Immune system-related plasma extracellular vesicles in healthy aging.

Objectives: To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. Methods: EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Results: Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1ß, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1ß, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Conclusion: Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

AAV-mediated hepatic LPL expression ameliorates severe hypertriglyceridemia and acute pancreatitis in Gpihbp1 deficient mice and rats.

GPIHBP1 plays an important role in the hydrolysis of triglyceride (TG) lipoproteins by lipoprotein lipases (LPLs). However, Gpihbp1 knockout mice did not develop hypertriglyceridemia (HTG) during the suckling period but developed severe HTG after weaning on a chow diet. It has been postulated that LPL expression in the liver of suckling mice may be involved. To determine whether hepatic LPL expression could correct severe HTG in Gpihbp1 deficiency, liver-targeted LPL expression was achieved via intravenous administration of the adeno-associated virus (AAV)-human LPL gene, and the effects of AAV-LPL on HTG and HTG-related acute pancreatitis (HTG-AP) were observed. Suckling Gpihbp1-/- mice with high hepatic LPL expression did not develop HTG, whereas Gpihbp1-/- rat pups without hepatic LPL expression developed severe HTG. AAV-mediated liver-targeted LPL expression dose-dependently decreased plasma TG levels in Gpihbp1-/- mice and rats, increased post-heparin plasma LPL mass and activity, decreased mortality in Gpihbp1-/- rat pups, and reduced the susceptibility and severity of both Gpihbp1-/- animals to HTG-AP. However, the muscle expression of AAV-LPL had no significant effect on HTG. Targeted expression of LPL in the liver showed no obvious adverse reactions. Thus, liver-targeted LPL expression may be a new therapeutic approach for HTG-AP caused by GPIHBP1 deficiency.

Comprehensive characterization of pathogenic synovial fluid extracellular vesicles from knee osteoarthritis.

Synovial fluid (SF) extracellular vesicles (EVs) play a pathogenic role in osteoarthritis (OA). However, the surface markers, cell and tissue origins, and effectors of these EVs are largely unknown. We found that SF EVs contained 692 peptides that were positively associated with knee radiographic OA severity; 57.4% of these pathogenic peptides were from 46 proteins of the immune system, predominantly the innate immune system. CSPG4, BGN, NRP1, and CD109 are the major surface markers of pathogenic SF EVs. Genes encoding surface marker CSPG4 and CD109 were highly expressed by chondrocytes from damaged cartilage, while VISG4, MARCO, CD163 and NRP1 were enriched in the synovial immune cells. The frequency of CSPG4+ and VSIG4+ EV subpopulations in OA SF was high. We conclude that pathogenic SF EVs carry knee OA severity-associated proteins and specific surface markers, which could be developed as a new source of diagnostic biomarkers or therapeutic targets in OA.

Copper induced intestinal inflammation response through oxidative stress induced endoplasmic reticulum stress in Takifugu fasciatus.

Copper (Cu) pollution in aquaculture water has seriously threatened the healthy and sustainable development of the aquaculture industry. Recently, many researchers have studied the toxic effects of Cu exposure on fish. However, the relationship between endoplasmic reticulum stress (ERS) and the inflammatory response, as well as its possible mechanisms, remain unclear. Particularly, information related to fish intestines must be expanded. Our study initially investigated the mechanisms underlying intestinal toxicity and inflammation resulting from Cu-induced ERS in vivo and in vitro in Takifugu fasciatus. In vivo study, T. fasciatus were treated with different concentrations (control, 20, and 100 µg/L) of Cu exposure for 28 days, causing intestinal oxidative stress, ERS, inflammatory responses, and histopathological and ultrastructural damage. Transcriptomic data further showed that Cu exposure caused ERS, as well as inflammatory responses, in the intestinal tracts of T. fasciatus. In vitro experiments on the intestinal cells of T. fasciatus showed that Cu exposure treatment (7.5 µg/mL) for 24 h induced ERS and increased mitochondrial numbers and inflammatory responses. In contrast, the addition of 4-phenylbutyric acid (4-PBA) alleviated ERS and inflammatory response in the Cu-exposed group. Furthermore, the reactive oxygen species (ROS) inhibitor, N-Acetyl-l-cysteine (NAC), effectively alleviated Cu-induced ERS. In conclusion, our in vivo and in vitro studies have confirmed that oxidative stress triggers the ERS pathway, which is involved in the intestinal inflammatory response. Our study provides new insights into the relationship among Cu-induced oxidative stress, ERS, and inflammatory responses in fish, as well as for the healthy culture of fish in aqueous environments.

Causal analysis identifies small HDL particles and physical activity as key determinants of longevity of older adults.

BACKGROUND: The hard endpoint of death is one of the most significant outcomes in both clinical practice and research settings. Our goal was to discover direct causes of longevity from medically accessible data. METHODS: Using a framework that combines local causal discovery algorithms with discovery of maximally predictive and compact feature sets (the "Markov boundaries" of the response) and equivalence classes, we examined 186 variables and their relationships with survival over 27 years in 1507 participants, aged ≥71 years, of the longitudinal, community-based D-EPESE study. FINDINGS: As few as 8-15 variables predicted longevity at 2-, 5- and 10-years with predictive performance (area under receiver operator characteristic curve) of 0·76 (95% CIs 0·69, 0·83), 0·76 (0·72, 0·81) and 0·66 (0·61, 0·71), respectively. Numbers of small high-density lipoprotein particles, younger age, and fewer pack years of cigarette smoking were the strongest determinants of longevity at 2-, 5- and 10-years, respectively. Physical function was a prominent predictor of longevity at all time horizons. Age and cognitive function contributed to predictions at 5 and 10 years. Age was not among the local 2-year prediction variables (although significant in univariable analysis), thus establishing that age is not a direct cause of 2-year longevity in the context of measured factors in our data that determine longevity. INTERPRETATION: The discoveries in this study proceed from causal data science analyses of deep clinical and molecular phenotyping data in a community-based cohort of older adults with known lifespan. FUNDING: NIH/NIA R01AG054840, R01AG12765, and P30-AG028716, NIH/NIA Contract N01-AG-12102 and NCRR 1UL1TR002494-01.

The transcription factor PAX8 promotes angiogenesis in ovarian cancer through interaction with SOX17.

PAX8 is a master transcription factor that is essential during embryogenesis and promotes neoplastic growth. It is expressed by the secretory cells lining the female reproductive tract, and its deletion during development results in atresia of reproductive tract organs. Nearly all ovarian carcinomas express PAX8, and its knockdown results in apoptosis of ovarian cancer cells. To explore the role of PAX8 in these tissues, we purified the PAX8 protein complex from nonmalignant fallopian tube cells and high-grade serous ovarian carcinoma cell lines. We found that PAX8 was a member of a large chromatin remodeling complex and preferentially interacted with SOX17, another developmental transcription factor. Depleting either PAX8 or SOX17 from cancer cells altered the expression of factors involved in angiogenesis and functionally disrupted tubule and capillary formation in cell culture and mouse models. PAX8 and SOX17 in ovarian cancer cells promoted the secretion of angiogenic factors by suppressing the expression of SERPINE1, which encodes a proteinase inhibitor with antiangiogenic effects. The findings reveal a non-cell-autonomous function of these transcription factors in regulating angiogenesis in ovarian cancer.

Transcriptional, secondary metabolic, and antioxidative investigations elucidate the rapid response mechanism of Pontederia cordata to cadmium.

Pontederia cordata is previously demonstrated a cadmium (Cd) tolerant plant, and also a candidate for the phytoremediation of heavy-metal-contaminated wetlands. A hydroponic experiment was used to investigate variations in photosynthetic gas exchange parameters, antioxidative activities, chlorophyll and secondary metabolite contents, and transcriptome in leaves of the plant exposed to 0.44 mM Cd2+ for 0 h, 24 h, and 48 h. Under Cd2+ exposure for 24 h, the plant presented a favorable photosynthesis by maintaining relatively higher antioxidant activity. Cd2+ exposure for 48 h accelerated membrane peroxidation, declined photosynthetic pigment content, and increased polyphenol oxidase activity, thus interfering with photosynthesis. The phenylpropane pathway served as a chemical rather than physical defense against Cd2+ in the plant leaves. A total of 20,998, 4743, and 4413 differentially expressed genes (DEGs) were identified in the groups of 0 h vs 24 h, 0 h vs 48 h, and 24 h vs 48 h, respectively. The primary metabolic pathways of the DEGs were mainly enriched in nitrogen metabolism, starch and sucrose metabolism, fructose and mannose metabolism, as well as pentose-phosphate pathway, contributing to a stable cell structure and function. Flavonoid biosynthesis directly or indirectly played an antioxidative role against Cd2+ in the leaves. Forty-nine transcription factor (TF) families were identified, and 8 TF families were shared among the three groups. The present study provides a theoretical foundation for investigating tolerance mechanisms of wetland plants to Cd stress in terms of secondary metabolism and transcriptional regulation.

Correction of Familial LCAT Deficiency by AAV-hLCAT Prevents Renal Injury and Atherosclerosis in Hamsters-Brief Report.

[Figure: see text].

Chinese herbal fumigant and lotion for postoperative complication in surgical wound of anal fistula: A protocol for a systematic review and meta-analysis.

BACKGROUND: Surgery is the most common and effective therapy for anal fistula, while the postoperative complication, such as pain, edema, pruritus, turgescence, and exudation in surgical wound, can have serious impact on wound healing and patients' quality of life. Chinese herbal fumigant and lotion have been commonly used in postoperative treatment and achieved satisfied effect in China. However, clinical evidence-based literature of Chinese herbal fumigant and lotion for postoperative anal fistula is not sufficient. This protocol is described for a systematic review to investigate the beneficial effects. METHODS: A systematic search will be conducted in database involving PubMed, the Cochrane library, Embase, Web of Science, Google Scholar, SinoMed, China National Knowledge Infrastructure(CNKI), VIP, Wanfang Database, CiNii(National Institute of Informatics), and KISS(Koreanstudies Information Service System) from inceptions to December 31, 2019. We will include randomized controlled trials (RCT) regarding Chinese herbal fumigant and lotion in the treatment of complication in surgical wound of anal fistula. Quality of included RCTs will be assessed according to the Cochrane Handbook 5.1.0. GRADE will be used to assess the quality of evidence. The summary results will be pooled using the random-effects model or fixed-effects model according to the heterogeneity of included studies. RESULTS: After peer-review, the study will be disseminated in scientific journals and conferences. CONCLUSION: This systematic review will provide evidence for the efficacy of Chinese herbal fumigant and lotion for curing postoperative complication of anal fistula. In addition, it might provide suggestions for Chinese medicine clinical practice or guideline. PROSPERO REGISTRATION: CRD42020164975.

AAV-Mediated ApoC2 Gene Therapy: Reversal of Severe Hypertriglyceridemia and Rescue of Neonatal Death in ApoC2-Deficient Hamsters.

Apolipoprotein C2 (ApoC2) is a key activator of lipoprotein lipase for plasma triglyceride metabolism. ApoC2-deficient patients present with severe hypertriglyceridemia and recurrent acute pancreatitis, for whom the only effective treatment is the infusion of normal plasma containing ApoC2. However, since ApoC2 has a fast catabolic rate, a repeated infusion is required, which limits its clinical use. To explore a safe and efficient approach for ApoC2 deficiency, we herein established an adeno-associated virus expressing human ApoC2 (AAV-hApoC2) to evaluate the efficacy and safety of gene therapy in ApoC2-deficient hypertriglyceridemic hamsters. Administration of AAV-hApoC2 via jugular or orbital vein in adult and neonatal ApoC2-deficient hamsters, respectively, could prevent the neonatal death and effectively improve severe hypertriglyceridemia of ApoC2-deficient hamsters without side effects in a long-term manner. Our novel findings in the present study demonstrate that AAV-hApoC2-mediated gene therapy will be a promising therapeutic approach for clinical patients with severe hypertriglyceridemia caused by ApoC2 deficiency.

Cadmium phytotoxicity, related physiological changes in Pontederia cordata: antioxidative, osmoregulatory substances, phytochelatins, photosynthesis, and chlorophyll fluorescence.

Pontederia cordata is a heavy metal accumulator, while the heavy metal tolerance mechanisms of this plant are not well understood. Hydroponic experiments were used to assess the effects of Cd2+ on antioxidative activities, osmoregulatory substances and photosynthesis in leaves. Exposure of 5 mg L-1 Cd2+ for 7 days, the photosynthetic apparatus functioned normally and sustained a relatively high photosynthetic rate, and good growth was observed. Under 50 and 75 mg L-1 Cd2+, accelerated lipid peroxidation and increased peroxidase activity (POD; E.C.1.11.1.7) were detected, while no significant differences were observed in superoxide dismutase (SOD; E.C.1.15.1.1) and catalase (CAT; E.C.1.11.1.6) activities, as well as in lutein, ascorbic acid, and glutathione contains of leaves. Proline content increased, while soluble sugar and soluble protein contents decreased under 75 mg L-1 Cd2+. Cd2+ at different concentrations induced a reduction in carotenoid, total carotenoid, and ascorbic acid-dehydroascorbate contents. A significant increase in phytochelatin content was induced by 75 mg L-1. Chlorophyll content decreased under Cd stress and disturbed photosynthesis, causing dramatic reductions in photosynthetic parameters. Stomatal closure was responsible for a reduced photosynthetic rate under Cd2+ exposure. Cd2+ concentrations of no less than 25 mg L-1 disorganized the photosynthetic apparatus, induced the partial closure, and decreased activity of the photosystem II (PS II) reaction center, thus disturbing light conversion and utilization, thereby decreasing the photosynthetic efficiency in PS II.

Bariatric Surgery is Associated With Decreased Progression of Nonalcoholic Fatty Liver Disease to Cirrhosis: A Retrospective Cohort Analysis.

OBJECTIVE: This study sought to compare trends in the development of cirrhosis between patients with NAFLD who underwent bariatric surgery and a well-matched group of nonsurgical controls. SUMMARY OF BACKGROUND DATA: Patients with NAFLD who undergo bariatric surgery generally have improvements in liver histology. However, the long-term effect of bariatric surgery on clinically relevant liver outcomes has not been investigated. METHODS: From a large insurance database, patients with a new NAFLD diagnosis and at least 2 years of continuous enrollment before and after diagnosis were identified. Patients with traditional contraindications to bariatric surgery were excluded. Patients who underwent bariatric surgery were identified and matched 1:2 with patients who did not undergo bariatric surgery based on age, sex, and comorbid conditions. Kaplan-Meier analysis and Cox proportional hazards modeling were used to evaluate differences in progression from NAFLD to cirrhosis. RESULTS: A total of 2942 NAFLD patients who underwent bariatric surgery were identified and matched with 5884 NAFLD patients who did not undergo surgery. Cox proportional hazards modeling found that bariatric surgery was independently associated with a decreased risk of developing cirrhosis (hazard ratio 0.31, 95% confidence interval 0.19-0.52). Male gender was associated with an increased risk of cirrhosis (hazard ratio 2.07, 95% confidence interval 1.31-3.27). CONCLUSIONS: Patients with NAFLD who undergo bariatric surgery are at a decreased risk for progression to cirrhosis compared to well-matched controls. Bariatric surgery should be considered as a treatment strategy for otherwise eligible patients with NAFLD. Future bariatric surgery guidelines should include NAFLD as a comorbid indication when determining eligibility.

Dilution of Molecular-Pathologic Gene Signatures by Medically Associated Factors Might Prevent Prediction of Resection Status After Debulking Surgery in Patients With Advanced Ovarian Cancer.

PURPOSE: Predicting surgical outcome could improve individualizing treatment strategies for patients with advanced ovarian cancer. It has been suggested earlier that gene expression signatures (GES) might harbor the potential to predict surgical outcome. EXPERIMENTAL DESIGN: Data derived from high-grade serous tumor tissue of FIGO stage IIIC/IV patients of AGO-OVAR11 trial were used to generate a transcriptome profiling. Previously identified molecular signatures were tested. A theoretical model was implemented to evaluate the impact of medically associated factors for residual disease (RD) on the performance of GES that predicts RD status. RESULTS: A total of 266 patients met inclusion criteria, of those, 39.1% underwent complete resection. Previously reported GES did not predict RD in this cohort. Similarly, The Cancer Genome Atlas molecular subtypes, an independent de novo signature and the total gene expression dataset using all 21,000 genes were not able to predict RD status. Medical reasons for RD were identified as potential limiting factors that impact the ability to use GES to predict RD. In a center with high complete resection rates, a GES which would perfectly predict tumor biological RD would have a performance of only AUC 0.83, due to reasons other than tumor biology. CONCLUSIONS: Previously identified GES cannot be generalized. Medically associated factors for RD may be the main obstacle to predict surgical outcome in an all-comer population of patients with advanced ovarian cancer. If biomarkers derived from tumor tissue are used to predict outcome of patients with cancer, selection bias should be focused on to prevent overestimation of the power of such a biomarker.See related commentary by Handley and Sood, p. 9.

Extracting Pathway-level Signatures from Proteogenomic Data in Breast Cancer Using Independent Component Analysis.

Recent advances in the multi-omics characterization necessitate knowledge integration across different data types that go beyond individual biomarker discovery. In this study, we apply independent component analysis (ICA) to human breast cancer proteogenomics data to retrieve mechanistic information. We show that as an unsupervised feature extraction method, ICA was able to construct signatures with known biological relevance on both transcriptome and proteome levels. Moreover, proteome and transcriptome signatures can be associated by their respective correlation with patient clinical features, providing an integrated description of phenotype-related biological processes. Our results demonstrate that the application of ICA to proteogenomics data could lead to pathway-level knowledge discovery. Potential extension of this approach to other data and cancer types may contribute to pan-cancer integration of multi-omics information.

Developing a Clinico-Molecular Test for Individualized Treatment of Ovarian Cancer: The interplay of Precision Medicine Informatics with Clinical and Health Economics Dimensions.

We report recent progress in the development of a precision test for individualized use of the VEGF-A targeting drug bevacizumab for treating ovarian cancer. We discuss the discovery model stage (i.e., past feasibility modeling and before conversion to the production test). Main results: (a) Informatics modeling plays a critical role in supporting driving clinical and health economic requirements. (b) The novel computational models support the creation of a precision test with sufficient predictivity to reduce healthcare system costs up to $30 billion over 10 years, and make the use of bevacizumab affordable without loss of length or quality of life.

Combination of AAV-TRAIL with miR-221-Zip Therapeutic Strategy Overcomes the Resistance to TRAIL Induced Apoptosis in Liver Cancer.

TNF-related apoptosis-inducing ligand (TRAIL) possesses the capacity to induce apoptosis in a wide variety of tumor cells without affecting most normal cells. However, it has now emerged that many primary cancer cells are resistant to TRAIL monotherapy. Overcoming the intrinsic or acquired TRAIL resistance is desirable for TRAIL-mediated cancer therapy. In this study, we found that the miR-221/222 cluster was up-regulated in TRAIL-resistant liver cancer cells. Specific inhibitors of miR-221 and/or miR-222, called sponge, TuD and miR-Zip were constructed, and their ability to overcome TRAIL resistance was compared. Among them, AAV-mediated gene therapy using co-expression of TRAIL with miR-221-Zip showed the most synergistic activity in the induction of apoptosis in vitro. In vivo treatment of nude mice bearing human TRAIL-resistant liver cancer xenografts with AAV-TRAIL-miR-221-Zip also led to growth inhibition. This sensitizing effect of miR-221-Zip was associated with increased expression of PTEN, the miR-221 target, as well as with decreasing levels of Survivin. Moreover, miR-221 expression was concomitant with promotion of Survivin expression and suppression of PTEN expression. TRAIL sensitivity of cancer cells isolated from liver cancer tissues or from patients was significantly correlated with miR-221 expression. And miR-221 blood expression levels in liver cancer patients were correlated with TRAIL sensitivity, thus it had the potential to be a predictor of TRAIL sensitivity in liver cancer. These data suggested the potential of combining AAV-TRAIL with miR-221-Zip as a therapeutic intervention for liver cancer.

In Situ Hot-Spot Assembly as a General Strategy for Probing Single Biomolecules.

Single-molecule detection using surface-enhanced Raman spectroscopy (SERS) has attracted increasing attention in chemical and biomedical analysis. However, it remains a major challenge to probe single biomolecules by means of SERS hot spots owing to the small volume of hot spots and their random distribution on substrates. We here report an in situ hot-spot assembly method as a general strategy for probing single biomolecules. As a proof-of-concept, this proposed strategy was successfully used for the detection of single microRNA-21 (miRNA-21, a potential cancer biomarker) at the single-cell level, showing great capability in differentiating the expression of miRNA-21 in single cancer cells from normal cells. This approach was further extended to single-protein detection. The versatility of the strategy opens an exciting avenue for single-molecule detection of biomarkers of interest and thus holds great promise in a variety of biological and biomedical applications.

Interference-Free Surface-Enhanced Raman Scattering Tags for Single-Cell Molecular Imaging with a High Signal-to-Background Ratio.

An interference-free surface-enhanced Raman scattering tag is constructed to profile the expression of cancer biomarkers at the single-cell level. The Raman tags present a strong and sharp peak in the cellular Raman-silent region, significantly diminishing the background interference. Moreover, the reporters are embedded in the layered gold nanoparticles, avoiding desorption and enzymatic degradation in physiological conditions.

Prussian Blue as a Highly Sensitive and Background-Free Resonant Raman Reporter.

We report a surprising discovery that Prussian blue (PB) can be employed as a highly sensitive and background-free resonant Raman reporter. Conventional Raman reporters show multiple spectral bands in the fingerprint region, which are generally overlapped with those from dominant endogenous biomolecules, and are thus difficult to be separated. Herein, we found that PB only possesses a strong and sharp single-band in the cellular Raman-silent region, where no Raman signals from biological species were observed. Therefore, the Raman spectra from PB and endogenous biomolecules are completely resolved without resorting to complicated spectral unmixing. Moreover, PB holds a strong UV-vis absorption band between 500 and 900 nm, which is resonant with the incident detection lasers, providing extremely high sensitivity. Through assembly of PB onto plasmonic cores, a new surface-enhanced resonance Raman scattering (SERRS) probe was achieved with a high signal-to-background ratio (SBR). We demonstrated the performance of the PB-based SERRS tags for high-sensitivity immunoassay and cancer cell imaging.

Human transposon insertion profiling: Analysis, visualization and identification of somatic LINE-1 insertions in ovarian cancer.

Mammalian genomes are replete with interspersed repeats reflecting the activity of transposable elements. These mobile DNAs are self-propagating, and their continued transposition is a source of both heritable structural variation as well as somatic mutation in human genomes. Tailored approaches to map these sequences are useful to identify insertion alleles. Here, we describe in detail a strategy to amplify and sequence long interspersed element-1 (LINE-1, L1) retrotransposon insertions selectively in the human genome, transposon insertion profiling by next-generation sequencing (TIPseq). We also report the development of a machine-learning-based computational pipeline, TIPseqHunter, to identify insertion sites with high precision and reliability. We demonstrate the utility of this approach to detect somatic retrotransposition events in high-grade ovarian serous carcinoma.