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ObjectiveTo study the phenomenon of pulmonary hypostasis in corpses of various causes of death， and to explore the potential value of this phenomenon in assisting forensic pathological diagnosis of drowning.MethodsA total of 235 cases with clear cause of death through systematic autopsy were collected from January 2011 to June 2021 in Guangzhou. According to the location of body discovery, the cases were divided into the water body group （97 cases） and the non-water body group （138 cases）, and the water body group was further divided into the water drowning group （90 cases） and the water non-drowning group （7 cases）. Non-water body group was further divided into the non-water drowning group （1 case） and the non-water non-drowning group （137 cases）. Three senior forensic pathologists independently reviewed autopsy photos to determine whether there was hypostasis in the lungs. The detection rate of pulmonary hypostasis was calculated.ResultsThe detection rate of pulmonary hypostasis in the water drowning group （90 cases） was 0， and the negative rate was 100%. The detection rate of pulmonary hypostasis in the water non-drowning group （7 cases） was 100% and the negative rate was 0. The detection rate of pulmonary hypostasis in the water body group and in the non-water body group （after excluding 2 cases, 136 cases were calculated） was 7.22% and 87.50%， respectively. There were statistically significant differences in the detection rate of pulmonary hypostasis between water body group and non-water body group, and between water drowning group and water non-drowning group （P

MicroRNAs (miRNA) are believed to play a crucial role in the cause and treatment of temporal lobe epilepsy (TLE) by controlling gene expression in different stages of the disease. To investigate role of miRNA in the latent stage following status epilepticus, we first compared microRNA expression profiles in mice hippocampus at 1 week after pilocarpine-induced status epilepticus (SE) vs. controls in hippocampal tissues using Exiqon miRCURY LNA™ miRNAs Array. Then, the target genes of altered miRNAs were predicted using both TargetScan 7.1 and miRDB V5, and were further selected by intersecting with another independent mRNA expression profile dataset from the samples at the same time point. We found out 14 common genes as down miRNA target (up-mRNA) and 4 common genes as up miRNA target (down mRNA) in SE mice. miR-669m-3p-TRHR (thyrotropin releasing hormone receptor), miR-669m-3p-B3galt2 (β-1,3-Galactosyltransferase 2), miR-105-PDPN (Podoplanin) and miR-883b-3p-CLEC-2 (C-type-lectin-like-2) were found to be potential molecular mechanisms to modulate the calcium signaling pathway, glycosylation pathways and chemokine mediated inflammatory processes in mice hippocampus at 1 week after pilocarpine-induced SE, respectively. Our results offered potential novel insights into the cellular events in the mice hippocampus mediated by miRNASs-target genes that shape SE-evoked epileptogenesis. • At 1 w after pilocarpine-induced SE, 1 up-regulated miRNA and 8 down-regulated miRNAs were identified in the mice hippocampus. • 4 genes were selected as up miRNA target and 14 genes were selected as down miRNA target respectively. • miR-669m-3p-TRHR, miR-669m-3p-B3galt2 and miR-105-PDPN may be involved in SE-evoked epileptogenesis [ABSTRACT FROM AUTHOR]

Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). Method The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (A 0X1) promoter and expressed intracellularly. The expression products in the soluble ceil extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEY specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg. Conclusion The chimeric HEY/HEY VLPs produced in this study may have potential to be a recombinant HEY/HEY bivalent vaccine candidate. [ABSTRACT FROM AUTHOR]

The autophagy adaptor protein SQSTM1/p62 is overexpressed in breast cancer and has been identified as a metastasis-related protein. However, the mechanism by which SQSTM1/p62 contributes to breast cancer progression and tumor microenvironment remains unclear. This study revealed that silencing SQSTM1/p62 expression suppressed breast cancer progression via regulating cell proliferation and reshaping the tumor microenvironment (TME). Here, we found that SQSTM1/ p 62 was overexpressed in multiple human cancer tissue types and that was correlated with poor patient overall survival (OS) and disease-free survival (DFS). Moreover, we found that short-hairpin RNA (shRNA)-mediated knockdown of p62 expression significantly inhibited cell proliferation, migration, and invasion, and promoted cell death in vitro , as well as suppressed breast cancer growth and lung metastasis in vivo . In addition, flow cytometry analysis of splenocytes and tumor infiltrating lymphocytes (TILs) indicated that the numbers of CD8α + interferon (IFN)-γ + cells (CTLs) and CD4 + IFN-γ + (Th1) cells were increased while those of CD4 + IL-4 + (Th2) cells, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) were decreased. RT-PCR analyses showed that the gene expression of Th1/Th2 cytokines changed in the tumor microenvironment. Silencing SQSTM1 / p 62 suppressed tumor cell lung metastasis. Together, our results provide strong evidence that silencing tumor cell SQSTM1 / p 62 inhibited tumor growth and metastasis through cell cycle arrest and TME regulation. This finding provides a novel molecular therapeutic strategy for breast cancer progression and metastasis treatment., (© 2021 Chongqing Medical University. Production and hosting by Elsevier B.V.)

Multidrug resistant (MDR) pathogen infections are serious threats to hospitalized patients because of the limited therapeutic options. A novel group of antibiotic candidates, antimicrobial peptides (AMPs), have recently shown powerful activities against both Gram-negative and Gram-positive bacteria. Unfortunately, the viability of using these AMPs in clinical settings remains to be seen, since most still need to be evaluated prior to clinical trials and not all of AMPs are potent against MDR clinical isolates. To find a connection between the characteristics of several of these AMPs and their effects against MDR pathogens, we selected 14 AMPs of animal origin with typical structures and evaluated their in vitro activities against clinical strains of extensive drug-resistant Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, extended spectrum β-lactamase-producing Pseudomonas aeruginosa and extended spectrum β-lactamase-producing Escherichia coli. Our results showed that these peptides' hydrophilic/hydrophobic characteristics, rather than their secondary structures, may explain their antibacterial effects on these clinical isolates. Peptides that are amphipathic along the longitudinal direction seemed to be effective against Gram-negative pathogens, while peptides with hydrophilic terminals separated by a hydrophobic intermediate section appeared to be effective against both Gram-negative and Gram-positive pathogens. Among these, cathelicidin-BF was found to inhibit all of the Gram-negative pathogens tested at dosages of no more than 16 mg/L, killing a pandrug-resistant A. baumannii strain within 2 h at 4×MICs and 4 h at 2×MICs. Tachyplesin III was also found capable of inhibiting all Gram-negative and Gram-positive pathogens tested at no more than 16 mg/L, and similarly killed the same A. baumannii strain within 4 h at 4×MICs and 2×MICs. These results suggest that both cathelicidin-BF and tachyplesin III are likely viable targets for the development of AMPs for clinical uses.

Objective: To investigate the expression of fusion protein c-Abeta-c, a fusion protein from the beta amyloid peptide (Abeta) and the Hepatitis B core antigen/ Major immunodominant region (HbcAg/MIR), in the BL21/pET28 prokaryotic expression system and to immunize mice with the expressed fusion protein and evaluate the immunogenicity and biological effects of the serum in vitro., Methods: The recombinant prokaryotic expression plasmid PET-28a /c-Abeta-c was constructed by molecular cloning technique and the c-Abeta-c fusion gene expression was induced in E. coli BL21 by isopropyl-1-thio-b-Dgalactopyranoside (IPTG). The expressed fusion protein was analyzed by SDS-PAGE. Intraperitoneal injection (i.p.) of the purified c-Abeta-c fusion protein was given to the BALB/c mice. The anti-Abeta effect of the immune serum was detected by indirect ELISA. The biological effect of the immune serum on Alzheimer's disease (AD) transgeneic cells was assessed by MTT assay and flow cytometer., Results: The c-Abeta-c fusion protein was found in the sediment of the isolated bacteria. The expressed protein comprised more than 30% of the total proteins in the bacteria sediment. The anti-Abeta antibody in the serum of the immunized mice reached 1:16000. The antiserum reduced the cytotoxicity of Abeta peptide in the AD transgeneic cells and significantly decreased the apoptosis of cells., Conclusion: The c-Abeta-c fusion protein has good Abeta immunogenicity and the animal immune serum efficiently inhibits the cytotoxicity of Abeta peptide.

Objective: To achieve expression of human brain-derived neurotrophic factor (hBDNF) mediated by recombinant adeno-associated virus (rAAV) and explore the mechanism of its neuroprotective effects in rat neurons against beta-amyloid-induced Alzheimer's disease., Methods: Using molecular cloning technique, rAAV vector containing hBDNF gene (AAV-hBDNF) was constructed to transfect SD rat hippocampal neurons exposed to beta-amyloid treatment. The changes in cell apoptosis were observed by MTT assay and flow cytometry, and the expression of hBDNF and Bcl-2 protein were determined by immunocytochemical staining. Laser scanning confocal microscopy (LSCM) was used to observe the changes of [Ca(2+)](i)., Results: The cultured rat hippocampal neurons were effectively transfected with AAV-hBDNF and expression of BDNF protein was obviously increased. hBNDF expression showed significant protective effects against beta-amyloid-induced neuronal damage, and the expression of Bcl-2 protein was increased significantly and the balance of [Ca(2+)](i) was maintained in BDNF-treated cells with beta-amyloid exposure., Conclusion: hBDNF expression can effectively protect cultured rat hippocampal cells from beta-amyloid-induced apoptosis through inhibiting the intracellular calcium overload and increasing the expression of Bcl-2 protein.

Objective: To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation., Methods: The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10., Results: Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response., Conclusions: The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.

Objective: To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta)., Methods: Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR., Results: Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus., Conclusions: The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.