In silico designed novel multi-epitope mRNA vaccines against Brucella by targeting extracellular protein BtuB and LptD.

Brucella, a gram-negative intracellular bacterium, causing Brucellosis, a zoonotic disease with a range of clinical manifestations, from asymptomatic to fever, fatigue, loss of appetite, joint and muscle pain, and back pain, severe patients have developed serious diseases affecting various organs. The mRNA vaccine is an innovative type of vaccine that is anticipated to supplant traditional vaccines. It is widely utilized for preventing viral infections and for tumor immunotherapy. However, research regarding its effectiveness in preventing bacterial infections is limited. In this study, we analyzed the epitopes of two proteins of brucella, the TonB-dependent outer membrane receptor BtuB and the LPS assembly protein LptD, which is involved in nutrient transport and LPS synthesis in Brucella. In order to effectively stimulate cellular and humoral immunity, we utilize a range of immunoinformatics tools such as VaxiJen, AllergenFPv.1.0 and SignalP 5.0 to design proteins. Finally, five cytotoxic T lymphocyte (CTL) cell epitopes, ten helper T lymphocyte (HTL) cell epitopes, and eight B cell epitopes were selected to construct the vaccine. Computer simulations are also used to verify the immune response of the vaccine. The codon optimization, in silico cloning showed that the vaccine can efficiently transcript and translate in E. coli. The secondary structure of mRNA vaccines and the secondary and tertiary structures of vaccine peptides were predicted and then docked with TLR-4. Finally, the stability of the developed vaccine was confirmed through molecular dynamics simulation. These analyses showed that the design the multi-epitope mRNA vaccine could potentially target extracellular protein of prevalent Brucella, which provided novel strategies for developing the vaccine.

Impact of cascade reservoirs on nutrients transported downstream and regulation method based on hydraulic retention time.

Cascade reservoirs construction has modified the nutrients dynamics and biogeochemical cycles, consequently affecting the composition and productivity of river ecosystems. The Jinsha River, as the predominant contributor to runoff, suspended sediment (SS), and nutrients production within the Yangtze River, is a typical cascade reservoir region with unclear transport patterns and retention mechanisms of nutrients (nitrogen and phosphorus). Furthermore, how to regulate nutrients delivery in the cascade reservoirs region is also an urgent issue for basin water environment study. Therefore, we monitored monthly variations in nitrogen and phosphorus concentrations from November 2021 to October 2022 in the cascade reservoirs of the Jinsha River. The results indicated that the concentrations and fluxes of total phosphorus (TP) and particulate phosphorus (PP) decreased along the cascade of reservoirs, primarily due to PP deposited with SS, while opposing trends for total nitrogen (TN) and dissolved total nitrogen (DTN), which might be the consequences of human inputs and the increase of dissolved inorganic nitrogen discharged from the bottom of the reservoirs. Moreover, the positive average annual retention ratios for TP and PP were 10% and 16%, respectively, in contrast to the negative averages of -8 % for TN and -11% for particulate nitrogen (PN). The variability in runoff-sediment and hydraulic retention time (HRT) of cascade reservoirs played crucial roles in the retention of TP and PP. A regulatory threshold of HRT = 5.3 days in the flood season was obtained for controlling the balance of TP based on the stronger relationship between HRT and TP retention ratio. Consequently, the HRT of these reservoirs could be managed to control nutrients delivery, which was of particular significance for basin government institutions. This study enhances our comprehension of how cascade reservoirs influence the distribution and transport patterns of nutrients, offering a fresh perspective on nutrients delivery regulation.

The role of the Notch signaling pathway in bacterial infectious diseases.

The Notch signaling pathway is the most crucial link in the normal operation and maintenance of physiological functions of mammalian life processes. Notch receptors interact with ligands and this leads to three cleavages and goes on to enter the nucleus to initiate the transcription of target genes. The Notch signaling pathway deeply participates in the differentiation and function of various cells, including immune cells. Recent studies indicate that the outcomes of Notch signaling are changeable and highly dependent on different bacterial infection. The Notch signaling pathway plays a different role in promoting and inhibiting bacterial infection. In this review, we focus on the latest research findings of the Notch signaling pathway in bacterial infectious diseases. The Notch signaling pathway is critically involved in a variety of development processes of immunosuppression of different APCs. The Notch signaling pathway leads to functional changes in epithelial cells to aggravate tissue damage. Specifically, we illustrate the regulatory mechanism of the Notch signaling pathway in various bacterial infections, such as Mycobacterium tuberculosis, Mycobacterium avium paratuberculosis, Mycobacterium leprae, Helicobacter pylori, Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, Ehrlichia chaffeensis and sepsis. Collectively, this review will not only help beginners intuitively and systematically understand the Notch signaling pathway in bacterial infectious diseases but also help experts to generate fresh insight in this field.

Real-world progression in the survival of de novo Metastatic prostate cancer over the past decade.

BACKGROUND: The impact of evolving treatment strategies for metastatic prostate cancer (mPCa) on real-world survival is not well understood. We analyzed changes in mPCa survival over the past decade and discussed the potential driving factors behind these changes. METHODS: Our study involved 43,228 mPCa patients (2004-2020) from the SEER database, divided into 4 diagnostic periods. We used a multivariate Cox proportional hazards model to evaluate diagnostic periods' influence on overall mortality (OM) and prostate cancer-specific mortality (PSM), and calculated relative median survival improvements between adjacent periods. Subgroup analyses based on age and distant metastasis sites were conducted. RESULTS: Patients diagnosed in 2016 to 2020 experienced significantly reduced mortality risk compared to those in 2004 to 2007 (HR 0.64 for OM, HR 0.62 for CSM, both P < 0.001). The study period witnessed an absolute improvement in median overall survival (OS) and prostate cancer-specific survival (PCSS), 17 months (54.8%) and 25 months (67.6%) respectively. The most rapid relative survival improvement occurred post-2016, with a 29.7% increase in median OS and a 37.8% increase in PCSS compared to 2012 to 2015. There was a significant reduction in mortality risk throughout the study period in both age groups (age <75 and ≥75), but absolute survival gains were smaller in the older group (24 months [68.6%] vs. 8 months [32%] for OS, 36 months [90.0%] vs. 11 months [33.3%] for PCSS), with lower relative survival improvements after 2016 (37.2% vs. 17.9% for OS, 49% vs. 22.2% for PCSS). All metastasis site subgroups (except M1a) exhibited a significant reduction in mortality risk (all P < 0.001). Absolute survival improvements were 58 months (134.9%) for M1a, 16 months (50.0%) for M1b, and 17 months (54.8%) for M1c. CONCLUSION: The survival of mPCa have significantly improved over the past decade, although the progress is slower in elderly patients. Investigating the underlying reasons for survival differences among various patient profiles can further refine mPCa treatment strategies.

Design of multi-epitope vaccine candidate against Brucella type IV secretion system (T4SS).

Brucellosis is a common zoonosis, which is caused by Brucella infection, and Brucella often infects livestock, leading to abortion and infertility. At present, human brucellosis remains one of the major public health problems in China. According to previous research, most areas in northwest China, including Xinjiang, Tibet, and other regions, are severely affected by Brucella. Although there are vaccines against animal Brucellosis, the effect is often poor. In addition, there is no corresponding vaccine for human Brucellosis infection. Therefore, a new strategy for early prevention and treatment of Brucella is needed. A multi-epitope vaccine should be developed. In this study, we identified the antigenic epitopes of the Brucella type IV secretion system VirB8 and Virb10 using an immunoinformatics approach, and screened out 2 cytotoxic T lymphocyte (CTL) epitopes, 9 helper T lymphocyte (HTL) epitopes, 6 linear B cell epitopes, and 6 conformational B cell epitopes. These advantageous epitopes are spliced together through different linkers to construct a multi-epitope vaccine. The silico tests showed that the multi-epitope vaccine was non-allergenic and had a strong interaction with TLR4 molecular docking. In immune simulation results, the vaccine construct may be useful in helping brucellosis patients to initiate cellular and humoral immunity. Overall, our findings indicated that the multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for the development of a Brucella vaccine.

Contriving a novel of CHB therapeutic vaccine based on IgV_CTLA-4 and L protein via immunoinformatics approach.

Chronic infection induced by immune tolerance to hepatitis B virus (HBV) is one of the most common causes of hepatic cirrhosis and hepatoma. Fortunately, the application of therapeutic vaccine can not only reverse HBV-tolerance, but also serve a potentially effective therapeutic strategy for treating chronic hepatitis B (CHB). However, the clinical effect of the currently developed CHB therapeutic vaccine is not optimistic due to the weak immunogenicity. Given that the human leukocyte antigen CTLA-4 owns strong binding ability to the surface B7 molecules (CD80 and CD86) of antigen presenting cell (APCs), the immunoglobulin variable region of CTLA-4 (IgV_CTLA-4) was fused with the L protein of HBV to contrive a novel therapeutic vaccine (V_C4HBL) for CHB in this study. We found that the addition of IgV_CTLA-4 did not interfere with the formation of L protein T cell and B cell epitopes after analysis by means of immunoinformatics approaches. Meanwhile, we also found that the IgV_CTLA-4 had strong binding force to B7 molecules through molecular docking and molecular dynamics (MD) simulation. Notably, our vaccine V_C4HBL showed good immunogenicity and antigenicity by in vitro and in vivo experiments. Therefore, the V_C4HBL is promising to again effectively activate the cellular and humoral immunity of CHB patients, and provides a potentially effective therapeutic strategy for the treatment of CHB in the future.Communicated by Ramaswamy H. Sarma.

Butyrate inhibits iILC2-mediated lung inflammation via lung-gut axis in chronic obstructive pulmonary disease (COPD).

BACKGROUND: The study investigated the effects and underlying mechanisms of intestinal flora metabolite butyrate on inflammatory ILC2 cells (iILC2s)-mediated lung inflammation in chronic obstructive pulmonary disease (COPD). METHODS: Mouse models of COPD and acute exacerbation of COPD (AECOPD) were established. Flow cytometry was used to detect natural ILC2 cells (nILC2s) and iILC2s in lung and colon tissues. The 16s rRNA and GC-MS were used to detect microbial flora and short chain fatty acids (SCFAs) in feces. ELISA was used to detect IL-13 and IL-4. Western blot and qRT-PCR were used to detect the relative protein and mRNA levels, respectively. In vitro experiments were performed with sorted ILC2s from colon tissues of control mice. Mice with AECOPD were treated with butyrate. RESULTS: The nILC2s and iILC2s in lung and colon tissues of AECOPD mice were significantly higher than control groups. The abundance of the flora Clostridiaceae was significantly reduced, and the content of SCFAs, including acetate and butyrate, was significantly reduced. The in vitro experiments showed that butyrate inhibited iILC2 cell phenotype and cytokine secretion. Butyrate treatment reduced the proportion of iILC2 cells in the colon and lung tissues of mice with AECOPD. CONCLUSIONS: The nILC2s and iILC2s in the colon tissues are involved in the course of COPD. Decreased Clostridiaceae and butyrate in AECOPD mice caused the accumulation of iILC2 cells in the intestines and lungs. Supplementation of butyrate can reduce iILC2 in the intestine and lung tissues. Our data may provide new ideas for prevention and treatment of COPD.

N6-methyladenosine-methylomic landscape of lung tissues of mice with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), a common respiratory disease, can be divided into stable phase and acute exacerbation phase (AECOPD) and is characterized by inflammation and hyper-immunity. Methylation of N6-methyladenosine (m6A) is an epigenetic modification that regulates the expression and functions of genes by influencing post-transcriptional RNA modifications. Its influence on the immune regulation mechanism has attracted great attention. Herein, we present the m6Amethylomic landscape and observe how the methylation of m6A participates in the pathological process of COPD. The m6A modification of 430 genes increased and that of 3995 genes decreased in the lung tissues of mice with stable COPD. The lung tissues of mice with AECOPD exhibited 740 genes with hypermethylated m6A peak and 1373 genes with low m6A peak. These differentially methylated genes participated in signaling pathways related to immune functions. To further clarify the expression levels of differentially methylated genes, RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing data were jointly analyzed. In the stable COPD group, 119 hypermethylated mRNAs (82 upregulated and 37 downregulated mRNAs) and 867 hypomethylated mRNAs (419 upregulated and 448 downregulated mRNAs) were differentially expressed. In the AECOPD group, 87 hypermethylated mRNAs (71 upregulated and 16 downregulated mRNAs) and 358 hypomethylated mRNAs (115 upregulated and 243 downregulated mRNAs) showed differential expression. Many mRNAs were related to immune function and inflammation. Together, this study provides important evidence on the role of RNA methylation of m6A in COPD.

Neutrophilic granulocyte percentage is associated with anxiety in Chinese hospitalized heart failure patients.

BACKGROUND: In patients with heart failure, anxiety disorder is common and associated with adverse prognosis. This study intended to find more confounding factors of Chinese heart failure patients. METHODS: We enrolled 284 hospitalized heart failure patients, whose New York Heart Association (NYHA) classed as II-IV and left ventricular ejection fraction (LVEF) ≤ 45%. All the patients were scaled in Hamilton Rating Scale for Anxiety (14-items) (HAM-A14). Ordinal logistic regression analysis was performed to examine the association of correlated factors with anxiety disorder. RESULTS: There were 184 patients had anxiety accounting for 64.8% of all 284 hospitalized heart failure patients. The neutrophilic granulocyte percentage, urea nitrogen, total bilirubin and brain natriuretic peptide were positively associated with HAM-A14 score, meanwhile, the hemoglobin, red blood cells counts, albumin and LVEF were negatively associated with HAM-A14 score (All P < 0.05). After the adjustments of sex, hemoglobin, urea nitrogen, total bilirubin, albumin and brain natriuretic peptide, the neutrophilic granulocyte percentage was significantly associated with anxiety (OR = 43.265, P = 0.012). The neutrophilic granulocyte percentage was 0.616 ± 0.111, 0.640 ± 0.102, 0.681 ± 0.106 and 0.683 ± 0.113 in heart failure patients with no anxiety, possible anxiety, confirmed anxiety and obvious anxiety, respectively. CONCLUSIONS: Neutrophilic granulocyte percentage as well as the traditional risk factors such as sex, urea nitrogen and brain natriuretic peptide is associated with anxiety in hospitalized heart failure patients.

Design of a multi-epitope vaccine candidate against Brucella melitensis.

Brucella is a typical facultative intracellular bacterium that can cause zoonotic infections. For Brucella, it is difficult to eliminate with current medical treatment. Therefore, a multi-epitope vaccine (MEV) should be designed to prevent Brucella infection. For this purpose, we applied the reverse vaccinology approach from Omp10, Omp25, Omp31 and BtpB. Finally, we obtained 13 cytotoxic T lymphocyte (CTL) epitopes, 17 helper T lymphocyte (HTL) epitopes, 9 linear B cell epitopes, and 2 conformational B cell epitopes for further study. To keep the protein folded normally, we linked AAY, GPGPG, and KK to CTL epitopes, HTL epitopes, and B cell epitopes, respectively. The N-terminal of the vaccine peptide is supplemented with appropriate adjuvants to enhance immunogenicity. To evaluate its immunogenicity, stability, safety, and feasibility, a final MEV containing 806 amino acids was constructed by linking linkers and adjuvants. In addition, molecular docking and molecular dynamics simulations were performed to verify the affinity and stability of the MEV-TLR4. Then, codon adaptation and in silico cloning studies were carried out to identify the possible codons for expressing the MEV. In animal experiments, the results demonstrated that the MEV had high immunogenicity. Collectively, this study provided a theoretical basis for the development of a Brucella vaccine.

Tim-3/Galectin-9 signaling pathway is involved in the cytokine changes in mice with alveolar echinococcosis.

BACKGROUND: Tim-3/Galectin-9 is involved in the immune escape of many pathogens. However, the role of Tim-3/Galectin-9 in persistent infection of Echinococcus multilocularis (Em), which is related to immune escape, is still unclear. OBJECTIVE: To investigate the role of Tim-3/Galectin-9 and related cytokines in mice with persistent infection of Em. METHODS: Em infection model was established by injecting the protoscoleces. Serum was collected at days 2, 8, 30, 60, 90, 180 and 270 after infection. Lymphocytes were isolated from liver tissue samples with Ficoll. Tim-3 + CD4 + T percentage was analyzed by flow cytometry. CD4 + T cells were isolated from liver tissues of Em infected mice and cultured in vitro. The mRNA levels of Tim-3, Galectin-9, IFN-γ and IL-4 were detected by qRT-PCR. Cytokine levels in serum and culture supernatant (IFN-γ and IL-4) were analyzed by cytometric bead array. RESULTS: The expression of Tim-3 and Galectin-9 mRNA significantly increased after 30 days of infection, reached peak on day 90, and then decreased slightly on days 180-270. The expression of IFN-γ mRNA, increased on day 2 and 8 after infection, slightly decreased on days 30-60, and obvious decreased on days 90-270, but were still higher than those of the control group. The expression of IL-4 mRNA gradually increased along with the time of infection. In serum of Em infected mice, level of IFN-γ peaked at day 30 and then gradually decreased; whereas IL-4 level peaked at day 90 and then gradually decreased. In vitro experiment found that Tim-3/Galectin-9 directly caused the changes in the levels of IFN-γ and IL-4. CONCLUSIONS: Tim-3/Galectin-9 signaling pathway may be involved in the development of persistent infection of Em by regulating the production of Th1 and Th2 cytokines.

Design of a Recombinant Multivalent Epitope Vaccine Based on SARS-CoV-2 and Its Variants in Immunoinformatics Approaches.

The development of an effective multivalent vaccine against SARS-CoV-2 variants is an important means to improve the global public health situation caused by COVID-19. In this study, we identified the antigen epitopes of the main global epidemic SARS-CoV-2 and mutated virus strains using immunoinformatics approach, and screened out 8 cytotoxic T lymphocyte epitopes (CTLEs), 17 helper T lymphocyte epitopes (HTLEs), 9 linear B-cell epitopes (LBEs) and 4 conformational B-cell epitopes (CBEs). The global population coverage of CTLEs and HTLEs was 93.16% and 99.9% respectively. These epitopes were spliced together by corresponding linkers and recombined into multivalent vaccine. In silico tests, the vaccine protein was a non-allergen and the docking with TLR-3 molecule showed a strong interaction. The results of immune simulation showed that the vaccine may be helpful to initiate both cellular and humoral immunity against all VOC. The optimistic immunogenicity of the vaccine was confirmed in vivo and in vitro finally. Therefore, our vaccine may have potential protection against SARS-CoV-2 and its variants.

Immmunoinformatics-based design of a multi-epitope vaccine with CTLA-4 extracellular domain to combat Helicobacter pylori.

In view of the high infection rate of Helicobacter pylori, a safe and effective vaccine is urgently needed. Recent trends in vaccine design have shifted toward safe and specific epitope-based vaccines. In this study, by using different immunoinformatics approaches, a total of eight linear B cell epitopes, four HTL and three CTL epitopes of FlaA and UreB proteins of H. pylori G27 strain were screened out, we also predicted the conformational epitopes of the two proteins. Then, the dominant epitopes were sequentially linked by appropriate linkers, and the cytotoxic T lymphocyte-associated antigen 4 extracellular domain was attached to the N-terminal of the epitope sequence. Meanwhile, molecular docking, molecular dynamics simulations and principal component analysis were performed to show that the multi-epitope vaccine structure had strong interactions with B7 (B7-1, B7-2) and Toll-like receptors (TLR-2, -4). Eventually, the effectiveness of the vaccine was validated using in silico cloning. These analyses suggested that the designed vaccine could target antigen-presenting cells and had high potency against H. pylori, which could provide a reference for the future development of efficient H. pylori vaccines.

Urinary exosome-based androgen receptor-variant 7 detection in metastatic castration-resistant prostate cancer patients.

Background: Androgen receptor variant 7 (AR-V7) detection provides important information for the clinical management of abiraterone in metastatic castration-resistant prostate cancer (mCRPC). We performed a non-invasive urine-derived exosomal AR-V7 analysis of mCRPC patients. Methods: A total of 34 mCRPC patients were recruited including 16 patients treated with abiraterone (ABI) with stable prostate-specific antigen (PSA)/radiograph response (the ABI-Sta group) and 18 were resistant to abiraterone (the ABI-Res group). Urine was collected from patients and healthy control patients for the analysis. Exosomal ribonucleic acid was isolated from urine. Urinary exosome-based androgen receptor-variant 7 was detected by quantitative real-time polymerase chain reaction assay. Characteristics of patients and survival data were collected. The correlation between AR-V7 expression and the therapeutic effect/survival outcomes of abiraterone was analyzed. Results: Urine is the ideal biological sample for exosome separation and AR full-length analysis. Positive urine-derived exosomal AR-V7 was detected in 32.4% (11 of 34) of the mCRPC patients' urine samples. Positive AR-V7 was more common in the ABI-Res patients than the ABI-Sta patients (50.0% vs. 12.5%, respectively; P=0.009), and was associated with a higher PSA progression rate and poorer overall survival (OS) (P=0.0031, and P=0.0012, respectively). Conclusions: The present study showed that the detection of urine-derived exosomal AR-V7 provides a sensitive and feasible clinical workflow. The predicting role of urine-derived exosomal AR-V7 in mCRPC patients should be further verified using studies with greater sample sizes.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

Serum levels of inhibitory costimulatory molecules and correlations with levels of innate immune cytokines in patients with pulmonary tuberculosis.

OBJECTIVE: To analyze serum levels of inhibitory costimulatory molecules and their correlations with innate immune cytokine levels in patients with pulmonary tuberculosis (PTB). METHODS: Data for 280 PTB patients and 280 healthy individuals were collected. Serum levels of immune molecules were measured using ELISA. Univariate, multivariate, subgroup, matrix correlation, and receiver operating characteristic curve analyses were performed. RESULTS: Host, environment, lifestyle, clinical features, and medical history all influenced PTB. Serum levels of soluble programmed death ligand 1 (sPD-L1), soluble T-cell immunoglobulin- and mucin-domain-containing molecule 3 (sTim-3), soluble galectin-9 (sGal-9), interleukin (IL)-4, and IL-33 were significantly higher in patients with PTB, while levels of IL-12, IL-23, IL-18, and interferon (IFN)-γ were significantly lower. Serum levels of sTim-3 were higher in alcohol users. Levels of sTim-3 were negatively correlated with those of IL-12. Levels of IL-12, IL-23, and IL-18 were positively correlated with those of IFN-γ, while levels of IL-12 were negatively correlated with those of IL-4. The areas under the curve of sPD-L1, sTim-3, sGal-9, IL-12, IL-23, IL-18, IFN-γ, IL-4, and IL-33 for identifying PTB were all >0.77. CONCLUSIONS: Inhibitory costimulatory molecules may be targets for controlling PTB. Immune molecules may be helpful for diagnosis of PTB.

ILC2 Cells Promote Th2 Cell Differentiation in AECOPD Through Activated Notch-GATA3 Signaling Pathway.

This study is to investigate the capacity of type 2 innate lymphoid cells (ILC2s) in regulating the Th2 type adaptive immune response of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The study enrolled healthy people, stable chronic obstructive pulmonary disease (COPD) patients, and AECOPD patients. Flow cytometry was used to detect Th2 and ILC2 cells in the peripheral blood. In addition, ILC2s from the peripheral blood of AECOPD patients were stimulated with PBS, IL-33, Jagged1, DAPT, IL-33+Jagged1, IL-33+DAPT, and IL-33+Jagged-1+DAP in vitro. The levels of cytokines in the culture supernatant were detected by ELISA and the culture supernatant was used to culture CD4 + T cells. The mRNA and protein levels of Notch1, hes1, GATA3, RORα, and NF-κB of ILC2s were detected by real-time PCR and Western blot. The proportion of Th2 and ILC2s was significantly increased in the peripheral blood of AECOPD patients, alone with the increased Notch1, hes1, and GATA3 mRNA levels. In vitro results showed that the mRNA and protein levels of Notch1, hes1, GATA3 and NF-κB were significantly increased after stimulation with Notch agonist, meanwhile, the level of type 2 cytokines were increased in the supernatant of cells stimulated with Notch agonist, and significantly promoted differentiation of Th2 cells in vitro. Disruption of Notch pathway weakened GATA3 expression and cytokine production, and ultimately affected the differentiation of Th2 cells. In conclusion, our results suggest that ILC2s can promote Th2 cell differentiation in AECOPD via activated Notch-GATA3 signal pathway.

A Multi-Epitope Chitosan Nanoparticles Vaccine of Canine Against Echinococcus granulosus.

Cystic Echinococcosis (CE) is caused by Echinococcus granulosus (Eg), which endangers the health of the intermediate host. Therefore, effective canid vaccines against Eg infection are urgently needed to reduce the incidence of this disease. In the present work, the aim was to predict epitopes in four vaccine candidate antigens (VCAs) in Eg as a basis to design a multi-epitope canine-directed vaccine. This vaccine is based on chitosan nanoparticles (CS-NPs) and is directed against Eg infection in the definitive host. The canine-directed vaccine was designed based on Eg antigens EgM9, Eg_10196, EgA31 and EgG1Y162. Several tools in online servers were used to predict VCAs information, which was combined with B cell, CTL and Th epitopes. Considering that acquiring experimental information in canids is difficult, and that it may be possible to perform future experiments in mice, we predicted both canine and murine T cell epitopes. The multi-epitope vaccine was synthetically prepared by ionic crosslinking method, and CS-NPs was used as adjuvant. The mice were immunized by oral gavage and laser scanning confocal microscopy was used to localize the fluorescein- labeled multi-epitope peptide in the intestinal tract. The final multi-epitope vaccine was construct consist of Co1 targeting peptide, four B-cell epitopes, four canine-directed CTL epitopes and four murine-directed Th epitopes. It has been proven experimentally by this research that multi-epitope antigen concentration merged with microfold cells was high in the CS-NPs vaccine group. The present bioinformatics study is a first step towards the construction of a canine-specific multiepitope vaccine against Eg with twelve predicted epitopes. CS-NPs is a potential adjuvant with relatively safe penetration enhancement delivery and a potent immunostimulant.

Use of Circulating Tumor DNA for the Clinical Management of Metastatic Castration-Resistant Prostate Cancer: A Multicenter, Real-World Study.

BACKGROUND: This study aimed to describe the aberrations of DNA damage repair genes and other important driving genes in Chinese patients with metastatic castration-resistant prostate cancer (mCRPC) using circulating tumor (ctDNA) sequencing and to evaluate the associations between the clinical outcomes of multiple therapies and key genomic alterations in mCRPC, especially DNA damage repair genes. PATIENTS AND METHODS: A total of 292 Chinese patients with mCRPC enrolled from 8 centers. Multigene targeted sequencing was performed on 306 ctDNA samples and 23 matched tumor biopsies. The frequency of genomic alterations were compared with the Stand Up to Cancer-Prostate Cancer Foundation (SU2C-PCF) cohort. The Kaplan-Meier method was used to evaluate progression-free survival (PFS) following standard systemic treatments for mCRPC. Cox regression analyses were performed to determine prognostic factors associated with PFS resulting from treatments for mCRPC. RESULTS: In total, 33 of 36 (91.7%) mutations were found consistently between ctDNA and paired biopsy samples. The most common recurrent genomic alterations were found in AR (34.6%), TP53 (19.5%), CDK12 (15.4%), BRCA2 (13%), and RB1 (5.8%). The frequency of CDK12 alterations (15.4%) in our cohort was significantly higher than that in Western populations (5%-7%). AR amplification and TP53 and/or RB1 alterations were associated with resistance to abiraterone or docetaxel. Patients with a CDK12 defect showed rapid disease progression after abiraterone treatment. However, the clinical outcome after docetaxel treatment was similar between patients with and without CDK12 defects. In multivariate Cox regression analysis, a CDK12 defect was significantly associated with inferior PFS after abiraterone treatment. Patients with a BRCA2 defect showed marked response to both PARP inhibitors and platinum-based chemotherapy. CONCLUSIONS: Our study explored the genomic landscape of Chinese patients with mCRPC at different treatment stages using minimally invasive methods and evaluated the clinical implications of the driver genomic alterations on patients' response to the most widely used therapies for mCRPC. We observed a significantly higher alteration frequency of CDK12 in our cohort compared with the SU2C-PCF cohort.

Design of a new multi-epitope vaccine against Brucella based on T and B cell epitopes using bioinformatics methods.

Brucellosis is one of the most serious and widespread zoonotic diseases, which seriously threatens human health and the national economy. This study was based on the T/B dominant epitopes of Brucella outer membrane protein 22 (Omp22), outer membrane protein 19 (Omp19) and outer membrane protein 28 (Omp28), with bioinformatics methods to design a safe and effective multi-epitope vaccine. The amino acid sequences of the proteins were found in the National Center for Biotechnology Information (NCBI) database, and the signal peptides were predicted by the SignaIP-5.0 server. The surface accessibility and hydrophilic regions of proteins were analysed with the ProtScale software and the tertiary structure model of the proteins predicted by I-TASSER software and labelled with the UCSF Chimera software. The software COBEpro, SVMTriP and BepiPred were used to predict B cell epitopes of the proteins. SYFPEITHI, RANKpep and IEDB were employed to predict T cell epitopes of the proteins. The T/B dominant epitopes of three proteins were combined with HEYGAALEREAG and GGGS linkers, and carriers sequences linked to the N- and C-terminus of the vaccine construct with the help of EAAAK linkers. Finally, the tertiary structure and physical and chemical properties of the multi-epitope vaccine construct were analysed. The allergenicity, antigenicity and solubility of the multi-epitope vaccine construct were 7.37-11.30, 0.788 and 0.866, respectively. The Ramachandran diagram of the mock vaccine construct showed 96.0% residues within the favoured and allowed range. Collectively, our results showed that this multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for future laboratory experiments.