HDAC1-Smad3-mSin3A complex is required for Smad3-induced transcriptional inhibition of hepatocyte growth factor receptor in human lung cancers.

c-Met hyperactivity has been observed in numerous neoplasms. Several researchers have shown that the abnormal activation of c-Met is mainly caused by transcriptional activation. However, the molecular mechanism behind this transcriptional regulation is poorly understood. Here, we suggest that Smad3 negatively regulates the expression and activation of c-Met via a transcriptional mechanism. We explore the molecular mechanisms that underlie Smad3-induced c-Met transcription inhibition. We found in contrast to the high expression of c-Met, Smad3 showed low protein and mRNA levels. Smad3 and c-Met expressions were inconsistent between lung cancer tissues and cell lines. We also found that Smad3 overexpression suppresses whereas Smad3 knockdown significantly promotes Epithelial-Mesenchymal Transition and production of the angiogenic factors VEGF, CTGF and COX-2 through the ERK1/2 pathway. In addition, Smad3 overexpression decreases whereas Smad3 knockdown significantly increases protein and mRNA levels of invasion-related ß-catenin and FAK through the PI3K/Akt pathway. Furthermore, using the chromatin immunoprecipitation analysis method, we demonstrate that a transcriptional regulatory complex consisting of HDAC1, Smad3 and mSin3A binds to the promoter of the c-Met gene. By either silencing endogenous mSin3A expression with siRNA or by pretreating cells with a specific HDAC1 inhibitor (MS-275), Smad3-induced transcriptional suppression of c-Met could be effectively attenuated. These results demonstrate that Smad3-induced inhibition of c-Met transcription depends on of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the c-Met promoter. Collectively, our findings reveal a new regulatory mechanism of c-Met signaling, and suggest a potential molecular target for the development of anticancer drugs.

Inferring spatial patterns and drivers of population divergence of Neolitsea sericea (Lauraceae), based on molecular phylogeography and landscape genomics.

The relative roles of geography, climate and ecology in driving population divergence and (incipient) speciation has so far been largely neglected in studies addressing the evolution of East Asia's island flora. Here, we employed chloroplast and ribosomal DNA sequences and restriction site-associated DNA sequencing (RADseq) loci to investigate the phylogeography and drivers of population divergence of Neolitsea sericea. These data sets support the subdivision of N. sericea populations into the Southern and Northern lineages across the 'Tokara gap'. Two distinct sublineages were further identified for the Northern lineage of N. sericea from the RADseq data. RADseq was also used along with approximate Bayesian computation to show that the current distribution and differentiation of N. sericea populations resulted from a combination of relatively ancient migration and successive vicariant events that likely occurred during the mid to late Pleistocene. Landscape genomic analyses showed that, apart from geographic barriers, barrier, potentially local adaptation to different climatic conditions appears to be one of the major drivers for lineage diversification of N. sericea.

Evolution of East Asia's Arcto-Tertiary relict Euptelea (Eupteleaceae) shaped by Late Neogene vicariance and Quaternary climate change.

BACKGROUND: The evolutionary origin and historical demography of extant Arcto-Tertiary forest species in East Asia is still poorly understood. Here, we reconstructed the evolutionary and population demographic history of the two extant Euptelea species in China (E. pleiosperma) and Japan (E. polyandra). Chloroplast/nuclear DNA sequences and microsatellite loci were obtained from 36 Euptelea populations to explore molecular structure and diversity in relation to past and present distributions based on ecological niche modelling (ENM). Time-calibrated phylogenetic/phylogeographic inferences and niche-identity tests were used to infer the historical process of lineage formation. RESULTS: Euptelea pleiosperma diverged from E. polyandra around the Late Miocene and experienced significant ecological differentiation. A near-simultaneous diversification of six phylogroups occurred during the mid-to-late Pliocene, in response to the abrupt uplift of the eastern Tibetan Plateau and an increasingly cooler and drier climate. Populations of E. pleiosperma seem to have been mostly stationary through the last glacial cycles, while those of E. polyandra reflect more recent climate-induced cycles of range contraction and expansion. CONCLUSIONS: Our results illustrate how Late Neogene climatic/tectonic changes promoted speciation and lineage diversification in East Asia's Tertiary relict flora. They also demonstrate for the first time a greater variation in such species' responses to glacial cycles in Japan when compared to congeners in China.

[Characterization of Metal Elements in Atmospheric Fine Particulate Matter and Their Sources in Winter in the Southern Sichuan Urban Agglomeration].

To investigate the concentration characteristics and sources of metal elements in PM2.5 during winter heavy pollution in the southern Sichuan urban agglomeration (Zigong, Luzhou, Neijiang, and Yibin), the metal elements in PM2.5 were measured using membrane sampling methods from December 30, 2018 to January 14, 2019, and the enrichment factor method (EF) and positive matrix factorization(PMF) were applied to investigate the sources of metal elements. The metal element observation data of Zigong in the same period of 2015 were also used to investigate the changes in metal element pollution and enrichment in Zigong in the middle and end of the implementation of China's Air Pollution Prevention and Control Action Plan. The main findings were as follows:① The concentrations and percentages of metal elements in particulate matter in different cities did not differ significantly. The elements with higher concentrations in the four cities showed similarities, with Al, Sb, and Fe at the top. From the comparison of different observation periods in Zigong, the concentrations of all elements except Tl changed. ② The results of the enrichment factor calculation showed that the enrichment of the elements Cr (Zigong and Yibin), Ni, Cu, As, Se, Ag, Cd, Sb, Tl, and Pb in the urban agglomeration was high. The comparison of the enrichment levels of elements in Zigong for different observation periods showed that the enrichment levels of all elements, except Cu, tended to decrease in the winter observation period of 2018. ③ The results of PMF source analysis showed that the metal elements in each city mainly originated from dust sources, coal-fired sources, industrial sources, and traffic sources, whereas there was a mixed contribution among the sources. The contribution of the main sources differed among cities, in which Zigong was dominated by traffic dust sources and mixed sources, Luzhou was dominated by industrial sources, Neijiang had a similar contribution from different sources, and Yibin was dominated by traffic sources.

[Characteristics of Carbonaceous Species in PM2.5 in Chengdu Under the Background of Emission Reduction].

The Air Pollution Prevention and Control Action Plan and Three-year Plan on Defending the Blue Sky promulgated by the State Council of the People's Republic of China have played an important role in the overall improvement of air quality in China. However, few studies have evaluated the implementation effects of these two policies in Sichuan Basin and the new characteristics of PM2.5 chemical components after the implementation of these policies. The key periods for evaluating the implementation effects of these two pollution reduction policies are 2017 and 2020, respectively. In order to study the atmospheric PM2.5 and carbonaceous species in Chengdu during these two periods, this study sampled the PM2.5 in Chengdu from October 2016 to July 2017 and December 2020, respectively, and the organic carbon (OC) and elemental carbon (EC) were analyzed. The results showed that the annual ρ(PM2.5) from 2016-2017 in Chengdu was (114.0±76.4) µg·m-3. The maximum value of the ρ(PM2.5) appeared in winter[(193.3±98.5) µg·m-3], and the minimum value appeared in spring[(73.8±32.3) µg·m-3]. By contrast, the ρ(PM2.5) in winter decreased significantly in 2020, with a value of (96.0±39.3) µg·m-3. The annual ρ(OC) and ρ(EC) from 2016-2017 were (21.1±16.4) µg·m-3 and (1.9±1.3) µg·m-3, which accounted for 18.5% and 1.7% of the PM2.5 mass, respectively. The seasonal variation characteristic of ρ(OC) was:winter[(40.6±21.5) µg·m-3]>autumn[(17.0±7.0) µg·m-3]>summer[(14.4±3.9) µg·m-3]>spring[(12.6±6.0) µg·m-3], whereas the ρ(EC) in the four seasons were close, ranging from 1.3 to 2.4 µg·m-3. The annual ρ(SOC) was (9.4±9.1) µg·m-3, which accounted for 44.5% of the OC mass. Compared with that in winter 2016, the ρ(OC) decreased by 52.7% in winter 2020, whereas the ρ(EC) increased by 26.1%. With the aggravation of pollution, the change trends of carbon species and their contributions were different. Compared with that in winter 2016, the variation in the contribution of OC with the aggravation of pollution in winter 2020 was more stable, whereas the proportion of SOC increased more obviously. There were obvious differences in the direction of air masses and the potential source area of pollutants in each season. Although there was no significant change in the direction of air masses in winter 2020 compared with those in winter 2016, the pollutant concentrations corresponding to each cluster decreased significantly, and the potential source area of pollutants expanded significantly to the eastern area.

Flavonoid and chromone-rich extract from Euscaphis Konishii Hayata leaf attenuated alcoholic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Euscaphis konishii Hayata is a traditional medicinal plant in China, and its leaves are usually used to make dishes for hepatic or gastrointestinal issues by Chinese She nationality. Pharmacological analysis showed that E. konishii leaves contain high levels of flavonoids and chromones with favorable anti-hepatoma effect. AIM OF THE STUDY: The extract from E. konishii leaves was detected to evaluate its chemical composition, and the alcoholic liver injury mice model was adopted to elucidate its hepatoprotective effects. MATERIALS AND METHODS: The total leaf extract from E. konishii was separated by polyamide column to get the flavonoid and chromone-rich extract (FCE). Single compounds from FCE was purified by gel and Sephadex LH-20 chromatography and analyzed by nuclear magnetic resonance (NMR). The chemical component of FCE was confirmed and quantified by HPLC-MS. The OH·, O2-, DPPH and ABTS + free radical assays were adopted to estimate the antioxidant activity of FCE in vitro. The alcohol-fed model mice were established to assess the hepatoprotective capacity of FCE in vivo, through biochemical determination, histopathological analysis, mitochondrial function measurement, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) detection and Western blot determination. RESULTS: 8 flavonoids and 2 chromones were recognized in the FCEextract by both NMR and HPLC-MS. FCE represented strong free radicals scavenging activity in vitro. With oral administration, FCE (50, 100 and 200 mg/kg) dose-dependently decreased the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in alcohol-fed mice. FCE gradually reduced the malondialdehyde (MDA) content, increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the alcohol-treated liver tissues. FCE also alleviated the hepatic inflammation, inhibited the hepatocyte apoptosis and lessened the alcohol-induced histological alteration and lipid accumulation in the liver tissues. FCE administration inhibited the overexpression of endoplasmic reticulum (ER) chaperones signaling and unfolded protein response (UPR) pathways to defense the ER-induced apoptosis. Pretreatment with FCE also restored the mitochondrial membrane potentials andadenosine triphosphate (ATP) levels, which in turn suppressed the Cytochrome C release and mitochondria-induced apoptosis. CONCLUSIONS: FCE conferred great protection against alcoholic liver injury, which might be associated with its viability through suppressing reactive oxygen species (ROS) stress and hepatocyte apoptosis.

Genomic insights into historical population dynamics, local adaptation, and climate change vulnerability of the East Asian Tertiary relict Euptelea (Eupteleaceae).

The warm-temperate and subtropical climate zones of East Asia are a hotspot of plant species richness and endemism, including a noticeable number of species-poor Tertiary relict tree genera. However, little is understood about when East Asian Tertiary relict plants diversified, how they responded demographically to past environmental change, and to what extent their current genomic composition (and adaptive capacity) might mitigate the effects of global warming. Here, we obtained genomic (RAD-SNP) data for 171 samples from two extant species of Euptelea in China (24 E. pleiosperma populations) and Japan (11 E. polyandra populations) to elucidate their divergence and demographic histories, genome-wide associations with current environmental variables, and genomic vulnerability to future climate change. Our results indicate that Late Miocene changes in climate and/or sea level promoted species divergence, whereas Late Pliocene uplifting in southwest China likely fostered lineage divergence within E. pleiosperma. Its subsequent range expansion into central/east (CE) China bears genomic signatures of climate-driven selection, yet extant CE populations are predicted to be most vulnerable to future climate change. For E. polyandra, geography was the only significant predictor of genomic variation. Our findings indicate a profound impact of Late Neogene geological and climate change on the evolutionary history of Euptelea, with much stronger signals of local adaptation left in China than in Japan. This study deepens our understanding of the complex evolutionary forces that influence the distribution of genetic variation of Tertiary relict trees, and provides insights into their susceptibility to global change and potential for adaptive responses. Our results lay the groundwork for future conservation and restoration programs for Euptelea.

Protective Effect of the Total Triterpenes of Euscaphis konishii Hayata Pericarp on Bacillus Calmette-Guérin Plus Lipopolysaccharide-Induced Liver Injury.

BACKGROUND: Liver injury has been recognized as a primary cause of hepatic morbidity and mortality. Euscaphis konishii Hayata, also called Euscaphis fukienensis Hsu, is usually used as a detumescent and analgesic agent to improve liver function in South China, but its mechanism of action and chemical composition are unclear. OBJECTIVE: The main aim of the study was to investigate the constituent and potential hepatoprotective mechanism of the total triterpenes of E. konishii pericarp (TTEP). METHODS: The constituent of TTEP was analyzed by a series of silica gel column to get single compounds and then identified by NMR and MS. In vitro assays were conducted to test the free radical scavenging activity of TTEP. The BCG/LPS-induced immunological livery injury mice model was established to clarify the hepatoprotective effect of TTEP in vivo. RESULTS: 8 pentacyclic triterpene acids were separated and identified by NMR and MS. TTEP treatment (50, 100, and 200 mg/Kg) improved the immune function of the BCG/LPS-infected mice, dose-dependently alleviated the BCG/LPS-induced inflammation and oxidative stress, and ameliorated the hepatocyte apoptosis in the liver tissue. CONCLUSION: The pericarp of E. konishii may be further considered as a potent natural food for liver disease treatment.

Assessing the real-time activation of the cannabinoid CB1 receptor and the associated structural changes using a FRET biosensor.

The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.

iTRAQ-based proteomics screen for potential regulators of wheat (Triticum aestivum L.) root cell wall component response to Al stress.

OBJECTIVE: The inhibition of Aluminum (Al)-induced root tip cell elongation is a major cause of plant root elongation suppression. The inhibition of root tip cell elongation is caused by a disruption of cell wall component metabolism, growth signaling, or cellular damage. The aim of this study was to identify the proteins involved in the metabolism of the root cell wall components under Al stress in the Al-tolerant wheat (Triticum aestivum L.) cultivar ET8. METHODS: Differentially expressed proteins of Al-tolerant wheat roots were screened via isobaric tags for relative and absolute quantification (iTRAQ). Furthermore, their expression changes were detected via RT-PCR analysis. The contents of major components of the cell wall and their changes in metabolic enzyme activities were also investigated. RESULTS: A total of 97 differentially expressed proteins from Al-tolerant wheat roots were screened and nine of these 97 proteins were root cell wall component related. The known nucleic acid sequences of proteins were 14-3-3 protein, the plasm membrane (PM) H+-ATPase, phospholipase D, peroxidase, and glycosyltransferase. For 14-3-3 protein, phospholipase D and peroxidase, the protein expression and mRNA expression were consistent with Al-treatment; however, for PM H+-ATPase and glycosyltransferase, the protein expression and mRNA expression were inconsistent under Al-stress. Furthermore, both cellulase activity and callase activity were down-regulated by Al stress, while the phenylalanineammonialyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) activities were up-regulated. Furthermore, the PM H+-ATPase activity was decreased in response to Al stress. In addition, the contents of callose, cellulose, lignin, and H2O2 varied significantly. CONCLUSIONS: The cell wall components, relative metabolism enzymes activity, and gene expression also changed followed by protein expression changed in response to Al stress. The results suggest that Al stress leads to marked variations in metabolic enzyme activity, carbohydrate content, followed by changes of root cell components in wheat roots.

Visualization of the activation of the histamine H3 receptor (H3R) using novel fluorescence resonance energy transfer biosensors and their potential application to the study of H3R pharmacology.

Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands.