Conditional, Tissue-Specific CRISPR/Cas9 Vector System in Zebrafish Reveals the Role of Nrp1b in Heart Regeneration.

BACKGROUND: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. Neuropilins play crucial roles in zebrafish heart regeneration, heart failure in mice, and electrical remodeling after myocardial infarction in rats. But the cell-specific functions of nrp1 have not been described before. In this study, we have investigated the role of nrp1 isoforms, including nrp1a and nrp1b, in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts. METHODS: In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cmlc2 promoter drives Cas9 expression to silence the nrp1 gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts. RESULTS: We observed that both the isoforms (nrp1a and nrp1b) are upregulated after the cryoinjury. Interestingly, the nrp1b knockout significantly delayed heart regeneration and impaired cardiac function in the adult zebrafish after cryoinjury, demonstrated by reduced heart rate, ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury. CONCLUSIONS: To our knowledge, this study is novel where we have reported a heat-shock-mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.

Non-destructive sugar content assessment of multiple cultivars of melons by dielectric properties.

BACKGROUND: Non-destructive determination of the internal quality of fruit with a thick rind and of a large size is always difficult and challenging. To investigate the feasibility of the dielectric spectroscopy technique with respect to determining the sugar content of melons during the postharvest stage, three cultivars of melon samples (160 melons for each cultivar) were used to acquire dielectric spectra over the frequency range 20-4500 MHz. The three cultivars of melons were divided separately into a calibration set and a prediction set in a ratio of 3:1 by a joint x-y distance algorithm. Partial least squares (PLS) and extreme learning machine (ELM) methods were applied to develop individual-cultivar and multi-cultivar models based on full frequencies (FFs) and effective dielectric frequencies (EDFs) selected by the successive projection algorithm (SPA). RESULTS: The results showed that ELM models demonstrated a better performance than PLS models for the same input dielectric variables. Most of the models built based on the EDFs selected by SPA had a slightly worse performance compared to those based on FFs. For both PLS and ELM methods, the models for multi-cultivars demonstrated a worse calibration and prediction performance compared to those for individual cultivars. When individual-cultivar and multi-cultivar samples were used to build sugar content determination models, the best model was FFs-ELM (Rp  = 0.887, RMSEP = 0.986), FFs-ELM (Rp  = 0.870, RMSEP = 1.028), FFs-PLS (Rp  = 0.882, RMSEP = 1.010) and FFs-ELM (Rp  = 0.849, RMSEP = 1.085) for 'Hongyanliang', 'Xinzaomi', 'Manao' and multi-cultivar melons, respectively. CONCLUSION: The present study indicates that it is possible to develop both individual-cultivar and multi-cultivar models for determining the sugar content of melons based on the dielectric spectroscopy technique. © 2021 Society of Chemical Industry.

Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2ß.

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2ß to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2ß up-regulation decreases VEGFR-2 expression and that loss of AP2ß enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2ß knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2ß at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2ß nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2ß nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2ß increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.

VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.

Aggregated amyloid ß (Aß) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aß peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aß1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aß oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aß1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aß1-42 monomers, and 5 µM Aß1-42 fibrils, respectively. Brain ECs treated with Aß1-42 oligomers showed increased senescence-associated ß-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aß1-42 monomer or Aß1-42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aß1-42 oligomer-treated ECs, and these cells showed significantly increased VEGFR-1 expression and decreased VEGFR-2 levels. Overexpression of VEGFR-1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR-1 signaling events in this paradigm. More importantly, small interfering RNA-mediated knockdown of VEGFR-1 expression in brain ECs was able to prevent up-regulation of p21 protein expression and significantly reduced induction of senescence following Aß1-42 oligomer treatment. Our studies show that exposure to Aß1-42 oligomers may impair vascular functions by altering VEGFR-1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR-1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.-Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.

Neuropilin-1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II-induced hypertension.

Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin-1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA-mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen-inducible endothelial cell-specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR-219-5p in HUVECs. Although the endothelial cell-specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low-dose angiotensin II infusion-induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low-dose angiotensin II-induced increases in blood pressure.-Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin-1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II-induced hypertension.

Predicting seizure freedom with AED treatment in newly diagnosed patients with MRI-negative epilepsy: A large cohort and multicenter study.

OBJECTIVE: We developed and validated a prediction score for predicting the probability of 6-month and 12-month seizure freedom of antiepileptic drug (AED) treatment in newly diagnosed patients with magnetic resonance imaging (MRI)-negative epilepsy. METHODS: The development cohort included 543 consecutive patients from the Epilepsy Center of Henan Provincial People's Hospital, while the validation cohorts included 493 consecutive patients in two independent cohorts. Univariate analysis and a forward and backward elimination of multivariate Cox regression analysis were used to select predictive factors. The performance of the score was evaluated with C-index, calibration plots, and decision curve analysis. The risk stratification was also performed. RESULTS: The score included five routinely available predictors including Circadian rhythms, Electroencephalography before AED treatment, Neuropsychiatric disorders, Perinatal brain injury, and History of central nervous system infection (CENPH score). When applied to the external validation cohort, the score showed good discrimination with C-index (development group: 0.83; validation group: 0.78), and calibration plots indicated well calibration, as well as the decision curve analysis showed good predictive accuracy and clinical values in four cohorts. The points of the score were categorized to the following three probability levels for predicting seizure freedom: high probability (0-83.11 points), medium probability (83.11-122.71 points), and low probability (>122.71 points). And online calculator was established to make this score easily applicable in clinical practice. CONCLUSIONS: We established a simple, practical, and evidence-based prediction score for predicting seizure freedom with AEDs to aid in the clinical consultation and treatment decision for the newly diagnosed patients with MRI-negative epilepsy.

Mapping tissue pH in an experimental model of acute stroke - Determination of graded regional tissue pH changes with non-invasive quantitative amide proton transfer MRI.

pH-weighted amide proton transfer (APT) MRI is sensitive to tissue pH change during acute ischemia, complementing conventional perfusion and diffusion stroke imaging. However, the currently used pH-weighted magnetization transfer (MT) ratio asymmetry (MTRasym) analysis is of limited pH specificity. To overcome this, MT and relaxation normalized APT (MRAPT) analysis has been developed that to homogenize the background signal, thus providing highly pH conspicuous measurement. Our study aimed to calibrate MRAPT MRI toward absolute tissue pH mapping and determine regional pH changes during acute stroke. Using middle cerebral artery occlusion (MCAO) rats, we performed lactate MR spectroscopy and multi-parametric MRI. MRAPT MRI was calibrated against a region of interest (ROI)-based pH spectroscopy measurement (R2 = 0.70, P < 0.001), showing noticeably higher correlation coefficient than the simplistic MTRasym index. Capitalizing on this, we mapped brain tissue pH and semi-automatically segmented pH lesion, in addition to routine perfusion and diffusion lesions. Tissue pH from regions of the contralateral normal, perfusion/diffusion lesion mismatch and diffusion lesion was found to be 7.03 ±â€¯0.04, 6.84 ±â€¯0.10, 6.52 ±â€¯0.19, respectively. Most importantly, we delineated the heterogeneous perfusion/diffusion lesion mismatch into perfusion/pH and pH/diffusion lesion mismatches, with their pH being 7.01 ±â€¯0.04 and 6.71 ±â€¯0.12, respectively (P < 0.05). To summarize, our study calibrated pH-sensitive MRAPT MRI toward absolute tissue pH mapping, semi-automatically segmented and determined graded tissue pH changes in ischemic tissue and demonstrated its feasibility for refined demarcation of heterogeneous metabolic disruption following acute stroke.

Determination of multipool contributions to endogenous amide proton transfer effects in global ischemia with high spectral resolution in vivo chemical exchange saturation transfer MRI.

PURPOSE: Chemical exchange saturation transfer (CEST) MRI has been used for quantitative assessment of dilute metabolites and/or pH in disorders such as acute stroke and tumor. However, routine asymmetry analysis (MTRasym ) may be confounded by concomitant effects such as semisolid macromolecular magnetization transfer (MT) and nuclear Overhauser enhancement. Resolving multiple contributions is essential for elucidating the origins of in vivo CEST contrast. METHODS: Here we used a newly proposed image downsampling expedited adaptive least-squares fitting on densely sampled Z-spectrum to quantify multipool contribution from water, nuclear Overhauser enhancement, MT, guanidinium, amine, and amide protons in adult male Wistar rats before and after global ischemia. RESULTS: Our results revealed the major contributors to in vivo T1 -normalized MTRasym (3.5 ppm) contrast between white and gray matter (WM/GM) in normal brain (-1.96%/second) are pH-insensitive macromolecular MT (-0.89%/second) and nuclear Overhauser enhancement (-1.04%/second). Additionally, global ischemia resulted in significant changes of MTRasym , being -2.05%/second and -1.56%/second in WM and GM, which are dominated by changes in amide (-1.05%/second, -1.14%/second) and MT (-0.88%/second, -0.62%/second). Notably, the pH-sensitive amine and amide effects account for nearly 60% and 80% of the MTRasym changes seen in WM and GM, respectively, after global ischemia, indicating that MTRasym is predominantly pH-sensitive. CONCLUSION: Combined amide and amine effects dominated the MTRasym changes after global ischemia, indicating that MTRasym is predominantly pH-sensitive and suitable for detecting tissue acidosis following acute stroke.

Design and Evaluation of PEGylated Liposomal Formulation of a Novel Multikinase Inhibitor for Enhanced Chemosensitivity and Inhibition of Metastatic Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates among cancers. Chemotherapy is the standard first-line treatment, but only modest survival benefits are observed. With the advent of targeted therapies, epidermal growth factor receptor (EGFR) has been acknowledged as a prospective target in PDAC since it is overexpressed in up to 60% of cases. Similarly, the tyrosine-protein kinase Met (cMET) is also overexpressed in PDAC (27-60%) and is a prognostic marker for poor survival. Interestingly, EGFR and cMET share some common signaling pathways including PI3K/Akt and MAPK pathways. Small molecule inhibitors or bispecific antibodies that can target both EGFR and cMET are therefore emerging as novel options for cancer therapy. We previously developed a dual EGFR and cMET inhibitor (N19) that was able to inhibit tumor growth in nonsmall cell lung cancer models resistant to EGFR tyrosine kinase inhibitors (TKI). Here, we report the development of a novel liposomal formulation of N19 (LN19) and showed significant growth inhibition and increased sensitivity toward gemcitabine in the pancreatic adenocarcinoma orthotopic xenograft model. Taken together, our results suggest that LN19 can be valued as an effective combination therapy with conventional chemotherapy such as gemcitabine for PDAC patients.

Tumor selective uptake of drug-nanodiamond complexes improves therapeutic outcome in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.

Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells.

Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1-CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1-IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.

Direct saturation-corrected chemical exchange saturation transfer MRI of glioma: Simplified decoupling of amide proton transfer and nuclear overhauser effect contrasts.

PURPOSE: Chemical exchange saturation transfer (CEST) MRI has shown promise in tissue characterization in diseases like stroke and tumor. However, in vivo CEST imaging such as amide proton transfer (APT) MRI is challenging because of concomitant factors such as direct water saturation, macromolecular magnetization transfer, and nuclear overhauser effect (NOE), which lead to a complex contrast in the commonly used asymmetry analysis (MTRasym). Here, we propose a direct saturation-corrected CEST (DISC-CEST) analysis for simplified decoupling and quantification of in vivo CEST effects. METHODS: CEST MRI and relaxation measurements were carried out on a classical 2-pool creatine-gel CEST phantom and normal rat brains (N = 6) and a rat model of glioma (N = 8) at 4.7T. The proposed DISC-CEST quantification was carried out and compared with conventional MTRasym and the original three-offset method. RESULTS: We demonstrated that the DISC-CEST contrast in the phantom had much stronger correlation with MTRasym than the three-offset method, which showed substantial underestimation. In normal rat brains, the DISC-CEST approach revealed significantly stronger APT effect in gray matter and higher NOE effect in white matter. Furthermore, the APT and NOE maps derived from DISC-CEST showed significantly higher APT effect in the tumors than contralateral normal tissue but no apparent difference in NOE. CONCLUSION: The proposed DISC-CEST method, by correction of nonlinear direct water saturation effect, serves as a promising alternative to both the commonly used MTRasym and the simplistic three-offset analyses. It provides simple yet reliable in vivo CEST quantification such as APT and NOE mapping in brain tumor, which is promising for clinical translation. Magn Reson Med 78:2307-2314, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Glucocorticoid Receptor-Targeted Liposomal Codelivery of Lipophilic Drug and Anti-Hsp90 Gene: Strategy to Induce Drug-Sensitivity, EMT-Reversal, and Reduced Malignancy in Aggressive Tumors.

Many cancers including the late stage ones become drug-resistant and undergo epithelial-to-mesenchymal transition (EMT). These lead to enhanced invasion, migration, and metastasis toward manifesting its aggressiveness and malignancy. One of the key hallmarks of cancer is its overdependence on glycolysis as its preferred energy metabolism pathway. The strict avoidance of alternate energy pathway gluconeogenesis by cancer cells points to a yet-to-be hoisted role of glucocorticoid receptor (GR) especially in tumor microenvironment, where cells are known to become drug-sensitive through induction of gluconeogenesis. However, since GR is involved in metabolism, anti-inflammatory reactions, immunity besides inducing gluconeogenesis, a greater role of GR in tumor microenvironment is envisaged. We have shown previously that GR, although ubiquitously expressed in all cells; afford to be an effective cytoplasmic target for killing cancer cells selectively. Herein, we report the therapeutic use of a newly developed GR-targeted liposomal concoction (DXE) coformulating a lipophilic drug (ESC8) and an anti-Hsp90 anticancer gene against aggressive tumor models. This induced drug-sensitivity and apoptosis while reversing EMT in tumor cells toward effective retardation of aggressive growth in pancreas and skin tumor models. Additionally, the ESC8-free lipid formulation upon cotreatment with hydrophilic drugs, gemcitabine and doxorubicin, could effectively sensitize and kill pancreatic cancer and melanoma cells, respectively. The formulation-triggered EMT-reversal was GR-dependent. Overall, we found a new strategy for drug sensitization that led to the advent of new GR-targeted anticancer therapeutics.

Cardiomyopathy and Worsened Ischemic Heart Failure in SM22-α Cre-Mediated Neuropilin-1 Null Mice: Dysregulation of PGC1α and Mitochondrial Homeostasis.

OBJECTIVE: Neuropilin-1 (NRP-1) is a multidomain membrane receptor involved in angiogenesis and development of neuronal circuits, however, the role of NRP-1 in cardiovascular pathophysiology remains elusive. APPROACH AND RESULTS: In this study, we first observed that deletion of NRP-1 induced peroxisome proliferator-activated receptor γ coactivator 1α in cardiomyocytes and vascular smooth muscle cells, which was accompanied by dysregulated cardiac mitochondrial accumulation and induction of cardiac hypertrophy- and stress-related markers. To investigate the role of NRP-1 in vivo, we generated mice lacking Nrp-1 in cardiomyocytes and vascular smooth muscle cells (SM22-α-Nrp-1 KO), which exhibited decreased survival rates, developed cardiomyopathy, and aggravated ischemia-induced heart failure. Mechanistically, we found that NRP-1 specifically controls peroxisome proliferator-activated receptor γ coactivator 1 α and peroxisome proliferator-activated receptor γ in cardiomyocytes through crosstalk with Notch1 and Smad2 signaling pathways, respectively. Moreover, SM22-α-Nrp-1 KO mice exhibited impaired physical activities and altered metabolite levels in serum, liver, and adipose tissues, as demonstrated by global metabolic profiling analysis. CONCLUSIONS: Our findings provide new insights into the cardioprotective role of NRP-1 and its influence on global metabolism.

Abnormal Neural Network of Primary Insomnia: Evidence from Spatial Working Memory Task fMRI.

BACKGROUND: Contemporary functional MRI (fMRI) methods can provide a wealth of information about the neural mechanisms associated with primary insomnia (PI), which centrally involve neural network circuits related to spatial working memory. METHODS: A total of 30 participants diagnosed with PI and without atypical brain anatomy were selected along with 30 age- and gender-matched healthy controls. Subjects were administered the Pittsburgh Sleep Quality Index (PSQI), Hamilton Rating Scale for Depression and clinical assessments of spatial working memory, followed by an MRI scan and fMRI in spatial memory task state. RESULTS: Statistically significant differences between PSQI and spatial working memory were observed between PI patients and controls (p < 0.01). Activation of neural networks related to spatial memory task state in the PI group was observed at the left temporal lobe, left occipital lobe and right frontal lobe. Lower levels of activation were observed in the left parahippocampal gyrus, right parahippocampal gyrus, bilateral temporal cortex, frontal cortex and superior parietal lobule. CONCLUSION: Participants with PI exhibited characteristic abnormalities in the neural network connectivity related to spatial working memory. These results may be indicative of an underlying pathological mechanism related to spatial working memory deterioration in PI, analogous to recently described mechanisms in other mental health disorders.

Inhibition of tumor growth and metastasis by a self-therapeutic nanoparticle.

Although biomedical applications of nanotechnology, which typically involve functionalized nanoparticles, have taken significant strides, biological characterization of unmodified nanoparticles remains underinvestigated. Herein we demonstrate that unmodified gold nanoparticles (AuNPs) inhibit the proliferation of cancer cells in a size- and concentration-dependent manner by abrogating MAPK-signaling. In addition, these AuNPs reverse epithelial-mesenchymal transition (EMT) in cancer cells by reducing secretion of a number of proteins involved in EMT, up-regulating E-Cadherin, and down-regulating Snail, N-Cadherin, and Vimentin. Inhibition of MAPK signaling and reversal of EMT upon AuNP treatment inhibits tumor growth and metastasis in two separate orthotopic models of ovarian cancer. Western blot analyses of tumor tissues reveal up-regulation of E-Cadherin and down-regulation of Snail and phospho-MAPK, confirming the reversal of EMT and inhibition of MAPK signaling upon AuNP treatment. The ability of a single self-therapeutic nanoparticle to abrogate signaling cascades of multiple growth factors is distinctive and purports possible medical applications as potential antitumor and antimetastatic agent.

[Application of spatial working memory task fMRI in evaluation of primary insomnia patient's cognitive dysfunction].

OBJECTIVE: To explore abnormal brain activation of spatial working memory in primary insomnia and its potential neuromechanism. METHODS: we recruited 30 cases primary insomnia (PI) patients and 30 cases age, gender matched healthy control (HC) subjects from July 2013 to December 2013, the diagnosis of primary insomnia matched the diagnosis criterion of DSM-IV and Classification and diagnostic criteria of mental disorders in China third edition (CCMD-3). All the subjects attended the tests of PSQI, HAMA, HAMD and index of spatial working memory. And then, we collected the data of routine MRI and spatial working memory task fMRI on 3.0 T MRI scanner. After that, we used SPM8 and REST1.8 to analyze the fMRI data, compared difference of PSQI, HAMA, HAMD, index of spatial working memory and brain activation of spatial working memory between PI group and HC group. RESULTS: There were significant difference between PI group and HC group in PSQI, HAMA, HAMD and index of spatial working memory (P < 0.05). In the spatial working memory related activate brain region, compared with HC group, left temporal lobe, occipital lobe and right frontal lobe activation increased and bilateral parahippocampalis, temporal cortex, frontal cortex and superior parietal lobule activation reduced in PI group. CONCLUSION: Spatial working memory task fMRI revealed the pathological mechanisms of cognitive dysfunction of clinical spatial working memory and emotional disorder in primary insomnia patients.

NHERF-2 maintains endothelial homeostasis.

The Na(+)/H(+) exchanger regulatory factor-2 (NHERF-2) is an integral component of almost all endothelial cells (ECs), yet its endothelial function is not known. Here, we found that NHERF-2, is a key regulator of endothelial homeostasis because NHERF-2-silenced ECs proliferate at a much higher rate even in the absence of mitogens such as VEGF compared with control ECs. We further show that the hyperproliferation phenotype of NHERF-2-silenced EC is because of an accelerated cell cycle that is probably caused by a combination of the following factors: increased cytoplasmic calcium, increased expression of c-Myc, increased expression of cyclin D1, and reduced expression of p27. Using an experimental mouse model of human hemangioma, we found that the endothelial neoplasms derived from NHERF-2-silenced cells were much larger in volume than those derived from control cells. Thus, NHERF-2 is a negative regulator of endothelial proliferation and may have important roles in endothelial homeostasis and vascular modeling.

[Default mode network in childhood absence epilepsy by 3.0T magnetic resonance imaging].

OBJECTIVE: To explore the default mode network (DMN) in childhood absence epilepsy (CAE) patients and examine their correlations between functional connectivity (FC) and clinical characteristics. METHODS: Fourteen CAE patients and 14 healthy volunteers were prospectively recruited from Henan Provincial People's Hospital from September 2012 to June 2014. FC in DMN of each group, between-group comparison of DMN FC and their relationships with clinical characteristics were respectively analyzed with 3.0T resting-state functional magnetic resonance imaging (fMRI) FC analysis seeding at bilateral precuneus/posterior cingulate cortex (PCC). RESULTS: Seeding at bilateral precuneus/posterior cingulate cortex (PCC), positive connection was found in bilateral angular gyrus, bilateral superior parietal gyrus, bilateral superior and middle frontal gyrus, bilateral superior medial frontal gyrus, bilateral middle temporal gyrus and bilateral superior and middle occipital gyrus in controls. However, positive connection in CAE patients was observed in bilateral superior parietal gyrus and bilateral superior occipital gyrus. Between-group analysis of DMN connectivity revealed a reduction of DMN FC in bilateral medial orbitofrontal cortex, bilateral anterior cingulate cortex, bilateral superior frontal gyrus, bilateral middle frontal gyrus and left caudate in CAE patients. Moreover, increased DMN FC was present in right paracentral lobule and right middle cingulate gyrus. FC between PCC and bilateral medial orbitofrontal cortex or bilateral superior/middle frontal gyrus correlated negatively with disease duration, but there was no correlation with seizure frequency or initial age. CONCLUSION: Brain's default mode network in childhood absence epilepsy is impaired, presumably, as a result of unconsciousness and cognitive impairment during absence seizure. Abnormal DMN activities may be a biomaker of disease progress in absence epilepsy.

Surface Chemistry of Gold Nanoparticles Modulates Cytokines and Nanomechanical Properties in Pancreatic Cancer Cell Lines: A Correlative Study.

Surface chemistry of nanoparticles play significant role in their cellular interaction. Along with other group, we previously demonstrated that dynamic alteration of cell membrane during uptake of gold nanoparticles can be thoroughly probed by nanomechanical properties of cell membrane. Additionally, endocytosis influences intracellular cytokines expression that also impact membrane stiffness. Hence, we have hypothesized that surface chemistry of gold nanoparticles influences intracellular cytokines which in turn imparts dynamic alteration of nanomechanical properties of cellular membrane of pancreatic cancer cells. Various gold nanoparticles decorated with targeting peptide, polyethylene glycol or their combinations have been used to treat two pancreatic cancer cell lines, Panc-1 and AsPC1, for 1 and 24 hours. Atomic force microscope is used to measure linear and nonlinear nanomechanical properties of cell membrane. Intracellular cytokine has been measured using real time polymeric chain reaction. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and different timepoints, to deduce correlations between cytokines and nanomechanical attributes. We have identified unique relationship pro-tumorigenic cytokines with both linear and non-linear nanomechanical properties of Panc-1 and AsPC1 cell membrane during uptake of pristine gold nanoparticles or for PEGylation and for targeting peptide conjugation at the nanoparticle surface.