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.

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.

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.

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.

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.

Genetic and Cross Neutralization Analyses of Coxsackievirus A16 Circulating in Taiwan from 1998 to 2021 Suggest Dominant Genotype B1 can Serve as Vaccine Candidate.

Coxsackievirus A16 (CVA16) is well known for causing hand-foot-and-mouth disease (HFMD) and outbreaks were frequently reported in Taiwan in the past twenty years. The epidemiology and genetic variations of CVA16 in Taiwan from 1998 to 2021 were analyzed in this study. CVA16 infections usually occurred in early summer and early winter, and showed increased incidence in 1998, 2000-2003, 2005, 2007-2008, and 2010 in Taiwan. Little or no CVA16 was detected from 2017 to 2021. CVA16 infection was prevalent in patients between 1 to 3 years old. A total of 69 isolates were sequenced. Phylogenetic analysis based on the VP1 region showed that CVA16 subgenotype B1 was dominantly isolated in Taiwan from 1998 to 2019, and B2 was identified only from isolates collected in 1999 and 2000. There was a high frequency of synonymous mutations in the amino acid sequences of the VP1 region among CVA16 isolates, with the exception of position 145 which showed positive selection. The recombination analysis of the whole genome of CVA16 isolates indicated that the 5'-untranslated region and the non-structural protein region of CVA16 subgenotype B1 were recombined with Coxsackievirus A4 (CVA4) and enterovirus A71 (EVA71) genotype A, respectively. The recombination pattern of subgenotype B2 was similar to B1, however, the 3D region was similar to EVA71 genotype B. Cross-neutralization among CVA16 showed that mouse antisera from various subgenotypes viruses can cross-neutralize different genotype with high neutralizing antibody titers. These results suggest that the dominant CVA16 genotype B1 can serve as a vaccine candidate for CVA16.

BCL-2 inhibitor synergizes with PI3Kδ inhibitor and overcomes FLT3 inhibitor resistance in acute myeloid leukaemia.

Inhibitors targeting the antiapoptotic molecule BCL-2 have therapeutic potential for the treatment of acute myeloid leukaemia (AML); however, BCL-2 inhibitors such as venetoclax exhibit limited monotherapy efficacy in relapsed or refractory human AML. PI3Kδ/AKT signalling has been shown to be constitutively active in AML patients. Here, we demonstrate that the combination of BCL-2 and PI3Kδ inhibitors exerts synergistic antitumour effects both in vitro and in vivo in AML. Cotreatment with venetoclax and the specific PI3Kδ inhibitor idelalisib significantly enhanced antiproliferative effects and induced caspase-dependent apoptosis in a panel of AML cell lines. The synergistic effects were mechanistically based on the inactivation of AKT/4E-BP-1 signalling and the reduction of MCL-1 expression, which diminished the binding of Bim to MCL-1. Notably, compared with the parental FLT3-ITD-positive MV-4-11, the acquired FLT3 inhibitor quizartinib-resistant xenograft model carrying the F691L mutation, exhibited a markedly higher sensitivity to venetoclax. Furthermore, venetoclax combined with idelalisib led to tumour regression in all animals in this quizartinib-resistant AML model. Thus, these data indicate that combined inhibition of BCL-2 and PI3Kδ may be a promising strategy in AML, especially for patients with FLT3-ITD and/or FLT3-TKD mutations.

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.

[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.

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.

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.

Treatment of Whole-Plant Corn Silage With Lactic Acid Bacteria and Organic Acid Enhances Quality by Elevating Acid Content, Reducing pH, and Inhibiting Undesirable Microorganisms.

We investigated the variation in microbial community and fermentation characteristics of whole-plant corn silage after treatment with lactic acid bacteria (LAB) and organic acids. The fresh corn forages were treated with a combination of L. acidophilus and L. plantarum (106 CFU/g fresh material) or a 7:1:2 ratio of formic acid, acetic acid, and propionic acid (6 mL/g fresh material) followed by 45 or 90 days of ensiling. Silages treated with LAB showed increased lactic acid content and decreased pH after 45 days. Although treatment with LAB or organic acids decreased the common and unique operational taxonomic units, indicating a reduction in microbial diversity, the relative abundance of Lactobacillus was elevated after 45 and 90 days compared with control, which was more distinct in the organic acid groups. Moreover, we found higher levels of acetic acid and increased abundance of Acetobacter in silages treated with organic acids whereas undesirable microorganisms such as Klebsiella, Paenibacillus, and Enterobacter were reduced. In summary, the quality of corn silages was improved by LAB or organic acid treatment in which LAB more effectively enhanced lactic acid content and reduced pH while organic acid inhibited the growth of undesirable microorganisms.

Author Correction: G9a regulates breast cancer growth by modulating iron homeostasis through the repression of ferroxidase hephaestin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Assessing the application of a pseudovirus system for emerging SARS-CoV-2 and re-emerging avian influenza virus H5 subtypes in vaccine development.

BACKGROUND: Highly pathogenic emerging and re-emerging viruses continuously threaten lives worldwide. In order to provide prophylactic prevention from the emerging and re-emerging viruses, vaccine is suggested as the most efficient way to prevent individuals from the threat of viral infection. Nonetheless, the highly pathogenic viruses need to be handled in a high level of biosafety containment, which hinders vaccine development. To shorten the timeframe of vaccine development, the pseudovirus system has been widely applied to examine vaccine efficacy or immunogenicity in the emerging and re-emerging viruses. METHODS: We developed pseudovirus systems for emerging SARS coronavirus 2 (SARS-CoV-2) and re-emerging avian influenza virus H5 subtypes which can be handled in the biosafety level 2 facility. Through the generated pseudovirus of SARS-CoV-2 and avian influenza virus H5 subtypes, we successfully established a neutralization assay to quantify the neutralizing activity of antisera against the viruses. RESULTS: The result of re-emerging avian influenza virus H5Nx pseudoviruses provided valuable information for antigenic evolution and immunogenicity analysis in vaccine candidate selection. Together, our study assessed the potency of pseudovirus systems in vaccine efficacy, antigenic analysis, and immunogenicity in the vaccine development of emerging and re-emerging viruses. CONCLUSION: Instead of handling live highly pathogenic viruses in a high biosafety level facility, using pseudovirus systems would speed up the process of vaccine development to provide community protection against emerging and re-emerging viral diseases with high pathogenicity.

Genome survey and SSR analysis of Apocynum venetum.

Apocynum venetum is an eco-economic plant that exhibits high stress resistance. In the present paper, we carried out a whole-genome survey of A. venetum in order to provide a foundation for its whole-genome sequencing. High-throughput sequencing technology (Illumina NovaSep) was first used to measure the genome size of A. venetum, and bioinformatics methods were employed for the evaluation of the genome size, heterozygosity ratio, repeated sequences, and GC content in order to provide a foundation for subsequent whole-genome sequencing. The sequencing analysis results indicated that the preliminary estimated genome size of A. venetum was 254.40 Mbp, and its heterozygosity ratio and percentage of repeated sequences were 0.63 and 40.87%, respectively, indicating that it has a complex genome. We used k-mer = 41 to carry out a preliminary assembly and obtained contig N50, which was 3841 bp with a total length of 223949699 bp. We carried out further assembly to obtain scaffold N50, which was 6196 bp with a total length of 227322054 bp. We performed simple sequence repeat (SSR) molecular marker prediction based on the A. venetum genome data and identified a total of 101918 SSRs. The differences between the different types of nucleotide repeats were large, with mononucleotide repeats being most numerous and hexanucleotide repeats being least numerous. We recommend the use of the '2+3' (Illumina+PacBio) sequencing combination to supplement the Hi-C technique and resequencing technique in future whole-genome research in A. venetum.

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.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells.

Myelin-associated glycoprotein (MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase (ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Glycan-binding preferences and genetic evolution of human seasonal influenza A(H3N2) viruses during 1999-2007 in Taiwan.

It is generally agreed that human influenza virus preferentially binds to α-2,6-linked sialic acid-containing receptors, and mutations that change the binding preference may alter virus infectivity and host tropism. Limited information is available on the glycan-binding specificity of epidemic influenza viruses. In this study, we systemically investigated the glycan-binding preferences of human influenza A(H3N2) viruses isolated from 1999 to 2007 in Taiwan using a high-throughput carbohydrate array. The binding patterns of 37 H3N2 viruses were classified into three groups with significant binding-pattern variations. The results showed that the carbohydrate-binding patterns of H3N2 varied over time. A phylogenetic analysis of the hemagglutinin gene also revealed progressive drift year to year. Of note, the viruses that caused large outbreaks in 1999 and 2003 showed glycan-binding preferences to both α-2,3 and α-2,6 sialylated glycans. Twenty amino acid substitutions were identified primarily at antigenic sites that might contribute to H3N2 virus evolution and the change in the glycan-binding patterns. This study provides not only a systematic analysis of the receptor-binding specificity of influenza clinical isolates but also information that could help to monitor the outbreak potential and virus evolution of influenza viruses.

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.

The Effect of Botulinum Neurotoxin Serotype a Heavy Chain on the Growth Related Proteins and Neurite Outgrowth after Spinal Cord Injury in Rats.

(1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (p < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (p < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.