulated MCL-1 and BCL-XL, correlating with reduced CD40 surface expression. Moreover, ETC inhibition equaled mTOR1/2 but not mTOR1 inhibition alone for venetoclax resistance, and all three pathways were linked to control of general protein translation. In line with this, ETC plus mTOR inhibition synergistically counteracted venetoclax resistance. These findings link oxidative CLL metabolism to CD40 expression and cellular signaling, and may hold clinical potential.

Clinicopathological characteristics, surgical treatments, and oncological outcomes of localized primary unifocal urothelial carcinoma involving the ureterovesical junction.

OBJECTIVE: To investigate clinicopathological characteristics, surgical treatments, and oncological outcomes of patients with localized primary unifocal urothelial carcinoma involving the ureterovesical junction (UC-UVJ). PATIENTS AND METHODS: Localized primary unifocal UC-UVJ cases in patients admitted to our hospital from March 2013 to August 2021 were reviewed. Clinicopathological parameters, perioperative data, and oncological outcomes were compared between patients grouped by tumor location and surgical treatment. RESULTS: A total of 130 patients with localized primary unifocal UC-UVJ were enrolled in this study. These included 72 cases of bladder cancer (BC) involving the ureteral orifice, and 58 cases of upper urinary tract urothelial carcinoma (UTUC) involving the intramural ureter. The proportion of male patients, hydronephrosis, flank pain/abdominal pain, and tumor size differed significantly between the BC and UTUC groups (all P < 0.05). During the median follow-up period of 32.9 months, 49 cases (37.7%) recurred and 29 (22.3%) died from urothelial carcinoma (UC), though no statistical difference in recurrence (P = 0.436) or cancer-specific mortality (P = 0.653) was observed between the BC and UTUC groups. Cox proportional hazards regression analysis identified age, tumor grade, and lymphovascular invasion (LVI) as independent predictors of cancer-specific survival (CSS), and sex, T stage, tumor grade, and LVI as independent predictors of recurrence-free survival (RFS). CONCLUSION: Owing to positional properties, patients with localized primary unifocal UC-UVJ exhibited significant heterogeneity, leading to varied treatment strategies. No statistically significant differences in CSS or RFS were observed between the BC and UTUC groups. Furthermore, age, sex, T stage, tumor grade, and LVI should be carefully considered in clinical practice because of their associations with CSS and RFS.

Changes of Temporomandibular Joint Morphology and Symptoms in Class II Malocclusion Patients With Bilateral Sagittal Split Ramous Osteotomy.

This study included 46 patients with class II malocclusion ranging in age from 19 to 39 years old treated with bilateral sagittal split ramous osteotomy (BSSRO). Left and right temporomandibular joints (TMJs) of each subject were evaluated independently with cone-beam computed tomography (CBCT) before operation (T1), 1 week after operation (T2), and 1 year after operation (T3) and assessed the effects of orthognathic surgery (OGS) on the temporomandibular joint disease (TMD) symptoms. Temporomandibular joint morphology evaluation included condylar volume, condylar area, cortical bone thickness, depth of the mandibular fossa, fossa thickness, joint nodule angle, joint space, and condyle-fossa relationship, which were calculated by using the Mimics software and 3-matic software. Data were statistically analyzed with SPSS software (P <0.05 means statistically significant). In our study, bilateral TMJs have no difference in T3. Bilateral sagittal split ramous osteotomy had no significant effect on the articular fossa. The condyle volume and surface area decreased from T1 to T3, but the cortical thickness of the bone did not change significantly. More anterior condyle positions in T1 and more posterior in T3.21 patients had at least 1 sign or symptom of TMD in T1 and 27 patients in T3. Four patients who were asymptomatic in T1 developed pain after surgery, 10 developed noises, 12 showed limited mouth opening, and 8 had abnormal opening patterns. It is concluded that more condylar posterior position after BSSRO and the reduction of condyle may be related to the enlargement of anterior space. The number of patients with joint symptoms increased postoperative, and the impact of BSSRO on TMD may be negative.

Cryo-EM structures of human SPCA1a reveal the mechanism of Ca2+/Mn2+ transport into the Golgi apparatus.

Secretory pathway Ca2+/Mn2+ ATPase 1 (SPCA1) actively transports cytosolic Ca2+ and Mn2+ into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the ATP2C1 gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo-electron microscopy structures of human SPCA1a in the ATP and Ca2+/Mn2+-bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca2+ and Mn2+ share the same metal ion-binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca2+-binding site in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca2+/Mn2+ transport.

Pan-Cancer Analysis of the TRP Family, Especially TRPV4 and TRPC4, and Its Expression Correlated with Prognosis, Tumor Microenvironment, and Treatment Sensitivity.

BACKGROUND: Transient receptor potential (TRP) channels are involved in various physiological, pathological, and tumorigenesis-related processes. However, only a few studies have comprehensively analyzed TRP family members and their association with prognosis and tumor microenvironment (TME) in various cancers. Thus, in this study, we focused on TRP channels in pan-cancer and screened two typical TRP channels, TRPV4 and TRPC4, as examples. METHODS: Based on the latest public databases, we evaluated the expression level and prognostic value of TRP family genes in pan-cancer tissues via various bioinformatic analytical methods, and investigated the relationship between the expression of TRP family genes with TME, stemness score, immune subtype, drug sensitivity, and immunotherapy outcome in pan-cancer tissues. RESULTS: Pan-cancer analysis revealed that the TRP family genes were differentially expressed in tumor and para-carcinoma tissues. A significant correlation existed between the expression of TRP family genes and prognosis. The expression of TRP family genes was significantly correlated with stromal, immune, RNA stemness, and DNA stemness scores in pan-cancer tissues. Our results indicated that the expression of TRP family genes correlated with the sensitivity to various drugs including PLX-4720, SB-590885, and HYPOTHEMYCIN, immunotherapy outcome, and immune-activation-related genes. Immunohistochemical analysis revealed significant differential expression of TRPV4 in bladder and para-carcinoma tissues. CONCLUSIONS: Our study elucidated the possible role of TRP family genes in cancer progression and provided insights for further studies on TRP family genes as potential pan-cancer targets to develop diagnostic and therapeutic strategies.

The Combination of Chrysin and Cisplatin Induces Apoptosis in HepG2 through Down-regulation of cFLIP and Activity of Caspase.

AIM: The study aims to investigate the combined effects of chrysin and cisplatin on hepatoma(HepG2) cell lines in vivo and in vitro. OBJECTIVE: Studies have suggested that chrysin can enhance the sensitivity of tumor cells to apoptosis. Drug resistance in tumor cells reduced the effectiveness of chemotherapy drugs such as cisplatin. We investigated whether the combination of chrysin and cisplatin can induce more apoptosis than chrysin alone and cisplatin alone. METHODS: HepG2 cells were pretreated with chrysin for 2 h, followed by the addition of cisplatin for another 24 h. The morphologic changes were observed under inverted microscope and the cell viability was measured using the MTT test. The protein and cleavage of caspase-3,8,9, PARP, and cFLIP were determined by Western blotting. RESULTS: The cell viability of the HepG2 cell can be reduced by the combination of chrysin pretreatment for 2 h and cisplatin addition for 24 h; Caspase-3,8,9 and PARP were cleaved after 12 h treatment with chrysin and cisplatin; Pancaspase inhibitor, Z-VAD-fmk, could reverse the apoptosis induced by chrysin and cisplatin in HepG2 cells; cFLIP was down-regulated by the combination of chrysin and cisplatin, and could be reversed by Z-VAD-fmk; the xenografted HepG2 cells formed a tumor in one week; At the end of the experiment, there were significant differences in relative tumor volume (RTV) and relative tumor proliferation rate between the combined group and the control group, the chrysin group and the cisplatin group; Western blotting showed that the levels of PARP, cFLIP, and caspase-3 proteins in isolated tumor tissues also decreased under the combined action of chrysin and cisplatin. CONCLUSION: The combination of chrysin and cisplatin induces apoptosis of hepatic tumor in vivo and in vitro. It downregulates cFLIP and then activates caspase-8, which triggers caspase-mediated apoptosis of HepG2 cell.

Senescence Rewires Microenvironment Sensing to Facilitate Antitumor Immunity.

Cellular senescence involves a stable cell-cycle arrest coupled to a secretory program that, in some instances, stimulates the immune clearance of senescent cells. Using an immune-competent liver cancer model in which senescence triggers CD8 T cell-mediated tumor rejection, we show that senescence also remodels the cell-surface proteome to alter how tumor cells sense environmental factors, as exemplified by type II interferon (IFNγ). Compared with proliferating cells, senescent cells upregulate the IFNγ receptor, become hypersensitized to microenvironmental IFNγ, and more robustly induce the antigen-presenting machinery-effects also recapitulated in human tumor cells undergoing therapy-induced senescence. Disruption of IFNγ sensing in senescent cells blunts their immune-mediated clearance without disabling the senescence state or its characteristic secretory program. Our results demonstrate that senescent cells have an enhanced ability to both send and receive environmental signals and imply that each process is required for their effective immune surveillance. SIGNIFICANCE: Our work uncovers an interplay between tissue remodeling and tissue-sensing programs that can be engaged by senescence in advanced cancers to render tumor cells more visible to the adaptive immune system. This new facet of senescence establishes reciprocal heterotypic signaling interactions that can be induced therapeutically to enhance antitumor immunity. See related article by Marin et al., p. 410. This article is highlighted in the In This Issue feature, p. 247.

High-Throughput Electrosynthesis of Gradient Polypyrrole Film Using a Single-Electrode Electrochemical System.

As an effective approach for materials synthesis, bipolar electrochemistry has been earning a renewed interest nowadays thanks to its unique features compared to conventional electrochemistry. Indeed, the wireless mode of electrode reactions and the generation of a gradient potential distribution above the bipolar electrode are among the most appealing qualities of bipolar electrochemistry. In particular, the gradient potential distribution is a highly attractive characteristic for the fabrication of surfaces with gradients in their chemical properties or molecular functionalities. Herein, we report the high-throughput electrosynthesis of gradient polypyrrole films by means of a new electrochemical cell design named the single-electrode electrochemical system (SEES). SEESs are made by attaching an inert plastic board with holes onto an indium tin oxide electrode, constructing multiple microelectrochemical cells on the same electrode. This type of arrangement enables parallel electrochemical reactions to be carried out simultaneously and controlled in a contactless manner by a single electrode. Several experimental conditions for polypyrrole film growth were extensively investigated. Furthermore, the gradient property of the polymer films was evaluated by thickness determination, surface morphology analysis, and contact angle measurements. The use of SEES has been demonstrated as a convenient and cost-effective strategy for high-throughput electrosynthesis and electroanalytical applications and has opened up a new door for gradient film preparation via a rapid condition screening process.

The association between serum cadmium and diabetes in the general population: A cross-sectional study from NHANES (1999-2020).

Background: Associations between serum cadmium and diabetes had been reported in previous studies, however there was still considerable controversy regarding associations. Studies in general population that investigated the effects of serum cadmium on diabetes were currently lacking. We designed this cross-sectional study among U.S. adults under high and low cadmium exposure to assess associations between serum cadmium and diabetes. Methods: This cross-sectional study analyzed 52,593 adults who aged more than 20 years and participated in the National Health and Nutrition Examination Survey (NHANES), 1999-2020. The missing values and extreme values in the covariables were filled by multiple interpolation. Univariate logistics regression, multivariate logistics regression and smooth fitting curves were used to analyze the association between serum cadmium and diabetes. Simultaneously, sensitivity analysis was carried out by converting the serum cadmium from continuous variable to categorical variable. The stratification logistics regression model was used to analyze whether there were special groups in each subgroup to test the stability of the results. Results: In this cross-sectional study, serum cadmium levels were negatively correlated with the occurrence of diabetes in the low serum cadmium exposure group (OR = 0.811, 95% CI 0.698, 0.943; P = 0.007). There was no association between serum cadmium level and the occurrence of diabetes in the high serum cadmium exposure group (OR = 1.01, 95% CI 0.982, 1.037; P = 0.511). These results were consistent across all the subgroups (P for interaction >0.05). Conclusion: Serum cadmium was negatively associated diabetes among the representative samples of the whole population in the United States under the normal level of serum cadmium exposure. However, there was no association between serum cadmium level and the occurrence of diabetes in the high serum cadmium exposure group. This study promoted an update of new preventative strategy targeting environment for the prevention and control of diabetes in the future.

Prognosis Risk Model Based on Necroptosis-Related Signature for Bladder Cancer.

BACKGROUND: Bladder cancer(BLCA) is the ninth most common cancer. In recent years, necroptosis was found to be related to the occurrence and development of tumors. In this study, we aimed to construct a model based on a necroptosis-related signature to evaluate the potential prognostic application in BLCA. METHODS: A total of 67 necroptosis-related genes were used to select the ideal cluster numbers, and it was found that there were four necroptosis-related patterns. Then, we compared the gene expression levels among all of the groups and established a necroptosis-related prognostic model. We made the following enrichment analysis of function and built a novel scoring system, the NEC score, to evaluate the state of necroptosis according to the expression level of necroptosis-related genes. RESULTS: A total of 67 necroptosis-related genes were used to define four distinct necroptosis-related patterns: NEC cluster1-4. Each NEC cluster exhibited different patterns of survival and immune infiltration. Based on univariate Cox regression analyses and least absolute shrinkage and selection operator (Lasso) regression, 14 necroptosis-related genes were established to develop the NEC score. Patients were divided into two groups based on the NEC score. Patients in the high NEC score group had a significantly poorer overall survival than those in the low NEC score group. We further confirmed the correlation of clinical characteristics, as well as the immunotherapy outcome, with the NEC score, and confirmed the potential of the NEC score to be an independent prognostic factor. Furthermore, we compared the expression levels of eight potential biomarker genes between our own BLCA tissues and para-carcinoma tissue. CONCLUSION: We developed a novel NEC score that has a potential prognostic value in BLCA patients and may help personalized immunotherapy counselling.

Integrated Analysis of the Fecal Metagenome and Metabolome in Bladder Cancer in a Chinese Population.

Bladder cancer (BLCA) is a common malignancy of the urinary system. The gut microbiome produces various metabolites that play functional roles in tumorigenesis and tumor progression. However, the integrative analysis of the gut microbiome and metabolome in BLCA has still been lacking. Thus, the aim of this study was to identify microbial and functional characteristics and metabolites in BLCA in a Chinese population. Metagenomics, targeted metabolomics, bioinformatics, and integrative analysis were used in fecal samples of BLCA patients and healthy individuals. We found gut microbiomes were significantly dysregulated in BLCA patients, including Bifidobacterium, Lactobacillus, Streptococcus, Blautia, and Eubacterium. We also found 11Z-eicosenoic acid, 3-methoxytyrosine, abrine, aniline-2-sulfonate, arachidic acid, conjugated linoleic acids, elaidic acid, glycylleucine, glycylproline, leucyl-glycine, linoelaidic acid, linoleic acid, nicotinamide hypoxanthine dinucleotide, oleic acid, petroselinic acid, and ricinoleic acid to be significantly decreased, while cholesterol sulfate was significantly increased in BLCA patients. Integration of metagenomics and metabolomics revealed interactions between gut microbiota and metabolites and the host. We identified the alterations of gut microbiomes and metabolites in BLCA in a Chinese population. Moreover, we preliminarily revealed the associations between specific gut microbiomes and metabolites. These findings determined potential causative links among gut dysbiosis, dysregulated metabolites, and BLCA.

A Novel tiRNA-Gly-GCC-1 Promotes Progression of Urothelial Bladder Carcinoma and Directly Targets TLR4.

BACKGROUND: Patients with urothelial bladder carcinoma (UBC) have a poor prognosis and a high risk of progression. Recently, tRNA-derived small RNAs (tsRNAs), a novel type of noncoding RNA, have been identified. In our previous study, we found tiRNA-Gly-GCC-1 was significantly upregulated in UBC tissue and might target the predicted target gene toll-like receptor 4 (TLR4) to play a regulatory role in UBC. Thus, the aim of this study was to identify the functional roles of tiRNA-Gly-GCC-1 and the relationship between tiRNA-Gly-GCC-1 and TLR4. METHODS: After lentiviral transfection in 5637 and T24 cell lines, quantitative reverse transcription-PCR, Cell Counting Kit-8, IncuCyte ZOOM™ live cell imaging, flow cytometry, Transwell assays, scratch assay, and luciferase assay were performed. RESULTS: The results showed down-regulation of tiRNA-Gly-GCC-1 inhibits cell proliferation, migration and invasion, promotes cell apoptosis, and affects the cell cycle. Besides, tiRNA-Gly-GCC-1 was found to inhibit TLR4 expression by directly targeting its 3'UTR. CONCLUSIONS: Our study demonstrated that tiRNA-Gly-GCC-1 promotes the progression of UBC and directly targets TLR4. This study provides novel insights for future investigations to explore the mechanisms and therapeutic targets for UBC.

Characterization of metabolic alterations of chronic lymphocytic leukemia in the lymph node microenvironment.

Altered metabolism is a hallmark of both cell division and cancer. Chronic lymphocytic leukemia (CLL) cells circulate between peripheral blood (PB) and lymph nodes (LNs), where they receive proliferative and prosurvival signals from surrounding cells. However, insight into the metabolism of LN CLL and how this may relate to therapeutic response is lacking. To obtain insight into CLL LN metabolism, we applied a 2-tiered strategy. First, we sampled PB from 8 patients at baseline and after 3-month ibrutinib (IBR) treatment, which forces egress of CLL cells from LNs. Second, we applied in vitro B-cell receptor (BCR) or CD40 stimulation to mimic the LN microenvironment and performed metabolomic and transcriptomic analyses. The combined analyses indicated prominent changes in purine, glucose, and glutamate metabolism occurring in the LNs. CD40 signaling mostly regulated amino acid metabolism, tricarboxylic acid cycle (TCA), and energy production. BCR signaling preferably engaged glucose and glycerol metabolism and several biosynthesis routes. Pathway analyses demonstrated opposite effects of in vitro stimulation vs IBR treatment. In agreement, the metabolic regulator MYC and its target genes were induced after BCR/CD40 stimulation and suppressed by IBR. Next, 13C fluxomics performed on CD40/BCR-stimulated cells confirmed a strong contribution of glutamine as fuel for the TCA cycle, whereas glucose was mainly converted into lactate and ribose-5-phosphate. Finally, inhibition of glutamine import with V9302 attenuated CD40/BCR-induced resistance to venetoclax. Together, these data provide insight into crucial metabolic changes driven by the CLL LN microenvironment. The prominent use of amino acids as fuel for the TCA cycle suggests new therapeutic vulnerabilities.

Differential Expression Profiles and Bioinformatics Analysis of tRNA-Derived Small RNAs in Muscle-Invasive Bladder Cancer in a Chinese Population.

Muscle-invasive bladder cancer (MIBC) leads to a large societal burden. Recently, tRNA-derived small RNAs (tsRNAs), a novel type of noncoding RNA (ncRNAs), have been identified. However, the expression patterns and functions of tsRNAs in MIBC have not yet been identified. Here, RNA sequencing, bioinformatics, and quantitative reverse transcription- polymerase chain reaction (qRT-PCR) were used to screen the expression profiles and predict the potential roles of tsRNAs in MIBC. Of 406 tsRNAs differentially expressed in MIBC tissues, 91 tsRNAs were significantly differentially expressed. Then, four candidate tsRNAs, tiRNA-1:34-Val-CAC-2, tiRNA-1:33-Gly-GCC-1, tRF-1:32-Gly-GCC-1, and tRF-+1:T20-Ser-TGA-1, were selected. Next, a bioinformatics analysis showed the potential target genes and tsRNA-mRNA network. The most significant and meaningful terms of gene ontology were the positive regulation of the phosphate metabolic process, lamellipodium, and protein-cysteine S-acyltransferase activity in the biological process, cellular component, and molecular function, respectively. In addition, the top four pathways were predicted by the Kyoto Encyclopedia of Genes and Genomes database (KEGG). Finally, qRT-PCR demonstrated a similar expression pattern compared to sequencing data for the candidate tsRNAs. In short, we find differential expression profiles and predict that tiRNA-1:33-Gly-GCC-1, tRF-1:32-Gly-GCC-1, and tRF-+1:T20-Ser-TGA-1 are very likely to engage in the pathophysiological process of MIBC via regulating the target genes in the key pathways.

Upregulation of transient receptor potential cation channel subfamily M member-3 in bladder afferents is involved in chronic pain in cyclophosphamide-induced cystitis.

ABSTRACT: The transient receptor potential cation channel subfamily M member-3 (TRPM3) channel is a recently recognized noxious heat sensor that is involved in inflammatory thermal hyperalgesia. To examine its involvement in the development of hyperalgesia in interstitial cystitis/painful bladder syndrome (IC/PBS), rats with cyclophosphamide (CYP)-induced chronic cystitis were used as a model of IC/PBS. Mechanical and thermal hyperalgesia in lower abdominal region overlying the bladder in CYP rats were measured using von Frey filaments and radiant heat, respectively. Transient receptor potential cation channel subfamily M member-3 expression at the mRNA, protein, and functional levels in dorsal root ganglion neurons innervating the bladder was detected using RNA in situ hybridization (RNAscope), Western blotting, immunohistochemistry, and Ca 2+ imaging, respectively. Transient receptor potential cation channel subfamily M member-3 channels were expressed on most of the bladder primary afferent nerve terminals containing calcitonin gene-related peptide and their cell bodies in L6-S1 dorsal root ganglion. Activation of TRPM3 in the bladder wall by its specific agonist pregnenolone sulphate or CIM0216 induced spontaneous bladder pain, calcitonin gene-related peptide release, and neurogenic inflammation that was evidenced by edema, plasma extravasation, inflammatory cell accumulation, and mast cell infiltration. In CYP rats, pretreatment with the TRPM3 antagonist primidone (2 mg/kg, i.p.) significantly alleviated the mechanical and thermal hyperalgesia, bladder submucosal edema, mast cell infiltration, and bladder hyperactivity. Cyclophosphamide-induced cystitis was associated with TRPM3 upregulation at the mRNA, protein, and functional levels in bladder afferent neurons. Our results suggest that upregulation of TRPM3 channels is involved in the development of chronic pain in CYP-induced cystitis, and targeting TRPM3 may be a pharmacological strategy for treating bladder pain in IC/PBS.

The immunoregulatory landscape of human tuberculosis granulomas.

Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-ß, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.