VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

143D, a novel selective KRASG12C inhibitor exhibits potent antitumor activity in preclinical models.

The KRASG12C mutant has emerged as an important therapeutic target in recent years. Covalent inhibitors have shown promising antitumor activity against KRASG12C-mutant cancers in the clinic. In this study, a structure-based and focused chemical library analysis was performed, which led to the identification of 143D as a novel, highly potent and selective KRASG12C inhibitor. The antitumor efficacy of 143D in vitro and in vivo was comparable with that of AMG510 and of MRTX849, two well-characterized KRASG12C inhibitors. At low nanomolar concentrations, 143D showed biochemical and cellular potency for inhibiting the effects of the KRASG12C mutation. 143D selectively inhibited cell proliferation and induced G1-phase cell cycle arrest and apoptosis by downregulating KRASG12C-dependent signal transduction. Compared with MRTX849, 143D exhibited a longer half-life and higher maximum concentration (Cmax) and area under the curve (AUC) values in mouse models, as determined by tissue distribution assays. Additionally, 143D crossed the blood‒brain barrier. Treatment with 143D led to the sustained inhibition of KRAS signaling and tumor regression in KRASG12C-mutant tumors. Moreover, 143D combined with EGFR/MEK/ERK signaling inhibitors showed enhanced antitumor activity both in vitro and in vivo. Taken together, our findings indicate that 143D may be a promising drug candidate with favorable pharmaceutical properties for the treatment of cancers harboring the KRASG12C mutation.

Dysregulation of iron homeostasis by TfR-1 renders EZH2 wild type diffuse large B-cell lymphoma resistance to EZH2 inhibition.

EZH2 has been regarded as an efficient target for diffuse large B-cell lymphoma (DLBCL), but the clinical benefits of EZH2 inhibitors (EZH2i) are limited. To date, only EPZ-6438 has been approved by FDA for the treatment of follicular lymphoma and epithelioid sarcoma. We have discovered a novel EZH1/2 inhibitor HH2853 with a better antitumor effect than EPZ-6438 in preclinical studies. In this study we explored the molecular mechanism underlying the primary resistance to EZH2 inhibitors and sought for combination therapy strategy to overcome it. By analyzing EPZ-6438 and HH2853 response profiling, we found that EZH2 inhibition increased intracellular iron through upregulation of transferrin receptor 1 (TfR-1), ultimately triggered resistance to EZH2i in DLBCL cells. We demonstrated that H3K27ac gain by EZH2i enhanced c-Myc transcription, which contributed to TfR-1 overexpression in insensitive U-2932 and WILL-2 cells. On the other hand, EZH2i impaired the occurrence of ferroptosis by upregulating the heat shock protein family A (Hsp70) member 5 (HSPA5) and stabilizing glutathione peroxidase 4 (GPX4), a ferroptosis suppressor; co-treatment with ferroptosis inducer erastin effectively overrode the resistance of DLBCL to EZH2i in vitro and in vivo. Altogether, this study reveals iron-dependent resistance evoked by EZH2i in DLBCL cells, and suggests that combination with ferroptosis inducer may be a promising therapeutic strategy.

Enterovirus A71 Containing Codon-Deoptimized VP1 and High-Fidelity Polymerase as Next-Generation Vaccine Candidate.

Enterovirus A71 (EV-A71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD), which occasionally results in severe neurological complications. In this study, we developed four EV-A71 (rgEV-A71) strains by reverse genetics procedures as possible vaccine candidates. The four rgEV-A71 viruses contained various codon-deoptimized VP1 capsid proteins (VP1-CD) and showed replication rates and antigenicity similar to that of the wild-type virus, while a fifth virus, rg4643C4VP-CD, was unable to form plaques but was still able to be examined by median tissue culture infectious dose (TCID50) titers, which were similar to those of the others, indicating the effect of CD on plaque formation. However, the genome stability showed that there were some mutations which appeared during just one passage of the VP1-CD viruses. Thus, we further constructed VP1-CD rgEV-A71 containing high-fidelity determinants in 3D polymerase (CD-HF), and the number of mutations in CD-HF rgEV-A71 was shown to have decreased. The CD-HF viruses showed less virulence than the parental strain in a mouse infection model. After 14 days postimmunization, antibody titers had increased in mice infected with CD-HF viruses. The mouse antisera showed similar neutralizing antibody titers against various CD-HF viruses and different genotypes of EV-A71. The study demonstrates the proof of concept that VP1 codon deoptimization combined with high-fidelity 3D polymerase decreased EV-A71 mutations and virulence in mice but retained their antigenicity, indicating it is a good candidate for next-generation EV-A71 vaccine development.IMPORTANCE EV-A71 can cause severe neurological diseases with fatality in infants and young children, but there are still no effective drugs to date. Here, we developed a novel vaccine strategy with the combination of CD and HF substitutions to generate the genetically stable reverse genetics virus. We found that CD combined with HF polymerase decreased the virulence but maintained the antigenicity of the virus. This work demonstrated the simultaneous introduction of CD genome sequences and HF substitutions as a potential new strategy to develop attenuated vaccine seed virus. Our work provides insight into the development of a low-virulence candidate vaccine virus through a series of genetic editing of virus sequences while maintaining its antigenicity and genome stability, which will provide an additional strategy for next-generation vaccine development of EV-A71.

Attenuation of TRPV1 by AMG-517 after nerve injury promotes peripheral axonal regeneration in rats.

Aims The main objective was to investigate the effects of the transient receptor potential cation channel subfamily V member 1 (TRPV1) on nerve regeneration following sciatic transection injury by functional blockage of TRPV1 using AMG-517, a specific blocker of TRPV1. Methods AMG-517 was injected into the area surrounding ipsilateral lumbar dorsal root ganglia 30 min after unilateral sciatic nerve transection. The number of sciatic axons and the expression of growth-associated protein-43 (GAP-43) and glial fibrillary acidic protein was examined using semithin sections, Western blot, and immunofluorescence analyses. Results Blockage of TRPV1 with AMG-517 markedly promoted axonal regeneration, especially at two weeks after sciatic injury; the number of axons was similar to the uninjured control group. After sciatic nerve transection, expression of glial fibrillary acidic protein was decreased and GAP-43 was increased at the proximal stump. However, the expression of both glial fibrillary acidic protein and GAP-43 increased significantly in AMG-517-treated groups. Conclusions TRPV1 may be an important therapeutic target to promote peripheral nerve regeneration after injury.

Cell surface nucleolin facilitates enterovirus 71 binding and infection.

UNLABELLED: Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens. IMPORTANCE: Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.

Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells.

AIM: Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy. METHODS: The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg(-1)·d(-1)) was intraperitoneally injected to BEL-7402-bearing mice for 21 days. RESULTS: Platycodin D (5-40 µmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 µmol/L at 24, 48 and 72 h, respectively. Platycodin D (5-20 µmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5-20 µmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 µmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 µmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg(-1)•d(-1)) significantly reduced relative tumor volume with decreased body weight. CONCLUSION: Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors.

Baicalein Triggers Autophagy and Inhibits the Protein Kinase B/Mammalian Target of Rapamycin Pathway in Hepatocellular Carcinoma HepG2 Cells.

Baicalein (BA), isolated from the Chinese medicinal herb Scutellariae radix (Huangqin in Chinese), is a flavonoid with various pharmacological effects. Herein, we found that BA only slightly reduced the cell viability on HepG2 cells after 24-h treatment as determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. However, BA (50 µM) effectively blocked the colony formation. Meanwhile, BA remarkably induced the formation of autophagosomes after 24-h treatment as determined by immunofluorescence with monodansylcadaverine staining as well as transmission electron microscopy, respectively. Moreover, BA obviously up-regulated the expression of microtubule-associated protein 1A/1B-light chain 3-II in concentration-dependent and time-dependent manners in HepG2 cells. When combined with the autophagy inhibitor chloroquine and BA, the cell viability and colony formation were significantly decreased, indicating that BA triggered protective autophagy, which prevented cell death. Further study showed that BA concentration-dependently and time-dependently decreased the expression of p-AKT (S473), p-ULK1 (S757) and p-4EBP1 (T37 and S65), suggesting the involvement of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) in BA-triggered autophagy.

[Expression of SFRP5 mRNA in Rat Skeletal Muscle after Contusion].

OBJECTIVE: To investigate the relationship between the expression of secreted frizzled-related protein 5 (SFRP5) mRNA and the time interval after skeletal muscle injury in rats by real-time PCR. METHODS: A total of ninety SD rats were randomly divided into the contusion groups at different times including 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h after contusion, incision groups at different times including 4h and 8h after incision and the control group. The samples were taken from the contused zone at different time points. The total RNA was isolated from the samples and reversely transcribed to analyze the expression levels of SFRP5 mRNA. RESULTS: Compared to the control group, the expression of SFRP5 mRNA in contusion groups were down-regulated within 48 h after contusion and reached the lowest level at 20 h, and the expression of SFRP5 mRNA gradually increased from 20 h to 48 h after contusion. The expression of SFRP5 mRNA in the incised groups were significantly lower than that of the contusion groups at 4 h after injury. At the time of 8 h, the expression levels between the contusion and incision groups showed no statistically significant difference. CONCLUSION: It is suggested that SFRP5 mRNA analysis may show regular expression and can be a marker for estimation of skeletal muscle injury age.

Animal models of enterovirus 71 infection: applications and limitations.

Human enterovirus 71 (EV71) has emerged as a neuroinvasive virus that is responsible for several outbreaks in the Asia-Pacific region over the past 15 years. Appropriate animal models are needed to understand EV71 neuropathogenesis better and to facilitate the development of effective vaccines and drugs. Non-human primate models have been used to characterize and evaluate the neurovirulence of EV71 after the early outbreaks in late 1990s. However, these models were not suitable for assessing the neurovirulence level of the virus and were associated with ethical and economic difficulties in terms of broad application. Several strategies have been applied to develop mouse models of EV71 infection, including strategies that employ virus adaption and immunodeficient hosts. Although these mouse models do not closely mimic human disease, they have been applied to determine the pathogenesis of and treatment and prevention of the disease. EV71 receptor-transgenic mouse models have recently been developed and have significantly advanced our understanding of the biological features of the virus and the host-parasite interactions. Overall, each of these models has advantages and disadvantages, and these models are differentially suited for studies of EV71 pathogenesis and/or the pre-clinical testing of antiviral drugs and vaccines. In this paper, we review the characteristics, applications and limitation of these EV71 animal models, including non-human primate and mouse models.

Epidemiology and seroepidemiology of human enterovirus 71 among Thai populations.

BACKGROUND: Human enterovirus 71 (EV71) is an important pathogen caused large outbreaks in Asian-Pacific region with severe neurological complications and may lead to death in young children. Understanding of the etiological spectrum and epidemic changes of enterovirus and population's immunity against EV71 are crucial for the implementation of future therapeutic and prophylactic intervention. RESULTS: A total of 1,182 patients who presented with the symptoms of hand foot and mouth disease (67.3%) or herpangina (HA) (16.7%) and admitted to the hospitals during 2008-2013 were tested for enterovirus using pan-enterovirus PCR targeting 5'-untranslated region and specific PCR for viral capsid protein 1 gene. Overall, 59.7% were pan-enterovirus positive comprising 9.1% EV71 and 31.2% coxsackievirus species A (CV-A) including 70.5% CV-A6, 27.6% CV-A16, 1.1% CV-A10, and 0.8% CV-A5. HFMD and HA occurred endemically during 2008-2011. The number of cases increased dramatically in June 2012 with the percentage of the recently emerged CV-A6 significantly rose to 28.4%. Co-circulation between different EV71 genotypes was observed during the outbreak. Total of 161 sera obtained from healthy individuals were tested for neutralizing antibodies (NAb) against EV71 subgenotype B5 (EV71-B5) using microneutralization assay. The seropositive rate of EV71-B5 was 65.8%. The age-adjusted seroprevalence for individuals was found to be lowest in children aged >6 months to 2 years (42.5%). The seropositive rate remained relatively low in preschool children aged > 2 years to 6 years (48.3%) and thereafter increased sharply to more than 80% in individuals aged > 6 years. CONCLUSIONS: This study describes longitudinal data reflecting changing patterns of enterovirus prevalence over 6 years and demonstrates high seroprevalences of EV71-B5 NAb among Thai individuals. The rate of EV71 seropositive increased with age but without gender-specific significant difference. We identified that relative lower EV71 seropositive rate in early 2012 may demonstrate widely presented of EV71-B5 in the population before account for a large outbreak scale epidemic occurred in 2012 with due to a relatively high susceptibility of the younger population.

The molecular epidemiology of a dengue virus outbreak in Taiwan: population wide versus infrapopulation mutation analysis.

Dengue virus (DENV) causes approximately 390 million dengue infections worldwide every year. There were 22,777 reported DENV infections in Tainan, Taiwan in 2015. In this study, we sequenced the C-prM-E genes from 45 DENV 2015 strains, and phylogenetic analysis based on C-prM-E genes revealed that all strains were classified as DENV serotype 2 Cosmopolitan genotype. Sequence analysis comparing different DENV-2 genotypes and Cosmopolitan DENV-2 sequences prior to 2015 showed a clade replacement event in the DENV-2 Cosmopolitan genotype. Additionally, a major substitution C-A314G (K73R) was found in the capsid region which may have contributed to the clade replacement event. Reverse genetics virus rgC-A314G (K73R) showed slower replication in BHK-21 and C6/36 cells compared to wildtype virus, as well as a decrease in NS1 production in BHK-21-infected cells. After a series of passaging, the C-A314G (K73R) mutation reverted to wildtype and was thus considered to be unstable. Next generation sequencing (NGS) of three sera collected from a single DENV2-infected patient at 1-, 2-, and 5-days post-admission was employed to examine the genetic diversity over-time and mutations that may work in conjunction with C-A314G (K73R). Results showed that the number of haplotypes decreased with time in the DENV-infected patient. On the fifth day after admission, two new haplotypes emerged, and a single non-synonymous NS4A-L115I mutation was identified. Therefore, we have identified a persistent mutation C-A314G (K73R) in all of the DENV-2 isolates, and during the course of an infection, a single new non-synonymous mutation in the NS4A region appears in the virus population within a single host. The C-A314G (K73R) thus may have played a role in the DENV-2 2015 outbreak while the NS4A-L115I may be advantageous during DENV infection within the host.

Modified stent for the treatment of tracheomegaly combined with a tracheoesophageal fistula: a case reportStent.

Tracheoesophageal fistulas (TEFs) are common in clinical practice and we address them in different ways according to their etiologies. Herein, we present a case of tracheomegaly combined with a TEF after long-term tracheotomy. We placed a modified silicone stent into the trachea to simultaneously cover the fistula and maintain an artificial airway for ventilation. After migration of the modified stent, we replaced it with a prolonged tracheotomy tube. This modified stent is a novel clinical attempt at addressing TEFs that should be more thoroughly explored.

Las fístulas traqueoesofágicas son frecuentes en la práctica clínica y las abordamos de diferentes formas según sus etiologías. Aquí, presentamos un caso de traqueomegalia combinada con una fístula traqueoesofágica después de una traqueotomía a largo plazo. Colocamos un stent de silicona modificado en la tráquea para cubrir simultáneamente la fístula y mantener una vía aérea artificial para la ventilación. Después de la migración del stent modificado, lo reemplazamos con un tubo de traqueotomía prolongado. Este stent modificado es un intento clínico novedoso para abordar las fístulas traqueoesofágicas que debe explorarse más a fondo.

Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells.

BACKGROUND: Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear. RESULTS: We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA) and Sambucus Nigra (SNA), could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation. CONCLUSIONS: In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.

Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice.

Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)-6, monocyte chemoattractant protein-1, tumor necrosis factor, and IFN-γ. EV 71 infection abolished both poly (I:C)- and CB3-induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71-infected RAW264.7 cells produced significantly lower amount of type I IFNs than non-infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over-expression systems in both mice and RAW264.7 cells. The 3C over-expression, however, did not interfere with poly (I:C)-induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein.

Characterization of glycan binding specificities of influenza B viruses with correlation with hemagglutinin genotypes and clinical features.

The carbohydrate binding specificities are different among avian and human influenza A viruses and may affect the tissue tropism and transmission of these viruses. The glycan binding biology for influenza B, however, has not been systematically characterized. Glycan binding specificities of influenza B viral isolates were analyzed and correlated to hemagglutinin (HA) genotypes and clinical manifestations. A newly developed solution glycan array was applied to characterize the receptor binding specificities of influenza B virus clinical isolates from 2001 to 2007 in Taiwan. Thirty oligosaccharides which include α-2,3 and α-2,6 linkage glycans were subjected to analysis. The glycan binding patterns of 53 influenza B isolates could be categorized into three groups and were well correlated to their HA genotypes. The Yamagata-like strains predominantly bound to α-2,6-linkage glycan (24:29, 83%) while Victoria-like strains preferentially bound to both α-2,3- and α-2,6-linkage glycans (13:24, 54%). A third group of viruses bound to sulfated glycans and these all belonged to Victoria-like strains. Based on the HA sequences, Asn-163, Glu-198, Ala-202, and Lys-203 were conserved among Victoria-like strains which may influence their carbohydrate recognition. The viruses bound to dual type glycans were more likely to be associated with the development of bronchopneumonia and gastrointestinal illness than those bound only to α-2,6 sialyl glycans (P < 0.05). Glycan binding analyses provide additional information to monitor the antigenic shift, tissue tropism, and transmission capability of influenza B viruses, and will contribute to virus surveillance and vaccine strain selection.

Investigation of Avian Influenza H5N6 Virus-like Particles as a Broad-Spectrum Vaccine Candidate against H5Nx Viruses.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses have been reported to be the source of infections in several outbreaks in the past decades. In a previous study, we screened out a broad-spectrum virus strain, H5N6-Sichuan subtype, by using a lentiviral pseudovirus system. In this project, we aimed to investigate the potential of H5N6 virus-like particles (VLPs) serving as a broad-spectrum vaccine candidate against H5Nx viruses. We cloned the full-length M1 gene and H5, N6 genes derived from the H5N6-Sichuan into pFASTBac vector and generated the VLPs using the baculovirus-insect cell system. H5N6 VLPs were purified by sucrose gradient centrifugation, and the presence of H5, N6 and M1 proteins was verified by Western blot and SDS-PAGE. The hemagglutination titer of H5N6 VLPs after purification reached 5120 and the particle structure remained as viewed by electron microscopy. The H5N6 VLPs and 293T mammalian cell-expressed H5+N6 proteins were sent for mice immunization. Antisera against the H5+N6 protein showed 80 to 320 neutralizing antibody titers to various H5Nx pseudoviruses. In contrast, H5N6 VLPs not only elicited higher neutralizing antibody titers, ranging from 640 to 1280, but also induced higher IL-2, IL-4, IL-5, IFN-γ and TNF production, thus indicating that H5N6 VLPs may be a potential vaccine candidate for broad-spectrum H5Nx avian influenza vaccines.

[Effects of the removal of invasive Moso bamboo on soil microbial biomass and enzyme activities in subtropical forests]. / å ¥ä¾µæ¯›ç«¹çš†ä¼å¯¹äºšçƒ­å¸¦æ£®æž—åœŸå£¤å¾®ç”Ÿç‰©ç”Ÿç‰©é‡å’Œé ¶æ´»æ€§çš„å½±å“.

Removal of invasive plant species is the first step to restoring the invaded ecosystems. The soil microbial biomass and extracellular enzyme activities were measured in Moso bamboo (Phyllostachys edulis) pure forest (completely invasion), invasive P. edulis removal forest (secondary succession 5 years after clear cutting), and the evergreen broadleaved forest (no invasion) in Tianmu Mountain. The results showed that compared with P. edulis pure forest, invasive P. edulis removal significantly increased the contents of soil organic carbon (SOC), nitrate nitrogen, available phosphorus and potassium, as well as microbial biomass carbon (MBC) and microbial biomass phosphorus (MBP), while significantly decreased microbial biomass nitrogen (MBN). The activities of α-glucosidase (AG), ß-glucosidase (BG), leucine aminopeptidase (LAP) and phenol oxidase (POX) in the forest with removal of invasive P. edulis were significantly higher than those in P. edulis pure forest, while invasive P. edulis removal did not change the activities of cellodisaccharide hydrolase (CBH), ß-N-acetyl-glucosaminopeptidase (NAG), acid phosphatase (ACP) and peroxidase (PER). Furthermore, the activities of AG, BG and LAP were positively correlated with SOC and MBC, while the increase in POX activity was positively correlated with soil nitrate content. In addition, MBC, MBN and MBP, and activities of AG, BG, NAG, LAP and ACP in P. edulis removal forest forest were significantly higher than those in evergreen broadleaved forests. Taken together, the removal of invasive P. edulis could increase soil nutrient contents, microbial biomass and extracellular enzyme activities, thus could be considered as an effective way to restore the invaded forests. Our results provide important theoretical basis for controlling P. edulis invasion in subtropical forests.

Assessment of the Risk of Severe Dengue Using Intrahost Viral Population in Dengue Virus Serotype 2 Patients via Machine Learning.

Dengue virus, a positive-sense single-stranded RNA virus, continuously threatens human health. Although several criteria for evaluation of severe dengue have been recently established, the ability to prognose the risk of severe outcomes for dengue patients remains limited. Mutant spectra of RNA viruses, including single nucleotide variants (SNVs) and defective virus genomes (DVGs), contribute to viral virulence and growth. Here, we determine the potency of intrahost viral population in dengue patients with primary infection that progresses into severe dengue. A total of 65 dengue virus serotype 2 infected patients in primary infection including 17 severe cases were enrolled. We utilized deep sequencing to directly define the frequency of SNVs and detection times of DVGs in sera of dengue patients and analyzed their associations with severe dengue. Among the detected SNVs and DVGs, the frequencies of 9 SNVs and the detection time of 1 DVG exhibited statistically significant differences between patients with dengue fever and those with severe dengue. By utilizing the detected frequencies/times of the selected SNVs/DVG as features, the machine learning model showed high average with a value of area under the receiver operating characteristic curve (AUROC, 0.966 ± 0.064). The elevation of the frequency of SNVs at E (nucleotide position 995 and 2216), NS2A (nucleotide position 4105), NS3 (nucleotide position 4536, 4606), and NS5 protein (nucleotide position 7643 and 10067) and the detection times of the selected DVG that had a deletion junction in the E protein region (nucleotide positions of the junction: between 969 and 1022) increased the possibility of dengue patients for severe dengue. In summary, we demonstrated the detected frequencies/times of SNVs/DVG in dengue patients associated with severe disease and successfully utilized them to discriminate severe patients using machine learning algorithm. The identified SNVs and DVGs that are associated with severe dengue will expand our understanding of intrahost viral population in dengue pathogenesis.

Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer.

Tumor associated macrophages (TAMs) play an important role in tumorigenesis, development and anti-cancer drug therapy. However, very few epigenetic compounds have been elucidated to affect tumor growth by educating TAMs in the tumor microenvironment (TME). Herein, we identified that EZH2 performs a crucial role in the regulation of TAMs infiltration and protumoral polarization by interacting with human breast cancer (BC) cells. We showed that EZH2 inhibitors-treated BC cells induced M2 macrophage polarization in vitro and in vivo, while EZH2 knockdown exhibited the opposite effect. Mechanistically, inhibition of EZH2 histone methyltransferase alone by EZH2 inhibitors in breast cancer cells could reduce the enrichment of H3K27me3 on CCL2 gene promoter, elevate CCL2 transcription and secretion, contributing to the induction of M2 macrophage polarization and recruitment in TME, which reveal a potential explanation behind the frustrating results of EZH2 inhibitors against breast cancer. On the contrary, EZH2 depletion led to DNA demethylation and subsequent upregulation of miR-124-3p level, which inhibited its target CCL2 expression in the tumor cells, causing arrest of TAMs M2 polarization. Taken together, these data suggested that EZH2 can exert opposite regulatory effects on TAMs polarization through its enzymatic or non-enzymatic activities. Our results also imply that the effect of antitumor drugs on TAMs may affect its therapeutic efficacy, and the combined application with TAMs modifiers should be warranted to achieve great clinical success.