Search

Introduction: Mature adipocyte-derived dedifferentiated fat cells (DFATs) are mesenchymal stem cell (MSC)-like cells with high proliferative ability and multilineage differentiation potential. In this study, we first examined whether DFATs can be prepared from infrapatellar fat pad (IFP) and then compared phenotypic and functional properties of IFP-derived DFATs (IFP-DFATs) with those of subcutaneous adipose tissue (SC)-derived DFATs (SC-DFATs). Methods: Mature adipocytes isolated from IFP and SC in osteoarthritis patients (n = 7) were cultured by ceiling culture method to generate DFATs. Obtained IFP-DFATs and SC-DFATs were subjected to flow cytometric and microarray analysis to compare their immunophenotypes and gene expression profiles. Cell proliferation assay and adipogenic, osteogenic, and chondrogenic differentiation assays were performed to evaluate their functional properties. Results: DFATs could be prepared from IFP and SC with similar efficiency. IFP-DFATs and SC-DFATs exhibited similar immunophenotypes (CD73+, CD90+, CD105+, CD31-, CD45-, HLA-DR-) and tri-lineage (adipogenic, osteogenic, and chondrogenic) differentiation potential, consistent with the minimal criteria for defining MSCs. Microarray analysis revealed that the gene expression profiles in IFP-DFATs were very similar to those in SC-DFATs, although there were certain number of genes that showed different levels of expression. The proliferative activity in IFP-DFATs was significantly (p

Mass spectrometry imaging (MSI) has emerged as a rapidly expanding field in the MS community. The analysis of large spectral data is further complicated by the added spatial dimension of MSI. A plethora of resources exist for expert users to begin parsing MSI data in R, but there is a critical lack of guidance for absolute beginners. This tutorial is designed to serve as a one-stop guide to start using R with MSI data and describe the possibilities that data science can bring to MSI analysis.

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING–TBK1–IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68+ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy., Graphical abstract SHP2-mediated tumor immunosuppression in colon cancer was defined by scRNA-seq. SHP2 negatively regulates type I interferon signaling. SHP2 allosteric inhibition remolds the anti-tumor TME, indicating SHP2 is a promising target for colon cancer immunotherapy.Image 1

Background & Aims Microbiota dysbiosis and mucosa-associated bacteria are involved in colorectal cancer progression. We hypothesize that an interaction between virulent pathobionts and epithelial defense promotes tumorigenesis. Methods Chemical-induced CRC mouse model was treated with antibiotics at various phases. Colonic tissues and fecal samples were collected in a time-serial mode and analyzed by gene microarray and 16S rRNA sequencing. Intraepithelial bacteria were isolated using a gentamicin resistance assay, and challenged in epithelial cultures. Results Our study showed that antibiotic treatment at midphase but not early or late phase reduced mouse tumor burden, suggesting a time-specific host–microbe interplay. A unique antimicrobial transcriptome profile showing an inverse relationship between autophagy and oxidative stress genes was correlated with a transient surge in microbial diversity and virulence emergence in mouse stool during cancer initiation. Gavage with fimA/fimH/htrA-expressing invasive Escherichia coli isolated from colonocytes increased tumor burden in recipient mice, whereas inoculation of bacteria deleted of htrA or triple genes did not. The invasive E.coli suppressed epithelial autophagy activity through reduction of microtubule-associated protein 1 light-chain 3 transcripts and caused dual oxidase 2–dependent free radical overproduction and tumor cell hyperproliferation. A novel alternating spheroid culture model was developed for sequential bacterial challenge to address the long-term changes in host–microbe interaction for chronic tumor growth. Epithelial cells with single bacterial encounter showed a reduction in transcript levels of autophagy genes while those sequentially challenged with invasive E.coli showed heightened autophagy gene expression to eliminate intracellular microbes, implicating that bacteria-dependent cell hyperproliferation could be terminated at late phases. Finally, the presence of bacterial htrA and altered antimicrobial gene expression were observed in human colorectal cancer specimens. Conclusions Invasive pathobionts contribute to cancer initiation during a key time frame by counterbalancing autophagy and oxidative stress in the colonic epithelium. Monitoring gut microbiota and antimicrobial patterns may help identify the window of opportunity for intervention with bacterium-targeted precision medicine., Graphical abstract

Proteins and peptides (PPs) have gradually become more attractive therapeutic molecules than small molecular drugs due to their high selectivity and efficacy, but fewer side effects. Owing to the poor stability and limited permeability through gastrointestinal (GI) tract and epithelia, the therapeutic PPs are usually administered by parenteral route. Given the big demand for oral administration in clinical use, a variety of researches focused on developing new technologies to overcome GI barriers of PPs, such as enteric coating, enzyme inhibitors, permeation enhancers, nanoparticles, as well as intestinal microdevices. Some new technologies have been developed under clinical trials and even on the market. This review summarizes the history, the physiological barriers and the overcoming approaches, current clinical and preclinical technologies, and future prospects of oral delivery of PPs., Graphical abstract The current strategies for improving oral absorption of proteins and peptides (PPs) include stabilization, absorption enhancement and mucus-related technologies. Most of marketed oral PP products employ two or more strategies.Image 1

Highlights • CLIC2 is highly expressed in benign, less invasive and less metastatic tumors. • Forced expression of CLIC2 prevents metastasis and invasion in animal tumor models. • CLIC2 is associated with decreased vascular permeability in tumor masses. • CLIC2, a secretable soluble protein, can bind to and inhibit MMP14. • Extracellular CLIC2 can suppress malignant cell invasion., The abilities to invade surrounding tissues and metastasize to distant organs are the most outstanding features that distinguish malignant from benign tumors. However, the mechanisms preventing the invasion and metastasis of benign tumor cells remain unclear. By using our own rat distant metastasis model, gene expression of cells in primary tumors was compared with that in metastasized tumors. Among many distinct gene expressions, we have focused on chloride intracellular channel protein 2 (CLIC2), an ion channel protein of as-yet unknown function, which was predominantly expressed in the primary tumors. We created CLIC2 overexpressing rat glioma cell line and utilized benign human meningioma cells with naturally high CLIC2 expression. CLIC2 was expressed at higher levels in benign human brain tumors than in their malignant counterparts. Moreover, its high expression was associated with prolonged survival in the rat metastasis and brain tumor models as well as with progression-free survival in patients with brain tumors. CLIC2 was also correlated with the decreased blood vessel permeability likely by increased contents of cell adhesion molecules. We found that CLIC2 was secreted extracellularly, and bound to matrix metalloproteinase (MMP) 14. Furthermore, CLIC2 prevented the localization of MMP14 in the plasma membrane, and inhibited its enzymatic activity. Indeed, overexpressing CLIC2 and recombinant CLIC2 protein effectively suppressed malignant cell invasion, whereas CLIC2 knockdown reversed these effects. Thus, CLIC2 suppress invasion and metastasis of benign tumors at least partly by inhibiting MMP14 activity., Graphical abstract Image, graphical abstract

Background Diabetes mellitus (DM) is a leading cause of preventable cardiovascular disease, but the metabolic changes from prediabetes to diabetes have not been fully clarified. This study implemented a metabolomics profiling platform to investigate the variations of metabolites and to elucidate their global profiling from metabolic syndrome to DM. Methods: Male Sprague-Dawley rats (n = 44) were divided into four groups. Three groups were separately fed with a normal diet, a high-fructose diet (HF), or a high-fat (HL) diet while one group was treated with streptozotocin. The HF and HL diet were meant to induce insulin resistance, obesity, and dyslipidemia, which known to induce DM. Results: The most significant metabolic variations in the DM group’s urine samples were the reduced release of citric acid cycle intermediates, the increase in acylcarnitines, and the decrease in urea excretion, all of which indicated energy metabolism abnormalities and mitochondrial dysfunction. Overall, the metabolic analysis revealed tryptophan metabolic pathway variations in the prediabetic phase, even though the mitochondrial function remains unaffected. Conclusion: This study show that widespread methylations and impaired tryptophan metabolism occur in metabolic syndrome and are then followed by a decline in citric acid cycle intermediates, indicating mitochondrial dysfunction in diabetes.

Background & Aims We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn’s disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. Methods RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. Results We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. Conclusions PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes., Graphical abstract

Quality by Design (QbD) and chemometric models are different sides of the same coin. While QbD models utilize experimentally designed settings for optimization of some quality attributes, these settings can also be utilized for chemometric prediction of the same attributes. We aimed to synchronize optimization of comparative dissolution results of carvedilol immediate release tablets with chemometric prediction of dissolution profile and content uniformity of the product. As an industrial application, selection of variables for optimization was done by performing risk assessment utilizing the archived product records at the pharmaceutical site. Experimental tablets were produced with 20 different settings with the variables being contents of sucrose, sodium starch glycolate, lactose monohydrate, and avicel Ph 101. Contents of the excipients were modelled with F1 dissimilarity factor and F2 similarity factor in HCL, acetate, and USP dissolution media to determine the design space. We initiatively utilized Partial Least Square based Structural Equation Modelling (PLS-SEM) to explore how the excipients and their NIR records explained dissolution of the product. Finally, the optimized formula was utilized with varied content of carvedilol for chemometric prediction of the content uniformity.

The recent discovery of activator compounds binding to an allosteric site on the NAD+-dependent protein lysine deacetylase, sirtuin 6 (SIRT6) has attracted interest and presents a pharmaceutical target for aging-related and cancer diseases. However, the mechanism underlying allosteric activation of SIRT6 by the activator MDL-801 remains largely elusive because no major conformational changes are observed upon activator binding. By combining molecular dynamics simulations with biochemical and kinetic analyses of wild-type SIRT6 and its variant M136A, we show that conformational rotation of 2-methyl-4-fluoro-5-bromo substituent on the right phenyl ring (R-ring) of MDL-801, which uncovers previously unseen hydrophobic interactions, contributes to increased activating deacetylation activity of SIRT6. This hypothesis is further supported by the two newly synthesized MDL-801 derivatives through the removal of the 5-Br atom on the R-ring (MDL-801-D1) or the restraint of the rotation of the R-ring (MDL-801-D2). We further propose that the 5-Br atom serves as an allosteric driver that controls the ligand allosteric efficacy. Our study highlights the effect of allosteric enzyme catalytic activity by activator binding and provides a rational approach for enhancing deacetylation activity., Graphical abstract Sirtuin 6 (SIRT6), a NAD+-dependent protein lysine deacetylase, has attracted interest and presents a pharmaceutical target for aging-related and cancer diseases. By combining molecular dynamics simulations, compound synthesis, and biochemical and kinetic analyses of wild-type SIRT6 and its variants, we elucidate the allosteric activation mechanism of SIRT6 by small-molecule compounds.Image 1

Graphical abstract, Highlights • “Multiblock metabolomics” elucidates the global metabolic network in a whole body. • “Multiblock metabolomics” combines LC/MS-based metabolomics with multiblock PCA. • “Multiblock metabolomics” highlights and elicits organ-specific metabolism. • TGs with less unsaturated fatty acids were highly accumulated in the diabetic liver., Principal component analysis (PCA) is a useful tool for omics analysis to identify underlying factors and visualize relationships between biomarkers. However, this approach is limited in addressing life complexity and further improvement is required. This study aimed to develop a new approach that combines mass spectrometry-based metabolomics with multiblock PCA to elucidate the whole-body global metabolic network, thereby generating comparable metabolite maps to clarify the metabolic relationships among several organs. To evaluate the newly developed method, Zucker diabetic fatty (ZDF) rats (n = 6) were used as type 2 diabetic models and Sprague Dawley (SD) rats (n = 6) as controls. Metabolites in the heart, kidney, and liver were analyzed by capillary electrophoresis and liquid chromatography mass spectrometry, respectively, and the detected metabolites were analyzed by multiblock PCA. More than 300 metabolites were detected in the heart, kidney, and liver. When the metabolites obtained from the three organs were analyzed with multiblock PCA, the score and loading maps obtained were highly synchronized and their metabolism patterns were visually comparable. A significant finding in this study was the different expression patterns in lipid metabolism among the three organs; notably triacylglycerols with polyunsaturated fatty acids or less unsaturated fatty acids showed specific accumulation patterns depending on the organs.

Despite advances in therapeutics, the progression of melanoma to metastasis still confers a poor outcome to patients. Nevertheless, there is a scarcity of biological models to understand cellular and molecular changes taking place along disease progression. Here, we characterized the transcriptome profiles of a multi-stage murine model of melanoma progression comprising a nontumorigenic melanocyte lineage (melan-a), premalignant melanocytes (4C), nonmetastatic (4C11-) and metastasis-prone (4C11+) melanoma cells. Clustering analyses have grouped the 4 cell lines according to their differentiated (melan-a and 4C11+) or undifferentiated/“mesenchymal-like” (4C and 4C11-) morphologies, suggesting dynamic gene expression patterns associated with the transition between these phenotypes. The cell plasticity observed in the murine melanoma progression model was corroborated by molecular markers described during stepwise human melanoma differentiation, as the differentiated cell lines in our model exhibit upregulation of transitory and melanocytic markers, whereas “mesenchymal-like” cells show increased expression of undifferentiated and neural crest-like markers. Sets of differentially expressed genes (DEGs) were detected at each transition step of tumor progression, and transcriptional signatures related to malignancy, metastasis and epithelial-to-mesenchymal transition were identified. Finally, DEGs were mapped to their human orthologs and evaluated in uni- and multivariate survival analyses using gene expression and clinical data of 703 drug-naïve primary melanoma patients, revealing several independent candidate prognostic markers. Altogether, these results provide novel insights into the molecular mechanisms underlying the phenotypic switch taking place during melanoma progression, reveal potential drug targets and prognostic biomarkers, and corroborate the translational relevance of this unique sequential model of melanoma progression.

Background: Gut microbiota mainly function in the biotransformation of primary ginsenosides into bioactive metabolites. Herein, we investigated the effects of three prebiotic fibers by targeting gut microbiota on the metabolism of ginsenoside Rb1 in vivo. Methods: Sprague Dawley rats were administered with ginsenoside Rb1 after a two-week prebiotic intervention of fructooligosaccharide, galactooligosaccharide, and fibersol-2, respectively. Pharmacokinetic analysis of ginsenoside Rb1 and its metabolites was performed, whilst the microbial composition and metabolic function of gut microbiota were examined by 16S rRNA gene amplicon and metagenomic shotgun sequencing. Results: The results showed that peak plasma concentration and area under concentration time curve of ginsenoside Rb1 and its intermediate metabolites, ginsenoside Rd, F2, and compound K (CK), in the prebiotic intervention groups were increased at various degrees compared with those in the control group. Gut microbiota dramatically responded to the prebiotic treatment at both taxonomical and functional levels. The abundance of Prevotella, which possesses potential function to hydrolyze ginsenoside Rb1 into CK, was significantly elevated in the three prebiotic groups (P

Highlights • Review on tools and databases linking miRNA and its mRNA targetome. • Databases show little overlap in miRNA targetome predictions suggesting strong contextual effects. • Deconvolution and deep learning approaches are promising new approaches to improve miRNA targetome predictions., Advanced sequencing technologies such as RNASeq provide the means for production of massive amounts of data, including transcriptome-wide expression levels of coding RNAs (mRNAs) and non-coding RNAs such as miRNAs, lncRNAs, piRNAs and many other RNA species. In silico analysis of datasets, representing only one RNA species is well established and a variety of tools and pipelines are available. However, attaining a more systematic view of how different players come together to regulate the expression of a gene or a group of genes requires a more intricate approach to data analysis. To fully understand complex transcriptional networks, datasets representing different RNA species need to be integrated. In this review, we will focus on miRNAs as key post-transcriptional regulators summarizing current computational approaches for miRNA:target gene prediction as well as new data-driven methods to tackle the problem of comprehensively and accurately dissecting miRNome-targetome interactions.

Graphical abstract THQ of Cr in the meat was more than one. HI in chicken was more than one. CR was neglectable in the case of both Cd and Pb., Highlights • All of the samples were contaminated with Cd, Cr, Cu, Ni and Pb. • The highest concentration of Pb (0.11 mg/kg) was in the gizzard. • The concentration of Cd and Cu were highest in the liver, while the highest contents of Cr and Ni were in the chicken. • THQs of Cr in the muscle were more than one. • Consumption of chicken meat in both of adults and children may pose a significant health risk for consumers., The present study aimed to determine the average concentration of some metals, including cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni) and lead (Pb) in the chicken, hen’s liver, and gizzard in the east of Iran. Estimated daily intake (EDI), target hazard quotient (THQ), hazard index (HI) and carcinogenic risk (CR) were calculated. In this cross-sectional study, fifty one samples including chicken, hen’s liver and gizzard were obtained from Birjand, Iran. Measurement of Cd, Cr, Cu, Ni, and Pb was carried out by using an Inductively Coupled Plasma-Optic Emission Spectroscopy (ICP-OES). All of the measured metals were detected in 100 % of the samples. The metals had a different distribution pattern. The highest concentration of Cd and Cu was in the liver samples while the Cr and Ni had the highest levels in the chicken. Pb concentration was at the highest level in the gizzard. The least amount of Cr, Ni, and Pb was found in the liver while Cu had the least content in the muscle. EDI had an acceptable level, but the highest daily intake of all studied metals was through muscle. Cr had the highest THQ and it was more than one in the meat. HI in chicken was more than one. Liver and gizzard of hens had a neglectable HI. CR was neglectable in the case of both Cd and Pb, but it was considerable for Cr and Ni. The consumption of chicken in both adults and children may pose a significant health risk for consumers.

Background & Aims Intestinal macrophages adopt a hyporesponsive phenotype through education by local signals. Lack of proper macrophage maturation in patients with ulcerative colitis (UC) in remission may initiate gut inflammation. The aim, therefore, was to determine the effects of fecal luminal factors derived from healthy donors and UC patients in remission on macrophage phenotype and function. Methods Fecal supernatants (FS) were extracted from fecal samples of healthy subjects and UC patients in remission. Monocytes were matured into macrophages in the presence of granulocyte-macrophage colony-stimulating factor without/with FS, stimulated with lipopolysaccharide, and macrophage phenotype and function were assessed. Fecal metabolomic profiles were analyzed by gas-chromatography/mass-spectrometry. Results Fecal luminal factors derived from healthy donors were effective in down-regulating Toll-like receptor signaling, cytokine signaling, and antigen presentation in macrophages. Fecal luminal factors derived from UC patients in remission were less potent in inducing lipopolysaccharide hyporesponsiveness and modulating expression of genes involved in macrophage cytokine and Toll-like receptor signaling pathways. Although phagocytic and bactericidal abilities of macrophages were not affected by FS treatment, healthy FS-treated macrophages showed a greater ability to suppress cluster of differentiation 4+ T-cell activation and interferon γ secretion compared with UC remission FS-treated counterparts. Furthermore, metabolomic analysis showed differential fecal metabolite composition for healthy donors and UC patients in remission. Conclusions Our data indicate that UC patients in remission lack luminal signals able to condition macrophages toward a hyporesponsive and tolerogenic phenotype, which may contribute to their persistent vulnerability to relapse., Graphical abstract

Background Inflammatory bowel disease (IBD) is a complex disease with variable presentation, progression, and response to therapies. Current disease classification is based on subjective clinical phenotypes. The peripheral blood immunophenome can reflect local inflammation, and thus we measured 39 circulating immune cell types in a large cohort of IBD and control subjects and performed immunotype:phenotype associations. Methods We performed fluorescence-activated cell sorting or CyTOF analysis on blood from 728 Crohn’s disease, 464 ulcerative colitis, and 334 non-IBD patients, with available demographics, endoscopic and clinical examinations and medication use. Results We observed few immune cell types commonly affected in IBD (lowered natural killer cells, B cells, and CD45RA– CD8 T cells). Generally, the immunophenome was distinct between ulcerative colitis and Crohn’s disease. Within disease subtype, there were further distinctions, with specific immune cell types associating with disease duration, behavior, and location. Thiopurine monotherapy altered abundance of many cell types, often in the same direction as disease association, while anti-tumor necrosis factor (anti-TNF) monotherapy demonstrated an opposing pattern. Concomitant use of an anti-TNF and thiopurine was not synergistic, but rather was additive. For example, thiopurine monotherapy use alone or in combination with anti-TNF was associated with a dramatic reduction in major subclasses of B cells. Conclusions We present a peripheral map of immune cell changes in IBD related to disease entity and therapies as a resource for hypothesis generation. We propose the changes in B cell subsets could affect antibody formation and potentially explain the mechanism behind the superiority of combination therapy through the impact of thiopurines on pharmacokinetics of anti-TNFs., Graphical abstract

Graphical abstract, Highlights • Uranium and arsenic co-occur as contaminants in water sources in the Southwestern US. • Exposure to metals may alter gene expression and cause immune dysregulation in CD4+ T-cells. • RNA-seq analysis on CD4+ T-cells treated with UA prior to activation led to no significant alterations in gene expression. • A mixture of UA and As demonstrated unique alterations in gene expression compared to As alone., Communities in the western region of the United States experience environmental exposure to metal mixtures from living in proximity to numerous unremediated abandoned uranium mines. Metals including arsenic and uranium co-occur in and around these sites at levels higher than the United States Environmental Protection Agency maximum contaminant levels. To address the potential effect of these metals on the activation of CD4+ T-cells, we used RNA sequencing methods to determine the effect of exposure to sodium arsenite (1 μM and 10 μM), uranyl acetate (3 μM and 30 μM) or a mixture of sodium arsenite and uranyl acetate (1 μM sodium arsenite + 3 μM uranyl acetate). Sodium arsenite induced a dose dependent effect on activation associated gene expression; targeting immune response genes at the lower dose. Increases in oxidative stress gene expression were observed with both sodium arsenite doses. While uranyl acetate alone did not significantly alter activation associated gene expression, the mixture of uranyl acetate with sodium arsenite demonstrated a combined effect relative to sodium arsenite alone. The results demonstrate the need to investigate metal and metalloid mixtures at environmentally relevant concentrations to better understand the toxicological impact of these mixtures on T-cell activation, function and immune dysregulation.

Tumor necrosis factor (TNF) and its receptors are widely expressed in non-small cell lung cancer (NSCLC). TNF has an established role in inflammation and also plays a key role in inflammation-induced cancer. TNF can induce cell death in cancer cells and has been used as a treatment in certain types of cancer. However, TNF is likely to play an oncogenic role in multiple types of cancer, including NSCLC. TNF is a key activator of the transcription factor NF-κB. NF-κB, in turn, is a key effector of TNF in inflammation-induced cancer. Data from The Cancer Genome Atlas database suggest that TNF could be a biomarker in NSCLC and indicate a complex role for TNF and its receptors in NSCLC. Recent studies have reported that TNF is rapidly upregulated in NSCLC in response to targeted treatment with epidermal growth factor receptor (EGFR) inhibition, and this upregulation leads to NF-κB activation. The TNF upregulation and consequent NF-κB activation play a key role in mediating both primary and secondary resistance to EGFR inhibition in NSCLC, and a combined inhibition of EGFR and TNF can overcome therapeutic resistance in experimental models. TNF may mediate the toxic side effects of immunotherapy and may also modulate resistance to immune checkpoint inhibitors. Drugs inhibiting TNF are widely used for the treatment of various inflammatory and rheumatologic diseases and could be quite useful in combination with targeted therapy of NSCLC and other cancers.

Background & Aims Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. Methods We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. Results Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2–4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5–8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2–4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. Lay summary MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy., Graphical abstract, Highlights • Serum miRNA was sequenced in 183 NAFLD cases of varying severity and 10 population controls. • Plasma levels of miR-193a-5p were significantly increased in patients with advanced fibrosis, high NAS scores, or high SAF scores. • Other miRNAs including miR378d and miR378e were also significantly increased in certain comparisons. • The findings for miR-193a-5p were replicated in a cohort of 372 additional NAFLD cases.

Visual Abstract, Highlights • Differences in the clinical presentation and physiology of aortic stenosis in men and women complicate the management of the condition. • By combining traditional inferential statistics, artificial intelligence predictive modeling, and genetic pathway analysis, one can gain further insight into sex-specific gene expression patterns, potentially driving the valvular phenotype differences between the sexes. • Results from this study, implementing a mixed and comprehensive methodological approach, offer a foundation for further exploration of potential drug targets., Summary Male and female aortic stenosis patients have distinct valvular phenotypes, increasing the complexities in the evaluation of valvular pathophysiology. In this study, we present cutting-edge artificial intelligence analyses of transcriptome-wide array data from stenotic aortic valves to highlight differences in gene expression patterns between the sexes, using both sex-differentiated transcripts and unbiased gene selections. This approach enabled the development of efficient models with high predictive ability and determining the most significant sex-dependent contributors to calcification. In addition, analyses of function-related gene groups revealed enriched fibrotic pathways among female patients. Ultimately, we demonstrate that artificial intelligence models can be used to accurately predict aortic valve calcification by carefully analyzing sex-specific gene transcripts.

Highlights • Biological process-associated scores generated from mass spectrometry of serum. • Scores demonstrated acceptable levels of reproducibility. • Scores associated with biological processes and clinical outcome in cancer patients. • Possible application to biomarker studies for treatment or monitoring of disease. • Multiple biological processes assessed from 3 µL of serum., Introduction Most diseases involve a complex interplay between multiple biological processes at the cellular, tissue, organ, and systemic levels. Clinical tests and biomarkers based on the measurement of a single or few analytes may not be able to capture the complexity of a patient’s disease. Novel approaches for comprehensively assessing biological processes from easily obtained samples could help in the monitoring, treatment, and understanding of many conditions. Objectives We propose a method of creating scores associated with specific biological processes from mass spectral analysis of serum samples. Methods A score for a process of interest is created by: (i) identifying mass spectral features associated with the process using set enrichment analysis methods, and (ii) combining these features into a score using a principal component analysis-based approach. We investigate the creation of scores using cohorts of patients with non-small cell lung cancer, melanoma, and ovarian cancer. Since the circulating proteome is amenable to the study of immune responses, which play a critical role in cancer development and progression, we focus on functions related to the host response to disease. Results We demonstrate the feasibility of generating scores, their reproducibility, and their associations with clinical outcomes. Once the scores are constructed, only 3 µL of serum is required for the assessment of multiple biological functions from the circulating proteome. Conclusion These mass spectrometry-based scores could be useful for future multivariate biomarker or test development studies for informing treatment, disease monitoring and improving understanding of the roles of various biological functions in multiple disease settings.

Graphical abstract, The effective non-invasive diagnosis and prognosis are critical for cancer treatment. The plasma cell-free DNA (cfDNA) provides a good material for cancer liquid biopsy and its worth in this field is increasingly explored. Here we describe a new pipeline for effectively finding new cfDNA-based biomarkers for cancers by combining SALP-seq and machine learning. Using the pipeline, 30 cfDNA samples from 26 esophageal cancer (ESCA) patients and 4 healthy people were analyzed as an example. As a result, 103 epigenetic markers (including 54 genome-wide and 49 promoter markers) and 37 genetic markers were identified for this cancer. These markers provide new biomarkers for ESCA diagnosis, prognosis and therapy. Importantly, these markers, especially epigenetic markers, not only shed important new insights on the regulatory mechanisms of this cancer, but also could be used to classify the cfDNA samples. We therefore developed a new pipeline for effectively finding new cfDNA-based biomarkers for cancers by combining SALP-seq and machine learning. In this study, we also discovered new clinical worth of cfDNA distinct from other reported characters.

Graphical abstract By repeatedly selecting and pooling the most rapidly outgrowing clones during multiple single cell subcloning rounds, the time required for subcloning was reduced from 3 to 2 weeks and the cloning efficiency significantly increased. Transcriptome analyses of the resulting host cell lines revealed a diverse set of pathways differentially enriched in each host cell line treated, with the only shared DE pathway related to changes in extracellular matrix. This indicates that cells struggle with the lack of cell-to-cell communication in isolation during subcloning., Chinese Hamster Ovary (CHO) cells are the working horse of the pharmaceutical industry. To obtain high producing cell clones and to satisfy regulatory requirements single cell cloning is a necessary step in cell line development. However, it is also a tedious, labor intensive and expensive process. Here we show an easy way to enhance subclonability using subcloning by single cell sorting itself as the selection pressure, resulting in improved subcloning performance of three different host cell lines. These improvements in subclonability also lead to an enhanced cellular growth behavior during standard batch culture. RNA-seq was performed to shed light on the underlying mechanisms, showing that there is little overlap in differentially expressed genes or associated pathways between the cell lines, each finding their individual strategy for optimization. However, in all three cell lines pathways associated with the extracellular matrix were found to be enriched, indicating that cells struggle predominantly with their microenvironment and possibly lack of cell-to-cell contact. The observed small overlap may hint that there are multiple ways for a cell line to achieve a certain phenotype due to numerous genetic and subsequently metabolic redundancies.

Background & Aims Organ-level research using an animal model lacking Il2rg, the gene responsible for X-linked severe combined immunodeficiency (X-SCID), is clinically unavailable and would be a powerful tool to gain deeper insights into the symptoms of patients with X-SCID. Methods We used an X-SCID animal model, which was first established in our group by the deletion of Il2rg gene in pigs, to understand the clinical signs from multiple perspectives based on pathology, immunology, microbiology, and nutrition. We also treated the X-SCID pigs with bone marrow transplantation (BMT) for mimicking a current therapeutic treatment for patients with X-SCID and investigated the effect at the organ-level. Moreover, the results were confirmed using serum and fecal samples collected from patients with X-SCID. Results We demonstrated that X-SCID pigs completely lacked Peyer’s patches (PPs) and IgA production in the small intestine, but possessed some dysfunctional intestinal T and B cells. Another novel discovery was that X-SCID pigs developed a heterogeneous intestinal microflora and possessed abnormal plasma metabolites, indicating that X-SCID could be an immune disorder that affects various in vivo functions. Importantly, the organogenesis of PPs in X-SCID pigs was not promoted by BMT. Although a few isolated lymphoid follicles developed in the small intestine of BMT-treated X-SCID pigs, there was no evidence that they contributed to IgA production and microflora formation. Consistently, most patients with X-SCID who received BMT possessed abnormal intestinal immune and microbial environments regardless of the presence of sufficient serum IgG. Conclusions These results indicate that the current BMT therapies for patients with X-SCID may be insufficient to induce the organogenesis of intestinal lymphoid tissues that are associated with numerous functions in vivo., Graphical abstract

Background & Aims Epidemiological and animal studies have indicated an inverse correlation between the rising prevalence of obesity and metabolic syndrome and exposure to helminths. Whether helminth-induced immune response contributes to microbiota remodeling in obesity remains unknown. The aim of this study is to explore the immune-regulatory role of helminth in the prevention of HFD-induced obesity through remodeling gut microbiome. Methods C57BL/6J WT and STAT6-/- mice were infected with Heligmosomoides polygyrus and followed by high fat diet (HFD) feeding for 6 weeks. The host immune response, body weight, and fecal microbiota composition were analyzed. We used adoptive transfer of M2 macrophages and microbiota transplantation approaches to determine the impact of these factors on HFD-obesity. We also examined stool microbiota composition and short chain fatty acids (SCFAs) concentration and determined the expression of SCFA-relevant receptors in the recipient mice. Results Helminth infection of STAT6-/- (Th2-deficient) mice and adoptive transfer of helminth-induced alternatively activated (M2) macrophages demonstrated that the helminth-associated Th2 immune response plays an important role in the protection against obesity and induces changes in microbiota composition. Microbiota transplantation showed that helminth-induced, Th2-dependent alterations of the gut microbiota are sufficient to confer protection against obesity. Collectively, these results indicate that helminth infection protects against HFD-induced obesity by Th2-dependent, M2 macrophage-mediated alterations of the intestinal microbiota. Conclusion Our findings provide new mechanistic insights into the complex interplay between helminth infection, the immune system and the gut microbiota in a HFD-induced obesity model and holds promise for gut microbiome-targeted immunotherapy in obesity prevention., Graphical abstract

Background & Aims The epithelia of the intestine and colon turn over rapidly and are maintained by adult stem cells at the base of crypts. Although the small intestine and colon have distinct, well-characterized physiological functions, it remains unclear if there are fundamental regional differences in stem cell behavior or region-dependent degenerative changes during aging. Mesenchyme-free organoids provide useful tools for investigating intestinal stem cell biology in vitro and have started to be used for investigating age-related changes in stem cell function. However, it is unknown whether organoids maintain hallmarks of age in the absence of an aging niche. We tested whether stem cell–enriched organoids preserved the DNA methylation-based aging profiles associated with the tissues and crypts from which they were derived. Methods To address this, we used standard human methylation arrays and the human epigenetic clock as a biomarker of age to analyze in vitro–derived, 3-dimensional, stem cell–enriched intestinal organoids. Results We found that human stem cell–enriched organoids maintained segmental differences in methylation patterns and that age, as measured by the epigenetic clock, also was maintained in vitro. Surprisingly, we found that stem cell–enriched organoids derived from the small intestine showed striking epigenetic age reduction relative to organoids derived from colon. Conclusions Our data validate the use of organoids as a model for studying human intestinal aging and introduce methods that can be used when modeling aging or age-onset diseases in vitro., Graphical abstract

Background A distinct serum metabonomic pattern has been previously revealed to be associated with various forms of liver disease. Here, we aimed to apply mass spectrometry to obtain serum metabolomic profiles from individuals with cholangiocarcinoma and benign hepatobiliary diseases to gain an insight into pathogenesis and search for potential early-disease biomarkers. Methods Serum samples were profiled using a hydrophilic interaction liquid chromatography platform, coupled to a mass spectrometer. A total of 47 serum specimens from 8 cholangiocarcinoma cases, 20 healthy controls, 8 benign disease controls (bile duct strictures) and 11 patients with hepatocellular carcinoma (as malignant disease controls) were included. Data analysis was performed using univariate and multivariate statistics. Results The serum metabolome disparities between the metabolite profiles from healthy controls and patients with hepatobiliary disease were predominantly related to changes in lipid and lipid-derived compounds (phospholipids, bile acids and steroids) and amino acid metabolites (phenylalanine). A metabolic pattern indicative of inflammatory response due to cirrhosis and cholestasis was associated with the disease groups. The abundance of phospholipid metabolites was altered in individuals with liver disease, particularly cholangiocarcinoma, but no significant difference was seen between profiles from patients with benign biliary strictures and cholangiocarcinoma. Conclusion The serum metabolome in cholangiocarcinoma exhibited changes in metabolites related to inflammation, altered energy production and phospholipid metabolism. This study serves to highlight future avenues for biomarker research in large-scale studies.

Graphical abstract, Cancer proteomics has become a powerful technique for characterizing the protein markers driving transformation of malignancy, tracing proteome variation triggered by therapeutics, and discovering the novel targets and drugs for the treatment of oncologic diseases. To facilitate cancer diagnosis/prognosis and accelerate drug target discovery, a variety of methods for tumor marker identification and sample classification have been developed and successfully applied to cancer proteomic studies. This review article describes the most recent advances in those various approaches together with their current applications in cancer-related studies. Firstly, a number of popular feature selection methods are overviewed with objective evaluation on their advantages and disadvantages. Secondly, these methods are grouped into three major classes based on their underlying algorithms. Finally, a variety of sample separation algorithms are discussed. This review provides a comprehensive overview of the advances on tumor maker identification and patients/samples/tissues separations, which could be guidance to the researches in cancer proteomics.

Graphical abstract, Background In this paper we propose a novel framework for the definition of Personas for healthcare workers based on an online survey, with the aim of highlighting different levels of risk of developing mental disorders induced by COVID-19 and tailor psychological support interventions. Methods Data were gathered from Italian healthcare workers between April and May 2020. Information about socio-demographic characteristics, current lifestyle, occupational, COVID-19 infection, and psychological indexes (Maslach Burnout Inventory, Impact of Event Scale and Patient Health Questionnaire) was collected. Respondents were divided in four subgroups based on their health profession: physicians (P), nurses (N), other medical professionals (OMP) and technical-administrative (TA). For each sub-group, collected variables (46) were reduced using Principal Component Analysis and clustered by means of k-medoids clustering. Statistical analysis was then applied to define which variables were able to differentiate among the k clusters, leading to the generation of a Persona card (i.e., a template with textual and graphical information) for each of the obtained clusters. Results From the 538 respondents (153 P, 175 N, 176 OMP, 344 TA), the highest stress level, workload impact and risk of mental disorders were found in the N subgroup. Two clusters were identified for P, three clusters for N, two for OMP and one for TA. Conclusions The proposed framework was able to stratify different risk levels of possible development of mental health issues in healthcare workers due to COVID-19. This approach could represent the first step towards the development of mobile health tools to tailor psychological interventions in pandemic situations.

Background & Aims The I148M Patatin-like Phospholipase Domain-containing 3 (PNPLA3), the rs641738 in the Membrane bound O-acyltransferase domain containing 7-transmembrane channel-like 4 (MBOAT7-TMC4) locus, and the E167K Transmembrane 6 Superfamily Member 2 (TM6SF2) polymorphisms represent the main predisposing factors to nonalcoholic fatty liver disease (NAFLD) development and progression. We previously generated a full knockout of MBOAT7 in HepG2 cells (MBOAT7-/-), homozygous for I148M PNPLA3. Therefore, we aimed to investigate the synergic impact of the 3 at-risk variants on liver injury and hepatocellular carcinoma (HCC) in a large cohort of NAFLD patients, and create in vitro models of genetic NAFLD by silencing TM6SF2 in both HepG2 and MBOAT7-/- cells. Methods NAFLD patients (n = 1380), of whom 121 had HCC, were stratified with a semiquantitative score ranging from 0 to 3 according to the number of PNPLA3, TM6SF2, and MBOAT7 at-risk variants. TM6SF2 was silenced in HepG2 (TM6SF2-/-) and MBOAT7-/- (MBOAT7-/-TM6SF2-/-) through Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Results In NAFLD patients, the additive weight of these mutations was associated with liver disease severity and an increased risk of developing HCC. In HepG2 cells, TM6SF2 silencing altered lipid composition and induced the accumulation of microvesicular lipid droplets (LDs), whereas the MBOAT7-/-TM6SF2-/- cells showed a mixed microvesicular/macrovesicular pattern of LDs. TM6SF2 deletion strongly affected endoplasmic reticulum and mitochondria ultrastructures, thus increasing endoplasmic reticulum/oxidative stress. The mitochondrial number was increased in both TM6SF2-/- and MBOAT7-/-TM6SF2-/- models, suggesting an unbalancing in mitochondrial dynamics, and the silencing of both MBOAT7 and TM6SF2 impaired mitochondrial activity with a shift toward anaerobic glycolysis. MBOAT7-/-TM6SF2-/- cells also showed the highest proliferation rate. Finally, the re-overexpression of MBOAT7 and/or TM6SF2 reversed the metabolic and tumorigenic features observed in the compound knockout model. Conclusions The co-presence of the 3 at-risk variants impacts the NAFLD course in both patients and experimental models, affecting LD accumulation, mitochondrial functionality, and metabolic reprogramming toward HCC., Graphical abstract

Graphical abstract, Highlights • Effect of ultrasound and the antioxidants of bamboo leaves (AOB) on the quality of scallop were evaluated. • UAOB-350 was the best treatment conditions on the quality maintenance of scallop. • UAOB-350 treatment could effectively retard protein and lipid oxidation. • UAOB-350 treatment could inhibit bacterial growth and texture deterioration. • UAOB-350 treatment prolonged the shelf life of scallop adductor muscles by 2 days., The combined effects of ultrasound and the antioxidants of bamboo leaves (AOB) on the quality maintenance of the adductor muscle of scallops (AMSs) during cold storage was investigated. Ultrasound power at 350 W coupled with AOB solution (2% w/v) (UAOB-350) was applied to treat the AMSs according to Taylor diagram analysis. The microstructure, oxidative changes (lipid and protein oxidation), total numbers of colonies, total volatile basic nitrogen, and texture of the AMSs during 6 days of cold storage were analysed. The results indicated that UAOB-350 treatment could effectively retard protein and lipid oxidation and bacterial growth and maintain better microstructure and texture characteristics than AOB solution treatment alone, prolonging the shelf life of the AMSs by 2 days during storage at 4 °C. These results indicate that the UAOB-350 combination method has promising potential to maintain the quality and extend the shelf life of AMSs during cold storage.

Background Recently, the distinction between left- and right-sided colon cancer (LCC and RCC) has been brought into focus. RCC is associated with an inferior overall survival and progression-free survival. We aimed to perform a detailed analysis of the diversity of extracellular vesicles (EV) between LCC and RCC using quantitative proteomics and to identify for new diagnostic and prognostic biomarkers. Methods We isolated EVs from patients with LCC, RCC and healthy volunteers, and treated colorectal cancer cell line with serum-derived EVs. We then performed a quantitative proteomics analysis of the serum-derived EVs and cell line treated with EVs. Proteomic data are available via ProteomeXchange with the identifiers PXD012283 and PXD012304. In addition, we assessed the performance of EV SPARC and LRG1 as diagnosis and prognosis biomarkers in colon cancer. Findings The expression profile of the serum EV proteome in patients with RCC was different from that of patients with LCC. Serum-derived EVs in RCC promoted cellular mobility more significantly than EVs derived from LCC. EV SPARC and LRG1 expression levels demonstrated area under the receiver-operating characteristic curve values of 0.95 and 0.93 for discriminating patients with colon cancer from healthy controls. Moreover, the expression levels of SPARC and LRG1 correlated with tumour sidedness and were predictive of tumour recurrence. Interpretation We identified differences in EV protein profiles between LCC and RCC. Serum-derived EVs of RCC may promote metastasis via upregulation of extracellular matrix (ECM)-related proteins, especially SPARC and LRG1, which may serve as diagnosis and prognosis biomarkers in colon cancer.

Background: Alzheimer's Disease (AD) is a complex and multifactorial disease and novel approaches are needed to illuminate the underlying pathology. Metabolites comprise the end-product of genes, transcripts, and protein regulations and might reflect disease pathogenesis. Blood is a common biofluid used in metabolomics; however, since extracellular vesicles (EVs) hold cell-specific biological material and can cross the blood-brain barrier, their utilization as biological material warrants further investigation. We aimed to investigate blood- and EV-derived metabolites to add insigts to the pathological mechanisms of AD.Methods: Blood samples were collected from 10 AD and 10 Mild Cognitive Impairment (MCI) patients, and 10 healthy controls. EVs were enriched from plasma using 100,000×g, 1 h, 4 °C with a wash. Metabolites from serum and EVs were measured using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Multivariate and univariate analyses were employed to identify altered metabolites in cognitively impaired individuals.Results: While no significant EV-derived metabolites were found differentiating patients from healthy individuals, six serum metabolites were found important; valine (p = 0.001, fold change, FC = 0.8), histidine (p = 0.001, FC = 0.9), allopurinol riboside (p = 0.002, FC = 0.2), inosine (p = 0.002, FC = 0.3), 4-pyridoxic acid (p = 0.006, FC = 1.6), and guanosine (p = 0.004, FC = 0.3). Pathway analysis revealed branched-chain amino acids, purine and histidine metabolisms to be downregulated, and vitamin B6 metabolism upregulated in patients compared to controls.Conclusion: Using a combination of LC-MS and NMR methodologies we identified several altered mechanisms possibly related to AD pathology. EVs require additional optimization prior to their possible utilization as a biological material for AD-related metabolomics studies.

Geranylgeranoic acid (GGA) was developed as a preventative agent against second primary hepatoma, and was reported to induce cell death in human hepatoma cells via Toll-like receptor 4 (TLR4)-mediated pyroptosis. We recently reported that GGA is enzymatically biosynthesized from mevalonic acid in human hepatoma-derived HuH-7 cells and that endogenous GGA is found in most rat organs including the liver. An unbiased metabolomics analysis of ice-cold 50% acetonitrile extracts from control and GGA-treated cells was performed in this study to characterize the intracellular metabolic changes in GGA-induced pyroptosis and to analyze their relationship with the mechanism of GGA-induced cell death. The total positive ion chromatograms of the cellular extracts in ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry were apparently unchanged after GGA treatment, but an orthogonal partial least squares-discriminant analysis score plot clearly discriminated the intracellular metabolite profiles of GGA-treated cells from that of control cells. S-plot analysis revealed 15 potential biomarkers up-regulated by 24-h GGA treatment according to their variable importance in the projection value of more than 1, and the subsequent metabolomics analysis identified nine of these metabolites as a group of lysophospholipids containing lysophosphatidylcholine with C16:0, C20:4, or C20:3 fatty acids. The possible roles of these lysophospholipids in GGA-induced pyroptosis are discussed., Highlights • Metabolomics analysis was performed on geranylgeranoic acid (GGA)-treated cells. • Total positive ion chromatograms were apparently similar after GGA treatment. • The OPLS-DA score plot distinguished the GGA-treated cells from control cells. • The S-plot analysis revealed GGA-induced upregulation of lysophospholipids. • The possible roles of lysophospholipids in GGA-induced pyroptosis are discussed.

Graphical abstract, Highlights • Reported Cd, Cr, Cu, Fe, Ni, Pb, and Zn contents in orthodox and CTC green teas. • CTC green tea had a higher content of Cr, Cu, Fe, Ni, and Zn than orthodox one. • Extraction of elements in CTC green tea was significantly higher than orthodox. • In both types of tea hazard quotient and hazard index of analyte element were, This study reported the content of selected metals, viz. cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), nickel (Ni), lead (Pb) and zinc (Zn) as well as non-carcinogenic risks of orthodox green tea and CTC (crush, tear and curl) green tea (Camellia sinensis L.) in India. Results revealed that significantly higher amount of Cr (1.26–10.48 mg kg−1), Cu (13.40–22.73 mg kg−1), Fe (54.14–99.65 mg kg−1), Ni (3.43–7.09 mg kg−1), and Zn (25.04–38.04 mg kg−1) in CTC green tea than orthodox one. However, no definite trend was observed for Cd and Pb, with overall contents ranged from 6.68 to 23.32 µg kg−1 and 0.04 to 0.13 mg kg−1, respectively. The extraction of the elements in tea infusion was higher for CTC green tea. The hazard quotient and hazard index values of all the studied metals were less than unity, confirming no significant health effect for consumers assuming drinking of 750 mL tea infusion prepared from 10 g green tea per day per person.

Highlights • We investigated network dynamics in the tauopathy progressive supranuclear palsy • Abnormal temporal properties of large-scale networks are related to phenotype • Progressive supranuclear palsy paradoxically increases frontoparietal state time • Reductions in neural signal complexity relate to altered network dynamics • Dynamic network and topological changes occur distally to primary sites of atrophy, The clinical syndromes of Progressive Supranuclear Palsy (PSP) may be mediated by abnormal temporal dynamics of brain networks, due to the impact of atrophy, synapse loss and neurotransmitter deficits. We tested the hypothesis that alterations in signal complexity in neural networks influence short-latency state transitions. Ninety-four participants with PSP and 64 healthy controls were recruited from two independent cohorts. All participants underwent clinical and neuropsychological testing and resting-state functional MRI. Network dynamics were assessed using hidden Markov models and neural signal complexity measured in terms of multiscale entropy. In both cohorts, PSP increased the proportion of time in networks associated with higher cognitive functions. This effect correlated with clinical severity as measured by the PSP-rating-scale, and with reduced neural signal complexity. Regional atrophy influenced abnormal brain-state occupancy, but abnormal network topology and dynamics were not restricted to areas of atrophy. Our findings show that the pathology of PSP causes clinically relevant changes in neural temporal dynamics, leading to a greater proportion of time in inefficient brain-states.

Funder: European Commission, BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a progressive liver disease with potentially severe complications including cirrhosis and hepatocellular carcinoma. Previously, we have identified circulating lipid signatures associating with liver fat content and non-alcoholic steatohepatitis (NASH). Here, we develop a metabolomic map across the NAFLD spectrum, defining interconnected metabolic signatures of steatosis (non-alcoholic fatty liver, NASH, and fibrosis). METHODS: We performed mass spectrometry analysis of molecular lipids and polar metabolites in serum samples from the European NAFLD Registry patients (n = 627), representing the full spectrum of NAFLD. Using various univariate, multivariate, and machine learning statistical approaches, we interrogated metabolites across 3 clinical perspectives: steatosis, NASH, and fibrosis. RESULTS: Following generation of the NAFLD metabolic network, we identify 15 metabolites unique to steatosis, 18 to NASH, and 15 to fibrosis, with 27 common to all. We identified that progression from F2 to F3 fibrosis coincides with a key pathophysiological transition point in disease natural history, with n = 73 metabolites altered. CONCLUSIONS: Analysis of circulating metabolites provides important insights into the metabolic changes during NAFLD progression, revealing metabolic signatures across the NAFLD spectrum and features that are specific to NAFL, NASH, and fibrosis. The F2-F3 transition marks a critical metabolic transition point in NAFLD pathogenesis, with the data pointing to the pathophysiological importance of metabolic stress and specifically oxidative stress. CLINICAL TRIALS REGISTRATION: The study is registered at Clinicaltrials.gov (NCT04442334). LAY SUMMARY: Non-alcoholic fatty liver disease is characterised by the build-up of fat in the liver, which progresses to liver dysfunction, scarring, and irreversible liver failure, and is markedly increasing in its prevalence worldwide. Here, we measured lipids and other small molecules (metabolites) in the blood with the aim of providing a comprehensive molecular overview of fat build-up, liver fibrosis, and diagnosed severity. We identify a key metabolic 'watershed' in the progression of liver damage, separating severe disease from mild, and show that specific lipid and metabolite profiles can help distinguish and/or define these cases.

Highlights • BSP increased production of SCFAs and CO2 while reduced the ammonia concentrations. • BSP fermentation increased the diversity of the bacterial communities. • BSP fermented by intestinal microbiota to produce beneficial organic acid metabolites. • BSP with potential prebiotic properties were beneficial to human health., The effects of Moso bamboo (Phyllostachys edulis) shoot polysaccharide (BSP) on the human gut microbiota composition and volatile metabolite components were investigated by in vitro fermentation. After fermentation for 48 h, BSP utilization reached 40.29% and the pH of the fermentation solution decreased from 6.89 to 4.57. Moreover, the total short-chain fatty acid concentration significantly (P

Highlights • Gait and balance impairments correlated to various imaging abnormalities. • Stride length, cadence and balance varied independently in PSP. • PSP phenotype affects the associations between gait and imaging., Progressive supranuclear palsy is a neurodegenerative disorder characterized primarily by tau inclusions and neurodegeneration in the midbrain, basal ganglia, thalamus, premotor and frontal cortex. Neurodegenerative change in progressive supranuclear palsy has been assessed using MRI. Degeneration of white matter tracts is evident with diffusion tensor imaging and PET methods have been used to assess brain metabolism or presence of tau protein deposits. Patients with progressive supranuclear palsy present with a variety of clinical syndromes; however early onset of gait impairments and postural instability are common features. In this study we assessed the relationship between multimodal imaging biomarkers (i.e., MRI atrophy, white matter tracts degeneration, flortaucipir-PET uptake) and laboratory-based measures of gait and balance abnormalities in a cohort of nineteen patients with progressive supranuclear palsy, using univariate and multivariate statistical analyses. The PSP rating scale and its gait midline sub-score were strongly correlated to gait abnormalities but not to postural imbalance. Principal component analysis on gait variables identified velocity, stride length, gait stability ratio, length of gait phases and dynamic stability as the main contributors to the first component, which was associated with diffusion tensor imaging measures in the posterior thalamic radiation, external capsule, superior cerebellar peduncle, superior fronto-occipital fasciculus, body and splenium of the corpus callosum and sagittal stratum, with MRI volumes in frontal and precentral regions and with flortaucipir-PET uptake in the precentral gyrus. The main contributor to the second principal component was cadence, which was higher in patients presenting more abnormalities on mean diffusivity: this unexpected finding might be related to compensatory gait strategies adopted in progressive supranuclear palsy. Postural imbalance was the main contributor to the third principal component, which was related to flortaucipir-PET uptake in the left paracentral lobule and supplementary motor area and white matter disruption in the superior cerebellar peduncle, putamen, pontine crossing tract and corticospinal tract. A partial least square model identified flortaucipir-PET uptake in midbrain, basal ganglia and thalamus as the main correlate of speed and dynamic component of gait in progressive supranuclear palsy. Although causality cannot be established in this analysis, our study sheds light on neurodegeneration of brain regions and white matter tracts that underlies gait and balance impairment in progressive supranuclear palsy.