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Background: Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has high affinity for the human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for production of recombinant murine TRAIL has not been reported. Objective: In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein. Methods: Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Ni-chelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo. Results: Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, Zn2+ supplement was essential to produce soluble murine TRAILs in E.coli BL21 (DE3). The two purified soluble mTRAILs showed similar cytotoxicity to cancer cells, moreover, mT_N99 also showed a good anti-tumor effect in vivo and is more suitable for the treatment of murine tumor models. Conclusion: A production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

The safety, biodistribution, and pharmacokinetics of any new therapeutic tumor DNA vaccine must be evaluated in preclinical studies. We previously developed the DNA vaccine (CpDV-IL2-sPD1/MUC1 and survivin), which showed excellent antitumor effects in a variety of tumor models. In this study, we demonstrate the safety and biodistribution after immunization with naked DNA vaccine (10 mg/kg) by electroporation in a mice model. All mice reached the end of the study with good body conditions. By established and validated QPCR method, we found high-copy plasmid DNA at the injection site (muscle) on day 1 in all eight animals, followed by a downward trend. By day 49, a small amount of plasmid DNA was still detectable, but only in one mouse. On reproductive safety, no plasmids existed in the ovary at any time point. Also, only two of the 16 testis samples could detect a very small amount of DNA on days 7 and 14. The most important thing was that plasmids were cleared from almost all organs (heart, liver, spleen, lung, kidney, stomach, blood, thymus, intestine) on day 49. In summary, the results of our experiments demonstrate that the DNA vaccine delivered by electroporation was shown to be safe and merits further development for cancer treatment.

Traditional anticancer treatments directly target tumor cells. In contrast, cancer immunotherapy fortifies host immunity. Nanoparticles that incorporate both immunomodulatory and chemotherapeutic agents regulate the tumor microenvironment by activating immune cells and enhancing antitumor immunity. Nanoparticle-based cancer immunotherapy has received considerable attention and has been extensively studied in recent years. In this study, we developed a targeted drug delivery system to enhance immunotherapeutic efficacy and overcome drug resistance by inducing tumor apoptosis and immunogenic cell death (ICD), and activating immune cells. Periodic mesoporous organosilica nanoparticles (PMOs) bore tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on their surfaces, and their inner cores were loaded with doxorubicin (DOX). TRAIL enhanced the nanoparticle-targeting capacity and worked synergistically with DOX against breast cancer cells in vitro and in vivo. Furthermore, we revealed for the first time the ability of PMOs to activate dendritic cells (DCs) and elevate ICD levels of DOX in vitro, and TRAIL further enhances the immunomodulatory function of PMOs. Systemic exposure to DOX@PMO-hT induced an immune response, activated DCs and CD4

Preclinical studies have shown that the induction of secretory IgA (sIgA) in mucosa and neutralizing antibodies (NAbs) in sera is essential for designing vaccines that can effectively block the transmission of HIV-1. We previously showed that a vaccine consisting of bacterium-like particles (BLPs) displaying Protan-gp120AE-MTQ (PAM) could induce mucosal immune responses through intranasal (IN) immunization in mice and NAbs through intramuscular (IM) immunization in guinea pigs. Here, we evaluated the ability of this vaccine BLP-PAM to elicit HIV-1-specific mucosal and systemic immune responses through IN and IM immunization combination strategies in rhesus macaques. First, the morphology, antigenicity and epitope accessibility of the vaccine were analysed by transmission electron microscopy, bio-layer interferometry and ELISA. In BLP-PAM-immunized macaques, HIV-1-specific sIgA were rapidly induced through IN immunization in situ and distant mucosal sites, although the immune responses are relatively weak. Furthermore, the HIV-1-specific IgG and IgA antibody levels in mucosal secretions were enhanced and maintained, while production of serum NAbs against heterologous HIV-1 tier 1 and 2 pseudoviruses was elicited after IM boost. Additionally, situ mucosal responses and systemic T cell immune responses were improved by rAd2-gp120AE boost immunization via the IN and IM routes. These results suggested that BLP-based delivery in combination with the IN and IM immunization approach represents a potential vaccine strategy against HIV-1.

The pH-induced aggregation and cysteine-induced reduction of [Mo154] in tumor cells lead to PTT enhancement and improved antitumor effects.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFβ) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFβ to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFβ affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFβ target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFβ to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFβ interaction may be promising in the development of a new AIDS therapeutic strategy.

HIV-1 infection-induced cGAS–STING–TBK1–IRF3 signaling activates innate immunity to produce type I interferon (IFN). The HIV-1 nonstructural protein viral infectivity factor (Vif) is essential in HIV-1 replication, as it degrades the host restriction factor APOBEC3G. However, whether and how it regulates the host immune response remains to be determined. In this study, we found that Vif inhibited the production of type I IFN to promote immune evasion. HIV-1 infection induced the activation of the host tyrosine kinase FRK, which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif (ITIM) of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN. Mechanistically, the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162. However, the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection. These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING. These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection.

Background Progressive neuronal death is the key pathological feature of Alzheimer’s disease (AD). However, the molecular mechanisms underlying the neuronal death in AD patients have not been fully elucidated. Necroptosis reportedly activates and induces neuronal death in patients with Alzheimer’s disease (AD); however, the main mediators and mechanisms underlying necroptosis induction in AD remain elusive. Methods The function of hyperphosphorylated tau (pTau) in inducing necroptosis in neuronal cell was examined using Western blotting, RT-PCR and flow cytometry. Tau-induced inflammation was identified via RNA sequencing and transwell assay. Pharmacological methods and CRISPR–Cas9 technology were used to verify the role of necrosome proteins in pTau-stimulated neuronal death and inflammation. TauP301S model mice were treated with Nec-1 s to evaluate the role of necroptosis in tau pathology. Results Hyperphosphorylated tau could induce necroptosis in neuronal cells by promoting the formation of the RIPK1/RIPK3/MLKL necrosome. In addition, pTau significantly stimulated cell-autonomous overexpression of cytokines and chemokines via the intracellular nuclear factor kappa B (NF-κB) signaling pathway. Importantly, the RIPK1/RIPK3/MLKL axis was essential for the pTau-mediated NF-κB activation and cytokine storm. Furthermore, necroptosis stimulation, NF-κB activation, and cytokine induction have been detected in TauP301S mice and blocking necroptosis markedly ameliorated behavioral defects and excessive neuroinflammation in AD mice. Conclusions Our study, for the first time, revealed that pTau contributes to neuronal death by inducing necroptosis and inflammation, mediated by activating the RIPK1/RIPK3/MLKL and NF-κB pathways, thereby delineating the hierarchical molecular network of neuronal necroptosis induction in AD.

The incidence of diabetes worldwide continues to rise, placing a huge economic and medical burden on human society. More than 90% of diabetic cases are type 2 diabetes (T2D). At present, the pathogenesis of T2D is not yet fully understood. Metabolomics uses high-resolution analytical techniques (typically NMR and MS) to help identify biomarkers associated with the risk of T2D and reveal potential pathogenesis. Many metabolites such as branched-chain amino acids (BCAAs), aromatic amino acids, glycine, 2-hydroxybutyric acid (2-HB), lysophosphatidylcholine (LPC) (18:2), and trehalose have proven to be biomarkers of T2D. Insulin resistance (IR) induced by BCAA in T2D mice is related to the activation of mammalian target of rapamycin (mTOR) and phosphorylation of insulin receptor substrate-1 (IRS1). Incomplete LCFA [Formula: see text]-oxidation promote acylcarnitine byproduct accumulation and stimulates proinflammatory NF[Formula: see text]B-related pathways to inhibit insulin action. Traditional Chinese Medicine (TCM) presents unique advantages in the treatment of T2D. Multiple metabolites and metabolic pathways have been identified in the treatment of TCM, providing valuable biomarkers and novel targets for drug therapy and pharmacological mechanism. Therefore, this paper reviews the modern achievements of metabolomics in T2D research and the progress of TCM management in recent years, in order to provide valuable information for related research.

HIV remains a health challenge worldwide, partly because of the continued development of resistance to drugs. Therefore, it is urgent to find new HIV inhibitors and targets. Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3) are important host restriction factors that inhibit HIV-1 replication by their cytidine deaminase activity. HIV-1 viral infectivity factor (Vif) promotes proteasomal degradation of APOBEC3 proteins by recruiting the E3 ubiquitin ligase complex, in which core-binding factor β (CBFβ) is a necessary molecular chaperone. Interrupting the interaction between Vif and CBFβ can release APOBEC3 proteins to inhibit HIV-1 replication and may be useful for developing new drug targets for HIV-1. In this study, we identified a potent small molecule inhibitor CBFβ/Vif-3 (CV-3) of HIV-1 replication by employing structure-based virtual screening using the crystal structure of Vif and CBFβ (PDB: 4N9F) and validated CV-3's antiviral activity. We found that CV-3 specifically inhibited HIV-1 replication (IC(50) = 8.16 µm; 50% cytotoxic concentration >100 µm) in nonpermissive lymphocytes. Furthermore, CV-3 treatment rescued APOBEC3 family members (human APOBEC3G (hA3G), hA3C, and hA3F) in the presence of Vif and enabled hA3G packaging into HIV-1 virions, which resulted in Gly-to-Ala hypermutations in viral genomes. Finally, we used FRET to demonstrate that CV-3 inhibited the interaction between Vif and CBFβ by simultaneously forming hydrogen bonds with residues Gln-67, Ile-102, and Arg-131 of CBFβ. These findings demonstrate that CV-3 can effectively inhibit HIV-1 by blocking the interaction between Vif and CBFβ and that this interaction can serve as a new target for developing HIV-1 inhibitors.

Although many vaccines have been designed to induce effective mucosal immune responses against HIV-1, designing an effective HIV-1 vaccine remains a challenge. Bacterium-like particles (BLPs) are a new type of vector used to induce mucosal immune responses, and have already been used for some vaccines against respiratory tract viruses. In this study, we designed a mucosal vaccine against HIV-1 based on BLPs. The vaccine was used to immunize both mice and guinea pigs via intramuscular (i.m.) injection or intranasal (i.n.) drip. We found that gp120 trimers bound to BLPs delivered via i.n. drip successfully induced Env-specific secretory IgA (sIgA) at mucosal sites in mice. Furthermore, nasal washes from guinea pigs immunized via i.n. drip showed neutralizing activity against HIV-1 tier 1 pseudoviruses. Thus, gp120 trimers bound to BLPs may be an effective vaccine strategy against HIV-1.

Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has a high affinity for human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for the production of recombinant murine TRAIL has not been reported.In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein.Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Nichelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo.Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, ZnA production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

Understanding maturation pathways of broadly neutralizing antibodies (bnAbs) against HIV-1 can be highly informative for HIV-1 vaccine development. A lineage of J038 bnAbs is now obtained from a long-term SHIV-infected macaque. J038 neutralizes 54% of global circulating HIV-1 strains. Its binding induces a unique "up" conformation for one of the V2 loops in the trimeric envelope glycoprotein and is heavily dependent on glycan, which provides nearly half of the binding surface. Their unmutated common ancestor neutralizes the autologous virus. Continuous maturation enhances neutralization potency and breadth of J038 lineage antibodies via expanding antibody-Env contact areas surrounding the core region contacted by germline-encoded residues. Developmental details and recognition features of J038 lineage antibodies revealed here provide a new pathway for elicitation and maturation of V2-targeting bnAbs.

In this study, we investigate the synergistic effect of gemcitabine (Gem) and a novel DNA vaccine in the treatment of pancreatic cancer in mice and explore the anti-tumor mechanism of this combination therapy. Fibroblast activation protein α-expressing cancer-associated fibroblasts (FAPα

Soluble tumor necrosis factor related apoptosis-inducing ligand (sTRAIL) is a promising candidate for antitumor protein drugs. However, the low stability and poor pharmacokinetic profile significantly limit its clinical application. In this work, a novel strategy was used to improve sTRAIL performance by combining genetic engineering with coaxial electrospinning. We first obtained a stable homotrimer via genetic engineering using the adenovirus knobless fiber motif to modify sTRAIL. In vitro studies showed that the engineered sTRAIL was more stable and had higher antitumor activity than wild sTRAIL. This new recombinant sTRAIL was then encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanofibers by coaxial electrospinning to further enhance the stability and achieve the controlled release of sTRAIL. Surprisingly, the recombinant sTRAIL maintained its biological activity during the electrospinning process and exhibited a good cumulative release. The establishment of incorporation treatment of breast c...

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential therapeutic anti-cancer drug with selective cytotoxicity in cancer cells. However, in multiple clinical trials, the therapeutic effect of TRAIL is limited owing to tumor resistance. The combination of small molecules or other drugs may represent a suitable strategy to overcome TRAIL resistance. This study found that 20(s)-ginsenoside Rh2 sensitized non-sensitive human hepatocellular carcinoma cells to TRAIL-induced apoptosis. The combination of TRAIL and Rh2 decreased cell viability and increased caspase cascade-induced apoptosis in several liver cancer cell lines. Moreover, we found that Rh2 reduced the apoptosis-related protein XIAP and Survivin, a negative regulator of the apoptosis pathway. At the same time, Rh2 can further enhance TRAIL-induced apoptosis by upregulating the death receptor 5 (DR5), thereby significantly enhancing its anti-tumor effect. Furthermore, Rh2 enhanced the therapeutic efficacy of TRAIL in mouse xenograft models, suggesting that Rh2 also sensitizes TRAIL in vivo. Taken together, our study indicates that Rh2 may act as a sensitizer in combination with TRAIL to increase the efficacy of its anti-tumor activity.

This study reports a unique phenomenon, the opposite color response of a giant polyoxometalate, (NH4)42[Mo132O372(CHCOO)30] (H2O)72 ([Mo132]), to the existing states of human papillomavirus (HPV) major capsid protein, L1-pentamer (L1-p) and virus-like particles (VLPs). The color responses originate from the different assembly forms between [Mo132] and the capsid protein, which were inspected and separated by using CsCl gradient centrifugation, and validated in detail by sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and transmission electron microscopy (TEM) images. The intrinsic mechanism difference between them were investigated in depth by using the XPS-based semi-quantitative analysis and the well designed peptides, which reveal the key points of L1 determining the charge-transfer ratio between Mo(V) to Mo(VI), and consequently the levels of [Mo132] hypochromicity in different assemblies. Such unique phenomenon is significant as it supplies a colorimetry approach to directly distinguish the existing states of HPV capsid protein and would be significant in the quality assay of HPV vaccine and existing states of other virus in future.

Glioblastoma, the most common human brain tumor, is highly invasive and difficult to cure using conventional cancer therapies. As an alternative, adenovirus-mediated virotherapies represent a popular and maturing technology. However, the cell surface coxsackievirus and adenovirus receptor (CAR)-dependent infection mechanism limits the infectivity and oncolytic effects of Adenovirus type 5. To address this limitation, in this study we aimed to develop a novel oncolytic adenovirus for enhanced infectivity and therapeutic efficacy toward glioblastoma. We developed a novel genetically modified oncolytic adenovirus vector with dual capsid modifications to facilitate infection and specific cytotoxicity toward glioma cells. Modification of the adenoviral capsid proteins involved the incorporation of a synthetic leucine zipper-like dimerization domain into the capsid protein IX (pIX) of human adenovirus serotype 5 (Ad5) and the exchange of the fiber knob from Ad37. The virus infection mechanism and anti-tumor efficacy of modified vectors were evaluated in both in vitro (cell) and in vivo (mouse) models. Ad37-knob exchange efficiently promoted the virus infection and replication-induced glioma cell lysis by oncolytic Ad5. We also found that gene therapy mediated by the dual-modified oncolytic Ad5 vector coupled with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibited significantly enhanced anti-tumor efficacy in vitro and in vivo. This genetically modified oncolytic adenovirus provides a promising vector for future use in glioblastoma gene-viral-based therapies.

An integrase-defective SIV (idSIV) vaccine delivered by a DNA prime and viral particle boost approach can suppress viral loads (VLs) during the acute infection stage after intravenous SIVmac239 challenge. This study investigated how idSIV DNA and viral particle immunization alone contributed to the suppression of VLs in Chinese rhesus macaques after SIV challenge. Two macaques were immunized with idSIV DNA five times and two macaques were immunized with idSIV viral particles three times. Cellular and humoral immune responses were measured in the vaccinated macaques after immunization. The VLs and CD4+ T cell counts were monitored for 28 weeks after the intravenous SIVmac239 challenge. The SIV-specific T cell responses were only detected in the DNA-vaccinated macaques. However, binding and neutralizing antibodies against autologous and heterologous viruses were moderately better in macaques immunized with viral particles than in macaques immunized with DNA. After the challenge, the mean peak viremia in the DNA group was 2.3 logs lower than that in the control group, while they were similar between the viral particle immunization and control groups. Similar CD4+ T cell counts were observed among all groups. These results suggest that idSIV DNA immunization alone reduces VLs during acute infection after SIV challenge in macaques and may serve as a key component in combination with other immunogens as prophylactic vaccines.

Cancer-associated fibroblasts (CAFs), major components of the tumor microenvironment (TME), promote tumor growth and metastasis and inhibit the anti-tumor immune response. We previously constructed a DNA vaccine expressing human FAPα, which is highly expressed by CAFs, to target these cells in the TME, and observed limited anti-tumor effects in the 4T1 breast cancer model. When the treatment time was delayed until tumor nodes formed, the anti-tumor effect of the vaccine completely disappeared. In this study, to improve the safety and efficacy, we constructed a new FAPα-targeted vaccine containing only the extracellular domain of human FAPα with a tissue plasminogen activator signal sequence for enhanced antigen secretion and immunogenicity. The number of CAFs was more effectively reduced by CD8+ T cells induced by the new vaccine. This resulted in decreases in CCL2 and CXCL12 expression, leading to a significant decrease in the ratio of myeloid-derived suppressor cells in the TME. Moreover, when mice were treated after the establishment of tumors, the vaccine could still delay tumor growth. To facilitate the future application of the vaccine in clinical trials, we further optimized the gene codons and reduced the homology between the vaccine and the original sequence, which may be convenient for evaluating the vaccine distribution in the human body. These results indicated that the new FAPα-targeted vaccine expressing an optimized secreted human FAPα induced enhanced anti-tumor activity by reducing the number of FAPα+ CAFs and enhancing the recruitment of effector T cells in the 4T1 tumor model mice.

Conventional DNA vaccine strategies usually employ a regimen of immunizations at 2-week or longer intervals to induce effective memory cell-dependent immune responses. Clinical cancer treatment requires a faster immunization strategy to contend with tumor progression. In this study, a novel fast immunization strategy was established, wherein a DNA vaccine was intramuscularly administered on days 0, 2, and 5 in a murine lung cancer model. Effector cells peaked 7 to 10 days after the last vaccination. Compared with traditional 2-week-interval immunization strategies, antigen-specific cytolysis and INF-γ secretion were significantly enhanced under the fast vaccination approach. As a result, the rapidly administered DNA vaccine elicited stronger and more prompt antitumor effects. The probable underlying mechanism of fast immunization was the accumulation of CD8+CD11c+ antigen-presenting cells at the injection site, which enhanced subsequent antigen presentation. In conclusion, the fast DNA vaccination strategy shortened vaccination time to 5 days and elicited a stronger antitumor immune response.

Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

(1) Background: The EMT plays a crucial role in tumor metastasis, which is the major cause for colorectal carcinoma-related mortality. However, the underlying regulators and mechanisms of EMT in CRC metastasis are still poorly understood; (2) Methods: The transcriptional regulators of EMT in CRC and their functions were examined using RT2212PCR, Western blotting, and luciferase reporter assay. The components of ZEB2/TWIST1 complex and their mutual interactions were identified via affinity purification, mass spectrometry, co-immunoprecipitation, and pull-down experiments. The functional mechanisms of ZEB2/TWIST1/PRMT5/NuRD axis were determined by chromatin immunoprecipitation and luciferase reporter assay. The contribution of ZEB2/TWIST1/PRMT5/NuRD complex in the CRC metastasis was investigated using wound healing, transwell assay, and in vivo xenograft mouse model; (3) Results: We found that ZEB2 and TWIST1 were both significantly upregulated in CRC tissues and EMT of CRC cells. ZEB2 could recruit TWIST1 to the E-cadherin promoter and synergistically repressed its transcription. In addition, ZEB2 physically interacted with TWIST1, PRMT5, and the nucleosome remodeling and deacetylase (NuRD) complex to form a novel repressive multicomplex, leading to epigenetic silencing of E-cadherin in CRC cells. Notably, the combined inhibition of ZEB2 and TWIST1 and epigenetic inhibition markedly reduced CRC metastasis in mice; (4) Conclusions: We revealed for the first time that ZEB2 could recruit TWIST1, PRMT5, and NuRD to form a repressive multicomplex and epigenetically suppresses the transcription of E-cadherin, thereby inducing the EMT process and metastasis in CRC. Our results also confirmed the therapeutic potential of epigenetic inhibitors in CRC.

We present the calibration strategy for the 20 kton liquid scintillator central detector of the Jiangmen Underground Neutrino Observatory (JUNO). By utilizing a comprehensive multiple-source and multiple-positional calibration program, in combination with a novel dual calorimetry technique exploiting two independent photosensors and readout systems, we demonstrate that the JUNO central detector can achieve a better than 1% energy linearity and a 3% effective energy resolution, required by the neutrino mass ordering determination. [Figure not available: see fulltext.], 0, info:eu-repo/semantics/published

Background: Adjuvants are important components of vaccines and effectively enhance the immune response of specific antigens. However, the role of adjuvants or combinations of adjuvants in stimulating immunogenicity of the amyloid-β (Aβ) vaccine, as well as molecular mechanisms underlying such stimulation still remain unclear. A previous study of ours developed a norovirus P particle-based active Aβ epitope vaccine, PP-3copy-Aβ1-6-loop123, which stimulates a high titer of Aβ-specific antibodies in mouse Alzheimer’s disease (AD) models. Objective: The most effective and safe adjuvant that maximizes the immunogenicity of our protein vaccine was determined. Methods: We investigated four adjuvants (CpG, AS02, AS03, and MF59), and combinations of those, for capacity to enhance immunogenicity, and performed transcriptome analysis to explore mechanisms underlying the role of these in AD immunotherapy. Results: Addition of the adjuvant, AS02, remarkably improved the immunogenicity of the PP-3copy-Aβ1-6-loop123 vaccine without triggering an Aβ-specific T-cell response. Combinations of adjuvants, particularly CpG + AS02 and CpG + AS03, elicited a significantly elevated and prolonged Aβ-specific antibody response. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that a combination of two adjuvants was more effective in activating immune-related pathways, thereby enhancing the immunogenicity of PP-3copy-Aβ1-6-loop123. Conclusion: These findings demonstrated that adjuvants can be used as enhancers in AD protein vaccination, and that a combination of CpG and AS-related adjuvants may be a very effective adjuvant candidate suitable for further clinical trials of the PP-3copy-Aβ1-6-loop123 vaccine. Our studies also revealed potential mechanisms underlying the stimulation of immune response of protein vaccines by adjuvants.

Human SAMHD1 (hSAM) restricts lentiviruses at the reverse transcription step through its dNTP triphosphohydrolase (dNTPase) activity. Besides humans, several mammalian species such as cats and cows that carry their own lentiviruses also express SAMHD1. However, the intracellular distribution of feline and bovine SAMHD1 (fSAM and bSAM) and its significance in their lentiviral restriction function is not known. Here, we demonstrated that fSAM and bSAM were both predominantly localized to the nucleus and nuclear localization signal ((11)KRPR(14))-deleted fSAM and bSAM relocalized to the cytoplasm. Both cytoplasmic fSAM and bSAM retained the antiviral function against different lentiviruses and cytoplasmic fSAM could restrict Vpx-encoding SIV and HIV-2 more efficiently than its wild-type (WT) protein as cytoplasmic hSAM. Further investigation revealed that cytoplasmic fSAM was resistant to Vpx-induced degradation like cytoplasmic hSAM, while cytoplasmic bSAM was not, but they all demonstrated the same in vitro dNTPase activity and all could interact with Vpx as their WT proteins, indicating that cytoplasmic hSAM and fSAM can suppress more SIV and HIV-2 by being less sensitive to Vpx-mediated degradation. Our results suggested that fSAM- and bSAM-mediated lentiviral restriction does not require their nuclear localization and that fSAM shares more common features with hSAM. These findings may provide insights for the establishment of alternative animal models to study SAMHD1 in vivo. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-021-00351-5) contains supplementary material, which is available to authorized users.

Castration‐resistant progression of prostate cancer is a major cause of prostate cancer mortality, and increased expression and activity of the full‐length and the splice variants of androgen receptor (AR) have been indicated to drive castration resistance. Consequently, there is an urgent need to develop agents that can target both the full‐length and the splice variants of AR for more effective treatment of prostate cancer. In the present study, we showed that raddeanin A (RA), an oleanane‐type triterpenoid saponin, suppresses the transcriptional activities of both the full‐length and the splice variants of AR. This is attributable to their decreased expression as a result of RA induction of proteasome‐mediated degradation and inhibition of the transcription of the AR gene. We further showed the potential of using RA to enhance the growth inhibitory efficacy of docetaxel, the first‐line chemotherapy for prostate cancer. This study identifies RA as a new agent to target both the full‐length and the splice variants of AR and provides a rationale for further developing RA for prostate cancer treatment.

Anti-viral therapy has made remarkable strides in improving the quality of life of infected individuals over the past few decades. However, the emergence of adverse side effects and drug resistance highlights the need for alternate drugs with either different structures or distinct mechanisms of action. The virus capsid is an attractive therapeutic target owing to its high sequence conservation and essential role in the virus life cycle. The capsid protein can be affected by small molecules at different positions in several combinations, both in early and late steps in the cycles of virus infection and replication. Therefore, a variety of small molecules that bind to the capsid proteins have been developed, and some of them have shown promise for pre-clinical therapeutic development. In this review, we focus on the recent advances on small molecules targeting the capsid protein of several clinically related spherical viruses such as the hepatitis B virus (HBV), human immunodeficiency virus (HIV) and human papillomavirus (HPV), together with filoviruses such as Ebola in several distinct classes of (i) synthetic organics, (ii) natural products and their derivatives, and (iii) peptides. Hopefully, some of these preclinical capsid inhibitors will be approved for therapy within the next few years.

Snail2 is a repressor of E-cadherin during carcinogenesis; however, the specific mechanisms involved in this process remain largely unknown. Here, we determined that Snail2 was highly increased during TGF-β-induced EMT process in lung cells. H3K9 methylation was up-regulated and H3K4/H3K56 acetylation were down-regulated at the E-cadherin promoter. Snail2 interacted with G9a and HDACs to exert suppression of E-cadherin transcription. Overexpression of Snail2 enhanced the migration and invasion ability, whereas G9a and HDACs inhibition significantly reversed this effect. Our study demonstrated the importance of G9a- and HDACs-mediated regulation during Snail2-induced E-cadherin repression and metastasis during LC progression.

The immune status of the tumor microenvironment is a key indicator determining the antitumor effect of immunotherapy. Oncolytic viruses directly target tumor cells or indirectly modulate the tumor microenvironment (TME) especially when properly armed. It was previously demonstrated that conditionally replicating adenovirus serotype 5 (CRAd5) encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had outstanding antitumor effects in different human cancer cells xenograft models; however, its antitumor immune mechanism has not been evaluated in immunocompetent preclinical mouse models. We first explored the antitumor activity of CRAd5-TRAIL in several murine tumor models and found that the expression of TRAIL induced increases or activation in tumor-infiltrating T cells. To further improve the antitumor effects, mouse CD40 ligand (mCD40L) as an immune activator expressed by recombinant Ad5 vector was firstly used in combination with CRAd5-TRAIL for tumor immunotherapy. Both in vitro and in vivo studies demonstrated that mCD40L effectively activated dendritic cells (DCs), B cells, and tumor-infiltrating T cells, and also promoted tumor cell apoptosis by increasing the expression of TRAIL receptors, thereby significantly enhancing the antitumor activity of oncolytic adenoviruses in CT26 and B16 tumor-bearing models. Although affected by the restriction of oncolytic adenovirus replication in mouse cells, the combination treatment failed to completely eliminate tumor cells, our research still provided a promising strategy for oncolytic adenovirus-mediated solid tumor immunotherapy.

Background: Mice co-expressing human amyloid precursor protein with the Swedish mutation (APPswe) and exon-9-deleted presenilin (PS1dE9) has become one of the most widely used mouse models for studying Alzheimer’s disease (AD) pathogenesis and preclinical studies of AD therapeutic approaches. Objective: In this study, we systematically investigated cognitive decline, amyloid-β (Aβ) deposition and cerebral or Aβ serum levels as well as the relationships among these measures in APPswe/PS1dE9 transgenic mice. Method: APPswe/PS1dE9 mice were separated into four equal age cohorts (4, 6, 9, and 12 months). We assessed cognitive capacity, deposited plaques, and the levels of Aβ40/Aβ42 in brain tissue and serum of mice at different ages. Results: APPswe/PS1dE9 mice exhibited declined memory beginning at 6 months of age, with cognitive capacity remarkably impaired at 12-months. Coincidently, amyloid deposits began to develop in transgenic mice brain at 6-months and increased with age. In addition, Aβ42 levels in brains of APPswe/ PS1dE9 mice increased with age with no parallel increase in Aβ40. The concentration of serum Aβ42 declined from 4 to 6 months of age, but a similar age-dependent decrease was not observed for Aβ40. Conclusion: APPswe/PS1dE9 transgenic mice began to develop amyloid plaques at 6 months of age and exhibited a corresponding impairment of spatial learning capacity. Serum Aβ42 level decreased remarkably from 4 to 6 months, at which stage Aβ42 began to accumulate in the brain and deposit as plaques.

A smart supramolecular nanosystem integrating targeting, chemotherapy, and photothermal therapy was constructed based on carboxylatopillar[5]arene (CP[5]A)-functionalized CuS nanoparticles (CuS@CP NPs). CuS@CP NPs with good monodispersibility and strong near-infrared absorption were synthesized in aqueous solution through a facile one-pot supramolecular capping method, followed by surface installation of a liver cancer-targeted galactose derivative through host-guest binding interaction. The resulting smart supramolecular nanosystem, namely, CuS@CPG, exhibited excellent photothermal ablation capability to HepG2 cells upon irradiation with laser at 808 nm. Chemotherapeutic drug, doxorubicin hydrochloride (DOX), was further loaded on CuS@CPG via electrostatic interactions between positively charged DOX and negatively charged CP[5]A to give CuS@CPG-DOX with a high drug-loading capacity up to 48.4%. The weakening of DOX-CP[5]A interactions in an acidic environment promoted the pH-responsive drug release from CuS@CPG-DOX. Significantly, this multifunctional supramolecular nanosystem showed a remarkably enhanced therapeutic effect through the combination of targeted chemotherapy and photothermal therapy upon in vitro cell study. Moreover, preliminary in vivo study demonstrated that CuS@CPG and CuS@CPG-DOX had good biocompatibility and excellent tumor inhibition effects upon near-infrared laser irradiation.

The oxidation behavior at 1073 K of in-situ TiC/Ni composites prepared by reactive sintering obey parabolic kinetics. The oxidation activation energy is 210 ± 30 kJ•mol−1, closed to that of O diffusion through rutile. Oxide scale formed on composites includes two layers: continuous outer layer consisted by upper TiO2 +NiTiO3 +NiO and nether TiO2, and inner layer composed of TiO2 islands dispersed in Ni matrix. The oxidation process is determined by outward diffusion of Ti and Ni and inward diffusion of O, and the later one plays a predominant role, leading to the faster growth rate of inner layer than outer layer.

Nonhuman primates (NHPs) are the only animal model that can be used to evaluate protection efficacy of HIV-1 envelope vaccines. However, whether broadly neutralizing antibodies (bnAbs) can be elicited in NHPs infected with simian/human immunodeficiency virus (SHIV) has not been fully understood. The objective of this study is to investigate whether broad neutralization activities were developed in SHIV-infected macaques after long-term infection as in humans.Neutralization breadth and specificities in plasmas from SHIV-infected macaques were determined by analyzing a panel of tier 2 viruses and their mutants.Forty-four Chinese macaques infected with SHIV1157ipd3N4, SHIVSF162P3 or SHIVCHN19P4 were followed for 54-321 weeks. Archived plasmas from 19 macaques were used to determine neutralization breadth and specificities against 17 tier 2 envelope-pseudoviruses.Longitudinal plasma from three SHIVSF162P3-infected macaques and three SHIV1157ipd3N4-infected macaques rarely neutralized viruses (25%) within 1 year of infection. The neutralization breadth in two SHIV1157ipd3N4-infected macaques significantly increased (≥65%) by year 6. Four of six SHIV1157ipd3N4-infected macaques could neutralize 50-75% viruses, whereas none of macaques infected with SHIVSF162P3 or SHIVCHN19P4 could neutralize more than 25% of viruses after 6 years of infection (P = 0.035). Neutralization specificity analysis showed mutations resistant to bnAbs in V2, V3 or CD4bs regions could abrogate neutralization by year-6 plasma from three SHIV1157ipd3N4-infected macaques.These results demonstrate that bnAbs targeting common HIV-1 epitopes can be elicited in SHIV1157ipd3N4-infected macaques as in humans after 4-6 years of infection, and SHIV/NHP can serve as an ideal model to study bnAb maturation.

In an effort to improve both the stability of virus-like particles (VLPs) and the medical activity of polyoxometalates (POMs), a new hybrid assembly system between human papillomavirus (HPV) capsid protein L1 and a europium-containing POM (EuW10) has been constructed, for the first time, via the electrostatic interactions between them. The co-assembly of EuW10 and HPV 16 L1-pentamer (L1-p) in buffer solution resulted in the encapsulation of POMs in the cavity of VLPs, which was further confirmed by cesium chloride (CsCl) gradient ultracentrifugation, SDS-PAGE, dynamic light scattering, and transmission electron microscopy, whereas the post-assembly of EuW10 with the as-prepared VLPs leads to the adsorption of POMs only on the external surface of particles, and both cases improved the thermal and storage stabilities of VLPs obviously. Particularly, the encapsulation of POMs in VLPs largely improved the antibacterial activity of EuW10, and thereby, the present study will be significant for both the stabilit...

BCA2/RNF115/Rabring7 is a RING type E3 ubiquitin ligase that is overexpressed in human breast tumors and is important for regulating breast cancer cell migration. In the present investigation, feline BCA2 (fBCA2) was identified and characterized. Compared with its human counterpart, the fBCA2 cDNA was confirmed to be 918 base pairs in length showing 92.6% consensus and identity positions, encoding a protein of 305 amino acids with 96.7% consensus and 93.1% identity positions. The fBCA2 protein contains a RING domain at the C-terminus, which was found to be essential for its autoubiquitination.

The TiC/Ni composites with 0 wt%, 55 wt% and 70 wt% Ni contents were fabricated by pressureless reactive sintering to study the effect of Ni on the formation mechanism of composites. The results show that the solid reaction between Ti and graphite is controlled by the diffusion of C through TiC layer, and the morphology of TiC layer is similar to the graphite sheet in sample without Ni. By contrast, graphite would dissolve into Ni in samples with Ni, and Ni particles would form continuous skeleton around Ti particles by solid phase sintering. Meanwhile, Ni diffuses into Ti particles to form Ni Ti compound. Therefore, graphite and C in Ni skeleton reacts with Ti to form TiC layer on whole surface of Ni Ti particles, resulted in the similar morphology of TiC layers to original Ti particles. Moreover, Ni plays one key role in the liquid phase sintering of TiC/Ni composites by forming Ni Ti C eutectic phase. The relative density of TiC/Ni composites are higher than 96%, while the samples without Ni shows no obvious shrinkage. The increase of Ni also contributes to the decrease of sintering temperature by enhancing the solution-precipitation process.

Background Tau hyper-phosphorylation has been considered a major contributor to neurodegeneration in Alzheimer’s disease (AD) and related tauopathies, and has gained prominence in therapeutic development for AD. To elucidate the pathogenic mechanisms underlying AD and evaluate therapeutic approaches targeting tau, numerous transgenic mouse models that recapitulate critical AD-like pathology have been developed. Tau P301S transgenic mice is one of the most widely used mouse models in AD research. Extensive studies have demonstrated that sex significantly influences AD pathology, behavioral status, and therapeutic outcomes, suggesting that studies using mouse models of AD must consider sex- and age-related differences in neuropathology, behavior, and plasma content. Method We systematically investigated differences in tau P301S transgenic mice (PS19 line) and wildtype littermates of different sex behavioral performance, tau neuropathology, and biomarkers in plasma and brain. Results Male P301S transgenic mice exhibited significant changes in weight loss, survival rate, clasping, kyphosis, composite phenotype assessment, nest building performance, tau phosphorylation at Ser202/Thr205, and astrocyte activation compared to that of wild-type littermates. In contrast, female P301S transgenic mice were only sensitive in the Morris water maze and open field test. In addition, we characterized the absence of macrophage-inflammatory protein (MIP-3α) and the upregulation of interferon (IFN)-γ, interleukin (IL)-5, and IL-6 in the plasma of P301S transgenic mice, which can be served as potential plasma biomarkers in P301S Tg mice. Male P301S transgenic mice expressed more monokine induced by IFN-γ (MIG), tumor necrosis factor-α (TNF-α), IL-10, and IL-13 than those of female P301S mice. Conclusion Our findings highlight sexual dimorphism in the behavior, neuropathology, and plasma proteins in tau P301S transgenic AD mice, indicating that the use of male P301S transgenic mice may be more suitable for assessing anti-phosphorylated tau therapeutic strategies for AD and related tauopathies, and the MIP-3α may be a new potential plasma biomarker.

Additional file 3. The original western blot figures of Fig. 4.

Therapeutic cancer vaccines aim to induce an effective immune response against cancer, and the effectiveness of these vaccines is influenced by the choice of immunogen, vaccine type, and immunization strategy. Although treatment with cancer vaccines can improve tumor burden and survival, in most animal studies, it is challenging to achieve a complete response against tumor growth and recurrence, without the use of other therapies in combination. Here, we present a novel approach where dual antigens (survivin and MUC1) are co-targeted using three DNA vaccines, followed by a single booster of a recombinant modified vaccinia Ankara (MVA) vaccine. This heterologous vaccination strategy induced higher levels of interferon (IFN)-γ-secretion and stronger antigen-specific T-cell responses than those induced individually by the DNA vaccines and the MVA vaccine in mice. This strategy also increased the number of active tumor-infiltrating T cells that efficiently inhibit tumor growth in tumor-bearing mice. Heterologous DNA prime-MVA boost immunization was capable of inducing a robust antigen-specific immune-memory, as seen from the resistance to subsequent survivin- and MUC1-expressing tumors. Moreover, the therapeutic effects of DNA prime-MVA boost and DNA prime-adenovirus boost strategies were compared. DNA prime-MVA boost immunization performed better, as indicated by the T effector ratio and the induction of Th1 immunity. This study provides the basis for the use of heterologous DNA prime-MVA boost vaccination regime targeting two antigens simultaneously as a promising immunotherapeutic strategy against cancer.

Tau hyper-phosphorylation has been considered a major contributor to neurodegeneration in Alzheimer’s disease (AD) and related tauopathies, and has gained prominence in therapeutic development for AD. To elucidate the pathogenic mechanisms underlying AD and evaluate therapeutic approaches targeting tau, numerous transgenic mouse models that recapitulate critical AD-like pathology have been developed. Tau P301S transgenic mice is one of the most widely used mouse models in AD research. Extensive studies have demonstrated that sex significantly influences AD pathology, behavioral status and therapeutic outcomes, suggesting that studies using mouse models of AD must consider sex- and age-related differences in neuropathology, behavior, and plasma content. Method: We systematically investigated differences in tau P301S transgenic mice (PS19 line) and wildtype littermates of different sex behavioral performance, tau neuropathology and biomarkers in plasma and brain. Results: Male P301S transgenic mice exhibited significant changes in weight loss, survival rate, clasping, kyphosis, composite phenotype assessment, nest building performance, tau phosphorylation at Ser202/Thr205 and astrocyte activation compared to that of wild type littermates. In contrast, female P301S transgenic mice were only sensitive in the Morris Water Maze and open field test. In addition, we characterized the upregulation of interferon (IFN)-γ, interleukin (IL)-5, and IL-6 and the absense of macrophage-inflammatory protein (MIP)-3α. Male P301S transgenic mice expressed more plasma biomarkers than those of female P301S mice. Conclusion: Our findings highlight sexual dimorphism in the behavior, neuropathology, and biomarkers in tau P301S transgenic AD mice, indicating that the use of male P301S transgenic mice may be more suitable for assessing anti-phosphorylated tau therapeutic strategies for AD and related tauopathies.

Summary Objective Viral fitness plays an important role in HIV-1 evolution, transmission and pathogenesis. However, how mutations accumulated during early infection affect viral fitness has not been well studied. Methods Paired infectious molecular clones (IMCs) for transmitted/founder (T/F) and 6-month (6-mo) viruses post infection were generated from 10 infected individuals to investigate the impact of accumulated mutations on viral fitness by comparing 6-mo viruses to their cognate T/F viruses. Results All ten 6-mo viruses were less fit than their cognate T/F viruses. Moreover, the fitness losses of the 6-mo viruses correlated with the decrease in viral loads from the peak of viremia. Conclusion These results show that the mutations accumulated during half a year post infection collectively reduce viral fitness and thereby contribute to lowering viral loads.

Functional E‐cadherin loss, a hallmark of epithelial‐mesenchymal transition (EMT), is important for metastasis. However, the mechanism of Snail2 in hepatocellular carcinoma (HCC) EMT and metastasis remains unclear. Here, we showed that Snail2 was upregulated in primary HCC, and significantly increased during transforming growth factor‐β‐induced liver cell EMT. Snail2‐overexpressing and knockdown cell lines have been established to determine its function in EMT in HCC. H3K9 methylation was upregulated and H3K4 and H3K56 acetylation were downregulated at the E‐cadherin promoter in Snail2‐overexpressing cancer cells. Furthermore, Snail2 interacted with G9a and histone deacetylases (HDACs) to form a complex to suppress E‐cadherin transcription. Snail2 overexpression enhanced migration and invasion in HCC cells, whereas G9a and HDAC inhibition significantly reversed this effect. Moreover, Snail2 overexpression in cancer cells increased tumor metastasis and shortened survival time in mice, whereas G9a and HDAC inhibitors extended survival. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT but also suggests novel treatment strategies for HCC.

The abnormal deposition of amyloid-β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

The Norovirus (NoV) P domain, with three surface loops for foreign antigen insertion, has been demonstrated as an excellent platform for antigen presentation and novel vaccine development. The P domain alone can self-assemble into a P dimer, 12-mer small particle or 24-mer P particle, and vaccines based on those particles may elicit different levels of immunogenicity. Currently, P particles are generally produced in soluble expression systems in Escherichia coli, mainly in the 24-mer form. However, the low yield of the soluble protein has hindered further clinical applications of P particle-based protein vaccines. In this study, we inserted the Alzheimer's disease (AD) immunogen Aβ1-6 into the three loops of the P particle to generate an AD protein vaccine. To increase the yield of this chimeric protein, we tested the generation of proteins in a soluble expression system and an inclusion body expression system separately in E. coli. The result showed that the inclusion body expression system could greatly enhance the product yield of the chimeric protein compared with the soluble expression system. The refolded protein from the inclusion bodies was mainly in the 12-mer form, while the protein generated from the soluble supernatant was mainly in the 24-mer form. Moreover, the immunogenicity of soluble proteins was significantly stronger than that of the refolded proteins. Thus, comparisons between the two expression methods suggested that the soluble expression system generated chimeric P particles with better immunogenicity, while inclusion body expression system yielded more P particle proteins.

Based on the helix4-exchanged HPV16 L1 and HPV18 L1, HPV16 L1 Bi and HPV18 L1 Bi, we have successfully realized the controlled hybrid-assembly of HPV16/18 L1 Bi VLPs (bihybrid-VLPs) in vitro. The bihybrid-VLPs were further confirmed by fluorescence resonance energy transfer (FRET) and complex-immunoprecipitation (Co-IP) assays. The ratio of 16 L1 Bi and 18 L1 Bi in bihybrid-VLPs was verified to be 3:5 based on a modified magnetic Co-IP procedure, when mixing 1 equiv pentamer in assembly buffer solution, but it changed with conditions. In addition, the bihybrid-VLPs showed identical thermal stability as that of normal VLPs, suggesting high potential in practical applications. The present study is significant because it modified one of the vital steps of virus life cycle at the stage of virus assembly, supplying a new approach not only to deepen structural insights but also a possibility to prepare stable, low-cost, bivalent antivirus vaccine. Furthermore, the controlled hybrid-assembly of bihybrid-VLPs in vitro provides suggestions for the design of effective multivalent hybrid-VLPs, being a potential to develop broad-spectrum vaccines for the prevention of infection with multiple types of HPV.