The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

Injectable, self-healing hyaluronic acid-based hydrogels for spinal cord injury repair.

The cystic cavity that develops following spinal cord injury is a major obstacle for repairing spinal cord injury (SCI). The injectable self-healing biomaterials treatment is a promising strategy to enhance tissue repair after traumatic spinal cord injury. Herein, a natural extracellular matrix (ECM) biopolymer hyaluronic acid-based hydrogel was developed based on multiple dynamic covalent bonds. The hydrogels exhibited excellent injectable and self-healing properties, could be effectively injected into the injury site, and filled the lesion cavity to accelerate the tissue repair of traumatic SCI. Moreover, the hydrogels were compatible with cells and various tissues and possessed proper stiffness matched with nervous tissue. Additionally, when implanted into the injured spinal cord site, the hyaluronic acid-based hydrogel promoted axonal regeneration and functional recovery by accelerating remyelination, axon regeneration, and angiogenesis. Overall, the injectable self-healing hyaluronic acid-based hydrogels are ideal biomaterials for treating traumatic SCI.

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.

Porcine circovirus type 2 induces CHOP-ERO1α-ROS-mediated apoptosis in PK-15 cells.

Porcine circovirus type 2 (PCV2) infection induces endoplasmic reticulum (ER) stress and oxidative stress. These cellular responses could be connected with apoptosis. However, the mechanisms that link ER stress and oxidative stress in PCV2-induced apoptosis are poorly characterized. Here, we demonstrate that PCV2 infection increased expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of CHOP by RNA silencing or inhibition of ERO1α by short hairpin RNA or EN460 repressed PCV2-induced reactive oxygen species (ROS) generation, cytosolic calcium level, and apoptotic rate in PK-15 cells. Overexpression of ERO1α enhanced PCV2-induced oxidative stress, caspase-3 cleavage, and apoptosis rate. Treatment of PCV2-infected cells with ROS scavenger N-acetyl-L-cysteine downregulated PCV2-induced ROS production, cytosolic calcium level, and apoptosis rate, but intriguingly decreased expression of CHOP and ERO1α. Thus, we propose that PCV2 induces apoptosis through ER Stress via CHOP-ERO1α-ROS signaling in host cells.

Degradable Hydrogel Adhesives with Enhanced Tissue Adhesion, Superior Self-Healing, Cytocompatibility, and Antibacterial Property.

Degradable hydrogel adhesives with multifunctional advantages are promising to be candidates as hemostatic agents, surgical sutures, and wound dressings. In this study, hydrogel adhesives are constructed by catechol-conjugated gelatin from natural resource, iron ions (Fe3+ ), and a synthetic polymer. Specifically, the latter is prepared by the radical ring-opening copolymerization of a cyclic ketene acetal monomer 5,6-benzo-2-methylene-1,3-dioxepane and N-(2-ethyl p-toluenesulfonate) maleimide. By the incorporation of ester bonds in the backbone and the combination with quaternary ammonium salt pendants in the polymer, it exhibits excellent degradability and antibacterial property. Remarkably, doping the synthetic polymer into the 3,4-dihydroxyphenylacetic acid-modified gelatin network forms a semi-interpenetrating polymer network which can effectively improve the rigidity, tissue adhesion, and antibacterial property of fabricated hydrogel adhesives. Moreover, non-covalent bonds from coordination interaction between catechol and Fe3+ contribute to the fast self-healing of the developed hydrogel adhesives. These hydrogel adhesives with the multiple merits including the degradability, enhanced tissue adhesion, superior self-healing, good cytocompatibility, and antibacterial property show the great potential to be used as tissue adhesives in biomedical fields.

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.

Self-healing hyaluronic acid hydrogels based on dynamic Schiff base linkages as biomaterials.

Natural hydrogels are widely investigated for biomedical applications because of their structures similar to extracellular matrix of native tissues, possessing excellent biocompatibility and biodegradability. However, they are often susceptible to mechanical disruption. In this study, self-healing hyaluronic acid (HA) hydrogels are fabricated through a facile dynamic covalent Schiff base reaction. Dialdehyde-modified HA (AHA) precursor was synthesized, and then the AHA/cystamine dihydrochloride (AHA/Cys) hydrogels were formed by blending AHA and Cys at acidic pH levels. By varying Cys to AHA ratio, the hydrogel morphology, swelling and kinetics of gelation could be controlled. Gelation occurred fast, which was predominantly attributed to Schiff base reaction between the dialdehyde groups on AHA and amimo groups on Cys. The hydrogel exhibited improved mechanical properties with increase in Cys content. Furthermore, due to dynamic imine bonds, this hydrogel demonstrated excellent self-healing ability based on the stress after mechanical disruption. Also, it was found to be pH-responsive and injectable. Taken together, this kind of hyaluronic acid hydrogel can provide promising future for various biomedical applications in drug delivery, bioprinting, smart robots and tissue regeneration.

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.

Deletion of glutaredoxin promotes oxidative tolerance and intracellular infection in Listeria monocytogenes.

Thiol-disulfide glutaredoxin systems of bacterial cytoplasm favor reducing conditions for the correct disulfide bonding of functional proteins, and therefore were employed by bacteria to defend against oxidative stress. Listeria monocytogenes has been shown to encode a putative glutaredoxin, Grx (encoded by lmo2344), while the underlying roles remain unknown. Here we suggest an unexpected role of L. monocytogenes Grx in oxidative tolerance and intracellular infection. The recombinant Grx was able to efficiently catalyze the thiol-disulfide oxidoreduction of insulin in the presence of DTT as an election donor. Unexpectedly, the deletion of grx resulted in a remarkably increased tolerance and survival ability of this bacteria when exposed to various oxidizing agents, including diamide, and copper and cadmium ions. Furthermore, loss of grx significantly promoted bacterial invasion and proliferation in human epithelial Caco-2 cells and murine macrophages, as well as a notably increasing invasion but not cell-to-cell spread in the murine fibroblasts L929 cells. More importantly, L. monocytogenes lacking the glutaredoxin exhibited more efficient proliferation and recovery in the spleens and livers of the infected mice, and hence became more virulent by upregulating the virulence factors, InlA and InlB. In summary, we here for the first time demonstrated that L. monocytogenes glutaredoxin plays a counterintuitive role in bacterial oxidative resistance and intracellular infection, which is the first report to provide valuable evidence for the role of glutaredoxins in bacterial infection, and more importantly suggests a favorable model to illustrate the functional diversity of bacterial Grx systems during environmental adaption and host infection.

Photopolymerizable thiol-acrylate maleiated hyaluronic acid/thiol-terminated poly(ethylene glycol) hydrogels as potential in-situ formable scaffolds.

Despite their potential in various biomedical applications, photocrosslinkable hyaluronate hydrogels have often been limited by weak formation and unsatisfied mechanical strength which can be attributed to insufficient substitution of photoactive groups on the hyaluronate backbone and the oxygen inhibition effect. In this study, a new approach for the production of hyaluronic acid (MHA) with high acrylate group substitution (i.e. 2.27) is developed. It is based on the reaction of sodium hyaluronate and maleic anhydride in dimethyl sulfoxide, which has never been reported previously. Furthermore, the thiol-acrylate photopolymerization approach is employed to prepare maleiated hyaluronic acid/thiol-terminated poly(ethylene glycol) (MHA/TPEG) hydrogels which can overcome the oxygen inhibition effect. And the hydrogels possess porous structures, high swelling ratio, and tunable degradation rate. Specifically, the hydrogels could gel quickly within 15 s and demonstrate improved stiff (G' = 4100 Pa). The in vitro cytotoxic evaluation demonstrates that the hydrogels are cytocompatible to L929 cells. As a result, the in-situ formable hydrogel scaffolds exhibit great potential for medical applications.

Thermal-oxidative effect of Kraft lignin antioxidant in polypropylene: Uncovering the key factor using correlation analysis model.

Which factors are crucial to improving lignin antioxidant ability in polymers has been debated over years. Here, the structural effect of natural antioxidant from technical Kraft lignin (KL) on the oxidation induction time (OIT) of KL/polypropylene (PP) was quantitatively investigated using correlation analysis model instead of traditional linear fitting model. The correlation coefficient and significance value of their relationship clearly demonstrated the dominating role of non-condensed Phenolic OH (Ph-OH) in improving KL/PP thermal-oxidation stability, subsequently followed by M¯n, aliphatic OH/total Ph-OH, condensed Ph-OH and polydispersity. OIT is positive with non-condensed and total Ph-OH, while it declines with increase of the above other factors. Remarkably, the longest OIT (∼90min) was obtained even at extreme loading (0.5wt%), almost ∼911% and ∼201% huge increase in contrast to that of pure PP and crude KL/PP composite, respectively. Meanwhile, the mechanical properties of KL fractions/PP blends can be maintained at the same or better level than that of pure PP, due to strong interfacial adhesion strength.

Porcine teschovirus 2 induces an incomplete autophagic response in PK-15 cells.

Autophagy is a homeostatic process that has been shown to be vital in the innate immune defense against pathogens. However, little is known about the regulatory role of autophagy in porcine teschovirus 2 (PTV-2) replication. In this study, we found that PTV-2 infection induces a strong increase in GFP-LC3 punctae and endogenous LC3 lipidation. However, PTV-2 infection did not enhance autophagic protein degradation. When cellular autophagy was pharmacologically inhibited by wortmannin or 3-methyladenine, PTV-2 replication increased. The increase in virus yield via autophagy inhibition was further confirmed by silencing atg5, which is required for autophagy. Furthermore, PTV-2 replication was suppressed when autophagy was activated by rapamycin. Together, the results suggest that PTV-2 infection activates incomplete autophagy and that autophagy then inhibits further PTV-2 replication.

Selective aminolysis of acetylated lignin: Toward simultaneously improving thermal-oxidative stability and maintaining mechanical properties of polypropylene.

Even with outstanding radical capturing ability, the utilization of lignin as a natural antioxidant in polypropylene (PP) still has been pended. Usually, the compatibility of its blends is improved based on the reaction of hydroxyl content, thus leading to the decreasing content of phenolic hydroxyl (Ph-OH) group and inferior thermal-oxidative stability of lignin blends. Here, the selective aminolysis of acetylated Kraft lignin (pyr-KL) was investigated, which structures were characterized using FTIR, 31P-NMR and GPC. The Ph-OH group of acetylated KL could be released by the addition of pyrrolidine; however the aliphatic hydroxyl group is still blocked. With the control of reaction conditions, the highest oxidation induction time of pyr-KL/PP (0.5wt% loading) reaches up to 22.6min, almost 2.6 times than that of pure PP. More importantly, the mechanical properties of PP were also maintained under the loading of pyr-KL, which is much better than that of curde KL/PP.

Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis.

Porcine circovirus type 2 (PCV2) has recently been reported to elicit the unfolded protein response (UPR) via activation of the PERK/eIF2α (RNA-activated protein kinase-like endoplasmic reticulum (ER) kinase/eukaryotic initiation factor 2α) pathway. This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein. By transient expression, we found that both replicase (Rep) and capsid (Cap) proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eIF2α-ATF4 (activating transcription factor 4)-CHOP (CCAAT/enhancer-binding protein homologous protein) axis. Cap expression, but not Rep, significantly reduced anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage, possibly due to increased expression of CHOP. Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression, caspase-3 cleavage, and apoptotic cell death possibly by partially rescuing Bcl-2 expression, we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eIF2α/ATF4/CHOP/Bcl-2 pathway. This study, together with our earlier studies, provides insight into the mechanisms underlying PCV2 pathogenesis.

Effects of Dietary Bacillus licheniformis on Gut Physical Barrier, Immunity, and Reproductive Hormones of Laying Hens.

Previous study showed that dietary Bacillus licheniformis (B. licheniformis) administration contributes to the improvement of laying performance and egg quality in laying hens. In this study, we aimed to further evaluate its underlying mechanisms. Three hundred sixty Hy-Line Variety W-36 hens (28 weeks of age) were randomized into four groups, each group with six replications (n = 15). The control group received the basal diet and the treatment groups received the same basal diets supplemented with 0.01, 0.03, and 0.06% B. licheniformis powder (2 × 1010 cfu/g) for an 8-week trial. The results demonstrate that B. licheniformis significantly enhance the intestinal barrier functions via decreasing gut permeability, promoting mucin-2 transcription, and regulating inflammatory cytokines. The systemic immunity of layers in B. licheniformis treatment groups is improved through modulating the specific and non-specific immunity. In addition, gene expressions of hormone receptors, including estrogen receptor α, estrogen receptor ß, and follicle-stimulating hormone receptor, are also regulated by B. licheniformis. Meanwhile, compared with the control, B. licheniformis significantly increase gonadotropin-releasing hormone level, but markedly reduce ghrelin and inhibin secretions. Overall, our data suggest that dietary inclusion of B. licheniformis can improve the intestinal barrier function and systemic immunity and regulate reproductive hormone secretions, which contribute to better laying performance and egg quality of hens.

Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.

Porcine Circovirus Type 2 Activates CaMMKß to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium.

Porcine circovirus type 2 (PCV2) induces autophagy via the 5' adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKß) by increasing cytosolic Ca(2+) via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca(2+)) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKß then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca(2+) both in PK-15 cells and PCV2-targeted primary cells from pigs.

N-carboxyethyl chitosan fibers prepared as potential use in tissue engineering.

To improve the hydrophilicity of chitosan fiber, N-carboxyethyl chitosan fiber was prepared through Michael addition between chitosan fiber with acrylic acid. The structure was studied by (1)H NMR. The degree of N-substitution, measured via (1)H NMR, was easily varied from 0.10 to 0.51 by varying the molar ratio of acrylic acid to chitosan. Series of properties of N-carboxyethyl chitosan fiber including mechanical property, crystallinity, thermal property and in vitro degradation were investigated by Instron machine, X-ray diffraction and differential scanning calorimetry and thermogravimetric analysis, respectively. The results showed that, introducing the carboxyethyl group into the backbone chain of chitosan fiber destroyed the intra/intermolecular hydrogen bonding, leading to loss of the intra/intermolecular hydrogen bonding and improvement of hydrophilicity. Indirect cytotoxicity assessment of carboxyethyl chitosan fibers was investigated using a L929 cell line. And the obtained results clearly suggested that N-carboxyethyl chitosan fiber was nontoxic to L929 cells. The N-carboxyethyl chitosan fibers are potential as tissue engineering scaffolds.

Porcine circovirus type 2 induces autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in PK-15 cells.

Porcine circovirus type 2 (PCV2) uses autophagy machinery to enhance its replication in PK-15 cells. However, the underlying mechanisms are unknown. By the use of specific inhibitors, RNA interference, and coimmunoprecipitation, we show that PCV2 induces autophagy in PK-15 cells through a pathway involving the kinases AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2), the tumor suppressor protein TSC2, and the mammalian target of rapamycin (mTOR). AMPK and ERK1/2 positively regulate autophagy through negative control of the mTOR pathway by phosphorylating TSC2 in PCV2-infected PK-15 cells. Thus, PCV2 might induce autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in the host cells, representing a pivotal mechanism for PCV2 pathogenesis.