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Weimin Luan,1,2,* Xiqian Qi,3,* Feng Liang,2 Xiaotao Zhang,2 Ziyang Jin,2 Ligen Shi,2 Benyan Luo,1 Xuejiao Dai1 1Department of Neurology, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 2Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Department of Neurology, Ningbo Municipal Hospital of T.C.M., Ningbo, Zhejiang, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xuejiao Dai; Benyan LuoDepartment of Neurology, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang, 312000, People’s Republic of ChinaTel/Fax +86-571-87235101Email x.dai@zju.edu.cn; luobenyan@zju.edu.cnPurpose: Age-related increase in myelin loss may be responsible for brain atrophy, and the mechanism is not completely understood. We aim to comprehensively delineate oligodendrocyte heterogeneity in young and aged mice and to reveal the underlying mechanism for myelin loss during aging.Methods: Diffusion tensor imaging and immunofluorescent staining were performed to verify the demyelination in the aged brains of both rodents and human. Further, the single-cell RNA sequencing data of all brain cells from young and aged mice were deeply analyzed to identify the subsets of oligodendrocyte lineage cells. Cell-to-cell interaction analysis was performed to detect the mechanism of observed changes in oligodendrocyte generation.Results: Oligodendrocytes were observed to up-regulate several senescence associated genes in aged brain. Four clusters of oligodendrocyte precursor cells (OPCs) were identified in both young and aged brains. The number of those OPCs in basal state was significantly increased, while the OPCs in the procedure of differentiation were immensely decreased in aged brain. Furthermore, it was identified that activated microglia in the aged brain released inflammatory factors to suppress OPC differentiation. Stat1 might be a potential target to transform senescent microglia into tissue repair type to promote oligodendrocyte generation.Conclusion: These results provided a perspective on how age activated microglia could impede remyelination and might give a new therapeutic target for age-related remyelinating diseases.Keywords: remyelination, oligodendrocyte precursor cells, microglia, oligodendrocytes, single cell RNA sequencing

This study was conducted to investigate the effect of in ovo administration of a mixture of Astragalus polysaccharide (APS) and Newcastle disease vaccine (NDV) on growth performance, intestinal development, and mucosal immunity in newly hatched chicks. Six hundred specific-pathogen-free (SPF) Leghorn fertilised eggs were incubated in a commercial hatchery and divided into four groups: (a) control group injected with 1 ml of 0.9% physiological saline, (b) APS group injected with 1 ml of 1 mg/ml APS solution, and (c) NDV group injected with 1 ml of 10

The immunomodulatory effect of Acanthopanax senticosus polysaccharide (ASPS) on immunosuppressed chickens induced by cyclophosphamide (Cy) was observed in this study. Four hundred 7-day-old chickens were randomly divided into 4 groups: vaccinated control group (VC group), Cy-challenged control group (Cy group), Cy-challenged + low-dose ASPS group (ASPSL + Cy group), and Cy-challenged + high-dose ASPS group (ASPSH + Cy group). All groups except the VC group were injected with Cy at a dose of 80 mg/kg/day of BW for 3 successive days to induce immunosuppression. At the age of 10 d, the ASPSL + Cy group and ASPSH + Cy group were intramuscularly injected with 0.2 mL of ASPS at the dose of 100 and 200 mg/mL/day, respectively, once a day for 3 successive days. The Cy group was injected with saline solution in the same way as the 2 ASPS groups. At the age of 14 d, the chickens were vaccinated with Newcastle disease (ND) vaccine in all groups. On day 7, 14, 21, and 28 after the vaccination, BW, lymphocyte proliferation, the serum antibody titers of the ND vaccine, the proportion of CD4+ and CD8+ T lymphocytes, and the concentrations of interferon gamma and IL-2 were determined. The results showed that chickens were injected with Cy at a dose of 80 mg/kg of BW for 3 d displayed lower immune responses than the control group, indicating that the immunosuppressive model was successfully established. At most time points, both high and low doses of ASPS could significantly promote lymphocyte proliferation; enhance BW, antibody titers, and the proportion of CD4+ and CD8+ T lymphocytes; and raised the concentrations of interferon gamma and IL-2 in Cy-treated chickens compared with those in the Cy control group (P < 0.05). These results indicated that ASPS could resist immunosuppression induced by Cy and may be a new-type immune adjuvant to improve vaccination in normal and immunosuppressed chickens.

This study was conducted to investigate the effections of in ovo injection with Newcastle disease vaccine combined with Astragalus polysaccharide on the mucosal immunity of jejunum. Four hundred fertilized eggs were randomly divided into four groups (n=100/group), and embryonic immunization with solutions of APS, ND vaccine, ND vaccine combined with APS, and 0.9% physiological saline into their amniotic fluid on d 18.5 of incubation. Significant improvement of the mucosal immunity of jejunum was displayed in those vaccinated with ND vaccine combined with APS, manifested as enhanced villus height and the ratio of villus height to crypt depth. Moreover, chicks immunized with ND vaccine plus APS significantly elevated the levels of slgA in the jejunum during the embryonic period. Overall results indicated embryonic immunization with ND vaccine combined with APS could stimulate stronger humoral immunity in the jejunum of newly hatched chicks.

Newcastle disease virus (NDV) is one of the most important diseases in poultry. The present study generated recombinant surface-displayed Lactobacillus casei (L. casei) expressing the hemagglutinin-neuraminidase (HN) of NDV (Lc-pPG-HN) and a live pPG vector (Lc-pPG) and evaluated their immunogenicity. A 1670 bp HN gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in the resulting recombinant Lc-pPG-HN (surface displayed) strain was verified using Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, and the molecular weight of the corresponding protein was 63 kDa. A fluorescent signal for Lc-pPG-HN was observed using fluorescence microscopy. A total of 270 healthy chicks were divided into three treatment groups. Five replicates were used for each treatment, while six chicks were used per replicate. The following three treatment groups were used: physiological saline group (Control), Lc-pPG group and recombinant vaccine group (Lc-pPG-HN). The primary immunization and booster immunization of the chicks were performed via oral administration on 1 and 10 days old. Tissue and blood samples were collected from chickens that received oral recombinant L. casei strains on 1, 7, 14 and 21 days post-immunization for immune-related index analyses. Chickens orally immunized with Lc-pPG-HN showed significantly increased body weights and immune organ indices. Oral immunization with Lc-pPG-HN also enhanced the concentrations of serum interleukin-2 (IL-2), interferon-γ (IFN-γ), intestinal lavage fluid secretory immunoglobulin A (SIgA) and histomorphological development of the small intestine. Our results also indicated that recombinant L. casei significantly increased Lactobacillus and Bifidobacterium colonization and decreased the relative abundance of Escherichia coli (E. coli) in the chicken caecum. Similar enhancement effects from hemagglutination inhibition were also observed in the antibody titers. Oral administration of Lc-pPG-HN effectively protected against NDV and alleviated the symptoms of the NDV challenge. In summary, recombinant L. casei had positive impacts on the performance, immunological function, gut development, and microbiota of growing chicks and may be a potential therapeutic candidate against NDV.

Backgroud: Newcastle disease virus (NDV) is considered one of the most important diseases among chickens. In this study, we generated recombinant surface-displayed Lactobacillus casei (L.casei) expressing the hemagglutinin-neuraminidase (HN) of NDV and a live vector pPG alone (named Lc-pPG-HN and Lc-pPG), and evaluated their effects on early growth development, intestinal health and protection against NDV challenge in chickens. 270 chickens were randomly divided into three groups: Lc-pPG-HN, Lc-pPG and physiological saline (control) group, and chickens from each group were respectively immunized with Lc-pPG-HN, Lc-pPG and physiological saline on 1 and 10 days. Results: Recombinant L.casei expressing the HN protein of NDV (Lc-pPG-HN) was successfully constructed. Orally immunized with Lc-pPG-HN could significantly increase body weight (BW) and immune organs index. Moreover, Lc-pPG-HN improved secretory immunoglobulin A (SIgA) in jejunum, the relative abundance of flora in cecum, histomorphological development of small intestine. In addition, the similar enhancement effects were also observed with hemaggluti-nation inhibition (HI) antibody titer and the expression of cytokines in the serum. The oral administration of Lc-pPG-HN also provided effective protection and alleviated the symptoms of NDV challenge.Conclusions: Thus, a recombinant L.casei vaccine expressing HN may be a potential therapeutic candidate against NDV and improve chickens growth and development.

In this study, immunogenic efficacies of in ovo administration of Astragalus polysaccharide (APS) along with live Newcastle disease vaccine (live ND vaccine) (live VG/GA strain) were evaluated. Four hundred fertilized eggs were randomly divided into four groups (n = 100/group), and vaccinated in ovo, respectively, with solutions of APS, live ND vaccine, live ND vaccine combined with APS, and 0.9% physiological saline into their amniotic fluid on d 18.5 of incubation. Significant improvement of chicks' development was displayed in those vaccinated with live ND vaccine adjuvanted with APS in ovo, manifested as enhanced hatchability and gaining weight. Moreover, in ovo administration of live NDV vaccine plus APS could significantly enhance the serum anti-NDV antibody titres and interferon gamma (IFN-γ), interleukin (IL)-2, IL-4 and IL-6 concentrations, promote lymphocyte proliferative capability as well as improve the frequencies of CD4+ and CD8+ T cells in peripheral blood. Overall results indicated in ovo administration of live ND vaccine adjuvanted with APS could stimulate stronger humoral and cellular responses in newly hatched chicks.

Cyprinid herpesvirus 3 (CyHV-3) infection in carp causes a fatal and highly contagious disease that results in huge economic losses in common and koi carp aquaculture worldwide. Thus the development of an effective vaccine to protect carp stocks against the CyHV3 virus is imperative. In this study, we immunized common carps with a DNA vaccine consisting of a plasmid that co-expresses the CyHV-3 envelope protein ORF25 and the carp IL-1β gene in order to evaluate the adjuvant potential of IL-1β. Our result shows that antibodies specific to ORF25 can be detected as early as one week after intramuscular injection of the DNA vaccine at low dosage. Moreover, the co-expression of IL-1β can enhance the potency of the vaccine, as demonstrated by a higher antibody level after the third immunizations. Importantly, the DNA vaccine reduced mortality in carps when they were immunized prior to a CyHV-3 challenge, as compared to negative control groups. However, despite being able to induce higher neutralizing antibody titres, the co-expression of IL-1β in the DNA vaccine did not significantly improve the overall survival of immunized fish following virus challenge. Furthermore, the DNA vaccine can protect carps from tissue damage and histopathological alteration caused by viral infection. These strongly suggests that the vaccine can efficiently elicit protective immunity against CyHV-3 infection. In conclusion, the DNA vaccine formulated with the pIRES-ORF25-IL-1β DNA construct can protect carp against CyHV-3 infection and has potential applicability in the aquaculture industry.

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1β, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P

SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (PPTrim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

TRIM28 is a co-repressor, which interacts with HP1 proteins and chromatin repressive complexes leading to gene expression silencing. In this study, after we used Lipofectamine3000-mediating siRNA and restructured p EGFP-IRES2-TRIM28 to transfect bovine fibroblasts, the expression levels of TRIM28, HP1BP3, DNMT, SETDB1, TP53, BAX, Bcl and SOD were detected by real-time polymerase chain reaction (PCR). In addition, the influence of p EGFP-IRES2-TRIM28 on histone H3K9me3 was also observed. In the siRNA transfected group, TRIM28 was down-regulated (P less than 0.01), which inhibited cell proliferation and reduced the level of H3K9me3 expression. In the p EGFP-IRES2-TRIM28 transfected group, TRIM28 was over-expressed (P less than 0.01), and the pro-apoptosis gene BAX was significantly decreased (P ess tahn 0.01), but there was no significant change in other genes. Our results demonstrate that TRIM28 plays an important role in bovine fibroblasts and might be a valuable biomolecule for proliferation, apoptosis, and histone H3K9 trimethylation.

The lysine is considered as the most important essential amino acid, because it is the most limiting in the cereals grains. In this study, a lysine-rich (LR) gene, and the expression vector pcDNA3.1-LR and pBC1-LR were constructed. The LR was expressed in 293T cells driven by the vector pcDNA3.1-LR and checked by RT-PCR and WB. The mammary gland tissue-specific expression vector carrying the LR was injected directly into the lactating mammary glands of cows and the milk samples were checked by a complete amino acid analysis. The results showed that the LR protein was expressed successfully in cells and in cow milk; the expression of LR lasted for 6 d, and the lysine level of the injection group was significantly higher than that of negative controls (p Lass Than 0.05). This study provide a better understanding of how mammary gland expression systems increase the lysine content of milk that can be applied to transgenic dairy cow.

Astragalus polysaccharides (APS) are a traditional Chinese medicine with a therapeutic effect by enhancing immune function; however, the underlying functional mechanism is still unclear. The aim of the present study was to determine the effect of oral administration of APS on jejunum mucosal immunity in chickens vaccinated against Newcastle disease (ND). One-day-old Hy-Line male chickens were divided into five groups of 20 chicks each: three APS groups, one vaccinated control (VC) group and one non-vaccinated negative control (NC) group. On d 10, the APS groups were orally administered 0.5 mL of APS at doses of 1 mg/mL (APSL), 2 mg/mL (APSM) and 4 mg/mL (APSH) daily for 4 consecutive days. The chicks in the control groups were administered 0.5 mL saline for those 4 days. All groups except NC were administered a ND virus (NDV) vaccine on day 14. The jejunum was removed from 4 randomly selected chickens of each group at 1, 7, 14 and 28 days after vaccination. The jejunal villus height (VH) and crypt depth (CD) were measured and the VH:CD ratio calculated. Immunohistochemistry was used to analyze the differences of IgA+ cells in the jejunum. NDV specific secretory IgA (sIgA) levels in jejunal contents were detected using an indirect ELISA. At most time points, VH:CD ratios, number of IgA+ cells, and sIgA levels were significantly higher in the APS groups than those in VC and NC groups, but there were little differences among the three doses of APS groups. These results indicate that oral administration of APS could enhance the intestinal mucosal immune function of chickens, and APS could be used as a vaccine enhancer.

Genomic imprinting is a mechanism of differentially epigenetic modification that restricts monoallelic expression to either the maternally or paternally inherited copy of the gene during gametogenesis. Imprinted methylation undergoes a process of erasure, acquisition, and maintenance during gametogenesis and early embryogenesis. Disruptions in any of these steps may lead to imprinting disorders, resulting in the aberrant development of embryogenesis, placentation and postnatal growth. Recent studies have shown that maternal-effect proteins are important for the regulation of imprinted gene during the development of preimplantation embryos. In order to obtain a better understanding for the mechanism of maternal-effect proteins in the maintenance of genomic imprints, the recent study progress of maternal-effect proteins, such as DPPA3, ZFP57, TRIM28 and DNMT1, are summarized, and the regulation mechanism of these maternal-effect proteins for genomic imprints are discussed.

Major royal jelly protein 1 (MRJP1, the most abundant protein of honeybee royal jelly) has been recognized as a valuable functional foods and therapeutic protein for human beings. There is great potential using transgenic technology to express recombinant MRJP1 in milk in order to improve the human health conditions. For expression of MRJP1 in milk, mammary gland-specific expression vector (pBC1-MRJP1-NEOr) carrying the expression cassette composed of e-casein promoter, cDNA encoding MRJP1 (with bovine e-casein signal peptide) and a Neor selection marker was constructed, and expressed in human MCF-7 cells. To examine the expression of MRJP1, a His-tag protein cDNA fragments were fused on the 5' end of MRJP1 gene using PCR amplification. This vector was shown to be expressed successfully, thus potentially enabling the generation of transgenic cows to be used as mammary gland bioreactors.