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Ischemic stroke is a major cause of death and long-term disability. We demonstrate that middle cerebral artery occlusion (MCAO) in mice leads to a strong decline in dendritic arborization of penumbral neurons. These defects were subsequently repaired by an ipsilateral recovery process requiring the actin nucleator Cobl. Ischemic stroke and excitotoxicity, caused by calpain-mediated proteolysis, significantly reduced Cobl levels. In an apparently unique manner among excitotoxicity-affected proteins, this Cobl decline was rapidly restored by increased mRNA expression and Cobl then played a pivotal role in poststroke dendritic arbor repair in peri-infarct areas. In Cobl knockout (KO) mice, the dendritic repair window determined to span day 2 to 4 poststroke in wild-type (WT) strikingly passed without any dendritic regrowth. Instead, Cobl KO penumbral neurons of the primary motor cortex continued to show the dendritic impairments caused by stroke. Our results thereby highlight a powerful poststroke recovery process and identified causal molecular mechanisms critical during poststroke repair.

Recent studies have indicated that DNA methylation plays an important role in the development of alcohol abuse. 5-Aza-2'-deoxycytidine (5-Aza-dc), an inhibitor of DNA methyltransferases, was FDA approved for myelodysplastic syndrome treatment. However, it is unclear whether 5-Aza-dc is involved in alcohol abuse. In this study, using a chronic alcohol exposure model in rats, 5-Aza-dc was injected into the medial prefrontal cortex (mPFC). Alcohol-drinking behavior and the anxiety related behavior were evaluated by two-bottle choice and open field test. We found that 5-Aza-dc injection into the mPFC significantly decreased alcohol consumption and alcohol preference in alcohol-exposure rats, corresponding to the reduced blood alcohol levels. Although 5-Aza-dc potentiated the anxiety-like behavior of alcohol-exposure rats, it had no effect on the locomotor activity. Moreover, both of the mRNA and protein levels of DNA Methyltransferase 3A (DNMT3A) and DNMT3B in the mPFC were upregulated after 35 days of alcohol exposure and this upregulation could be reversed by 5-Aza-dc treatment. Additionally, 5-Aza-dc reversed the alcohol-induced downregulation of neurotrophin-3 (Ntf3), correspondingly the expression of its receptor-TrkC was reduced. These findings identified a functional role of 5-Aza-dc in alcohol-related behavioral phenotypes and one of the potential target genes, Ntf3. We also provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcohol abuse.

Baicalein, a widely used Chinese herbal medicine, has historically been used in anti-inflammatory and anti-cancer therapies. However, the anti-metastatic effect and molecular mechanism(s) of baicalein on hepatocellular carcinoma (HCC) remain poorly understood. Therefore, the purpose of this study was to assess the anti-metastatic effects of baicalein and related mechanism(s) on HCC. Based on assays utilized in both HCC cell lines and in an animal model, we found that baicalein inhibited tumor cell metastasis in vivo and in vitro. Furthermore, after treatment with baicalein for 24 hours, there was a decrease in the levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and urokinase-type plasminogen activator (u-PA) expression as well as proteinase activity in hepatocellular carcinoma MHCC97H cells. Meanwhile, the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 were increased in a dose-dependent fashion. Moreover, baicalein treatment dramatically decreased the levels of the phosphorylated forms of MEK1 and ERK1/2. MEK1 overexpression partially blocked the anti-metastatic effects of baicalein. Combined treatment with an ERK inhibitor (U0126) and baicalein resulted in a synergistic reduction in MMP-2, MMP-9 and u-PA expression and an increase in TIMP-1 and TIMP-2 expression; the invasive capabilities of MHCC97H cells were also inhibited. In conclusion, baicalein inhibits tumor cell invasion and metastasis by reducing cell motility and migration via the suppression of the ERK pathway, suggesting that baicalein is a potential therapeutic agent for HCC.

ObjectiveAldehyde dehydrogenase (ALDH) has recently been reported as a marker of cancer stem-like cells in ovarian cancer. However, the prognostic role of ALDH in ovarian cancer still remains controversial. In this study, we aimed to evaluate the association between the expression of ALDH and the outcome of ovarian cancer patients by performing a meta-analysis.MethodsWe systematically searched for studies investigating the relationships between ALDH expression and outcome of ovarian cancer patients. Only articles in which ALDH expression was detected by immunohistochemical staining were included. A meta-analysis was performed to generate combined hazard ratios (HRs) with 95% confidence intervals (CIs) for overall survival (OS) and disease-free survival (DFS).ResultsA total of 1,258 patients from 7 studies (6 articles) were included in the analysis. Our results showed that high ALDH expression in patients with ovarian cancer was associated with poor prognosis in terms of Os (HR, 1.25; 95% CI, 1.07-1.47; P = 0.005) and DFS (HR, 1.58; 95% CI, 1.00-2.49; P = 0.052), though the difference for DFS was not statistically significant. In addition, there was no evidence of publication bias as suggested by Begg's and Egger's tests (Begg's test, P = 0.707; Egger's test, P = 0.355).ConclusionThe present meta-analysis indicated that elevated ALDH expression was associated with poor prognosis in patients with ovarian cancer.

ObjectiveThis systematic review aimed to clarify if acupuncture is more effective for menopausal insomnia compared with sham acupuncture, standard care (sedative hypnotics and/or MHT) or waitlist control.MethodsSeven literature databases were searched on April 30, 2024, to identify RCTs assessing the effectiveness of acupuncture. The methodological quality was assessed by the Cochrane Collaboration, and meta-analyses were conducted to calculate comparative effects using Rev Man software.Results28 RCTs were analyzed. Six sham acupuncture-controlled RCTs were notable because of their high quality, and they showed that acupuncture significantly lowered PSQI scores, increased TST, sleep efficiency, and reduced WASO. The effect of acupuncture was maintained at a 4-week follow-up. Sixteen RCTs compared acupuncture with standard care, which showed acupuncture significantly reduced PSQI scores, KI scores, HAMD and HAMA scores. However, the subgroup analysis showed that there was no obviously difference between acupuncture and western medication in the treatment duration >8 weeks. Five RCTs assessed acupuncture combined with standard care and showed a favorable reduction in the PSQI score than standard care. One RCT showed that acupuncture significantly reduced PSQI and KI scores than a waitlist control. The GRADE assessment demonstrated that the level of evidence was very low to moderate, probably for the poor methodological quality and substantial heterogeneity among studies.ConclusionThe results showed that acupuncture may play a positive role in patients with menopausal insomnia.

Current approaches to high-field functional MRI (fMRI) provide 2 means to map hemodynamics at the level of single vessels in the brain. One is through changes in deoxyhemoglobin in venules, i.e., blood oxygenation level-dependent (BOLD) fMRI, while the second is through changes in arteriole diameter, i.e., cerebral blood volume (CBV) fMRI. Here, we introduce cerebral blood flow-related velocity-based fMRI, denoted CBFv-fMRI, which uses high-resolution phase contrast (PC) MRI to form velocity measurements of flow. We use CBFv-fMRI in measure changes in blood velocity in single penetrating microvessels across rat parietal cortex. In contrast to the venule-dominated BOLD and arteriole-dominated CBV fMRI signals, CBFv-fMRI is comparable from both arterioles and venules. A single fMRI platform is used to map changes in blood pO2 (BOLD), volume (CBV), and velocity (CBFv). This combined high-resolution single-vessel fMRI mapping scheme enables vessel-specific hemodynamic mapping in animal models of normal and diseased states and further has translational potential to map vascular dementia in diseased or injured human brains with ultra-high-field fMRI.

The complement system is activated in tuberculous pleural effusion (TPE), with increased levels of the anaphylatoxins stimulating pleural mesothelial cells (PMCs) to secrete chemokines, which recruit nonclassical monocytes to the pleural cavity. The differentiation and recruitment of naive CD4+ T cells are induced by pleural cytokines and PMC-produced chemokines in TPE. However, it is unclear whether anaphylatoxins orchestrate CD4+ T cell response via interactions between PMCs and monocytes in TPE. In this study, CD16+ and CD16- monocytes isolated from TPE patients were cocultured with PMCs pretreated with anaphylatoxins. After removing the PMCs, the conditioned monocytes were cocultured with CD4+ T cells. The levels of the cytokines were measured in PMCs and monocyte subsets treated separately with anaphylatoxins. The costimulatory molecules were assessed in conditioned monocyte subsets. Furthermore, CD4+ T cell response was evaluated in different coculture systems. The results indicated that anaphylatoxins induced PMCs and CD16+ monocytes to secrete abundant cytokines capable of only inducing Th17 expansion, but Th1 was feeble. In addition, costimulatory molecules were more highly expressed in CD16+ than in CD16- monocytes isolated from TPE. The interactions between monocytes and PMCs enhanced the ability of PMCs and monocytes to produce cytokines and that of monocytes to express HLA-DR, CD40, CD80 and CD86, which synergistically induced Th17 expansion. In the above process, anaphylatoxins enhanced the interactions between monocytes and PMCs by increasing the level of the cytokines IL-1β, IL-6, IL-23 and upregulating the phenotype of CD40 and CD80 in CD16+ monocytes. Collectively, these data indicate that anaphylatoxins play a central role in orchestrating Th17 response mainly via interactions between CD16+ monocytes and PMCs in TPE.

BACKGROUND:The psychological health (PH) of doctors affects the quality of medical service and is related to the safety of patients. The serious problems with the doctor-patient relationship in China can lead to long-term imbalances in doctor PH, and the poor PH status of doctors has raised scholars' concern. Current research mainly focuses on how factors such as social support and the impact of the residential environment correlate with individual PH. We continue this direction of research to see how the mechanism of social support impacts physician PH, also investigating the moderating effect of demographic indicators on physician PH. METHODS:Based on a survey of 399 physicians, a descriptive analysis of measured data was done using SPSS 19.0. Pearson correlation coefficient analysis was used to examine the correlations between PH and the social support rating scale (SSRS) and the demographic variables. KMO and Bartlett methods were used to examine the correlations between PH and SDS (a scale to measure depression) and between PH and SAS (a scale to measure anxiety). The method of factor analysis was used for multicollinearity tests, and multiple stepwise regression analysis was used to explore the demographic factors correlated with PH and SSRS. Two-way interactions in moderated multiple regression were used to test the moderating effect of education level and title on SSRS, SDS, and SAS. RESULTS:Our results indicate that the level of PH is influenced by the age, education, and title of a doctor. A physician's title is significantly and positively correlated with PH, but age and education are significantly negatively related. Age, education, and title also affect the level of SSRS in physicians. SSRS is positively correlated with age, education, and title, and SSRS positively influences PH. Education and title had significant effects on the moderating influences of SSRS, SDS, SAS, and PH. CONCLUSION:The factors directly affecting PH include SSRS, age, and title, while education was found to be an indirect influencing factor. To meet goals expressed in Chinese government policy related to these issues, we suggest strengthening the guidance of the media, introducing laws and regulations on doctor-patient risk management and control, reforming the review mechanism of hospital job titles, improving the education level of doctors, building a comprehensive evaluation system of "practice performance + doctor-patient satisfaction", and strengthening doctor-patient empathy. Through such measures, the level of PH in physicians will improve.

Hepatitis E virus (HEV) infection is relatively high in the southern regions of China. Yunnan, located in southwestern China, has the highest number of ethnic groups. However, HEV infection in the ethnic population is largely unknown. Therefore, we aimed to investigate the seropositive rate, risk factor, and clinical impact of HEV infection in the ethnic groups of Yunnan. We recruited 1912 individuals from four minority groups in three prefectures of Yunnan province. Epidemiological records on potential risk factors for exposure to HEV and blood biochemical index were analyzed. All the serum samples were tested for anti-HEV IgM/IgG by enzyme-linked immunosorbent assay, and the IgM-positive samples were subjected to nested reverse transcription-PCR to detect HEV RNA. Overall, 1273 individuals (66.58%) were positive for anti-HEV IgG, 16 (0.84%) for anti-HEV IgM, and 64 (3.35%) for anti-HEV IgG and IgM both; none of them had detectable HEV RNA. Multivariate analysis revealed a strong statistical association between ethnic origin and HEV IgG seroprevalence. Anti-HEV IgG reactivity in the Hani ethnic (82.3%; 401/487) population was higher than that in the Naxi (71.9%, 340/473), Bulang (65.1%; 302/464), and Wa (60.2%; 294/488) ethnic populations (p < 0.0001). Older age and male sex were independently associated with the risk of past HEV infection. Moreover, anti-HEV IgG-positive individuals showed significantly higher levels of total and direct bilirubin and alanine amino transferase but significantly lower levels of globulin and low-density lipoprotein, than the respective levels in anti-HEV IgG-negative individuals. Thus, the seroprevalence of HEV infection is high in the ethnic populations of Yunnan, China. It is therefore necessary to increase the surveillance of specific risk groups and raise awareness about the possible infectious diseases to help limit the HEV transmission here.

BackgroundThe tension in doctor-patient relationships is becoming progressively greater due to the high expectations of patients and the physicians' work pressure. Recent studies have addressed factors which affect the tension of doctor-patient relationships, and our study continues this trend by looking at the influence of resiliency and physician trust in the patient (PTP), that is, how much the doctor trusts the patient.MethodsBased on a survey of 329 physicians, a descriptive analysis of measured data was done using SPSS 19.0. Pearson correlation coefficient analysis was used to examine the correlation between PTP and resilience and the demographic variables. KMO and Bartlett methods were used to examine the correlation between PTPS and resilience. The method of factor analysis was used for multicollinearity tests, and multiple stepwise regression analysis was used to explore the demographic factors correlated with PTP and resilience.ResultOur results indicate that the level of PTP is influenced by the age, education, and income of the doctors. Physician age and income are significantly and positively correlated with PTP, but education is significantly and negatively related. Age, education, and income also affect the level of psychological resilience of physicians. Resilience is positively correlated with age and education but is negatively related to income. Resilience positively influences PTP.ConclusionThe direct factors of PTP include resilience, age, education, and income, while gender, title, and hospital department were found to be indirect influencing factors. To meet goals expressed in Chinese government policy related to these issues, we suggest improving the level of education of the doctors, providing reasonable annual salary increases for doctors, easing the tensions involved in medical treatment, reducing the physicians' work pressure, improving the physicians' work environment, and enhancing the physicians' professional sympathy. Through such measures, the level of PTP will be enhanced.

In many computer vision and machine learning applications, the data sets distribute on certain low-dimensional subspaces. Subspace clustering is a powerful technology to find the underlying subspaces and cluster data points correctly. However, traditional subspace clustering methods can only be applied on data from one source, and how to extend these methods and enable the extensions to combine information from various data sources has become a hot area of research. Previous multi-view subspace methods aim to learn multiple subspace representation matrices simultaneously and these learning task for different views are treated equally. After obtaining representation matrices, they stack up the learned representation matrices as the common underlying subspace structure. However, for many problems, the importance of sources and the importance of features in one source both can be varied, which makes the previous approaches ineffective. In this paper, we propose a novel method called Robust Auto-weighted Multi-view Subspace Clustering (RAMSC). In our method, the weight for both the sources and features can be learned automatically via utilizing a novel trick and introducing a sparse norm. More importantly, the objective of our method is a common representation matrix which directly reflects the common underlying subspace structure. A new efficient algorithm is derived to solve the formulated objective with rigorous theoretical proof on its convergency. Extensive experimental results on five benchmark multi-view datasets well demonstrate that the proposed method consistently outperforms the state-of-the-art methods.

ABA has been widely acknowledged to regulate ethylene biosynthesis and signaling during fruit ripening, but the molecular mechanism underlying the interaction between these two hormones are largely unexplored. In the present study, exogenous ABA treatment obviously promoted fruit ripening as well as ethylene emission, whereas NDGA (Nordihydroguaiaretic acid, an inhibitor of ABA biosynthesis) application showed the opposite biological effects. Combined RNA-seq with time-course RT-PCR analysis, our study not only helped to illustrate how ABA regulated itself at the transcription level, but also revealed that ABA can facilitate ethylene production and response probably by regulating some crucial genes such as LeACS4, LeACO1, GR and LeETR6. In addition, investigation on the fruits treated with 1-MCP immediately after ABA exposure revealed that ethylene might be essential for the induction of ABA biosynthesis and signaling at the onset of fruit ripening. Furthermore, some specific transcription factors (TFs) known as regulators of ethylene synthesis and sensibility (e.g. MADS-RIN, TAGL1, CNR and NOR) were also observed to be ABA responsive, which implied that ABA influenced ethylene action possibly through the regulation of these TFs expression. Our comprehensive physiological and molecular-level analysis shed light on the mechanism of cross-talk between ABA and ethylene during the process of tomato fruit ripening.

Excessive mechanical loads on the temporomandibular joint (TMJ) can cause mandibular cartilage degradation and subchondral bone erosion, but the treatment of these conditions remains challenging. Salubrinal, which target eukaryotic translation initiation factor 2 alpha, has been shown to have multiple beneficial effects on skeletal tissue. Here, we examined the effect of a Salubrinal injection on the mandibular cartilage and subchondral bone of the TMJ under various compressive stresses. We conducted in vivo analyses in rat models using various compressive stresses (40 g and 80 g), and we observed time-related degeneration and pathological changes in the cartilage and subchondral bone of the TMJ at days 1, 3 and 7 through histological measurements, subcellular observation, and changes in proliferation and apoptosis. After the Salubrinal injection, the thickness of the cartilage recovered, and the pathological change was alleviated. In the Salubrinal/light (Sal/light) compressive stress group, the drug altered the proliferation and apoptosis of chondrocytes most significantly at day 1. In the Salubrinal/heavy (Sal/heavy) compressive stress group, the drug increased the proliferation of chondrocytes most significantly at day 1 and reduced the apoptosis of chondrocytes most significantly at day 7. Salubrinal also increased the area of the bone trabeculae and suppressed inflammatory responses and pathological change in the subchondral bone of the TMJ. Together, these results indicate that the administration of Salubrinal reduces apoptosis and strengthens the proliferation of chondrocyte to varying degrees at days 1, 3 and 7 under various compressive mechanical stresses, both of which contribute to the recovery of cartilage thickness and the alleviation of pathological change. Salubrinal also suppresses inflammatory responses and pathological change in the subchondral bone of the TMJ.

Resting-state functional magnetic resonance imaging (R-fMRI) has been intensively used to assess alterations of inter-regional functional connectivity in patients with stroke, but the regional properties of brain activity in stroke have not yet been fully investigated. Additionally, no study has examined a frequency effect on such regional properties in stroke patients, although this effect has been shown to play important roles in both normal brain functioning and functional abnormalities. Here we utilized R-fMRI to measure the amplitude of low-frequency fluctuations (ALFF) and regional homogeneity (ReHo), two major methods for characterizing the regional properties of R-fMRI, in three different frequency bands (slow-5: 0.01-0.027 Hz; slow-4: 0.027-0.73 Hz; and typical band: 0.01-0.1 Hz) in 19 stroke patients and 15 healthy controls. Both the ALFF and ReHo analyses revealed changes in brain activity in a number of brain regions, particularly the parietal cortex, in stroke patients compared with healthy controls. Remarkably, the regions with changed activity as detected by the slow-5 band data were more extensive, and this finding was true for both the ALFF and ReHo analyses. These results not only confirm previous studies showing abnormality in the parietal cortex in patients with stroke, but also suggest that R-fMRI studies of stroke should take frequency effects into account when measuring intrinsic brain activity.

Although European Society of Urogenital Radiology proposed the potential of multiparametric magnetic resonance imaging (MP-MRI) as a tool in the diagnostic pathway for prostate cancer (PCa) and published a unified scoring system named Prostate Imaging Reporting and Data System (PI-RADS version 1), these still need to be validated by real-life studies.To evaluate the role of MP-MRI in detection and prediction of PCa.Patients with clinical suspicion of PCa who underwent prebiopsy MP-MRI from 2002 to 2009 were recruited. MP-MRI results were retrospectively assigned as overall scores using PI-RADS by two radiologists. Patients were followed and the end point was the diagnosis of PCa. Receiver operating characteristics (ROC) curve was performed to test diagnostic efficacy of MP-MRI, under results of biopsy within three months. The cox proportional hazards model was used to identify independent variables for the detection of PCa.Finally, 1113 of the 1806 enrolled patients were included for analysis. The median follow-up was 56.0 months (1-137 mo). For 582 patients biopsied within three months, area under the curve for the detection of PCa with MP-MRI was 0.88 (95% confidence interval [CI], 0.75-1.00) in group of baseline prostate specific antigen (PSA) 0.01-4.00 ng/ml (n = 31), 0.90 (95% CI, 0.84-0.95) in PSA 4.01-10.00 ng/ml (n = 142), and 0.91 (95% CI, 0.87-0.94) in PSA >10.00 ng/ml (n = 409), respectively. In the cox model adjusted for age and baseline PSA level, for the detection rate of PCa, compared with PI-RADS 1-2 (reference), the hazard ratio was 6.43 (95% CI, 4.29-9.65) for PI-RADS 3, 18.58 (95% CI, 13.36-25.84) for PI-RADS 4-5 (p < 0.001).Prebiopsy MP-MRI with PI-RADS is demonstrated as a valuable diagnostic and predictive tool for PCa.

We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.

Neutrophil plays an essential role in host defense against infection, but uncontrolled neutrophilic infiltration can cause inflammation and severe epithelial damage. We recently showed that CXCR2 formed a signaling complex with NHERF1 and PLC-2, and that the formation of this complex was required for intracellular calcium mobilization and neutrophilic transepithelial migration. To uncover the structural basis of the complex formation, we report here the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal sequence of CXCR2 at 1.16 Å resolution. The structure reveals that the CXCR2 peptide binds to PDZ1 in an extended conformation with the last four residues making specific side chain interactions. Remarkably, comparison of the structure to previously studied PDZ1 domains has allowed the identification of PDZ1 ligand-specific interactions and the mechanisms that govern PDZ1 target selection diversities. In addition, we show that CXCR2 can bind both NHERF1 PDZ1 and PDZ2 in pulldown experiments, consistent with the observation that the peptide binding pockets of these two PDZ domains are highly structurally conserved. The results of this study therefore provide structural basis for the CXCR2-mediated neutrophilic migration and could have important clinical applications in the prevention and treatment of numerous neutrophil-dependent inflammatory disorders.

PURPOSE: The mechanism underlying the decline in milk quality during periods of feeding high-concentrate diets to dairy ruminants is not well documented. The aim of this study was to investigate the metabolic changes in the liver that contribute to the input of substrate precursors to the mammary gland after feeding a high-concentrate diet to lactating goats for a long period. EXPERIMENTAL DESIGN: Eight mid-lactating goats with rumen cannulas were randomly assigned to two groups. For 9 weeks, the treatment group was fed a high-concentrate diet (60% concentrate of dry matter, HC) and the control group was fed a low-concentrate diet (40% concentrate of dry matter, LC). Ruminal fluid, plasma, and liver tissues were sampled, microarray techniques and real-time polymerase chain reaction were used to evaluate metabolic parameters and gene expression in liver. RESULTS: Feeding a 60%-concentrate diet for 9 weeks resulted in a significant decrease in rumen pH. Changes in fat and protein content also occurred, which negatively affected milk quality. Plasma levels of leptin (p = 0.058), non-esterified fatty acid (p = 0.071), and glucose (p = 0.014) increased markedly in HC group. Plasma cortisol concentration was significantly elevated in the treatment group (p

NHERF1 is a PDZ adaptor protein that scaffolds the assembly of diverse signaling complexes and has been implicated in many cancers. However, little is known about the mechanism responsible for its scaffolding promiscuity or its ability to bind to multiple targets. Computational studies have indicated that PDZ promiscuity may be attributed to its conformational dynamics, but experimental evidence for this relationship remains very limited. Here we examine the conformational flexibility of the NHERF1 PDZ1 domain using crystal lattice trapping via solving PDZ1 structure of a new crystal form. The structure, together with prior PDZ1 structures of a different space group, reveals that 4 of 11 ligand-interacting residues undergo significant crystal packing-induced structural changes. Most of these residues correspond to the residues involved in allosteric transition when a peptide ligand binds. In addition, a subtle difference in ligand conformations causes the same peptide to bind in slightly different modes in different crystal forms. These findings indicate that substantial structural flexibility is present in the PDZ1 peptide-binding pocket, and the structural substate trapped in the present crystal form can be utilized to represent the conformational space accessible to the protein. Such knowledge will be critical for drug design against the NHERF1 PDZ1 domain, highlighting the continued need for experimentally determined PDZ1-ligand complexes.

Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER) that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF) physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.

SmyD2 belongs to a new class of chromatin regulators that control gene expression in heart development and tumorigenesis. Besides methylation of histone H3 K4, SmyD2 can methylate non-histone targets including p53 and the retinoblastoma tumor suppressor. The methyltransferase activity of SmyD proteins has been proposed to be regulated by autoinhibition via the intra- and interdomain bending of the conserved C-terminal domain (CTD). However, there has been no direct evidence of a conformational change in the CTD. Here, we report two crystal structures of SmyD2 bound either to the cofactor product S-adenosylhomocysteine or to the inhibitor sinefungin. SmyD2 has a two-lobed structure with the active site located at the bottom of a deep crevice formed between the CTD and the catalytic domain. By extensive engagement with the methyltransferase domain, the CTD stabilizes the autoinhibited conformation of SmyD2 and restricts access to the catalytic site. Unexpectedly, despite that the two SmyD2 structures are highly superimposable, significant differences are observed in the first two helices of the CTDs: the two helices bend outwards and move away from the catalytic domain to generate a less closed conformation in the sinefungin-bound structure. Although the overall fold of the individual domains is structurally conserved among SmyD proteins, SmyD2 appear to be a conformational "intermediate" between a close form of SmyD3 and an open form of SmyD1. In addition, the structures reveal that the CTD is structurally similar to tetratricopeptide repeats (TPR), a motif through which many cochaperones bind to the heat shock protein Hsp90. Our results thus provide the first evidence for the intradomain flexibility of the TPR-like CTD, which may be important for the activation of SmyD proteins by Hsp90.