A rational designed multi-epitope vaccine elicited robust protective efficacy against Klebsiella pneumoniae lung infection.

BACKGROUND: The emergence of drug-resistant strains of Klebsiella pneumoniae (K. pneumoniae) has become a significant challenge in the field of infectious diseases, posing an urgent need for the development of highly protective vaccines against this pathogen. METHODS AND RESULTS: In this study, we identified three immunogenic extracellular loops based on the structure of five candidate antigens using sera from K. pneumoniae infected mice. The sequences of these loops were linked to the C-terminal of an alpha-hemolysin mutant (mHla) from Staphylococcus aureus to generate a heptamer, termed mHla-EpiVac. In vivo studies confirmed that fusion with mHla significantly augmented the immunogenicity of EpiVac, and it elicited both humoral and cellular immune responses in mice, which could be further enhanced by formulation with aluminum adjuvant. Furthermore, immunization with mHla-EpiVac demonstrated enhanced protective efficacy against K. pneumoniae channeling compared to EpiVac alone, resulting in reduced bacterial burden, secretion of inflammatory factors, histopathology and lung injury. Moreover, mHla fusion facilitated antigen uptake by mouse bone marrow-derived cells (BMDCs) and provided sustained activation of these cells. CONCLUSIONS: These findings suggest that mHla-EpiVac is a promising vaccine candidate against K. pneumoniae, and further validate the potential of mHla as a versatile carrier protein and adjuvant for antigen design.

Analysis of the risk factors of radiation pneumonitis in patients after radiotherapy for esophageal squamous cell carcinoma.

Objective: To predict the risk factors of radiation pneumonitis (RP) in patients with esophageal squamous cell carcinoma (ESCC) who received radiotherapy. Methods: From January 2015 to October 2021, 477 ESCC patients were enrolled and were assessed retrospectively. All these patients received radiotherapy for primary lesions or mediastinal metastatic lymph nodes. Clinical efficacy and adverse events (AEs) were observed. Univariate analysis identified clinical and dosimetric factors associated with the development of RP, and multivariate logistic regression analysis identified independent potential risk factors associated with the development of RP. Nomograms were constructed to predict RP based on the results of multivariate logistic regression analysis. Results: Among the 477 ESCC patients, the incidence of RP was 22.2%, and the incidence of grade 4 or higher RP was 1.5%. Univariate analysis indicated that chronic obstructive pulmonary disease (COPD), pulmonary infection, leucopenia, PTV volume, V5, V20, V30 and MLD affected the occurrence of RP. The multivariate logistic regression analysis indicated that COPD (OR:1.821, 95%CI:1.111-2.985; P=0.017), pulmonary infection (OR:2.528, 95%CI:1.530-4.177; P<0.001), higher V20 (OR: 1.129, 95% CI:1.006-1.266; P=0.029) were significant independent predictors of RP in ESCC patients. COPD, pulmonary infection, V20 have been integrated for the RP nomogram. The rate of RP was significantly reduced in the V20<21.45% group. Further analysis indicated that the old age, diabetes, higher V20, and higher MLD were risk factors for grade 4 or higher RP. The area under the curve (AUC) value for V20 was 0.73 (95% CI, 0.567-0.893, P < 0.05). Conclusion: We have determined the risk factors of RP and grade 4 or higher RP in ESCC patients after radiotherapy. MLD, V20, COPD were independent factors for RP. It was necessary to take measures to reduce or avoid the occurrence of RP for patients with these risk factors at the early stage.

Denosumab Induces Neoplastic Stromal Cell Apoptosis via p62 Downregulation Dependent on Autophagy Pathway in Giant Cell Tumour of Bone.

BACKGROUND: As the only humanized monoclonal antibody against receptor activator of nuclear factor-κB ligand (RANKL) for giant cell tumour of bone (GCTB) therapy, denosumab has limited antitumour effect on neoplastic stromal cells. Nevertheless, its mechanism of action has not yet been clarified. A previous study has revealed that p62 may play an important role in the antitumour activity of denosumab. OBJECTIVE: The study aimed to investigate if the mechanism by which denosumab inhibits GCTB neoplastic stromal cells growth is via p62 modulation and other related mechanisms. METHODS: p62 expression before and after denosumab therapy was analysed by RT‒qPCR, western blot, ELISA, and immunohistochemical assays. Two primary neoplastic stromal cells were isolated from fresh GCTB tumour tissue (L cell) and metastatic tissue (M cell). Cell proliferation, migration, apoptosis, and autophagy were investigated in p62 knockdown neoplastic stromal cells transfected by short hairpin RNA lentivirus in vitro. Tumor growth was evaluated in the chick chorioallantoic membrane model in vivo. RESULTS: p62 expression was found to be downregulated following denosumab therapy. The patients with a decrease in p62 expression had lower recurrence-free survival rates. The proliferation of M cells was not inhibited by denosumab therapy, but it was restored by p62 knockdown. Moreover, p62 knockdown inhibited tumour growth in vivo. Denosumab induced M cell apoptosis and arrested the cell cycle at the G1/G0 transition and these effects were also enhanced by p62 knockdown. Autophagic flux assays revealed p62 modulation to be dependent on autophagy following denosumab incubation. CONCLUSION: Denosumab induced neoplastic stromal cells apoptosis via p62 downregulation dependent on autophagy pathway. The combination of p62 and RANKL knockdown might be a better strategy than RANKL knockdown alone for GCTB targeted therapy.

A high-performance TPGDA/PETEA composite gel polymer electrolyte for lithium metal batteries.

A gel polymer electrolyte (GPE) supported by a polyimide (PI) nanofiber membrane with Li6.5La3Zr1.5Ta0.5O12 (LLZTO) nanoparticles (PI/LLZTO/GPE) shows excellent flexibility and electrochemical properties, the ionic conductivity is 1.87 mS cm-1 and the Li+ transfer number is 0.64 at room temperature. The assembled Li metal battery with a LiFePO4 (LFP) cathode retains a 56.4 mA h g-1 discharge capacity at 10C.

Bone defect reconstruction using Masquelet technique for calcaneal chondroblastoma: a case report.

Masquelet technique demonstrated superiority in reconstructing long bone defect after trauma or infection. However, reports in foot tumor were rare. A 24-year-old male diagnosed with calcaneal chondroblastoma who had a defect of calcaneal after intralesional curettage. We reconstructed the defect by Masquelet technique. This is the first case as far as we know that reported Masquelet technique for calcaneal tumor. The technique to treat irregular bone defects after operation can be considered in other similar situations.

A miRNA-205-based model for prediction of the recurrence of endometrioid endometrial cancer.

AIM: To develop a miRNA-205 based model for prediction of the recurrence of endometrial cancer. METHODS: The FIGO (International Federation of Gynecology and Obstetrics) stage, grading, myometrial infiltration, lymph node status and miRNA-205 expression levels were extracted from 90 endometrioid endometrial cancer patients, recurrence related risk factors were analyzed by Cox regression analysis. A risk model was then developed. RESULTS: A total of 90 endometrial cancer patients were retrospectively included for the analysis. The FIGO stage and expression levels of miRNA 205 were independently associated with the recurrence-free survival of the patients. The FIGO stage and expression levels of miRNA 205 were used for a prognostic model of recurrence-free survival. The c-index of the model reached 0.764, and the output of the model (risk score) could stratify the patients into different groups on the risk of recurrence. CONCLUSION: A miRNA-205 based model could predict the risk of recurrence for endometrioid endometrial cancer, and the model could provide a risk stratification of patients by recurrence risk.

Opposite evolution of pathogenicity driven by in vivo wzc and wcaJ mutations in ST11-KL64 carbapenem-resistant Klebsiella pneumoniae.

AIMS: To investigate the in vivo evolution of the mucoid-phenotype of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from the same patients and gain insights into diverse evolution and biology of these strains. METHODS: Whole genome sequencing and bioinformatic analysis were used to determine the mutation involved in the mucoid phenotype of ST11-KL64 CRKP. Gene knockout, bacterial morphology and capsular polysaccharides (CPS) extraction were used to verify the role of wzc and wcaJ in the mucoid phenotypes. Antimicrobial susceptibility, growth assay, biofilm formation, host cell adhesion and virulence assay were used to investigate the pleiotropic role of CPS changes in ST11-KL64 CRKP strains. RESULTS: Mutation of wzc S682N led to hypermucoid phenotype, which had negative impact on bacterial fitness and resulted in reduced biofilm formation and epithelial cell adhesion; while enhanced resistance to macrophage phagocytosis and virulence. Mutations of wcaJ gene led to non-mucoid phenotype with increased biofilm formation and epithelial cell adhesion, but reduced resistance of macrophage phagocytosis and virulence. Using virulence gene knockout, we demonstrated that CPS, rather than the pLVPK-like virulence plasmid, has a greater effect on mucoid phenotypic changes. CPS could be used as a surrogate marker of virulence in ST11-KL64 CRKP strains. CONCLUSIONS: ST11-KL64 CRKP strains sacrifice certain advantages to develop pathogenicity by changing CPS with two opposite in vivo evolution strategies.

Host Immune Response to Clinical Hypervirulent Klebsiella pneumoniae Pulmonary Infections via Transcriptome Analysis.

Klebsiella pneumoniae (K. pneumoniae), especially those with hypervirulence, is becoming a global concern and posing great threat to human health. Studies on individual immune cells or cytokines have partially revealed the function of the host immune defense against K. pneumoniae pulmonary infection. However, systematic immune response against K. pneumoniae has not been fully elucidated. Herein, we report a transcriptome analysis of the lungs from a mouse pneumonia model infected with a newly isolated K. pneumoniae clinical strain YBQ. Total RNA was isolated from the lungs of mice 48 hours post infection to assess transcriptional alteration of genes. Transcriptome data were analyzed with KEGG, GO, and ICEPOP. Results indicated that upregulated transcription level of numerous cytokines and chemokines was coordinated with remarkably activated ribosome and several critical immune signaling pathways, including IL-17 and TNF signaling pathways. Notably, transcription of cysteine cathepsin inhibitor (stfa1, stfa2, and stfa3) and potential cysteine-type endopeptidase inhibitor (cstdc4, cstdc5, and cstdc6) were upregulated. Results of ICEPOP showed neutrophils functions as the most essential cell type against K. pneumoniae infection. Critical gene alterations were further validated by rt-PCR. Our findings provided a global transcriptional perspective on the mechanisms of host defense against K. pneumoniae infection and revealed some unique responding genes.

α-Hemolysin-Aided Oligomerization of the Spike Protein RBD Resulted in Improved Immunogenicity and Neutralization Against SARS-CoV-2 Variants.

The increase in confirmed COVID-19 cases and SARS-CoV-2 variants calls for the development of safe and broad cross-protective vaccines. The RBD of the spike protein was considered to be a safe and effective candidate antigen. However, the low immunogenicity limited its application in vaccine development. Herein, we designed and obtained an RBD heptamer (mHla-RBD) based on a carrier protein-aided assembly strategy. The molecular weight of mHla-RBD is up to 450 kDa, approximately 10 times higher than that of the RBD monomer. When formulated with alum adjuvant, mHla-RBD immunization significantly increased the immunogenicity of RBD, as indicated by increased titers of RBD-specific antibodies, neutralizing antibodies, Th2 cellular immune response, and pseudovirus neutralization activity, when compared to RBD monomer. Furthermore, we confirmed that RBD-specific antibodies predominantly target conformational epitopes, which was approximately 200 times that targeting linear epitopes. Finally, a pseudovirus neutralization assay revealed that neutralizing antibodies induced by mHla-RBD against different SARS-CoV-2 variants were comparable to those against the wild-type virus and showed broad-spectrum neutralizing activity toward different SARS-CoV-2 variants. Our results demonstrated that mHla-RBD is a promising candidate antigen for development of SARS-CoV-2 vaccines and the mHla could serve as a universal carrier protein for antigen design.

Antibiotic Combined with Epitope-Specific Monoclonal Antibody Cocktail Protects Mice Against Bacteremia and Acute Pneumonia from Methicillin-Resistant Staphylococcus aureus Infection.

PURPOSE: We previously reported that monoclonal antibody (mAb) cocktail improves survival in Staphylococcus aureus infection. In this study, we used acute pneumonia model and lethal sepsis model to investigate the efficacy of antibiotic combined with epitope-specific mAb cocktail in treating MRSA252 infection. METHODS: MRSA252 was challenged by tail vein injection or tracheal intubation to establish sepsis model or pneumonia model. One hour after infection, the mice received a single intravenous injection of normal saline, vancomycin, and vancomycin combined monoclonal antibody, linezolid alone or linezolid combined monoclonal antibody. Daily record survival rate (total 7 days), bacterial load, histology, cytokine analysis of serum and alveolar lavage fluid, and in vitro determination of the neutralizing ability of antibodies to SEB toxin and Hla toxin explained the mechanism of antibody action. RESULTS: The mAb cocktail combined with low doses of vancomycin or linezolid improved survival rates in acute pneumonia model (70%, 80%) and lethal sepsis model (80%, 80%). Epitope-specific monoclonal antibodies reduced bacterial colonization in the kidneys and lungs of mice and inhibited the biological functions of the toxins Hla and SEB in vitro. Compared to the antibiotic alone or PBS groups, the combination group had higher levels of IL-1α, IL-1ß and IFN-γ and lower levels of IL-6, IL-10, TNF-α. Further, the combination of antibiotic and mAb cocktail improved infection survival against the clinical MRSA isolates in a lethal sepsis model. CONCLUSION: This study demonstrates a novel method to treat people with low immunity against drug-resistant S. aureus infections.

Pore-forming alpha-hemolysin efficiently improves the immunogenicity and protective efficacy of protein antigens.

Highly immunogenic exotoxins are used as carrier proteins because they efficiently improve the immunogenicity of polysaccharides. However, their efficiency with protein antigens remains unclear. In the current study, the candidate antigen PA0833 from Pseudomonas aeruginosa was fused to the α-hemolysin mutant HlaH35A from Staphylococcus aureus to form a HlaH35A-PA0833 fusion protein (HPF). Immunization with HPF resulted in increased PA0833-specific antibody titers, higher protective efficacy, and decreased bacterial burden and pro-inflammatory cytokine secretion compared with PA0833 immunization alone. Using fluorescently labeled antigens to track antigen uptake and delivery, we found that HlaH35A fusion significantly improved antigen uptake in injected muscles and antigen delivery to draining lymph nodes. Both in vivo and in vitro studies demonstrated that the increased antigen uptake after immunization with HPF was mainly due to monocyte- and macrophage-dependent macropinocytosis, which was probably the result of HPF binding to ADAM10, the Hla host receptor. Furthermore, a transcriptome analysis showed that several immune signaling pathways were activated by HPF, shedding light on the mechanism whereby HlaH35A fusion improves immunogenicity. Finally, the improvement in immunogenicity by HlaH35A fusion was also confirmed with two other antigens, GlnH from Klebsiella pneumoniae and the model antigen OVA, indicating that HlaH35A could serve as a universal carrier protein to improve the immunogenicity of protein antigens.

Immunodominance of Epitopes and Protective Efficacy of HI Antigen Are Differentially Altered Using Different Adjuvants in a Mouse Model of Staphylococcus aureus Bacteremia.

HI, a fusion protein that consists of the alpha-toxin (Hla) and the N2 domain of iron surface determinant B (IsdB), is one of the antigens in the previously reported S. aureus vaccine rFSAV and has already entered phase II clinical trials. Previous studies revealed that HI is highly immunogenic in both mice and healthy volunteers, and the humoral immune response plays key roles in HI-mediated protection. In this study, we further investigated the protective efficacy of immunization with HI plus four different adjuvants in a mouse bacteremia model. Results showed that HI-mediated protection was altered in response to different adjuvants. Using antisera from immunized mice, we identified seven B-cell immunodominant epitopes on Hla and IsdB, including 6 novel epitopes (Hla1-18, Hla84-101, Hla186-203, IsdB342-359, IsdB366-383, and IsdB384-401). The immunodominance of B-cell epitopes, total IgG titers and the levels of IFN-γ and IL-17A from mice immunized with HI plus different adjuvants were different from each other, which may explain the difference in protective immunity observed in each immunized group. Thus, our results indicate that adjuvants largely affected the immunodominance of epitopes and the protective efficacy of HI, which may guide further adjuvant screening for vaccine development and optimization.

An Immunodominant Epitope-Specific Monoclonal Antibody Cocktail Improves Survival in a Mouse Model of Staphylococcus aureus Bacteremia.

To date, no vaccine or monoclonal antibody (mAb) against Staphylococcus aureus has been approved for use in humans. Our laboratory has developed a 5-antigen S. aureus vaccine (rFSAV), which is now under efficacy evaluation in a phase 2 clinical trial. In the current study, using overlapping peptides and antiserum from rFSAV-immunized volunteers, we identified 7 B-cell immunodominant epitopes on 4 antigens in rFSAV, including 5 novel epitopes (Hla48-65, IsdB402-419, IsdB432-449, SEB78-95, and MntC7-24). Ten immunodominant epitope mAbs were generated against these epitopes, and all of them exhibited partial protection in a mouse sepsis model. Four robust mAbs were used together as an mAb cocktail to prevent methicillin-resistant S. aureus strain 252 infection. The results showed that the mAb cocktail was efficient in combating S. aureus infection and that its protective efficacy correlated with a reduced bacterial burden and decreased infection pathology, which demonstrates that the mAb cocktail is a promising S. aureus vaccine candidate.

Upregulation of Tubulointerstitial nephritis antigen like 1 promotes gastric cancer growth and metastasis by regulating multiple matrix metallopeptidase expression.

BACKGROUND AND AIM: Tubulointerstitial nephritis antigen-like 1 (TINAGL1), as a novel matricellular protein, has been demonstrated to participate in cancer progression, whereas the potential function of TINAGL1 in gastric cancer (GC) remains unknown. METHODS: The expression pattern of TINAGL1 in GC was examined by immunohistochemistry, ELISA, real-time polymerase chain reaction, and Western blot. Correlation between TINAGL1 and matrix metalloproteinases (MMPs) was analyzed by the GEPIA website and Kaplan-Meier plots database. The lentivirus-based TINAGL1 knockdown, CCK-8, and transwell assays were used to test the function of TINAGL1 in vitro. The role of TINAGL1 was confirmed by subcutaneous xenograft, abdominal dissemination, and lung metastasis model. Microarray experiments, ELISA, real-time polymerase chain reaction, and Western blot were used to identify molecular mechanism. RESULTS: TINAGL1 was increased in GC tumor tissues and associated with poor patient survival. Moreover, TINAGL1 significantly promoted GC cell proliferation and migration in vitro as well as facilitated GC tumor growth and metastasis in vivo. TINAGL1 expression in GC cells was accompanied with increasing MMPs including MMP2, MMP9, MMP11, MMP14, and MMP16. GEPIA database revealed that these MMPs were correlated with TINAGL1 in GC tumors and that the most highly expressed MMP was MMP2. Mechanically, TINAGL1 regulated MMP2 through the JNK signaling pathway activation. CONCLUSIONS: Our data highlight that TINAGL1 promotes GC growth and metastasis and regulates MMP2 expression, indicating that TINAGL1 may serve as a therapeutic target for GC.

Innate Immune Effectors Play Essential Roles in Acute Respiratory Infection Caused by Klebsiella pneumoniae.

Innate immune effectors constitute the first line of host defense against pathogens. However, the roles of these effectors are not clearly defined during Klebsiella pneumoniae (K. pneumoniae) respiratory infection. In the current study, we established an acute pneumonia model of K. pneumoniae respiratory infection in mice and confirmed that the injury was most severe 48 h post infection. Flow cytometric assay demonstrated that alveolar macrophages were the predominant cells in BALF before infection, and neutrophils were quickly recruited after infection, and this was in consistent with the kinetics of chemokine expression. Further, we depleted neutrophils, macrophages, and complement pathways in vivo and challenged these mice with a sublethal dose of K. pneumonia, the result showed that 80%, 60%, and 40% of mice were died in these groups, respectively, while no deaths occurred in the control group. Besides, innate immune effector depleted mice showed higher bacterial burdens in lungs and blood, companied with more severe lung damage and increased levels of cytokine/chemokine expression. These results demonstrated that the innate immune effectors are critical in the early controlling of K. pneumoniae infection, and neutrophils are the most important. Thus, alternative strategies targeting these innate immune effectors may be effective in controlling of K. pneumoniae respiratory infection.

Genome Sequence of Klebsiella pneumoniae YBQ, a Clinical Strain Isolated from the Sputum of a Patient with Severe Pneumonia.

We report here the genome of Klebsiella pneumoniae YBQ, a clinical strain isolated from the sputum of a patient with acute Klebsiella pneumoniae infection. The genome consists of a 5,119,471-bp circular chromosome and a 184,347-bp plasmid. Genome annotation predicted 5,028 coding DNA sequences (CDSs), 84 tRNAs, 25 rRNAs, and 47 small RNAs (sRNAs).

TrkC Overexpression Protects Sevoflurane-Induced Neurotoxicity in Human Induced Pluripotent Stem Cell-Derived Neurons.

BACKGROUND: Inhaled anesthetic sevoflurane (SEVO) may induce cortical neurotoxicity and memory dysfunction in both animals and humans. In this study, we investigated the toxic effects of SEVO on human induced pluripotent stem cell (iPS)-derived neurons. METHODS: Human iPS-derived neurons were exposed to SEVO in vitro. SEVO-induced toxic effects were examined with the viability, live caspase 3/7, and neurite density assays, respectively. The effects of SEVO on the receptors of the tyrosine kinases TrkA, TrkB, and TrkC were assessed by qRT-PCR. TrkA, TrkB, and TrkC were ectopically overexpressed in human iPS-derived neurons. Their functional effects on SEVO-induced human iPS-derived neuron toxicity were further investigated. RESULTS: SEVO induced dose-dependent cell death, caspase 3/7 elevation, neurite degeneration, and the downregulation of Trk receptors in human iPS-derived neurons. Adenovirus-mediated Trk receptor overexpression selectively upregulated endogenous TrkA, TrkB, or TrkC gene expressions in human iPS-derived neurons. Specifically, TrkC overexpression, but not TrkA or TrkB overexpression was found to overcome the neurotoxic effects of SEVO in human iPS-derived neurons. CONCLUSIONS: SEVO may induce neurotoxicity in human iPS-derived neurons, and its neurotoxic damage could be protected by the overexpression of TrkC.

Oligomerization of IC43 resulted in improved immunogenicity and protective efficacy against Pseudomonas aeruginosa lung infection.

IC43, a truncate form of outer membrane proteins OprF190-342 and OprI21-83 from Pseudomonas aeruginosa, is a promising candidate antigen and exists as monomer in solution. In this study, we generated the heptamer of IC43 by carrier protein aided oligomerization, which was confirmed by gel-filtration and chemical cross-linking analysis. The carrier protein naturally exists as a homo-heptamer, and IC43 was displayed on the surface of the carrier protein in the fusion protein. Immunization with this fusion protein resulted in increased level of antigen specific IgG antibodies and higher survival rate after infection. The improved efficacy was correlated with lower bacteria burden, inflammation and tissue damage in the lungs of immunized mice. Further studies revealed that immunization with this fusion protein resulted in increased levels of IL-4 and antigen specific IgG1, suggesting a stronger Th2 immune response was induced. The improved immunogenicity may be attributed to the exposure of more epitopes on the antigen, which was confirmed by results from immune-dominant peptide mapping and passive immunization. These results demonstrated a possible strategy to improve the immunogenicity of an antigen by carrier protein aided oligomerization.

Genetic characterization and fine mapping for multi-inflorescence in Brassica napus L.

KEY MESSAGE: A major QTL for multi-inflorescence was mapped to a 27.18-kb region on A05 in Brassica napus by integrating QTL mapping, microarray analysis and whole-genome sequencing. Multi-inflorescence is a desirable trait for the genetic improvement of rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait is not well understood. In the present study, a doubled haploid (DH) population derived from a cross between single- and multi-inflorescence lines was investigated for the penetrance of multi-inflorescence across 3 years and genotyped with 257 simple sequence repeat and sequence-related amplified polymorphism loci. A major quantitative trait locus (QTL) for penetrance of multi-inflorescence was mapped to a 9.31-Mb region on chromosome A05, explaining 45.81% of phenotypic variance on average. Subsequently, 13 single-inflorescence and 15 multi-inflorescence DH lines were genotyped with the Brassica microarray, and the QTL interval of multi-inflorescence was narrowed to a 0.74-Mb region with 37 successive single nucleotide polymorphisms between single- and multi-inflorescence groups. A 27.18-kb QTL interval was detected by screening 420 recessive F2 individuals with genome-specific markers. These results will be valuable for gene cloning and molecular breeding of multi-inflorescence in rapeseed.