A novel GARP humanized mouse model for efficacy assessment of GARP-targeting therapies.

Although breakthroughs have been achieved with immune checkpoint inhibitors (ICI) therapy, some tumors do not respond to those therapies due to primary or acquired resistance. GARP, a type I transmembrane cell surface docking receptor mediating latent transforming growth factor-ß (TGF-ß) and abundantly expressed on regulatory T lymphocytes and platelets, is a potential target to render these tumors responsive to ICI therapy, and enhancing anti-tumor response especially combined with ICI. To facilitate these research efforts, we developed humanized mouse models expressing humanized GARP (hGARP) instead of their mouse counterparts, enabling in vivo assessment of GARP-targeting agents. We created GARP-humanized mice by replacing the mouse Garp gene with its human homolog. Then, comprehensive experiments, including expression analysis, immunophenotyping, functional assessments, and pharmacologic assays, were performed to characterize the mouse model accurately. The Tregs and platelets in the B-hGARP mice (The letter B is the first letter of the company's English name, Biocytogen.) expressed human GARP, without expression of mouse GARP. Similar T, B, NK, DCs, monocytes and macrophages frequencies were identified in the spleen and blood of B-hGARP and WT mice, indicating that the humanization of GARP did not change the distribution of immune cell in these compartments. When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP/TGF-ß1 complexes demonstrated enhanced in vivo anti-tumor activity compared to monotherapy with either agent. The novel hGARP model serves as a valuable tool for evaluating human GARP-targeting antibodies in immuno-oncology, which may enable preclinical studies to assess and validate new therapeutics targeting GARP. Furthermore, intercrosses of this model with ICI humanized models could facilitate the evaluation of combination therapies.

Signatures of selection in Mulinia lateralis underpinning its rapid adaptation to laboratory conditions.

The dwarf surf clam, Mulinia lateralis, is considered as a model species for bivalves because of its rapid growth and short generation time. Recently, successful breeding of this species for multiple generations in our laboratory revealed its acquisition of adaptive advantages during artificial breeding. In this study, 310 individuals from five different generations were genotyped with 22,196 single nucleotide polymorphisms (SNPs) with the aim of uncovering the genetic basis of their adaptation to laboratory conditions. Results revealed that M. lateralis consistently maintained high genetic diversity across generations, characterized by high observed heterozygosity (H o: 0.2733-0.2934) and low levels of inbreeding (F is: -0.0244-0.0261). Population analysis indicated low levels of genetic differentiation among generations of M. lateralis during artificial breeding (F st <0.05). In total, 316 genomic regions exhibited divergent selection, with 168 regions under positive selection. Furthermore, 227 candidate genes were identified in the positive selection regions, which have functions including growth, stress resistance, and reproduction. Notably, certain selection signatures with significantly higher F st value were detected in genes associated with male reproduction, such as GAL3ST1, IFT88, and TSSK2, which were significantly upregulated during artificial breeding. This suggests a potential role of sperm-associated genes in the rapid evolutionary response of M. lateralis to selection in laboratory conditions. Overall, our findings highlight the phenotypic and genetic changes, as well as selection signatures, in M. lateralis during artificial breeding. This contributes to understanding their adaptation to laboratory conditions and underscores the potential for using this species to explore the adaptive evolution of bivalves.

Neurotoxicological mechanisms of carbon quantum dots in a new animal model Dugesia japonica.

As luminescent nanomaterials, the carbon quantum dots (CQDs) research focused on emerging applications since their discovery. However, their toxicological effects on the natural environment are still unclear. The freshwater planarian Dugesia japonica is distributed extensively in aquatic ecosystems and can regenerate a new brain in 5 days after amputation. Therefore it can be used as a new model organism in the field of neuroregeneration toxicology. In our study, D. japonica was cut and incubated in medium treated with CQDs. The results showed that the injured planarian lost the neuronal ability of brain regeneration after treatment with CQDs. Its Hh signalling system was interfered with at Day 5, and all cultured pieces died on or before Day 10 due to head lysis. Our work reveals that CQDs might affect the nerve regeneration of freshwater planarians via the Hh signalling pathway. The results of this study improve our understanding of CQD neuronal development toxicology and can aid in the development of warning systems for aquatic ecosystem damage.

Inhaled Lipid Nanoparticles Alleviate Established Pulmonary Fibrosis.

Pulmonary fibrosis, a sequela of lung injury resulting from severe infection such as severe acute respiratory syndrome-like coronavirus (SARS-CoV-2) infection, is a kind of life-threatening lung disease with limited therapeutic options. Herein, inhalable liposomes encapsulating metformin, a first-line antidiabetic drug that has been reported to effectively reverse pulmonary fibrosis by modulating multiple metabolic pathways, and nintedanib, a well-known antifibrotic drug that has been widely used in the clinic, are developed for pulmonary fibrosis treatment. The composition of liposomes made of neutral, cationic or anionic lipids, and poly(ethylene glycol) (PEG) is optimized by evaluating their retention in the lung after inhalation. Neutral liposomes with suitable PEG shielding are found to be ideal delivery carriers for metformin and nintedanib with significantly prolonged retention in the lung. Moreover, repeated noninvasive aerosol inhalation delivery of metformin and nintedanib loaded liposomes can effectively diminish the development of fibrosis and improve pulmonary function in bleomycin-induced pulmonary fibrosis by promoting myofibroblast deactivation and apoptosis, inhibiting transforming growth factor 1 (TGFß1) action, suppressing collagen formation, and inducing lipogenic differentiation. Therefore, this work presents a versatile platform with promising clinical translation potential for the noninvasive inhalation delivery of drugs for respiratory disease treatment.

[Determination of organophosphate diesters in facility vegetable soils using ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry].

Organophosphate diesters (Di-OPEs) are biotic or abiotic degradation products of organophosphate esters (OPEs). Current analytical methods focus on detecting Di-OPEs in human urine. Human exposure to Di-OPEs in environmental matrices has not been systematically studied. Soil plays an important role in the environmental migration and transformation of organic pollutants. Previous studies found that OPEs are ubiquitous in soil. However, few studies reported OPEs metabolite pollution in soil, especially in facility vegetable soil. In this study, an ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UPHLC-Orbitrap HRMS) method was developed for the determination of five Di-OPEs (bis(2-chloroethyl) phosphate (BCEP), bis(1,3-dichloro-2-propyl) phosphate (BDCP), di-n-butyl phosphate (DnBP), diphenyl phosphate (DPhP), and bis(2-ethylhexyl) phosphate (DEHP)) in the facility vegetable soil. The pretreatment process and chromatographic and mass spectrometric conditions were optimized in the present study. Comparative study of the purification effects of different solid-phase extraction columns showed that Oasis WAX cartridge had best purification efficiency for the five Di-OPEs. The cartridge was first activated using 3 mL methanol, 3 mL methanol containing 5% (v/v) ammonia, and 3 mL 0.1 mol/L sodium acetate-acetic acid buffer solution. Then, the cartridge was rinsed with 3 mL of 30% (v/v) methanol aqueous solution, and finally eluted using 8 mL methanol containing 5% (v/v) ammonia. The effects of mobile phase (with respect to solvent composition and flow rate) and column temperature on the shape and intensity of chromatographic peaks were studied. The optimized UHPLC conditions were as follows: chromatographic column, Thermo Accucore RP-MS; column temperature, 30 ℃; mobile phase, 0.2 mmol/L ammonium acetate aqueous solution and methanol; flow rate, 0.2 mL/min. In the UHPLC-Orbitrap HRMS experiment, the five Di-OPEs were analyzed in full MS mode with negative ionization. Instrumental parameters, such as sheath gas and auxiliary gas, were optimized to determine the MS conditions. The optimized Orbitrap HRMS conditions were as follows: heating electrospray ionization source (HESI), full MS mode with negative ionization; scan range, m/z 100-500; ion transfer tube temperature, 320 ℃; automatic gain control of target particle count, 1×106; sheath gas flow rate, 8.58 L/min; auxiliary gas flow rate, 17.40 L/min; spray voltage, 3.2 kV; and S-lens voltage, 50 V. The limits of detection and quantification were 0.001-0.047 ng/g and 0.004-0.156 ng/g, respectively. The correlation coefficients of the calibration curve were 0.9985-0.9999. At three spiked levels, 5.0, 25.0, and 50.0 ng/g, the recoveries of the five Di-OPEs ranged from 56.9% to 133.0% with relative standard deviations of 4.4%-18.9%. The established method was applied to the analysis of the five Di-OPEs in 16 facility vegetable soils. The detection frequencies of the five Di-OPEs exceeded 60% in all soil samples, indicating that the Di-OPEs were ubiquitous in the facility vegetable soil. The contents of the five Di-OPEs in the facility vegetable soil samples ranged from 2.53-6.94 ng/g. DnBP (1.37-3.20 ng/g) and DPhP (0.47-2.44 ng/g) were the predominant congeners in the facility vegetable soil samples, accounting for 23.4%-68.8% and 16.3%-35.9% of the five Di-OPEs, respectively. The developed method is simple, sensitive, and reproducible and can be used effectively for the determination of Di-OPEs in soil. The results of this study will be helpful for understanding the environmental behavior of Di-OPEs and their human exposure in facility vegetable soils.

Pollution profiles, influencing factors, and source apportionment of target and suspect organophosphate esters in ambient air: A case study in a typical city of Northern China.

Organophosphate esters (OPEs) are attracting attention because they pose risks to biota, including humans. Little research has been performed into the environmental fates of OPEs in the atmosphere. Here, target/suspect OPEs were determined in 122 atmosphere samples (gas phase (n = 31), PM2.5 (n = 30), PM10 (n = 30), and total suspended particles (n = 31)) from a city in Northern China. Pollution profiles were established, influencing factors identified, and sources apportioned. We found 12 target OPEs and 29 suspect OPEs. The target and suspect OPE concentrations in the ambient air samples were 2.2-172.5 and 0.7-53.9 ng/m3, respectively. Tris(chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, and tris(2,4-di-t-butylphenyl) phosphate were the dominant OPEs in all samples. The OPEs were not in equilibrium, indicated by a multi-parameter linear free energy relationship model. The air quality index and OPE concentrations significantly correlated, indicating that OPE pollution is often more serious during weather with worse air quality. The target and suspect screening strategy and a positive matrix factorization model allowed OPE sources to be apportioned, improving our understanding of OPE sources. The four dominant sources were (1) construction, (2) indoor emissions, (3) the plastic industry and industrial activities, and (4) traffic emissions, textiles, and foam products.

Evaluation of Tourism Food Safety and Quality with Neural Networks.

Food safety issues are inextricably linked to people's lives and, in extreme cases, endanger public safety and social stability. People are becoming increasingly concerned about food safety issues in a modern society with high-quality economic development. People's incomes are increasing day by day as the economy continues to grow, and the tourism industry has grown by leaps and bounds. However, many problems arose, such as the issue of food safety in tourism. Tourism food safety issues affect not only the development of the food industry but also the development of tourism. Food safety oversight of tourist attractions has always been a relatively concerning issue in the country, and it is also something that the general public is concerned about. It can be said that food safety supervision of tourist attractions is the most important thing in food safety supervision. In this context, it becomes an important task to evaluate the safety of tourist food. This work proposes a multiscale convolutional neural network (AMCNN) combined with neural networks and attention layers to realize the safety and quality evaluation of tourist food. The algorithm uses the lightweight Xception network as a basic model and utilizes multiscale depth-separable convolution modules of different sizes for feature extraction and fusion to extract richer food safety feature information. Furthermore, the convolutional attention module (CBAM) is embedded on the basis of the multiscale convolutional neural network, which makes the network model focus more on discriminative features.

The establishment and application of CD3E humanized mice in immunotherapy.

In the field of cancer immunotherapy, monoclonal antibody drugs, bispecific antibodies, and antibody-conjugated drugs have become the focus of current research, and gene-edited animal models play an essential role in the entire drug development process. In this study, CD3E humanized mice were established by replacing the second to the seventh exon of the Cd3e mouse gene with the same exon of the human gene. The expression of human CD3E in CD3E humanized mice was detected by RT-PCR as well as flow cytometry, also a tumor model was established based on CD3E humanized mice, and the pharmacodynamic effects of CD3E monoclonal antibodies were evaluated. The results showed that CD3E humanized mice expressed only human CD3E, and the proportion of each lymphocyte in the thymus and spleen was not significantly changed compared with wild-type mice. CD3E monoclonal antibody could promote tumor growth after treatment, which may be related to the activation-induced cell death effect caused by this CD3E antibody. In contrast, Bispecific antibody blinatumomab inhibited tumor growth significantly. Thus, the CD3E humanized mice provided an adequate animal model for evaluating the efficacy and safety of CD3E antibody drugs.

ß-elemonic acid inhibits growth and triggers apoptosis in human castration-resistant prostate cancer cells through the suppression of JAK2/STAT3/MCL-1 and NF-ĸB signal pathways.

Castration-resistant prostate cancer (CRPC) has become a significant problem in the current treatment of prostate cancer (PCa) with the characteristics of high metastatic potential, resistance and easy recurrence. The abnormal activation of JAK2/STAT3/MCL-1 and NF-κB has been confirmed as the main reason for the development of CRPC. We previously found that ß-elemonic acid (ß-EA) as a natural triterpene has potential anti-inflammatory and anti-osteosarcoma effects with lower toxicity. But it remains unknown whether it had effects on CRPC. The present research in vitro and in vivo systematically investigates anti-cancer effects and mechanisms of ß-EA on human CRPC. ß-EA treatment resulted in apoptotic cell death in human PCa cells by mitochondrial apoptotic pathways (including up-regulation of cleaved caspase-3, cleaved PARP, and Bax or down-regulation of Bcl-2). Besides, ß-EA at relatively lower levels inhibited colony-forming, the migration and invasion potential of PCa cells, indicating its anti-proliferation and anti-metastasis activities. After exploring the potential mechanism, our results suggested that it subsequently inhibited the activation of JAK2/STAT3/MCL-1 and NF-κB signaling pathway by the administration of ß-EA. The silencing of NF-κB/p65, JAK2 and STAT3, respectively, increased the sensitivity of the PCa cells to ß-EA induced apoptosis. Moreover, ß-EA exhibited a strong affinity with its essential proteins JAK2, RELA/p65, NF-κBIα/IκBα by molecular docking analysis. Importantly, ß-EA retards tumor growth in a murine xenograft model, consistent with our study in vitro. Taken together, findings from this study reveal for the first time the potential role and mechanisms of ß-EA on CRPC.

ß-Elemonic acid inhibits the growth of human Osteosarcoma through endoplasmic reticulum (ER) stress-mediated PERK/eIF2α/ATF4/CHOP activation and Wnt/ß-catenin signal suppression.

BACKGROUND: Osteosarcoma (OS) is a significant threat to the lives of children and young adults. Although neoadjuvant chemotherapy is the first choice of treatment for OS, it is limited by serious side-effects and cancer metastasis. ß-Elemonic acid (ß-EA), an active component extracted from Boswellia carterii Birdw., has been reported to exhibit potential anti-inflammatory and anticancer activities. However, the anti-tumor effects and underlying mechanisms on OS as well as pharmacokinetic characteristics of ß-EA remain unknown. PURPOSE: This study was aimed to investigating the anti-tumor effects of ß-EA on human OS, the underlying mechanisms, and the pharmacokinetic and tissue distribution characteristics. STUDY DESIGN AND METHODS: Cell viability and colony formation assays were performed to determine the effect of ß-EA cell on cell proliferation. Apoptosis rates, mitochondrial membrane potential and cell cycle features were analyzed by flow cytometry. qRT-PCR, Western blot, immunofluorescence and immunohistochemical assays were conducted to evaluate the expression levels of genes or proteins related to the pathways affected by ß-EA in vitro and in vivo. Cell migration and invasion were evaluated in wound healing and Transwell chamber assays. The effects and pharmacokinetic characteristics of ß-EA in vivo were evaluated by analyzing tumor suppression, pharmacokinetics and tissue distribution. RESULTS: Explorations indicated that endoplasmic reticulum (ER) stress conditions provoked by ß-EA activated the PERK/eIF2α/ATF4 branch of the unfolded protein reaction (UPR), stimulating C/EBP homologous protein (CHOP)-regulated apoptosis and inducing Ca2+ leakage leading to caspase-dependent apoptosis. Furthermore, ß-EA induced G0/G1 cell cycle arrest and inhibited metastasis of HOS and 143B cells by attenuating Wnt/ß-catenin signaling effects, which included decreased levels of p-Akt(Ser473), p-Gsk3ß (Ser9), Wnt/ß-catenin target genes (c-Myc and CyclinD1) along with a decline in nuclear ß-catenin accumulation. The fast absorption, short elimination half-life, and linear pharmacokinetic characteristics of ß-EA were also revealed. The distribution of ß-EA was detected in the tumor and bone tissues. CONCLUSIONS: Overall, both in vitro and in vivo investigations showed the potential of ß-EA for the treatment of human OS. The pharmacokinetic profile and considerable distribution in the tumor and bone tissues warrant further preclinical or even clinical studies.

Mitochondria-Targeted Two-Photon Fluorescent Photosensitizers for Cancer Cell Apoptosis via Spatial Selectability.

Organelle-targeted photosensitizers have been reported to be effective cell apoptosis agents. Mitochondria is recognized as an ideal target for cancer treatment due to its central role in oxidative metabolism and apoptosis. Meanwhile, two-photon (TP) fluorescence microscopy has become a powerful tool for fluorescence imaging in biological events based on its minimizing photodamage/photobleaching and intrinsic 3D resolution in deep tissues and in vivo. In this study, a series of novel mitochondrial-targeted TP fluorescent photosensitizers (TP-tracers) are designed, synthesized, and systematically investigated. These TP-tracers exhibit extraordinary anti-interference capability among different cations, anions, and amino acids as well as the insensitivity to the changes of pH and complex biological environments. TP-tracers are further used in fluorescence living cells, Drosophila brains, and zebrafish imaging with low cytotoxicity, excellent mitochondria-targeting, and TP properties. The results demonstrate efficient mitochondria-targeting cell selective apoptosis based on TP-activated cancer cells with highly single cell selectivity, and the pharmacokinetic study reveals that MitoY2 does not have accumulation in rats. It is believed that these molecules hold great potential in TP-related smart phototherapy.

Luteolin Ameliorates Testis Injury and Blood-Testis Barrier Disruption through the Nrf2 Signaling Pathway and by Upregulating Cx43.

SCOPE: Luteolin, a natural flavonoid, displays protective activities to testicular tissue. However, the molecular mechanisms are still unclear. In this study, the aim is to identify the protective effects and underlying mechanisms of luteolin against triptolide (TP)-induced damage of testicular tissue. METHODS AND RESULTS: Pre-incubation of Sertoli cells (SCs) with luteolin results in a significant reduction of TP-induced apoptotic cells, which occurs concomitantly with the effective inhibition of reactive oxygen species accumulation. Luteolin results in a significant reduction in testicular damage and spermatogenesis dysfunction in a mouse model of testicular damage. Mechanistic studies reveal that luteolin significantly triggers Nrf2 translocation, increases antioxidant response element-luciferase reporter activity, and induces antioxidant enzyme expression. Nrf2 siRNA reduces luteolin-induced protection in SCs. Besides inhibiting apoptosis, luteolin recovers the blood-testis barrier (BTB) integrity by upregulating connexin43 (Cx43) expression. Moreover, specifically blocked Cx43 activity completely blocks repairmen of luteolin to BTB values. In accordance with in vitro results, luteolin suppresses testicular injury and spermatogenesis dysfunction by activation of Nrf2 and Cx43 in a testicular injury model. CONCLUSION: Luteolin is identified as a novel active ingredient that contributes to the protective activity in testicular damage through activating the Nrf2 signaling pathway and by upregulating Cx43.

Corosolic acid, a natural triterpenoid, induces ER stress-dependent apoptosis in human castration resistant prostate cancer cells via activation of IRE-1/JNK, PERK/CHOP and TRIB3.

BACKGROUND: The development of potent non-toxic chemotherapeutic drugs against castration resistant prostate cancer (CRPC) remains a major challenge. Corosolic acid (CA), a natural triterpenoid, has anti-cancer activity with limited side effects. However, CA anti-prostate cancer activities and mechanisms, particularly in CRPC, are not clearly understood. In this study, we investigated CA anti-tumor ability against human CRPC and its mechanism of action. METHODS: The cell apoptosis and proliferation effects were evaluated via MTT detection, colony formation assay and flow cytometry. Western blot, gene transfection and immunofluorescence assay were applied to investigate related protein expression of Endoplasmic reticulum stress. A xenograft tumor model was established to investigate the inhibitory effect of CA on castration resistant prostate cancer in vivo. RESULTS: The results showed that CA inhibited cell growth and induced apoptosis in human prostate cancer cell (PCa) line PC-3 and DU145, as well as retarded tumor growth in a xenograft model, exerting a limited toxicity to normal cells and tissues. Importantly, CA activated endoplasmic reticulum (ER) stress-associated two pro-apoptotic signaling pathways, as evidenced by increased protein levels of typical ER stress markers including IRE-1/ASK1/JNK and PERK/eIF2α/ATF4/CHOP. IRE-1, PERK or CHOP knockdown partially attenuated CA cytotoxicity against PCa cells. Meanwhile, CHOP induced expression increased Tribbles 3 (TRIB3) level, which lead to AKT inactivation and PCa cell death. CHOP silencing resulted in PCa cells sensitive to CA-induced apoptosis. CONCLUSION: Our data demonstrated, for the first time, that CA might represent a novel drug candidate for the development of an anti-CRPC therapy.

Raddeanin A inhibits growth and induces apoptosis in human colorectal cancer through downregulating the Wnt/ß-catenin and NF-κB signaling pathway.

AIMS: Colorectal cancer (CRC) remains one of the most lethal human malignancies with high incidence and lack of effective therapy. Raddeanin A (RA), an active triterpenoid saponins, has been demonstrated the ability to inhibit the growth of tumor. But the therapeutic effects and mechanisms of RA in CRC remain elusive. Here, we investigated the efficacy and mechanism of RA in CRC both in vitro and in vivo. MAIN METHODS: Cell viability was investigated to evaluate cytotoxic activity by MTT method. Apoptosis induced by RA was studied using Annexin V-FITC/PI binding and JC-1 staining by flow cytometry analysis. The xenograft mouse model of CRC was used to investigate anti-tumor effects in vivo. The key proteins involved in mitochondrial apoptotic, Wnt/ß-catenin and NF-κB pathway were detected by Western blotting, Immunofluorescence, and Immunohistochemistry. KEY FINDINGS: RA induced apoptosis and inhibited cell proliferation of SW480 and LOVO cells in a concentration-dependent manner. Moreover, RA efficiently inhibited tumor growth in xenograft mouse model. RA could down regulate the Wnt/ß-catenin signaling to display anti-tumor effects via suppression of p-LRP6, induction of AKT inactivation, removal of GSK-3ß inhibition and attenuation of ß-catenin. Meanwhile, RA also suppressed the NF-κB pathway by decreasing the phosphorylation of IKBα to induce subsequently mitochondrial apoptotic pathway. SIGNIFICANCE: In summary, RA suppressed the growth and triggered the apoptosis of CRC through discontinuing Wnt/ß-catenin signaling and inhibiting the NF-κB pathway. These findings suggested that RA may hold a promise as a novel therapeutic agent for CRC therapy.

Estimation of personal PM2.5 and BC exposure by a modeling approach - Results of a panel study in Shanghai, China.

BACKGROUND: Epidemiologic studies of PM2.5 (particulate matter with aerodynamic diameter ≤2.5 µm) and black carbon (BC) typically use ambient measurements as exposure proxies given that individual measurement is infeasible among large populations. Failure to account for variation in exposure will bias epidemiologic study results. The ability of ambient measurement as a proxy of exposure in regions with heavy pollution is untested. OBJECTIVE: We aimed to investigate effects of potential determinants and to estimate PM2.5 and BC exposure by a modeling approach. METHODS: We collected 417 24 h personal PM2.5 and 130 72 h personal BC measurements from a panel of 36 nonsmoking college students in Shanghai, China. Each participant underwent 4 rounds of three consecutive 24-h sampling sessions through December 2014 to July 2015. We applied backwards regression to construct mixed effect models incorporating all accessible variables of ambient pollution, climate and time-location information for exposure prediction. All models were evaluated by marginal R2 and root mean square error (RMSE) from a leave-one-out-cross-validation (LOOCV) and a 10-fold cross-validation (10-fold CV). RESULTS: Personal PM2.5 was 47.6% lower than ambient level, with mean (±Standard Deviation, SD) level of 39.9 (±32.1) µg/m3; whereas personal BC (6.1 (±2.8) µg/m3) was about one-fold higher than the corresponding ambient concentrations. Ambient levels were the most significant determinants of PM2.5 and BC exposure. Meteorological and season indicators were also important predictors. Our final models predicted 75% of the variance in 24 h personal PM2.5 and 72 h personal BC. LOOCV analysis showed an R2 (RMSE) of 0.73 (0.40) for PM2.5 and 0.66 (0.27) for BC. Ten-fold CV analysis showed a R2 (RMSE) of 0.73 (0.41) for PM2.5 and 0.68 (0.26) for BC. CONCLUSION: We used readily accessible data and established intuitive models that can predict PM2.5 and BC exposure. This modeling approach can be a feasible solution for PM exposure estimation in epidemiological studies.

Raddeanin A, a natural triterpenoid saponin compound, exerts anticancer effect on human osteosarcoma via the ROS/JNK and NF-κB signal pathway.

Osteosarcoma (OS) is the most frequent and high mortality primary bone tumor in the adolescent. And it is well-known for poor prognosis due to high incidence of metastasis. Raddeanin A (RA), an active component of Anemone raddeana Regel, showed potential anti-cancer activities. However, the anti-tumor effect and molecular mechanism(s) of RA on osteosarcoma are still unclear. The present research is the first in vitro and in vivo investigate systematically anticancer of RA on human osteosarcoma. Our study demonstrated that RA induced mitochondria-dependent apoptosis in osteosarcoma cell lines and markedly suppressed the metastasis of osteosarcoma cells in vitro. And, RA treatment markedly inhibits tumor growth in vivo. Further mechanism study demonstrated that RA caused a significant enhance reactive oxygen species (ROS) level to stimulate phosphorylation of JNK. Moreover, RA led to decrease of p-IκBα level in the cytosol and reduction of p65 level in the nucleus, which was associated with the inhibition of NF-κB transcriptional activity. When NF-κB signaling was inhibited by siRNA targeting p65, a significant increase in cell apoptosis activity was observed. In addition, non-toxic RA concentrations (0.25, 0.5 and 1 µM) inhibited the migration and invasion of OS by suppressing MMP-2/9 expression associated with NF-κB-dependent transcription in vitro. The silencing of p65 increased the sensitivity of the osteosarcoma cells to RA suppressed migration and invasion. These findings suggest RA induces apoptosis and inhibits metastasis in OS cells, involved in provoking ROS/JNK and inhibiting NF-κB signaling pathways. Therefore, it may be a potential anti-metastatic and anti-proliferative therapeutic agent for human osteosarcoma.

Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response.

Triptolide (TP), an oxygenated diterpene, has a variety of beneficial pharmacodynamic activities but its clinical applications are restricted due to severe testicular injury. This study aimed to delineate the molecular mechanisms of TP-induced testicular injury in vitro and in vivo. TP (5-50000 nmol/L) dose-dependently decreased the viability of TM4 Sertoli cells with an IC50 value of 669.5-269.45 nmol/L at 24 h. TP (125, 250, and 500 nmol/L) dose-dependently increased the accumulation of ROS, the phosphorylation of JNK, mitochondrial dysfunction and activation of the intrinsic apoptosis pathway in TM4 cells. These processes were attenuated by co-treatment with the antioxidant N-acetyl cysteine (NAC, 1 mmol/L). Furthermore, TP treatment inhibited the translocation of Nrf2 from cytoplasm into the nucleus as well as the expression of downstream genes NAD(P)H quinone oxidoreductase1 (NQO1), catalase (CAT) and hemeoxygenase 1 (HO-1), thus abrogating Nrf2-mediated defense mechanisms against oxidative stress. Moreover, siRNA knockdown of Nrf2 significantly potentiated TP-induced apoptosis of TM4 cells. The above results from in vitro experiments were further validated in male mice after oral administration of TP (30, 60, and 120 mg·kg-1·d-1, for 14 d), as evidenced by the detected indexes, including dose-dependently decreased SDH activity, increased MDA concentration, altered testicle histomorphology, elevated caspase-3 activation, apoptosis induction, increased phosphorylation of JNK, and decreased gene expression of NQO1, CAT and HO-1 as well as nuclear protein expression of Nrf2 in testicular tissue. Our results demonstrate that TP activates apoptosis of Sertoli cells and injury of the testis via the ROS/JNK-mediated mitochondrial-dependent apoptosis pathway and down-regulates Nrf2 activation.

Estimation of residential fine particulate matter infiltration in Shanghai, China.

Ambient concentrations of fine particulate matter (PM2.5) concentration is often used as an exposure surrogate to estimate PM2.5 health effects in epidemiological studies. Ignoring the potential variations in the amount of outdoor PM2.5 infiltrating into indoor environments will cause exposure misclassification, especially when people spend most of their time indoors. As it is not feasible to measure the PM2.5 infiltration factor (Finf) for each individual residence, we aimed to build models for residential PM2.5Finf prediction and to evaluate seasonal Finf variations among residences. We repeated collected paired indoor and outdoor PM2.5 filter samples for 7 continuous days in each of the three seasons (hot, cold and transitional seasons) from 48 typical homes of Shanghai, China. PM2.5-bound sulfur on the filters was measured by X-ray fluorescence for PM2.5Finf calculation. We then used stepwise-multiple linear regression to construct season-specific models with climatic variables and questionnaire-based predictors. All models were evaluated by the coefficient of determination (R2) and root mean square error (RMSE) from a leave-one-out-cross-validation (LOOCV). The 7-day mean (±SD) of PM2.5Finf across all observations was 0.83 (±0.18). Finf was found higher and more varied in transitional season (12-25 °C) than hot (>25 °C) and cold (<12 °C) seasons. Air conditioning use and meteorological factors were the most important predictors during hot and cold seasons; Floor of residence and building age were the best transitional season predictors. The models predicted 60.0%-68.4% of the variance in 7-day averages of Finf, The LOOCV analysis showed an R2 of 0.52 and an RMSE of 0.11. Our finding of large variation in residential PM2.5Finf between seasons and across residences within season indicated the important source of outdoor-generated PM2.5 exposure heterogeneity in epidemiologic studies. Our models based on readily available data may potentially improve the accuracy of estimates of the health effects of PM2.5 exposure.

[H2O2 alters metabolism in TM4 Sertoli cells in the mouse].

OBJECTIVE: To explore the mechanisms of oxidative stress-induced damage to TM4 Sertoli cells in the mouse using metabolomics techniques based on gas chromatography-mass spectrometry (GC-MS). METHODS: We established the model of oxidative stress-induced damage to mouse TM4 Sertoli cells by treatment with H2O2. Then, we detected the survival rate and apoptosis rate of the TM4 cells by MTT and flow cytometry respectively, measured the concentration of ROS in the TM4 cells with the DCFH-DA fluorescent probe, and determined the levels of endogenous metabolites in the TM4 cells by GC-MS after H2O2 intervention. RESULTS: After 2 hours of treatment with H2O2 at 600 µmol/L, the survival rate of the TM4 cells was reduced to about 50%, and the total apoptosis rates in the low- (100 µmol/L), medium- (300 µmol/L), and high-dose (600 µmol/L) groups were (19.45 ± 0.53), (20.12 ± 0.58), and (37.13 ± 0.35)%, respectively, increased in a dose-dependent manner as compared with (10.28 ± 0.35)% in the blank control (P <0.05). The ROS level was significantly higher in the medium- and high-dose groups than in the control (ï¼»1.27 ± 0.10ï¼½ vs ï¼»1.00 ± 0.08ï¼½%, P <0.05; ï¼»2.07 ± 0.09ï¼½ vs ï¼»1.00 ± 0.08ï¼½%, P <0.01). Compared with the blank control group, the high-dose H2O2 group showed evident changes in the levels of amino acid and carbohydrates in the TM4 cells, more significantly in the levels of valine, norvaline, leucine, glutamic acid, arabinose, fructose, and 5-serotonin cholesterol (VIP >1, P <0.05). CONCLUSIONS: Oxidative stress-induced damage and apoptosis of TM4 Sertoli cells are closely associated with the metabolism of amino acid, glucose, and energy in the cells.

DNA hypomethylation and its mediation in the effects of fine particulate air pollution on cardiovascular biomarkers: A randomized crossover trial.

BACKGROUND: Short-term exposure to fine particulate matter (PM2.5) air pollution has been associated with altered DNA methylation in observational studies, but it remains unclear whether this change mediates the effects on cardiovascular biomarkers. OBJECTIVE: To examine the impact of ambient PM2.5 on gene-specific DNA methylation and its potential mediation in the acute effects of PM2.5 on cardiovascular biomarkers. METHODS: We designed a randomized, double-blind crossover trial using true or sham air purifiers for 48h among 35 healthy college students in Shanghai, China, in 2014. We measured blood global methylation estimated in long interspersed nucleotide element-1 (LINE­1) and Alu repetitive elements, methylation in ten specific genes, and ten cardiovascular biomarkers. We used linear mixed-effect models to examine the associations between PM2.5 and methylation. We also performed causal mediation analyses to evaluate the potential mediation of methylation in the associations between PM2.5 and biomarkers. RESULTS: Air purification increased DNA methylation in repetitive elements and all candidate genes. An IQR increase (64µg/m(3)) in PM2.5 was significantly associated with reduction of methylation in LINE-1 (1.44%), one pro-inflammatory gene (CD40LG, 9.13%), two pro-coagulant genes (F3, 15.20%; SERPINE1, 3.69%), and two pro-vasoconstriction genes (ACE, 4.64%; EDN1, 9.74%). There was a significant mediated effect (17.82%, P=0.03) of PM2.5 on sCD40L protein through CD40LG hypomethylation. Hypomethylation in other candidate genes generally showed positive but non-significant mediation. CONCLUSIONS: This intervention study provided robust human evidence that ambient PM2.5 could induce rapid decreases in DNA methylation and consequently partly mediate its effects on cardiovascular biomarkers.