Npro of classical swine fever virus enhances HMGB1 acetylation and its degradation by lysosomes to evade from HMGB1-mediated antiviral immunity.

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral Npro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or Npro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV Npro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent Npro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote Npro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its Npro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.

Porcine Circovirus 2 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Prenatal exposure to air pollution and the risk of preterm birth in rural population of Henan Province.

Due to the poor living and healthcare conditions, preterm birth (PTB) in rural population is a pressing health issue. However, PTB studies in rural population are rare. To explore the effects of air pollutants on PTB in rural population, we collected 697,316 medical records during 2014-2016 based on the National Free Preconception Health Examination Project. Logistic regression models were used to estimate the association between air pollutants and PTB and the modifying effects of demographic characteristics. Relative contribution and principal component analysis-generalized linear model (PCA-GLM) analysis were used to explore the most significant air pollutant and gestational period. Our results demonstrated that PTB risk is positively associated with exposure to air pollutants including PM10, PM2.5, SO2, NO2, and CO, while negatively associated with O3 exposure (P < 0.05). In addition, we found that NO2 was the largest contributor to the risk of PTB caused by air pollutants (26.5%). The third trimester of pregnancy was the most sensitive exposure window. PCA-GLM analysis showed that the first component (a combination of PM, SO2, NO2, and CO) increased the risk of PTB. Moreover, we found that rural women who are younger, had higher educated, multi-parity, or smoke appeared to be more sensitive to the association between air pollutants exposure and PTB (P-interaction<0.05). Our findings suggested that increased air pollutants except O3 were associated with elevated PTB risk, especially among vulnerable mothers. Therefore, the effects of air pollutants exposure on PTB should be mitigated by restricting emission sources of NO2 and SO2 in rural population, especially during the third trimester.

DNA methylation and fluoride exposure in school-age children: Epigenome-wide screening and population-based validation.

Excessive fluoride exposure and epigenetic change can induce numerous adverse health outcomes, but the role of epigenetics underneath the harmful health effects induced by fluoride exposure is unclear. In such gap, we evaluated the associations between fluoride exposure and genome-wide DNA methylation, and identified that novel candidate genes associated with fluoride exposure. A total of 931 school-age children (8-12 years) in Tongxu County of Henan Province (China) were recruited in 2017. Urinary fluoride (UF) concentrations were measured using the national standardized ion selective electrode method. Participants were divided into a high fluoride-exposure group (HFG) and control group (CG) according to the UF concentrations. Candidate differentially methylated regions (DMRs) were screened by Infinium-Methylation EPIC BeadChip of DNA samples collected from 16 participants (eight each from each group). Differentially methylated genes (DMGs) containing DMRs associated with skeletal and neuronal development influenced by fluoride exposure were confirmed using MethylTarget™ technology from 100 participants (fifty each from each group). DMGs were verified by quantitative methylation specific PCR from 815 participants. Serum levels of hormones were measured by auto biochemical analyzer. The mediation analysis of methylation in the effect of fluoride exposure on hormone levels was also performed. A total of 237 differentially methylated sites (DMSs) and 212 DMRs were found in different fluoride-exposure groups in the epigenome-wide phase. Methylation of the target sequences of neuronatin (NNAT), calcitonin-related polypeptide alpha (CALCA) and methylenetetrahydrofolate dehydrogenase 1 showed significant difference between the HFG and CG. Each 0.06% (95% CI: -0.11%, -0.01%) decreased in NNAT methylation status correlated with each increase of 1.0 mg/L in UF concentration in 815 school-age children using QMSP. Also, each 1.88% (95% CI: 0.04%, 3.72%) increase in CALCA methylation status correlated with each increase of 1.0 mg/L in UF concentration. The mediating effect of NNAT methylation was found in alterations of ACTH levels influenced by fluoride exposure, with a ß value of 11.7% (95% CI: 3.4%, 33.4%). In conclusion, long-term fluoride exposure affected the methylation pattern of genomic DNA. NNAT and CALCA as DMGs might be susceptible to fluoride exposure in school-age children.

Porcine Circovirus 2 Manipulates the PERK-ERO1α Axis of the Endoplasmic Reticulum To Favor Its Replication by Derepressing Viral DNA from HMGB1 Sequestration within Nuclei.

Porcine circovirus type 2 (PCV2) causes several disease syndromes in grower pigs. PCV2 infection triggers endoplasmic reticulum (ER) stress, autophagy, and oxidative stress, all of which support PCV2 replication. We have recently reported that nuclear HMGB1 is an anti-PCV2 factor by binding to viral genomic DNA. However, how PCV2 manipulates host cell responses to favor its replication has not been explored. Here, we demonstrate that PCV2 infection increased expression of ERO1α, generation of reactive oxygen species (ROS), and nucleocytoplasmic migration of HMGB1 via protein kinase R-like endoplasmic reticulum kinase (PERK) activation in PK-15 cells. Inhibition of PERK or ERO1α repressed ROS production in PCV2-infected cells and increased HMGB1 retention within nuclei. These findings indicate that PCV2-induced activation of the PERK-ERO1α axis would lead to enhanced generation of ROS sufficient to decrease HMGB1 retention in the nuclei, thus derepressing viral DNA from HMGB1 sequestration. The viral Rep and Cap proteins were able to induce PERK-ERO1α-mediated ROS accumulation. Cysteine residues 107 and 305 of Rep or 108 of Cap played important roles in PCV2-induced PERK activation and distribution of HMGB1. Of the mutant viruses, only the mutant PCV2 with substitution of all three cysteine residues failed to activate PERK with reduced ROS generation and decreased nucleocytoplasmic migration of HMGB1. Collectively, this study offers novel insight into the mechanism of enhanced viral replication in which PCV2 manipulates ER to perturb its redox homeostasis via the PERK-ERO1α axis, and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei-hence the release of HMGB1-bound viral DNA for replication. IMPORTANCE Considering the fact that clinical porcine circovirus-associated diseases (PCVAD) mostly results from activation of latent PCV2 infection by confounding factors such as coinfection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals, where PCV2 in infected cells might utilize the elevated reactive oxygen species (ROS) to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication, and severity of PCVAD. This will help decide the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD. Further investigation could examine if similar strategies are employed by DNA viruses, such as PCV3 and BFDV and if there is cross talk among endoplasmic reticulum (ER) stress, autophagy/mitophagy, and mitochondrial-sourced ROS in favor of PCV2 replication.

Effects of ambient temperature on the risk of preterm birth in offspring of adolescent mothers in rural henan, China.

BACKGROUND: A number of studies have explored the association between ambient temperature and preterm birth (PTB), but rarely among adolescent mothers. OBJECTIVES: To estimate the effects of ambient temperature on the risk of PTB and gestational age of newborns delivered by adolescent mothers in rural areas of Henan province. METHODS: We obtained 5394 medical records of adolescent mothers with results of pre-pregnancy physical examination and pregnancy outcomes from the National Free Preconception Health Examination Project (NFPHEP) in Henan province. Meteorological information was obtained from the China Meteorological Data Sharing Service System. Individual exposure levels were evaluated with an inverse distance-weighted model. A multiple logistic regression model and multiple linear regression model were used to estimate the effects of ambient temperature on the risk of PTB and gestational age, respectively. Stratified and interaction analyses were also performed. RESULTS: Of newborns in this study, 3.45% (186/5394) were PTB. Mean, maximum and minimum temperature during the entire pregnancy, especially the last 1-4 weeks of pregnancy, were positively associated with the risk of PTB and negatively associated with gestational age (P < 0.05). Nevertheless, a masking effect was observed that gestational age was positively associated with ambient temperature during the first trimester of pregnancy, due to the strongly inverse correlation between ambient temperature during the early and late stages of pregnancy. Stratified analyses showed that increasing temperature during the last 1-4 weeks of pregnancy increased the risk of PTB and decreased gestational age in newborns born in the cold season (P < 0.05). Furthermore, interaction analyses showed that birth season modified the effects of temperature on the gestational age (Pinteraction < 0.10). CONCLUSIONS: Elevated ambient temperature can decrease gestational age and increase the risk of PTB in offspring of adolescent mothers in rural areas. The birth season may modify the effects of temperature on gestational age.

ESRα Promoter Methylation May Modify the Association Between Lipid Metabolism and Type 2 Diabetes in Chinese Farmers.

Objective: This study is aimed to explore the potential association among the estrogen receptor alpha (ESRα) promoter methylation, lipid metabolism and the risk of type 2 diabetes mellitus (T2DM). Methods: A total of 1143 rural residents were recruited randomly from Henan Province, China. The circulating methylation levels in ESRα promoter region were determined by quantitative methylation-specific polymerase chain reaction. Serum high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglyceride (TG), total cholesterol (TC) and fasting plasma-glucose (FPG) were measured. Results: The ESRα promoter methylation levels were negatively associated with HDL-C levels whether gender stratification was performed (P < 0.05) and positively correlated with LDL-C in men (P < 0.05). Each unit standard deviation (SD) increment in TG was associated with a 43% increase (95% CI: 1.25, 1.64) in the risks of T2DM in all participants, a 36% increase (95% CI: 1.13, 1.64) in the risks of T2DM in men and a 49% increase (95% CI: 1.21, 1.83) in the risks of T2DM in women. Furthermore, each SD increment in HDL-C was associated with a reduction of 25% (OR = 0.75, 95% CI: 0.58, 0.97) in the risks of T2DM in men, and the risk of T2DM in men may be more susceptible to HDL-C than that in women (P for interaction < 0.05). Additionally, we found that the risk of T2DM in participants with lower methylation levels (≤4.07%) were more susceptible to HDL-C (P for interaction < 0.05). Conclusions: These findings suggested that lipid metabolism was associated with ESRα promoter methylation levels and the risk of T2DM. Besides, the levels of ESRα promoter methylation and gender can modify the association of HDL-C and T2DM.

Preconception ambient temperature and preterm birth: a time-series study in rural Henan, China.

Changes in the preconception ambient temperature (PAT) can affect the gametogenesis, disturbing the development of the embryo, but the health risks of PAT on the developing fetus are still unclear. Here, based on the National Free Preconception Health Examination Project in the rural areas of Henan Province, we evaluate the effects of PAT on preterm birth (PTB). Data of 1,231,715 records from self-reported interviews, preconception physical examination, early gestation follow-up, and postpartum follow-up were collected from 1 January 2013 to 31 December 2016. Generalized additive models were used to assess the cumulative and lag effects of PAT upon PTB. The significant cumulative effects of mean temperature within 2 weeks and 3 weeks on the risk of PTB, especially upon late PTB (34-36 weeks) (P < 0.05), were observed. Exposure to extreme heat (> 90th percentile) within 2 weeks (RR = 1.470) and 3 weeks (RR = 1.375) before conception could increase the risk of PTB. After stratifying PTB, exposure to extreme heat within 2 weeks before conception can increase the risks of early (< 34 weeks) and late PTB (P < 0.05). Besides, exposure to extreme cold (< 10th percentile) within 3 weeks or longer before conception can elevate the risk of PTB, especially late PTB. The significant lag effects of temperature changes on the risk of early PTB (lag-8 days or earlier) were observed. In conclusion, the risk of PTB was susceptible to PAT changes within 2 weeks or longer before conception. Our findings provide (i) guidance for rural couples to make pregnancy plans and (ii) scientific evidence for the government to formulate policies to prevent PTB.

Association between low-to-moderate fluoride exposure and bone mineral density in Chinese adults: Non-negligible role of RUNX2 promoter methylation.

Bone mineral density (BMD) changes were reported to be associated with excessive fluoride exposure and abnormal expression of RUNX2. However, whether the alteration of methylation status, a most commonly used marker for the alteration of gene expression in epidemiological investigation, of RUNX2 is associated with low-to-moderate fluoride exposure and BMD changes has not been reported. Our study aims to explore the role of RUNX2 promoter methylation in BMD changes induced by low-to-moderate fluoride exposure. A total of 1124 adults (413 men and 711 women) were recruited from Kaifeng City in 2017. We measured BMD using ultrasound bone densitometer. Concentrations of urinary fluoride (UF) were measured using ion-selective electrode, and the participants were grouped into control group (CG) and excessive fluoride group (EFG) according to the concentration of UF. We extracted DNA from fasting peripheral blood samples and then detected the promoter methylation levels of RUNX2 using quantitative methylation-specific PCR. Relationships between UF concentration, RUNX2 promoter methylation and BMD changes were analyzed using generalized linear model and logistic regression. Results showed in EFG (UF concentration > 1.6 mg/L), BMD was negatively correlated with UF concentration (ß: -0.14; 95%CI: -0.26, -0.01) and RUNX2 promoter methylation (ß: -0.13; 95%CI: -0.22, -0.03) in women. The methylation rate of RUNX2 promoter increased by 2.16% for each 1 mg/L increment in UF concentration of women in EFG (95%CI: 0.37, 3.96). No any significant associations between UF concentration, RUNX2 promoter methylation, and BMD were observed in the individuals in CG. Mediation analysis showed that RUNX2 promoter methylation mediated 18.2% (95% CI: 4.2%, 53.2%) of the association between UF concentration and BMD of women in EFG. In conclusion, excessive fluoride exposure (>1.6 mg/L) is associated with changes of BMD in women, and this association is mediated by RUNX2 promoter methylation.

Fluoride exposure and CALCA methylation is associated with the bone mineral density of Chinese women.

Excessive exposure to fluoride has been reported to affect bone mineral density (BMD). CALCA expression plays a critical part in bone formation. However, the role of CALCA in the association between fluoride and BMD is not known. We conducted a cross-sectional study and recruited 722 women in rural areas of Henan Province, China, to assess the relationship between fluoride exposure, CALCA methylation, and BMD. Urinary levels of fluoride, CALCA methylation, and BMD were measured by a fluoride ion-selective electrode, standalone ultrasound bone densitometer, and quantitative methylation-specific polymerases chain reaction, respectively. The association among fluoride exposure, CALCA methylation, and BMD was age-specific. Specifically, BMD was negatively correlated with methylation (ß: -0.008; 95% CI: -0.016, 0.000) and fluoride exposure (ß: -0.063; 95% CI: -0.129, -0.002) in women over 45 years and 50-54 years of age, respectively, whereas methylation was positively correlated with fluoride exposure (ß: 4.953; 95% CI: 1.162, 8.743) in women aged 40-44 years. Besides, increased BMD in women aged 45-49 years induced by the interactive effect of the highest methylation of CALCA exon 1 (tertile 3) and fluoride exposure was observed (P for interaction < 0.05). Our findings suggest an age-specific association between exposure to excessive fluoride, CALCA methylation, and BMD in a rural population of women in China. Notably, the susceptibility of BMD to fluoride exposure may be modified by CALCA methylation.

PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication.

Porcine circovirus type 2 (PCV2) is an important swine pathogen that causes significant economic losses to the pig industry. PCV2 interacts with host cellular factors to regulate its replication. High-mobility-group box 1 (HMGB1) protein, a major nonhistone protein in the nucleus, was recently discovered to participate in viral infections. Here, we demonstrate that nuclear HMGB1 negatively regulated PCV2 replication as shown by overexpression of HMGB1 or blockage of its nucleocytoplasmic translocation with ethyl pyruvate. The B box domain was essential in restricting PCV2 replication. Nuclear HMGB1 restricted PCV2 replication by sequestering the viral genome via binding to the Ori region. However, PCV2 infection induced translocation of HMGB1 from cell nuclei to the cytoplasmic compartment. Elevation of reactive oxygen species (ROS) induced by PCV2 infection was closely associated with cytosolic translocation of nuclear HMGB1. Treatment of PCV2-infected cells with ethyl pyruvate or N-acetylcysteine downregulated PCV2-induced ROS production, suppressed nucleocytoplasmic HMGB1 translocation, and decreased PCV2 replication. Collectively, these findings offer new insight into the mechanism of the PCV2 evasion strategy: PCV2 manages to escape restriction of its replication by nuclear HMGB1 by inducing ROS to trigger the nuclear-to-cytoplasmic translocation of HMGB1.IMPORTANCE Porcine circovirus type 2 (PCV2) is a small DNA virus that depends heavily on host cells for its infection. This study reports the close relationship between subcellular localization of host high-mobility-group box 1 (HMGB1) protein and viral replication during PCV2 infection. Restriction of PCV2 replication by nuclear HMGB1 is the early step of host defense at the host-pathogen interface. PCV2 then upregulates host reactive oxygen species (ROS) to prevent sequestration of its genome by expelling nuclear HMGB1 into the cytosol. It will be interesting to study if a similar evasion strategy is employed by other circoviruses such as beak and feather disease virus, recently discovered PCV3, and geminiviruses in plants. This study also provides insight into the justification and pharmacological basis of antioxidants as an adjunct therapy in PCV2 infection or possibly other diseases caused by the viruses that deploy the ROS-HMGB1 interaction favoring their replication.

Porcine Circovirus 2 Induction of ROS Is Responsible for Mitophagy in PK-15 Cells via Activation of Drp1 Phosphorylation.

Mitochondrial dynamics is essential for the maintenance of cell homeostasis. Previous studies have shown that porcine circovirus 2 (PCV2) infection decreases the mitochondrial membrane potential and causes the elevation of reactive oxygen species (ROS), which may ultimately lead to mitochondrial apoptosis. However, whether PCV2 induce mitophagy remains unknown. Here we show that PCV2-induced mitophagy in PK-15 cells via Drp1 phosphorylation and PINK1/Parkin activation. PCV2 infection enhanced the phosphorylation of Drp1 and its subsequent translocation to mitochondria. PCV2-induced Drp1 phosphorylation could be suppressed by specific CDK1 inhibitor RO-3306, suggesting CDK1 as its possible upstream molecule. PCV2 infection increased the amount of ROS, up-regulated PINK1 expression, and stimulated recruitment of Parkin to mitochondria. N-acetyl-L-cysteine (NAC) markedly decreased PCV2-induced ROS, down-regulated Drp1 phosphorylation, and lessened PINK1 expression and mitochondrial accumulation of Parkin. Inhibition of Drp1 by mitochondrial division inhibitor-1 Mdivi-1 or RNA silencing not only resulted in the reduction of ROS and PINK1, improved mitochondrial mass and mitochondrial membrane potential, and decreased mitochondrial translocation of Parkin, but also led to reduced apoptotic responses. Together, our study shows that ROS induction due to PCV2 infection is responsible for the activation of Drp1 and the subsequent mitophagic and mitochondrial apoptotic responses.

Porcine Circovirus Type 2 Induces ORF3-Independent Mitochondrial Apoptosis via PERK Activation and Elevation of Cytosolic Calcium.

Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316-323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca2+ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca2+ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca2+ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS.IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca2+ and ROS levels. Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ levels and cellular ROS levels as a result of activation of the PERK pathway.

Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples.

We developed and tested a prototype of an antibody microarray immunoassay for simultaneous quantitative detection of four typical mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, and fumonisin B1) in corn samples. The test kit consisted of a nitrocellulose membrane layered with immobilized monoclonal antibodies against mycotoxins. During the assay, the mycotoxin-protein conjugates were biotinylated. The signal detection was enhanced by a combination of the biotin-streptavidin system and enhanced chemiluminescence (ECL). This improved the sensitivity of the assay. Under the optimized conditions, four calibration curves with goodness of fit (R² > 0.98) were plotted. The results showed that the detection limits for aflatoxin B1, ochratoxin A, zearalenone, and fumonisin B1 were 0.21, 0.19, 0.09, and 0.24 ng/mL, with detection ranges of 0.47⁻55.69, 0.48⁻127.11, 0.22⁻31.36, and 0.56⁻92.57 ng/mL, respectively. The limit of detection (LOD) of this antibody microarray for aflatoxin B1, ochratoxin A, zearalenone, and fumonisin B1 in corn was 5.25, 4.75, 2.25, and 6 µg/kg, respectively. The recovery rates from the spiked samples were between 79.2% and 113.4%, with coefficient of variation <10%. The results of the analysis of commercial samples for mycotoxins using this new assay and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) were comparable and in good agreement. This assay could also be modified for the simultaneous detection of other multiple mycotoxins, as well as low-weight analytes, hazardous to human health.

Development of a Magnetic Nanoparticles-Based Screen-Printed Electrodes (MNPs-SPEs) Biosensor for the Quantification of Ochratoxin A in Cereal and Feed Samples.

A rapid and sensitive electrochemical biosensor based on magnetic nanoparticles and screen-printed electrodes (MNPs-SPEs sensor) was developed for the detection of ochratoxin A (OTA) in cereal and feed samples. Different types of magnetic nanoparticles-based ELISA (MNPs-ELISA) were optimized, and the signal detection, as well as sensitivity, was enhanced by the combined use of screen-printed electrodes (SPEs). Under the optimized conditions, the calibration curve of the MNPs-SPEs sensor was y = 0.3372x + 0.8324 (R² = 0.9805). The linear range of detection and the detection limit were 0.01⁻0.82 ng/mL and 0.007 ng/mL, respectively. In addition, 50% inhibition (IC50) was detectable at 0.10 ng/mL. The limit of detection (LOD) of this MNPs-SPEs sensor in cereal and feed samples was 0.28 µg/kg. The recovery rates in spiked samples were between 78.7% and 113.5%, and the relative standard deviations (RSDs) were 3.6⁻9.8%, with the coefficient of variation lower than 15%. Parallel analysis of commercial samples (corn, wheat, and feedstuff) showed a good correlation between MNPs-SPEs sensor and liquid chromatography tandem mass spectrometry (LC/MS-MS). This new method provides a rapid, highly sensitive, and less time-consuming method to determine levels of ochratoxin A in cereal and feedstuff samples.

Development and evaluation of height diameter at breast models for native Chinese Metasequoia.

Accurate tree height and diameter at breast height (dbh) are important input variables for growth and yield models. A total of 5503 Chinese Metasequoia trees were used in this study. We studied 53 fitted models, of which 7 were linear models and 46 were non-linear models. These models were divided into two groups of single models and multivariate models according to the number of independent variables. The results show that the allometry equation of tree height which has diameter at breast height as independent variable can better reflect the change of tree height; in addition the prediction accuracy of the multivariate composite models is higher than that of the single variable models. Although tree age is not the most important variable in the study of the relationship between tree height and dbh, the consideration of tree age when choosing models and parameters in model selection can make the prediction of tree height more accurate. The amount of data is also an important parameter what can improve the reliability of models. Other variables such as tree height, main dbh and altitude, etc can also affect models. In this study, the method of developing the recommended models for predicting the tree height of native Metasequoias aged 50-485 years is statistically reliable and can be used for reference in predicting the growth and production of mature native Metasequoia.

Complete mitochondrial genome of the Pigeye Shark Carcharhinus amboinensis (Carcharhiniformes: Carcharhinidae).

In this manuscript we describe the first complete mitochondrial sequence for the Data Deficient Pigeye Shark Carcharhinus amboinensis. The mitogenome is 16,704 bp long and consists of 1 control region, 2 rRNA genes, 22 tRNA genes and 13 protein-coding genes with an overall base composition of 31.6% A, 24.9% C, 13.1% G and 30.4% T. The gene arrangement pattern and transcriptional direction were typical for a vertebrate species. The tRNA-Ser2 lacks the dihydrouridine arm and forms a simple loop, therefore it cannot be folded into the typical cloverleaf secondary structures like other tRNAs.

Complete mitochondrial genome of the Yellow-spotted skate Okamejei hollandi (Rajiformes: Rajidae).

The complete mitochondrial genome of the Yellow-spotted skate Okamejei hollandi was determined in this study. It is 16,974 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one putative control region. The overall base composition is 30.5% A, 27.8% C, 14.0% G, and 27.8% T. There are 28 bp short intergenic spaces located in 12 gene junctions and 31 bp overlaps located in nine gene junctions in the whole mitogenome. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were used in the protein-coding genes. The lengths of 22 tRNA genes range from 68 (tRNA-Ser2) to 75 (tRNA-Leu1) bp. The origin of L-strand replication (OL) sequence (37 bp) was identified between the tRNA-Asn and tRNA-Cys genes. The control region is 1311 bp in length with high A + T and poor G content.

An improvement of shotgun proteomics analysis by adding next-generation sequencing transcriptome data in orange.

BACKGROUND: Shotgun proteomics data analysis usually relies on database search. Because commonly employed protein sequence databases of most species do not contain sufficient protein information, the application of shotgun proteomics to the research of protein sequence profile remains a big challenge, especially to the species whose genome has not been sequenced yet. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we present a workflow with integrated database to partly address this problem. First, we downloaded the homologous species database. Next, we identified the transcriptome of the sample, created a protein sequence database based on the transcriptome data, and integtrated it with homologous species database. Lastly, we developed a workflow for identifying peptides simultaneously from shotgun proteomics data. CONCLUSIONS/SIGNIFICANCE: We used datasets from orange leaves samples to demonstrate our workflow. The results showed that the integrated database had great advantage on orange shotgun proteomics data analysis compared to the homologous species database, an 18.5% increase in number of proteins identification.