Peptidome analysis reveals critical roles for peptides in a rat model of intestinal ischemia/reperfusion injury.

Intestinal ischemia/reperfusion injury (IIRI) has the potential to be life threatening and is associated with significant morbidity and serious damage to distant sites in the body on account of disruption of the intestinal mucosal barrier. In the present study, we have explored this line of research by comparing and identifying peptides that originated from the intestinal segments of IIRI model rats by using liquid chromatography-mass spectrometry (LC-MS). We also analyzed the basic characteristics, cleavage patterns, and functional domains of differentially expressed peptides (DEPs) between the IIRI model rats and control (sham-operated) rats and identified bioactive peptides that are potentially associated with ischemia reperfusion injury. We also performed bioinformatics analyses in order to identify the biological roles of the DEPs based on their precursor proteins. Enrichment analysis demonstrated the role of several DEPs in impairment of the intestinal mucosal barrier caused by IIRI. Based on the results of comprehensive ingenuity pathway analysis, we identified the DEPs that were significantly correlated with IIRI. We identified a candidate precursor protein (Actg2) and seven of its peptides, and we found that Actg2-6 had a more significant difference in its expression, a longer half-life, and better lipophilicity, hydrophobicity, and stability than the other candidate Actg2 peptides examined. Furthermore, we observed that Actg2-6 might play critical roles in the protection of the intestinal mucosal barrier during IIRI. In summary, our study provides a better understanding of the peptidomics profile of IIRI, and the results indicate that Actg2-6 could be a useful target in the treatment of IIRI.

MDL-800, the SIRT6 Activator, Suppresses Inflammation via the NF-κB Pathway and Promotes Angiogenesis to Accelerate Cutaneous Wound Healing in Mice.

Sirtuin 6 (SIRT6) is an NAD+-dependent deacetylase belonging to the sirtuin family. It has been shown to participate in wound healing and some inflammation-related disorders. However, the effect of MDL-800, a highly efficient and selective SIRT6 activator, on wound healing and inflammation has not been reported. Therefore, this study investigated whether MDL-800 confers anti-inflammatory effects and promotes wound healing and uncovered the molecular mechanisms involved. This was achieved using mouse models of full-thickness wounds. Results showed that MDL-800 significantly downregulated inflammation by attenuating the release of inflammatory mediators and improved collagen deposition and neovascularization of wounds, thereby accelerating cutaneous wound healing. Furthermore, MDL-800 significantly downregulated expression levels of TNF-α and IL-6 in the dorsal skin tissue of mice via the NF-κB pathway. These results demonstrated that MDL-800 exerted anti-inflammatory and prohealing effects, indicating that the SIRT6/NF-κB/IκB signaling pathway may play an important role in wound healing.

Surface design of g-C3N4 quantum dot-decorated TiO2(001) to enhance the photodegradation of indoor formaldehyde by experimental and theoretical investigation.

Formaldehyde (CHOH), a common volatile organic compound, causes many adverse effects on human health. The highly exposed TiO2(001) facet possesses a high photodegradation efficiency of CHOH due to its excellent ability to trap photogenerated holes and high density of surface unsaturated Ti atoms (Ti5c) to bind CHOH. However, the rapid recombination of photoinduced electron-hole pairs of TiO2(001) limits the photodegradation efficiency. We adopted a strategy of decorating TiO2(001) with g-C3N4 quantum dots (QDs), exploiting the quantum effect of g-C3N4QDs and their combined staggered band structure. This decoration improves the photocatalytic activity of TiO2(001). Moreover, the chemical configuration of g-C3N4QDs/TiO2(001) and the combination mode between the g-C3N4QDs and TiO2(001) support were explored in detail using high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculations. Following the physiochemical characteristic results, the transport mechanism of photoinduced carriers was further analyzed by ultraviolet photoelectron spectroscopy (UPS), electron paramagnetic resonance (EPR), and Heyd-Scuseria-Ernzerh (HSE) exchange-correlation functional calculations. Finally, the performance and reaction mechanism of the photodegradation of CHOH by TiO2(001) and g-C3N4QDs/TiO2(001) were thoroughly investigated. The results show that the g-C3N4QDs were composed of an N-defect tri-s-triazine supported by TiO2(001) via a strong C-O-Ti chemical bond, which accelerated the separation of photoinduced carriers through a Z-scheme route. The photodegradation and mineralization efficiencies of CHOH were significantly promoted by 30% and 60% for g-C3N4QDs/TiO2(001) compared with those of TiO2(001). The photodegradation mechanism proceeded as CHOH - dioxymethylene - formate - carbonate - CO2. This study provides a surface engineering means to design highly active modified TiO2 for CHOH photodegradation.

Correlation between 25-Hydroxyvitamin D, sFlt-1, PLGF, and Hypertension in Pregnancy.

Gestational hypertension is a common disease in clinical practice, which does great harm to the mother and infant. The purpose of this study was to investigate the relationship between 25-hydroxyvitamin D, sFlt-1, and PLGF and hypertensive disorder complicating pregnancy. Specimen preparation: after delivery or placental caesarean section, in order to avoid calcification and necrosis in the middle of the placenta, an area of about 1.5 cm × 1.5 cm × 1.5 cm should be separated immediately. After dehydration, use a Citadel 2000 dryer to dry it and place it in a block of saline for xylene immunohistochemical staining. Statistical processing was performed according to the proportion of positive cells in each part and the depth of staining. Placental tissue collection and treatment: within 20 minutes after the delivery of the placenta, two pieces of the placental tissue (about 1.0 cm × 1.0 cm × 1.0 cm) were taken from the central zone of the placental maternal surface without obvious bleeding and calcification. They were rinsed repeatedly in normal saline, fixed in 10% neutral formaldehyde solution for 24 hours, dehydrated using an automatic dehydrator, and embedded in paraffin for detection. Before the study, 20 ml of distilled water was added to the sample to stand for 20 minutes; the Cobas E610 immunoanalyzer was turned on, and sFlt-1 and PLGF (placental growth factor) were selected. The serum 25-hydroxyvitamin D level of pregnant women was detected in the fasting state at 24-28 weeks of gestation, and the best collection time was 8 : 00-11 : 00 in the morning. 5 ml of the whole blood sample without anticoagulant was collected and stored at 0-4°C in a cold storage and dark environment. The serum was obtained by high-speed centrifugation within 24 hours after collection. The method is suitable for the quantitative determination of 25-OH-vitamin D in human serum. In the hypertensive pregnancy group, the level of 25-hydroxyvitamin D was 18.44 ± 3.48 ng/ml, and the sFlt-1/PLGF level was highest at 0-5 weeks followed by 5-10 weeks, 10-15 weeks, and 15-20 weeks. This study provides new ideas and experimental clues for the prevention and treatment of pre-eclampsia.

Curcumin promotes the survival of ischemic random skin flaps via autophagy.

Random skin flaps have been widely applied in reconstructive and plastic surgery; however, necrosis usually happens due to insufficient blood supply in the ischemic area of flaps. Curcumin (CUR) is a primary bioactive compound of turmeric (Curcuma longa, L.), which has been proven to be effective on anticancer, decreasing oxidative stress and apoptosis through activating autophagy, and promoting angiogenesis in ischemic tissue. Therefore, the potential therapeutic effect of CUR on promoting survival of ischemic random skin flaps and its underlying mechanism associated with autophagy were investigated. After establishment of dorsal random skin flaps, sixty mice were randomly divided into three groups: Control, CUR or CUR+3-methyladenine (3-MA, an autophagy inhibitor). The results showed that CUR increased the viability area and blood flow as well as relieved the edema of skin flaps through promoting angiogenesis, decreasing oxidative stress, and inhibiting apoptosis of the ischemic area. Further study confirmed that CUR activated autophagy in the random skin flaps, and 3-MA effectively reversed the effect on viability, neovascularization, oxidative stress and apoptosis, suggesting autophagy played a vital role in these CUR's protective effect on random skin flaps. Moreover, this CUR-induced autophagy should be mediated through downregulating the PI3K/AKT/mTOR signaling pathway. Together with secondary response of increased angiogenesis, reduced oxidative stress and apoptosis, CUR effectively improved survival of random skin flaps in vivo. To sum up, our research showed the great potential of CUR using as a promising flap protective therapy for random skin flap survival and regeneration.

Investigation of Nrf2, AhR and ATF4 Activation in Toxicogenomic Databases.

Toxicological responses to chemical insult are largely regulated by transcriptionally activated pathways that may be independent, correlated and partially or fully overlapping. Investigating the dynamics of the interactions between stress responsive transcription factors from toxicogenomic data and defining the signature of each of them is an additional step toward a system level understanding of perturbation driven mechanisms. To this end, we investigated the segregation of the genes belonging to the three following transcriptionally regulated pathways: the AhR pathway, the Nrf2 pathway and the ATF4 pathway. Toxicogenomic datasets from three projects (carcinoGENOMICS, Predict-IV and TG-GATEs) obtained in various experimental conditions (in human and rat in vitro liver and kidney models and rat in vivo, with bolus administration and with repeated doses) were combined and consolidated where overlaps between datasets existed. A bioinformatic analysis was performed to refine pathways' signatures and to create chemical activation capacity scores to classify chemicals by their potency and selectivity of activation of each pathway. With some refinement such an approach may improve chemical safety classification and allow biological read across on a pathway level.

Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures.

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells.

Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.

Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations.

Cancer drug screening in patient-derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix-dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy-based assay to resolve drug-induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug-induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.