Vaccination with a trivalent Klebsiella pneumoniae vaccine confers protection in a murine model of pneumonia.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen and the major cause of healthcare-associated infections, which are increasingly complicated by the prevalence of highly invasive and hyper-virulent K. pneumoniae strains, necessitating the development of alternative strategies for combatting infections caused by this bacterium. In this study, we successfully constructed a fusion antigen called KP-Ag1, comprising three antigens (GlnH, FimA, and KPN_00466) that were previously identified through reverse vaccinology. Immunization with KP-Ag1 formulated with Al(OH)3 adjuvant elicited robust humoral and cellular immune response in mice, and conferred protective immunity in a murine model of K. pneumoniae lung infection. Further analysis of serum IgG subtypes from mice immunized with KP-Ag1 revealed a predominant IgG1 response, indicating that KP-Ag1 predominantly induces a Th2-biased immune response. Additionally, opsonophagocytic killing assay suggested that humoral immune responses play a pivotal role in mediating protection conferred by KP-Ag1. Moreover, KP-Ag1 was found to promote the activation and maturation of BMDCs in vitro, which is essential for subsequent efficient antigen presentation. More importantly, vaccination with KP-Ag1 demonstrated cross-protective efficacy against clinical isolates of K. pneumoniae varying in serotypes, antibiotic resistance, and virulence profiles. Therefore, KP-Ag1 holds promise as a candidate for K. pneumoniae vaccine development.

Dimeric product formation in the self-reaction of small peroxy radicals using synchrotron radiation vacuum ultraviolet photoionization mass spectrometry.

Peroxy radicals (RO2) are key reactive intermediates in atmospheric oxidation processes and yet their chemistry is not fully unraveled. Little is known about their structures and the structures of the dimeric products (ROOR) in the self-reaction of small RO2, which are among the most abundant RO2 in the atmosphere. The product branching ratios of ROOR and their atmospheric roles are still in controversy. Here, the self-reaction of propyl peroxy radicals (C3H7O2), a typical small RO2 radical in the atmosphere, has been studied using synchrotron radiation vacuum ultraviolet photoionization mass spectrometry. Both radical (C3H7O) and closed-shell molecular (C3H6O, C3H7OH, C3H7OOC3H7) products in the self-reaction are observed in photoionization mass spectra and their elusive isomers are definitely identified in mass-selected photoionization spectra. Three isomers of the C3H7OOC3H7 dimeric products, R1OOR1, R1OOR2, and R2OOR2 (R1 and R2 represent 1-C3H7 and 2-C3H7, respectively), as well as their complex structures have been determined for the first time. Kinetic experiments are performed and compared with chemical simulations to reveal the sources of specific products. The branching ratio of the C3H7OOC3H7 dimeric channel is measured at 10 ± 5%. This work demonstrates that the dimeric product formation in the self-reaction of small RO2 radicals is non-negligible and should provide valuable new insight into atmospheric modelling.

Type VII secretion system extracellular protein B targets STING to evade host anti-Staphylococcus aureus immunity.

Staphylococcus aureus (S. aureus) can evade antibiotics and host immune defenses by persisting within infected cells. Here, we demonstrate that in infected host cells, S. aureus type VII secretion system (T7SS) extracellular protein B (EsxB) interacts with the stimulator of interferon genes (STING) protein and suppresses the inflammatory defense mechanism of macrophages during early infection. The binding of EsxB with STING disrupts the K48-linked ubiquitination of EsxB at lysine 33, thereby preventing EsxB degradation. Furthermore, EsxB-STING binding appears to interrupt the interaction of 2 vital regulatory proteins with STING: aspartate-histidine-histidine-cysteine domain-containing protein 3 (DHHC3) and TNF receptor-associated factor 6. This persistent dual suppression of STING interactions deregulates intracellular proinflammatory pathways in macrophages, inhibiting STING's palmitoylation at cysteine 91 and its K63-linked ubiquitination at lysine 83. These findings uncover an immune-evasion mechanism by S. aureus T7SS during intracellular macrophage infection, which has implications for developing effective immunomodulators to combat S. aureus infections.

Effect of incubation temperature on identification of key odorants of sewage sludge using headspace GC analysis.

Currently, headspace gas chromatography-mass spectrometry is a widely used method to identify the key odorants of sludge. However, the effect of incubation temperature on the generation and emission of key odorants from sludge was still uncertain. Thus, in this paper, headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) and headspace gas chromatography-coupled ion mobility spectrometry (HS-GC-IMS) were carried out to analyze the volatiles emitted from the sludge incubated at different temperatures (30 °C, 50 °C, 60 °C, and 80 °C). The results indicated that the total volatile concentration of the sludge increased with temperatures, which affected the identified proportion of sludge key odorants to a certain extent. Differently from the aqueous solutions, the variation of volatile emission from the sludge was inconsistent with temperature changes, suggesting a multifactorial influence of incubation temperature on the identification of sludge odorants. The microbial community structure and adenosine triphosphate (ATP) metabolic activity of the sludge samples were analyzed at the initial state, 30 °C, and 80 °C. Although no significant effect of incubation temperature on the microbial community structure of the sludge, the incubation at 80 °C led to a noticeable decrease in microbial ATP metabolic activity, accompanied by a significant change in the proportion of odor-related microorganisms with low relative abundances. Changes in the composition and activity of these communities jointly contributed to the differences in odor emission from sludge at different temperatures. In summary, the incubation temperature affects the production and emission of volatiles from sludge through physicochemical and biochemical mechanisms, by which the microbial metabolism playing a crucial role. Therefore, when analyzing the key odorants of sludge, these factors should be considered.

Online quantification of nicotine in e-cigarette aerosols by vacuum ultraviolet photoionization mass spectrometry.

The growing popularity of e-cigarettes and the associated risks of nicotine addiction present a new challenge to global public health security. Measuring the nicotine levels in e-cigarette aerosols is essential to assess the safety of e-cigarettes. In this study, a rapid in situ method was developed for online quantification of nicotine in e-cigarette aerosols by using a homemade vacuum ultraviolet photoionization aerosol mass spectrometer (VUV-AMS). E-cigarette liquids with different nicotine concentrations were prepared to generate aerosols containing different levels of nicotine, which were employed as the calibration sources for nicotine quantification by VUV-AMS. The results showed that the mass concentration of nicotine in e-cigarette aerosols has a good linear relationship with its signal intensity in the mass spectrum, and the limits of detection and quantitation of nicotine by VUV-AMS were found to be 2.0 and 6.2 µg per puff respectively. Then the online method was utilized to measure five commercial e-cigarettes, and their nicotine yields were determined to be between 31 and 188 µg per puff with the nicotine fluxes from 7.7 to 70 µg s-1, agreeing with the results of the gas chromatography with a flame ionization detector (GC-FID). This study demonstrated the feasibility and advantages of VUV-AMS for quick quantification of nicotine in e-cigarette aerosols within seconds.

KDELR2 promotes bone marrow mesenchymal stem cell osteogenic differentiation via GSK3ß/ß-catenin signaling pathway.

Nonunion is a challenging complication of fractures for the surgeon. Recently the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum protein retention receptor 2 (KDELR2) has been found that involved in osteogenesis imperfecta. However, the exact mechanism is still unclear. In this study, we used lentivirus infection and mouse fracture model to investigate the role of KDELR2 in osteogenesis. Our results showed that KDELR2 knockdown inhibited the osteogenic differentiation of mBMSCs, whereas KDELR2 overexpression had the opposite effect. Furthermore, the levels of active-ß-catenin and phospho-GSK3ß (Ser9) were upregulated by KDELR2 overexpression and downregulated by KDELR2 knockdown. In the fracture model, mBMSCs overexpressing KDELR2 promoted healing. In conclusion, KDELR2 promotes the osteogenesis of mBMSCs by regulating the GSK3ß/ß-catenin signaling pathway.

CD39 expression defines exhausted CD4+ T cells associated with poor survival and immune evasion in human gastric cancer.

Objectives: CD4+ T cell helper and regulatory function in human cancers has been well characterised. However, the definition of tumor-infiltrating CD4+ T cell exhaustion and how it contributes to the immune response and disease progression in human gastric cancer (GC) remain largely unknown. Methods: A total of 128 GC patients were enrolled in the study. The expression of CD39 and PD-1 on CD4+ T cells in the different samples was analysed by flow cytometry. GC-infiltrating CD4+ T cell subpopulations based on CD39 expression were phenotypically and functionally assessed. The role of CD39 in the immune response of GC-infiltrating T cells was investigated by inhibiting CD39 enzymatic activity. Results: In comparison with CD4+ T cells from the non-tumor tissues, significantly more GC-infiltrating CD4+ T cells expressed CD39. Most GC-infiltrating CD39+CD4+ T cells exhibited CD45RA-CCR7- effector-memory phenotype expressing more exhaustion-associated inhibitory molecules and transcription factors and produced less TNF-α, IFN-γ and cytolytic molecules than their CD39-CD4+ counterparts. Moreover, ex vivo inhibition of CD39 enzymatic activity enhanced their functional potential reflected by TNF-α and IFN-γ production. Finally, increased percentages of GC-infiltrating CD39+CD4+ T cells were positively associated with disease progression and patients' poorer overall survival. Conclusion: Our study demonstrates that CD39 expression defines GC-infiltrating CD4+ T cell exhaustion and their immunosuppressive function. Targeting CD39 may be a promising therapeutic strategy for treating GC patients.

A Respiratory Motion Prediction Method Based on LSTM-AE with Attention Mechanism for Spine Surgery.

Respiratory motion-induced vertebral movements can adversely impact intraoperative spine surgery, resulting in inaccurate positional information of the target region and unexpected damage during the operation. In this paper, we propose a novel deep learning architecture for respiratory motion prediction, which can adapt to different patients. The proposed method utilizes an LSTM-AE with attention mechanism network that can be trained using few-shot datasets during operation. To ensure real-time performance, a dimension reduction method based on the respiration-induced physical movement of spine vertebral bodies is introduced. The experiment collected data from prone-positioned patients under general anaesthesia to validate the prediction accuracy and time efficiency of the LSTM-AE-based motion prediction method. The experimental results demonstrate that the presented method (RMSE: 4.39%) outperforms other methods in terms of accuracy within a learning time of 2 min. The maximum predictive errors under the latency of 333 ms with respect to the x, y, and z axes of the optical camera system were 0.13, 0.07, and 0.10 mm, respectively, within a motion range of 2 mm.

Regional distribution prevalence of heterotopic ossification in the elbow joint: a 3D study of patients after surgery for traumatic elbow injury.

BACKGROUND: Heterotopic ossification (HO) is a common complication after elbow fracture surgery and can lead to severe upper extremity disability. The radiographic localization of postoperative HO has been reported previously. However, there is no literature examining the distribution of postoperative HO at the three-dimensional (3D) level. This study aimed to investigate 1) the distribution characteristics of postoperative HO and 2) the possible risk factors affecting the severity of postoperative HO at a 3D level. METHODS: A retrospective review was conducted of patients who presented to our institution with HO secondary to elbow fracture between 13 January 2020 and 16 February 2023. Computed tomography scans of 56 elbows before elbow release surgery were reconstructed in 3D. HO was identified using density thresholds combined with manual identification and segmentation. The elbow joint and HO were divided into six regions according to three planes: the transepicondylar plane, the lateral ridge of the trochlear plane, and the radiocapitellar joint and coronoid facet plane. The differences in the volume of regional HO associated with different initial injuries were analyzed. RESULTS: Postoperative HO was predominantly present in the medial aspect of the capsule in 52 patients (93%), in the lateral aspect of the capsule in 45 patients (80%), in the medial supracondylar in 32 patients (57%), and in the lateral supracondylar, radial head, and ulnar region in the same number of 28 patients (50%). The median and interquartile range volume of total postoperative HO was 1683 (777-4894) mm3. The median and interquartile range volume of regional postoperative HO were: 584 (121-1454) mm3 at medial aspect of capsule, 207 (5-568) mm3 at lateral aspect of capsule, 25 (0-449) mm3 at medial supracondylar, 1 (0-288) at lateral supracondylar, 2 (0-478) at proximal radius and 7 (0-203) mm3 at the proximal ulna. In the subgroups with Injury Severity Score > or = 16, Gustilo-Anderson II, normal uric acid levels, elevated alkaline phosphatase, and body mass index > or = 24, the median HO volume exceeds that of the respective control groups. CONCLUSION: The medial aspect of the capsule was the area with the highest frequency and median volume of postoperative HO among all initial elbow injury types. Patients with higher Gustilo-Anderson grade, Injury Severity Score, alkaline phosphatase or Body Mass Index had higher median volume of postoperative HO.

Jasminoidin reduces ischemic stroke injury by regulating microglia polarization via PASK-EEF1A1 axis.

Jasminoidin (JAS) can alleviate ischemic stroke (IS) injury, but its molecular mechanism remains undefined. The polarization of microglia affects IS process. This research is powered to probe whether the molecular mechanism of JAS for IS treatment is coupled with microglia polarization. IS modeling in mice was accomplished by middle cerebral artery occlusion (MCAO) and model mice were injected with 25 and 50 mg/mL JAS, followed by determination of infarct volume, brain water content, and histological changes in mouse brains. The microglia modeling was performed by 1-h oxygen-glucose deprivation and 24-h reoxygenation. Oxygen-glucose deprivation/reoxygenation (OGD/R)-induced microglia were treated with JAS and transfected with Per-Arnt-Sim kinase (PASK)-overexpressing plasmid, subsequent to which cell viability and lactate dehydrogenase (LDH) level were determined. The mRNA or protein expressions of examined genes in microglia and brain tissues were detected by quantitative real-time polymerase chain reaction or western blot. MCAO-induced massive infarction, edema, and injury in mouse brain tissues, upregulated interleukin-1 beta (IL-1ß), FcγRIIB (CD32), tumor necrosis factor alpha (TNF-α), PASK, p-eukaryotic elongation factor 1A1 (EEF1A1), and p-EEF1A1/EEF1A1 levels, but downregulated mannose receptor 1 (CD206), arginase-1 (Arg-1) and interleukin-10 (IL-10), and EEF1A1 expressions, which was reversed by JAS. OGD/R treatment decreased microglial viability as well as expressions of CD206, Arg-1, IL-10, and EEF1A1, yet increased cytotoxicity and levels of IL-1ß, CD32, TNF-α, PASK, p-EEF1A1, and p-EEF1A1/EEF1A1, which was reversed by JAS. PASK overexpression reversed the effects of JAS on microglia. JAS reduces IS injury by regulating microglia polarization via PASK-EEF1A1 axis.

Carbon dioxide activation by discandium dioxide cations in the gas phase: a combined investigation of infrared photodissociation spectroscopy and DFT calculations.

We present a combined computational and experimental study of CO2 activation at the Sc2O2+ metal oxide ion center in the gas phase. Density functional theory calculations on the structures of [Sc2O2(CO2)n]+ (n = 1-4) ion-molecule complexes reveal a typical end-on binding motif as well as bidentate and tridentate carbonate-containing configurations. As the number of attached CO2 molecules increases, activated forms tend to dominate the isomeric populations. Distortion energies are unveiled to account for the conversion barriers from molecularly bound isomers to carbonate structures, and show a monotonically decreasing trend with successive CO2 ligand addition. The infrared photodissociation spectra of target ion-molecule complexes were recorded in the 2100-2500 cm-1 frequency region and interpreted by comparison with simulated IR spectra of low-lying isomers representing distinct configurations, demonstrating a high possibility of carbonate structure formation in current experiments.

Artificial intelligence aided precise detection of local recurrence on MRI for nasopharyngeal carcinoma: a multicenter cohort study.

Background: MRI is the routine examination to surveil the recurrence of nasopharyngeal carcinoma, but it has relatively lower sensitivity than PET/CT. We aimed to find if artificial intelligence (AI) could be competent pre-inspector for MRI radiologists and whether AI-aided MRI could perform better or even equal to PET/CT. Methods: This multicenter study enrolled 6916 patients from five hospitals between September 2009 and October 2020. A 2.5D convolutional neural network diagnostic model and a nnU-Net contouring model were developed in the training and test cohorts and used to independently predict and visualize the recurrence of patients in the internal and external validation cohorts. We evaluated the area under the ROC curve (AUC) of AI and compared AI with MRI and PET/CT in sensitivity and specificity using the McNemar test. The prospective cohort was randomized into the AI and non-AI groups, and their sensitivity and specificity were compared using the Chi-square test. Findings: The AI model achieved AUCs of 0.92 and 0.88 in the internal and external validation cohorts, corresponding to the sensitivity of 79.5% and 74.3% and specificity of 91.0% and 92.8%. It had comparable sensitivity to MRI (e.g., 74.3% vs. 74.7%, P = 0.89) but lower sensitivity than PET/CT (77.9% vs. 92.0%, P < 0.0001) at the same individual-specificities. The AI model achieved moderate precision with a median dice similarity coefficient of 0.67. AI-aided MRI improved specificity (92.5% vs. 85.0%, P = 0.034), equaled PET/CT in the internal validation subcohort, and increased sensitivity (81.9% vs. 70.8%, P = 0.021) in the external validation subcohort. In the prospective cohort of 1248 patients, the AI group had higher sensitivity than the non-AI group (78.6% vs. 67.3%, P = 0.23), albeit nonsignificant. In future randomized controlled trials, a sample size of 3943 patients in each arm would be required to demonstrate the statistically significant difference. Interpretation: The AI model equaled MRI by expert radiologists, and AI-aided MRI by expert radiologists equaled PET/CT. A larger randomized controlled trial is warranted to demonstrate the AI's benefit sufficiently. Funding: The Sun Yat-sen University Clinical Research 5010 Program (2015020), Guangdong Basic and Applied Basic Research Foundation (2022A1515110356), and Guangzhou Science and Technology Program (2023A04J1788).

Eicosapentaenoic acid supplementation modulates the osteoblast/osteoclast balance in inflammatory environments and protects against estrogen deficiency-induced bone loss in mice.

BACKGROUND: An imbalance of osteoblasts (OBs) and osteoclasts (OCs) in a chronic inflammatory microenvironment is an important pathological factor leading to osteoporosis. Eicosapentaenoic acid (EPA) has been shown to suppress inflammation in macrophages and adipocytes. However, the effect of EPA on OBs and OCs has yet to be fully elucidated. AIMS: We explored the roles of EPA in the differentiation of OBs and OCs, as well as the coupling between OBs and OCs in an inflammatory microenvironment. The effects of EPA on estrogen deficiency-induced osteoporosis were also evaluated. METHODS: Mouse bone marrow mesenchymal stem cells (mBMSCs) and mouse bone marrow-derived macrophages (mBMMs) were used for in vitro OBs and OCs differentiation. TNF-α was used to create an inflammatory microenvironment. We examined the effects of EPA on osteoblastogenesis in the absence or presence of TNF-α and collect OBs' culture medium as the conditioned medium (CM). Then we examined the effects of EPA and CM on RANKL-induced osteoclastogenesis. The in vivo effects of EPA were determined using an ovariectomized (OVX) mouse model treated with EPA or vehicle. RESULTS: High-dose EPA was shown to promote osteoblastogenesis in an inflammatory environment in vitro, as well as upregulate expression of OBs-specific proteins and genes. ARS and ALP staining also showed that high-dose EPA-treated groups restored mBMSCs' impaired osteogenic capacity caused by TNFa. Mechanistically, EPA suppressed the NF-κB pathway activated by TNF-α in mBMSCs and rescued TNF-α-mediated inhibition of osteoblastogenesis. EPA was also shown to inhibit expression of RANKL and decrease the RANKL/OPG ratio in OBs in an inflammatory environment. CM from TNF-α-stimulated OBs promoted osteoclastogenesis of mBMMs; EPA-treated CM prevented this. In the OVX mouse model, EPA supplementation prevented bone loss in an estrogen deficiency-induced inflammatory environment. CONCLUSIONS: EPA was demonstrated for the first time to restore mBMSCs' impaired osteogenic capacity caused by TNFa-induced inflammation and rescue the OBs/OCs balance via regulation of RANKL and OPG expression in OBs. EPA showed a remarkable ability to prevent bone loss in OVX mice, suggesting a potential application of EPA in postmenopausal osteoporosis.

Molecular properties and biotoxicity of dissolved organic matter leached from microplastic (MP-DOM) during typical hydrothermal treatment of sewage sludge.

Microplastic-derived dissolved organic matter (MP-DOM) is crucial for assessing ecological and environmental impact of microplastics. However, the factors that influence the ecological effects of MP-DOM are yet to be determined. This study investigated the influence of plastic type and leaching conditions (thermal hydrolysis, TH; hydrothermal carbonization, HTC) on the molecular properties and toxicity of MP-DOM using spectroscopy and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Results revealed that plastic type was the main factor affecting the chemodiversity of MP-DOM, compared to leaching conditions. Polyamide 6 (PA6) dissolved the largest number of DOM due to the presence of heteroatoms, followed by polypropylene (PP) and polyethylene (PE). From TH to HTC processes, the molecular composition of PA-DOM remained constant, with CHNO compounds being dominant, and labile compounds (lipids-like, and protein/amino sugar-like compounds) accounting for >90 % of the total compounds. In polyolefin-sourced DOM, CHO compounds were dominant, and the relative concentration of labile compounds decreased dramatically, resulting in the higher degree of unsaturation and humification than PA-DOM. The mass difference network analysis showed that the main reaction in PA-DOM and PE-DOM was oxidation while in PP-DOM, it was the carboxylic acid reaction. However, plastic type and leaching conditions jointly influenced the toxic effects of MP-DOM. PA-DOM was bioavailable, while polyolefin-sourced DOM leached under HTC treatment exhibited toxicity, with lignin/CRAM-like compounds being the dominant toxic compounds. Notably, the 2-fold higher relative intensity of the toxic compounds and the 6-fold higher abundance of highly unsaturated and phenolic-like compounds in PP-DOMHTC resulted in the higher inhibition rate than PE-DOMHTC. Toxic molecules in PE-DOMHTC mainly directly dissolved from PE polymers, while almost 20 % of toxic molecules in PP-DOMHTC resulted from molecular transformation, where dehydration (-H2O) was the core reaction. These findings offer advanced insights into the management and treatment of MPs in sludge.

Incidence and clinical features of HHV-7 detection in lower respiratory tract in patients with severe pneumonia: a multicenter, retrospective study.

PURPOSE: The significance of detecting human herpesvirus 7 (HHV-7) in the lower respiratory tract of patients with severe pneumonia is unclear. This study aims to evaluate the clinical characteristics and prognosis of detecting HHV-7 in the lower respiratory tract of patients with severe pneumonia. METHODS: Patients with severe pneumonia requiring invasive mechanical ventilation and underwent commercial metagenomic next-generation sequencing (mNGS) testing of bronchoalveolar lavage fluid from January 2019 to March 2023 were enrolled in 12 medical centers. Clinical data of patients were collected retrospectively, and propensity score matching was used for subgroup analysis and mortality assessment. RESULTS: In a total number of 721 patients, 45 cases (6.24%) were identified with HHV-7 positive in lower respiratory tract. HHV-7 positive patients were younger (59.2 vs 64.4, p = 0.032) and had a higher rate of co-detection with Cytomegalovirus (42.2% vs 20.7%, p = 0.001) and Epstein-Barr virus (35.6% vs 18.2%, p = 0.008). After propensity score matching for gender, age, SOFA score at ICU admission, and days from ICU admission to mNGS assay, there was no statistically significant difference in the 28-day mortality rate between HHV-7 positive and negative patients (46.2% vs 36.0%, p = 0.395). Multivariate Cox regression analysis adjusting for gender, age, and SOFA score showed that HHV-7 positive was not an independent risk factor for 28-day mortality (HR 1.783, 95%CI 0.936-3.400, p = 0.079). CONCLUSION: HHV-7 was detected in the lungs of 6.24% of patients with severe pneumonia. The presence of HHV-7 in patients with severe pneumonia requiring invasive mechanical ventilation is associated with a younger age and co-detected of Cytomegalovirus and Epstein-Barr virus. While HHV-7 positivity was not found to be an independent risk factor for mortality in this cohort, this result may have been influenced by the relatively small sample size of the study.

PTPRC promoted CD8+ T cell mediated tumor immunity and drug sensitivity in breast cancer: based on pan-cancer analysis and artificial intelligence modeling of immunogenic cell death-based drug sensitivity stratification.

Background: Immunogenic cell death (ICD) is a result of immune cell infiltration (ICI)-mediated cell death, which is also a novel acknowledgment to regulate cellular stressor-mediated cell death, including drug therapy and radiotherapy. Methods: In this study, TCGA and GEO data cohorts were put into artificial intelligence (AI) to identify ICD subtypes, and in vitro experiments were performed. Results: Gene expression, prognosis, tumor immunity, and drug sensitivity showed significance among ICD subgroups, Besides, a 14-gene-based AI model was able to represent the genome-based drug sensitivity prediction, which was further verified in clinical trials. Network analysis revealed that PTPRC was the pivotal gene in regulating drug sensitivity by regulating CD8+ T cell infiltration. Through in vitro experiments, intracellular down-regulation of PTPRC enhanced paclitaxel tolerance in triple breast cancer (TNBC) cell lines. Meanwhile, the expression level of PTPRC was positively correlated with CD8+ T cell infiltration. Furthermore, the down-regulation of PTPRC increased the level of TNBC-derived PD-L1 and IL2. Discussion: ICD-based subtype clustering of pan-cancer was helpful to evaluate chemotherapy sensitivity and immune cell infiltration, and PTPRC was a potential target to against drug resistance of breast cancer.

The pretherapeutic systemic inflammation score is a prognostic predictor for elderly patients with oesophageal cancer: a case control study.

BACKGROUND: The systemic inflammation score (SIS), based on serum albumin (Alb) and lymphocyte-to-monocyte ratio (LMR), is a novel prognostic tool for some tumours. Studies indicate that the SIS can be used as a postoperative prognostic marker. However, its predictive value in elderly oesophageal squamous cell carcinoma (ESCC) patients treated with radiotherapy is unclear. METHODS: In total, 166 elderly ESCC patients who received radiotherapy with or without chemotherapy were included. Based on different combinations of Alb and LMR levels, the SIS was divided into 3 groups, SIS = 0 (n = 79), SIS = 1 (n = 71) and SIS = 2 (n = 16). The Kaplan-Meier method was used for survival analysis. Univariate and multivariate analyses were performed to assess prognosis. Time-dependent receiver operating characteristic (t-ROC) curves were used to compare the prognostic accuracy of the SIS with that of Alb, LMR, neutrophil-to lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and systemic immune-inflammatory index (SII). RESULTS: Decreased Alb and LMR were both associated with shorter OS, whereas a lower SIS was significantly associated with better outcomes. The OS of SIS = 0, SIS = 1 and SIS = 2 was 28.0 ± 2.9, 16.0 ± 2.8 and 10.0 ± 7.0 months, respectively (p = 0.000). Similar results were also observed for PFS. Multivariate analysis of the model with SIS revealed that the SIS was a significant independent biomarker for predicting OS and PFS. The nomogram showed that the C-index was improved to 0.677 when the SIS factor was incorporated. Furthermore, the 3-year OS rates for patients in the SIS-high group (SIS = 1 and SIS = 2) undergoing concurrent radiotherapy with a single agent (CCRT-1) and concurrent radiotherapy with two agents (CCRT-2) were 42% and 15%, respectively (p = 0.039). The t-ROC curve showed that the SIS was more sensitive than other prognostic factors for predicting overall survival. CONCLUSION: The SIS may be a useful prognostic marker in elderly patients with ESCC receiving radiotherapy alone or chemoradiotherapy. The SIS showed a better predictive ability for OS than the continuous variable Alb and could stratify patient prognosis in different therapeutic regimens. CCRT-1 may be the best treatment for SIS-high patients.

LAMP2A regulates the balance of mesenchymal stem cell adipo-osteogenesis via the Wnt/ß-catenin/GSK3ß signaling pathway.

Chaperone-mediated autophagy (CMA) plays multiple roles in cell metabolism. We found that lysosome-associated membrane protein type 2A (LAMP2A), a crucial protein of CMA, plays a key role in the control of mesenchymal stem cell (MSC) adipo-osteogenesis. We identified a differentially expressed CMA gene (LAMP2) in GEO datasets (GSE4911 and GSE494). Further, we performed co-expression analyses to define the relationships between CMA components genes and other relevant genes including Col1a1, Runx2, Wnt3 and Gsk3ß. Mouse BMSCs (mMSCs) exhibiting Lamp2a gene knockdown (LA-KD) and overexpression (LA-OE) were created using an adenovirus system; then we investigated LAMP2A function in vitro by Western blot, Oil Red staining, ALP staining, ARS staining and Immunofluorescence analysis. Next, we used a modified mouse model of tibial fracture to investigate LAMP2A function in vivo. LAMP2A knockdown in mMSCs decreased the levels of osteogenic-specific proteins (COL1A1 and RUNX2) and increased those of the adipogenesis markers PPARγ and C/EBPα; LAMP2A overexpression had the opposite effects. The active-ß-catenin and phospho-GSK3ß (Ser9) levels were upregulated by LAMP2A overexpression and downregulated by LAMP2A knockdown. In the mouse model of tibial fracture, mMSC-overexpressing LAMP2A improved bone healing, as demonstrated by microcomputed tomography and histological analyses. In summary, LAMP2A positively regulates mMSC osteogenesis and suppresses adipo-osteogenesis, probably via Wnt/ß-catenin/GSK3ß signaling. LAMP2A promoted fracture-healing in the mouse model of tibial fracture. KEY MESSAGES: • LAMP2 positively regulates the mBMSCs osteogenic differentiation. • LAMP2 negatively regulates the mBMSCs adipogenic differentiation. • LAMP2 regulates mBMSCs osteogenesis via Wnt/ß-catenin/GSK3ß signaling pathway. • LAMP2 overexpression mBMSCs promote the fracture healing.

MFG-E8 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells through GSK3ß/ß-catenin signaling pathway.

Fracture nonunion and bone defects are challenging for orthopedic surgeons. Milk fat globule-epidermal growth factor 8 (MFG-E8), a glycoprotein possibly secreted by macrophages in a fracture hematoma, participates in bone development. However, the role of MFG-E8 in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. We investigated the osteogenic effect of MFG-E8 in vitro and in vivo. The CCK-8 assay was used to assess the effect of recombinant human MFG-E8 (rhMFG-E8) on the viability of hBMSCs. Osteogenesis was investigated using RT-PCR, Western blotting, and immunofluorescence. Alkaline phosphatase (ALP) and Alizarin red staining were used to evaluate ALP activity and mineralization, respectively. An enzyme-linked immunosorbent assay was conducted to evaluate the secretory MFG-E8 concentration. Knockdown and overexpression of MFG-E8 in hBMSCs were established via siRNA and lentivirus vector transfection, respectively. Exogenous rhMFG-E8 was used to verify the in vivo therapeutic effect in a tibia bone defect model based on radiographic analysis and histological evaluation. Endogenous and secretory MFG-E8 levels increased significantly during the early osteogenic differentiation of hBMSCs. Knockdown of MFG-E8 inhibited the osteogenic differentiation of hBMSCs. Overexpression of MFG-E8 and rhMFG-E8 protein increased the expression of osteogenesis-related genes and proteins and enhanced calcium deposition. The active ß-catenin to total ß-catenin ratio and the p-GSK3ß protein level were increased by MFG-E8. The MFG-E8-induced enhanced osteogenic differentiation of hBMSCs was partially attenuated by a GSK3ß/ß-catenin signaling inhibitor. Recombinant MFG-E8 accelerated bone healing in a rat tibial-defect model. In conclusion, MFG-E8 promotes the osteogenic differentiation of hBMSCs by regulating the GSK3ß/ß-catenin signaling pathway and so, is a potential therapeutic target.

EGFL7 Secreted By Human Bone Mesenchymal Stem Cells Promotes Osteoblast Differentiation Partly Via Downregulation Of Notch1-Hes1 Signaling Pathway.

BACKGROUND: Epidermal growth factor-like domain protein 7 (EGFL7) is a secreted protein that is differentially expressed in the bone microenvironment; however, the effect of EGFL7 on the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) is largely unknown. METHODS: EGFL7 expression in the fracture microenvironment was analyzed based on the Gene Expression Omnibus (GEO) database. Knockdown of EGFL7 by small interfering RNA (siRNA) and in vitro stimulation with recombinant human EGFL7 (rhEGFL7) protein were used to assess alterations in downstream signaling and changes in the osteogenic differentiation and proliferation of hBMSCs. A γ-secretase inhibitor was used to further explore whether inhibition of Notch signaling rescued the osteogenic-inhibitory effect of EGFL7 knockdown in hBMSCs. A femoral defect model was established to verify the effect of recombinant mouse EGFL7 on bone healing in vivo. RESULTS: EGFL7 expression increased during hBMSC osteogenesis. Knockdown of EGFL7 impaired hBMSC osteogenesis and activated Notch1/NICD/Hes1 signaling. rhEGFL7 promoted hBMSC osteogenesis and downregulated Notch1 signaling. The osteoblast-inhibitory effect of EGFL7 knockdown was rescued by Notch1 signaling inhibition. Recombinant EGFL7 led to enhanced bone healing in mice with femoral defects. CONCLUSIONS: EGFL7 promotes osteogenesis of hBMSCs partly via downregulation of Notch1 signaling.