Identification and characterization of a marine bacterium extract from Mameliella sp. M20D2D8 with antiviral effects against influenza A and B viruses.

Despite significant improvements in vaccines and chemotherapeutic drugs, pathogenic RNA viruses continue to have a profound impact on the global economy and pose a serious threat to animal and human health through emerging and re-emerging outbreaks of diseases. To overcome the challenge of viral adaptation and evolution, increased vigilance is required. Particularly, antiviral drugs derived from new, natural sources provide an attractive strategy for controlling problematic viral diseases. In this antiviral study, we discovered a previously unknown bacterium, Mameliella sp. M20D2D8, by conducting an antiviral screening of marine microorganisms. An extract from M20D2D8 exhibited antiviral activity with low cytotoxicity and was found to be effective in vitro against multiple influenza virus strains: A/PR8 (IC50 = 2.93 µg/mL, SI = 294.85), A/Phil82 (IC50 = 1.42 µg/mL, SI = 608.38), and B/Yamagata (IC50 = 1.59 µg/mL, SI = 543.33). The antiviral action was found to occur in the post-entry stages of viral replication and to suppress viral replication by inducing apoptosis in infected cells. Moreover, it efficiently suppressed viral genome replication, protein synthesis, and infectivity in MDCK and A549 cells. Our findings highlight the antiviral capabilities of a novel marine bacterium, which could potentially be useful in the development of drugs for controlling viral diseases.

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target.

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

7-MEGA™ inhibits adipogenesis in 3T3-L1 adipocytes and suppresses obesity in high-fat-diet-induced obese C57BL/6 mice.

BACKGROUND: Overweight, often known as obesity, is the abnormal and excessive accumulation of fat that exposes the health of a person at risk by increasing the likelihood that they may experience many chronic conditions. Consequently, obesity has become a global health threat, presenting serious health issues, and attracting a lot of attention in the healthcare profession and the scientific community. METHOD: This study aims to explore the anti-adipogenic properties of 7-MEGA™ in an attempt to address obesity, using both in vitro and in vivo research. The effects of 7MEGA™ at three distinct concentrations were investigated in obese mice who were given a high-fat diet (HFD) and 3T3-L1 adipocytes. RESULTS: 7MEGA™ decreased the total fat mass, overall body weight, and the perirenal and subcutaneous white adipose tissue (PWAT and SWAT) contents in HFD mice. Additionally, 7MEGA™ showed promise in improving the metabolic health of individuals with obesity and regulate the levels of insulin hormone, pro-inflammatory cytokines and adipokines. Furthermore, Peroxisome proliferator-activated receptors (PPAR) α and γ, Uncoupling Protein 1 (UCP-1), Sterol Regulatory Element-Binding Protein 1 (SREBP-1), Fatty Acid-Binding Protein 4 (FABP4), Fatty Acid Synthase (FAS), Acetyl-CoA Carboxylase (ACC), Stearoyl-CoA Desaturase-1 (SCD-1) and CCAAT/Enhancer-Binding Protein (C/EBPα) were among the adipogenic regulators that 7MEGA™ could regulate. CONCLUSION: In summary, this study uncovered that 7MEGA™ demonstrates anti-adipogenic and anti-obesity effects, suggesting its potential in combating obesity.

Concurrent primary splenic lymphoma and mammary gland tumour with polycystic ovaries in a dog.

Here, we report a rare case of concurrent primary splenic lymphoma and mammary gland tumour (MGT) with polycystic ovaries in a 10-year-old, intact female Jindo dog. The dog was presented with multiple masses in the fourth left mammary gland, the largest of which measured 6 cm in diameter, along with enlargement of the left inguinal lymph node on physical examination. Ultrasonography, radiography, and computed tomography scans revealed polycystic ovaries and a mass in the tail of the spleen, after total splenectomy and mastectomy with ovariohysterectomy, histopathological examination identified splenic diffuse large B cell lymphoma and malignant myoepithelioma of the mammary gland was found. To our knowledge, this is the first report of the concurrent occurrence of splenic lymphoma, MGT, and polycystic ovaries in a dog.

Canine multiple primary tumours: Mammary tubular carcinoma, uterine leiomyosarcoma, and facial sebaceous epithelioma.

Multiple primary malignant tumours (MPMTs) are multiple neoplasms with independent pathogenetic origins, placing great importance on the tumorigenesis and clinical treatment. However, due to the rare occurrence and diagnostic confusion, MPMTs have rarely been investigated in veterinary medicine. In this report, a 10-year-old intact female Maltese dog had MPMTs, consisting of two malignant tumours and one benign tumour each derived from a topographically different site: tubular carcinoma in the mammary glands, leiomyosarcoma in the uterus and sebaceous epithelioma in the cheek. The unique combination of MPMTs would be the first case in veterinary research to give insight into the diagnosis, disease characteristics, and surgical treatment.

Opposite Effects of Apoptotic and Necroptotic Cellular Pathways on Rotavirus Replication.

Group A rotavirus (RVA), one of the leading pathogens causing severe acute gastroenteritis in children and a wide variety of young animals worldwide, induces apoptosis upon infecting cells. Though RVA-induced apoptosis mediated via the dual modulation of its NSP4 and NSP1 proteins is relatively well studied, the nature and signaling pathway(s) involved in RVA-induced necroptosis are yet to be fully elucidated. Here, we demonstrate the nature of RVA-induced necroptosis, the signaling cascade involved, and correlation with RVA-induced apoptosis. Infection with the bovine NCDV and human DS-1 RVA strains was shown to activate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL), the key necroptosis molecules in virus-infected cells. Using an immunoprecipitation assay, RIPK1 was found to bind phosphorylated RIPK3 (pRIPK3) and pMLKL. pMLKL, the major executioner molecule in the necroptotic pathway, was translocated to the plasma membrane of RVA-infected cells to puncture the cell membrane. Interestingly, transfection of RVA NSP4 also induced necroptosis through the RIPK1/RIPK3/MLKL necroptosis pathway. Blockage of each key necroptosis molecule in the RVA-infected or NSP4-transfected cells resulted in decreased necroptosis but increased cell viability and apoptosis, thereby resulting in decreased viral yields in the RVA-infected cells. In contrast, suppression of RVA-induced apoptosis increased necroptosis and virus yields. Our findings suggest that RVA NSP4 also induces necroptosis via the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, necroptosis and apoptosis-which have proviral and antiviral effects, respectively-exhibited cross talk in RVA-infected cells. These findings significantly increase our understanding of the nature of RVA-induced necroptosis and the cross talk between RVA-induced necroptosis and apoptosis. IMPORTANCE Viral infection usually culminates in cell death through apoptosis, necroptosis, and, rarely, pyroptosis. Necroptosis is a form of programmed necrosis that is mediated by signaling complexes of the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Although apoptosis induction by rotavirus and its NSP4 protein is well known, rotavirus-induced necroptosis is not fully understood. Here, we demonstrate that rotavirus and also its NSP4 protein can induce necroptosis in cultured cells through activation of the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, rotavirus-induced necroptosis and apoptosis have opposite effects on viral yield, i.e., they function as proviral and antiviral processes, respectively, and counterbalance each other in rotavirus-infected cells. Our findings provide important insights for understanding the nature of rotavirus-induced necroptosis and the development of novel therapeutic strategies against infection with rotavirus and other RNA viruses.

Rotavirus exploits SREBP pathway for hyper lipid biogenesis during replication.

Species A rotavirus (RVA) is one of the pathogens causing severe acute gastroenteritis in young children and animals worldwide. RVA replicates and assembles its immature particle within electron dense compartments known as viroplasm. Despite the importance of lipid droplet (LD) formation in the RVA viroplasm, the upstream molecules modulating LD formation have remained elusive. Here, we demonstrate that RVA infection reprogrammes sterol regulatory element binding proteins (SREBPs)-dependent lipogenic pathways in virus-infected cells. Interestingly, silencing of SREBPs significantly reduced RVA protein synthesis, genome replication and progeny virus production. Moreover, knockout of SREBP-1c gene conferred resistance to RVA-induced diarrhoea, reduction of RVA replication, and mitigation of small intestinal pathology in mice. This study identifies SREBPs-mediated lipogenic reprogramming in RVA-infected host cells for facilitating virus replication and SREBPs as a potential target for developing therapeutics against RVA infection.

Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of RhoA/ROCK/MLC Signaling Pathway.

Tight junctions (TJs) are a major barrier and also an important portal of entry for different pathogens. Porcine sapovirus (PSaV) induces early disruption of the TJ integrity of polarized LLC-PK cells, allowing it to bind to the buried occludin co-receptors hidden beneath the TJs on the basolateral surface. However, the signaling pathways involved in the PSaV-induced TJ dissociation are not yet known. Here, we found that the RhoA/ROCK/MLC signaling pathway was activated in polarized LLC-PK cells during the early infection of PSaV Cowden strain in the presence of bile acid. Specific inhibitors of RhoA, ROCK, and MLC restored PSaV-induced reduction of transepithelial resistance, increase of paracellular flux, intracellular translocation of occludin, and lateral membrane lipid diffusion. Moreover, each inhibitor significantly reduced PSaV replication, as evidenced by a reduction in viral protein synthesis, genome copy number, and progeny viruses. The PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, known to dissociate TJs, were not activated during early PSaV infection. Among the above signaling pathways, the RhoA/ROCK/MLC signaling pathway was only activated by PSaV in the absence of bile acid, and specific inhibitors of this signaling pathway restored early TJ dissociation. Our findings demonstrate that PSaV binding to cell surface receptors activates the RhoA/ROCK/MLC signaling pathway, which in turn disrupts TJ integrity via the contraction of the actomyosin ring. Our study contributes to understanding how PSaV enters the cells and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.IMPORTANCEPorcine sapovirus (PSaV), one of the most important enteric pathogens, is known to disrupt tight junction (TJ) integrity to expose its buried co-receptor occludin in polarized LLC-PK cells. However, the cellular signaling pathways that facilitate TJ dissociation are not yet completely understood. Here, we demonstrate that early infection of PSaV in polarized LLC-PK cells in either the presence or absence of bile acids activates the RhoA/ROCK/MLC signaling pathway, whose inhibitors reverse the early PSaV infection-induced early dissociation of TJs and reduce PSaV replication. However, early PSaV infection did not activate the PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, which are also known to dissociate TJs. This study provides a better understanding of the mechanism involved in early PSaV infection-induced disruption of TJs, which is important for controlling or preventing PSaV and other calicivirus infections.

Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor.

The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor.IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

Dual Recognition of Sialic Acid and αGal Epitopes by the VP8* Domains of the Bovine Rotavirus G6P[5] WC3 and of Its Mono-reassortant G4P[5] RotaTeq Vaccine Strains.

Group A rotaviruses, an important cause of severe diarrhea in children and young animals, initiate infection via interactions of the VP8* domain of the VP4 spike protein with cell surface sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is also used in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for the VP8* domain of WC3 and its reassortant strains have not yet been identified. In the present study, HBGA- and saliva-binding assays showed that both G6P[5] WC3 and mono-reassortant G4P[5] strains recognized the αGal HBGA. The infectivity of both P[5]-bearing strains was significantly reduced in αGal-free MA-104 cells by pretreatment with a broadly specific neuraminidase or by coincubation with the α2,6-linked SA-specific Sambucus nigra lectin, but not by the α2,3-linked specific sialidase or by Maackia amurensis lectin. Free NeuAc and the αGal trisaccharide also prevented the infectivity of both strains. This indicated that both P[5]-bearing strains utilize α2,6-linked SA as a ligand on MA104 cells. However, the two strains replicated in differentiated bovine small intestinal enteroids and in their human counterparts that lack α2,6-linked SA or αGal HBGA, suggesting that additional or alternative receptors such as integrins, hsp70, and tight-junction proteins bound directly to the VP5* domain can be used by the P[5]-bearing strains to initiate the infection of human cells. In addition, these data also suggested that P[5]-bearing strains have potential for cross-species transmission.IMPORTANCE Group A rotaviruses initiate infection through the binding of the VP8* domain of the VP4 protein to sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is used as the backbone in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for their P[5] VP8* domain has remained elusive. Using a variety of approaches, we demonstrated that the WC3 and bovine-human mono-reassortant G4P[5] vaccine strains recognize both α2,6-linked SA and αGal HBGA as ligands. Neither ligand is expressed on human small intestinal epithelial cells, explaining the absence of natural human infection by P[5]-bearing strains. However, we observed that the P[5]-bearing WC3 and G4P[5] RotaTeq vaccine strains could still infect human intestinal epithelial cells. Thus, the four P[5] RotaTeq vaccine strains potentially binding to additional alternative receptors may be efficient and effective in providing protection against severe rotavirus disease in human.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

Molecular Regulation of α3ß4 Nicotinic Acetylcholine Receptors by Lupeol in Cardiovascular System.

Cardiovascular disease (CVD) occurs globally and has a high mortality rate. The highest risk factor for developing CVD is high blood pressure. Currently, natural products are emerging for the treatment of hypertension to avoid the side effects of drugs. Among existing natural products, lupeol is known to be effective against hypertension in animal experiments. However, there exists no study regarding the molecular physiological evidence against the effects of lupeol. Consequently, we investigated the interaction of lupeol with α3ß4 nicotinic acetylcholine receptors (nAChRs). In this study, we performed a two-electrode voltage-clamp technique to investigate the effect of lupeol on the α3ß4 nicotine acetylcholine receptor using the oocytes of Xenopus laevis. Coapplication of acetylcholine and lupeol inhibited the activity of α3ß4 nAChRs in a concentration-dependent, voltage-independent, and reversible manner. We also conducted a mutational experiment to investigate the influence of residues of the α3 and ß4 subunits on lupeol binding with nAChRs. Double mutants of α3ß4 (I37A/N132A), nAChRs significantly attenuated the inhibitory effects of lupeol compared to wild-type α3ß4 nAChRs. A characteristic of α3ß4 nAChRs is their effect on transmission in the cardiac sympathetic ganglion. Overall, it is hypothesized that lupeol lowers hypertension by mediating its effects on α3ß4 nAChRs. The interaction between lupeol and α3ß4 nAChRs provides evidence against its effect on hypertension at the molecular-cell level. In conclusion, the inhibitory effect of lupeol is proposed as a novel therapeutic approach involving the antihypertensive targeting of α3ß4 nAChRs. Furthermore, it is proposed that the molecular basis of the interaction between lupeol and α3ß4 nAChRs would be helpful in cardiac-pharmacology research and therapeutics.

Acyclic Triterpenoid Isolated from Alpinia katsumadai Alleviates Formalin-Induced Chronic Mouse Paw Inflammation by Inhibiting the Phosphorylation of ERK and NF-κB.

Chronic and excessive inflammation can destroy host organs and cause inflammatory diseases such as inflammatory bowel disease, asthma, and rheumatoid arthritis. In this study, we investigated the anti-inflammatory effects of Alpinia katsumadai seed-derived 2,3,5,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-tetracosa-6,10,14,18-tetraene (PHT) using lipopolysaccharide (LPS)-stimulated J774 cells and a formalin-induced chronic paw inflammation mouse model. The in vitro results showed that PHT exhibited no cytotoxicity and decreased LPS-induced NO secretion. Additionally, PHT inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygenase 2 (COX2) protein expression. The quantitative real-time PCR results showed that PHT downregulated the gene expression of the proinflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) but not tumor necrosis factor α (TNF-α). PHT inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB). In a mouse model, oral administration of 50 mg/kg PHT significantly alleviated both mouse paw thickness and volume. These results indicate that PHT has potential anti-inflammatory effects and should be considered a possible functional material.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells.

Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Excretion of Toxoplasma gondii oocysts from Feral Cats in Korea.

Sporulated oocysts from the feces of infected cats with Toxoplasma gondii can cause detrimental disease in both humans and animals. To investigate the prevalence of feral cats that excrete T. gondii oocysts in the feces, we examined fecal samples of 563 feral cats over a 3-year period from 2009 to 2011. Oocysts of T. gondii excreted into the feces were found from 4 of 128 cats in 2009 (3.1%) and one of 228 (0.4%) in 2010 while none of the 207 cats in 2010 were found positive with oocysts in their feces, resulting in an overall prevalence rate of 0.89% (5/563) between 2009 and 2011. Among the 5 cats that tested positive with T. gondii oocysts, 4 of the cats were male and 1 was a female with an average body weight of 0.87 kg. Numerous tissue cysts of 60 µm in diameter with thin (<0.5 µm) cyst walls were found in the brain of one of the 5 cats on necropsy 2 months after the identification of oocysts in the feces. A PCR amplification of the T. gondii-like oocysts in the feces of the positive cats using the primer pairs Tox-5/Tox-8 and Hham34F/Hham3R confirmed the presence of T. gondii oocysts in the feces. This study provides a good indication of the risk assessment of feral cats in the transmission of T. gondii to humans in Korea.

Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II.

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-ß-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.

Transcatheter Arterial Embolization for Gastrointestinal Bleeding Associated with Gastric Carcinoma: Prognostic Factors Predicting Successful Hemostasis and Survival.

PURPOSE: To evaluate outcomes of transcatheter arterial embolization (TAE) for gastric cancer-related gastrointestinal (GI) bleeding and factors associated with successful TAE and improved survival after TAE. MATERIALS AND METHODS: This retrospective study included 43 patients (34 men; age 60.6 y ± 13.6) with gastric cancer-related GI bleeding undergoing angiography between January 2000 and December 2015. Clinical course, laboratory findings, and TAE characteristics were reviewed. Technical success of TAE was defined as target area devascularization, and clinical success was defined as bleeding cessation with hemodynamic stability during 72 hours after TAE. Student t test was used for comparison of continuous variables, and Fisher exact test was used for categorical variables. Univariate and multivariate analysis were performed to identify predictors of successful TAE and 30-day survival after TAE. RESULTS: TAE was performed in 40 patients. Technical and clinical success rates of TAE were 85.0% and 65.0%, respectively. Splenic infarction occurred in 2 patients as a minor complication. Rebleeding after TAE occurred in 7 patients. Death related to bleeding occurred in 5 patients. Active bleeding (P = .044) and higher transfusion requirement (3.3 U ± 2.6 vs 1.8 U ± 1.7; P = .039) were associated with TAE failure. Successful TAE predicted improved 30-day survival after TAE on univariate and multivariate analysis (P = .018 and P = .022; odds ratio, 0.132). CONCLUSION: TAE for gastric cancer-associated GI bleeding may be a lifesaving procedure. Severe bleeding with a higher transfusion requirement and active bleeding on angiography predicted TAE failure.

Whole genomic characterization of Korean porcine G8P[7] reassortant rotaviruses.

This study analyzed eleven genomic segments of three Korean porcine G8P[7] group A rotavirus (RVA) strains. Phylogenetically, these strains contained two bovine-like and nine porcine-like genomic segments. Eight genes (VP1, VP2, VP6 and NSP1-NSP5) of strains 156-1 and 42-1 and seven genes (VP1, VP2, VP6 and NSP2-NSP5) of strain C-1 clustered closely with porcine and porcine-like animal strains and distantly from typical human Wa-like strains. The VP3-M2 genotype of these strains clustered closely with bovine-like strains, but distantly with typical human DS-1-like strains. These data indicate that multiple reassortments involving porcine and bovine RVA strains in Korea must have occurred.

CREB5 promotes the proliferation and self-renewal ability of glioma stem cells.

Glioblastoma multiforme (GBM) is the most fatal form of brain cancer in humans, with a dismal prognosis and a median overall survival rate of less than 15 months upon diagnosis. Glioma stem cells (GSCs), have recently been identified as key contributors in both tumor initiation and therapeutic resistance in GBM. Both public dataset analysis and direct differentiation experiments on GSCs have demonstrated that CREB5 is more highly expressed in undifferentiated GSCs than in differentiated GSCs. Additionally, gene silencing by short hairpin RNA (shRNA) of CREB5 has prevented the proliferation and self-renewal ability of GSCs in vitro and decreased their tumor forming ability in vivo. Meanwhile, RNA-sequencing, luciferase reporter assay, and ChIP assay have all demonstrated the closely association between CREB5 and OLIG2. These findings suggest that targeting CREB5 could be an effective approach to overcoming GSCs.