Lung Fibroblasts Take up Breast Cancer Cell-derived Extracellular Vesicles Partially Through MEK2-dependent Macropinocytosis.

Extracellular vesicles (EV) have emerged as critical effectors in the cross-talk between cancer and normal cells by transferring intracellular materials between adjacent or distant cells. Previous studies have begun to elucidate how cancer cells, by secreting EVs, adapt normal cells at a metastatic site to facilitate cancer cell metastasis. In this study, we utilized a high-content microscopic screening platform to investigate the mechanisms of EV uptake by primary lung fibroblasts. A selected library containing 90 FDA-approved anticancer drugs was screened for the effect on fibroblast uptake of EVs from MDA-MB-231 breast cancer cells. Among the drugs identified to inhibit EV uptake without exerting significant cytotoxicity, we validated the dose-dependent effect of Trametinib (a MEK1/2 inhibitor) and Copanlisib (a PI3K inhibitor). Trametinib suppressed macropinocytosis in lung fibroblasts and inhibited EV uptake with a higher potency comparing with Copanlisib. Gene knockdown and overexpression studies demonstrated that uptake of MDA-MB-231 EVs by lung fibroblasts required MEK2. These findings provide important insights into the mechanisms underlying lung fibroblast uptake of breast cancer cell-derived EVs, which could play a role in breast cancer metastasis to the lungs and suggest potential therapeutic targets for preventing or treating this deadly disease. SIGNIFICANCE: Through a phenotypic screen, we found that MEK inhibitor Trametinib suppressed EV uptake and macropinocytosis in lung fibroblasts, and that EV uptake is mediated by MEK2 in these cells. Our results suggest that MEK2 inhibition could serve as a strategy to block cancer EV uptake by lung fibroblasts.

Cystic renal mass screening: machine-learning-based radiomics on unenhanced computed tomography.

PURPOSE: The present study compares the diagnostic performance of unenhanced computed tomography (CT) radiomics-based machine learning (ML) classifiers and a radiologist in cystic renal masses (CRMs). METHODS: Patients with pathologically diagnosed CRMs from two hospitals were enrolled in the study. Unenhanced CT radiomic features were extracted for ML modeling in the training set (Guangzhou; 162 CRMs, 85 malignant). Total tumor segmentation was performed by two radiologists. Features with intraclass correlation coefficients of >0.75 were screened using univariate analysis, least absolute shrinkage and selection operator, and bidirectional elimination to construct random forest (RF), decision tree (DT), and k-nearest neighbor (KNN) models. External validation was performed in the Zhuhai set (45 CRMs, 30 malignant). All images were assessed by a radiologist. The ML models were evaluated using calibration curves, decision curves, and receiver operating characteristic (ROC) curves. RESULTS: Of the 207 patients (102 women; 59.1 ± 11.5 years), 92 (41 women; 58.0 ± 13.7 years) had benign CRMs, and 115 (61 women; 59.8 ± 11.4 years) had malignant CRMs. The accuracy, sensitivity, and specificity of the radiologist's diagnoses were 85.5%, 84.2%, and 91.1%, respectively [area under the (ROC) curve (AUC), 0.87]. The ML classifiers showed similar sensitivity (94.2%-100%), specificity (94.7%-100%), and accuracy (94.3%-100%) in the training set. In the validation set, KNN showed better sensitivity, accuracy, and AUC than DT and RF but weaker specificity. Calibration and decision curves showed excellent and good results in the training and validation set, respectively. CONCLUSION: Unenhanced CT radiomics-based ML classifiers, especially KNN, may aid in screening CRMs.

Molecular characterization and immune protective efficacy of 3 Eimeria tenella antigens.

Avian coccidiosis caused by Eimeria is a serious parasitic disease that poses a threat to the poultry industry. Currently, prevention and treatment mainly rely on the administration of anticoccidials and live oocyst vaccines. However, the prevalence of drug resistance and the inherent limitations of live vaccines have driven the development of novel vaccines. In this study, the surface protein (Et-SAG14), a previously annotated rhoptry protein (Eten5-B), and a gametocyte phosphoglucomutase (Et-PGM1) were characterized and the vaccine potential of the recombinant proteins were evaluated. Et-SAG14 was dispersed in the form of particles in the sporozoite and merozoite stages, whereas Et-PGM1 was distributed in the apical part of the sporozoite and merozoite stages. The previously annotated rhoptry Eten5-B was found not to be located in the rhoptry but distributed in the cytoplasm of sporozoites and merozoites. Immunization with rEten5-B significantly elevated host interferon gamma (IFN-γ) and interleukin 10 (IL-10) transcript levels and exhibited moderate anticoccidial effects with an anticoccidial index (ACI) of 161. Unexpectedly, both recombinant Et-SAG14 and Et-PGM1 immunization significantly reduced host IFN-γ and IL-10 transcription levels, and did not show protection against E. tenella challenge (ACI < 80). These results suggest that the rEten5-B protein can trigger immune protection against E. tenella and may be a potential and effective subunit vaccine for the control of coccidiosis in poultry.

Revisiting maize Brittle endosperm-2 reveals new insights in BETL development and starchy endosperm filling.

Rerouting the starch biosynthesis pathway in maize can generate specialty types, like sweet corn and waxy corn, with a drastically increasing global demand. Hence, a fine-tuning of starch metabolism is relevant to create diverse maize cultivars for end-use applications. Here, we characterized a new maize brittle endosperm mutant, referred to as bt1774, which exhibited decreased starch content but a dramatic increase of soluble sugars at maturity. Both endosperm and embryo development was impaired in bt1774 relative to the wild-type (WT), with a prominently arrested basal endosperm transfer layer (BETL). Map-based cloning revealed that BRITTLE ENDOSPERM2 (Bt2), which encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase), is the causal gene for bt1774. A MuA2 element was found to be inserted into intron 2 of Bt2, leading to a severe decrease of its expression, in bt1774. This is in line with the irregular and loosely packed starch granules in the mutant. Transcriptome of endosperm at grain filling stage identified 1,013 differentially expressed genes in bt1774, which were notably enriched in the BETL compartment, including ZmMRP1, Miniature1, MEG1, and BETLs. Gene expression of the canonical starch biosynthesis pathway was marginally disturbed in bt1774. Combined with the residual 60 % of starch in this nearly null mutant of Bt2, this data strongly suggests that an AGPase-independent pathway compensates for starch synthesis in the endosperm. Consistent with the BETL defects, zein accumulation was impaired in bt1774. Co-expression network analysis revealed that Bt2 probably has a role in intracellular signal transduction, besides starch synthesis. Altogether, we propose that Bt2 is likely involved in carbohydrate flux and balance, thus regulating both the BETL development and the starchy endosperm filling.

Sweet potato extract alleviates high-fat-diet-induced obesity in C57BL/6J mice, but not by inhibiting pancreatic lipases.

Scope and aim: Sweet potato is widely consumed as a healthy and nutritive vegetable containing bioactive constituents for health promotion. This study investigated the beneficial impact of white-fleshed sweet potato extract (SPE) on high fat diet (HFD)-induced obese mice. Methods and results: First, SPE, in which resin glycoside was found as the dominant constituent, was suggested as a potential anti-obesity agent, because 20-70% pancreatic lipase (PL) inhibition was measured with SPE by in vitro turbidity assay and pNPP assay. Hence, next, the effect of SPE on obese mice was detected by oral administration of HFD supplemented with 6% SPE on C57BL/6J mice for 9 weeks. Surprisingly, being the opposite of what was typically observed from a lipase inhibitor such as orlistat, the fecal fat content in SPE-fed obese mice was decreased (p < 0.01). Meanwhile, 6% SPE supplement indeed significantly ameliorated HFD-induced obesity in mice, including body weight gain, fat accumulation, adipocyte enlargement, insulin resistance, and hepatic steatosis (p < 0.05). The improved liver steatosis was found associated with a down-regulating action of SPE on nuclear factor kappa B activation in HFD-fed mice. The anti-obesity influence of SPE was also confirmed on the HepG2 cell model for non-alcoholic fatty liver disease (NAFLD). Conclusion: These results indicate that SPE, as a dietary supplement, has the great potential for weight control and treating hepatic steatosis, possibly through a different action mechanism from that of orlistat.

Oxygen mediated oxidative couplings of flavones in alkaline water.

Catalyzed oxidative C-C bond coupling reactions play an important role in the chemical synthesis of complex natural products of medicinal importance. However, the poor functional group tolerance renders them unfit for the synthesis of naturally occurring polyphenolic flavones. We find that molecular oxygen in alkaline water acts as a hydrogen atom acceptor and oxidant in catalyst-free (without added catalyst) oxidative coupling of luteolin and other flavones. By this facile method, we achieve the synthesis of a small collection of flavone dimers and trimers including naturally occurring dicranolomin, philonotisflavone, dehydrohegoflavone, distichumtriluteolin, and cyclodistichumtriluteolin. Mechanistic studies using both experimental and computational chemistry uncover the underlying reasons for optimal pH, oxygen availability, and counter-cations that define the success of the reaction. We expect our reaction opens up a green and sustainable way to synthesize flavonoid dimers and oligomers using the readily available monomeric flavonoids isolated from biomass and exploiting their use for health care products and treatment of diseases.

Resin glycoside extracts from Ipomoea aquatica retard lipid digestibility of high-fat food in vitro.

Due to the considerable increase in the prevalence of obesity, there is an increased interest in developing safe and effective anti-obesity treatments from fruits and vegetables. In this study, Ipomoea aquatica, commonly known as Kang Kong in Southeast Asia was first reported to contain potent pancreatic lipase (PL) inhibitors due to resin glycosides (RG). Ipomoea aquatica extract demonstrated a dose-dependent inhibitory activity against PL with an Orlistat equivalent (OE) value of 6.86 ± 0.51 × 10-4. In vitro lipolysis study showed that consuming RG in tandem with high-fat food (butter & salad dressing) was effective in delaying enzymatic fat digestion by inhibiting PL. Pre-incubation of PL with RG extract before substrate addition also significantly enhanced their inhibitory activity. However, RG was unstable when subjected to high heat treatments (90 °C). Overall, these results provided useful knowledge of RG as PL inhibitors for body weight management.

Resin glycosides in aerial parts of Ipomoea batatas are potent lipase inhibitors: potential upcycling of sweet potato by-products to combat obesity.

Sweet potato (Ipomoea batatas) is a staple food crop that is cultivated globally. While the tubers are widely utilised in the food industry, 95-98% of sweet potato leaves (SPL) are disposed during harvesting. Hence, there is great potential to convert SPL into high-value food products through various processing techniques. In this regard, different varieties of Ipomoea batatas (Blackie, Blackheart, and Margarita) that were grown in Singapore were tested for their pancreatic lipase (PL) inhibitory activity using a high-throughput screening assay. Among them, the Margarita variety showed the highest PL inhibitory activity with an Orlistat Equivalent (OE) value of 3.83 ± 0.36 × 10-4. The kinetic study of the Margarita extract revealed a non-competitive inhibition mechanism. Overall, a series of resin glycosides (RG), responsible for the PL inhibitory activity, was characterised in Margarita Ipomoea batatas. To evaluate the effectiveness of RG under gastrointestinal conditions with high-fat food, in vitro digestion with salad dressing was performed. The in vitro digestion model showed that the consumption of RG with salad dressing was effective in delaying enzymatic fat digestion in a dose-dependent manner. The findings strongly suggested that the aerial parts of Ipomoea batatas have great potential to be upcycled into functional food to combat obesity.

Therapeutic efficacy and immunoregulatory effect of Qiangji Jianli Capsule for patients with myasthenia gravis: Study protocol for a series of randomized, controlled N-of-1 trials.

INTRODUCTION: Myasthenia gravis (MG) is an autoimmune disease in which antibodies directly target components of the neuromuscular junction, causing neuromuscular conduction damage that leads to muscle weakness. The current pharmaceutical treatment for MG is still not ideal to address the problems of disease progression, high recurrence rate, and drug side effects. Clinical observations suggest that traditional Chinese medicine (TCM) can strengthen immunity and improve symptoms of MG patients, delay the progression of the disease, reduce or even prevent the need for immunosuppressive therapy when used in combination with acetylcholinesterase inhibitors or low-dose prednisone, as well as improve the quality of life of patients. The Qiangji Jianli Capsule (QJC) is a combination of medicinal herbs which is used in traditional Chinese medicine. Since MG is a rare disorder, randomized controlled trials comparing large cohorts are difficult to conduct. Therefore, we proposed to aggregate data from a small series of N-of-1 trials to assess the effect of the Chinese medical prescription QJC, which strengthens the spleen and nourishes Qi, as an add-on treatment for MG with spleen and stomach Qi deficiency syndrome. METHODS AND ANALYSIS: Single-center, randomized, double-blind, multiple crossover N-of-1 studies will compare QJC versus placebo in 5 adult MG patients with spleen and stomach Qi deficiency syndrome. Patients will undergo 3 cycles of two 4-week intervention periods. According to the treatment schedule, patients will continue to be treated with pyridine bromide tablets, prednisone acetate, tablets and/or tacrolimus capsules throughout the entire trial. Each period consisting of 4-week oral add-on treatment with QJC will be compared with 4-week add-on treatment with a placebo. The primary endpoints are quantitative myasthenia gravis (QMG) test; measurement of the amount of Treg cells and cytokines such as interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-17A (IL-17A), and transforming growth factor-ß (TGF-ß); and corticosteroid or immunosuppressive agent dosage. Secondary outcome measures: Clinical: Evaluation of the effect of TCM syndromes; MG-activities of daily living (MG-ADL) scales; adverse events. ETHICS AND DISSEMINATION: This study was approved by The First Affiliated Hospital of Guangzhou University of Chinese Medicine (GZUCM), No. ZYYECK[2019]038. The results will be published in a peer-reviewed publication. Regulatory stakeholders will comment on the suitability of the trial for market authorization and reimbursement purposes. Trial registration: Chinese Clinical Trial Register, ID: ChiCTR2000033516. Registered on 3 June 2020, http://www.chictr.org.cn/showprojen.aspx?proj=54618.

Characterizations of the endogenous starch hydrolase inhibitors in acorns of Quercus fabri Hance.

The acorns of Quercus fabri Hance tree, or the mandarin duck fruits have been consumed by locals in the form of noodle, yet there is little scientific study. We found that they have much lower digestibility compared with that of rice flour. Solvent extraction using hexane, dichloromethane, and acetone-ethanol-water (AEW) mixture yielded three fractions but the starch hydrolase inhibitors were only found in the AEW fraction. Using assay-guided fractionation of the extracts, we were able to further separate the active compounds by using Sephadex LH-20 column and characterize the inhibitor chemical identities by LC-MS(n). The main active compounds are ellagitannins including pedunculagin, punicalagin, castalagin, and eucalbanin. Our results indicate that the acorns from Quercus fabri Hance have potential for preparation of low glycemic index foods due to the endogenous starch hydrolase inhibitors.

Hydroxyapatite/mesoporous silica coated gold nanorods with improved degradability as a multi-responsive drug delivery platform.

In this study, gold nanorods/mesoporous silica/hydroxyapatite (Au/SiO2/HAP) hybrid nanoparticles with AuNR core and SiO2/HAP hybrid inorganic shell for multi-responsive drug delivery had been prepared. The morphology and structure of the nanoparticles were characterized by TEM, XPS, XRD, FT-IR and N2 adsorption-desorption isotherms. The degradation of the hybrid nanoparticles could be significantly improved by the dissolution of HAP from the hybrid skeleton in acid environment. In vitro drug release results indicated that Au/SiO2/HAP nanoparticles exhibited high drug loading efficiency, excellent near-infrared (NIR)- and pH-responsive drug release properties. Compared to the drug release of Au/SiO2 nanoparticles over 12h (about 6.35%), Au/SiO2/HAP nanoparticles displayed a higher drug release of 37.62% upon NIR irradiation at pH4.5 due to the NIR-responsiveness of AuNRs and the pH-responsiveness of HAP in acid media. The cell viability results also indicated that the Au/SiO2/HAP nanoparticles exhibited the excellent biocompatibility. The present paper provides a facile route to fabricate a hybrid drug carrier with high drug loading efficiency, excellent pH sensitivity, NIR-sensitivity, biodegradability and biocompatibility.

Metal-free oxysulfenylation of alkenes with 1-(arylthio)pyrrolidine-2,5-diones and alcohols.

ß-Alkoxy sulfides are widely used as versatile building blocks in organic synthesis. Therefore, it is highly desirable to develop a convenient and efficient method for oxysulfenylation of alkenes. In this communication, an easy and efficient metal-free approach to ß-alkoxy sulfides has been developed. The protocol uses readily available 1-(arylthio)pyrrolidine-2,5-diones and alcohols as the oxysulfenylating agents, chloroform as the solvent, and no ligand, additive and exclusion of air were required. Therefore, the present method provides a useful strategy for synthesis of ß-alkoxy sulfides.