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ObjectiveThis study aimed to establish an effective prognostic model based on triglyceride and inflammatory markers, including neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR), to predict overall survival (OS) in patients with nasopharyngeal carcinoma (NPC). Additionally, we aimed to explore the interaction and mediation between these biomarkers in their association with OS.MethodsA retrospective review was conducted on 259 NPC patients who had blood lipid markers, including triglyceride and total cholesterol, as well as parameters of peripheral blood cells measured before treatment. These patients were followed up for over 5 years, and randomly divided into a training set (n=155) and a validation set (n=104). The triglyceride-inflammation (TI) score was developed using the random survival forest (RSF) algorithm. Subsequently, a nomogram was created. The performance of the prognostic model was measured by the concordance index (C-index), time-dependent receiver operating characteristic (ROC) curve, and decision curve analysis (DCA). The interaction and mediation between the biomarkers were further analyzed. Bioinformatics analysis based on the GEO dataset was used to investigate the association between triglyceride metabolism and immune cell infiltration.ResultsThe C-index of the TI score was 0.806 in the training set, 0.759 in the validation set, and 0.808 in the entire set. The area under the curve of time-dependent ROC of TI score in predicting survival at 1, 3, and 5 years were 0.741, 0.847, and 0.871 respectively in the training set, and 0.811, 0.837, and 0.758 in the validation set, then 0.771, 0.848, and 0.862 in the entire set, suggesting that TI score had excellent performance in predicting OS in NPC patients. Patients with stage T1-T2 or M0 had significantly lower TI scores, NLR, and PLR, and higher LMR compared to those with stage T3-T3 or M1, respectively. The nomogram, which integrated age, sex, clinical stage, and TI score, demonstrated good clinical usefulness and predictive ability, as evaluated by the DCA. Significant interactions were found between triglyceride and NLR and platelet, but triglyceride did not exhibit any medicating effects in the inflammatory markers. Additionally, NPC tissues with active triglyceride synthesis exhibited high immune cell infiltration.ConclusionThe TI score based on RSF represents a potential prognostic factor for NPC patients, offering convenience and economic advantages. The interaction between triglyceride and NLR may be attributed to the effect of triglyceride metabolism on immune response.

BackgroundHuman milk contains a complex mixture of triacylglycerols (TAG), making it challenging to recreate using common ingredients.ObjectiveThe study aimed to develop an innovative fermentation technique to produce essential human milk TAG, effectively tackling a significant hurdle in infant nutrition.MethodAn in-depth analysis of the literature has been conducted to identify the specific TAG to be targeted. We used a microalgal oil production platform and a two-step procedure to modify its fatty acid and TAG composition. The palmitic acid (16:0) content has been increased by classical strain improvement techniques, followed by a step involving the expression of a lysophosphatidic acid acyltransferase (LPAAT) sequence capable of esterifying 16:0 specifically at the internal position (sn-2 palmitate) of TAG. Once the strain was stabilized, the fermentation was scaled up in a 50-L reactor to yield several kilograms of biomass. Subsequently, the oil was extracted and refined using standard oil processing conditions. Liquid chromatography-mass spectrometry was employed to monitor the TAG profile and the region specificity of 16:0 at the internal position (sn-2 palmitate) of TAG.ResultsThe initial strain had a 16:0 level of 25% of total fatty acids, which was increased to 30% by classical strain improvement. Simultaneously, the oleic acid level decreased from 61% to 57% of total fatty acids. Upon expression of an exogenous LPAAT gene, the level of the 16:0 esterified in the internal position of the TAG (sn-2 palmitate) increased by a factor of 10, to reach 73% of total palmitic acid. Consequently, the concentration of oleic acid in the internal position decreased from 81% to 22% of total fatty acids, with TAG analysis confirming that the primary TAG species in the oil was 1,3-dioleoyl-2-palmitoyl-glycerol (OPO). The 50-L-scale fermentation trial confirmed the strain's ability to produce oil with a yield of >150 g of oil per liter of fermentation broth in a timeframe of 5 days, rendering the process scalable for larger-scale industrialization.ConclusionWe have demonstrated the feasibility of producing a suitable TAG composition that can be effectively integrated into the formulations of infant nutrition in combination with other fats and oils to meet the infant feeding requirements.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2–vaccinated and SARS-CoV-2–infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

Lipid metabolism reprogramming is one of the most prominent metabolic anomalies in cancer, wherein cancer cells undergo dysregulation of lipid metabolism to acquire adequate energy, cell membrane building blocks, as well as signaling molecules essential for cell proliferation, survival, invasion, and metastasis. These adaptations enable cancer cells to effectively respond to challenges posed by the tumor microenvironment, leading to cancer therapy resistance and poor cancer prognosis. Head and neck cancer, ranking as the seventh most prevalent cancer, exhibits numerous abnormalities in lipid metabolism. Nevertheless, the precise role of lipid metabolic rewiring in head and neck cancer remains unclear. In line with the LIPID MAPS Lipid Classification System and cancer risk factors, the present review delves into the dysregulated molecules and pathways participating in the process of lipid uptake, biosynthesis, transportation, and catabolism. We also present an overview of the latest advancements in understanding alterations in lipid metabolism and how they intersect with the carcinogenesis, development, treatment, and prognosis of head and neck cancer. By shedding light on the significance of metabolic therapy, we aspire to improve the overall prognosis and treatment outcomes of head and neck cancer patients.

Loss of cell–cell adhesions is the indispensable first step for cancer cells to depart from the primary tumor mass to metastasize. Metastasis suppressor 1 (MTSS1) is frequently lost in metastatic tissues, correlating to advanced tumor stages and poor prognosis across a variety of cancers. Here we explore the anti-metastatic mechanisms of MTSS1, which have not been well understood. We found that MTSS1 is downregulated in NPC tissues. Lower levels of MTSS1 expression correlate to worse prognosis. We show that MTSS1 suppresses NPC cell migration and invasion in vitro through cytoskeletal remodeling at cell–cell borders and assembly of E-cadherin/β-catenin/F-actin in adherens junctions. The I-BAR domain of MTSS1 was both necessary and sufficient to restore this formation of E-cadherin/β-catenin/F-actin–mediated cell adherens junctions.

Reducing chemical exposure is a crucial principle in alternatives assessment (AA) frameworks. Since the release of the report, A Framework to Guide Selection of Chemical Alternatives by the National Research Council in 2014, comparative exposure assessment (CEA) has been increasingly viewed as an essential part of selecting safer alternatives to chemicals of concern in consumer products. However, CEA has not been fully integrated into existing AA frameworks. CEA remains merely a technical step, disconnected from other AA components. This paper advocates for the integration of CEA as an essential part of AA, providing a holistic approach to identifying safer alternatives. The paper aims to illustrate the connection between CEA and other AA components, such as problem formulation, hazard assessment, life cycle assessment, economic assessment, and decision-making. It suggests systematic integration of CEA with cross-cutting AA considerations, including transparency, uncertainty, chemical mixtures, and sensitive receptors. This integration will enable the selection of a fit-for-purpose CEA approach based on the decision context and foster a more comprehensive approach to identifying safer alternatives. While the examples provided are not exhaustive, they aim to encourage further discussion on the integration of CEA into AA.

Dietary fibers (DFs) and their metabolites attract significant attention in research on health and disease, attributing to their effects on regulating metabolism, proliferation, inflammation, and immunity. When fermented by gut microbiota, DFs mainly produce short-chain fatty acids (SCFAs), such as acetic acid, propionic acid, and butyric acid. As the essential nutrients for intestinal epithelial cells, SCFAs maintain intestinal homeostasis and play essential roles in a wide range of biological functions. SCFAs have been found to inhibit histone deacetylase, activate G protein-coupled receptors, and modulate the immune response, which impacts cancer and anti-cancer treatment. Notably, while extensive studies have illuminated the roles of SCFAs in colorectal cancer development, progression, and treatment outcomes, limited evidence is available for other types of cancers. This restricts our understanding of the complex mechanisms and clinical applications of SCFAs in tumors outside the intestinal tract. In this study, we provide a comprehensive summary of the latest evidence on the roles and mechanisms of SCFAs, with a focus on butyric acid and propionic acid, derived from microbial fermentation of DFs in cancer. Additionally, we recapitulate the clinical applications of SCFAs in cancer treatments and offer our perspectives on the challenges, limitations, and prospects of utilizing SCFAs in cancer research and therapy.

Epstein-Barr virus (human herpesvirus 4, EBV) is a linear double-stranded DNA virus that infects over 90% of the population worldwide. However, our understanding of EBV’s contribution to tumorigenesis of EBV-associated GC (EBVaGC) remains incomplete. Recent advancements in EBVaGC research have highlighted that EBV-encoded microRNAs (miRNAs) play prominent roles in critical cellular processes such as migration, cell cycle, apoptosis, cell proliferation, immune response, and autophagy. Notably, the largest group of EBV-encoded miRNAs, known as BamHI-A rightward transcripts (BARTs), exhibit bidirectional effects in EBVaGC. For instance, they present both anti-apoptotic and pro-apoptotic functions and enhance chemosensitivity while also conferring resistance to 5-fluorouracil. Despite these findings, the comprehensive mechanisms through which miRNAs contribute to EBVaGC are yet to be fully elucidated. In this work, we summarize the current evidence of the roles of miRNA in EBVaGC, particularly with the application of multi-omic techniques. Additionally, we discuss the application of miRNA in EBVaGC in retrospective analyses and provide novel perspectives on the use of miRNA in EBVaGC in translational medicine.

Substantial improvement in prognosis among metastatic renal cell carcinoma (mRCC) patients has been achieved, owing to the rapid development and utilization of immunotherapy. In particular, immune checkpoint inhibitors (ICIs) have been considered the backbone of systemic therapy for patients with mRCC alongside multi-targeted tyrosine kinase inhibitors (TKIs) in the latest clinical practice guidelines. However, controversies and challenges in optimal individualized treatment regarding immunotherapy remains still About 2/3 of the patients presented non-response or acquired resistance to ICIs. Besides, immune-related toxicities, namely immune-related adverse events, are still elusive and life-threatening. Thus, reliable biomarkers to predict immunotherapeutic outcomes for mRCC patients are needed urgently. Tumor microenvironment (TME), consisting of immune cells, vasculature, signaling molecules, and extracellular matrix and regulates tumor immune surveillance and immunological evasion through complex interplay, plays a critical role in tumor immune escape and consequently manipulates the efficacy of immunotherapy. Various studied have identified the different TME components are significantly associated with the outcome of mRCC patients receiving immunotherapy, making them potential valuable biomarkers in therapeutic guidance. The present review aims to summarize the latest evidence on the associations between the components of TME including immune cells, cytokines and extracellular matrix, and the therapeutic responses among mRCC patients with ICI-based treatment. We further discuss the feasibility and limitation of these components as biomarkers.

BackgroundAlthough the gut microbiota may be involved in obesity onset and progression, the exact association of the gut microbiota in metabolically healthy obesity (MHO) remains largely unknown.MethodsAn integrated paired-sample metagenomic analysis was conducted to investigate the gut microbial network and biomarkers of microbial species from the MHO and healthy non-obese subjects in the GMrepo database. Further explorations were performed in the MHO mice model using a multiomics analysis to detect changes in the composition and function of the intestinal microbiome and associated metabolites.ResultsIn the human study, 314 matched metagenomic data were qualified for the final analysis. We identified seven significantly changed species possibly involved in MHO pathogenesis (MHO-enriched: Bacteroides vulgatus, Megamonas sp; MHO-depleted: Butyrivibrio crossotus, Faecalibacterium prausnitzii, Bacteroides cellulosilyticus; Eubacterium siraeum; Bacteroides massiliensis). In the murine study, we found 79 significantly-changed species which may have possible associations with the MHO phenotype. The depletion of Bacteroides cellulosilyticus was commonly recognized in the human and murine MHO phenotype. Consistent with the metagenomic data, liquid chromatography-mass spectrometry (LC/MS) revealed significantly changed gut metabolites, which may promote MHO pathogenesis by altering the amino acids and lipid metabolic pathways. In the microbe-metabolites interaction analysis, we identified certain fatty acids (Dodecanedioic acid, Arachidic Acid, Mevalonic acid, etc.) that were significantly correlated with the MHO-enriched or depleted species.ConclusionThis study provides insights into identifying specific microbes and metabolites that may involve in the development of obesity without metabolic disorders. Future modalities for MHO intervention may be further validated by targeting these bacteria and metabolites.

ObjectiveTo conduct a cost-effectiveness analysis of drug-eluting beads transcatheter arterial chemoembolization (DEB-TACE) and conventional transcatheter arterial chemoembolization (cTACE) for first-line treatment of hepatocellular carcinoma (HCC) from the perspective of the Chinese healthcare system.MethodsBased on the real-world clinical data of HCC patients receiving interventional therapy, a partitioned survival model was constructed for cost-effectiveness analysis. The model period is 1 month, and the research time limit is 10 years. The incremental cost-effectiveness ratio (ICER) is used as the evaluation index. One-way sensitivity analysis and probabilistic sensitivity analysis were used to analyze the uncertainty of parameters to test the stability of the model results.ResultsThe ICER of the DEB-TACE group was 11,875.62 $/QALYs, which was lower than the willingness to pay threshold (WTP) of 31,499.23 $/QALYs. One-way sensitivity analysis suggested that the utility value of progression-free survival (PFS) in the DEB-TACE group had the greatest impact. Probabilistic sensitivity analysis showed that at the level of WTP of 31,499.23 $/QALYs, DEB-TACE had a cost-effective probability of 92%.ConclusionUnder the current economic level in my country, DEB-TACE is more cost-effective than cTACE in the treatment of HCC patients.

BackgroundDelayed post-polypectomy bleeding (DPPB) is the most common complication which occurs within 30 days after polypectomy, it has become rather common with the widespread of colorectal cancer screening. It is important to clarified predictors of DPPB and identify patients at high risk.Materials and methodsThis was a real-world retrospective study based on medical records from The First Affiliated Hospital of Nanjing Medical University. Cases of patients who underwent colonoscopic polypectomy between January 2016 and December 2020 were reviewed to identify risk factors of DPPB. We use the LASSO-Logistic regression analysis model to identify independent predictors and create a predictive model. The model finally got visualized by developing a nomogram.ResultsColonoscopic polypectomy was done on 16,925 patients in our study. DPPB occurred in 125 (0.74%) of these instances. In multivariate analysis, age, sex, hypertension, polyp location, polyp size, and operative modality were found to be independent risk factors and were integrated for the construction of a nomogram. The model’s C-index is 0.801 (95%CI: 0.761–0.846). We also found polyps located at the right semicolon and polyp ≥ 1 cm associated with active bleeding under the therapeutic colonoscopy.ConclusionYoung age, male, hypertension, polyp ≥ 1 cm, proximal colon location and operative modality were finally identified as significant predictors of DPPB. We developed and validated a nomogram which performs well in predicting the incidence of DPPB, the model we established can be used as a valuable screening tool to identify patients who are at high risk of bleeding.

Previous researches have emphasized a trypsin-centered theory of acute pancreatitis (AP) for more than a century. With additional studies into the pathogenesis of AP, new mechanisms have been explored. Among them, the role of immune response bears great importance. Pro-inflammatory substances, especially damage-associated molecular patterns (DAMPs), play an essential role in activating, signaling, and steering inflammation. Meanwhile, activated neutrophils attach great importance to the immune defense by forming neutrophil extracellular traps (NETs), which cause ductal obstruction, premature trypsinogen activation, and modulate inflammation. In this review, we discuss the latest advances in understanding the pathological role of DAMPs and NETs in AP and shed light on the flexible crosstalk between these vital inflammatory mediators. We, then highlight the potentially promising treatment for AP targeting DAMPs and NETs, with a focus on novel insights into the mechanism, diagnosis, and management of AP.

Commensal microbes cross talk with their colonized mucosa. We show that microbes and their cell wall components induce an inflammatory response in cultured human mucosal cells derived from the nonmalignant nasopharyngeal epithelium (NNE) cells in vitro. NNE cells show significant induction of NF-κB with nuclear shuttling and inflammatory gene response when exposed to Gram-positive bacteria (streptococci) or peptidoglycan (PGN), a component of the Gram-positive bacterial cell wall. This response is abrogated in nasopharyngeal carcinoma (NPC)–derived cell lines. The inflammatory response induced by NF-κB signaling was blocked at two levels in the tumor-derived cells. We found that NF-κB was largely trapped in lipid droplets (LDs) in the cytoplasm of the NPC-derived cells, while the increased expression of lysine-specific histone demethylase 1 (LSD1, a repressive nuclear factor) reduces the response mediated by remaining NF-κB at the promoters responding to inflammatory stimuli. This refractory response in NPC cells might be a consequence of long-term exposure to microbes in vivo during carcinogenic progression. It may contribute to the decreased antitumor immune responses in NPC, among others despite heavy T-helper cell infiltration, and thus facilitate tumor progression.

BackgroundPeripherally inserted central catheter (PICC) and centrally inserted totally implanted access port (PORT) are two types of intravenous infusion devices that are widely used in clinical practice. PORTs are more expensive to insert than PICCs but have fewer complications. Two cost-utility analyses of PICCs and PORTs in China have been published, but had conflicting findings. This study aimed to compare the cost-utility of PICCs and PORTs.MethodsWe conducted a prospective observational trial including 404 patients with cancer and a cross-sectional study to calculate cost and complications of a PICC and PORT. Utility was measured using the EuroQol five-dimensional questionnaire (EQ-5D-5L). A cost-utility analysis was performed from a healthcare system perspective in China.ResultsThe average total cost of PICCs and PORTs were ¥ 4,091.7 and ¥ 4,566.8, which yielded 0.46 and 0.475 quality-adjusted life-years (QALYs) in a 6-month dwell time, respectively. The incremental cost-utility ratio (ICUR) was ¥ 31,670.9 per QALY. A one-way sensitivity analysis showed that the base-case results were robust, and the probabilistic sensitivity analysis showed that at a willingness-to-pay (WTP) threshold of ¥ 80,976 per QALY (China's per capita GDP in 2021) the probability of a PORT being cost-effective was 96%.ConclusionPORTs were more cost-effective than PICCs for a 6 and 12-month dwell time. The total cost for a PORT was also less than that of a PICC. PORT is therefore recommended as a medium to long-term intravenous delivery device in clinical practice.

Plasma lipidomics has been commonly used for biomarker discovery. Studies in cancer have suggested a significant alteration of circulating metabolite profiles which is correlated with cancer characteristics and treatment outcome. However, the lipidomics characteristics of nasopharyngeal carcinoma (NPC) have rarely been studied. We previously described the phenomenon of lipid droplet accumulation in NPC cells and showed that such accumulation could be regulated by latent infection of Epstein–Barr virus (EBV). Here, we compared the plasma lipidome of NPC patients to that of healthy controls by liquid chromatography-tandem mass spectrometry (LC–MS/MS). We found 19 lipids (e.g., phosphatidylinositols 18:0/20:4 and 18:0/18:2 and free fatty acid 22:6) to be remarkably decreased, whereas 2 lipids (i.e., diacylglycerols 16:0/16:1 and 16:0/20:3) to be increased, in the plasma of NPC patients, compared with controls. Different lipid profiles were also observed between patients with different titers of EBV antibodies (e.g., EA-IgA and VCA-IgA) as well as between patients with and without lymph node or distant organ metastasis. In conclusion, plasma lipidomics might help to differentiate NPC cases from controls, whereas EBV infection might influence the risk and prognosis of NPC through modulating lipid metabolism in both tumor cells and peripheral blood.

Background and AimsThis study aimed to develop an interpretable random forest model for predicting severe acute pancreatitis (SAP).MethodsClinical and laboratory data of 648 patients with acute pancreatitis were retrospectively reviewed and randomly assigned to the training set and test set in a 3:1 ratio. Univariate analysis was used to select candidate predictors for the SAP. Random forest (RF) and logistic regression (LR) models were developed on the training sample. The prediction models were then applied to the test sample. The performance of the risk models was measured by calculating the area under the receiver operating characteristic (ROC) curves (AUC) and area under precision recall curve. We provide visualized interpretation by using local interpretable model-agnostic explanations (LIME).ResultsThe LR model was developed to predict SAP as the following function: -1.10-0.13×albumin (g/L) + 0.016 × serum creatinine (μmol/L) + 0.14 × glucose (mmol/L) + 1.63 × pleural effusion (0/1)(No/Yes). The coefficients of this formula were utilized to build a nomogram. The RF model consists of 16 variables identified by univariate analysis. It was developed and validated by a tenfold cross-validation on the training sample. Variables importance analysis suggested that blood urea nitrogen, serum creatinine, albumin, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, calcium, and glucose were the most important seven predictors of SAP. The AUCs of RF model in tenfold cross-validation of the training set and the test set was 0.89 and 0.96, respectively. Both the area under precision recall curve and the diagnostic accuracy of the RF model were higher than that of both the LR model and the BISAP score. LIME plots were used to explain individualized prediction of the RF model.ConclusionsAn interpretable RF model exhibited the highest discriminatory performance in predicting SAP. Interpretation with LIME plots could be useful for individualized prediction in a clinical setting. A nomogram consisting of albumin, serum creatinine, glucose, and pleural effusion was useful for prediction of SAP.

Background and AimsThe aim of this study was to apply machine learning models and a nomogram to differentiate critically ill from non-critically ill COVID-19 pneumonia patients.MethodsClinical symptoms and signs, laboratory parameters, cytokine profile, and immune cellular data of 63 COVID-19 pneumonia patients were retrospectively reviewed. Outcomes were followed up until Mar 12, 2020. A logistic regression function (LR model), Random Forest, and XGBoost models were developed. The performance of these models was measured by area under receiver operating characteristic curve (AUC) analysis.ResultsUnivariate analysis revealed that there was a difference between critically and non-critically ill patients with respect to levels of interleukin-6, interleukin-10, T cells, CD4+ T, and CD8+ T cells. Interleukin-10 with an AUC of 0.86 was most useful predictor of critically ill patients with COVID-19 pneumonia. Ten variables (respiratory rate, neutrophil counts, aspartate transaminase, albumin, serum procalcitonin, D-dimer and B-type natriuretic peptide, CD4+ T cells, interleukin-6 and interleukin-10) were used as candidate predictors for LR model, Random Forest (RF) and XGBoost model application. The coefficients from LR model were utilized to build a nomogram. RF and XGBoost methods suggested that Interleukin-10 and interleukin-6 were the most important variables for severity of illness prediction. The mean AUC for LR, RF, and XGBoost model were 0.91, 0.89, and 0.93 respectively (in two-fold cross-validation). Individualized prediction by XGBoost model was explained by local interpretable model-agnostic explanations (LIME) plot.ConclusionsXGBoost exhibited the highest discriminatory performance for prediction of critically ill patients with COVID-19 pneumonia. It is inferred that the nomogram and visualized interpretation with LIME plot could be useful in the clinical setting. Additionally, interleukin-10 could serve as a useful predictor of critically ill patients with COVID-19 pneumonia.

Chloroplast development is crucial for photosynthesis and plant growth and many factors are involved in its regulation. The regulatory mechanism differs in different green tissues, and previous studies have focused on chloroplasts in leaves. In this study, a mutant with sepal-specific chlorophyll-deficiency was observed in Brassica napus and named as df74. Genetic analysis indicated that the phenotype was controlled by a single recessive nuclear gene. The gene was located on chromosome C08 by bulked-segregant analysis with whole-genome sequencing, which was designated as BnC08.cds. To fine-map the BnC08.cds, a F2 population was created from the cross of df74 and Zhongshuang11 (ZS11). Finally, the BnC08.cds was fine-mapped in the region between the single-nucleotide polymorphism (SNP) markers M5 and M6, corresponding to a 228.72 kb interval of the B. napus “ZS11” genome. Eighteen genes were predicted in the target region, and it was speculated that BnaC08G0442100ZS was the most likely candidate gene based on the results of transcriptome analyses and sequence variation analyses. These results provide a foundation to explore the regulation of chloroplast development in sepals.

BackgroundThe human microbiome has been reported to mediate the response to anticancer therapies. However, research about the influence of the oral microbiome on nasopharyngeal carcinoma (NPC) survival is lacking. We aimed to explore the effect of oral microbiota on NPC prognosis.MethodsFour hundred eighty-two population-based NPC cases in southern China between 2010 and 2013 were followed for survival, and their saliva samples were profiled using 16s rRNA sequencing. We analyzed associations of the oral microbiome diversity with mortality from all causes and NPC.ResultsWithin- and between-community diversities of saliva were associated with mortality with an average of 5.29 years follow-up. Lower Faith’s phylogenetic diversity was related to higher all-cause mortality [adjusted hazard ratio (aHR), 1.52 (95% confidence interval (CI), 1.06–2.17)] and NPC-specific mortality [aHR, 1.57 (95% CI, 1.07–2.29)], compared with medium diversity, but higher phylogenetic diversity was not protective. The third principal coordinate (PC3) identified from principal coordinates analysis (PCoA) on Bray–Curtis distance was marginally associated with reduced all-cause mortality [aHR, 0.85 (95% CI, 0.73–1.00)], as was the first principal coordinate (PC1) from PCoA on weighted UniFrac [aHR, 0.86 (95% CI, 0.74–1.00)], but neither was associated with NPC-specific mortality. PC3 from robust principal components analysis was associated with lower all-cause and NPC-specific mortalities, with HRs of 0.72 (95% CI, 0.61–0.85) and 0.71 (95% CI, 0.60–0.85), respectively.ConclusionsOral microbiome may be an explanatory factor for NPC prognosis. Lower within-community diversity was associated with higher mortality, and certain measures of between-community diversity were related to mortality. Specifically, candidate bacteria were not related to mortality, suggesting that observed associations may be due to global patterns rather than particular pathogens.

Lipid is the building block and an important source of energy, contributing to the malignant behavior of tumor cells. Recent studies suggested that lipid droplets (LDs) accumulations were associated with nasopharyngeal carcinoma (NPC) progression. Solute carrier family 27 member 6 (SLC27A6) mediates the cellular uptake of long-chain fatty acid (LCFA), a necessary lipid component. However, the functions of SLC27A6 in NPC remain unknown. Here, we found a significant reduction of SLC27A6 mRNA in NPC tissues compared with normal nasopharyngeal epithelia (NNE). The promoter methylation ratio of SLC27A6 was greater in NPC than in non-cancerous tissues. The demethylation reagent 5-aza-2’-deoxycytidine (5-aza-dC) remarkably restored the mRNA expression of SLC27A6, suggesting that this gene was downregulated in NPC owing to DNA promoter hypermethylation. Furthermore, SLC27A6 overexpression level in NPC cell lines led to significant suppression of cell proliferation, clonogenicity in vitro, and tumorigenesis in vivo. Higher SLC27A6 expression, on the other hand, promoted NPC cell migration and invasion. In particular, re-expression of SLC27A6 faciliated epithelial-mesenchymal transition (EMT) signals in xenograft tumors. Furthermore, we observed that SLC27A6 enhanced the intracellular amount of triglyceride (TG) and total cholesterol (T-CHO) in NPC cells, contributing to lipid biosynthesis and increasing metastatic potential. Notably, the mRNA level of SLC27A6 was positively correlated with cancer stem cell (CSC) markers, CD24 and CD44. In summary, DNA promoter hypermethylation downregulated the expression of SLC27A6. Furthermore, re-expression of SLC27A6 inhibited the growth capacity of NPC cells but strengthened the CSC markers. Our findings revealed the dual role of SLC27A6 in NPC and shed novel light on the link between lipid metabolism and CSC maintenance.

Gravity-flow can carry a large number of sediments and organic matters from shallow water to deep lakes with its strong transporting energy, directly or indirectly facilitating the formation of deep-water tight reservoirs and shale reservoirs. Therefore, studying the genetic types, dynamic mechanisms, and depositional models of gravity-flow deposits is essential in the exploration of unconventional petroleum in large lacustrine basins. This research studied the genetic types, dynamic mechanisms, and sedimentary models of the gravity-flow deposits of the Chang 6 oil group in the Heshui Area, Ordos Basin, China, aiming to reveal its petroleum geological significance. Core observation, microscopic thin section identification, particle size analysis, and determination of rare earth elements were carried out. As a result, three types of gravity-flow deposits are detected, namely, slide-slump, sandy debris flow, and turbidity current. A certain slope gradient in bed form is the necessary geomorphic condition for gravity flow formation, and determines its development level, distribution range, and flow transformation efficiency. Sufficient provenance lays the material foundation and determines its depositional composition and development type. Other factors include earthquakes, volcanoes, and floods, which serve as triggering forces. In addition, fragmentation, liquefaction, and fluid mixing are the main dynamic mechanisms driving flow transformation. Based on the flow type of gravity flow, particle size characteristics, gravity-flow transformation relations, development mechanism, and spatial distribution pattern, we distinguished two depositional gravity-flow models, i.e., slump turbidite body and sublacustrine fan. Re-portrait the spatial distribution of deep-water gravity flow in the study area. From the perspective of sedimentology, explain the genesis of sand bodies in the northeast and southwest. The sandy debris flow in the middle fan braided channel microfacies of the sublacustrine fan sways the development of thick massive sand bodies in the study area. Hybrid event beds formed by the fluid transformation in a slump turbidite are the potential dessert area for deep-water tight oil and gas.

Atopic dermatitis (AD) in early childhood is often the initial manifestation of allergic disease associated with high IgE. Accumulating evidences show that follicular helper T (Tfh) cells play a critical role in promoting B cell differentiation and IgE production, human regulatory B (Breg) cells participate in immunomodulatory processes and inhibition of allergic inflammation. However, the roles and interactions between IL-10-producing Breg cells and Tfh cells in childhood AD are unclear. In this study, we found that the percentage of CD19+IL-10+ Breg cells in children with extrinsic AD was significantly lower than that in age-matched healthy controls, and that it correlated negatively with enhanced CD4+CXCR5+PD-1+ICOS+ circulating Tfh cell responses and increased disease activity; however, there was no significant correlation with serum total IgE levels. A co-culture system revealed that Breg cells from patients with extrinsic AD cannot effectively inhibit differentiation of Tfh cells in an IL-10 dependent manner. Abnormal pSTAT3 signaling induced via Toll-like receptors (TLR), but not the B-cell receptor (BCR) signaling, might contribute to the defect of Breg cells in AD. Taken together, these observations demonstrate an important role for IL-10-producing Breg cells in inhibiting Tfh cell differentiation, and suggest that they may participate in the pathogenesis of AD.

The dysregulation of epigenetic modification and energy metabolism cooperatively contribute to the tumorigenesis of nasopharyngeal carcinoma (NPC). However, the detailed mechanisms underlying their joint contribution to NPC development and progression remain unclear. Here, we investigate the role of Acy1 Coenzyme A Acyltransferases1 (ACAT1), a key enzyme in the metabolic pathway of ketone bodies, in the proliferation and metastasis of NPC and to elucidate the underlying molecular mechanisms. Ketogenesis, plays a critical role in tumorigenesis. Previously, we reported two enzymes involved in ketone body metabolism mediate epigenetic silencing and act as tumor suppressor genes in NPC. Here, we identify another key enzyme, Acetyl-CoA acetyltransferase 1 (ACAT1), and show that its transcriptional inactivation in NPC is due to promoter hypermethylation. Ectopic overexpression of ACAT1 significantly suppressed the proliferation and colony formation of NPC cells in vitro. The migratory and invasive capacity of NPC cells was inhibited by ACAT1. The tumorigenesis of NPC cells overexpressing ACAT1 was decreased in vivo. Elevated ACAT1 in NPC cells was accompanied by an elevated expression of CDH1 and a reduced expression of vimentin and SPARC, strongly indicating that ACAT1 is involved in regulating epithelial-mesenchymal transition (EMT). We also found that ACAT1 contributes to increased intracellular levels of β-hydroxybutyrate (β-HB). Exogenously supplied β-HB significantly inhibits the growth of NPC cells in a dose-dependent manner. In summary, ACAT1 may function as a tumor suppressor via modulation of ketogenesis and could thus serve as a potential therapeutic target in NPC. In summary, our data suggest that regulation of ketogenesis may serve as adjuvant therapy in NPC.

The emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system (Cas) gene-editing system represents a promising tool for genome manipulation. However, its low intracellular delivery efficiency severely compromises its use and potency for clinical applications. Nanocarriers, such as liposomes, polymers, and inorganic nanoparticles, have shown great potential for gene delivery. The remarkable development of nanoparticles as non-viral carriers for the delivery of the CRISPR/Cas9 system has shown great promise for therapeutic applications. In this review, we briefly summarize the delivery components of the CRISPR/Cas9 system and report on the progress of nano-system development for CRISPR/Cas9 delivery. We also compare the advantages of various nano-delivery systems and their applications to deliver CRISPR/Cas9 for disease treatment. Nano-delivery systems can be modified to fulfill the tasks of targeting cells or tissues. We primarily emphasize the novel exosome-based CRISPR/Cas9 delivery system. Overall, we review the challenges, development trends, and application prospects of nanoparticle-based technology for CRISPR/Cas9 delivery.

Next-generation sequencing is effective for the molecular diagnosis of genetic diseases. However, the identification of the clinical significance of synonymous variants remains a challenge. Our previous study showed that some synonymous variants in ATP7B gene produced splicing disruptions, leading to Wilson disease (WD). To test the hypothesis that synonymous variants of ATP7B cause abnormal splicing by disrupting authentic splice sites or splicing regulatory elements, we used computational tools and minigene assays to characterize 253 naturally occurring ATP7B gene synonymous variants in this study. Human Splicing Finder (HSF) and ESE Finder 3.0 were used to predict the impact of these rare synonymous variants on pre-mRNA splicing. Then, we cloned 14 different wild-type Minigene_ATP7B_ex constructs for in vitro minigene assay, including 16 exons of ATP7B gene. After computational prediction, 85 candidate variants were selected to be introduced into the corresponding Minigene_ATP7B_ex constructs for splicing assays. Using this two-step procedure, we demonstrated that 11 synonymous variants in ExAc database (c.1620C>T, c.3888C>T, c.1554C>T, c.1677C>T, c.1830G>A, c.1875T>A, c.2826C>A, c.4098G>A, c.2994C>T, c.3243G>A, and c.3747G>A) disrupted RNA splicing in vitro, and two (c.1620C>T and c.3243G>A) of these caused a complete exon skipping. The results not only provided a reliable experimental basis for the genetic diagnosis of WD patients but also offered some new insights into the pathogenicity of synonymous variants in genetic diseases.

BackgroundEnterokinase deficiency (EKD) is a rare autosomal recessively inherited disorder mainly characterized by diarrhea, hypoproteinemia and failure to thrive in infancy. Loss-of-function variants in the TMPRSS15 gene cause EKD.MethodsWe report the clinical manifestations and molecular basis of EKD in a Chinese child. We investigated in vitro two TMPRSS15 variants: the c.1921G > A as a possible splicing variant by minigene assay; the c.2396T > A(p.Val799Asp) as a missense change by protein expression analysis, enterokinase activity and effect on cellular localization.ResultsThe proband presented with intractable diarrhea accompanied by vomiting, failure to thrive and hypoproteinemia in his second year. Genetic analysis showed that the patient was compound heterozygous for two variants in the TMPRSS15 gene: c.[1921G > A];[2396T > A]. The c.1921 G > A variant may change the glutamic acid 641 into lysine; this change is predicted to be benign by bioinformatics analysis. However, it was predicted to disrupt the splicing donor site. Our minigene assay revealed that c.1921G > A caused the skipping of exon 16. The c.2396T > A(p.Val799Asp) change in the serine protease domain predicted to be deleterious hitting an evolutionary conserved amino acid. Functional studies in vitro revealed that the p.Val799Asp variant decreased the total expression level of TMPRSS15 by 29%, and the enterokinase activity of p.Val799Asp mutants was decreased by 37%, compared with that of wild type.ConclusionWe reported an EKD patient with novel compound heterozygous variants in the TMPRSS15 gene, expanding the genotypic and phenotypic spectrum of EKD. The functional characterization in vitro demonstrated that the c.1921G > A variant alters pre-mRNA splicing and the p.Val799Asp variant leads to a decrease in protein expression and enzyme activity.

Kidney is an important organ for ketone body metabolism. However, the role of abnormal ketone metabolism and its possible function in tumorigenesis of clear cell renal cell carcinoma (ccRCC) have not yet been elucidated. Three differentially expressed key enzymes involved in ketone body metabolism, ACAT1, BDH2, and HMGCL, were screened out between ccRCC and normal kidney tissues using the GEO and TCGA databases.We confirmed that the transcription and protein expression of ACAT1, BDH2, and HMGCL were significantly lower in ccRCC by real-time RT-PCR and IHC assays. Those patients with lower expression of these three genes have a worse outcome. In addition, we demonstrated that ectopic expression of each of these genes inhibited the proliferation of ccRCC cells. The overexpressed ACAT1 and BDH2 genes remarkably impeded the migratory and invasive capacity of ccRCC cells. Furthermore, exogenous β-hydroxybutyrate suppressed the growth of ccRCC cells in vitro in a dose-dependent manner. Our findings suggest that ACAT1, BDH2, and HMGCL are potential tumor suppressor genes, and constitute effective prognostic biomarkers for ccRCC. Ketone body metabolism might thus be a promising target in a process for developing novel therapeutic approaches to treat ccRCC.

The aryl hydrocarbon receptor (AhR) is an important immune regulator with a role in inflammatory response. However, the role of AhR in IL-10 production by inflammatory macrophages is currently unknown. In this study, we investigated LPS-induced IL-10 expression in macrophages from AhR-KO mice and AhR-overexpressing RAW264.7 cells. AhR was highly expressed after LPS stimulation through NF-κB pathway. Loss of AhR resulted in reduced IL-10 expression in LPS-induced macrophages. Moreover, the IL-10 expression was elevated in LPS-induced AhR-overexpressing RAW264.7 cells. Maximal IL-10 expression was dependent on an AhR non-genomic pathway closely related to Src and STAT3. Furthermore, AhR-associated Src activity was responsible for tyrosine phosphorylation of STAT3 and IL-10 expression by inflammatory macrophages. Adoptive transfer of AhR-expressing macrophages protected mice against LPS-induced peritonitis associated with high IL-10 production. In conclusion, we identified the AhR-Src-STAT3-IL-10 signaling pathway as a critical pathway in the immune regulation of inflammatory macrophages, It suggests that AhR may be a potential therapeutic target in immune response.

Emerging resistance to the antimicrobial agents of choice for treatment of thermophilic Campylobacter infections is becoming a serious threat to public health. In this study, 548 Campylobacter (372 C. jejuni and 176 C. coli) isolates from diarrheal patients and poultry meat were subjected for antibiotic susceptibility analysis to ciprofloxacin, tetracycline, gentamicin, erythromycin and clindamycin. Among them, 151 Campylobacter (32 C. jejuni and 119 C. coli) were identified as multidrug resistant isolates. PFGE analysis was performed on the 151 multidrug resistant isolates to determine their genetic relatedness, and 103 PFGE genotypes were determined. Some isolates from both human and chicken belonged to identical genotypes, indicating these clones might be able to spread between human and chicken. Antibiotic resistant genes of the 151 isolates were identified. The numbers of isolates carried tet (O), aadE, ermB, and aadE-sat4-aphA were 148 (98%), 89 (58.9%), 31 (20.5%), and 10 (6.6%), respectively. Almost all (n = 150, 99.3%) had gyrA mutation at codon 86. And the 23s rRNA A2075G point mutation was found in 56 (37.1%) isolates. Gene mutations at the cmeR-cmeABC intergenic region may lead to the activation of CmeABC multidrug efflux pump, and in this study novel sequence types of the intergenic region were identified in both C. jejuni and C. coli. This study determined the genetic prerequisites for antibiotic resistance of multidrug resistant Campylobacter isolates from diarrheal patients and poultry meat in Shanghai, China.

Activated hepatic stellate cells (aHSCs) are now established as a central driver of fibrosis in human liver injury. In the presence of chronic or repeated injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) can occur, so there is interest in down-regulating aHSCs activity in order to treat these diseases. Here, we report that Vγ9Vδ2 T cells are reduced in patients with liver cirrhosis, stimulating us to investigate possible interactions between Vγ9Vδ2 T cells and aHSCs. We find that Vγ9Vδ2 T cells kill aHSCs and killing is enhanced when aHSCs are pretreated with BPH-1236, a lipophilic analog of the bone resorption drug zoledronate. Cytotoxicity is mediated by direct cell-to-cell contact as shown by Transwell experiments and atomic force microscopy, with BPH-1236 increasing the adhesion between aHSCs and Vγ9Vδ2 T cells. Mechanistically, BPH-1236 functions by inhibiting farnesyl diphosphate synthase, leading to accumulation of the phosphoantigen isopentenyl diphosphate and recognition by Vγ9Vδ2 T cells. The cytolytic process is largely dependent on the perforin/granzyme B pathway. In a Rag2−/−γc−/− immune-deficient mouse model, we find that Vγ9Vδ2 T cells home-in to the liver, and when accompanied by BPH-1236, kill not only orthotopic aHSCs but also orthotopic HCC tumors. Collectively, our results provide the first proof-of-concept of a novel immunotherapeutic strategy for the treatment of fibrosis–cirrhosis–HCC diseases using adoptively transferred Vγ9Vδ2 T cells, combined with a lipophilic bisphosphonate.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dualbarcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dualbarcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARSCoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigenspecific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences. [ABSTRACT FROM AUTHOR]