A porcine circovirus type 2d-based virus-like particle vaccine induces humoral and cellular immune responses and effectively protects pigs against PCV2d challenge.

The pathogenic porcine circovirus type 2 (PCV2) leads to significant economic losses in pig production. PCV2d is currently the dominant genotype causing porcine circovirus-associated disease (PCVAD) worldwide. Therefore, development of a recombinant PCV2d-based vaccine is required to elicit complete protection against PCV2d infection. In this study, we generated virus-like particles of PCV2d-based capsid protein (Bac-2dCP) using a baculovirus expression system and evaluated its protective efficacy against PCV2d infection in specific pathogen-free (SPF) pigs. Three-week-old SPF miniature pigs were intramuscularly immunized with purified Bac-2dCP and intranasally challenged with PCV2d at 4 weeks post-vaccination. The Bac-2dCP group showed significantly higher IgG levels and neutralizing antibodies against PCV2b and PCV2d genotypes, as well as increased interferon-γ levels, and increased body weight and average daily weight gain compared with positive (challenged) and negative (unchallenged) controls. In particular, the Bac-2dCP group showed almost complete absence of PCV2d DNA in serum, nasal, and rectal swabs and in lung, lymph node, and kidney tissue samples. However, the positive control group exhibited low levels of neutralizing antibody, and high levels of PCV2 DNA in serum, swab, and tissue samples, resulting in PCV2-associated pathological lesions. The results of this study demonstrated that a recombinant Bac-2dCP vaccine conferred complete protection against a PCV2d challenge in SPF miniature pigs.

Glucose Transport and Transporters in the Endomembranes.

Glucose is a basic nutrient in most of the creatures; its transport through biological membranes is an absolute requirement of life. This role is fulfilled by glucose transporters, mediating the transport of glucose by facilitated diffusion or by secondary active transport. GLUT (glucose transporter) or SLC2A (Solute carrier 2A) families represent the main glucose transporters in mammalian cells, originally described as plasma membrane transporters. Glucose transport through intracellular membranes has not been elucidated yet; however, glucose is formed in the lumen of various organelles. The glucose-6-phosphatase system catalyzing the last common step of gluconeogenesis and glycogenolysis generates glucose within the lumen of the endoplasmic reticulum. Posttranslational processing of the oligosaccharide moiety of glycoproteins also results in intraluminal glucose formation in the endoplasmic reticulum (ER) and Golgi. Autophagic degradation of polysaccharides, glycoproteins, and glycolipids leads to glucose accumulation in lysosomes. Despite the obvious necessity, the mechanism of glucose transport and the molecular nature of mediating proteins in the endomembranes have been hardly elucidated for the last few years. However, recent studies revealed the intracellular localization and functional features of some glucose transporters; the aim of the present paper was to summarize the collected knowledge.

Integrative analysis of DNA methylation and mRNA expression during differentiation of umbilical cord blood derived mononuclear cells to endothelial cells.

Differentiation of umbilical cord blood derived mononuclear cells to endothelial cells is accompanied by massive changes in gene expression. Although methylation and demethylation of DNA likely play crucial roles in regulating gene expression, their interplay during differentiation remains elusive. To address this question, we performed deep sequencing of DNA methylation and mRNA expression to profile global changes in promoter methylation and gene expression during differentiation from mononuclear cells to outgrowing cells. We identified 61 downregulated genes with hypermethylation, including CD74, VAV1, TLR8, and NCF4, as well as 21 upregulated genes with hypomethylation, including ECSCR, MCAM, PGF, and ARHGEF15. Interestingly, gene ontology analysis showed that downregulated genes with hypermethylation were enriched in immune-related functions, and upregulated genes with hypomethylation were enriched in the developmental process and angiogenesis, indicating the important roles of DNA methylation in regulating differentiation. We performed polymerase chain reaction analyses and bisulfite sequencing of representative genes (CD74, VAV1, ECSCR, and MCAM) to verify the negative correlation between DNA methylation and gene expression. Further, inhibition of DNA methyltransferase and demethylase activities using 5'-aza-dc and shRNAs, specific for TET1 and TET2 mRNAs, respectively, revealed that DNA methylation was the main regulator of the reversible expression of functionally important genes. Collectively, our findings implicate DNA methylation as a critical regulator of gene expression during umbilical cord blood derived mononuclear cells to endothelial cell differentiation.

Sac-1004, a vascular leakage blocker, reduces cerebral ischemia-reperfusion injury by suppressing blood-brain barrier disruption and inflammation.

BACKGROUND: Blood-brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring. RESULTS: Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1ß-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation. CONCLUSIONS: Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.

The endothelial E3 ligase HECW2 promotes endothelial cell junctions by increasing AMOTL1 protein stability via K63-linked ubiquitination.

Cell-to-cell junctions are critical for the formation of endothelial barriers, and its disorganization is required for sprouting angiogenesis. Members of the angiomotin (AMOT) family have emerged as key regulators in the control of endothelial cell (EC) junction stability and permeability. However, the underlying mechanism by which the AMOT family is regulated in ECs remains unclear. Here we report that HECW2, a novel EC ubiquitin E3 ligase, plays a critical role in stabilizing endothelial cell-to-cell junctions by regulating AMOT-like 1 (AMOTL1) stability. HECW2 physically interacts with AMOTL1 and enhances its stability via lysine 63-linked ubiquitination. HECW2 depletion in human ECs decreases AMOTL1 stability, loosening the cell-to-cell junctions and altering subcellular localization of yes-associated protein (YAP) from cytoplasm into the nucleus. Knockdown of HECW2 also results in increased angiogenic sprouting, and this effect is blocked by depletion of ANG-2, a potential target of YAP. These results demonstrate that HECW2 is a novel regulator of angiogenesis and provide new insights into the mechanisms coordinating junction stability and angiogenic activation in ECs.

Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2.

Angiogenesis is regulated by the dynamic interaction between endothelial cells (ECs). Hippo-Yes-associated protein (YAP) signalling has emerged as a key pathway that controls organ size and tissue growth by mediating cell contact inhibition. However, the role of YAP in EC has not been defined yet. Here, we show expression of YAP in the developing front of mouse retinal vessels. YAP subcellular localization, phosphorylation and activity are regulated by VE-cadherin-mediated-EC contacts. This VE-cadherin-dependent YAP phosphorylation requires phosphoinositide 3-kinase-Akt activation. We further identify angiopoietin-2 (ANG-2) as a potential transcriptional target of YAP in regulating angiogenic activity of EC in vitro and in vivo. Overexpression of YAP-active form in EC enhances angiogenic sprouting, and this effect is blocked by ANG-2 depletion or soluble Tie-2 treatment. These findings implicate YAP as a critical regulator in angiogenesis and provide new insights into the mechanism coordinating junctional stability and angiogenic activation of ECs.

TIS21(/BTG2/PC3) accelerates the repair of DNA double strand breaks by enhancing Mre11 methylation and blocking damage signal transfer to the Chk2(T68)-p53(S20) pathway.

DNA double strand breaks (DSBs) occur more frequently in TIS21(-/-) mouse embryo fibroblasts than that in wild type MEFs (wt-MEFs). Therefore, the role TIS21 plays in the DNA damage response was investigated. Adenoviral transduction of Huh7 tumor cells with the TIS21 gene accelerated the repair of DSBs induced by etoposide treatment as evaluated by clearance of γH2AX foci and the Comet assay. TIS21 increased methylation of Mre11 and protein arginine methyltransferase 1 (PRMT1) activity, leading to Mre11 activation in vitro and in vivo, as determined by immunoprecipitation and radiolabeling analyses. When downstream DNA damage response mediators were evaluated in various human cancer cells lines, TIS21 was found to strongly inhibit Chk2(T68) and p53(S20) phosphorylation by p-ATM(S1981) but not p53(S15). The loss of Chk2 activation after etoposide treatment reduced apoptosis in the cells by downregulating the expression of E2F1 and Bax. These data suggest that TIS21 regulates DSB repair and apoptosis. Expression of TIS21 promoted the repair of DSBs and reduced apoptosis by blocking the damage signal from p-ATM(S1981) to Chk2(T68)-p53(S20)via the activation of Mre11 and PRMT1.

Comparative study on the toxic effects of red tide flagellates Heterocapsa circularisquama and Chattonella marina on the short-necked clam (Ruditapes philippinarum).

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.