Simvastatin promotes NPC1-mediated free cholesterol efflux from lysosomes through CYP7A1/LXRα signalling pathway in oxLDL-loaded macrophages.

Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.

Concentration-Dependent Diversifcation Effects of Free Cholesterol Loading on Macrophage Viability and Polarization.

BACKGROUND/AIMS: The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol buildup in atherosclerosis, contradictory results have been generated, indicating that free cholesterol produces both pro- and anti-atherosclerosis effects in macrophages. This inconsistency might stem from the examination of only select concentrations of free cholesterol. In the present study, we sought to investigate the implication of excess free cholesterol loading in the pathophysiology of atherosclerosis across a broad concentration range from (in µg/ml) 0 to 60. METHODS: Macrophage viability was determined by measuring formazan formation and flow cytometry viable cell counting. The polarization of M1 and M2 macrophages was differentiated by FACS (Fluorescence-Activated Cell Sorting) assay. The secretion of IL-1ß in macrophage culture medium was measured by ELISA kit. Macrophage apoptosis was detected by flow cytometry using a TUNEL kit. RESULTS: Macrophage viability was increased at the treatment of lower concentrations of free cholesterol from (in µg/ml) 0 to 20, but gradually decreased at higher concentrations from 20 to 60. Lower free cholesterol loading induced anti-inflammatory M2 macrophage polarization. The activation of the PPARx03B3; (Peroxisome Proliferator-Activated Receptor gamma) nuclear factor underscored the stimulation of this M2 phenotype. Nevertheless, higher levels of free cholesterol resulted in pro-inflammatory M1 activation. Moreover, with the application of higher free cholesterol concentrations, macrophage apoptosis and secretion of the inflammatory cytokine IL-1ß increased significantly. CONCLUSION: These results for the first time demonstrate that free cholesterol could render concentration-dependent diversification effects on macrophage viability, polarization, apoptosis and inflammatory cytokine secretions, thereby reconciling the pros and cons of free cholesterol buildup in macrophages to the pathophysiology of atherosclerosis.

[Determination of four amide synthetic cannabinoid isomers by ultra-high performance liquid chromatography-high resolution mass spectrometry].

Isomerization commonly occurs in synthetic cannabinoids (SCs). Owing to the few differences in their structure and properties, it is difficult to simultaneously separate and identify them. Thus, the identification of synthetic cannabinoids is challenging, posing a threat to public security. This study aims to separate and identify four SCs, which are 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3,3-dimethylbutyrate methyl ester (5F-MDMB-PICA), 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3-methylbutyrate ethyl ester (5F-EMB-PICA), N-(1-amino-2,2-dimethyl-1-oxobutyl-2-yl)-1-butyl-1H-indazole-3-formamide (ADB-BINACA), N-(1-carbamoyl-2-methylpropyl)-1-pentyl indazole-3-formamide (AB-PINACA).Supercritical fluid chromatography-mass spectroscopy (SFC-MS) can realize the effective separation of some cannabinoid isomers. However, most laboratories are not equipped with SFC-MS systems. Ultra-high performance liquid chromatography-high resolution mass spectroscopy (UHPLC-HRMS) effectively combines the excellent efficient separation characteristics of liquid chromatography and the powerful qualitative ability of mass spectrometry. It is a commonly used technical method for the detection of amide synthetic cannabinoids and their metabolites in vivo and in vitro because of its advantages of high accuracy and efficiency. Liquid chromatography allows the separation of tested components by exploiting the difference in the partition coefficients between the mobile and stationary phases. When the two phases are in relative motion, the tested components are divided between the two phases, facilitating the separation and analysis of each component. Although the difference in the polarities of the tested amide synthetic cannabinoid isomeric substances is extremely small, liquid chromatography can induce a strong separation effect. The advantages of UHPLC-HRMS include high resolution imparted by mass spectrometry and high sensitivity, allowing its application in the qualitative analysis of various substances. Through UHPLC-HRMS, trace analytes at the nanogram scale as well as pure drugs and their metabolites in biosamples can be detected. This study proposed a method for the determination of two pairs of amide synthetic cannabinoid isomers-5F-EMB-PICA and 5F-MDMB-PICA, ADB-BINACA and AB-PINACA-through UHPLC-HRMS. A Hypersil GOLD C18 column (100 mm×2.1 mm, 1.9 µm) was selected for separation via liquid chromatography, and gradient elution was performed with methanol containing 0.1% formic acid and a 0.1% formic acid aqueous solution containing 10 mmol/L ammonium formate. Full scan/data-dependent secondary mass spectrometry (Full MS/dd-MS2) was conducted in the positive ion mode for detection. The results indicated that the four synthetic cannabinoid isomers could be accurately detected under the abovementioned conditions. The resolution between 5F-EMB-PICA and 5F-MDMB-PICA was 2.06, while that between ADB-BINACA and AB-PINACA was 1.22, indicating the effective separation and detection of both pairs. Furthermore, method validation was conducted to ensure the accuracy of the proposed method. The relationship of the four amide synthetic cannabinoid isomers exhibited excellent linearity. The correlation coefficients (R2) were >0.99. Moreover, the matrix effects of the four SCs in hair samples were between 88.67% and 111.76% and the recoveries were 96.23%-105.11%. The intra-day and inter-day precisions (RSDs) were <10%. The proposed method was used to identify the case materials. AB-PINACA was detected in a hair sample at a content of 0.73 µg/g. 5F-MDMB-PICA was detected in a tobacco sample at a content of 11.3 mg/g. The results indicate that the proposed method can be used for the examination of practical samples conducted by public security organizations. This study provides a reference method for the identification of synthetic cannabinoid isomers.

Comparison of feature selection and data fusion of Fourier transform infrared and Raman spectroscopy for identifying watercolor ink.

The identification of different kinds of watercolor inks is an important work in the field of forensic science. Four different kinds of watercolor ink Spectroscopy data fusion strategies (Fourier Transform Infrared spectroscopy and Raman spectroscopy) combined with a non-linear classification model (Extreme Learning Machine) were used to identify the brand of watercolor inks. The study chose Competitive Adaptive Reweighted Sampling (CARS), Random Frog (RF), Variable Combination Population Analysis-Genetic Algorithm (VCPA-GA), and Variable Combination Population Analysis-Iteratively Retains Informative Variables (VCPA-IRIV) to extract characteristic variables for mid-level data fusion. The Cuckoo Search (CS) algorithm is used to optimize the extreme learning machine classification model. The results showed that the classification capacity of the mid-level fusion spectra model was more satisfactory than that of single Infrared spectroscopy or Raman spectroscopy. The CS-ELM models based on infrared spectroscopy used to recognize the watercolor ink according to brands (ZHENCAI, DELI, CHENGUANG, and STAEDTLER) obtained an accuracy of 66.67% in the test set using all spectral datasets. The accuracy of CS-ELM models based on Raman spectroscopy was 67.39%. The characteristic wavelength selection algorithms effectively improved the accuracy of the CS-ELM models. The classification accuracy of the mid-level spectroscopy fusion model combined with the VCPA-IRIV algorithm was 100%. The data fusion method increased effectively spectral information. The method could satisfactorily identify different brands of watercolor inks and support the preservation of artifacts, paintings, and forensic document examination.

Application of terahertz Time-Domain spectroscopy and chemometrics-based whale optimization algorithm in PDE5 inhibitor detection.

Combating the illicit use of PDE5 inhibitor drugs is a focal point in forensic science research. In order to achieve rapid identification of such drugs, this study applies terahertz time-domain spectroscopy combined with chemometrics to establish a fast and accurate detection method for PDE5 inhibitors. The optimal detection method is determined by comparing the spectral performance of three optical parameters, namely absorption coefficient, refractive index, and dielectric constant. Linear discriminant models based on different spectral parameters, whale optimization algorithm optimized extreme learning machine models, and whale optimization algorithm optimized random forest models are established. The effectiveness and performance of principal component analysis and competitive adaptive reweighted sampling algorithm for spectral feature data selection are also investigated. The PDE5 inhibitor identification model based on the competitive adaptive reweighted sampling - whale optimization algorithm - random forest (CARS-WOA-RF) model achieves an accuracy of 98.61%, and the identification model for two concentrations of Sildenafil achieves 100% accuracy. The results demonstrate that terahertz time-domain spectroscopy combined with chemometrics can effectively detect various common types of PDE5 inhibitor drugs and different concentrations.

Cycasin derivative: a potential embryotoxic component of Atractylodes macrocephala rhizome for limb malformation.

Objective: The rhizome of Atractylodes macrocephala Koidz. (Asteraceae), called Atractylodes macrocephala rhizome (AMR) and known by its traditional name Bai Zhu, is a prominent Chinese herbal medicine employed for preventing miscarriage. However, our previous study revealed that high dosages of AMR administered during pregnancy could cause embryotoxicity but the specific embryotoxic components and their underlying mechanisms remain unclear. This study aimed to screen and identify the potential embryotoxic components of AMR. Methods: The AMR extracts and sub-fractions were analyzed by thin layer chromatography and subsequently screened by in vitro mouse limb bud micromass and mouse whole embryo culture bioassays. The embryotoxic fractions from AMR were further evaluated in vivo using a pregnant mouse model. The structures of the potential embryotoxic components were analyzed using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Results: In vitro and in vivo bioassays revealed that AMR glycoside-enriched sub-fractions (AMR-A-IIa and AMR-A-IIb) exhibited potential embryotoxicity. These sub-fractions, when administered to pregnant animals, increased the incidence of stillbirth and congenital limb malformations. MS spectrometry analysis identified cycasin derivatives in both sub-fractions, suggesting their possible role in the observed limb malformations. However, further experiments are necessary to validate this hypothesis and to elucidate the underlying mechanisms. Conclusions: Our study provides significant scientific evidence on the pharmacotoxicity of AMR, which is important for the safe clinical application of commonly used Chinese herbal medicines during pregnancy.

Number of positive lymph nodes is superior to neck stage of the 8th AJCC in predicting prognosis in parotid mucoepidermoid carcinoma.

BACKGROUND: To clarify the impact of the number of positive lymph nodes (LNs) on the prognosis of parotid mucoepidermoid carcinoma (MEC). METHODS: Patients who underwent neck dissection for parotid MEC were retrospectively enrolled. The primary outcome variable was overall survival (OS). Associations between OS and LN factors, including the AJCC N stage, intraparotid LN metastasis, number of positive LNs, LN size, and extranodal extension (ENE), were evaluated using Cox proportional hazard regression analyses. RESULTS: A total of 720 patients were included with a mean age of 56 ± 16 years. There was no additional survival compromise until two positive LNs were presented. After adjusting for the number of positive LNs, intraparotid LN metastasis, ENE, and LN size were not related to prognosis. Our proposed N stage based on the number of metastatic LNs (0/1 vs. 2-4 vs. 5+) showed a superior C-index to the AJCC N stage in OS prediction. CONCLUSION: Quantitative LN burden was an important determinant of prognosis, and the proposed N stage provided better OS stratification than the AJCC N stage.

Chinese herbal medicine for threatened miscarriage: An updated systematic review and meta-analysis.

Objective: To conduct an updated systematic review and meta-analysis on the efficacy and safety of Chinese herbal medicine (CHM) for threatened miscarriage. Data Sources: Electronic databases were searched from inception to 30 June 2022. Study Eligibility Criteria: Only randomized controlled trials (RCTs) that assessed the efficacy and safety of CHM or combined CHM and Western medicine (CHM-WM) and compared with other treatments for threatened miscarriage were included for analysis. Methods: Three review authors independently evaluated included studies, assessed the risk of bias and extracted data for meta-analysis (continuation of pregnancy after 28 gestational weeks, continuation of pregnancy after treatment, preterm birth, adverse maternal outcomes, neonatal death, TCM syndrome severity, ß-hCG levels after treatment), sensitivity analysis (ß-hCG level) and subgroup analysis (TCM syndrome severity, ß-hCG level). The risk ratio and 95% confidence interval were calculated by RevMan. Certainty of the evidence was assessed according to GRADE. Results: Overall, 57 RCTs involving 5,881 patients met the inclusion criteria. Compared with WM alone, CHM alone showed significant higher incidence of continuation of pregnancy after 28 gestational weeks (Risk Ratio (RR) 1.11; 95% CI 1.02 to 1.21; n = 1; moderate quality of evidence), continuation of pregnancy after treatment (RR 1.30; 95% CI 1.21 to 1.38; n = 10; moderate quality of evidence), higher ß-hCG level (Standardized Mean Difference (SMD) 6.88; 95% CI 1.74 to 12.03; n = 4) and lower Traditional Chinese medicine (TCM) syndrome severity (SMD -2.94; 95% CI -4.27 to -1.61; n = 2). Compared with WM alone, combined CHM-WM showed significant higher incidence of continuation of pregnancy after 28 gestational weeks (RR 1.21; 95% CI 1.16 to 1.27; n = 15; moderate quality of evidence), continuation of pregnancy after treatment (RR 1.19; 95% CI 1.16 to 1.23; n = 41; moderate quality of evidence), higher ß-hCG level (SMD 2.27; 95% CI 1.72 to 2.83; n = 37) and lower TCM syndrome severity (SMD -1.74; 95% CI -2.21 to -1.27; n = 15). No significant differences in reducing the adverse maternal outcomes and neonatal death were found in combined CHM-WM compared with WM alone (RR 0.97; 95% CI 0.62 to 1.52; n = 8; RR 0.39; 95% CI 0.12 to 1.21; n = 2). Conclusion: Current evidence supported CHM could be a potential treatment for threatened miscarriage. However, results should be interpreted with caution considering the low to moderate quality of the available evidence. Systematic Review Registration: [https://inplasy.com/inplasy-2022-6-0107/], identifier [INPLASY20220107].

In vitro tests to evaluate embryotoxicity and irritation of Chinese herbal medicine (Pentaherbs formulation) for atopic dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD) is a common chronic inflammatory skin disorder and its prevalence is increasing in the last few decades. No treatment can cure the condition. Pregnancy often worsens the clinical manifestation. There are considerable interests in Chinese Herbal Medicine (CHM) as an alternative treatment for AD. A well tolerated CHM formula (Pentaherbs formulation, PHF) has been proven efficacious in improving life quality and reducing topical corticosteroid use in children with moderate-to-severe AD. However, safety data of PHF are not available. AIM OF THE STUDY: Our study aimed to evaluate the safety of PHF and its 5 individual herbal extracts, including embryotoxicity by Embryonic Stem Cell Test (EST) and irritation by Skin Irritation Test (SIT). MATERIALS AND METHODS: Quality of 5 herbal extracts of PHF was confirmed by chromatography. In EST, mouse embryonic stem cell line (D3) and mouse fibroblast cell line (3T3) were used to study potential embryotoxicity. Three endpoints were assessed by concentration-response curves after 10 days' culture: 50% inhibition of D3 differentiation into beating cardiomyocytes (ID50D3), 50% cytotoxic effects on D3 (IC50D3) and on fibroblasts (IC503T3). A biostatistically based prediction model (PM) was applied to predict the embryotoxic potentials of each CHM. In SIT, epidermis equivalent commercially available kits (EpiDerm™) were used, and concentration-viability curves were obtained by MTT assay to detect skin irritations of each CHM. RESULTS: Chemical authentication confirmed that 5 test herbal extracts contained their main active compounds. EST results indicated that the formula PHF and its individual CHMs were non-embryotoxic, except one CHM, Amur Corktree Bark (Huang Bai, Phellodendron chinense C.K.Schneid), was weakly embryotoxic. SIT results showed that cell viability was above 50% after treatment with different concentrations of all tested CHMs. CONCLUSIONS: Our in vitro tests provided preliminary evidence for safety of the formula PHF in embryonic stem cell test and skin irritation model, but PHF shall be cautiously used in pregnant women with AD. Further studies are needed to support its clinical application as an alternative treatment for AD, especially to the patients who plan for pregnancy or at lactation stages.

Corrigendum: Rational design of a Gd(III)-Cu(II) nanobooster for chemodynamic therapy against cancer cells.

[This corrects the article DOI: 10.3389/fchem.2022.856495.].

Rational Design of a Gd(III)-Cu(II) Nanobooster for Chemodynamic Therapy Against Cancer Cells.

Copper (II) containing coordination complexes have attracted much attention for chemodynamic therapy (CDT) against cancer cells. In this study, the bimetallic nanobooster [Gd2Cu(L)2(H2O)10]·6H2O was prepared by a solvothermal method based on tetrazole carboxylic acid ligand H4L [H4L = 3,3-di (1H-tetrazol-5-yl) pentanedioic acid]. It showed considerable cytotoxicity toward three kinds of human cancer cells (HeLa, HepG2, and HT29). The MTT assay showed that the IC50 (half-maximal inhibitory concentration) of the complex NPs on HeLa cells (4.9 µg/ml) is superior to that of HepG2 (11.1 µg/ml) and HT29 (5.5 µg/ml). This result showed that [Gd2Cu(L)2(H2O)10]·6H2O NPs can inhibit cell proliferation in vitro and may be potential candidates for chemodynamic therapy. In addition, the cytotoxicity was also confirmed by the trypan blue staining experiment. The results promise the great potential of Gd(III)-Cu(II) for CDT against cancer cells.

The efficacy and safety of combined chinese herbal medicine and western medicine therapy for COVID-19: a systematic review and meta-analysis.

OBJECTIVE: To systematically review the clinical efficacy and safety of Chinese herbal medicine (CHM) with and without Western medicine (WM) for different severity of COVID-19. METHODS: CNKI, PubMed, Wanfang Database, ClinicalTrails.gov, Embase, ChiCTR and ICTRP were searched from 01 Jan, 2020 to 30 Jun, 2021. Two authors independently assessed all the randomized clinical trials (RCTs) for trial inclusion, data extraction and quality assessment. Meta-analysis was conducted using Review Manager software (RevMan 5.4.1). Evidence was assessed using Grading of Recommendations Assessment, Development, and Evaluation (GRADE). Primary outcomes included total effectiveness rate. Secondary outcomes included improvements in symptom improvement and total adverse event rate. Different severity of COVID-19 patients was assessed in subgroup analysis. This study was registered with INPLASY, INPLASY202210072. RESULTS: 22 high quality RCTs involving 1789 participants were included. There were no trial used CHM alone nor compare placebo or no treatment. Compared with WM, combined CHM and WM (CHM-WM) treatment showed higher total effectiveness rate, lower symptom scores of fever, cough, fatigue, dry throat and pharyngalgia, shorter mean time to viral conversion, better Computerized Tomography (CT) image and blood results, fewer total adverse events and worse conditions (P < 0.05). Subgroup analysis showed that the total effectiveness rate of combined CHM-WM group was significantly higher than WM group, especially for mild and moderate patients. No significant differences in mortality and adverse events were found between combined CHM-WM and WM treatment. No serious adverse events and long-term outcomes were reported. CONCLUSION: Current evidence supported the therapeutic effects and safety of combined CHM-WM treatment on COVID-19, especially for patients with mild and moderate symptoms. Long-term effects of therapy are worthy in further study.

Evidence on Efficacy and Safety of Chinese Medicines Combined Western Medicines Treatment for Breast Cancer With Endocrine Therapy.

BACKGROUND: Breast cancer, a malignant disorder, occurs in epithelial tissue of the breast glands and ducts. Endocrine therapy is commonly applied as an important adjuvant treatment for breast cancer, but it usually induces a variety of side effects. Chinese Medicines (CM) has therapeutic effect on reducing adverse effects of the endocrine therapy in many clinical studies. But strong evidence is still limited on the efficacy and safety of CM combined western medicines (CM-WM) for breast cancer. OBJECTIVE: To study the efficacy and safety of CM-WM as an adjuvant treatment for reducing side effects induced by endocrine therapy in breast cancer patients. METHOD: We searched relevant clinical studies in PubMed and the Chinese National Knowledge Infrastructure (CNKI) databases up to February 28, 2021 and only Randomized Controlled Trials (RCTs) were included. There were no limitations on the languages. We extracted data from the included RCTs, assessed study quality, conducted meta-analyses by RevMan 5.4 and compared the pooled Risk Ratios (RR) or Mean Difference (MD) with 95% CIs. RESULTS: In total 28 trials involving 1,926 participants were included. Six RCTs compared CM-WM with CM placebo-WM, while 22 RCTs compared CM-WM with WM alone. No study compared CM-WM with no treatment. Meta-analysis showed that CM-WM treatment significantly improved quality of life (MD = 0.73, 95% CI = 0.11-1.35, P = 0.02) when compared with CM placebo-WM treatment. When compared with WM treatment alone, CM-WM treatment significantly improved bone mineral density (MD = 0.24, 95% CI = 0.13-0.35, P <0.0001), TCM syndrome score (MD = -5.39, 95% CI = -8.81 to -1.97, P = 0.0002), Kupperman Scale (MD = 0.24, 95% CI = -2.76 to -1.94, P <0.0001), Karnofsky Performance Scale (MD = 3.76, 95% CI = 1.64-5.88, P = 0.0005), quality of life (MD = 3.01, 95% CI = 1.00-5.02, P = 0.003), and pain relief (MD = 2.10, 95% CI = 0.72-3.48, P <0.0001). Compared with WM, CM-WM significantly decreased incidence of TCM symptoms (nausea, vomiting, fatigue, etc.) (RR = 1.60, 95% CI = 1.40-1.84, P <0.0001). For safety, serum calcium, estradiol, ALP, and blood CD3, CD4 and CD8 counts were not significantly difference between two treatments (P >0.05). Serious side effects or reactions were not reported in all included studies. CONCLUSION: The adjunctive use of CM reduced the endocrine therapy associated adverse events, including bone mineral density loss, perimenopausal symptoms, poor quality of life, pain and impaired immune function. But large-scale and high quality RCTs are needed to support the application of CM-WM therapy.

Abnormal podocyte TRPML1 channel activity and exosome release in mice with podocyte-specific Asah1 gene deletion.

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1fl/fl/PodoCre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1fl/fl/PodoCre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1fl/fl/PodoCre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1fl/fl/PodoCre mice compared to WT/WT mice. In the podocytes from Asah1fl/fl/PodoCre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited in the podocytes of Asah1fl/fl/PodoCre mice. By GCaMP3 Ca2+ imaging, it was found that lysosomal Ca2+ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1fl/fl/PodoCre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1fl/fl/PodoCre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1fl/fl/PodoCre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.

Structural Transformation of Polyacrylonitrile (PAN) Fibers during Rapid Thermal Pretreatment in Nitrogen Atmosphere.

The modification before the stabilization process could tune the exothermic behavior and the structural evolution of PAN fibers during stabilization. In this study, we demonstrate that a rapid thermal pretreatment in nitrogen can effectively mitigate the exothermic behavior of PAN fibers, such as decreasing the initial temperature, broadening the exothermal peak, and decreasing the nominal heat release during heating the fibers in air. The color of fibers has shown gradual changes from white to light yellow, yellow and brown during thermal pretreatment in nitrogen with the increase of pretreating temperature and time. The differential scanning calorimetry (DSC), Fourier Transform Infrared Spectrometer (FTIR), X-ray diffraction (XRD), and Thermogravimetric Analysis (TG) characterization have been applied to analyze the thermal properties, chemical and physical structural difference between PAN, and thermally pretreated PAN fibers. The thermal pretreatment of PAN fibers in nitrogen could induce cyclization, dehydrogenation, and cross-linking reactions, in which the cyclization play an important role on improving the cyclization index of stabilized PAN fibers. Meanwhile, the pretreatment can result in noticeable changes of the aggregation structure of PAN fibers, as indicated by the increase of crystallinity and crystalline size. These structural modifications can benefit the main cyclization reaction during stabilization and enhance the carbon yield in resultant carbon fibers. The rapid thermal pretreatment in nitrogen could increase efficiency of modification on PAN fibers, and that could save much time and energy. It is beneficial to manufacture low-cost carbon fibers and to spread the applications of carbon fibers.

Distribution and population structure of endobacteria in arbuscular mycorrhizal fungi at North Atlantic dunes.

Arbuscular mycorrhizal fungi (AMF, Glomeromycotina), in addition to forming symbioses with the majority of land plants, harbor vertically transmitted endosymbiotic bacteria 'Candidatus Glomeribacter gigasporarum' (CaGg) and 'Candidatus Moeniiplasma glomeromycotorum' (CaMg). CaGg is a nonessential mutualist of AMF, whereas the lifestyle of CaMg is unknown. To start unraveling the interactions between AMF and their endosymbionts in nature, we examined diversity and distribution of AMF-associated endobacteria in North Atlantic dunes at Cape Cod. Of nearly 500 foredune AMF isolates successfully genotyped during a systematic study, 94% were classified as Gigasporaceae. Two percent of all AMF spores harbored CaGg, and 88% contained CaMg. CaGg was found only in the Gigasporaceae, whereas CaMg was present in Gigasporaceae, Acaulosporaceae, and Diversisporaceae. Incidence of CaGg across AMF was not affected by any of the environmental parameters measured, whereas distribution of CaMg in one of the fungal hosts was impacted by plant density. CaMg populations associated with AMF individuals displayed high levels of genetic diversity but no evidence of gene flow, suggesting that host physical proximity is not sufficient to facilitate horizontal transmission of CaMg. Finally, in addition to a novel lineage of CaGg, we discovered that AMF likely harbor Burkholderia-related bacteria with close phylogenetic affinity to free-living Burkholderia and endobacteria of other Mucoromycota fungi.

pH responsive poly amino-acid hydrogels formed via silk sericin templating.

Poly(amino acid) hydrogels have attracted a great deal of attention as biodegradable biomaterials that can limit products of synthetic polymer degradation. Here we report on a stimuli-responsive, porous, composite biomaterial based on the protein templating of the poly(amino acid) hydrogel from poly(aspartic acid) with the silk protein sericin. This low-cost, biocompatible and biodegradable hydrogel demonstrates a greatly increased porosity and improvement in volumetric swelling over networks formed from pure poly(aspartic acid). The swelling capacity measured over a range of pH values surrounding physiological pH 7.0 demonstrates a linear profile, in which hydrogel volume and mass increase to a maximum, with an increase as a function of higher sericin content. In comparison to pure poly(aspartic acid), this demonstrates a nearly 3-fold increase in retention volume at basic pH. The increase in swelling is also demonstrated by the increase in porosity and internal micro-architecture of the hydrogel networks. The biomaterial is then shown to perform well as a scaffold for cells with high mechanical strength and integrity. This protein- and homo poly(amino acid)-based super-swelling hydrogel has applications in drug delivery and tissue engineering as an economical and environmentally friendly biomaterial, in addition to ensuring the species incorporated maintain their biocompatibility during processing.