Search

Parental attitude is one of the important factors to promote the formation of the long-term mechanism of the double reduction policy (DRP). By exerting ABC Model, the questionnaire and interview are designed to investigate the attitudes and influencing factors from the parents of middle school students in Nanchong towards the DRP from the three dimensions. Three dimensions are cognition, behavior, and affect. Parental attitudes studied contributes to the formation of long-term mechanism of the DRP. The results show that on the affect dimension, the parental attitude of supporting the DRP policy has a significant positive impact; parental cognition of the DRP shows positive impact on students’ learning and school education; in behavioral indicators, parental attitude towards the DRP confirms positive supportive behavior. Three suggestions are proposed based on the findings.

Co-cultivation has been used as a promising tool to turn on or up-regulate cryptic biosynthetic pathways for microbial natural product discovery. Recently, a modified culturing strategy similar to co-cultivation was investigated, where heat-killed inducer cultures were supplemented to the culture medium of producer fermentations to induce cryptic pathways. In the present study, the repeatability and effectiveness of both methods in turning on cryptic biosynthetic pathways were unbiasedly assessed using UHPLC-HRESIMS-based metabolomics analysis. Both induction methods had good repeatability, and they resulted in very different induced metabolites from the tested producers. Co-cultivation generated more induced mass features than the heat-killed inducer cultures, while both methods resulted in the induction of mass features not observed using the other induction method. As examples, pathways leading to two new natural products, N-carbamoyl-2-hydroxy-3-methoxybenzamide (1) and carbazoquinocin G (5), were induced and up-regulated through co-culturing a producer Streptomyces sp. RKND-216 with inducers Alteromonas sp. RKMC-009 and M. smegmatis ATCC 120515, respectively.

Background and objectives Adipose tissue-derived mesenchymal stem cells (ASCs) are recognized as an advantaged source for the prevention and treatment of diverse diseases including type 2 diabetes mellitus (T2DM). However, alterations in characteristics of ASCs from the aforementioned T2DM patients are still obscure, which also hinder the rigorous and systematic illumination of progression and pathogenesis. Methods and results In this study, we originally isolated peripancreatic adipose tissue-derived mesenchymal stem cells from both human type 2 diabetic and non-diabetic donors (T2DM-ASCs, ND-ASCs) with the parental consent, respectively. We noticed that T2DM-ASCs exhibited indistinguishable immunophenotype, cell vitality, chondrogenic differentiation and stemness as ND-ASCs. Simultaneously, there's merely alterations in migration and immunoregulatory capacities in T2DM-ASCs. However, differing from ND-ASCs, T2DM-ASCs exhibited deficiency in adipogenic and osteogenic differentiation, and in particular, the delayed cell cycle and different cytokine expression spectrum. Conclusions The conservative alterations of T2DM-ASCs in multifaceted characteristics indicated the possibility of autologous application of ASCs for cell-based T2DM treatment in the future.

Objectives The present study aimed to investigate the dynamic change of α cells and β cells, and their ratios in prediabetes and type 2 diabetes in the Chinese population. Methods Pancreata from 27 nondiabetic (ND), 8 prediabetic (PreD), and 19 type 2 diabetic (T2D) organ donors were subjected to immunofluorescence staining with insulin and glucagon. Results The β to α ratio in islets (β/α) in PreD was significantly higher than that in ND, resulting from an increase of β cells and a decrease of α cells per islet, but that in T2D was significantly lower than that in ND, resulting from a decrease of β cells and an increase of α cells per islet. The β-cell percentage and β/α ratio positively correlated and α-cell percentage negatively correlated with HbA1c (glycated hemoglobin) in ND and PreD, but these correlations disappeared when T2D subjects were included. Conclusions The islet β to α ratio increased in PreD individuals because of a relative α-cell loss and β-cell compensation and decreased after T2D onset because of both β-cell loss and α-cell reexpansion.

Hypoxia affects the function of pancreatic β cells, and the molecular mechanism underlying hypoxia-related β cell dysfunction in human type 2 diabetes mellitus (T2DM) remains to be elucidated. In this study, by comparing the gene expression profiles of islets from nondiabetic and T2D subjects using gene chip array, we aimed to elucidate that hypoxia signaling pathways are activated in human T2DM islets. CoCl2 treatment, which was employed to mimic hypoxic stimulation in human islets, decreased insulin secretion, insulin content, and the functional gene expression of human islets. In parallel, the expression of mature β cell-disallowed genes was upregulated by CoCl2, including progenitor cell marker NGN3, β cell differentiation marker ALDH1A3, and genes that are typically inhibited in mature β cells, namely, GLUT1 and LDHA, indicating that CoCl2-mimicked hypoxia induced β cell dedifferentiation of human islets. This finding in human islets was confirmed in mouse β cell line NIT-1. By using Dimethyloxalylglycine (DMOG) to activate hypoxia-inducible factor-1α (HIF-1α) or siRNAs to knockdown HIF-1α, we found that HIF-1α was a key regulator of hypoxia-induced dedifferentiation of β cells by upregulating mature β cell-disallowed genes. Our findings suggested that HIF-1α activation might be an important contributor to β cell dedifferentiation in human T2DM islets, and HIF-1α-targeted therapies may have the potential to reverse β cell dedifferentiation of human T2DM islets.

Additional file 1: Supplementary Fig. 1. Correlations between NKX6.1 inactivation level in β cells and Age in non-diabetic subjects. a Correlation between NKX6.1 Nuc-Ins+ cells count and percentage with Aging in Non-diabetic subjects (n = 40). b Correlation between NKX6.1cytIns− cell count and percentage with Aging in non-diabetic subjects (n = 40). Supplementary Fig. 2. Correlations between NKX6.1 inactivation level in β cells and BMI in non-diabetic subjects. a Correlation between NKX6.1 Nuc-Ins+ cells count and percentage with BMI in Non-diabetic subjects (n = 40). b Correlation between NKX6.1cytIns− cell count and percentage with BMI in non-diabetic subjects (n = 40). Supplementary Fig. 3. Correlations between NKX6.1 inactivation level in β cells and HbA1c in non-diabetic subjects. a Correlation between NKX6.1 Nuc-Ins+ cells count and percentage with HbA1c in non-diabetic subjects (n = 40). b Correlation between NKX6.1cytIns− cell count and percentage with HbA1c in non-diabetic subjects (n = 40)

PI3Kα is a key lipid kinase in the PI3K/Akt pathway. Its frequent oncogenic mutations make it a primary drug target. Calmodulin (CaM) activates PI3Kα independently of extracellular signals, indicating a significant role in oncogenic PI3Kα activation. Here, we reveal the atomic-scale structures of CaM in complexes with the nSH2 and cSH2 domains of the regulatory p85α subunit of PI3Kα, and illustrate how CaM activates PI3Kα by targeting the “soft 1–5–10” CaM-binding motifs in both nSH2 and cSH2 domains. Experiment observed CaM binding cSH2 first, followed by nSH2 binding hours later. CaM typically prefers binding helical peptides. Here we observe that, unlike in cSH2, the CaM-binding motif in nSH2 populates a mixed β-sheet/α-helix/random coil structure. The population shift from a β-sheet toward CaM’s favored α-helical conformation explains why the nSH2 domain needs a longer time for CaM binding in the experiments. The “soft” CaM-binding motifs in both nSH2 and cSH2 domains establish strong CaM–PI3Kα interactions, collectively facilitating PI3Kα activation. This work uncovers the structural basis for CaM-driven PI3Kα activation.

Calmodulin (CaM) uniquely promotes signaling of oncogenic K-Ras; but not N-Ras or H-Ras. How CaM interacts with K-Ras and how this stimulates cell proliferation are among the most challenging questions in KRAS-driven cancers. Earlier data pointed to formation of a ternary complex consisting of K-Ras, PI3Kα and CaM. Recent data point to phosphorylated CaM binding to the SH2 domains of the p85 subunit of PI3Kα and activating it. Modeling suggests that the high affinity interaction between the phosphorylated CaM tyrosine motif and PI3Kα, can promote full PI3Kα activation by oncogenic K-Ras. Our up-to-date review discusses CaM's role in PI3K signaling at the membrane in KRAS-driven cancers. This is significant since it may help development of K-Ras-specific pharmacology.

The cyclooxygenase2 (COX-2) enzyme catalyzes the first step of prostanoid biosynthesis, and is known for its crucial role in the pathogenesis of several inflammatory diseases including type 2 diabetes mellitus (T2DM). Although a variety of studies revealed that COX-2 played a role in the IL-1β induced β cell dysfunction, the molecular mechanism remains unclear. Here, using a cDNA microarray and in silico analysis, we demonstrated that inflammatory responses were upregulated in human T2DM islets compared with non-diabetic (ND) islets. COX-2 expression was significantly enhanced in human T2DM islets, correlated with the high inflammation level. PGE2, the catalytic product of COX-2, downregulated the functional gene expression of PDX1, NKX6.1, and MAFA and blunted the glucose induced insulin secretion of human islets. Conversely, inhibition of COX-2 activity by a pharmaceutical inhibitor prevented the β-cell dysfunction induced by IL-1β. COX-2 inhibitor also abrogated the IL-1β autostimulation in β cells, which further resulted in reduced COX-2 expression in β cells. Together, our results revealed that COX-2/PGE2 signaling was involved in the regulation of IL-1β autostimulation, thus forming an IL-1β/COX-2/PGE2 pathway loop, which may result in the high inflammation level in human T2DM islets and the inflammatory impairment of β cells. Breaking this IL-1β/COX-2/PGE2 pathway loop provides a potential therapeutic strategy to improve β cell function in the treatment of T2DM patients.

Type 1 diabetes mellitus (T1DM) is a syndrome of loss of glucose homeostasis caused by the loss of β cell chronic autoimmunity against islet cells. Islet-specific epitopes coupled antigen presenting cells by Ethylenecarbodiimide (ECDI) is a promising strategy to induce antigen-specific tolerance. However, single epitope induced tolerance is insufficient to prevent the onset of T1DM. The aim of this study is to evaluate the efficacy of whole islet antigens in preventing the onset and progression of T1DM and identify the underlying immune mechanism in NOD mice. In this study, the whole islet antigens, derived from islet lysate isolated from BALB/c mice, were coupled to splenocytes of BALB/c mice by ECDI fixation (SP-Islet lysate), and then intravenously administrated to NOD mice. The results showed that, compared with control group, SP-Islet lysate group significantly decreased T1DM incidence and improved the survival of NOD mice. SP-Islet lysate treated mice had reduced insulitis score and autoantibody levels, and improved glucose tolerance and insulin/glucagon production. Furthermore, the effector memory T cells (TEMs) were downregulated and regulatory T cells (Tregs) were upregulated by the SP-Islet lysate treatment, with reduced populations of Th1&Th17 cells. In conclusion, ECDI-fixed splenocytes carrying whole islet antigens effectively prevented the onset of T1DM in NOD mice, via suppressing the production of autoantibodies and inducing anergy of autoreactive T cells.

Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) has been developed to provide comprehensive information featuring allosteric regulation. With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries.

Background: A physiological hallmark of patients with type 2 diabetes mellitus (T2DM) is β cell dysfunction. Despite adequate treatment, it is an irreversible process that follows disease progression. Therefore, the development of novel therapies that restore β cell function is of utmost importance. Methods: This study aims to unveil the mechanistic action of mesenchymal stem cells (MSCs) by investigating its impact on isolated human T2DM islets ex vivo and in vivo. Findings: We propose that MSCs can attenuate β cell dysfunction by reversing β cell dedifferentiation in an IL-1Ra-mediated manner. In response to the elevated expression of proinflammatory cytokines in human T2DM islet cells, we observed that MSCs was activated to secret IL-1R antagonist (IL-1Ra) which acted on the inflammed islets and reversed β cell dedifferentiation, suggesting a crosstalk between MSCs and human T2DM islets. The co-transplantation of MSCs with human T2DM islets in diabetic SCID mice and intravenous infusion of MSCs in db/db mice revealed the reversal of β cell dedifferentiation and improved glycaemic control in the latter. Interpretation: This evidence highlights the potential of MSCs in future cell-based therapies regarding the amelioration of β cell dysfunction. Keywords: Mesenchymal stem cells, Type 2 diabetes mellitus, β cell dysfunction, Inflammation, β cell dedifferentiation

Summary: Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a ‘Core set’ with 235 unique allosteric sites and a ‘Core-Diversity set’ with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand–protein interactions to guide allosteric drug design. Availability and implementation: The benchmarking sets are freely available at http://mdl.shsmu.edu.cn/asbench. Contact: jian.zhang@sjtu.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online

The alteration of p21-activated kinase 4 (PAK4) and transforming growth factor-beta (TGF-β) signaling effector Smad2/3 was detected in several types of tumors, which acts as oncogenic factor and tumor suppressor, but the relationship between these events has not been explored. Here, we demonstrate that PAK4 interacts with and modulates phosphorylation of Smad2/3 via both kinase-dependent and kinase-independent mechanisms, which attenuate Smad2/3 axis transactivation and TGF-β-mediated growth inhibition in gastric cancer cells. First, PAK4 interaction with Smad2/3, which is independent of PAK4 kinase activity, blocks TGF-β1-induced phosphorylation of Smad2 Ser465/467 or Smad3 Ser423/425 and the consequent activation. In addition, PAK4 phosphorylates Smad2 on Ser465, leading to the degradation of Smad2 through ubiquitin-proteasome-dependent pathway under hepatocyte growth factor (HGF) stimulation. Interestingly, PAK4 expression correlates negatively with phospho-Ser465/467 Smad2 but positively with phospho-Ser465 Smad2 in gastric cancer tissues. Furthermore, the expressions of HGF, phospho-Ser474 PAK4 and phospho-Ser465 Smad2 are markedly increased in gastric cancer tissues, and the expression of Smad2 is decreased in gastric cancer tissues. Our results document an oncogenic role of PAK4 in repression of Smad2/3 transactivation that involved in tumorigenesis, and suggest PAK4 as a potential therapeutic target for gastric cancer.

Superior cervical ganglia 10 (SCG10), as a microtubule (MT) destabilizer, maintains MT homeostasis and has a critical role in neuronal development, but its function in tumorigenesis has not been characterized. In the present study, we demonstrated that p21-activated kinase 4 (PAK4)-mediated SCG10 phosphorylation regulates MT homeostasis in metastatic gastric cancer. Our results indicate that SCG10 is a physiological substrate of PAK4, which is phosphorylated on serine 50 (Ser50) in a PAK4-dependent manner. Phosphorylated SCG10 regulated MT dynamics to promote gastric cancer cell migration and invasion in vitro and metastasis in a xenograft mouse models. Inhibiting PAK4, either by LCH-7749944 or RNA interference, resulted in the inhibition of Ser50 phosphorylation and a blockade to cell invasion, suggesting that PAK4-SCG10 signaling occurs in gastric cancer cell invasion. Moreover, we demonstrated a strong positive correlation between PAK4 and phospho-Ser50 SCG10 expression in gastric cancer samples. We also showed that high expression of SCG10 phospho-Ser50 is highly correlated to an aggressive phenotype of clinical gastric cancer. These findings revealed a novel function of SCG10 in promoting invasive potential of gastric cancer cells, suggesting that blocking PAK4-mediated SCG10 phosphorylation might be a potential therapeutic strategy for metastasis of gastric cancer.

The p21-activated kinases have been implicated in the control of cell cycle progression. However, the biological mechanism underlying the role of p21-activated kinase 4 (PAK4) in cell cycle control remains unknown. Here, by using quantitative RT–PCR and immunoblot analyses, we discovered that over-expression of PAK4 could suppress cyclin-dependent kinase inhibitor 1C (p57Kip2) expression in the MCF-7 human breast cancer cell line, whereas lentiviral vector-mediated small interfering RNA (siRNA) knockdown of PAK4 markedly promoted p57Kip2 expression in MCF-7 cells. Furthermore, PAK4-mediated down-regulation of p57Kip2 was reversed by MG132, a specific proteasome inhibitor. The ubiquitination assay confirmed that the activity of PAK4 attenuated p57Kip2 protein stability through the ubiquitin-proteasome pathway in MCF-7 cells. Moreover, a significant inverse correlation between PAK4 and p57Kip2 protein levels was observed in breast cancer tissues by immunohistochemical analysis. Taken together, our data demonstrate a novel function for PAK4 in regulating the stability of p57Kip2, possibly through the ubiquitin-proteasome pathway, leading to increased proliferation of breast cancer cells. Thus, PAK4 may be used as a potential diagnostic and therapeutic target for human breast cancer. Anat Rec, 296:1561–1567, 2013. © 2013 Wiley Periodicals, Inc.

Carbonic anhydrase IX(CA9)is a member of the carbonic anhydrase family that catalyzes the reversible hydration of carbon dioxide, and plays a key role in the regulation of pH. Although a large number of studies have shown that CA9 is strongly up-regulated by HIF1-α, little is known about the negative regulation mechanism of CA9 in cancer cells. Here we find that CREB is a key negative regulator of CA9 in gastric cancer. Over-expression of CREB can significantly repress the expression of CA9. Treating with anisomycin (ANS), an activator of p38, the phosphorylation and nuclear translocation of CREB are both promoted, while the transcription of CA9 is repressed. Besides, our results firstly identify that CREB can recruit SIRT1 (class III HDACS) by adaptor protein p300, then repress the expression of CA9. These findings may contribute to understand the negative regulation mechanisms of CA9 in gastric cancer.

Cell division cycle 42 (CDC42), an important member of the Ras homolog (Rho) family, plays a key role in regulating multiple cellular processes such as cell cycle progression, migration, cell cytoskeleton organization, cell fate determination and differentiation. Among the downstream effectors of CDC42, P21-activated kinases (PAKs) obtain the most attention. Although a large body of evidence indicates that CDC42/PAKs pathway plays important role in tumor growth, invasion and metastasis, the mechanism of their negative regulation remains unclear. Here, we identified CDC42, a PAKs activating factor, was a target of miR-133. Ectopic overexpression of miRNAs not only downregulated CDC42 expression and PAKs activation, but also inhibited cancer cell proliferation and migration. We also found that miR-133 was down-regulated in 180 pairs gastric cancer tissues. miR-133 expression was negatively associated with tumor size, invasion depth and peripheral organ metastasis. Besides, dysfunction of miR-133 was an independent prognosis factor for overall survival. Our findings could provide new insights into the molecular mechanisms of gastric carcinogenesis, and may help facilitating development of CDC42/PAK-based therapies for human cancer.

The p21-activated kinases have been implicated in the control of cell cycle progression. However, the biological mechanism underlying the role of p21-activated kinase 4 (PAK4) in cell cycle control remains unknown. Here, by using quantitative RT-PCR and immunoblot analyses, we discovered that over-expression of PAK4 could suppress cyclin-dependent kinase inhibitor 1C (p57(Kip2) ) expression in the MCF-7 human breast cancer cell line, whereas lentiviral vector-mediated small interfering RNA (siRNA) knockdown of PAK4 markedly promoted p57(Kip2) expression in MCF-7 cells. Furthermore, PAK4-mediated down-regulation of p57(Kip2) was reversed by MG132, a specific proteasome inhibitor. The ubiquitination assay confirmed that the activity of PAK4 attenuated p57(Kip2) protein stability through the ubiquitin-proteasome pathway in MCF-7 cells. Moreover, a significant inverse correlation between PAK4 and p57(Kip2) protein levels was observed in breast cancer tissues by immunohistochemical analysis. Taken together, our data demonstrate a novel function for PAK4 in regulating the stability of p57(Kip2) , possibly through the ubiquitin-proteasome pathway, leading to increased proliferation of breast cancer cells. Thus, PAK4 may be used as a potential diagnostic and therapeutic target for human breast cancer.