Tcap deficiency impedes striated muscle function and heart regeneration with elevated ROS and autophagy.

Telethonin/titin-cap (TCAP) encodes a Z-disc protein that plays important roles in sarcomere/T-tubule interactions, stretch-sensing and signaling. Mutations in TCAP are associated with muscular dystrophy and cardiomyopathy; however, the complete etiology and its roles in myocardial infarction and regeneration are not fully understood. Here, we generated tcap gene knockout zebrafish with CRISPR/Cas9 technology and observed muscular dystrophy-like phenotypes and abnormal mitochondria in skeletal muscles. The stretch-sensing ability was inhibited in tcap-/- mutants. Moreover, Tcap deficiency led to alterations in cardiac morphology and function as well as increases in reactive oxygen species (ROS) and mitophagy. In addition, the cardiac regeneration and cardiomyocyte proliferation ability of tcap-/- mutants were impaired, but these impairments could be rescued by supplementation with ROS scavengers or autophagy inhibitors. Overall, our study demonstrates the essential roles of Tcap in striated muscle function and heart regeneration. Additionally, elevations in ROS and autophagy may account for the phenotypes resulting from Tcap deficiency and could serve as novel therapeutic targets for muscular dystrophy and cardiomyopathy.

Embryonic dexamethasone exposure exacerbates hepatic steatosis and APAP-mediated liver injury in zebrafish.

Dexamethasone (DXMS), a synthetic glucocorticoid, is known for its pharmacological effects on anti-inflammation, stress response enhancement and immune suppression, and has been widely used to treat potential premature delivery and related diseases. However, emerging evidence has shown that prenatal DXMS exposure leads to increased susceptibility to multiple diseases. In the present study, we used zebrafish as a model to study the effects of embryonic DXMS exposure on liver development and disease. We discovered that embryonic DXMS exposure upregulated the levels of total cholesterol and triglycerides in the liver, increased the glycolysis process and ultimately caused hepatic steatosis in zebrafish larvae. Furthermore, DXMS exposure exacerbated hepatic steatosis in a zebrafish model of fatty liver disease. In addition, we showed that embryonic DXMS exposure worsened liver injury induced by paracetamol (N-acetyl-p-aminophenol, APAP), increased the infiltration of macrophages and neutrophils, and promoted the expression of inflammatory factors, leading to impeded liver regeneration. Taken together, our results provide new evidence that embryonic DXMS exposure exacerbates hepatic steatosis by activating glycolytic pathway, aggravates APAP-induced liver damage and impeded regeneration under a persistent inflammation, calling attention to DXMS administration during pregnancy with probable clinical implications for offspring.

Systemic inhibition of mitochondrial fatty acid ß-oxidation impedes zebrafish ventricle regeneration.

Unlike humans and other mammals, zebrafish demonstrate a remarkable capacity to regenerate their injured hearts throughout life. Mitochondrial fatty acid ß-oxidation (FAO) contributes to major energy demands of the adult hearts under physiological conditions; however, its functions in regulating cardiac regeneration and the underlying mechanisms are not completely understood. Different strategies targeting FAO have yield mixed outcomes. Here, we demonstrated that pharmacological inhibition of mitochondrial FAO with mildronate (MD) caused lipid accumulation in zebrafish larvae and suppressed ventricle regeneration. MD treatment impeded cardiogenic factor reactivation and cardiomyocyte (CM) proliferation, and impaired ventricle regeneration could be rescued by exogenous l-carnitine supplementation. Moreover, compared with the ablated hearts of wild-type fish, ventricle regeneration, cardiogenic factor reactivation and CM proliferation were significantly blocked in the ablated hearts of carnitine palmitoyltransferase-1b (cpt1b) knockout zebrafish. Further experiments suggested that NF-κB signaling and increased inflammation may be involved in the impediment of ventricle regeneration caused by systemic mitochondrial FAO inhibition. Overall, our study demonstrates the essential roles of mitochondrial FAO in zebrafish ventricle regeneration and reaffirms the sophisticated and multifaceted roles of FAO in heart regeneration with regard to different injury models and means of FAO inhibition.

Embryonic Amoxicillin Exposure Has Limited Impact on Liver Development but Increases Susceptibility to NAFLD in Zebrafish Larvae.

Amoxicillin is commonly used in clinical settings to target bacterial infection and is frequently prescribed during pregnancy. Investigations into its developmental toxicity and effects on disease susceptibility are not comprehensive. Our present study examined the effects of embryonic amoxicillin exposure on liver development and function, especially the effects on susceptibility to non-alcoholic fatty liver disease (NAFLD) using zebrafish as an animal model. We discovered that embryonic amoxicillin exposure did not compromise liver development, nor did it induce liver toxicity. However, co-treatment of amoxicillin and clavulanic acid diminished BESP expression, caused bile stasis and induced liver toxicity. Embryonic amoxicillin exposure resulted in elevated expression of lipid synthesis genes and exacerbated hepatic steatosis in a fructose-induced NAFLD model, indicating embryonic amoxicillin exposure increased susceptibility to NAFLD in zebrafish larvae. In summary, this research broadens our understanding of the risks of amoxicillin usage during pregnancy and provides evidence for the impact of embryonic amoxicillin exposure on disease susceptibility in offspring.

Visual and Rapid Detection of Porcine Epidemic Diarrhea Virus (PEDV) Using Reverse Transcription Loop-Mediated Isothermal Amplification Method.

Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/µL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis.

Low-toxic, fluorescent labeled and size-controlled graphene oxide quantum dots@polystyrene nanospheres as reference material for quantitative determination and in vivo tracing.

Polystyrene (PS) is selected as a representative nanoplastic and persistent pollutant for its difficult degradation and wide application. The environmental risk assessment of PS is obstructed by the toxic dye-based fluorescent PS, which false positives could be induced by the leakage of dye. For high biocompatibility, low toxicity, hydrophilicity, good water dispersibility, strong fluorescent stability, graphene oxide quantum dots (o-CQDs) are selected and embedded into PS microspheres, i.e., o-CQDs@PS, by microemulsion polymerization and denoted as CPS. Meanwhile, the sizes of CPS, e.g., 100, 150, and 200 nm, could be controlled by optimizing the type and number of water-soluble initiators. The anti-interference, low toxicity, and in vivo fluorescent tracing of CPS are proven by the coexistence of metals (including Fe2+, Fe3+, K+, Ba2+, Al3+, Zn2+, Mg2+, Ca2+, and Na+) on the fluorescence intensity of CPS, the growth of Chlorella pyrenoidosa and Artemia cysts as aquatic phytoplankton and zooplankton cultured with CPS, and the transfer of CPS from water into brine shrimp. In the concentration range of 0.1-100 mg/L, CPS can be quantitatively determined, which is suitable for coastal water and wastewater treatment plants. Therefore, CPS with standard size is suitable as reference material of PS.

Establishment of a porcine parvovirus (PPV) LAMP visual rapid detection method.

Porcine parvovirus (PPV) is one of the major causes of reproductive pig disease. Due to its serious nature, wide spread and consequent great damage to the swine industry, an effective, rapid and convenient method for its detection is needed. A loop-mediated isothermal amplification (LAMP) assay was established to detect PPV infection. Two pairs of primers were specifically designed to recognize the six different sequences of open reading frame1 (ORF1) gene. The optimized LAMP program was as follows: 50 min at 59 °C followed by 3 min at 80 °C.The amplified products were analyzed both by visual inspection after staining with SYBR Green I dye and by conventional agarose gel electrophoresis. Both methods showed the same sensitivity. The limit of detection (LOD) for PPV by LAMP was 10 copies, which is 100-fold lower than conventional PCR. Our LAMP assay did not cross-react with other viruses. We used the established LAMP system to test 1100 field samples and detected 660 positives. The LAMP detection method for PPV represents a visual, sensitive and rapid assay which can detect the virus in the field, offering an attractive alternative for the PPV detection methods currently in use.

Kinetics of viral load and antibody response in relation to COVID-19 severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.

Rapid visual detection of lily mottle virus using a loop-mediated isothermal amplification method.

Lily mottle virus (LMoV; genus Potyvirus, family Potyviridae) infects plants of the genus Lilium, causing a reduction in flower and bulb quality. A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed to detect the coat protein gene of LMoV. This LAMP method was highly specific for LMoV, with no cross-reaction with other lily viruses. The sensitivity of LMoV using the LAMP assay was 100 times more sensitive than that using conventional polymerase chain reaction. A reverse transcription LAMP (RT-LAMP) was then successfully applied to detect LMoV RNA. The newly established LAMP and one-step RT-LAMP provide an alternative method for detecting LMoV in lily plants.

[Correlation of follicular fluid human chorionic gonadotrophin level with oocyte maturity and early embryonic development].

OBJECTIVE: To explore the correlation of human chorionic gonadotrophin (hCG) level in the follicular fluid on oocyte retrieval day with the number of oocytes retrieved, maturation rate, embryonic development, and pregnancy outcome in controlled ovarian stimulation cycles. METHODS: The data of 311 IVF/ICSI-ET cycles from 2012 to 2013 was analyzed and stratified according to hCG level in follicular fluid on oocyte retrieval day (<7 nmol/L, 7-14 nmol/L, 14-21 nmol/L, and >21 nmol/L) determined with chemiluminescence method. The number of oocytes retrieved, oocyte maturation rate, fertilization rate, cleavage rate, available embryo rate and pregnancy rate were compared between the groups. RESULTS: In the IVF/ICSI-ET cycles, the cycles with hCG level of 14-21 nmol/L in the follicular fluid on the day of oocyte retrieval had significantly higher oocyte maturation rate and fertilization rate than those in the other 3 groups (P<0.05), but the number of oocytes retrieved, cleavage rate, available embryo rate and pregnancy rate, though slightly higher, showed no significant difference from the other 3 groups (P>0.05). In the group with hCG level >21 nmol/L, the oocyte maturation rate and fertilization rate were significantly lower than those in the other 3 groups (P<0.05), and the available embryo rate and pregnancy rate were slightly lower without significant differences from the other 3 groups (P<0.05). CONCLUSION: Follicular fluid hCG level on the day of oocyte retrieval is associated with oocyte maturation, fertilization, embryonic development potential, and IVF outcome. An excessively high follicular fluid hCG level on the day of oocyte retrieval may have negative effects on oocyte maturation and embryo development.

A new membrane re-anchored protein originating from GPC3 against hepatoma cells HepG2.

The aim of this study was to confirm the localization of recombinant pGPC3+afp-EGFP which expressed a new re-anchored protein named GPC3+afp-EGFP on the cytoplasmic membrane and to investigate its functions against hepatocellular carcinoma (HCC). EGFP expression in transfected HepG2 cells was observed using fluorescence and a confocal microscope. pGPC3+afp-EGFP expression was detected in membranous and soluble proteins extracted from transfected human embryonic kidney 293 cells by Western blot analysis using GPC3 mAb. The proliferation of transfected HepG2 cells with pGPC3+afp-EGFP (experimental group) was detected using SRB assay and compared to those of transfected HepG2 cells with pGPC3 (control group) and non-transfected HepG2 cells (blank group). Quantitative analysis of mRNA expression of the Fas gene was conducted by real-time PCR using the ß-actin housekeeping gene as the internal control at variable times. Apoptotic HepG2 cells in the three groups were counted and statistically analyzed by a contingency table Chi-square test using Spss 11.5 software and TUNEL assay. Production of both TNF-α and IFN-γ/IL2 was detected by ELISPOT after co-cultivation of transfected HepG2 cells with peripheral blood lymphocytes at different time-points in the experimental group. Green fluorescence was mainly found around the transfected HepG2 cell periphery through fluorescence and confocal microscopy. GPC3+afp-EGFP could not be detected in soluble protein but only in membranous protein. Proliferation curves showed that the proliferative quantities of transfected HepG2 cells in the experimental group decreased, whereas the mRNA expression of the Fas gene increased significantly compared to those of the other two groups. The numbers of apoptotic cells in the experimental group were significantly higher compared to those in the other two groups, as shown by statistical analysis. Both TNF-α and IFN-γ/IL2 were induced and were much higher in the experimental groups than in the diverse control groups at variable times. A new re-anchored protein GPC3+afp-EGFP expressed by recombinant pGPC3+afp-EGFP was localized on the cytoplasmic membrane, and had multiple functions against HCC, such as inhibition of transfected HepG2 cell proliferation, promotion of transfected HepG2 apoptosis and induction of antitumor cytokine excretion.

[Relation between insulin resistance and insulin receptor gene methylation in the endometrium of patients with polycystic ovary syndrome].

OBJECTIVE: To study the relationship between insulin resistance and methylation of insulin receptor (INSR) gene in the endometrium of women with polycystic ovary syndrome (PCOS). METHODS: Based on the HOMA index, 35 patients with PCOS were divided into insulin resistant group (IR group, n=18) and non-resistant group (NIR group, n=18). The patients age, serum estriol, testosterone, FSH and LH, fasting insulin and fasting blood glucose were compared between the two groups. The endometrial samples were obtained from the patients to examine DNA methylation status of INSR gene in the endometrial cells using methylation-specific PCR. RESULTS: The BMI, WHR, fasting glucose, fasting insulin, and HOMA index differed significantly between the two groups (P<0.05). PCR analysis showed partial methylation in the promoter region of INSR gene in 13 samples in IR group and 11 samples in NIR group, without detection of full methylation of the INSR gene in either group. The methylation status showed no significant difference between the two groups (P=0.328). CONCLUSION: Partial methylation of the INSR gene occurs in the endometria of PCOS patients, but this study does not provide a strong evidence supporting the relationship between insulin resistance and INSR gene methylation in women with PCOS.

[Correlations between preS1-antigen, HBV-DNA and HBV serum markers in patients with chronic hepatitis B].

OBJECTIVE: To study the correlations between preS1 antigen, HBV-DNA and hepatitis B virus (HBV) serum markers in patients with chronic hepatitis B. METHODS: The HBV markers, preS1 antigen and HBV-DNA were determined using enzyme- linked immunosorbent assay and quantitative PCR in 1158 patients with chronic hepatitis B. RESULTS: In these patients, the HBV-DNA positivity rate was 68.9%, significantly higher than preS1 antigen positivity (54.8%, chi2=53.24, P<0.005). The positivity rates of both HBV-DNA and PreS1-antigen were significantly higher in HBeAg-positive patients than in HBeAg-negative patients (P<0.005). The coincident rates of preS1-antigen and HBeAg with HBV-DNA were 56.9% and 63.3%, respectively. PreS1 antigen had higher sensitivity but lower specificity than HBeAg. The detection rates of preS1 antigen and HBeAg increased with the level of HBV-DNA, and preS1 antigen positivity was higher than that of HBeAg in patients with low HBV-DNA levels. CONCLUSION: Detection of HBV serum markers along with preS1 antigen and HBV-DNA may help assess the status of viral replication and therapeutic efficacy in patients with chronic hepatitis B. PreS1 antigen may serve as an auxiliary indicator in HBeAg-negative cases or when HBV-DNA detection is impossible.