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International Journal of Molecular... Jun 2024Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of...
Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.
Topics: Lipopolysaccharides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Animals; Mice; Macrophages; Lipid A; Cytokines; Endopeptidase K; Electrophoresis, Polyacrylamide Gel
PubMed: 38928052
DOI: 10.3390/ijms25126345 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To investigate the clinical significance, functional role and potential downstream mechanism of USP5 in acute myeloid leukemia (AML).
OBJECTIVE
To investigate the clinical significance, functional role and potential downstream mechanism of USP5 in acute myeloid leukemia (AML).
METHODS
The expression of in AML and normal tissues and its correlation with patients' survival were analyzed based on TCGA database. was knocked down and overexpressed in Jurkat and HL-60 cells using lentivirus. USP5 mRNA and protein expression were detected by RT-qPCR and Western blot, respectively. Cell proliferation and growth were measured by CCK-8 and methylcellulose colony-forming assay. Flow cytometry was used to analyze cell cycle and apoptosis.
RESULTS
was highly expression in AML compared with normal tissues. Up-regulation of was negatively correlated with the survival of AML patients. knockdown and overexpression inhibited and promoted the proliferation and colony growth of AML cells, respectively. Cell cycle arrest and apoptosis were induced in knockdown Jurkat and HL-60 cells. Furthermore, knockdown inhibited the phosphrylation of AKT, mTOR and 4EBP1.
CONCLUSION
Overexpression of predicts poor survival of AML patients. Targeting USP5 suppresses AKT/mTOR/4EBP1 signaling and reduces the proliferation and growth of AML cells.
Topics: Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Cell Proliferation; HL-60 Cells; Apoptosis; Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Jurkat Cells; Ubiquitin-Specific Proteases; Clinical Relevance
PubMed: 38926952
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.004 -
Scientific Reports Jun 2024Advancing healthcare for elderly men requires a deeper understanding of testicular aging processes. In this study, we conducted transcriptomic profiling of 43,323...
Advancing healthcare for elderly men requires a deeper understanding of testicular aging processes. In this study, we conducted transcriptomic profiling of 43,323 testicular single cells from young and old mice, shedding light on 1032 telocytes-an underexplored testicular cell type in previous research. Our study unveiled 916 age-related differentially expressed genes (age-DEGs), with telocytes emerging as the cell type harboring the highest count of age-DEGs. Of particular interest, four genes (Klk1b21, Klk1b22, Klk1b24, Klk1b27) from the Kallikrein family, specifically expressed in Leydig cells, displayed down-regulation in aged testes. Moreover, cell-type-level splicing analyses unveiled 1838 age-related alternative splicing (AS) events. While we confirmed the presence of more age-DEGs in somatic cells compared to germ cells, unexpectedly, more age-related AS events were identified in germ cells. Further experimental validation highlighted 4930555F03Rik, a non-coding RNA gene exhibiting significant age-related AS changes. Our study represents the first age-related single-cell transcriptomic investigation of testicular telocytes and Kallikrein genes in Leydig cells, as well as the first delineation of cell-type-level AS dynamics during testicular aging in mice.
Topics: Animals; Male; Mice; Alternative Splicing; Kallikreins; Testis; Aging; Gene Expression Profiling; Single-Cell Analysis; Transcriptome; Leydig Cells
PubMed: 38926537
DOI: 10.1038/s41598-024-65710-0 -
Anticancer Research Jul 2024The primary objective of this study was to identify predictors for biochemical recurrence (BCR) within 2 years following robot-assisted radical prostatectomy (RARP)....
BACKGROUND/AIM
The primary objective of this study was to identify predictors for biochemical recurrence (BCR) within 2 years following robot-assisted radical prostatectomy (RARP). Identifying predictors will enable insights that enhance personalized patient management and facilitate the ongoing refinement of postoperative therapy strategies.
PATIENTS AND METHODS
This retrospective study included patients undergoing RARP from September 2014 to January 2021. Exclusion criteria were preoperative endocrine therapy, BCR beyond 2 years post-surgery, and incomplete postoperative data. Multivariate analyses were conducted to evaluate predictors of BCR, focusing on preoperative prostate-specific antigen (PSA) level, pathological tumor (pT) stage, Gleason score (GS), extraprostatic extension (EPE), and surgical margin status.
RESULTS
Among 374 patients, 40 experienced BCR within 2 years. Significant predictors of early BCR included initial PSA level ≥10 ng/ml, pT3 or greater, GS ≥8, EPE, and positive surgical margins (RM1). Multivariate analysis identified pT3 or higher, GS ≥8, and RM1 as independent risk factors for early BCR.
CONCLUSION
Early BCR after RARP is significantly associated with advanced pathological stage, high GS, and positive surgical margins. These findings emphasize the need for tailored postoperative management strategies and highlight the importance of precision in surgical technique to improve patient outcomes.
Topics: Humans; Male; Prostatectomy; Prostate-Specific Antigen; Prostatic Neoplasms; Middle Aged; Neoplasm Grading; Robotic Surgical Procedures; Neoplasm Recurrence, Local; Aged; Retrospective Studies; Neoplasm Staging; Risk Factors; Margins of Excision
PubMed: 38925837
DOI: 10.21873/anticanres.17129 -
Anticancer Research Jul 2024The therapeutic landscape for metastatic castration-resistant prostate cancer (mCRPC) has changed dramatically with the introduction of several novel agents. However,...
BACKGROUND/AIM
The therapeutic landscape for metastatic castration-resistant prostate cancer (mCRPC) has changed dramatically with the introduction of several novel agents. However, limited data are available to determine whether the introduction of novel agents affected patient characteristics, treatment patterns, and outcomes compared with the period when these agents were not available. The objective of this study was to evaluate the impact of the introduction of novel mCRPC agents on patient characteristics, treatment patterns, and outcomes.
PATIENTS AND METHODS
Two cohorts of Japanese patients diagnosed with mCRPC between 2009 and 2014 (Epoch 1) and 2015 and 2019 (Epoch 2) were retrospectively analyzed.
RESULTS
A total of 125 treatment-naïve mCRPC patients, consisting of 42 patients in Epoch 1 and 83 patients in Epoch 2, were evaluated. We obtained the following results: (i) a dramatic shift in the first-line treatment from docetaxel to androgen receptor axis-targeted agents (ARATs), (ii) an age range expansion for first-line treatment, and (iii) an overall survival (OS) advantage in Eopch 2 compared to Epoch 1. Multivariate analysis in the overall population showed that Epoch 2 and low prostate-specific antigen (PSA) levels at the start of first-line treatment were independent prognostic factors for OS.
CONCLUSION
In real-world mCRPC practice, the introduction of novel agents has improved the prognosis of mCRPC while allowing more patients to receive mCRPC treatment across a broad age range. In addition, low PSA levels at the start of first-line treatment were associated with improved OS, indicating the importance of starting mCRPC treatment while PSA levels are low.
Topics: Humans; Male; Prostatic Neoplasms, Castration-Resistant; Aged; Aged, 80 and over; Retrospective Studies; Middle Aged; Treatment Outcome; Docetaxel; Prostate-Specific Antigen; Neoplasm Metastasis; Antineoplastic Combined Chemotherapy Protocols; Antineoplastic Agents
PubMed: 38925833
DOI: 10.21873/anticanres.17130 -
The Journal of Biological Chemistry Jun 2024The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and...
The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect hPSC-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide ChIP enrichment and mechanistic studies show that p38 MAPK-mediated CEBPD upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in MSCs exerts a negative regulatory effect on osteogenesis through a similar mechanism. ChIP-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.
PubMed: 38925326
DOI: 10.1016/j.jbc.2024.107494 -
FASEB Journal : Official Publication of... Jul 2024In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme,...
In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.
Topics: Animals; Hydrogen-Ion Concentration; Fibrinolysin; Polychaeta; Temperature; Molecular Weight; Enzyme Stability; Metals; Electrophoresis, Polyacrylamide Gel; Fibrinolytic Agents
PubMed: 38924451
DOI: 10.1096/fj.202400086RR -
Arteriosclerosis, Thrombosis, and... Jul 2024Most abdominal aortic aneurysms (AAAs) are small with low rupture risk (<1%/y) when diagnosed but slowly expand to ≥55 mm and undergo surgical repair. Patients and... (Review)
Review
CLINICAL PROBLEM
Most abdominal aortic aneurysms (AAAs) are small with low rupture risk (<1%/y) when diagnosed but slowly expand to ≥55 mm and undergo surgical repair. Patients and clinicians require medications to limit AAA growth and rupture, but drugs effective in animal models have not translated to patients.
RECOMMENDATIONS FOR INCREASING TRANSLATION FROM MOUSE MODELS
Use models that simulate human AAA tissue pathology, growth patterns, and rupture; focus on the clinically relevant outcomes of growth and rupture; design studies with the rigor required of human clinical trials; monitor AAA growth using reproducible ultrasound; and perform studies in both males and females.
SUMMARY OF STRENGTHS AND WEAKNESSES OF MOUSE MODELS
The aortic adventitial elastase oral β-aminopropionitrile model has many strengths including simulating human AAA pathology and modeling prolonged aneurysm growth. The Ang II (angiotensin II) model performed less well as it better simulates acute aortic syndrome than AAA. The elastase plus TGFβ (transforming growth factor-β) blocking antibody model displays a high rupture rate, making prolonged monitoring of AAA growth not feasible. The elastase perfusion and calcium chloride models both display limited AAA growth.
Topics: Animals; Aortic Aneurysm, Abdominal; Disease Models, Animal; Humans; Aortic Rupture; Pancreatic Elastase; Mice; Aorta, Abdominal; Female; Disease Progression; Male
PubMed: 38924435
DOI: 10.1161/ATVBAHA.124.320823 -
Arteriosclerosis, Thrombosis, and... Jul 2024
Topics: Proprotein Convertase 9; Humans; Cellular Senescence; Apoptosis; Cardiovascular Diseases; Animals; PCSK9 Inhibitors; Signal Transduction
PubMed: 38924434
DOI: 10.1161/ATVBAHA.124.320140 -
PloS One 2024The development of a cancer vaccine has become an essential focus in the field of medical biotechnology and immunology. In our study, the NY-SAR-35 cancer/testis antigen...
Bioinformatics design of a peptide vaccine containing sarcoma antigen NY-SAR-35 epitopes against breast cancer and evaluation of its immunological function in BALB/c mouse model.
The development of a cancer vaccine has become an essential focus in the field of medical biotechnology and immunology. In our study, the NY-SAR-35 cancer/testis antigen was targeted to design a novel peptide vaccine using bioinformatics tools, and BALB/c mice were used to evaluate the vaccine's immunological function. This evaluation involved assessing peptide-specific IgG levels in the serum via ELISA and measuring the levels of IFN-γ, IL-4, and granzyme B in the supernatant of cultured splenocytes. The final vaccine construct consisted of two T lymphocyte epitopes linked by the AAY linker. This construct displayed high antigenicity, non-allergenicity, non-toxicity, stability, and ability to induce IFN-γ and IL-4. It showed stable dynamics with both human MHC-I and II molecules, as well as mouse MHC-II molecules, and revealed strong Van der Waals and electrostatic energies. Emulsifying our peptide vaccine in incomplete Freund's adjuvant resulted in a remarkable increase in the levels of IgG. The splenocytes of mice that received the combination of peptide and adjuvant displayed a noteworthy increase in IFN-γ, IL-4, and granzyme B secretion. Additionally, their lymphocytes exhibited higher proliferation rates compared to the control group. Our data demonstrated that our vaccine could stimulate a robust immune response, making it a promising candidate for cancer prevention. However, clinical trials are necessary to assess its efficacy in humans.
Topics: Animals; Cancer Vaccines; Mice, Inbred BALB C; Mice; Female; Antigens, Neoplasm; Computational Biology; Humans; Vaccines, Subunit; Breast Neoplasms; Epitopes, T-Lymphocyte; Interleukin-4; Interferon-gamma; Immunoglobulin G; Granzymes; Disease Models, Animal; Protein Subunit Vaccines
PubMed: 38923980
DOI: 10.1371/journal.pone.0306117