Exposure to heavy metal elements may significantly increase serum prostate-specific antigen levels with overdosed dietary zinc.

BACKGROUND: Serum prostate-specific antigen (PSA) is a primary metric for diagnosis and prognosis of prostate cancer (PCa). Exposure to heavy metals, such as lead, cadmium, mercury, and zinc can impact PSA levels in PCa patients. However, it is unclear whether this effect also occurs in men without PCa, which may lead to the overdiagnosis of PCa. METHOD: Data on a total of 5089 American men who had never been diagnosed with PCa were obtained from the National Health and Nutrition Examination Survey performed from 2003-2010. The relationship between serum PSA levels (dependent variable) and concentrations of lead (µmol/L), cadmium (nmol/L), and mercury (µmol/L) were investigated with dietary zinc intake being used as a potential modifier or covariate in a weighted linear regression model and a generalized additive model. A series of bootstrapping analyses were performed to evaluate sensitivity and specificity using these models. RESULTS: Regression analyses suggested that, in general, lead, cadmium, or mercury did not show an association with PSA levels, which was consistent with the results of the bootstrapping analyses. However, in a subgroup of participants with a high level of dietary zinc intake (≥14.12 mg/day), a significant positive association between cadmium and serum PSA was identified (1.06, 95% CI, P = 0.0268, P for interaction=0.0249). CONCLUSIONS: With high-level zinc intake, serum PSA levels may rise in PCa-free men as the exposure to cadmium increases, leading to a potential risk of an overdiagnosis of PCa and unnecessary treatment. Therefore, environmental variables should be factored in the current diagnostic model for PCa that is solely based on PSA measurements. Different criteria for PSA screening are necessary based on geographical variables. Further investigations are needed to uncover the biological and biochemical relationship between zinc, cadmium, and serum PSA levels to more precisely diagnose PCa.

Low expression of GRM4 is associated with poor prognosis and tumor immune infiltration in glioma.

INTRODUCTION: The metabotropic glutamate receptor 4 (mGlu4, GRM4) exhibits significant expression within the central nervous system (CNS) and has been implicated to be correlated with a poor prognosis. OBJECTIVE: This study was aimed to elucidate the relationship between the expression profile of GRM4 and the prognosis of glioma patients. METHODS: RNA-sequencing datasets from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and China Glioma Genome Atlas (CGGA) repositories were used to evaluate the potential relationship. The value of clinical prognostic about GRM4 was assessed using clinical survival data from CGGA and TCGA. The GEPIA database was used to select genes like GRM4. PPI network was constructed by the database of (STRING), GO and KEGG analyses were performed. TargetScan, TarBase, miRDB, and starBase were used to explore miRNAs that could regulate GRM4 expression. EWAS Data Hub, MethSurv, and MEXPRESS were used for the analysis and relationship between DNA methylation and GRM4 expression and prognosis in glioma. TIMER2.0 and CAMOIP databases were used to assess the association between immune cell infiltration and GRM4. Human GBM cell lines were used to validate the function of GRM4. RESULTS: Our study shows that GRM4 is under expressed among gliomas and accompanied by poorer OS. Multivariate analysis showed that low mRNA expression of GRM4 was an independent factor of prognostic for shorter OS in all glioma patients. MiR-1262 affects the malignant phenotype of gliomas through GRM4. Methylation of DNA plays an important role in the instruction of GRM4 expression, the methylation level of GRM4 in glioma tissue is higher in comparison to normal tissue, and the higher methylation level was accompanied with the worse prognosis. Further analysis showed that GRM4 mRNA expression in GBM linked negatively with common lymphoid progenitor, Macrophage M1, Macrophage, and T cell CD4+ Th2, but not with the tumor purity. Overexpression of GRM4 prevents the migration of human GBM cell lines in vitro. CONCLUSION: GRM4 may have a substantial impact on the infiltration of immune cells and serve as a valuable prognostic biomarker in gliomas.

CRISPR-cas9 screening identified lethal genes enriched in Hippo kinase pathway and of predictive significance in primary low-grade glioma.

BACKGROUND: Low-grade gliomas (LGG) are a type of brain tumor that can be lethal, and it is essential to identify genes that are correlated with patient prognosis. In this study, we aimed to use CRISPR-cas9 screening data to identify key signaling pathways and develop a genetic signature associated with high-risk, low-grade glioma patients. METHODS: The study used CRISPR-cas9 screening data to identify essential genes correlated with cell survival in LGG. We used RNA-seq data to identify differentially expressed genes (DEGs) related to cell viability. Moreover, we used the least absolute shrinkage and selection operator (LASSO) method to construct a genetic signature for predicting overall survival in patients. We performed enrichment analysis to identify pathways mediated by DEGs, overlapping genes, and genes shared in the Weighted correlation network analysis (WGCNA). Finally, the study used western blot, qRT-PCR, and IHC to detect the expression of hub genes from signature in clinical samples. RESULTS: The study identified 145 overexpressed oncogenes in low-grade gliomas using the TCGA database. These genes were intersected with lethal genes identified in the CRISPR-cas9 screening data from Depmap database, which are enriched in Hippo pathways. A total of 19 genes were used to construct a genetic signature, and the Hippo signaling pathway was found to be the predominantly enriched pathway. The signature effectively distinguished between low- and high-risk patients, with high-risk patients showing a shorter overall survival duration. Differences in hub gene expression were found in different clinical samples, with the protein and mRNA expression of REP65 being significantly up-regulated in tumor cells. The study suggests that the Hippo signaling pathway may be a critical regulator of viability and tumor proliferation and therefore is an innovative new target for treating cancerous brain tumors, including low-grade gliomas. CONCLUSION: Our study identified a novel genetic signature associated with high-risk, LGG patients. We found that the Hippo signaling pathway was significantly enriched in this signature, indicating that it may be a critical regulator of tumor viability and proliferation in LGG. Targeting the Hippo pathway could be an innovative new strategy for treating LGG.

Overexpression of YTHDC2 contributes to the progression of prostate cancer and predicts poor outcomes in patients with prostate cancer.

YTH domain-containing protein 2 (YTHDC2), a member of N6-methyladenosine (m6A) readers, has been reported to be closely associated with multiple cancer types. However, very little is known about the YTHDC2 gene and its involvement in prostate cancer. YTHDC2 protein expression level was analyzed and correlated to clinical outcomes in prostate cancer patients who underwent prostatectomy in Guizhou Provincial People's Hospital. The YTHDC2 expression level was also detected in prostate cancer cell lines and an immortalized prostate epithelial cell line BPH-1 and RWPE1 by quantitative real-time reverse transcription polymerase chain reaction. Furthermore, we established stable cell lines (DU145 and PC-3) transfected with either empty vector or the full-length YTHDC2 gene and conducted cell function assays in vitro. Fisher's exact test and Pearson χ2 test were employed, Kaplan-Meier method was used for the survival analysis. Of 32 patient samples who enrolled in this study, YTHDC2 was significantly upregulated in prostate cancer (PCa) patients with higher Gleason scores and serum prostate-specific antigen levels. YTHDC2 expression was significantly elevated in all PCa cell lines compared to BPH-1 and RWPE1 (all p < 0.05). Functionally, the enforced expression of YTHDC2 markedly promoted cell growth, migration, and invasion efficacies in prostate cancer cells. Our data indicate that YTHDC2 upregulation may be potentially associated with the prognosis of prostate cancer patients.

Epidemiology of pediatric central nervous system tumors in Uyghur: experience from a single center.

PURPOSE: Retrospective analysis of clinical and epidemiological characteristics of central nervous system (CNS)tumors in Uyghur children from a single center in Xinjiang. METHODS: Between January 2013 and December 2021, 243 children (0-17 years old) with a clear pathological type of CNS tumor are collected and analyzed for tumor size, grade, and category, as well as their relationship with the child's gender, age, and region of origin according to the 2021 edition of the new WHO CNS tumor classification. OUTCOME: The 243 cases of CNS tumors in Uyghur children are predominantly from rural areas, with 144 cases (59.26%) of supratentorial tumors and 129 cases (53.09%) of low-grade tumors. With an overall male-to-female ratio of 1.43:1, a peak age of incidence of 6 to 8 years. CONCLUDING: The present study is based on a 9-year analysis of pediatric CNS data from a single center, and the center is the largest tertiary hospital in Xinjiang with large numbers of admitted patients, which may reflect some extent the clinical characteristics and epidemiological features characteristics of pediatric CNS tumors in Uyghur in Xinjiang.

Toxicological Effect and Molecular Mechanism of the Chiral Neonicotinoid Dinotefuran in Honeybees.

With the increasing demand for pollinating services, the wellness of honeybees has received widespread attention. Recent evidence indicated honeybee health might be posed a potential threat by widely used neonicotinoids worldwide. However, little is known about the molecular mechanism of these insecticides in honeybees especially at an enantiomeric level. In this study, we selected two species of bees, Apis mellifera (A. mellifera) and Apis cerana (A. cerana), to assess the toxicity and molecular mechanism of neonicotinoid dinotefuran and its enantiomers. The results showed that S-dinotefuran was more toxic than rac-dinotefuran and R-dinotefuran to honeybees by oral and contact exposures as much as 114 times. A. cerana was more susceptible to highly toxic enantiomer S-dinotefuran. S-dinotefuran induced the immune system response in A. cerana after 48 h exposure and significant changes were observed in the neuronal signaling of A. mellifera under three forms of dinotefuran exposure. Moreover, molecular docking also revealed that S-dinotefuran formed more hydrogen bonds than R-dinotefuran with nicotinic acetylcholine receptor, indicating the higher toxicity of S-dinotefuran. Data provided here show that R-dinotefuran may be a safer alternative to control pests and protect pollinators than rac-dinotefuran.

Construction of a novel rabbit model of ureteral calculi implanted with flowable resin.

BACKGROUND: The purpose of this study was to characterize the pathophysiological changes of hydronephrosis caused by ureteral calculi obstruction in a new rabbit ureteral calculi model by implanting flowable resin. METHODS: Forty New Zealand rabbits were randomly divided into two groups: the calculi group and the sham control group. In the calculi group (n = 20), rabbits were operated at left lower abdomen and the left ureter was exposed. Then flowable resin (flowable restorative dental materials) was injected into the left ureter using a 0.45 mm diameter intravenous infusion needle. Then light-cured for 40 s by means of a dental curing light to form calculi. In the sham control group, normal saline was injected into the ureter. Rabbits underwent X-ray and routine blood and urine tests preoperatively, as well as X-ray, CT, dissection, HE staining and routine blood and urine tests on 1, 3, 5 and 7 days postoperatively. Stone formation was assessed by X-ray and unenhanced CT scan after surgery. The pathophysiological changes were evaluated through dissection, HE staining and routine blood and urine tests. RESULTS: Ureteral calculi models were successfully constructed in 17 rabbits. In calculi group, high-density shadows were observed in the left lower abdomen on postoperative day 1st, 3rd, 5th and 7th by X-ray and CT scan. Dissection found obstruction formation of the left ureters, dilatation of the renal pelvis and upper ureter during 7 days after surgery. The renal long-diameters of the left ureters increased only on the 1st postoperative day. HE staining found ureteral and kidney damage after surgery. In calculi group and sham group,the serum creatinine, urea nitrogen, white blood cells and urine red blood cells were raised at day 1 after surgery. However, the indicators returned to normal at day 3, 5, and 7. CONCLUSIONS: This is a stable, less complicated operation and cost-effective ureteral calculi model by implanting flowable resin. And this novel model may allow us to further understand the pathophysiology changes caused by ureteral calculi obstruction.

Bivalirudin vs. Heparin on Radial Artery Thrombosis during Transradial Coronary Intervention: An Optical Coherence Tomography Study.

OBJECTIVES: This study aimed to evaluate the antithrombotic efficacy between bivalirudin and unfractionated heparin (UFH) on radial artery thrombosis (RAT) during transradial coronary intervention (TRI) by optical coherence tomography (OCT). METHODS AND RESULTS: We consecutively reviewed a total of 307 patients who underwent radial artery OCT inspection after TRI in our centre from October 2017 to January 2019; afterwards, 211 screened patients were divided into the UFH group (n = 144) and the bivalirudin group (n = 67) according to their anticoagulation strategy during TRI. The thrombosis in the radial artery was observed in 51 cases (24.17%) with a median thrombus volume of 0.054 mm3 (0.024, 0.334) and median thrombus score of 7 (4, 15). Thrombus occurred in 28 cases in the bivalirudin group with an incidence of 41.8%, which was significantly higher than that in the UFH group (n = 23, 16.0%, P < 0.001). This difference was even more remarkable after propensity score matching (bivalirudin group n = 22, 42.3% vs. UHF group n = 11, 13.9%, P < 0.001). Multivariate logistic analysis revealed that bivalirudin increased the RAT risk by 3.872 times (95% CI 2.006-8.354, P < 0.001) after adjustment for the other predictors. CONCLUSION: In this present study, the use of bivalirudin was associated with a higher risk of RAT than UFH. It highlighted UFH should be a more considerable choice to prevent radial artery access thrombosis in TRI.

Differentiation of isomeric cresols by silylation in combination with gas chromatography/mass spectrometry analysis.

RATIONALE: m-Cresol is listed as a priority controlled contaminant in many countries, but it is very difficult to accurately determine isomeric cresols due to their incomplete chromatographic separation on commercially available chromatographic columns and their nearly identical mass spectra. METHODS: Silylation of isomeric cresols was carried out by treatment with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The formed trimethyl(tolyloxy)silanes were analyzed by gas chromatography/mass spectrometry (GC/MS). Theoretical calculations were carried out with the Gaussian 03 program using the density functional theory (DFT) method at the B3LYP/6-311 + G(2d,p) level. RESULTS: The derivatives of three isomeric cresols and six isomeric xylenols have been completely separated on an HP-5MS capillary column within a GC run of only 10 minutes. In addition, the derivative o-cresol can be very easily differentiated from its isomers due to its characteristic base peak ion at m/z 91 in electron ionization (EI)-MS. DFT calculation results indicated that the formation of the abundant fragment ion at m/z 91 is attributed to a facile dissociation pathway involving the shift of a neighboring phenylmethyl hydrogen atom in EI-MS of trimethyl(o-tolyloxy)silane. CONCLUSIONS: Silylation provides a promising solution for simultaneous determination of isomeric cresols and isomeric xylenols.

Influencing factors of vascular endothelial function in patients with non-obstructive coronary atherosclerosis: a 1-year observational study.

BACKGROUND: Endothelial dysfunction may play a key role in non-obstructive coronary artery atherosclerosis. Our study aimed to evaluate the vascular endothelial function and its influencing factors in patients with non-obstructive coronary artery atherosclerosis. METHODS: A total of 131 consecutive patients with non-obstructive coronary artery atherosclerosis were enrolled. Flow-mediated dilatation (FMD) was measured at baseline and 1-year follow-up. Endothelial progenitor cells (EPCs) were counted by staining the fasting venous blood with antibodies against CD34 and vascular endothelial growth factor receptor 2. RESULTS: Systolic blood pressure, pulse pressure and the levels of HbA1c in participants with baseline FMD < 6% (n = 65) were significantly higher than those with baseline FMD ≥ 6% (n = 66). Baseline FMD was negatively associated with EPC counts (r = - 0.199, P < 0.05) and systolic blood pressure (r = - 0.315, P < 0.01). The 1-year FMD was significantly increased compared to the baseline FMD [(9.31 ± 5.62) % vs (7.31 ± 5.26) %, P < 0.001]. Independent predictors of FMD improvement included elevated EPC counts (OR = 1.104, 95% CI: 1.047-1.165, P < 0.001) and decreased levels of serum creatinine (OR = 0.915, 95% CI: 0.843-0.993, P = 0.034). CONCLUSIONS: Family history of premature cardiovascular diseases, hypertension, elevated systolic pressure, and HbA1c > 6.5% are independent risk factors for endothelial dysfunction in non-obstructive atherosclerotic patients. Elevated peripheral blood EPC counts and decreased levels of serum creatinine are independent predictors of endothelial function improvement.

Dietary phosphorus intake and serum prostate-specific antigen in non-prostate cancer American adults: A secondary analysis of the National Health and Nutrition Examination Survey (NHANES), 2003-2010.

BACKGROUND AND OBJECTIVES: Previous study has reported phosphorus intake is associated prostate cancer (PCa), but the association between phosphorus intake and serum prostate specific antigen (PSA) levels hasn't been reported in non-history of PCa population. Therefore, we performed a secondary data analysis based on existing data from the public Nutrition Examination Survey (NHANES) (2003-2010) database. METHODS AND STUDY DESIGN: Totally 6403 participants were selected from NHANES (2003-2010) database. The interested independent and dependent variables were considered as dietary phosphorus intake and PSA level, respectively. Covariates included demographic data, dietary data, physical examination data, and comorbidities. Weighted linear regression and generalized additive models were used to addressing the linear and non-linear link of phosphorus intake to PSA level. RESULTS: Linear association between phosphorus intake and PSA was not detected [ß=0.016 (95% Confidence Interval (CI) -0.012, 0.045)]. But we found an existing nonlinearity. By the recursive algorithm, the inflection point was 1151 mg. On the left side of the inflection point, we did not find the correlation between dietary phosphorus intake (per 100 change) and PSA level [ß=-0.04 (95% CI -0.11, 0.02), p=0.2155], while dietary phosphorus intake (per 100 change) positively associated with PSA [ß=0.05 (95% CI 0.01, 0.09) p=0.0293] on the right side of inflection point. CONCLUSIONS: There is a non-linear correlation between dietary phosphorus intake and PSA. Dietary phosphorus intake was positively associated with increased PSA when dietary phosphorus intake is beyond 1151 mg after adjusting other covariates. Over 1151 mg per day dietary phosphorus intake may be the risk factor for PSA increasing.

Curcumin inhibited the growth and invasion of human monocytic leukaemia SHI-1 cells in vivo by altering MAPK and MMP signalling.

Context: Curcumin, a polyphenolic compound extracted from the rhizome of the tropical plant Curcuma longa L. (Zingiberaceae), has been considered as a cancer chemopreventive drug by American National Cancer Institute.Objective: To examine the effect of curcumin on acute monocytic leukaemia SHI-1 cells in vivo.Materials and methods: The SHI-1 cells (1 × 106 cells in 0.1 mL PBS) were injected subcutaneously into the right flanks of the female SCID mice. Curcumin dissolved in olive oil (15 and 30 mg/kg) was administered (i.p.) to mice once a day for 15 days while the control group received olive oil injection. Tumour proliferation and apoptosis were examined by PCNA, TUNEL and cleaved caspase-3 staining. The expression of MAPK, NF-κB, MMP9, MMP2 and vimentin were confirmed by RT-PCR, immunohistochemistry or western blotting.Results: Administration of curcumin significantly inhibited tumour growth, as the tumour weight decreased from 0.67 g (control) to 0.47 g (15 mg/kg) and 0.35 g (30 mg/kg). Curcumin inhibited the expression of PCNA and increased the degree of TUNEL and cleaved caspase-3 staining in tumour tissue. The results of western blotting showed that curcumin treatment inhibited NF-κB and ERK signalling while activating p38 and JNK. Moreover, curcumin attenuated the mRNA transcription and protein expression of MMP2 and MMP9. Curcumin also suppressed the level of vimentin.Discussion and conclusions: Our study demonstrates that curcumin can inhibit the growth and invasion of human monocytic leukaemia in vivo, suggesting the possible use of curcumin for anti-metastasis in leukaemia and the value of determining its unique target.

Method development for analyzing ultratrace polyhalogenated carbazoles in soil and sediment.

In the past few years, polyhalogenated carbazoles (PHCZs) have been of increasing concern because their structure is similar to that of legacy POPs. In the present study, an analytical method, including intensive cleanup and fractionation procedures in combination with instrumental parameters, was developed to determine ultratrace polyhalogenated carbazoles (PHCZs) in soil and sediment. The eluting sorbents, volume and packing of the column were optimized. Our results showed that 5 g of florisil and 4 g of silica gel under 150 mL of hexane/DCM = 3:1 presented good performance in terms of recovery and repeatability. GC-HRMS, GC-MS/MS (EI-MRM) and GC-MS (EI-SIM) were applied to compare the performance of PHCZ analysis. For sensitivity, EI-MRM presents method detection limits comparable to those of GC-HRMS and much lower than those of EI-SIM. Regarding selectivity, GC-HRMS performed better than the other two techniques since GC-HRMS can reduce interference from perfluorokerosene (PFK) and DDX (DDT, DDE, and DDD) due to its high resolution. GC-HRMS was then further optimized by shortening the run time and modifying the SIM ion. The final method was successfully applied to determine PHCZs in soil and sediment, and the target compounds had almost 100% detection frequency in the samples. The ubiquitous presence of PHCZ in soil and sediment calls for a further investigation of its source, distribution and degradation in the environment.

Differentiation of isomeric methylanilines by imidization and gas chromatography/mass spectrometry analysis.

RATIONALE: Carcinogenic o-methylaniline is one of the banned aromatic amines in azo dyes, but it is very difficult to distinguish it from its noncarcinogenic isomers due to their identical retention time on chromatography and similar mass spectra. METHODS: Imidization of the isomeric methylanilines was carried out by treatment with benzaldehyde under mild conditions. The formed derivatives were analyzed by gas chromatography/mass spectrometry (GC/MS). Theoretical calculations were carried out on the Gaussian 03 program by using the density functional theory method at the B3LYP/6-311+G(d,p) level. RESULTS: Imidization of methylanilines occurred easily and gave rise to the corresponding N-methylbenzylidene benzenamines. The isomeric derivatives were completely separated by GC, and thus the three isomeric methylanilines could be determined simultaneously. Due to the ortho effect, the derivative from o-methylaniline has a characteristic fragment ion at m/z 118 with a stable bicyclic structure, and it could be easily differentiated from the meta- and para-isomers in electron ionization mass spectrometry. CONCLUSIONS: These results provided a promising solution for simultaneous determination of isomeric methylanilines.

Simultaneous determination of isomeric substituted anilines by imidization with benzaldehyde and gas chromatography-mass spectrometry.

The chromatographic separation of several isomeric anilines is a challenging issue. Herein, a simple method for the simultaneous determination of four groups of isomeric primary aromatic amines, including chloroanilines, methylanilines, methoxylanilines, and dimethylanilines, was presented. In this method, all of the 15 primary aromatic amines were easily transformed into the corresponding imine derivative by treatment with benzaldehyde under mild conditions. The formed isomeric imine derivatives were completely separated on a commercial capillary gas chromatography column. The effects of several derivatization parameters were investigated and optimized. Linearity in the optimized method ranged from 0.050 to 50 µg/mL with the squared correlation coefficients (R2 ) between 0.9981 and 0.9999. Reasonable reproducibility was obtained, with the intraday relative standard deviation (N = 5) ranging from 0.89 to 4.57% and interday relative standard deviation ranging from 2.26 to 7.69% at the concentration of 5.0 µg/mL. The developed method has been successfully applied to determine these isomeric aromatic amines in real samples.

Betanodavirus-like particles enter host cells via clathrin-mediated endocytosis in a cholesterol-, pH- and cytoskeleton-dependent manner.

Betanodavirus, also referred to nervous necrosis virus (NNV), is the causative agent of the fatal disease, viral nervous necrosis and has brought significant economic losses in marine and freshwater cultured fish, especially larvae and juveniles. Here, we used an established invasion model with virus-like particle (VLP)-cells, mimicking orange-spotted grouper nervous necrosis virus (OGNNV), to investigate the crucial events of virus entry. VLP were observed in the perinuclear regions of Asian sea bass (SB) cells within 1.5 h after attachment. VLP uptake was strongly inhibited when cells were pretreated with biochemical inhibitors (chlorpromazine and dynasore) blocking clathrin-mediated endocytosis (CME) or transfected with siRNA against clathrin heavy and light chains. Inhibitors against key regulators of caveolae/raft-dependent endocytosis and macropinocytosis had no effect on VLP uptake. In contrast, disruption of cellular cholesterol by methyl-ß-cyclodextrin or reduction of cholesterol fluidity by Cholera toxin B subunit significantly decreased VLP entry. Furthermore, VLP entry is dependent on low pH and cytoskeleton, demonstrated by inhibitor (chloroquine, ammonia chloride, cytochalasin D, wiskostatin, and nocodazole) perturbation. Therefore, OGNNV VLP enter SB cells via CME depending on dynamin-2, cholesterol and its fluidity, low pH, and cytoskeleton. In addition, ten more cell lines were screened for VLP entry and VLP can only enter NNV-sensitive cells, GB and SSN-1, via CME, indicating that CME is the common endocytosis pathway for VLP. These results may provide the data for NNV entry without the influence of the viral genome, an ideal model for exploring the behaviour of betanodavirus in cells, and valuable references to vaccine development.

The core ubiquitin system of mandarin fish, Siniperca chuatsi, can be utilized by infectious spleen and kidney necrosis virus.

The process of ubiquitination regulates various cellular processes. The ubiquitin-proteasome system (UPS) in fish, which is important for the generation of innate and adaptive immune responses to pathogens, is the target of aquatic viruses to achieve immune evasion. We cloned and characterized three genes, namely, a ubiquitin-activating enzyme (ScE1), a ubiquitin-conjugating enzyme (ScE2), and a HECT-type ubiquitin ligase (ScE3) of mandarin fish Siniperca chuatsi. The genes were expressed in all tissues and the highest levels were observed in the blood. In infectious spleen and kidney necrosis virus (ISKNV)-infected mandarin fish fry cells, the expression levels of the three genes in vitro were almost identical, and upregulated during the early stage and downregulated at the late stage. In the blood of ISKNV-infected mandarin fish, their expressions in vivo were downregulated equally although peaking at different timepoints, indicating the suppression of UPS by viral infection. Furthermore, these recombinant proteins were determined to function well in ubiquitination assays in vitro. Moreover, ScE1 and ScE2 can be utilized by four RING-type viral E3s (vE3s) that are encoded by ISKNV. The in vitro activity of vE3 was stronger than that of ScE3, suggesting that the fish UPS may be hijacked by ISKNV via E3 activity competition and expression modulation. The present study investigated the function of mandarin fish UPS as well as its response to iridovirus infection, providing insights to better understand the virus-host interactions and the mechanism of ISKNV in evading host immune responses.

Determination of trace polychlorinated biphenyls and organochlorine pesticides in water samples through large-volume stir bar sorptive extraction method with thermal desorption gas chromatography.

A fast and sensitive analytical method based on stir bar sorptive extraction technology with gas chromatography and mass spectrometry was developed to simultaneously analyze 18 kinds of polychlorinated biphenyls and 20 kinds of organochlorine pesticides in aqueous samples. A long adsorption time and small sample volume, which are problems encountered in conventional methods of stir bar sorptive extraction, were effectively solved by simultaneously using multiple stir bars for enrichment with sequential cryofocusing and merged injection. Optimized results showed good linear coefficients in the range of 10-500 ng/L and the method detection limits of 0.12-2.07 ng/L for polychlorinated biphenyls and organochlorine pesticides. The recovery ratios of the spiked samples at different concentrations were between 64.7 and 111.0%, and their relative standard deviations ranged from 0.9 to 17.6%. Four types of the studied compounds were determined in Qiantang River water samples, and their contents were between 0.82 and 5.00 ng/L.

Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle.

Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development.

Isomeric differentiation of chloroanilines by gas chromatography-mass spectrometry in combination with tosylation.

p-Chloroaniline is one of the banned aromatic amines in azo dyes, but it is very difficult to distinguish it from its isomers due to their identical retention time in chromatography and similar mass spectra. In this work, derivatization of the isomeric chloroanilines was carried out to yield the corresponding N-tosyl chloroanilines, which were completely separated by gas chromatography and also possessed clearly different electron ionization mass spectra. Thus, the three isomers could be differentiated and determined at the same time. Density functional theory calculation results indicated that the effect of the substituent pattern in electron ionization mass spectrometry is mainly due to the difference in the stability of the product ion (P2) at m/z 126, originating from the loss of tosyl radical from the precursor ion.