Spicy food consumption reduces the risk of ischaemic stroke: a prospective study.

Previous studies revealed that consuming spicy food reduced mortality from CVD and lowered stroke risk. However, no studies reported the relationship between spicy food consumption, stroke types and dose­response. This study aimed to further explore the association between the frequency of spicy food intake and the risk of stroke in a large prospective cohort study. In this study, 50 174 participants aged 30­79 years were recruited. Spicy food consumption data were collected via a baseline survey questionnaire. Outcomes were incidence of any stroke, ischaemic stroke (IS) and haemorrhagic stroke (HS). Multivariable-adjusted Cox proportional hazard models estimated the association between the consumption of spicy food and incident stroke. Restricted cubic spline analysis was used to examine the dose­response relationship. During the median 10·7-year follow-up, 3967 strokes were recorded, including 3494 IS and 516 HS. Compared with those who never/rarely consumed spicy food, those who consumed spicy food monthly, 1­2 d/week and 3­5 d/week had hazard ratio (HR) of 0·914 (95 % CI 0·841, 0·995), 0·869 (95 % CI 0·758, 0·995) and 0·826 (95 % CI 0·714, 0·956) for overall stroke, respectively. For IS, the corresponding HR) were 0·909 (95 % CI 0·832, 0·994), 0·831 (95 % CI 0·718, 0·962) and 0·813 (95 % CI 0·696, 0·951), respectively. This protective effect showed a U-shaped dose­response relationship. For obese participants, consuming spicy food ≥ 3 d/week was negatively associated with the risk of IS. We found the consumption of spicy food was negatively associated with the risk of IS and had a U-shaped dose­response relationship with risk of IS. Individuals who consumed spicy food 3­5 d/week had a significantly lowest risk of IS.

Gene Expression Nebulas (GEN): a comprehensive data portal integrating transcriptomic profiles across multiple species at both bulk and single-cell levels.

Transcriptomic profiling is critical to uncovering functional elements from transcriptional and post-transcriptional aspects. Here, we present Gene Expression Nebulas (GEN, https://ngdc.cncb.ac.cn/gen/), an open-access data portal integrating transcriptomic profiles under various biological contexts. GEN features a curated collection of high-quality bulk and single-cell RNA sequencing datasets by using standardized data processing pipelines and a structured curation model. Currently, GEN houses a large number of gene expression profiles from 323 datasets (157 bulk and 166 single-cell), covering 50 500 samples and 15 540 169 cells across 30 species, which are further categorized into six biological contexts. Moreover, GEN integrates a full range of transcriptomic profiles on expression, RNA editing and alternative splicing for 10 bulk datasets, providing opportunities for users to conduct integrative analysis at both transcriptional and post-transcriptional levels. In addition, GEN provides abundant gene annotations based on value-added curation of transcriptomic profiles and delivers online services for data analysis and visualization. Collectively, GEN presents a comprehensive collection of transcriptomic profiles across multiple species, thus serving as a fundamental resource for better understanding genetic regulatory architecture and functional mechanisms from tissues to cells.

Ectopic over-expression of HaFT-1, a 14-3-3 protein from Haloxylon ammodendron, enhances acquired thermotolerance in transgenic Arabidopsis.

Haloxylon ammodendron, an important shrub utilized for afforestation in desert areas, can withstand harsh ecological conditions such as drought, high salt and extreme heat. A better understanding of the stress adaptation mechanisms of H. ammodendron is vital for ecological improvement in desert areas. In this study, the role of the H. ammodendron 14-3-3 protein HaFT-1 in thermotolerance was investigated. qRT-PCR analysis showed that heat stress (HS) priming (the first HS) enhanced the expression of HaFT-1 during the second HS and subsequent recovery phase. The subcellular localization of YFP-HaFT-1 fusion protein was mainly detected in cytoplasm. HaFT-1 overexpression increased the germination rate of transgenic Arabidopsis seeds, and the survival rate of HaFT-1 overexpression seedlings was higher than that of wild-type (WT) Arabidopsis after priming-and-triggering and non-primed control treatments. Cell death staining showed that HaFT-1 overexpression lines exhibited significantly reduced cell death during HS compared to WT. Transcriptome analysis showed that genes associated with energy generation, protein metabolism, proline metabolism, autophagy, chlorophyll metabolism and reactive oxygen species (ROS) scavenging were important to the thermotolerance of HS-primed HaFT-1 transgenic plants. Growth physiology analysis indicated that priming-and-triggering treatment of Arabidopsis seedlings overexpressing HaFT-1 increased proline content and strengthened ROS scavenging activity. These results demonstrated that overexpression of HaFT-1 increased not only HS priming but also tolerance to the second HS of transgenic Arabidopsis, suggesting that HaFT-1 is a positive regulator in acquired thermotolerance.

Exploration of the Catalytic Cycle Dynamics of Vigna Radiata H+-Translocating Pyrophosphatases Through Hydrogen-Deuterium Exchange Mass Spectrometry.

Vigna radiata H+-translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.

Unraveling different influences of the fraction of the tetragonal phase in oxide films on the corrosion resistance of Zr alloys from the phase transition mechanism.

The mechanism of Sn and Nb influence on the fraction of tetragonal ZrO2 in oxide films on Zr alloys and their influence mechanism on corrosion resistance of Zr alloys, despite decades of research, are ambiguous due to the lack of kinetic knowledge of phase evolution of ZrO2 with doping. Using stochastic surface walking and density functional theory calculations, we investigate the influence of Nb and Sn on the stability of tetragonal (t) and monoclinic (m) ZrO2, and t-m phase transition in oxide films. We found that though Nb and Sn result in similar apparent variation trends in the t-phase fraction in oxide films, their influences on t-m phase transition differ significantly, which is the underlying origin of different influences of the t-phase fraction in oxide films on the corrosion resistance of Zr alloys with Sn and Nb alloying. These results clarify an important aspect of the relationship between the microstructure and corrosion resistance of Zr alloys.

Chlorogenic acid delays the progression of Parkinson's disease via autophagy induction in Caenorhabditis elegans.

OBJECTIVES: Parkinson's disease (PD) is the second most common neurodegenerative disease. Chlorogenic acid (CGA) is a polyphenolic substance derived from various medicinal plants. Although CGA is reported to have potential anti-PD effect, the beneficial effect and the underlying mechanism remain unclear. In this study, we aimed to further investigate the protective effect and clarify the mechanism of action of CGA in Caenorhabditis elegans (C. elegans) models of PD. METHODS: Measurements of a-synuclein aggregation, movement disorders, and lipid, ROS and malondialdehyde (MDA) contents were observed in NL5901 nematodes. Determinations of dopamine (DA) neuron degeneration, food perception, and ROS content were performed in 6-OHDA-exposed BZ555 nematodes. The autophagy activation of CGA was monitored using DA2123 and BC12921 nematodes. Meanwhile, RNAi technology was employed to knockdown the autophagy-related genes and investigate whether the anti-PD effect of CGA was associated with autophagy induction in C. elegans. RESULTS: CGA significantly reduced α-synuclein aggregation, improved motor disorders, restored lipid content, and decreased ROS and MDA contents in NL5901 nematodes. Meanwhile, CGA inhibited DA neuron-degeneration and improved food-sensing behavior in 6-OHDA-exposed BZ555 nematodes. In addition, CGA increased the number of GFP::LGG-1 foci in DA2123 nematodes and degraded p62 protein in BC12921 nematodes. Meanwhile, CGA up-regulated the expression of autophagy-related genes in NL5901 nematodes. Moreover, the anti-PD effect of CGA was closely related to autophagy induction via increasing the expression of autophagy-related genes, including unc-51, bec-1, vps-34, and lgg-1. CONCLUSIONS: The present study indicates that CGA exerts neuroprotective effect in C. elegans via autophagy induction.

Age at smoking initiation and smoking cessation influence the incidence of stroke in China: a 10-year follow-up study.

Our study aimed to explore the correlation between age at smoking initiation and smoking cessation for the risk for stroke in China. We investigated 50,174 participants from one of the urban areas of China Kadoorie Biobank (CKB) Study. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) for association between smoking and incidence of stroke were estimated using Cox regression model. During a median of 10.7 years of follow-up, 4370 total stroke cases were documented. Among men, comparing current smokers to never smokers, the HR of total stroke for current smokers was 1.279 (95% CI, 1.134-1.443) for total stroke. The HRs of total stroke were 1.344 (1.151-1.570) for those started smoking at age less than 20 years, 1.254 (1.090-1.443) for those started smoking at age 20-30 years, and 1.205 (1.012-1.435) for those started smoking at age 30 year and above, with a dose-response relation (P for trend, 0.004). Comparing former smokers to current smokers, in the low pack-year group, those stopped smoking at age less than 65 years had a 18.2% decreased risk for total stroke (0.818; 0.673-0.994). The decreased risk was not found in those stopped smoking at age 65 years and above. Similar results were observed in the high pack-year group. In conclusion, we found that current smokers had a higher stroke risk than never smokers, and the risk increased with a younger age at smoking initiation. Smoking cessation can reduce the risk for stroke, especially could benefit from cessation at a younger age.

Recapitulating the Drifting and Fusion of Two-Generation Spheroids on Concave Agarose Microwells.

Cells with various structures and proteins naturally come together to cooperate in vivo. This study used cell spheroids cultured in agarose micro-wells as a 3D model to study the movement of cells or spheroids toward other spheroids. The formation dynamics of tumor spheroids and the interactions of two batches of cells in the agarose micro-wells were studied. The results showed that a concave bottom micro-well (diameter: 2 mm, depth: 2 mm) prepared from 3% agarose could be used to study the interaction of two batches of cells. The initial tumor cell numbers from 5 × 103 cells/well to 6 × 104 cells/well all could form 3D spheroids after 3 days of incubation. Adding the second batch of DU 145 cells to the existing DU 145 spheroid resulted in the formation of satellite cell spheroids around the existing parental tumor spheroid. Complete fusion of two generation cell spheroids was observed when the parental spheroids were formed from 1 × 104 and 2 × 104 cells, and the second batch of cells was 5 × 103 per well. A higher amount of the second batch of cells (1 × 104 cell/well) led to the formation of independent satellite spheroids after 48 h of co-culture, suggesting the behavior of the second batch of cells towards existing parental spheroids depended on various factors, such as the volume of the parental spheroids and the number of the second batch cells. The interactions between the tumor spheroids and Human Umbilical Vein Endothelial Cells (HUVECs) were modeled on concave agarose micro-wells. The HUVECs (3 × 103 cell/well) were observed to gather around the parental tumor spheroids formed from 1 × 104, 2 × 104, and 3 × 104 cells per well rather than aggregate on their own to form HUVEC spheroids. This study highlights the importance of analyzing the biological properties of cells before designing experimental procedures for the sequential fusion of cell spheroids. The study further emphasizes the significant roles that cell density and the volume of the spheroids play in determining the location and movement of cells.

The transcriptomic responses of blunt snout bream (Megalobrama amblycephala) to acute hypoxia stress alone, and in combination with bortezomib.

BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia. A new blunt snout bream strain, "Pujiang No.2", was developed to overcome this shortcoming. As a proteasome inhibitor, bortezomib (PS-341) has been shown to affect the adaptation of cells to a hypoxic environment. In the present study, bortezomib was used to explore the hypoxia adaptation mechanism of "Pujiang No.2". We examined how acute hypoxia alone (hypoxia-treated, HN: 1.0 mg·L- 1), and in combination with bortezomib (hypoxia-bortezomib-treated, HB: Use 1 mg bortezomib for 1 kg fish), impacted the hepatic ultrastructure and transcriptome expression compared to control fish (normoxia-treated, NN). RESULTS: Hypoxia tolerance was significantly decreased in the bortezomib-treated group (LOEcrit, loss of equilibrium, 1.11 mg·L- 1 and 1.32 mg·L- 1) compared to the control group (LOEcrit, 0.73 mg·L- 1 and 0.85 mg·L- 1). The HB group had more severe liver injury than the HN group. Specifically, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the HB group (52.16 U/gprot, 32 U/gprot) were significantly (p < 0.01) higher than those in the HN group (32.85 U/gprot, 21. 68 U/gprot). In addition, more severe liver damage such as vacuoles, nuclear atrophy, and nuclear lysis were observed in the HB group. RNA-seq was performed on livers from the HN, HB and NN groups. KEGG pathway analysis disclosed that many DEGs (differently expressed genes) were enriched in the HIF-1, FOXO, MAPK, PI3K-Akt and AMPK signaling pathway and their downstream. CONCLUSION: We explored the adaptation mechanism of "Pujiang No.2" to hypoxia stress by using bortezomib, and combined with transcriptome analysis, accurately captured the genes related to hypoxia tolerance advantage.

Limitations of clinical trial sample size estimate by subtraction of two measurements.

In planning randomized clinical trials (RCTs) for diseases such as Alzheimer's disease (AD), researchers frequently rely on the use of existing data obtained from only two time points to estimate sample size via the subtraction of baseline from follow-up measurements in each subject. However, the inadequacy of this method has not been reported. The aim of this study is to discuss the limitation of sample size estimation based on the subtraction of available data from only two time points for RCTs. Mathematical equations are derived to demonstrate the condition under which the obtained data pairs with variable time intervals could be used to adequately estimate sample size. The MRI-based hippocampal volume measurements from the Alzheimer's Disease Neuroimaging Initiative (ADNI) and Monte Carlo simulations (MCS) were used to illustrate the existing bias and variability of estimates. MCS results support the theoretically derived condition under which the subtraction approach may work. MCS also show the systematically under- or over-estimated sample sizes by up to 32.27 % bias. Not used properly, such subtraction approach outputs the same sample size regardless of trial durations partly due to the way measurement errors are handled. Estimating sample size by subtracting two measurements should be treated with caution. Such estimates can be biased, the magnitude of which depends on the planned RCT duration. To estimate sample sizes, we recommend using more than two measurements and more comprehensive approaches such as linear mixed effect models.

Neuritin attenuates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by promoting autophagic flux.

The autophagy/apoptosis interaction has always been a focus of study in pathogenicity models. Neuritin is a neurotrophic factor that is highly expressed primarily in the central nervous system. Our previous study revealed that it protects against apoptosis in cortical neurons subjected to oxygen-glucose deprivation (OGD)/reoxygenation (OGD/R), and later animal experiments revealed that it can increase the expression of the autophagy-related protein LC3. Whether this neuroprotective effect is closely related to autophagy is still unclear. In this study, we hypothesized that neuritin can promote autophagic flux to protect nerve cells after OGD/R. To verify this hypothesis, we induced OGD/R in primary cortical neurons and assessed cell viability by the CCK8 and LDH assays. Cell apoptosis was assessed by Annexin V-FITC/PI, staining, and the contents and mRNA abundances of the autophagy-related proteins LC3 and p62, the apoptotic protein Caspase3 were quantified by Western blotting and RT-PCR. Autophagic flux was assessed by immunofluorescence after RFP-GFP-LC3 virus transfection, and ultrastructural changes in autophagosomes were observed by transmission electron microscopy (TEM). The results showed that cell viability was decreased, apoptosis was increased and autophagy was enhanced after OGD/R. Neuritin significantly increased cell viability, decreased apoptosis, further increased the expression of the autophagic flux-related protein LC3, further decreased p62 expression, and significantly increased the autophagosome number and autophagosome to lysosome ratio. Bafilomycin A1 (BafA1) is a late autophagy inhibitor, aggravated cell damage and apoptosis and counteracted the enhancement of autophagy activation and protective effects of neuritin. In conclusion, neuritin may promote the completion of autophagic flux by ameliorating neuronal damage after OGD/R.

Neobaicalein Inhibits Th17 Cell Differentiation Resulting in Recovery of Th17/Treg Ratio through Blocking STAT3 Signaling Activation.

Huangqin is the dried root of Scutellaria baicalensis Georgi, which has been widely utilized for heat-clearing (Qingre) and dewetting (Zaoshi), heat-killed (Xiehuo) and detoxifying (Jiedu) in the concept of Traditional Chinese Medicine and is used for treating inflammation and cancer in clinical formulas. Neobaicalein (NEO) is of flavonoid isolated from Huangqin and has been reported to possess prominent anti-inflammatory effects in published work. Th17/Treg balance shift to Th17 cells is an essential reason for autoimmune inflammatory diseases. However, the role NEO plays in Th17 and Treg and the underlying mechanism has not been elucidated yet. Network pharmacology-based study revealed that NEO predominantly regulated IL-17 signaling pathway. Moreover, our result shown that NEO (3-30 µmol/L) down-regulated Th17 differentiation and cellular supernatant and intracellular IL-17A level and tumor necrosis factor α production in a concentration-dependent manner. The further mechanism research revealed that NEO also specifically inhibited phosphorylation of STAT3(Tyr725) and STAT4 (Y693) without influence on activation of STAT5 and STAT6 in splenocytes. Immunofluorescence results illuminated that NEO effectively blocked STAT3 translocated into nucleus. Interestingly, NEO at appreciated dose could only inhibit Th17 cell differentiation and have no effect on Treg differentiation. The present study revealed that NEO effectively inhibited Th17 cell differentiation through specifically blocking the activation of STAT3 signaling without inactivation of STAT5 and STAT6. Additional inhibitory effect on activation of STAT4 by NEO also suggested the potential for antagonism against Th1 differentiation. All work suggested that NEO may be a potential candidate for immunoregulation and treating autoimmune inflammatory diseases through inhibiting immune cell viability and T cell differentiation.

DR-SIP: protocols for higher order structure modeling with distance restraints- and cyclic symmetry-imposed packing.

MOTIVATION: Quaternary structure determination for transmembrane/soluble proteins requires a reliable computational protocol that leverages observed distance restraints and/or cyclic symmetry (Cn symmetry) found in most homo-oligomeric transmembrane proteins. RESULTS: We survey 118 X-ray crystallographically solved structures of homo-oligomeric transmembrane proteins (HoTPs) and find that ∼97% are Cn symmetric. Given the prevalence of Cn symmetric HoTPs and the benefits of incorporating geometry restraints in aiding quaternary structure determination, we introduce two new filters, the distance-restraints (DR) and the Symmetry-Imposed Packing (SIP) filters. SIP relies on a new method that can rebuild the closest ideal Cn symmetric complex from docking poses containing a homo-dimer without prior knowledge of the number (n) of monomers. Using only the geometrical filter, SIP, near-native poses of 7 HoTPs in their monomeric states can be correctly identified in the top-10 for 71% of all cases, or 29% among 31 HoTP structures obtained through homology modeling, while ZDOCK alone returns 14 and 3%, respectively. When the n is given, the optional n-mer filter is applied with SIP and returns the near-native poses for 76% of the test set within the top-10, outperforming M-ZDOCK's 55% and Sam's 47%. While applying only SIP to three HoTPs that comes with distance restraints, we found the near-native poses were ranked 1st, 1st and 10th among 54 000 possible decoys. The results are further improved to 1st, 1st and 3rd when both DR and SIP filters are used. By applying only DR, a soluble system with distance restraints is recovered at the 1st-ranked pose. AVAILABILITY AND IMPLEMENTATION: https://github.com/capslockwizard/drsip. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lychee seed polyphenol protects the blood-brain barrier through inhibiting Aß(25-35)-induced NLRP3 inflammasome activation via the AMPK/mTOR/ULK1-mediated autophagy in bEnd.3 cells and APP/PS1 mice.

Blood-brain barrier (BBB) dysfunction has been implicated in Alzheimer's disease (AD) and is closely linked to the release of proinflammatory cytokines in brain capillary endothelial cells. We have previously reported that lychee seed polyphenols (LSP) exerted anti-neuroinflammatory effect. In this study, we aimed to explore the protective effect of LSP on BBB integrity. The monolayer permeability of bEnd.3 cells, and the mRNA level and protein expression of tight junction proteins (TJs), including Claudin 5, Occludin, and ZO-1, were examined. In addition, the inhibition of Aß(25-35)-induced NLRP3 inflammasome activation, and the autophagy induced by LSP were investigated by detecting the expression of NLRP3, caspase-1, ASC, LC3, AMPK, mTOR, and ULK1. Furthermore, the cognitive function and the expression of TJs, NLRP3, caspase-1, IL-1ß, and p62 were determined in APP/PS1 mice. The results showed that LSP significantly decreased the monolayer permeability and inhibited the NLRP3 inflammasome in Aß(25-35)-induced bEnd3 cells. In addition, LSP induced autophagy via the AMPK/mTOR/ULK1 pathway in bEnd.3 cells, and improved the spatial learning and memory function, increased the TJs expression, and inhibited the expression of NLRP3, caspase-1, IL-1ß, and p62 in APP/PS1 mice. Therefore, LSP protects BBB integrity in AD through inhibiting Aß(25-35)-induced NLRP3 inflammasome activation via the AMPK/mTOR/ULK1-mediated autophagy.

Tanshinone IIA regulates human AML cell proliferation, cell cycle, and apoptosis through miR-497-5p/AKT3 axis.

BACKGROUND: The roots of Salvia miltiorrhiza are used in traditional Chinese medicine (TCM) and have high medicinal value. Tanshinone IIA (Tan IIA) is the active ingredient of Salvia miltiorrhiza which can inhibit the growth of acute leukemia cell lines in vitro, although the mechanism remains unclear. METHODS: CCK-8 assays and BrdU stain were used to evaluate cell proliferation ability. Western blot analysis was used to detect protein expression. miR-497-5p expression level was detected by using qRT-PCR, and Annexin V-FITC/propidium iodide (PI) was used to detect cell apoptosis. RESULTS: Here we reported that Tan IIA could inhibit cell proliferation, induce cell cycle arrest, and promote cell apoptosis in acute myeloid leukemia (AML) cells. Thus, Tan IIA had the anti-cancer activity in AML cell lines, which was likely mediated by up-regulation of miR-497-5p expression. Our data further showed that in AML cells, the same effects were observed with overexpression of miR-497-5p by a miR-497-5p mimic. We demonstrated that Tan IIA could inhibit the expression of AKT3 by up-regulating the expression of miR-497-5p. We subsequently identified that AKT3 was the direct target of miR-497-5p, and that treatment with Tan IIA obviously reversed the effect of treatment with an miR-497-5p inhibitor under harsh conditions. In turn, PCNA expression was increased and cleaved Caspase-3 was suppressed, which contributed to the growth of AML cells. CONCLUSIONS: Our results showed that Tan IIA could inhibit cell proliferation in AML cells through miR-497-5p-mediated AKT3 downregulation pathway.

Daphnane Diterpenoids from Daphne genkwa Inhibit PI3K/Akt/mTOR Signaling and Induce Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells.

Seven new daphnane-type diterpenoids, daphgenkins A-G (1-7), and 15 known analogues (8-22) were isolated from the flower buds of Daphne genkwa. Their structures and absolute configurations were elucidated by spectroscopic data and calculated ECD analyses. The cytotoxicities of all daphnane-type diterpenoids (1-22) obtained were evaluated against three human colon cancer cell lines (SW620, RKO, and LoVo). Compounds 1, 12, and 13 exhibited cytotoxic effects against the SW620 and RKO cell lines, with IC50 values in the range of 3.0-9.7 µM. The most active new compound, 1, with an IC50 value of 3.0 µM against SW620 cells, was evaluated further for its underlying molecular mechanism. Compound 1 induced G0/G1 cell cycle arrest, leading to the induction of apoptosis in SW620 cells. Also, it induced cancer cell apoptosis by an increased ratio of Bax/Bcl-2, activated cleaved caspase-3 and caspase-9, and upregulated PARP. Finally, compound 1 significantly inhibited PI3K/Akt/mTOR signaling in SW620 cells. Together, the results suggest that compound 1 may be a suitable lead compound for further biological evaluation.

Differentiation of Tumorigenic C6 Glioma Cells Induced by Enhanced IL-6 Signaling.

Background and objectives: Cancer stem cells (CSCs) are obstacles to cancer therapy due to their therapeutic resistance, ability to initiate neoplasia, and roles in tumor relapse and metastasis. Efforts have been made to cure CSCs, such as the use of differentiation therapy, which induces cancer stem-like cells to undergo differentiation and decrease their tumorigenicity. Interleukin 6 (IL-6) upregulates the expression of glial fibrillary acidic protein (GFAP) in C6 glioma cells, indicating that it is able to induce the differentiation of these cells. The C6 glioma cell line forms a high percentage of cancer stem-like cells, leading us to speculate whether IL-6 signaling could modulate the differentiation of tumorigenic C6 glioma cells. However, we observed that IL-6 alone could not efficiently induce the differentiation of these cells. Therefore, different IL-6 signaling elicitors, including IL-6 alone, a combination of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), and tumor necrosis factor-α (TNF-α) plus IL-6/sIL-6R (TNF-α/IL-6/sIL-6R), were evaluated for their potential use in differentiation therapy. Materials and Methods: The potential of IL-6 signaling elicitors in differentiation therapy were examined by assessing changes in biomarker levels, the rate of cell proliferation, and tumorigenicity, respectively. Results: Enhanced IL-6 signaling could effectively induce C6 glioma cell differentiation, as determined by observed variations in the expression of differentiation, cell cycle, and stem cell biomarkers. Additionally, the total cell population and the tumorigenicity of glioma cells were all considerably reduced after TNF-α/IL-6/sIL-6R treatment. Conclusions: Our findings provide evidence that enhanced IL-6 signaling can efficiently promote tumorigenic C6 glioma cells to undergo differentiation.

Novel role of the clustered miR-23b-3p and miR-27b-3p in enhanced expression of fibrosis-associated genes by targeting TGFBR3 in atrial fibroblasts.

Atrial fibrillation (AF) is the most common type of arrhythmia in cardiovascular diseases. Atrial fibrosis is an important pathophysiological contributor to AF. This study aimed to investigate the role of the clustered miR-23b-3p and miR-27b-3p in atrial fibrosis. Human atrial fibroblasts (HAFs) were isolated from atrial appendage tissue of patients with sinus rhythm. A cell model of atrial fibrosis was achieved in Ang-II-induced HAFs. Cell proliferation and migration were detected. We found that miR-23b-3p and miR-27b-3p were markedly increased in atrial appendage tissues of AF patients and in Ang-II-treated HAFs. Overexpression of miR-23b-3p and miR-27b-3p enhanced the expression of collagen, type I, alpha 1 (COL1A1), COL3A1 and ACTA2 in HAFs without significant effects on their proliferation and migration. Luciferase assay showed that miR-23b-3p and miR-27b-3p targeted two different sites in 3'-UTR of transforming growth factor (TGF)-ß1 receptor 3 (TGFBR3) respectively. Consistently, TGFBR3 siRNA could increase fibrosis-related genes expression, along with the Smad1 inactivation and Smad3 activation in HAFs. Additionally, overexpression of TGFBR3 could alleviate the increase of COL1A1, COL3A1 and ACTA2 in HAFs after transfection with miR-23b-3p and miR-27b-3p respectively. Moreover, Smad3 was activated in HAFs in response to Ang-II treatment and inactivation of Smad3 attenuated up-regulation of miR-23b-3p and miR-27b-3p in Ang-II-treated HAFs. Taken together, these results suggest that the clustered miR-23b-3p and miR-27b-3p consistently promote atrial fibrosis by targeting TGFBR3 to activate Smad3 signalling in HAFs, suggesting that miR-23b-3p and miR-27b-3p are potential therapeutic targets for atrial fibrosis.

Potassium Stimulation of IAA Transport Mediated by the Arabidopsis Importer AUX1 Investigated in a Heterologous Yeast System.

Auxin regulates diverse processes involved in plant growth and development. AUX1 is the first identified and most widely investigated auxin importer, and plays an important role in root gravitropism and the development of lateral root and root hair. However, the regulation of auxin transport by AUX1 is still not well understood. In this study, we examined the effect of metal ions on AUX1 transport function and found that the activity could be specifically stimulated four times by K+. Further experiments revealed the preference of KF on the enhancement of transport activity of AUX1 over KCl, KBr, and KI. In addition, the interaction between K+ and AUX1 confers AUX1 more resistant to thermal stress but more vulnerable to proteolysis. Conventional chemical modification indicated that the extracellular acidic amino acids of AUX1 play a key role in the K+ stimulation. Site-specific mutagenesis showed that the replacement of Asp166, Asp293, and Asp312 of AUX1 to alanine deteriorated the K+-stimulated auxin transport. By contrast, when these residues were mutated to glutamate, lysine, or asparagine, only the D312E variant restored the IAA transport activity to the wild-type level. It is thus convinced that D312 is presumably the most promising residue for the K+ stimulation on AUX1.

Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confers increased resistance to bacterial wilt in tobacco.

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease affecting hundreds of plant species, yet the host factors remain poorly characterized. The leucine-rich repeat receptor-like kinase gene AhRLK1, characterized as CLAVATA1, was found to be up-regulated in peanut upon inoculation with R. solanacearum. The AhRLK1 protein was localized in the plasma membrane and cell wall. qPCR results showed AhRLK1 was induced in a susceptible variety but little changed in a resistant cultivar after inoculated with R. solanacearum. Hormones such as salicylic acid, abscisic acid, methyl jasmonate, and ethephon induced AhRLK1 expression. In contrast, AhRLK1 expression was down-regulated under cold and drought treatments. Transient overexpression of AhRLK1 led to a hypersensitive response (HR) in Nicotiana benthamiana. Furthermore, AhRLK1 overexpression in tobacco significantly increased the resistance to R. solanacearum. Besides, the transcripts of most representative defense responsive genes in HR and hormone signal pathways were significantly increased in the transgenic lines. EDS1 and PAD4 in the R gene signaling pathway were also up-regulated, but NDR1 was down-regulated. Accordingly, AhRLK1 may increase the defense response to R. solanacearum via HR and hormone defense signaling, in particular through the EDS1 pathway of R gene signaling. These results provide a new understanding of the CLAVATA1 function and will contribute to genetic enhancement of peanut.