PAQR8 promotes breast cancer recurrence and confers resistance to multiple therapies.

BACKGROUND: Breast cancer mortality is principally due to recurrent disease that becomes resistant to therapy. We recently identified copy number (CN) gain of the putative membrane progesterone receptor PAQR8 as one of four focal CN alterations that preferentially occurred in recurrent metastatic tumors compared to primary tumors in breast cancer patients. Whether PAQR8 plays a functional role in cancer is unknown. Notably, PAQR8 CN gain in recurrent tumors was mutually exclusive with activating ESR1 mutations in patients treated with anti-estrogen therapies and occurred in > 50% of both patients treated with anti-estrogen therapies and those treated with chemotherapy or anti-Her2 agents. METHODS: We used orthotopic mouse models to determine whether PAQR8 overexpression or deletion alters breast cancer dormancy or recurrence following therapy. In vitro studies, including assays for colony formation, cell viability, and relative cell fitness, were employed to identify effects of PAQR8 in the context of therapy. Cell survival and proliferation were quantified by immunofluorescence staining for markers of apoptosis and proliferation. Sphingolipids were quantified by liquid chromatography-high resolution mass spectrometry. RESULTS: We show that PAQR8 is necessary and sufficient for efficient mammary tumor recurrence in mice, spontaneously upregulated and CN gained in recurrent tumors that arise following therapy in multiple mouse models, and associated with poor survival following recurrence as well as poor overall survival in breast cancer patients. PAQR8 promoted resistance to therapy by enhancing tumor cell survival following estrogen receptor pathway inhibition by fulvestrant or estrogen deprivation, Her2 pathway blockade by lapatinib or Her2 downregulation, and treatment with chemotherapeutic agents. Pro-survival effects of PAQR8 were mediated by a Gi protein-dependent reduction in cAMP levels, did not require progesterone, and involved a PAQR8-dependent decrease in ceramide levels and increase in sphingosine-1-phosphate levels, suggesting that PAQR8 may possess ceramidase activity. CONCLUSIONS: Our data provide in vivo evidence that PAQR8 plays a functional role in cancer, implicate PAQR8, cAMP, and ceramide metabolism in breast cancer recurrence, and identify a novel mechanism that may commonly contribute to the acquisition of treatment resistance in breast cancer patients.

The ferroptosis-related long non-coding RNAs signature predicts biochemical recurrence and immune cell infiltration in prostate cancer.

BACKGROUND: Findings from numerous studies have revealed that ferroptosis is closely related to tumorigenesis and immune cell infiltration. Long non-coding RNAs (lncRNAs) are reportedly involved in the progression of various cancers, including prostate cancer (PCa). This study was designed to establish a ferroptosis-related lncRNA (frlncRNA) signature to predict PCa prognosis. METHODS: The frlncRNAs were identified by studying their expression by Pearson's correlation analysis. Differentially expressed prognosis related frlncRNAs were identified by the Wilcoxon test and univariate Cox regression analysis. The LASSO Cox regression model was used to build a model to predict biochemical recurrence (BCR) based on frlncRNAs. The GSEA software (version 4.1.0) was used to explore the enriched pathways in high- and low- risk groups. Patients with PCa were clustered into different subgroups by unsupervised clustering based on the frlncRNAs considered in the prognostic model. Real-time PCR and CCK8 assays were performed to verify the expression and function of frlncRNAs. RESULTS: We identified 35 differentially expressed prognosis related frlncRNAs based on data on PCa from TCGA. A risk signature based on five frlncRNAs (AP006284.1, AC132938.1, BCRP3, AL360181.4 and AL135999.1), was confirmed to perform well in predicting BCR. The high-risk group had higher disease grades and a greater number of infiltrating immune cells. Besides this, we found that the five frlncRNAs were connected with typical immune checkpoints. With respect to molecular mechanisms, several metabolic pathways were found to enriched in the low-risk group. Furthermore, patients could be classified into different subtypes with different PSA-free times using the five frlncRNAs. Notably, AP006284.1, AC132938.1, BCRP3 and AL135999.1 were upregulated in PCa cells and tissues, whereas AL360181.4 exhibited the opposite trend. The downregulation of BCRP3 and AP006284.1 impaired the proliferation of 22RV1 cells. CONCLUSION: We generated a prognostic model based on five frlncRNAs, with clinical usefulness, and thus provided a novel strategy for predicting the BCR of patients with PCa.

P2Y2 purinergic receptor modulates virus yield, calcium homeostasis, and cell motility in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. Our study reveals that levels of several components of the purinergic signaling system, including the P2Y2 and P2X5 receptors, are elevated in HCMV-infected fibroblasts. Knockdown and drug treatment experiments demonstrated that P2Y2 enhances the yield of virus, whereas P2X5 reduces HCMV production. The HCMV IE1 protein induces P2Y2 expression; and P2Y2-mediated signaling is important for efficient HCMV gene expression, DNA synthesis, and the production of infectious HCMV progeny. P2Y2 cooperates with the viral UL37x1 protein to regulate cystolic Ca2+ levels. P2Y2 also regulates PI3K/Akt signaling and infected cell motility. Thus, P2Y2 functions at multiple points within the viral replication cycle to support the efficient production of HCMV progeny, and it may facilitate in vivo viral spread through its role in cell migration.

Dicyandiamide-assisted HKUST-1 derived Cu/N-doped porous carbon nanoarchitecture for electrochemical detection of acetaminophen.

MOFs-derived metal/carbon materials have been considered as promising candidates for the electrochemical detection of micropollutants. However, the aggregation of metal nanoparticles and structure collapse of precursor MOFs during pyrolysis significantly hamper the improvement on detecting performance. Herein, a dicyandiamide-assisted strategy is utilized to synthesize well-dispersed Cu/N-doped porous carbon nanoarchitecture (CuNC) for the electrochemical detection of acetaminophen (AP). The constructed CuNC sensor exhibits excellent electro-analytical performance for monitoring AP with linear range from 0.01 µM to 921.2 µM, and the low detection limit of 2.46 nM (S/N = 3). The improved performance of CuNC sensor is ascribed to the introduction of dicyandiamide, which can prevent HKUST-1 framework breakage and reduce the aggregation tendency of Cu, leading to the evenly distributed small Cu nanoparticles, abundant N species, hierarchical channel structure, and high conductivity carbon framework. These advantages endow predominant repeatability, stability, and selectivity of CuNC sensor. This strategy provided a novel approach to preparing MOFs-derived carbon nanoarchitectures with excellent electroanalysis performance to monitor micropollutants.

Identification of four insertion sites for foreign genes in a pseudorabies virus vector.

BACKGROUND: Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRVΔTK&gE-AH02) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4 ~ 5 week-old PRV antibody negative piglets and provide rapid and 100 % protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVTK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. RESULTS: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S(UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 h post infection (P < 0.05). Moreover, at 12 h post infection, cells infected with PRV-S(UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)ΔTK/gE (P = 0.097) and PRV-S(UL46-27)ΔTK/gE (P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRVΔTK&gE-AH02). CONCLUSIONS: This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.

Polyfluoroalkyl chemicals and the risk of kidney stones in US adults: A population-based study.

The potential nephrotoxicity of polyfluoroalkyl chemicals (PFCs) have received extensive attention. However, the relationship between PFCs and the risk of kidney stones remain unclear. This study aimed to examine the level of PFCs in the US population and its relationship with the risk of kidney stones. We investigated the serum levels of six PFCs in 8453 adult participants (≥20 years) from the National Health and Nutrition Examination Survey (NHANES) between 2007 and 2016, including perfluorodecanoic acid (PFDE), perfluorohexane sulfonic acid (PFHS), 2-(N-methyl-perfluorooctane sulfonamido) acetate (MPAH), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUA), and perfluorododecanoic acid (PFDO). Logistic regression model was used to evaluate the correlation between PFCs and kidney stones. Of the 8453 participants, 787 self-reported a history of kidney stones. After adjusting for gender, age, race, education, marital status, body mass index (BMI), hypertension, diabetes and estimated glomerular filtration rate (eGFR), we found that total PFCs and PFHS were positively correlated with the risk of kidney stones. Compared with the lowest tertile, the odds ratios with 95% confidence intervals (CI) with increasing tertiles were 1.30 (95% CI,1.08-1.59, p = 0.007) and 1.25 (95 CI%,1.00-1.52, p = 0.024) for total PFCs and 1.24 (95 CI%,1.03-1.51, p = 0.032), and 1.35 (95 CI,1.10-1.68, p = 0.005) for PFHS. Our study shows that total PFCs and PFHS were associated with an increased risk of kidney stones.

[Hsa_circ_0005221 promotes prostate cancer progression through the miR-339-5p/STAT5a pathway].

OBJECTIVE: To investigate the molecular mechanism of hsa_circ_0005221 regulating the progression of PCa through the miR-339-5p/STAT5a pathway. METHODS: Localizations of hsa_circ_0005221 and miR-339-5p in cells were detected by nuclear-cytoplasmic isolation. MiRNA-339-5p was selected as the target miRNA bound to hsa_circ_0005221 by RNA pull-down assay. The binding site of the luciferase reporter gene was predicted by software and the binding capability of miR-339-5p validated by luciferase assay. The expression of hsa_circ_0005221 in the prostatic epithelial and PCa cells was determined by qPCR. The hsa_circ_0005221-overexpressed plasmid and siRNA were transfected into the PCa cells for measurement of their proliferation, invasion and migration abilities and the levels of epithelial-mesenchymal transformation (EMT) and apoptosis. After knockdown of hsa_circ_0005221 and transfection of miR-339-5p mimics and miR-339-5p inhibitor, the proliferation, invasion and migration abilities of the DU145 and LNCaP cells were detected, and so were the levels of the EMT signature protein, STAT5a and cell apoptosis. RESULTS: The expression of hsa_circ_0005221 was significantly higher in the PCa than in the prostatic epithelial cells. Nuclear-cytoplasmic isolation experiments showed that hsa_circ_0005221 and miR-339-5p were mainly located in the cytoplasm. The proliferation, invasion and migration abilities and EMT were decreased and the apoptosis increased in the DU145 and LNCaP cells with knockdown of hsa_circ_0005221, which was just the reverse in those with overexpressed hsa_circ_0005221. Among the top 5 miRNAs predicted by software, miR-339-5p, miR-17 and miR-520h were shown by pull-down assay to be bound to hsa_circ_0005221, with most obvious changes in miR-339-5p when hsa_circ_0005221 knocked down or overexpressed. Luciferase reporter gene assay showed the binding of hsa_circ_0005221 to miR-339-5p. Knockdown of hsa_circ_0005221 and transfection of miR-339-5p mimics into the DU145 and LNCaP cells significantly reduced the proliferation, invasion and migration abilities of the cells and the N-cad level, increased their apoptosis and E-cad level, and up-regulated the expression of STAT5a, while overexpression of hsa_circ_0005221 and transfection of miR-339-5p mimics induced just the opposite effects. CONCLUSIONS: Hsa_circ_0005221 enhances the progression of prostate cancer through the miR-339-5p/STAT5a pathway.

Efficient Removal of Organic Pollutants by Metal-organic Framework Derived Co/C Yolk-Shell Nanoreactors: Size-Exclusion and Confinement Effect.

Selective removal of organic pollutants from surface water with high efficiency is crucial in water purification. Here, yolk-shell Co/C nanoreactors (YSCCNs) are facilely synthesized via pyrolysis of controllably etched ZIF-67 by tannic acid, and their degradation performance on multiple pollutants is demonstrated. To present the structure-performance relationship between the designed nanocatalyst and the selective removal of organic pollutants, bisphenol A (BPA) was selected as the targeted pollutant with coexistence of humus acid (HA). For comparison, solid and hollow ZIF-67 derived Co/C nanoparticles denoted as SCCNs and HCCNs, were also tested. The results show that YSCCNs exhibit enhanced BPA degradation rate of 0.32 min-1, which is 23.1% and 45.4% higher than that of HCCNs and SCCNs in HA (10 ppm) system. The essential improvement can be ascribed to the synergetic effects from shell layer (size-exclusion) and core/shell (confinement effect). The degradation mechanism and pathway are further confirmed by radical quenching experiments and liquid chromatography-mass spectrograph (LC-MS), respectively. In addition, some influential factors, including reaction temperature, pH value, and peroxymonosulfate (PMS) dosage are investigated in detail. This work provides a possible way to selectively remove target contaminant from multiple pollutants in complex water system.

A gD&gC-substituted pseudorabies virus vaccine strain provides complete clinical protection and is helpful to prevent virus shedding against challenge by a Chinese pseudorabies variant.

BACKGROUND: Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gCS) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gCS was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7 days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions. RESULTS: The results showed that a BAC clone of Bartha-K61 and a B-gD&gCS clone were successfully generated. The growth kinetics of PRV B-gD&gCS strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gCS strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gCS and Bartha-K61 groups (n = 5 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gCS strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group. CONCLUSIONS: Our results suggest that PRV B-gD&gCS strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.

Decellularized and solubilized pancreatic stroma promotes the in vitro proliferation, migration and differentiation of BMSCs into IPCs.

Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into insulin-producing cells (IPCs). Bio-scaffolds derived from decellularized organs can act as a carrier for seed cells and may have broad applications in regenerative medicine. This study investigated the effect of native pancreatic stroma obtained from decellularized pancreas on the proliferation, migration and differentiation of BMSCs into IPCs, and explored the potential underlying molecular mechanism. The decellularized pancreas bio-scaffold was obtained by perfusion with Triton X-100/ammonium hydroxide, followed by digestion with a mixture of pepsin and hydrochloric acid to prepare the stroma solution. Islet-like cells were differentiated from BMSCs by a three-step induction method. The differences on the cytological behavior with or without stroma were evaluated by morphological observation, insulin release assay, qRT-PCR assay and western blot analysis. Our results showed that, stroma derived from decellularized pancreas could promote the proliferation and migration of BMSCs. Furthermore, the formation of IPCs could also be promoted, which possessed similar morphology to endogenous islets. During the induced differentiation process, the presence of stroma significantly increased the expression of insulin 1, insulin 2 and Pdx-1, as well as insulin release. This was accompanied by an increase in the phosphorylation of Akt and ERK in third stage cell clusters, which was prevented by the addition of the inhibitors PD98059 and LY294002, respectively. In summary, decellularized pancreatic stroma could promote the proliferation, migration and differentiation of BMSCs into IPCs, and this involved the activation of Akt and ERK signal pathways.

Th1/Th2 cytokines in early peripheral blood of patients with multiple injuries and its predictive value for SIRS: A bioinformatic analysis.

This study aims to evaluate the changes in helper T lymphocyte (Th)1/Th2 factor levels in peripheral blood of patients with severe multiple injuries and their prognostic value for nosocomial infection using bioinformatic analysis. The experimental group consisted of 180 patients with numerous injuries admitted to our hospital between January 2021 and June 2023, with 80 healthy volunteers serving as controls. Th1 cytokines (interleukin-2 and interferon-γ) and Th2 cytokines (IL-4 and IL-10) were evaluated 48 hours after admission using enzyme-linked immunosorbent assays. The experimental group was separated into two groups: those with systemic inflammatory response syndrome (SIRS) and those without SIRS, for cytokine analysis and SIRS incidence. Furthermore, the study examined Th1 and Th2 cytokine levels in trauma patients in various body locations within the experimental group. A receiver operating characteristic (ROC) curve analysis was performed to determine the predictive value of Th1/Th2 cytokines for SIRS incidence. The experimental group had lower IL-2 and IFN-γ levels compared to the control group, but greater levels of IL-4 and IL-10. There were no significant variations in Th1 and Th2 cytokine levels across the experimental groups. Patients with SIRS had lower levels of IL-2 and IFN-γ but greater levels of IL-4 and IL-10 compared to those without SIRS. Combined cytokine levels have a better predictive value for SIRS than individual cytokines alone. In conclusion, individuals with severe multiple injuries had a change from Th1 to Th2 cytokine profiles, which was most evident in those with SIRS. The combined cytokine levels had a substantial predictive value for SIRS incidence in this patient cohort.

Endoplasmic reticulum stress-related genes as prognostic and immunogenic biomarkers in prostate cancer.

BACKGROUND: The metastasis and aggressive nature of prostate cancer (PCa) has become a major malignancy related threat that concerns men's health. The efficacy of immune monotherapy against PCa is questionable due to its lymphocyte-suppressive nature. METHOD: Endoplasmic reticulum stress- (ERS-) and PCa-prognosis-related genes were obtained from the Molecular Signatures Database and the Cancer Genome Atlas database. The expression, prognosis and immune infiltration values of key genes were explored by "survival R package", "rms", "xCELL algorithm", and univariate-multivariate Cox and LASSO regression analyses. The "consensus cluster plus R package" was used for cluster analysis. RESULT: As ERS-related genes, ERLIN2 and CDK5RAP3 showed significant expressional, prognostic and clinic-pathologic values. They were defined as the key genes significantly correlated with immune infiltration and response. The nomogram was constructed with T-stage and primary treatment outcome, and the risk-prognostic model was constructed in the following way: Riskscore = (- 0.1918) * ERLIN2 + (0.5254) * CDK5RAP3. Subsequently, prognostic subgroups based on key genes classified the high-risk group as a pro-cancer subgroup that had lower mutation rates of critical genes (SPOP and MUC16), multiple low-expression immune-relevant molecules, and differences in macrophages (M1 and M2) expressions. Finally, ERLIN2 as an anti-oncogene and CDK5RAP3 as a pro-oncogene were further confirmed by cell phenotype assays and immunohistochemistry. CONCLUSION: We identified ERLIN2 and CDK5RAP3 as ERS-related genes with important prognostic and immunologic values, and classified patients between high- and low-risk subgroups, which provided new prognostic markers, immunotherapeutic targets, and basis for prognostic assessments.

Identification of an anaplastic subtype of prostate cancer amenable to therapies targeting SP1 or translation elongation.

Histopathological heterogeneity is a hallmark of prostate cancer (PCa). Using spatial and parallel single-nucleus transcriptomics, we report an androgen receptor (AR)-positive but neuroendocrine-null primary PCa subtype with morphologic and molecular characteristics of small cell carcinoma. Such small cell-like PCa (SCLPC) is clinically aggressive with low AR, but high stemness and proliferation, activity. Molecular characterization prioritizes protein translation, represented by up-regulation of many ribosomal protein genes, and SP1, a transcriptional factor that drives SCLPC phenotype and overexpresses in castration-resistant PCa (CRPC), as two potential therapeutic targets in AR-indifferent CRPC. An SP1-specific inhibitor, plicamycin, effectively suppresses CRPC growth in vivo. Homoharringtonine, a Food And Drug Administration-approved translation elongation inhibitor, impedes CRPC progression in preclinical models and patients with CRPC. We construct an SCLPC-specific signature capable of stratifying patients for drug selectivity. Our studies reveal the existence of SCLPC in admixed PCa pathology, which may mediate tumor relapse, and establish SP1 and translation elongation as actionable therapeutic targets for CRPC.

Diagnosis of photochemical O3 production of urban plumes in summer via developing the real-field IRs of VOCs: A case study in Beijing of China.

This study mainly developed an estimate method for photochemical ozone (O3) production from urban plumes in hot season, through simulating O3 evolution from precursors locally emitted and determining the real-field O3 increment reactivity (IR) of volatile organic compounds (VOCs) based on the box chemical model. Our simulation on June-2019 indicated that Beijing local emissions produced O3 at the rate of 0.7-9.2 ppb/h and led to an O3 increase of 48.9 ppb during 05:00-18:00, accounting for 68.3% of the observed O3 increase. The maximum level and production rate of simulated O3 showed a linear response to VOCs, therefore we can use VOCs levels in urban plumes to quantify O3 formation in summer. The IR (g O3 formed per g VOCs) was calculated on the actual precursor and meteorology condition of this megacity, 0.12-4.90 g/g for individual VOCs and 1.49 g/g for comprehensive TVOCs. The weighted average of individual IRs agreed well with that of TVOCs, but these IRs were 34.5% of MIR values that were widely used in references. It's noteworthy that these IRs had greater sensitivity to precursor levels, and broadly remained stable under the fixed VOCs:NOx. Considering the synchronous reductions of precursors in Beijing, we applied these IRs to quantify chemical O3 evolution from Beijing local emissions in summer of recent years, declining from 63.5 ppb in 2016 to 44.0 ppb in 2020 for June. The contributions of the diagnosed chemical O3 to Beijing O3 better matched with the atmospheric transport paths on daily basis, higher than 100% when the transport paths starting from the clean neighbor cities, but lower to 45%-66% when the transport paths originating from the highly-polluted neighbor cities. This consistence indicated the reliability of our IR calculation method for quickly estimating chemical O3 production of urban plumes in summer.

Improvement of peroxymonosulfate utilization efficiency for sulfamethazine degradation by photo-electron activating peroxymonosulfate: Performance and mechanism.

Enhancing the utilization efficiency of oxidant is of great importance for advanced oxidation processes (AOPs). Herein, nitrogen-doped titania dioxide/carbon (NTC7) catalyst was fabricated via pyrolyzing NH2-MIL-125 under nitrogen atmosphere at 700 °C. Excitation of NTC7 under visible light can successfully achieve efficient activation of peroxymonosulfate (PMS) (NTC7 + PMS + Vis). Degradation performance and PMS activation mechanism were systematically investigated using sulfamethazine (SMT) as the target pollutant. It was found that the photo-generated electrons excited from NTC7 under visible light played the dominant role in enhancing the productive consumption of PMS. Its utilization increased by 66 % (Δ[PMS]/Δ[SMT] = 7.0) in NTC7 + PMS + Vis process and the degradation rate was 2.14 times higher than that of NTC7 + PMS process. The ketonic CO groups and structural defects were responsible for the generation of 1O2 in dark activation while radicals (•OH, O2•-) were more inclined to be continuously produced in NTC7 + PMS + Vis process. The involved degradation pathways, intermediates, and toxicity assessment have been studied in detail. This work provides an effective approach to enhance the utilization efficiency of oxidant for pollutant degradation by AOPs.

A Novel Strategy of US3 Codon De-Optimization for Construction of an Attenuated Pseudorabies Virus against High Virulent Chinese Pseudorabies Virus Variant.

In this study, we applied bacterial artificial chromosome (BAC) technology with PRVΔTK/gE/gI as the base material to replace the first, central, and terminal segments of the US3 gene with codon-deoptimized fragments via two-step Red-mediated recombination in E. coli GS1783 cells. The three constructed BACs were co-transfected with gI and part of gE fragments carrying homologous sequences (gI+gE'), respectively, in swine testicular cells. These three recombinant viruses with US3 codon de-optimization ((PRVΔTK&gE-US3deop-1, PRVΔTK&gE-US3deop-2, and PRVΔTK&gE-US3deop-3) were obtained and purified. These three recombinant viruses exhibited similar growth kinetics to the parental AH02LA strain, stably retained the deletion of TK and gE gene fragments, and stably inherited the recoded US3. Mice were inoculated intraperitoneally with the three recombinant viruses or control virus PRVΔTK&gEAH02 at a 107.0 TCID50 dose. Mice immunized with PRVΔTK&gE-US3deop-1 did not develop clinical signs and had a decreased virus load and attenuated pathological changes in the lungs and brain compared to the control group. Moreover, immunized mice were challenged with 100 LD50 of the AH02LA strain, and PRVΔTK&gE-US3deop-1 provided similar protection to that of the control virus PRVΔTK&gEAH02. Finally, PRVΔTK&gE-US3deop-1 was injected intramuscularly into 1-day-old PRV-negative piglets at a dose of 106.0 TCID50. Immunized piglets showed only slight temperature reactions and mild clinical signs. However, high levels of seroneutralizing antibody were produced at 14 and 21 days post-immunization. In addition, the immunization of PRVΔTK&gE-US3deop-1 at a dose of 105.0 TCID50 provided complete clinical protection and prevented virus shedding in piglets challenged by 106.5 TCID50 of the PRV AH02LA variant at 1 week post immunization. Together, these findings suggest that PRVΔTK&gE-US3deop-1 displays great potential as a vaccine candidate.

Construction of pseudorabies virus variant attenuated vaccine: codon deoptimization of US3 and UL56 genes based on PRV gE/TK deletion strain.

Since 2011, pseudorabies based on the pseudorabies virus (PRV) variant has emerged as a serious health issue in pig farms in China. The PRV gE/TK or gE/gI/TK deletion strains protect against emerging PRV variants. However, these variants may cause lethal infections in newborn piglets without PRV antibodies. Previous studies have shown that codon deoptimization of a virulence gene causes virus attenuation. Accordingly, we deoptimized US3-S (US3 gene encoding a short isoform that represents approximately 95% of the total US3 transcription) and UL56 genes (first 10 or all codons) of PRV gE/TK deletion strain (PRVΔTK&gE-AH02) to generate six recombinant PRVs through bacterial artificial chromosome technology. In swine testicular cells, recombinant PRVs with all codon deoptimization of US3-S or UL56 genes were grown to lower titers than the parental virus. Notably, US3-S or UL56 with all codon deoptimization reduced mRNA and protein expressions. Subsequently, the safety and immunogenicity of recombinant PRVs with codon deoptimization of US3-S or UL56 are evaluated as vaccine candidates in mice and piglets. The mice inoculated with recombinant PRVs with codon deoptimization of US3-S or UL56 showed exceptional survival ability without severe clinical signs. All codons deoptimized (US3-S and UL56) significantly decreased virus load and attenuated pathological changes in the brains of the mice. Moreover, the protection efficiency offered by recombinant PRVs with codon deoptimization of US3-S or UL56 showed similar effects to PRVΔTK&gE-AH02. Remarkably, the 1-day-old PRV antibody-negative piglets inoculated with PRVΔTK&gE-US3-ST-CD (a recombinant PRV with all codon deoptimization of US3-S) presented no abnormal clinical symptoms, including fever. The piglets inoculated with PRVΔTK&gE-US3-ST-CD showed a high serum neutralization index against the PRV variant. In conclusion, these results suggest using codon deoptimization to generate innovative live attenuated PRV vaccine candidates.

YY1 complex in M2 macrophage promotes prostate cancer progression by upregulating IL-6.

BACKGROUND: Tumor-associated macrophages are mainly polarized into the M2 phenotype, remodeling the tumor microenvironment and promoting tumor progression by secreting various cytokines. METHODS: Tissue microarray consisting of prostate cancer (PCa), normal prostate, and lymph node metastatic samples from patients with PCa were stained with Yin Yang 1 (YY1) and CD163. Transgenic mice overexpressing YY1 were constructed to observe PCa tumorigenesis. Furthermore, in vivo and in vitro experiments, including CRISPR-Cas9 knock-out, RNA sequencing, chromatin immunoprecipitation (ChIP) sequencing, and liquid-liquid phase separation (LLPS) assays, were performed to investigate the role and mechanism of YY1 in M2 macrophages and PCa tumor microenvironment. RESULTS: YY1 was highly expressed in M2 macrophages in PCa and was associated with poorer clinical outcomes. The proportion of tumor-infiltrated M2 macrophages increased in transgenic mice overexpressing YY1. In contrast, the proliferation and activity of anti-tumoral T lymphocytes were suppressed. Treatment targeting YY1 on M2 macrophages using an M2-targeting peptide-modified liposome carrier suppressed PCa cell lung metastasis and generated synergistic anti-tumoral effects with PD-1 blockade. IL-4/STAT6 pathway regulated YY1, and YY1 increased the macrophage-induced PCa progression by upregulating IL-6. Furthermore, by conducting H3K27ac-ChIP-seq in M2 macrophages and THP-1, we found that thousands of enhancers were gained during M2 macrophage polarization, and these M2-specific enhancers were enriched in YY1 ChIP-seq signals. In addition, an M2-specific IL-6 enhancer upregulated IL-6 expression through long-range chromatin interaction with IL-6 promoter in M2 macrophages. During M2 macrophage polarization, YY1 formed an LLPS, in which p300, p65, and CEBPB acted as transcriptional cofactors. CONCLUSIONS: Phase separation of the YY1 complex in M2 macrophages upregulated IL-6 by promoting IL-6 enhancer-promoter interactions, thereby increasing PCa progression.

A noval strategy of deletion in PK gene for construction of a vaccine candidate with exellent safety and complete protection efficiency against high virulent Chinese pseudorabies virus variant.

A variant of pseudorabies virus (PRV) with enhanced pathogenicity have emerged in many vaccinated swine herds in China since 2011. PRVΔTK&gE-AH02, a previously described TK/gE deletion PRV strain arising from the PRV variant AH02LA, has been shown to be safe for PRV antibody positive piglets, and could provide protection against emerging PRV variants. However, inoculation of PRVΔTK&gE-AH02 into PRV antibody negative neonatal piglets caused lethal infection. In the study, in order to attenuate the virulence of PRVΔTK&gE-AH02, an additional deletion of 1∼x223C13 bp of US3 (the serine/threonine kinase, PK) gene was performed to generate a TK/PK/gE deletion PRV variant (PRVΔTK&PK&gE-AH02). We found that the growth kinetics of PRVΔTK&PK&gE-AH02 was similar to that of PRVΔTK&gE-AH02. Mice inoculated with PRVΔTK&PK&gE-AH02 in different dose (104.0∼x223C107.0 TCID50) survived and showed no observable clinical symptoms. No virus was detected in the brains or lungs of the mice inoculated with PRVΔTK&PK&gE-AH02. Moreover, mice inoculated with PRVΔTK&PK&gE-AH02 and PRVΔTK&gE-AH02 showed similar survival against virulent PRV AH02LA strain. Importantly, safety test showed no clinical symptoms in PRV antibody negative neonatal piglets that were intranasally inoculated with PRVΔTK&PK&gE-AH02 at a dose of 106.5 TCID50, indicating that the virulence of PRVΔTK&PK&gE-AH02 was significantly mitigated. Piglets immunized with PRVΔTK&PK&gE-AH02 exhibited a high serum neutralization index. All piglets inoculated intramuscularly (I.M.) with 1 mL (105.0 TCID50) PRVΔTK&PK&gE-AH02 were completely protected against challenge intranasally (I.N.) with 2LD50 (106.5TCID50) PRV AH02LA strain. In summary, our results indicate that deletion of 1∼x223C13 bp of US3 (PK) can provide a useful way for further attenuation of PRV and the PRVΔTK&PK&gE-AH02 might be a promising vaccine candidate for controlling of the virulent PRV variants in China.

ERR-activated GPR35 promotes immune infiltration level of macrophages in gastric cancer tissues.

Enhancer release and retargeting (ERR) events could activate disease-causing gene promoters for increasing the expression level of oncogenes. Meanwhile, class A orphan GPCRs (oGPCRs) are known as potential biomarkers or drug targets for various cancers, such as gastric cancer (GC). Hence, systemic investigation of ERR events for class A oGPCRs in GC could help to explore biomarkers for GC. In this study, ENCODE and GTEx eQTL data were utilized to define ERR events in GC. Only GPR35 was then detected that could be activated by ERR in GC based on these data and ChIP-seq. Then, activated GPR35 functional in GC cells were explored by flow cytometry, cell-based wound healing assay, Transwell migration assay, and M2 polarization of macrophages assay. Meanwhile, according to TCGA and GEO database, overall survival, immune-related gene expression, and immune cell infiltration level in different GPR35 expressions were calculated. Here, we found ERR event activate GPR35 results in GC cells proliferation and migration, and partly immune cells significance exhaustion (CD8 + T-cells and CD4 + memory T-cells) and/or infiltration (T-cells and macrophage). Meanwhile, high GRP35 level leads to a poor prognosis in GC patients, probably partly due to it promoting the immune infiltration level of macrophages and then inducing polarization of M2 macrophages. Notably, GPR35's high expression in CTSB+ and CD68 + macrophage could be a genetic indicator for early warning of primary GC. Hence, our findings provide a novel activation approach for oGPCRs, and GPR35 could be determined as a new drugable receptor and early genetic indicator for GC.