WSB1, a Hypoxia-Inducible E3 Ligase, Promotes Myofibroblast Accumulation and Attenuates Alveolar Epithelial Regeneration in Mouse Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive interstitial lung disease for which there is no curative therapy available. Repetitive alveolar epithelial injury repair, myofibroblast accumulation, and excessive collagen deposition are key pathologic features of idiopathic pulmonary fibrosis, eventually leading to cellular hypoxia and respiratory failure. The precise mechanism driving this complex maladaptive process remains inadequately understood. WD repeat and suppressor of cytokine signaling box containing 1 (WSB1) is an E3 ubiquitin ligase, the expression of which is associated strongly with hypoxia, and forms a positive feedback loop with hypoxia-inducible factor 1α (HIF-1α) under anoxic condition. This study explored the expression, cellular distribution, and function of WSB1 in bleomycin (BLM)-induced mouse lung injury and fibrosis. WSB1 expression was highly induced by BLM injury and correlated with the progression of lung fibrosis. Significantly, conditional deletion of Wsb1 in adult mice ameliorated BLM-induced pulmonary fibrosis. Phenotypically, Wsb1-deficient mice showed reduced lipofibroblast to myofibroblast transition, but enhanced alveolar type 2 proliferation and differentiation into alveolar type 1 after BLM injury. Proteomic analysis of mouse lung tissues identified caveolin 2 as a potential downstream target of WSB1, contributing to BLM-induced epithelial injury repair and fibrosis. These findings unravel a vital role for WSB1 induction in lung injury repair, thus highlighting it as a potential therapeutic target for pulmonary fibrosis.

Structural and functional prediction, evaluation, and validation in the post-sequencing era.

The surge of genome sequencing data has underlined substantial genetic variants of uncertain significance (VUS). The decryption of VUS discovered by sequencing poses a major challenge in the post-sequencing era. Although experimental assays have progressed in classifying VUS, only a tiny fraction of the human genes have been explored experimentally. Thus, it is urgently needed to generate state-of-the-art functional predictors of VUS in silico. Artificial intelligence (AI) is an invaluable tool to assist in the identification of VUS with high efficiency and accuracy. An increasing number of studies indicate that AI has brought an exciting acceleration in the interpretation of VUS, and our group has already used AI to develop protein structure-based prediction models. In this review, we provide an overview of the previous research on AI-based prediction of missense variants, and elucidate the challenges and opportunities for protein structure-based variant prediction in the post-sequencing era.

Communications between macrophages and cardiomyocytes.

The heart is a muscular organ that pumps blood throughout the body and is one of the most vital organs in human body. While cardiomyocytes are essential for maintaining the normal function of the heart, a variety of cardiovascular diseases such as coronary artery occlusion, arrhythmia, and myocarditis can lead to cardiomyocyte death, resulting in deterioration of heart function. The adult mammalian heart is incapable of regenerating sufficient cardiomyocytes following cardiac injuries, eventually leading to heart failure and death. Cardiac macrophages are ubiquitously distributed in the healthy heart and accumulated at the site of injury. Macrophages play essential roles in regulating homeostasis and proliferation of cardiomyocyte, promoting electrical conduction, and removing dead cardiomyocytes and debris through direct and indirect cell-cell crosstalk. In this review, we summarize the latest insights into the role of macrophages in maintaining cardiac homeostasis and the macrophage-cardiomyocyte crosstalk in both healthy and injured scenarios. Video Abstract.

Small extrachromosomal circular DNAs as biomarkers for multi-cancer diagnosis and monitoring.

BACKGROUND: Small extrachromosomal circular DNAs (eccDNAs) have the potential to be cancer biomarkers. However, the formation mechanisms and functions of small eccDNAs selected in carcinogenesis are not clear, and whether the small eccDNA profile in the plasma of cancer patients represents that in cancer tissues remains to be elucidated. METHODS: A novel sequencing workflow based on the nanopore sequencing platform was used to sequence naturally existing full-length small eccDNAs in tissues and plasma collected from 25 cancer patients (including prostate cancer, hepatocellular carcinoma and colorectal cancer), and from an independent validation cohort (including 7 cancer plasma and 14 healthy plasma). RESULTS: Compared with those in non-cancer tissues, small eccDNAs detected in cancer tissues had a significantly larger number and size (P = 0.040 and 2.2e-16, respectively), along with more even distribution and different formation mechanisms. Although small eccDNAs had different general characteristics and genomic annotation between cancer tissues and the paired plasma, they had similar formation mechanisms and cancer-related functions. Small eccDNAs originated from some specific genes had great multi-cancer diagnostic value in tissues (AUC ≥ 0.8) and plasma (AUC > 0.9), especially increasing the accuracy of multi-cancer prediction of CEA/CA19-9 levels. The high multi-cancer diagnostic value of small eccDNAs originated from ALK&ETV6 could be extrapolated from tissues (AUC = 0.804) to plasma and showed high positive predictive value (100%) and negative predictive value (82.35%) in a validation cohort. CONCLUSIONS: As independent and stable circular DNA molecules, small eccDNAs in both tissues and plasma can be used as ideal biomarkers for cost-effective multi-cancer diagnosis and monitoring.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

FGF10 mitigates doxorubicin-induced myocardial toxicity in mice via activation of FGFR2b/PHLDA1/AKT axis.

Doxorubicin is a common chemotherapeutic agent in clinic, but myocardial toxicity limits its use. Fibroblast growth factor (FGF) 10, a multifunctional paracrine growth factor, plays diverse roles in embryonic and postnatal heart development as well as in cardiac regeneration and repair. In this study we investigated the role of FGF10 as a potential modulator of doxorubicin-induced cardiac cytotoxicity and the underlying molecular mechanisms. Fgf10+/- mice and an inducible dominant negative FGFR2b transgenic mouse model (Rosa26rtTA; tet(O)sFgfr2b) were used to determine the effect of Fgf10 hypomorph or blocking of endogenous FGFR2b ligands activity on doxorubicin-induced myocardial injury. Acute myocardial injury was induced by a single injection of doxorubicin (25 mg/kg, i.p.). Then cardiac function was evaluated using echocardiography, and DNA damage, oxidative stress and apoptosis in cardiac tissue were assessed. We showed that doxorubicin treatment markedly decreased the expression of FGFR2b ligands including FGF10 in cardiac tissue of wild type mice, whereas Fgf10+/- mice exhibited a greater degree of oxidative stress, DNA damage and apoptosis as compared with the Fgf10+/+ control. Pre-treatment with recombinant FGF10 protein significantly attenuated doxorubicin-induced oxidative stress, DNA damage and apoptosis both in doxorubicin-treated mice and in doxorubicin-treated HL-1 cells and NRCMs. We demonstrated that FGF10 protected against doxorubicin-induced myocardial toxicity via activation of FGFR2/Pleckstrin homology-like domain family A member 1 (PHLDA1)/Akt axis. Overall, our results unveil a potent protective effect of FGF10 against doxorubicin-induced myocardial injury and identify FGFR2b/PHLDA1/Akt axis as a potential therapeutic target for patients receiving doxorubicin treatment.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Bacillus Calmette-Guérin Treatment Changes the Tumor Microenvironment of Non-Muscle-Invasive Bladder Cancer.

Background: Bacillus Calmette-Guérin (BCG) is currently the most effective intravesical therapy for non-muscle-invasive bladder cancer (NMIBC) as it can prevent disease recurrence and progression and lower mortality. However, the response rates to BCG vary widely and are dependent on a multitude of factors. Methods: We performed a systematic discovery by analyzing the whole exome sequence, expression profile, and immune repertoire sequence of treatment-naive and 5-year time-serial relapsed tumors from 24 NMIBC patients. Results: BCG therapy showed bidirectional effects on tumor evolution and immune checkpoint landscape, along with a significant reduction of the percentage of neoantigen burden. In addition, a remarkable proportion of subclonal mutations were unique to the matched pre- or post-treatment tumors, suggesting the presence of BCG-induced and/or spatial heterogeneity. In the relapsed tumors, we identified and validated a shift in the mutational signatures in which mutations associated with aristolochic acid (AA) exposure were enriched, implying AA may be associated with tumor recurrence. Enhanced expressions of immune checkpoint regulation genes were found in the relapsed tumors, suggesting that the combination of immune checkpoint with BCG treatment may be an effective strategy to treat NMIBC. TCR sequencing revealed treatment-associated changes in the T-cell repertoire in the primary and relapsed tumors. Conclusion: Our results provide insight into the genomic and immune dynamics of tumor evolution with BCG treatment, suggest new mechanisms of BCG resistance, and inform the development of clinically relevant biomarkers and trials of potential immune checkpoint inhibitor combination therapies.

Circular RNAs: Biomarkers of cancer.

Circular RNAs (circRNAs) are a class of single-stranded closed RNAs that are produced by the back splicing of precursor mRNAs. The formation of circRNAs mainly involves intron-pairing-driven circularization, RNA-binding protein (RBP)-driven circularization, and lariat-driven circularization. The vast majority of circRNAs are found in the cytoplasm, and some intron-containing circRNAs are localized in the nucleus. CircRNAs have been found to function as microRNA (miRNA) sponges, interact with RBPs and translate proteins, and play an important regulatory role in the development and progression of cancer. CircRNAs exhibit tissue- and developmental stage-specific expression and are stable, with longer half-lives than linear RNAs. CircRNAs have great potential as biomarkers for cancer diagnosis and prognosis, which is highlighted by their detectability in tissues, especially in fluid biopsy samples such as plasma, saliva, and urine. Here, we review the current studies on the properties and functions of circRNAs and their clinical application value.

Identification of differentially expressed circular RNAs associated with thymoma.

BACKGROUND: Thymomas and thymic carcinomas are the most common tumor types among anterior mediastinal lesions. However, the relationship between molecular aberrations and thymoma patients are poorly understood, especially abnormal changes in the expression profiles of circRNAs. The purpose of the present study was to investigate the expression profiles of circRNAs in thymoma patients and their possible roles in the pathogenesis of thymoma. METHODS: Diseased tissues and surrounding normal thymic tissues in two thymoma patients were collected for circRNA sequencing. The top four upregulated circRNAs were selected as candidates and further validated with RT-PCR in 20 thymoma patients. Gene ontology and signal transduction network analyses of circRNA-related mRNAs were performed to analyze the functional properties. Survival analysis of their parental genes were also carried out to evaluate the clinical value of differentially expressed circRNA. RESULTS: A total of 73 circRNAs were differentially expressed in thymoma tissues using high-throughput sequencing. Among these circRNAs, hsa_circ_0001173, hsa_circ_0007291, hsa_circ_0003550, and hsa_circ_0001947 were significantly upregulated in thymoma tissues compared with normal thymic tissues. We identified that these four circRNA-related mRNAs were involved in cell-cell adhesion, MAPK pathways, and TNF pathway, which may contribute to the pathological immune disorder in thymoma. Finally, we also found that SCAP (hsa_circ_0007291 parental gene) and AFF2 (hsa_circ_0001947 parental gene) were all significantly related with progression-free survival (PFS) of thymoma patients in a Kaplan-Meier plot (p-value <0.05). CONCLUSIONS: The expression levels of hsa_circ_0001173, hsa_circ_0007291, hsa_circ_0003550, and hsa_circ_0001947 were significantly upregulated and positively correlated with immune imbalance in thymoma patients.

Direct Determination of Coagulation Factor IIa and Plasmin Activities for Monitoring of Thrombotic State.

BACKGROUND: Current laboratory examinations for hypercoagulable diseases focus on the biomarker content of the activated coagulation cascade and fibrinolytic system. Direct detection of physiologically important protease activities in blood remains a challenge. This study aims to develop a general approach that enables the determination of activities of crucial coagulation factors and plasmin in blood. METHODS: This assay is based on the proteolytic activation of an engineered zymogen of l-phenylalanine oxidase (proPAO), for which the specific blood protease cleavage sites were engineered between the inhibitory and activity domains of proPAO. Specific cleavage of the recombinant proenzyme leads to the activation of proPAO, followed by oxidation and oxygenation of l-phenylalanine, resulting in an increase of chromogenic production when coupled with the Trinder reaction. RESULTS: We applied this method to determine the activities of both coagulation factor IIa and plasmin in their physiologically relevant basal state and fully activated state in sodium citrate-anticoagulated plasma respectively. Factor IIa and plasmin activities could be dynamically monitored in patients with thrombotic disease who were taking oral anticoagulants and used for assessing the hypercoagulable state in pregnant women. CONCLUSIONS: The high specificity, sensitivity, and stability of this novel assay not only makes it useful for determining clinically important protease activities in human blood and diagnosing thrombotic diseases but also provides a new way to monitor the effectiveness and safety of anticoagulant drugs.

Quantitative Evaluation of the Transcriptional Activity of Steroid Hormone Receptor Mutants and Variants Using a Single Vector With Two Reporters and a Receptor Expression Cassette.

Although the rapid development of high-throughput sequencing has led to the identification of a large number of truncated or mutated steroid hormone receptor (SHR) variants, their clinical relevance remains to be defined. A platform for functional analysis of these SHR variants in cells would be instrumental for better assessing their impact on normal physiology and SHR-associated diseases. Here we have developed a new reporter system that allows rapid and accurate assessment of the transcriptional activity of SHR variants in cells. The reporter is a single construct containing a firefly luciferase reporter gene, whose expression is under the control of a promoter with multiple steroid hormone responsive elements, and a Renilla luciferase reporter gene, that is constitutively expressed under the control of an internal ribosome entry site (IRES) and is not regulated by steroid hormones. The corresponding SHR (wildtype or mutant/variant) is also expressed from the same construct. Using this improved reporter system, we revealed a large spectrum of transactivation activities within a set of previously identified mutations and variations of the androgen receptor (AR), the estrogen receptor α (ERα) and the glucocorticoid receptor (GR). This novel reporter system enables functional analysis of SHR mutants and variants in physiological and pathological settings, offering valuable preclinical, or diagnostic information for the understanding and treatment of associated diseases.

Identification of Differentially Expressed Genes Associated with Papillary Thyroid Carcinoma.

OBJECTIVE: Next-generation sequencing (NGS) was performed to identify genes that were differentially expressed between normal thyroid tissue and papillary thyroid carcinoma (PTC). MATERIALS AND METHODS: Six candidate genes were selected and further confirmed with quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry in samples from 24 fresh thyroid tumors and adjacent normal tissues. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to investigate signal transduction pathways of the differentially expressed genes. RESULTS: In total, 1690 genes were differentially expressed between samples from patients with PTC and the adjacent normal tissue. Among these, SFRP4, ZNF90, and DCN were the top three upregulated genes, whereas KIRREL3, TRIM36, and GABBR2 were downregulated with the smallest p values. Several pathways were associated with the differentially expressed genes and involved in cellular proliferation, cell migration, and endocrine system tumor progression, which may contribute to the pathogenesis of PTC. Upregulation of SFRP4, ZNF90, and DCN at the mRNA level was further validated with RT-PCR, and DCN expression was further confirmed with immunostaining of PTC samples. CONCLUSION: These results provide new insights into the molecular mechanisms of PTC. Identification of differentially expressed genes should not only improve the tumor signature for thyroid tumors as a diagnostic biomarker but also reveal potential targets for thyroid tumor treatment.

Abiraterone and MDV3100 inhibits the proliferation and promotes the apoptosis of prostate cancer cells through mitophagy.

BACKGROUND: Abiraterone and MDV3100 are two effective anticancer agents for prostate cancer, however, the mechanism of their downstream action remains undefined. METHODS: A dual fluorescent biosensor plasmid was transfected in LNCaP cells to measure mitophagy. The DNA of LNCaP cells was extracted and performed with quantitative real-time PCR to detect mitochondrial DNA copy number. JC-1 staining was utilized to detect the mitochondrial membrane potential and electron microscope was performed to analyze mitochondrial morphology. Moreover, the protein levels of mitochondrial markers and apoptotic markers were detected by western blot. At last, the proliferation and apoptosis of LNCaP cells were analyzed with CCK-8 assay and flow cytometry after abiraterone or MDV3100 treatment. RESULTS: Mitophagy was induced by abiraterone and MDV3100 in LNCaP cells. The low expression level of mitochondrial DNA copy number and mitochondrial depolarization were further identified in the abiraterone or MDV3100 treatment groups compared with the control group. Besides, severe mitochondria swelling and substantial autophagy-lysosomes were observed in abiraterone- and MDV3100-treated LNCaP cells. The expression of mitochondria-related proteins, frataxin, ACO2 and Tom20 were significantly downregulated in abiraterone and MDV3100 treated LNCaP cells, whereas the expression level of inner membrane protein of mitochondria (Tim23) was significantly upregulated in the same condition. Moreover, the proliferation of LNCaP cells were drastically inhibited, and the apoptosis of LNCaP cells was increased in abiraterone or MDV3100 treatment groups. Meanwhile, the addition of mitophagy inhibitor Mdivi-1 (mitochondrial division inhibitor 1) could conversely elevate proliferation and constrain apoptosis of LNCaP cells. CONCLUSIONS: Our results prove that both abiraterone and MDV3100 inhibit the proliferation, promote the apoptosis of prostate cancer cells through regulating mitophagy. The promotion of mitophagy might enhance the efficacy of abiraterone and MDV3100, which could be a potential strategy to improve chemotherapy with these two reagents.

Palmitoylome profiling indicates that androgens regulate the palmitoylation of α­tubulin in prostate cancer­derived LNCaP cells and supernatants.

Prostate cancer is closely associated with constitutive transactivation of the androgen receptor (AR) signaling pathway. After treatment with androgen­deprivation therapy (ADT), the majority of patients develop castration­resistant prostate cancer within months or years. In order to investigate potential novel therapeutic targets in addition to ADT, the present study examined the regulatory mechanisms of the AR signaling pathway. In the present study, LNCaP cells were metabolically­labeled with Alk­C16, a palmitate probe. In addition, cells were treated with R1881, an androgen, or DMSO. Subsequently, click­chemistry­based palmitoylome profiling was performed in LNCaP cells and palmitoylated proteins were compared between cells treated with androgen and untreated cells. Androgen treatment was revealed to significantly increase the palmitoylation level of α­tubulin. In addition, the palmitoylation level of Ras­related protein Rab­7a (Rab7a) was enhanced by androgen treatment. Palmitoylation of α­tubulin and Rab7a were essential for cell proliferation. Notably, in the supernatant of LNCaP cells, the palmitoylation level of α­tubulin was also increased following androgen treatment. Palmitoylation of α­tubulin may provide a new potential target for the treatment of prostate cancer. In addition, the high level of α­tubulin palmitoylation in the supernatant may represent a biomarker for early­stage prostate cancer.

Alterations to the Lung Microbiome in Idiopathic Pulmonary Fibrosis Patients.

Lung microbiome ecosystem homeostasis in idiopathic pulmonary fibrosis (IPF) remains uncharacterized. The aims of this study were to identify unique microbial signatures of the lung microbiome and analyze microbial gene function in IPF patients. DNA isolated from BALF samples was obtained for high-throughput gene sequencing. Microbial metagenomic data were used for principal component analysis (PCA) and analyzed at different taxonomic levels. Shotgun metagenomic data were annotated using the KEGG database and were analyzed for functional and metabolic pathways. In this study, 17 IPF patients and 38 healthy subjects (smokers and non-smokers) were recruited. For the PCA, the first and the second principal component explained 16.3 and 13.4% of the overall variability, respectively. The ß diversity of microbiome was reduced in the IPF group. Signature of IPF's microbes was enriched of Streptococcus, Pseudobutyrivibrio, and Anaerorhabdus. The translocation of lung microbiome was shown that 32.84% of them were from oral. After analysis of gene function, ABC transporter systems, biofilm formation, and two-component regulatory system were enriched in IPF patients' microbiome. Here we shown the microbiology characteristics in IPF patients. The microbiome may participate in altering internal conditions and involving in generating antibiotic resistance in IPF patients.

cGMP interacts with tropomyosin and downregulates actin-tropomyosin-myosin complex interaction.

BACKGROUND: The nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH); however, its exact molecular mechanism remains undefined. METHODS: Biotin-cGMP pull-down assay was performed to search for proteins regulated by cGMP. The interaction between cGMP and tropomyosin was analyzed with antibody dependent pull-down in vivo. Tropomyosin fragments were constructed to explore the tropomyosin-cGMP binding sites. The expression level and subcellular localization of tropomyosin were detected with Real-time PCR, Western blot and immunofluorescence assay after the 8-Br-cGMP treatment. Finally, isothermal titration calorimetry (ITC) was utilized to detect the binding affinity of actin-tropomyosin-myosin in the existence of cGMP-tropomyosin interaction. RESULTS: cGMP interacted with tropomyosin. Isoform 4 of TPM1 gene was identified as the only isoform expressed in the human pulmonary artery smooth muscle cells (HPASMCs). The region of 68-208aa of tropomyosin was necessary for the interaction between tropomyosin and cGMP. The expression level and subcellular localization of tropomyosin showed no change after the stimulation of NO-sGC-cGMP pathway. However, cGMP-tropomyosin interaction decreased the affinity of tropomyosin to actin. CONCLUSIONS: We elucidate the downstream signal pathway of NO-sGC-cGMP. This work will contribute to the detection of innovative targeted agents and provide novel insights into the development of new therapies for PAH.

Screening of circular RNAs and validation of circANKRD36 associated with inflammation in patients with type 2 diabetes mellitus.

Circular RNAs (circRNAs) are an abundant class of endogenous non­coding RNAs and are associated with numerous diseases, including cancer, cardiovascular diseases, and type 2 diabetes mellitus (T2DM). However, the association between circRNAs and inflammation or inflammatory cytokines in patients with T2DM remains to be fully elucidated. The purpose of the present study was to investigate the expression profiles of circRNAs in peripheral leucocytes of patients with T2DM and their association with inflammatory cytokines. Peripheral blood from patients with T2DM (n=43) and healthy individuals (n=45) were collected for RNA sequencing and later verification. Reverse transcription­polymerase chain reaction (RT­PCR) and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analyses were used to detect the expression levels of circRNAs. The expression of inflammatory factors, including interleukin (IL)­1, (IL)­6, and tumor necrosis factor (TNF)­α were measured via enzyme­linked immunosorbent assay. Furthermore, the mRNA expression level of ankyrin repeat domain 36 (ANKRD36), a protein located at 2q11.2 that interacts with the GAPDH gene, was measured using RT­qPCR analysis. The circRNA/microRNA (miRNA) interaction was predicted using RegRNA and mirPath software. In total, 220 circRNAs were found to be differentially expressed between patients with T2DM and healthy individuals, of which 107 were upregulated and 113 were downregulated. Among the nine selected circRNAs, circANKRD36 was significantly upregulated in patients with T2DM compared with control subjects (P=0.02). The expression level of circANKRD36 was positively correlated with glucose and glycosylated hemoglobin (r=0.3250, P=0.0047 and r=0.3171, P=0.0056, respectively). The expression level of IL­6 was significantly different between the T2DM group and control group (P=0.028) and was positively correlated with circANKRD36. The difference of circANKRD36 host gene expression between patients with T2DM and healthy controls was significant (P=0.04). Taken together, circANKRD36 may be involved in T2DM and inflammation­associated pathways via interaction with miRNAs, including hsa­miR­3614­3p, hsa­miR­498, and hsa­miR­501­5p. The expression of circANKRD36 was up-regulated in peripheral blood leucocytes and was correlated with chronic inflammation in T2DM. Therefore, circANKRD36 can be used as a potential biomarker for screening chronic inflammation in patients with T2DM.

Identification of a low frequency missense mutation in MUC6 contributing to pulmonary artery hypertension by whole-exome sequencing.

Pulmonary arterial hypertension (PAH) represents a progressive disease characterized by abnormally high blood pressure in the pulmonary artery. Although mutations in the bone morphogenetic receptor 2 (BMPR2) are found in 80% of heritable, their low penetrance suggests that other unidentified genetic modifiers are required for this disease. In this report, whole-exome sequencing (WES) and a linkage analysis were performed on genomic DNA isolated from four affected relatives and one non-affected relative in two PAH families. By focusing on meaningful variants which were presented in the four affected family members, but not presented in the non-affected individual, 49 SNP and eight indel variants in 39 genes were identified as candidates. Further high-throughput multiplex genotyping and Sanger sequencing were carried out to confirm the putative causal mutations in 150 individuals (30 idiopathic PAH [IPAH] patients, 30 chronic thromboembolic pulmonary hypertension [CTEPH] patients, and 90 normal controls). A heterozygous and deleterious mutation in the gene MUC6 (p.Pro1716Ser) was confirmed in the IPAH group (20/30, 67%) and CTEPH group (1/30, 3.33%); no variant was detected in the 90 normal controls. MUC6, which is short for mucin 6, encodes high molecular weight glycoprotein produced by many epithelial tissues and forms an insoluble mucous barrier that protects the lumens. We re-confirmed this low frequency mutation with the 1000 Genomes database across all species; no population or frequency data of this allele were acquired. We also found that this mutation site was highly conserved in different species and predicted MUC6 has the protection function of the airway and pneumoangiogram based on genomic sequence data. The compound heterozygous MUC6 gene mutation (p.Pro1716Ser) suggests a novel disease mechanism leading to PAH.

Spatial Intratumor Genomic Heterogeneity within Localized Prostate Cancer Revealed by Single-nucleus Sequencing.

BACKGROUND: Prostate adenocarcinoma (PCa) is a complex genetic disease, and the implementation of personalized treatment in PCa faces challenges due to significant inter- and intrapatient tumor heterogeneities. OBJECTIVE: To systematically explore the genomic complexity of tumor cells with different Gleason scores (GSs) in PCa. DESIGN, SETTING, AND PARTICIPANTS: We performed single-cell whole genome sequencing of 17 tumor cells from localized lesions with distinct GS and matched four normal samples from two prostatectomy patients. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: All classes of genomic alterations were identified, including substitutions, insertions/deletions, copy number alterations, and rearrangements. RESULTS AND LIMITATIONS: Significant spatial, intra- and intertumoral heterogeneities were observed at the cellular level. In the patient 1, all cells shared the same TP53 driver mutation, implying a monoclonal origin of PCa. In the patient 2, only a subpopulation of cells contained the TP53 driver mutation, whereas other cells carried different driver mutations, indicating a typical polyclonal model with separate clonal cell expansions. The tumor cells from different sides of prostate owned various mutation patterns. Considerable neoantigens were predicted among different cells, implying unknown immune editing components helping prostate tumor cells escaping from immune surveillance. CONCLUSIONS: There is a significant spatial genomic heterogeneity even in the same PCa patient. Our study also provides the first genome-wide evidence at single-cell level, supporting that the origin of PCa could be either polyclonal or monoclonal, which has implications for treatment decisions for prostate cancer. PATIENT SUMMARY: We reported the first single-cell whole genomic data of prostate adenocarcinoma (PCa) from different Gleason scores. Identification of these genetic alterations may help understand PCa tumor progression and clonal evolution.