Landscape analysis of alternative splicing in kidney renal clear cell carcinoma and their clinical significance.

A growing number of studies reveal that alternative splicing (AS) is associated with tumorigenesis, progression, and metastasis. Systematic analysis of alternative splicing signatures in renal cancer is lacking. In our study, we investigated the AS landscape of kidney renal clear cell carcinoma (KIRC) and identified AS predictive model to improve the prognostic prediction of KIRC. We obtained clinical data and gene expression profiles of KIRC patients from the TCGA database to evaluate AS events. The calculation results for seven types of AS events indicated that 46276 AS events from 10577 genes were identified. Next, we applied Cox regression analysis to identify 5864 prognostic-associated AS events. We used the Metascape database to verify the potential pathways of prognostic-associated AS. Moreover, we constructed KIRC prediction systems with prognostic-associated AS events by the LASSO Cox regression model. AUCs demonstrated that these prediction systems had excellent prognostic accuracy simultaneously. We identified 34 prognostic associated splicing factors (SFs) and constructed homologous regulatory networks. Furthermore, in vitro experiments were performed to validate the favorable effect of SFs FMR1 in KIRC. In conclusion, we overviewed AS events in KIRC and identified AS-based prognostic models to assist the survival prediction of KIRC patients. Our study may provide a novel predictive signature to improve the prognostic prediction of KIRC, which might facilitate KIRC patient counseling and individualized management.

TRIM44 Promotes Rabies Virus Replication by Autophagy-Dependent Mechanism.

Tripartite motif (TRIM) proteins are a multifunctional E3 ubiquitin ligase family that participates in various cellular processes. Recent studies have shown that TRIM proteins play important roles in regulating host-virus interactions through specific pathways, but their involvement in response to rabies virus (RABV) infection remains poorly understood. Here, we identified that several TRIM proteins are upregulated in mouse neuroblastoma cells (NA) after infection with the rabies virus using RNA-seq sequencing. Among them, TRIM44 was found to regulate RABV replication. This is supported by the observations that downregulation of TRIM44 inhibits RABV replication, while overexpression of TRIM44 promotes RABV replication. Mechanistically, TRIM44-induced RABV replication is brought about by activating autophagy, as inhibition of autophagy with 3-MA attenuates TRIM44-induced RABV replication. Additionally, we found that inhibition of autophagy with rapamycin reverses the TRIM44-knockdown-induced decrease in LC3B expression and autophagosome formation as well as RABV replication. The results suggest that TRIM44 promotes RABV replication by an autophagy-dependent mechanism. Our work identifies TRIM44 as a key host factor for RABV replication, and targeting TRIM44 expression may represent an effective therapeutic strategy.

Additional Insertion of gC Gene Triggers Better Immune Efficacy of TK/gI/gE-Deleted Pseudorabies Virus in Mice.

In recent years, pseudorabies virus (PRV) variants have resulted in an epidemic in swine herds and huge economic losses in China. Therefore, it is essential to develop an efficacious vaccine against the spread of PRV variants. Here, the triple-gene-deletion virus and the triple-gene-deletion plus gC virus were constructed by homologous recombination (HR). And then, their growth capacity, proliferation ability, and immune efficacy were evaluated. The results showed that the growth kinetics of the recombinant viruses were similar to those of the parental strain PRV-AH. Compared with the triple-gene-deletion virus group, the more dominant level of neutralizing antibody (NA) can be induced in the triple-gene-deletion plus gC virus group with the same 106.0 TCID50 dose after 4 and 6 weeks post-initial immunization (PII) (p < 0.0001). In addition, the antibody titers in mice immunized with the triple-gene-deletion plus gC virus were significantly higher than those immunized with triple-gene deletion virus with the same 105.0 TCID50 dose after 6 weeks PII (p < 0.001). More importantly, in the triple-gene-deletion plus gC virus group with 105.0 TCID50, the level of NA was close to that in the triple-gene deletion virus group with 106.0 TCID50 at 6 weeks PII. Meanwhile, the cytokines IL-4 and IFN-γ in sera were tested by enzyme-linked immunosorbent assay (ELISA) in each group. The highest level of IL-4 or IFN-γ was also elicited in the triple-gene deletion plus gC virus group at a dose of 106.0 TCID50. After challenge with PRV-AH, the survival rates of the triple-gene deletion plus gC virus immunized groups were higher than those of other groups. In immunized groups with 105.0 TCID50, the survival rate shows a significant difference between the triple-gene deletion plus gC virus group (75%, 6/8) and the triple-gene deletion virus group (12.5%, 1/8). In general, the immune efficacy of the PRV TK/gI/gE-deleted virus can be increased with additional gC insertion in mice, which has potential for developing an attenuated vaccine candidate for PRV control.

Integrative Single Cell Atlas Revealed Intratumoral Heterogeneity Generation from an Adaptive Epigenetic Cell State in Human Bladder Urothelial Carcinoma.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

Autophagy and Apoptosis in Rabies Virus Replication.

Rabies virus (RABV) is a single-stranded negative-sense RNA virus belonging to the Rhabdoviridae family and Lyssavirus genus, which is highly neurotropic and can infect almost all warm-blooded animals, including humans. Autophagy and apoptosis are two evolutionarily conserved and genetically regulated processes that maintain cellular and organismal homeostasis, respectively. Autophagy recycles unnecessary or dysfunctional intracellular organelles and molecules in a cell, whereas apoptosis eliminates damaged or unwanted cells in an organism. Studies have shown that RABV can induce both autophagy and apoptosis in target cells. To advance our understanding of pathogenesis of rabies, this paper reviews the molecular mechanisms of autophagy and apoptosis induced by RABV and the effects of the two cellular events on RABV replication.

Rapid Genotyping of Campylobacter coli Strains from Poultry Meat by PFGE, Sau-PCR, and fla-DGGE.

The genotyping of Campylobacter coli was done using three methods, pulsed-field gel electrophoresis (PFGE), Sau-polymerase chain reaction (Sau-PCR), and denaturing gradient gel electrophoresis assay of flagellin gene (fla-DGGE) and the characteristics of these assays were compared. The results showed that a total of 53 strains of C. coli were isolated from chicken and duck samples in three markets. All isolates were clustered into 31, 33, and 15 different patterns with Simpson's index of diversity (SID) values of 0.972, 0.974, and 0.919, respectively. Sau-PCR assay was simpler, more rapid, and had higher discriminatory power than PFGE assay. Fla-DGGE assay could detect and illustrate the number of contamination types of C. jejuni and C. coli without cultivation, which saved more time and cost than Sau-PCR and PFGE assays. Therefore, Sau-PCR and fla-DGGE assays are both rapid, economical, and easy to perform, which have the potential to be promising and accessible for primary laboratories in genotyping C. coli strains.

Hypoxia-induced PLOD2 promotes clear cell renal cell carcinoma progression via modulating EGFR-dependent AKT pathway activation.

Clear cell renal cell carcinoma (ccRCC) is a type of kidney cancer that is both common and aggressive, with a rising incidence in recent decades. Hypoxia is a key factor that plays a vital role in the tumorigenesis and metastasis of malignancy. However, the precise mechanisms of hypoxia driving ccRCC progression were not totally uncovered. Our study found that hypoxia level was elevated in ccRCC and might be an independent risk factor of prognosis in ccRCC patients. We identified a key protein PLOD2 was induced under hypoxic conditions and strongly associated with poor prognosis in ccRCC patients. When PLOD2 was depleted, the proliferation and migration of ccRCC cells were reduced in vitro and in vivo, while overexpression of PLOD2 had the opposite effect. Mechanically, the study further revealed that PLOD2 was transcriptionally activated by HIF1A, which binds to a specific promoter region of the PLOD2 gene. PLOD2 was also shown to interact with EGFR, leading to the phosphorylation of the receptor. Furthermore, PLOD2 was responsible for binding to the extracellular domain of EGFR, which ultimately activated the AKT signaling pathway, thus promoting the malignant progression of ccRCC. Treatment with the PLOD2 inhibitor Minoxidil significantly suppressed ccRCC progression by inactivating the EGFR/AKT signaling axis. In summary, the findings of this study shed light on the molecular mechanisms behind PLOD2 expression in ccRCC and suggest that it may serve as a potential predictor and therapeutic target for the clinical prognosis and treatment of ccRCC.

TRIM25 Suppresses Rabies Virus Fixed HEP-Flury Strain Production by Activating RIG-1-Mediated Type I Interferons.

Rabies remains a great threat to public health worldwide. So far, the mechanism of rabies virus (RABV) infection is not fully understood, and there is no effective treatment for rabies. Identifying more host restriction factors of RABV will spur the development of novel therapeutic interventions against rabies. Accumulating studies suggest that tripartite motif-containing (TRIM) proteins have great effects on virus replication. TRIMs control the antiviral responses through either direct interaction with viral proteins or indirect regulation of innate immune signaling molecules in the host. The role of TRIM25 in rabies virus (RABV) infection is poorly understood. Using next-generation sequencing, we found that TRIM25 is upregulated during HEP-Flury infection. Knockdown of TRIM25 enhances HEP-Flury production, while overexpression of TRIM25 suppresses HEP-Flury replication. Knockdown of interferon α and interferon ß weakens the anti-RABV response induced by TRIM25 overexpression, and potentiates RABV production. Furthermore, we found that TRIM25 regulates type-I interferon response by targeting retinoic acid-inducible gene I (RIG-I) during HEP-Flury infection. Knockdown of RIG-I weakens the anti-HEP-Flury response induced by TRIM25 overexpression, indicating that TRIM25 regulates RABV production via the RIG-I-IFN axis. In addition, we observed that TRIM25 does not directly interact with HEP-Flury structural proteins, suggesting that TRIM25 regulates HEP-Flury production indirectly. Taken together, our work identifies TRIM25 as a new host factor involved in HEP-Flury infection, which may be a potential target for the development of antiviral drugs against RABV.

TRIM21 Promotes Rabies Virus Production by Degrading IRF7 through Ubiquitination.

Rabies, a highly fatal zoonotic disease, is a significant global public health threat. Currently, the pathogenic mechanism of rabies has not been fully elucidated, and no effective treatment for rabies is available. Increasing evidence shows that the tripartite-motif protein (TRIM) family of proteins participates in the host's regulation of viral replication. Studies have demonstrated the upregulated expression of tripartite-motif protein 21 (TRIM21) in the brain tissue of mice infected with the rabies virus. Related studies have shown that TRIM21 knockdown inhibits RABV replication, while overexpression of TRIM21 exerted the opposite effect. Knockdown of interferon-alpha and interferon-beta modulates the inhibition of RABV replication caused by TRIM21 knockdown and promotes the replication of the virus. Furthermore, our previous study revealed that TRIM21 regulates the secretion of type I interferon during RABV infection by targeting interferon regulatory factor 7 (IRF7). IRF7 knockdown reduced the inhibition of RABV replication caused by the knockdown of TRIM21 and promoted viral replication. TRIM21 regulates RABV replication via the IRF7-IFN axis. Our study identified TRIM21 as a novel host factor required by RABV for replication. Thus, TRIM21 is a potential target for rabies treatment or management.

NRP1 promotes prostate cancer progression via modulating EGFR-dependent AKT pathway activation.

Prostate cancer (PCa) is the most common malignant tumor with a high global incidence in males. The mechanism underlying PCa progression is still not clear. This study observed that NRP1 was highly expressed in PCa and associated with poor prognosis in PCa patients. Functionally, NRP1 depletion attenuated the proliferation and migration ability of PCa cells in vitro and in vivo, while NRP1 overexpression promoted PCa cell proliferation and migration. Moreover, it was observed that NRP1 depletion induced G1 phase arrest in PCa cells. Mechanistically, HIF1α is bound to the specific promoter region of NRP1, thereby regulating its transcriptional activation. Subsequently, NRP1 interacted with EGFR, leading to EGFR phosphorylation. This study also provided evidence that the b1/b2 domain of NRP1 was responsible for the interaction with the extracellular domain of EGFR. Moreover, EGFR mediated NRP1-induced activation of the AKT signaling pathway, which promoted the malignant progression of PCa. In addition, the administration of NRP1 inhibitor EG01377 significantly inactivated the EGFR/AKT signaling axis, thereby suppressing PCa progression. In conclusion, the findings from this study highlighted the molecular mechanism underlying NRP1 expression in PCa and provide a potential predictor and therapeutic target for clinical prognosis and treatment of PCa.

Intergenic region polymorphism analysis: a novel genotyping method for Klebsiella pneumoniae.

AIMS: The ability to distinguish between Klebsiella pneumoniae strains is critical for outbreak investigations. A new typing method, intergenic region polymorphism analysis (IRPA), was developed, validated, and the discriminatory power was determined by comparison with multiple-locus variable-number tandem repeat analysis (MLVA) in this study. METHODS AND RESULTS: This method is based on the idea that every IRPA locus (polymorphic fragment of intergenic regions present in one strain but not in other strains or different fragment sizes in other strains) could divide strains into different genotypes. A 9-loci IRPA scheme was designed to type 64 K. pneumoniae isolates. Five IRPA loci were identified that conferred the same level of discrimination as the 9-loci initially examined. Among these K. pneumoniae isolates, 7.81% (5/64), 6.25% (4/64), 4.96% (3/64), 9.38% (6/64), and 1.56% (1/64) were capsular serotypes K1, K2, K5, K20, and K54, respectively. The discriminatory power of the IRPA method was better than that of MLVA expressed in Simpson's index of diversity (SI) at 0.997 and 0.988, respectively. The congruent analysis of the IRPA method and MLVA showed moderate congruence between the two methods (AR = 0.378). The AW indicated that if IRPA data are availabl, one can accurately predict the MLVA cluster. CONCLUSION: The IRPA method was found to have higher discriminatory power than MLVA and allowed for simpler band profile interpretation. The IRPA method is a rapid, simple, and high-resolution technique for molecular typing of K. pneumoniae.

Comparison of pulsed-field gel electrophoresis and a novel amplified intergenic locus polymorphism method for molecular typing of Campylobacter jejuni.

Campylobacter is regarded as the leading cause of zoonotic diseases and Campylobacter jejuni (C. jejuni) is one of the predominant pathogenic species. To track C. jejuni infections, various genotyping methods have been used. In this study, amplified intergenic locus polymorphism (AILP) was used to type C. jejuni for the first time. To confirm its feasibility, pulsed-field gel electrophoresis (PFGE) was performed as a control, and the results obtained by the AILP and PFGE methods were compared. Fifty-one isolates were resolved into 34 and 29 different genotypes with Simpson's indices of 0.976 and 0.967 using the AILP and PFGE methods, respectively. The adjusted Rand coefficient of the two approaches was as high as 0.845. In summary, the data showed that the two genotyping methods were similar for discriminating isolates and were both appropriate methods to distinguish whether two isolates were indistinguishable, but the AILP was faster and less costly than PFGE. Therefore, the AILP is a reliable, rapid, and highly discriminative method to genotype C. jejuni collected from poultry meat, which is helpful to effectively monitor C. jejuni.

Subtyping of Campylobacter coli isolated from raw poultry meat in retail markets using amplified intergenic locus polymorphism - A novel rapid subtyping method.

In order to provide more phylogenetic information of Campylobacter coli in large-scale epidemiological investigation, this work was undertaken to develop a novel genotyping method based on amplified intergenic locus polymorphism (AILP), by using pulsed-field gel electrophoresis (PFGE; using SmaI enzymes) as control. Eleven pairs of primers were selected to type C. coli strains for this purpose. A total of 68 C. coli isolates recovered from 51 retail raw chicken and 37 retail raw duck were subtyped. The Simpson's index of diversity (SID) of AILP and PFGE, as well as the adjusted Rand index (AR) and the adjusted Wallace coefficient (AW) between AILP and PFGE, were calculated. The new AILP method differentiated 68 C. coli isolates into 55 different subtypes (SID = 0.993), compared with 46 different profiles obtained from PFGE (SID = 0.980). The SID value of the AILP method was improved with the increasing number of primers, and a combination of 7 loci was selected as the optimal combination. The congruent analysis of the AILP method and PFGE showed moderate congruence between the two methods (AR = 0.462). The AW indicated that if AILP data is the available one can confidently predict the PFGE cluster. The results of this study showed that the AILP method had higher discrimination than PFGE and also allowed for significant reduction in time and cost.

HIF1α lactylation enhances KIAA1199 transcription to promote angiogenesis and vasculogenic mimicry in prostate cancer.

Prostate cancer (PCa) is one of the most prevalent malignancies in adult males. However, PCa is resistant to multi-kinase inhibitors-based anti-angiogenic therapies, and the mechanism and effective targeting thereof remains unclear. In this study, single-cell and bulk-transcriptomic datasets analysis revealed that KIAA1199, a hyaluronic acid (HA) binding protein, was involved in glycolysis, hypoxia and angiogenesis pathways. Moreover, boosted KIAA1199 expression in PCa tissues was positively correlated with tumor stage, hypoxia-inducible factor (HIF)-1α overexpression, as well as angiogenesis markers. Tube formation, Western blot, enzyme-linked immunosorbent assay, and in vivo tumorigenesis results demonstrated that KIAA1199 silencing significantly inhibited angiogenesis and vasculogenic mimicry (VM), both in vitro and in vivo, by increasing semaphoring 3A (sema3A) expression while decreasing expressions of VEGFA, VE-cadherin, phosphorylated EphA2, and depolymerized HA levels. KIAA1199 overexpression was also found to promote angiogenesis and VM via increasing secretory VEGFA, however, this activity could be reversed by the HA biosynthesis inhibitor 4-methylumbelliferone (4MU). Furthermore, dual-luciferase and ChIP-PCR revealed that HIF1α is the transcriptional enhancer of KIAA1199, while lactate imported to PCa cells by monocarboxylate transporter 1 (MCT1) stabilizes HIF1α under normoxia via HIF1α lactylation. Our findings may provide a better understanding of angiogenesis and a promising therapeutic target of PCa.

Non-invasive diagnosis and surveillance of bladder cancer with driver and passenger DNA methylation in a prospective cohort study.

BACKGROUND: State-of-art non-invasive diagnosis processes for bladder cancer (BLCA) harbour shortcomings such as low sensitivity and specificity, unable to distinguish between high- (HG) and low-grade (LG) tumours, as well as inability to differentiate muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). This study investigates a comprehensive characterization of the entire DNA methylation (DNAm) landscape of BLCA to determine the relevant biomarkers for the non-invasive diagnosis of BLCA. METHODS: A total of 304 samples from 224 donors were enrolled in this multi-centre, prospective cohort study. BLCA-specific DNAm signature discovery was carried out with genome-wide bisulfite sequencing in 32 tumour tissues and 12 normal urine samples. A targeted sequencing assay for BLCA-specific DNAm signatures was developed to categorize tumour tissue against normal urine, or MIBC against NMIBC. Independent validation was performed with targeted sequencing of 259 urine samples in a double-blinded manner to determine the clinical diagnosis and prognosis value of DNAm-based classification models. Functions of genomic region harbouring BLCA-specific DNAm signature were validated with biological assays. Concordances of pathology to urine tumour DNA (circulating tumour DNA [ctDNA]) methylation, genomic mutations or other state-of-the-art diagnosis methods were measured. RESULTS: Genome-wide DNAm profile could accurately classify LG tumour from HG tumour (LG NMIBC vs. HG NMIBC: p = .038; LG NMIBC vs. HG MIBC, p = .00032; HG NMIBC vs. HG MIBC: p = .82; Student's t-test). Overall, the DNAm profile distinguishes MIBC from NMIBC and normal urine. Targeted-sequencing-based DNAm signature classifiers accurately classify LG NMIBC tissues from HG MIBC and could detect tumours in urine at a limit of detection of less than .5%. In tumour tissues, DNAm accurately classifies pathology, thus outperforming genomic mutation or RNA expression profiles. In the independent validation cohort, pre-surgery urine ctDNA methylation outperforms fluorescence in situ hybridization (FISH) assay to detect HG BLCA (n = 54) with 100% sensitivity (95% CI: 82.5%-100%) and LG BLCA (n = 26) with 62% sensitivity (95% CI: 51.3%-72.7%), both at 100% specificity (non-BLCA: n = 72; 95% CI: 84.1%-100%). Pre-surgery urine ctDNA methylation signature correlates with pathology and predicts recurrence and metastasis. Post-surgery urine ctDNA methylation (n = 61) accurately predicts recurrence-free survival within 180 days, with 100% accuracy. CONCLUSION: With the discovery of BLCA-specific DNAm signatures, targeted sequencing of ctDNA methylation outperforms FISH and DNA mutation to detect tumours, predict recurrence and make prognoses.

Interleukin-25 enhances humoral immune responses caused by the rabies virus.

Rabies is an important zoonotic disease caused by the rabies virus (RABV). Currently, no effective treatment is available for this condition. The prevention and control of rabies mainly depend on effective vaccination. Therefore, it is crucial to enhance the immune responses induced by the rabies vaccine. Virus neutralizing antibodies (VNA) induced by rabies vaccines are important for the clearance of RABV. Interleukin-25 (IL-25) has been demonstrated to activate T helper type 2 cells that contribute to humoral immune responses. The IL-25 gene was inserted into the genome of RABV, and the immunogenicity of recombinant RABV with IL-25 gene was investigated to develop more efficient rabies vaccines. Here, we found that the expression of IL-25 did not affect RABV production in vitro and pathogenicity in vivo. However, recombinant RABV expression of IL-25 induced a better VNA level than the parental virus in mice. In addition, expression of IL-25 enhanced the IgG1 level induced by RABV. Furthermore, mice immunized with recombinant RABV showed a higher survival rate and milder clinical signs than those immunized with the parent strain after challenge with CVS-11. Thus, these results showed that IL-25 could enhance the humoral immune responses induced by RABV, suggesting that IL-25 can be used as a viral vaccine adjuvant.

Erratum: EFEMP2 suppresses epithelial-mesenchymal transition via Wnt/ß-catenin signaling pathway in human bladder cancer: Erratum.

[This corrects the article DOI: 10.7150/ijbs.35541.].

USP26 promotes anaplastic thyroid cancer progression by stabilizing TAZ.

Anaplastic thyroid cancer (ATC) is one of the most lethal and aggressive human malignancies, with no effective treatment currently available. The Hippo tumor suppressor pathway is highly conserved in mammals and plays an important role in carcinogenesis. TAZ is one of major key effectors of the Hippo pathway. However, the mechanism supporting abnormal TAZ expression in ATC remains to be characterized. In the present study, we identified USP26, a DUB enzyme in the ubiquitin-specific proteases family, as a bona fide deubiquitylase of TAZ in ATC. USP26 was shown to interact with, deubiquitylate, and stabilize TAZ in a deubiquitylation activity-dependent manner. USP26 depletion significantly decreased ATC cell proliferation, migration, and invasion. The effects induced by USP26 depletion could be rescued by further TAZ overexpression. Depletion of USP26 decreased the TAZ protein level and the expression of TAZ/TEAD target genes in ATC, including CTGF, ANKRD1, and CYR61. In general, our findings establish a previously undocumented catalytic role for USP26 as a deubiquitinating enzyme of TAZ and provides a possible target for the therapy of ATC.