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Aims This study aimed to provide a safe, stable and efficient SARS-CoV-2 oral vaccine development strategy based on the type III secretion system of attenuated Salmonella and a reference for the development of a SARS-CoV-2 vaccine. Methods and Results The attenuated Salmonella mutant ΔhtrA-VNP was used as a vector to secrete the antigen SARS-CoV-2 based on the type III secretion system (T3SS). The Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS promoter (sifB) was screened to express heterologous antigens (RBD, NTD, S2), and the SPI-2-encoded secretion system (sseJ) was employed to secrete this molecule (psifB-sseJ-antigen, abbreviated BJ-antigen). Both immunoblotting and fluorescence microscopy revealed effective expression and secretion of the antigen into the cytosol of macrophages in vitro. The mixture of the three strains (BJ-RBD/NTD/S2, named AisVax) elicited a marked increase in the induction of IgA or IgG S-protein Abs after oral gavage, intraperitoneal and subcutaneous administration. Flow cytometric analysis proved that AisVax caused T-cell activation, as shown by a significant increase in CD44 and CD69 expression. Significant production of IgA or IgG N-protein Abs was also detected by using psifB-sseJ-N(FL), indicating the universality of this strategy. Conclusions Delivery of multiple SARS-CoV-2 antigens using the type III secretion system of attenuated Salmonella ΔhtrA-VNP is a potential COVID-19 vaccine strategy. Significance and Impact of the Study The attenuated Salmonella strain ΔhtrA-VNP showed excellent performance as a vaccine vector. The Salmonella SPI-2-encoded T3SS showed highly efficient delivery of SARS-COV-2 antigens. Anti-loss elements integrated into the plasmid stabilized the phenotype of the vaccine strain. Mixed administration of antigen-expressing strains improved antibody induction.

Glioma is the most common of all central nervous system (CNS) malignancies and is associated with a poor prognosis. Pyroptosis has been proven to be associated with the progression of multiple tumors and CNS diseases. However, the relationships between pyroptosis and clinical prognosis and immune cell infiltration are unclear in glioma. In this study, we conducted a comprehensive exploration of pyroptosis in glioma. First, prognosis-related genes were screened at each key regulatory locus in the pyroptosis pathway, and the prognostic ability and coexpression relationships of GSDMD and its upstream pathway genes NLRC4/CASP1/CASP4 were identified and well validated in multiple datasets. Tissue microarray-based immunohistochemistry results showed higher levels of NLRC4 and N-terminal GSDMD in high-grade gliomas, providing conclusive evidence of pyroptosis in gliomas. The robustness of the prognostic model based on these four genes was well validated in TCGA and CGGA cohorts. Bulk RNA-seq-based analysis showed that the group defined as the high-risk group according to the model showed activation of multiple inflammatory response pathways and impaired synaptic gene expression and had a higher infiltration of bone marrow-derived macrophages (BMDMs) and a hypersuppressed immune microenvironment. More importantly, three independent single-cell RNA-seq (scRNA-seq) datasets demonstrated that tumor-infiltrating macrophages, particularly BMDMs but not tissue-resident microglia, showed significant coexpression of the GSDMD and CASP genes, and BMDMs from high-grade gliomas accounted for a higher proportion of immune infiltrating cells and had higher expression of pyroptosis genes. Finally, we revealed the activation of pathways in response to LPS/bacteria and oxidative stress during BMDM development toward the pyroptosis cell fate by pseudotime trajectory analysis, suggesting potential BMDM pyroptosis initiators. The above results provide not only novel insights into the pathological mechanisms of glioma but also novel therapeutic targets for glioma, suggesting the potential application of pyroptosis inhibitors (e.g., disulfiram).

This study aimed to prepare edible films with cassava starch (CS) and sodium carboxymethyl cellulose (CMC) as basic raw materials and apple polyphenol (AP) as biologically active components. The addition of AP makes the flexibility of the film increase first and then decrease while the tensile strength decreases slightly and the barrier ability increases significantly. When the AP concentration was 70 mg/mL, the tensile strength of the film decreased from 5.61 ± 0.45 to 3.36 ± 0.19, the water vapor transmittance decreased from 7.17 ± 0.17 to 4.97 ± 0.07 and the peroxide value decreased from 1.896 ± 0.04 to 0.53 ± 0.04. By studying the microstructure of CS/CMC/AP-4 film, it was found that hydrogen bonds were formed between AP, CS, and CMC, and they showed high compatibility, thus improving the crystallinity. On this basis, the roughness of the film decreases and the compactness is enhanced. The increase in compactness is directly related to the increase in blocking ability. At the same time, the improvement of thermal stability is attributed to the increase in crystallinity. In addition, the application of CS/CMC/AP-4 film in chicken preservation also proved its antioxidant potential.

Aims Decomposition, a complicated process, depends on several factors, including carrion insects, bacteria and the environment. However, the composition of and variation in oral bacteria over long periods of decomposition remain unclear. The current study aims to illustrate the composition of oral bacteria and construct an informative model for estimating the post-mortem interval (PMI) during decomposition. Methods and Results Samples were collected from rats' oral cavities for 59 days, and 12 time points in the PMI were selected to detect bacterial community structure by sequencing the V3–V4 region of the bacterial 16S ribosomal RNA (16S rRNA) gene on the Ion S5 XL platform. The results indicated that microorganisms in the oral cavity underwent great changes during decomposition, with a tendency for variation to first decrease and then increase at day 24. Additionally, to predict the PMI, an informative model was established using the random forest algorithm. Three genera of bacteria (Atopostipes, Facklamia and Cerasibacillus) were linearly correlated at all 12 time points in the 59-day period. Planococcaceae was selected as the best feature for the last 6 time points. The R2 of the model reached 93.94%, which suggested high predictive accuracy. Furthermore, to predict the functions of the oral microbiota, PICRUSt results showed that energy metabolism was increased on day 3 post-mortem and carbohydrate metabolism surged significantly on days 3 and 24 post-mortem. Conclusions Overall, our results suggested that post-mortem oral microbial community data can serve as a forensic resource to estimate the PMI over long time periods. Significance and Impact of the Study The results of the present study are beneficial for estimating the PMI. Identifying changes in the bacterial community is of great significance for further understanding the applicability of oral flora in forensic medicine.

Tumor growth is an angiogenesis-dependent process and accompanied by the formation of hypoxic areas. Tumstatin is a tumor-specific angiogenesis inhibitor that suppresses the proliferation and induces the apoptosis of tumorous vascular endothelial cells. VNP20009, an attenuated Salmonella typhimurium strain, preferentially accumulates in the hypoxic areas of solid tumors. In this study, a novel Salmonella-mediated targeted expression system of tumstatin (VNP-Tum5) was developed under the control of the hypoxia-induced J23100 promoter to obtain anti-tumor efficacy in mice. Treatment with VNP-Tum5 effectively suppressed tumor growth and prolonged survival in the mouse model of B16F10 melanoma. VNP-Tum5 exhibited a higher efficacy in inhibiting the proliferation and inducing the necrosis and apoptosis of B16F10 cells in vitro and in vivo compared with VNP (control). VNP-Tum5 significantly inhibited the proliferation and migration of mouse umbilical vascular endothelial cells to impede angiogenesis. VNP-Tum5 downregulated the expression of anti-vascular endothelial growth factor A, platelet endothelial cell adhesion molecule-1, phosphorylated phosphoinositide 3 kinase, and phosphorylated protein kinase B and upregulated the expression of cleaved-caspase 3 in tumor tissues. This study is the first to use tumstatin-transformed VNP20009 as a tumor-targeted system for treatment of melanoma by combining anti-tumor and anti-angiogenic effects.

The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding.

Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.

Complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions. Activation of the terminal pathway of the complement system leads to the assembly of terminal complement complexes (C5b-9), which induces the characteristic complement-mediated cytolysis. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) is identified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum by RACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42% and 43% identity with human and Xenopus C9 respectively. Through sequence alignment and comparative analysis, the CpC9 protein was found well conserved, with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogenetic analysis places it in a clade with C9 orthologs in higher vertebrate and as a sister taxa to the Xenopus. Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much less or even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response, though it remains to be confirmed by further study.

Cocaine and amphetamine-regulated transcript (CART) is a neuropeptide that protects brains against ischemic injury in vivo and in vitro. By using small interference RNA against CART(CARTi), this study shows that CART knockdown by CARTi downregulated exogenous and endogenous CART mRNA and protein expression in vivo and in vitro. Consequently, CART knockdown exacerbated neuronal cell death induced by oxygen and glucose deprivation (OGD). It also showed that CART knockdown increased infarct size in a mouse middle cerebral artery occlusion model. CART's protective effects are most likely mediated through the ERK 1/2 pathway, since ERK 1/2 phosphorylation, not that of p38 or JNK is activated in CART-treated neurons after OGD. Furthermore, neuroprotection of CART is abolished by CART knockdown and by pretreatment with ERK antagonist PD98059 and U0126, but not with p38 or JNK antagonists SB203580 or SP600125. These results provide further evidence that CART is an endogenous neuroprotective peptide against cerebral ischemia and it does so through the MAPK/ERK signaling pathway. Therefore, CART may be developed into a therapeutic agent for stroke-related brain injury.

The presence of amyloid plaques and vascular amyloid deposits is one of the pathological features of Alzheimer's disease (AD). Amyloid plaques and vascular deposits mainly consist of amyloid-β (Aβ), which is a metabolic product of amyloid-β protein precursor cleaved by β- and γ-secretase. Soluble Aβ monomers readily aggregate into oligomers preceding the formation of insoluble fibrils, and Aβ oligomers are more toxic than fibrils. Intensive therapeutic efforts have been attempted in the treatment of AD targeting Aβ, including preventing Aβ generation, inhibiting Aβ aggregation, and promoting Aβ clearance. The results show that amyloid plaque burden is reduced together with improved cognition performance in AD. Gelsolin, a multifunctional actin-binding protein, exists intracellularly as a cytoplasmic form and extracellularly as a secreted form in blood/cerebrospinal fluid. Gelsolin is suggested to be implicated in AD, based on the findings that some changes of gelsolin are correlated with disease progression rate in AD patients. Gelsolin binds Aβ, inhibits its aggregation into fibrils, and protects cells from apoptosis induced by Aβ. More importantly, administration or overexpression of gelsolin results in significant reduction of amyloid load and decrease of Aβ level in AD transgenic mice. In this article, we review the most recent progress of gelsolin as a potential therapeutic strategy for treatment of AD, and discuss the possible mechanism involved in the clearance of amyloid plaques in AD.

Background gp340, a member of scavenger receptor cysteine rich family encoded by Deleted in Malignant Brain Tumors 1 (DMBT1), is an important component in innate immune defense. The first scavenger receptor cysteine rich domain (SRCR1) of gp340 has been shown to inhibit HIV-1 infection through binding to the N-terminal flank of the V3 loop of HIV-1 gp120. Results Through homology modeling and docking analysis of SRCR1 to a gp120-CD4-X5 antibody complex, we identified three loop regions containing polar or acidic residues that directly interacted with gp120. To confirm the docking prediction, a series of over-lapping peptides covering the SRCR1 sequence were synthesized and analyzed by gp120-peptide binding assay. Five peptides coincide with three loop regions showed the relative high binding index. An alanine substitution scan revealed that Asp34, Asp35, Asn96 and Glu101 in two peptides with the highest binding index are the critical residues in SRCR1 interaction with gp120. Conclusion We pinpointed the vital gp120-binding regions in SRCR1 and narrowed down the amino acids which play critical roles in contacting with gp120.

An energy-saving ethanol fermentation technology was developed using uncooked fresh sweet potato as raw material. A mutant strain of Aspergillus niger isolated from mildewed sweet potato was used to produce abundant raw starch saccharification enzymes for treating uncooked sweet potato storage roots. The viscosity of the fermentation paste of uncooked sweet potato roots was lower than that of the cooked roots. The ethanol fermentation was carried out by Zymomonas mobilis, and 14.4 g of ethanol (87.2% of the theoretical yield) was produced from 100g of fresh sweet potato storage roots. Based on this method, an energy-saving, high efficient and environment-friendly technology can be developed for large-scale production of fuel ethanol from sweet potato roots.