During metamorphic development from larva to adult, insect brains exhibit cell proliferation; however, the molecular mechanisms governing this process remain poorly understood. Employing the lepidopteran insect Helicoverpa armigera (cotton bollworm), an agricultural pest, as a model system, this study demonstrates that the steroid hormone 20-hydroxyecdysone (20E) regulates neural cell proliferation during imaginal brain development by upregulating the expression of SOX12 and DELTA via nuclear receptor EcRA. SOX12 exhibits significantly elevated expression and is localized in the brain during metamorphosis. Overexpression of SOX12-GFP in the H. armigera epidermal cell line (HaEpi) promotes cell proliferation. RNA interference (RNAi)-mediated knockdown of Sox12 in larvae leads to lethality, delayed pupation, impaired imaginal brain development, downregulation of cell proliferation-related genes, and reduced neural cell proliferation in the brain. CRISPR/Cas9 knockout of Sox12 can also impair imaginal brain development and reduce neural cell proliferation in the brain. Sox12 is upregulated by 20E through its nuclear receptor EcRA, which subsequently induces Delta expression to facilitate imaginal brain development during metamorphosis. Notably, Delta knockdown recapitulates the phenotypic effects observed on Sox12 silencing and knockout. Collectively, these findings establish that the steroid hormone 20E, acting through the EcRA-SOX12-DELTA regulatory axis, drives neural cell proliferation in the developing imaginal brain during insect metamorphosis.

In a recent Molecular Cell study,1 Zhou et al. elucidated how glycogenolysis-derived glucose-1-phosphate mediates source-specific routing of glucose-6-phosphate into the pentose phosphate pathway through allosteric activation of glucose-6-phosphate dehydrogenase and liquid-liquid phase separation-mediated metabolic compartments. This compartmentalized distribution enables efficient reduced nicotinamide adenine dinucleotide phosphate (NADPH) generation from glycogenolytic flux, promoting Tm cell persistence by maintaining redox homeostasis.

Objective:To analyze the relationship between Chemokine IP10 and its receptor CXCR3 during prion infection. Methods:We investigated the increases in IP10 signals, primarily localized in neurons within the brains of scrapie-infected mice, using western blotting, ELISA, co-immunoprecipitation, immunohistochemistry, immunofluorescence assays, and RT-PCR. Results:Both CXCR3 levels and activation were significantly higher in the brains of scrapie-infected mice and prion-infected SMB-S15 cells. Enhanced CXCR3 expression was predominantly observed in neurons and activated microglia. Morphological colocalization of PrP C/PrP Sc with IP10/CXCR3 was observed in scrapie-infected mouse brains using immunohistochemistry and immunofluorescence. immunohistochemistry (IHC) analysis of whole brain sections further revealed increased accumulation of IP10/CXCR3 specifically in brain regions with higher levels of PrP Sc deposits. Co-immunoprecipitation and biomolecular interaction assays revealed the molecular interactions between PrP and IP10/CXCR3. Notably, a significantly larger amount of IP10 accumulated within prion-infected SMB-S15 cells than in the normal partner cell line, SMB-PS. Importantly, resveratrol treatment effectively suppressed prion replication in SMB-S15 cells, thereby restoring the accumulation and secretion pattern of cellular IP10 similar to that observed in SMB-PS cells. Conclusion:Our data demonstrate that the activation of IP10/CXCR3 signaling in prion-infected brain tissues coincides with PrP Sc deposition. Modulation of IP10/CXCR3 signaling in the brain represents a potential therapeutic target for mitigating the progression of prion diseases.

Avermectin is a widely used insecticide, and it is mainly effective against animal parasites and insects. Given its extensive use in agriculture, a large amount of avermectin is accumulated in natural waters. Avermectin is a neurotoxin that affects the autonomous behavior of zebrafish and inhibits neurological responses in invertebrates via GABA-chloride channels. In this study, we used zebrafish as a model organism to explore the lethal teratogenic effects of different avermectin concentrations. We found that 50-μg/L avermectin could cause significant malformation abnormalities during the development of zebrafish heart, changes in heart rate, and significant reduction in hatching rate and body length. Transcriptome data revealed that 499 genes were upregulated and 877 genes were downregulated at 72 h post-fertilization (hpf), whereas 1805 genes were upregulated and 836 genes were downregulated at 120 hpf. According to gene ontology (GO) enrichment analysis, avermectin affected cardiac circulation and myocardial fiber development. KEGG analysis revealed that avermectin treatment significantly altered the activity of signal pathways associated with cardiac rhythm and vascular smooth muscle contraction. The main target of avermectin was identified as the heart, as it affected heart development and function by altering cardiac-related gene expression that led to a heart defect phenotype. Our findings indicate that developing zebrafish are sensitive to avermectin, which targets the heart.

Journal of the European Academy of Dermatology and VenereologyVolume 36, Issue 9 p. e695-e697 Letter to the Editor Leprosy: A life-changing disease Ying Zhang, Ying Zhang orcid.org/0000-0001-7793-7377 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Department of Dermatology, Chongqing Hospital of Chinese Medicine, Chongqing, ChinaSearch for more papers by this authorLin Feng, Lin Feng orcid.org/0000-0002-7281-9416 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Department of Dermatology, Chongqing Hospital of Chinese Medicine, Chongqing, ChinaSearch for more papers by this authorLin Wang, Corresponding Author Lin Wang lkzwl@126.com orcid.org/0000-0001-8704-1136 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, ChinaCorrespondence: L. Wang. E-mail: lkzwl@126.comSearch for more papers by this author Ying Zhang, Ying Zhang orcid.org/0000-0001-7793-7377 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Department of Dermatology, Chongqing Hospital of Chinese Medicine, Chongqing, ChinaSearch for more papers by this authorLin Feng, Lin Feng orcid.org/0000-0002-7281-9416 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, China Department of Dermatology, Chongqing Hospital of Chinese Medicine, Chongqing, ChinaSearch for more papers by this authorLin Wang, Corresponding Author Lin Wang lkzwl@126.com orcid.org/0000-0001-8704-1136 Department of Dermatovenereology, West China Hospital, Sichuan University, Chengdu, ChinaCorrespondence: L. Wang. E-mail: lkzwl@126.comSearch for more papers by this author First published: 12 April 2022 https://doi.org/10.1111/jdv.18144Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume36, Issue9September 2022Pages e695-e697 RelatedInformation

Abstract We constructed a ROP47 DNA vaccine combined with monophosphoryl lipid A (MPLA) and used it to immunize mice. Five groups of mice were injected every 2 weeks with PBS, empty vector, MPLA, pROP47, or pROP47 with MPLA. All mice were injected intramuscularly three times. Blood samples from all experimental mice were collected from the tail vein at 2, 4, 6, and 10 weeks after the first injection. The levels of IgG, IgG1, IgG2a, IFN-γ, IL-4, IL-10, and IL-12 in blood from all experimental mice were measured. The mice were challenged intragastrically with 20 cysts of Toxoplasma gondii PRU strain 2 weeks after the last vaccination. The mice immunized with MPLA/pROP47 produced higher levels of IgG, IgG2a, IFN-γ, and IL-12 compared with other groups. Furthermore, after a challenge with 20 cysts, higher numbers of cysts were present in the brains of other experimental mice compared with MPLA/pROP47-immunized mice. The pROP47 vaccine markedly stimulated humoral and cellular immune responses in immunized mice and MPLA enhanced the immune protection induced by the pROP47 DNA vaccine.

Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and macaques were immunized with an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine prepared by two inactivators. Various immunological indexes were tested, and viral challenges were performed on day 7 or 150 after booster immunization in monkeys. This inactivated SARS-CoV-2 vaccine was produced by sequential inactivation with formaldehyde followed by propiolactone. The various antibody responses and specific T cell responses to different viral antigens elicited in immunized animals were maintained for longer than 150 days. This comprehensive immune response could effectively protect vaccinated macaques by inhibiting viral replication in macaques and substantially alleviating immunopathological damage, and no clinical manifestation of immunopathogenicity was observed in immunized individuals during viral challenge. This candidate inactivated vaccine was identified as being effective against SARS-CoV-2 challenge in rhesus macaques.

With the relatively serious global epidemic outbreak of SARS-CoV-2 infection, public concerns focus on not only clinical therapeutic measures and public quarantine for this disease but also the development of vaccines. The technical design of our SARS-CoV-2 inactivated vaccine provides a viral antigen that enables the exposure of more than one structural protein based upon the antibody composition of COVID-19 patients’ convalescent serum. This design led to valid immunity with increasing neutralizing antibody titers and a CTL response detected post-immunization of this vaccine by two injections in rhesus macaques. Further, this elicited immunoprotection in macaques enables not only to restrain completely viral replication in tissues of immunized animals, compared to the adjuvant control and those immunized by an RBD peptide vaccine, but also to significantly alleviate inflammatory lesion in lung tissues in histo-pathologic detection, compared to the adjuvant control with developed interstitial pneumonia. The data obtained from these macaques immunized with the inactivated vaccine or RBD peptide vaccine suggest that immunity with a clinically protective effect against SARS-CoV-2 infection should include not only specific neutralizing antibodies but also specific CTL responses against at least the S and N antigens.

Background: Coronavirus disease 2019 (COVID-19) poses a great threat to human health and life. We performed a bioinformatics analysis to compare the sequence, structure, and epitopes of SARS-CoV-2 spike (S) protein in 10 different countries. Methods: The amino acid sequences of SARS-CoV-2 S protein were obtained from the NCBI database. We used DNASTAR Lasergene software to analyze the protein’s secondary structures. SWISS-MODEL combined with VMD software was used to construct a 3D model of SARS-CoV-2 S protein. DNASTAR Protean and the IEDB database were used to analyze the B cell epitopes and T cell epitopes, respectively. Results: The results of B cell epitopes analysis indicated that the epitopes of SARS-CoV-2 S protein in Korea and American increased, which suggested that the antigenicity of SARS-CoV-2 in Czech, Korea and American might be enhanced. A small number of B cell epitopes disappeared in the SARS-CoV-2 S protein sequence from Greece, Australia, Sweden and India, which suggested that the antigenicity of SARS-CoV-2 in Greece, Australia, Sweden and India may be weakened. T cell epitope analysis indicated that the antigenicity of SARS-CoV-2 in Czech, Korea and American was enhanced, while antigenicity of SARS-CoV-2 in Greece, Australia, Inida, Sweden and Thailand may be weakened. The sequence of SARS-CoV-2 S protein has changed as the virus has spread, and the structures and epitopes have changed accordingly. Conclusion: The mutation leads to a decrease in the antigenicity of SARS-CoV-2, which may be a mechanism for the virus to evade surveillance by the immune system.

Toxoplasma gondii is an intracellular parasite and causes a global epidemic parasitic disease. T gondii-infection could inhibit the growth of tumor. In this study, the transcriptomes of samples were detected by deep sequencing analysis. The transcriptome data was compared with reference genome to perform sequence alignment and the further analysis. The analyses of differential expression and the differentially expressed genes were performed in the present study. Genes involved in P53 signaling pathway, COLORECTAL cancer pathway, NON-SMALL CELL LUNG cancer signaling pathway, and BREAST cancer signaling pathway were up-regulated or down-regulated among the samples. The KEGG analysis indicated that the cancer pathways changed after infection of T. gondii. Furthermore, tumor-related mRNAs from different samples had a large difference, which suggested that the difference might provide important information in resisting cancer. The protein results indicated that tumor-related protein changes occurred after infection of T. gondii. In conclusion, the infection changed the cancer pathways, which could possibly inhibit the growth of tumor.

Toxoplasma gondii has a comprehensive impact on a great range of warm-blood mammals, in which one-third of the population all over the world is involved. Dense granular proteins, regarded as GRA family, mediating substantial interface between host cell cytoplasm and parasite, are widely studied for preventing the infection of T. gondii.As is handled in our study, the effect of intramuscularly injecting the genetic vaccine pEGFP-C1/GRA41 encoding a novel dense granule protein, GRA41, was evaluated.At the beginning, bioinformatics analysis was used to evaluate epitopes of both B cells and T cells on the GRA41 protein of T. gondii. Afterwards, recombinant plasmids (pEGFP-C1/GRA41) were injected into BALB/c mice and the quantity of IgG and its subclass IgG2a remarkably increased. IFN-γ, distinctive from the other cytokines (IL-4, and IL-10), was significant in growth. Afterwards, the intraperitoneal challenge was executed for recording survival time with tachyzoites with high virulence (in RH strain) and counting the number of brain cysts was carried out after the infection of PRU strain (low virulence).In pEGFP-C1/GRA41 group, the survival period was significantly longer (13.3 ± 3.37 days) after tachyzoites attack with the RH strain in high virulence, compared with the other groups (less than 8 days). Additionally, the cyst quantity is remarkably lower and the rate of reduction could reach 59.34%.All the results indicated effective protection of DNA vaccine encoding GRA41 against T. gondii.

SummaryAimsToxoplasma gondii is an intracellular parasite that causes a global epidemic parasitic disease. Studies using DNA vaccines for the control of toxoplasmosis have made considerable progress. ROP proteins were proven to be excellent candidates for T. gondii DNA vaccine development.Methods and ResultsHere, a ROP29 DNA vaccine was successfully produced and injected into mice in combination with R848 to evaluate its ability to provide protection against T. gondii challenge. Compared with other mice, the mice injected with R848/pROP29 produced higher levels of IgG, IgG2a, interleukin (IL)‐12, and interferon gamma (IFN‐γ). Moreover, after a challenge of 20 T. gondii cysts, the number of brain cysts was lower in the R848/pROP29‐immunized mice than in the other experimental mice.ConclusionsR848 could improve the productions of IL‐12 and IFN‐γ, thus enhancing the immune responses stimulated by the pROP29 DNA vaccine.

Lung cancer is the most common cause of cancer-related death. Non-small cell lung cancer (NSCLC) accounts for 80%-85% of total lung cancer cases. Dachshund homolog 1 (DACH1), is a protein encoded by the DACH1 gene in humans. DACH1 inhibits lung adenocarcinoma invasion and tumor growth but has a lower expression in NSCLC. To investigate the mechanisms of decreased DACH1 expression, its DNA methylation patterns were investigated. The results showed a higher methylation rate in NSCLC compared with the adjacent normal lung tissues. Cell transfection experiments showed that increased methylation impaired transcription factor transactivation. In vivo demethylation treatment and overexpression of DACH1 increased apoptosis and decreased migration and invasion in NSCLC A549 cells. Our research provides new insight into NSCLC pathogenesis and identifies a new therapeutic target.

Toxoplasma gondii infects almost all the warm-blooded animals. ROP20 protein is expressed in the rhoptry of Toxoplasma gondii. In this study, the secondary structure of ROP20 was analyzed using SMART software. We constructed and analyzed the 3D model of ROP20 protein using SWISS-MODEL online procedure and Visual Molecular Dynamics (VMD) software. The structure analysis fully indicated that ROP20 protein is an important member of the ROP family. Furthermore, We used DNASTAR software and Epitope Database online service to analyze liner-B cell epitopes and T-cell epitopes of ROP20 protein. All the analysis results of ROP20 protein can provide positive information on treatment and vaccine for toxoplasmosis. Moreover, ROP20 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-C1-ROP20) was constructed in the following study. After restriction enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result indicated that the recombinant plasmid could transcribe successfully in HEK 293-T cell. The results of western blotting indicated the expressed proteins can be recognized by anti-STAg mouse sera.

目的 构建弓形虫ROP31基因重组真核表达载体,对其编码的蛋白进行生物信息学分析. 方法 提取弓形虫RH株速殖子总RNA,采用PCR扩增ROP31基因,构建其真核表达载体pET30a-ROP31.通过生物信息学软件结合在线程序分析ROP31基因编码蛋白的理化性质、抗原表位、空间结构等. 结果 ROP31基因PCR扩增产物大小约1500 bp,与预期一致,构建的重组质粒经双酶切鉴定表明目的基因插入正确.生物信息学分析ROP31基大编码蛋白有多个磷酸化修饰位点,具备多个跨膜区域;二级结构和空间结构分析ROP31蛋白与ROP家族优秀DNA疫苗ROP5、ROP17、ROP18在结构上相似;IEDB在线程序及DNA MAN软件分析ROP31蛋白具备比SAG1更为优秀的线性T、B细胞表位. 结论 成功构建重组真核表达载体pROP31,生物信息学分析其编码的ROP31蛋白具备优秀的细胞表位,这将为弓形虫ROP31 DNA疫苗的研究提供理论依据.

Objective To construct and express the eukaryotic expression vector of ROP19 protein of Toxoplasma gondii PRU strain.Methods The physical and chemical characteristics of ROP19 protein were analyzed,and the B cell epitopes and T cell epitopes of ROP19 were compared with SAG1 using bioinformatics.The recombinant eukaryotic expression plasmid pROP19 was constructed by the molecular cloning technology.After being identified by PCR,restriction enzyme cleavage and sequencing,pROP19 was transfected into the HEK293T cells.The cells from different groups (control,pEGFP-C1 and pROP19) were respectively detected with fluorescence microscope under blue laser.The ROP19 protein was detected by Western blotting.Results The ROP19 protein was mainly located in the membrane and had better antigenic index than SAG1.The plasmid was constructed successfully.Western blotting showed that the expressed proteins could be recognized by anti-STAg mouse sera.Conclusion The eukaryotic expression vector pROP19 is constructed and expressed in eukaryotic cells.

BACKGROUND:Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4.METHODS:We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups (peptide, pROM4 and pROM4/peptide) and three control groups (PBS, pEGFP-C1 and pSAG1). Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain.RESULTS:Mice vaccinated with different immunization regimens (peptide, pROM4 and pROM4/peptide) elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls.CONCLUSION:A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization.

Toxoplasma gondii can lead to congenital infections in human. Surface antigen protein 4 (SAG4) of T. gondii is a potential stimulator for humoral and cellular immune responses. In the present study, a DNA vaccine encoding SAG4 from T. gondii was constructed and used to immunize BALB/c mice with peptide to evaluate the protective efficacy of the vaccine. The productions of IgG antibodies and cytokines (gamma interferon) from the vaccine (pSAG4/peptide) group were significantly higher than pSAG4 or peptide groups. After a lethal challenge by 1 × 104 tachyzoites from the I strain (RH), the survival time of mice immunized by pSAG4/peptide was longer than that of pSAG4 or peptide immunized mice or control mice. Moreover, after challenging by 20 cysts of the II strain (PRU) of T. gondii, the number of brain cysts from pSAG4/peptide vaccinated mice was only 31% of the number in PBS injected mice. The findings suggested the SAG4 DNA vaccine with peptide led significant immune responses and improved the protection against T. gondii challenges.

Toxoplasma gondii is defined as an obligate intracellular apicomplexan parasite and influences approximatelyone-third of the human all over the world. ROP54 protein is expressed in the rhoptry of Toxoplasma gondii. In the present study, we used SMART software to analyzethe secondary structure of ROP54. The 3D model of ROP54 protein was constructed and analyzed using SWISS-MODEL server and VMD software. The structure results fully showed that ROP54 proteinis an importantmember from the ROP family. Moreover, DNAMAN software and Epitope Database online service were used to analyze liner-B cell epitopes and Th-cell epitopes of the protein. The bioinformatics prediction of ROP54 protein could provide positive information on treatment and vaccine for toxoplasmosis. Furthermore, ROP54 gene was obtained from PCR, and a recombinant eukaryotic expression vector (pEGFP-ROP54) was constructed in the following study. After identification of enzyme digestion, the constructed plasmid was transfected into HEK 293-T cells. The RT-PCR result suggested that the recombinant plasmid could transcribe successfully in HEK 293-T cell.