Characterization of Human Norovirus Nonstructural Protein NS1.2 Involved in the Induction of the Filamentous Endoplasmic Reticulum, Enlarged Lipid Droplets, LC3 Recruitment, and Interaction with NTPase and NS4.

Human noroviruses (HuNVs) are the leading cause of gastroenteritis worldwide. NS1.2 is critical for HuNV pathogenesis, but the function is still unclear. The GII NS1.2 of HuNVs, unlike GI NS1.2, was localized to the endoplasmic reticulum (ER) and lipid droplets (LDs) and is accompanied by a distorted-filamentous ER morphology and aggregated-enlarged LDs. LC3 was recruited to the NS1.2-localized membrane through an autophagy-independent pathway. NS1.2, expressed from a cDNA clone of GII.4 norovirus, formed complexes with NTPase and NS4, which exhibited aggregated vesicle-like structures that were also colocalized with LC3 and LDs. NS1.2 is structurally divided into three domains from the N terminus: an inherently disordered region (IDR), a region that contains a putative hydrolase with the H-box/NC catalytic center (H-box/NC), and a C-terminal 251-330 a.a. region containing membrane-targeting domain. All three functional domains of NS1.2 were required for the induction of the filamentous ER. The IDR was essential for LC3 recruitment by NS1.2. Both the H-Box/NC and membrane-targeting domains are required for the induction of aggregated-enlarged LDs, NS1.2 self-assembly, and interaction with NTPase. The membrane-targeting domain was sufficient to interact with NS4. The study characterized the NS1.2 domain required for membrane targeting and protein-protein interactions, which are crucial for forming a viral replication complex.

Derivation and validation of a prediction model for neonate unplanned rehospitalization in a tertiary center in China.

BACKGROUND: To construct and externally validate a prediction model for neonate unplanned rehospitalization within 31 days of discharge. METHODS: A retrospective study was performed in the Department of Neonatology of the Children's Hospital of Fudan University. A binominal regression method was applied to construct and validate the prediction model. Analysis was performed on a total of 11,116 neonates with an index admission between 11/1/2016 and 12/31/2018. Neonates admitted from 11/1/2016 to 1/31/2018 were used for the selection of prognostic variables and construction of the model. Model validation was then performed with neonates admitted from 2/1/2018 to 12/31/2018. RESULTS: The rehospitalization rate for neonates was 3.27% (373/11,116). A total of 512 neonates were enrolled for the construction of the prediction model. Gestational age (GA), NICU length of stay (LOS), nonmedical order discharge and younger maternal age were strongly correlated with rehospitalization. By incorporating these 4 strong risk factors, we constructed a model to predict neonate unplanned rehospitalization within 31 days of discharge. The formula was turned into a nomogram for use in clinical practice. The nomogram has a total score of 180, with a predicted risk from 0 to 100%. Neonates are at high risk for rehospitalization if they have a total score greater than 39 points, according to the cutoff point established by the Youden index. The model was shown to have good discriminatory ability, with area under the receiver operating characteristic curves of 0.68 and 0.65 in the model construction and validation datasets, respectively. A total of 39 points is the cutoff for follow-up. CONCLUSIONS: The model is able to predict neonate unplanned rehospitalization well. A total score greater than 39 indicates that follow-up is necessary.

Prenatal chlorpyrifos exposure in association with PPARγ H3K4me3 and DNA methylation levels and child development.

BACKGROUND: Chlorpyrifos, one of the most widely used pesticides, can penetrate the placenta and affect fetal growth and neurodevelopment. Epigenetic regulation of peroxisome proliferator-activated receptor gamma (PPARγ), such as DNA methylation and trimethylation of lysine 4 of H3 (H3K4me3), may provide a potential mechanism for how fetal growth and development are impacted by chlorpyrifos exposure. The aims of the study were to investigate whether prenatal chlorpyrifos exposure was associated with H3K4me3 and DNA methylation levels of the PPARγ gene in the placenta and the related effects on birth outcomes and neurodevelopment. METHODS: Among 425 mother-infant pairs from the Taiwan Birth Panel Study, chlorpyrifos levels were measured in cord blood by using online SPE-LC/HESI/MS/MS; placental PPARγ H3K4me3 and DNA methylation levels were measured by ChIP-qPCR and pyrosequencing, respectively; the neonates' health outcomes were extracted from the medical records; and childhood neurodevelopment was evaluated by using the Comprehensive Developmental Inventory for Infants and Toddlers in 2-year-old children. Multivariable regression models were used to adjust for potential confounders. RESULTS: After controlling for potential confounders, each unit increase in the natural log-transformed prenatal chlorpyrifos exposure level was associated with an increase in the PPARγ DNA methylation level (adjusted ß (aß) = 0.77, p = 0.032) and poorer performance in the cognitive and language domains at 2 years old, especially in boys (aß = -1.66, p = 0.016, and aß = -1.79, p = 0.023, respectively). PPARγ H3K4me3 levels were positively associated with gestational age (aß = 0.16, p = 0.011), birth weight (aß = 30.52, p = 0.013), birth length (aß = 0.18, p = 0.003 and aß = 0.15, p = 0.042), and gross-motor performance (aß = 1.67, p = 0.036). CONCLUSIONS: Our findings suggested that prenatal chlorpyrifos exposure affected PPARγ DNA methylation levels and performance in the cognitive and language domains.

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein-Barr Virus Particles.

BGLF2 is a tegument protein of the Epstein-Barr virus (EBV). This study finds that BGLF2 is expressed in the late stage of the EBV lytic cycle. Microscopic investigations reveal that BGLF2 is present in both the nucleus and the cytoplasm and colocalized with BBLF1 and gp350 at juxtanuclear regions in the cytoplasm. This study also finds that the basic KKK69 motif of BGLF2 and acidic DYEE31 motif of BBLF1 are crucial for the interaction between BGLF2 and BBLF1, which is required for the recruitment of BGLF2 to the BBLF1 that is anchored on the trans-Golgi-network (TGN). In addition, BGLF2 in a density gradient is co-sedimented with un-enveloped capsids, revealing that BGLF2 associates with the EBV capsid before the final envelopment. The knockout of BGLF2 expression is demonstrated to reduce the numbers of infectious virions that are released into the culture medium, but they do not affect the expression of lytic proteins and viral DNA replication. The production of infectious viral particles by a BGLF2-knockout mutant can be rescued by exogenously expressed BGLF2 but only partially rescued by BGLF2-3KA, which is a mutant with reduced ability to interact with BBLF1 but does not affect its ability to activate the MAPK pathway and the expression of the EBV lytic proteins, suggesting that the interaction of BGLF2 with BBLF1 is important to the efficient production of infectious viral particles during the maturation. The results of this study improve our understanding of how BGLF2 promotes EBV viral production.

Association between IPTA gene polymorphisms and hematological abnormalities in hepatitis C virus-infected patients receiving combination therapy.

BACKGROUND/AIMS: Hematological abnormalities during hepatitis C virus (HCV) combination therapy with pegylated interferon α and ribavirin often necessitate dose reduction. Variants of the ITPA gene have been reported to protect against anemia during the early stages of HCV combina-tion treatments but have also been associated with larger decreases in platelet counts. We aimed to identify the as-sociation between specific ITPA gene polymorphisms and hematological abnormalities in patients undergoing HCV combination therapy. METHODS: In this retrospective study, 175 patients treated with HCV combination therapy were enrolled at St. Martin De Porres Hospital in Taiwan between 2006 and 2012. Two single nucleotide polymorphisms (SNP) within or adjacent to the ITPA gene (rs1127354, rs6051702) were genotyped. We investigated the effect of ITPA gene variants on hematological abnormalities during the therapy. RESULTS: The ITPA rs1127354 minor variants were significantly associated with protection against anemia at week 4 (p=1.86×10(-6)) and with more severe decreases in platelet counts during HCV combination therapy. SNP rs6051702 was not associated with the hemoglobin decline to >3 g/dL at week 4 in our study (p=0.055). CONCLUSIONS: The ITPA SNP rs1127354 is a useful predictor of ribavirin-induced anemia in Taiwanese patients and may be related to more severe decreases in platelet counts during the early stage of HCV combination therapy. (Gut Liver, 2015;9214-223).

Regulation of autophagic activation by Rta of Epstein-Barr virus via the extracellular signal-regulated kinase pathway.

Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development.

Characterization of Xanthomonas campestris pv. campestris heat shock protein A (HspA), which possesses an intrinsic ability to reactivate inactivated proteins.

hspA encodes a small heat shock protein (sHSP) in Xanthomonas campestris pv. campestris, the causative agent of black rot in cruciferous plants. In this study, two-dimensional gel electrophoresis, promoter activity assays, and Northern hybridization results revealed that HspA expression was induced by heat shock but not by other stresses, although low-level expression was detectable by reverse transcription-polymerase chain reaction (RT-PCR) under normal culture conditions. An hspA mutant exhibited reduced tolerance to heat, especially in the presence of MgSO4, but no change in pathogenicity. Results of size-exclusion chromatography indicated that purified HspA(his), containing six C-terminal histidine residues, formed two different size classes of oligomeric complexes--410 and 820 kDa. In contrast, HspA(ter), the unmodified protein translated from the original hspA gene, formed only the 820-kDa complex. These results suggest that the C-terminus of HspA is important for oligomerization. Both HspA820(his) and HspA410(his) were able to partially protect luciferase against heat-induced aggregation. Unlike other reported sHSPs that commonly capture denaturing proteins in refoldable states until refolded by adenosine triphosphate-dependent chaperone systems, HspA(his) alone was capable of reactivating heat-inactivated EcoRI. Thus, Xanthomonas campestris pv. campestris HspA has potential application as a protective agent during the storage of proteins.