ALBERT

Description: World Health Organization reports that methicillin-resistant Staphylococcus aureus (MRSA) is the origin of higher proportion of hospital acquired infections. In order to combat the effect of MRSA infection, an ideal drug should stimulate the allosteric exposure of active site, prompting penicillin binding proteins (PBP2a) to bind with that particular compound. Ceftaroline shows high binding affinity towards PBP2a and also confers resistance against degrading enzymes. Recently, two amino acid alterations in the allosteric site of PBP2a, asparagine (N) to lysine (K) at position 146 and glutamic acid (E) to lysine at position 150 are reported to confer resistance against ceftaroline resulting in the rise of ceftaroline-resistant MRSA strains. The present study focuses on the identification of potential ligands that can effectively bind with allosteric site of PBP2a, that leads to the access of active site and entry of a β-lactam antibiotic for effective inhibition. The results obtained from our study will be useful for designing effective compounds with potential therapeutic effects against ceftaroline resistant MRSA strains. This article is protected by copyright. All rights reserved

Description: The human protein kinase X gene (PRKX) and cAMP-dependent protein kinase (PKA) are both c-AMP-dependent serine/threonine protein kinases within the protein kinase AGC subgroup. Of all the protein kinases in this group, PRKX is the least studied. PRKX has been isolated from patients with chondrodysplasia punctate and is involved in numerous processes, including sexual differentiation and fertilization, normal kidney development, and autosomal dominant polycystic kidney disease (ADPKD), blood maturation, neural development and angiogenesis in vitro. Although the role of PRKX in development and disease has been reported recently, the underlying mechanism of PRKX activity is largely unknown. In addition, based on the expression pattern of PRKX and the extensive role of PKA in disease and development, PRKX might have additional crucial functions that have not been addressed in the literature. In this review, we summarize the characteristics and developmental functions of PRKX that have been reported by recent studies. In particular, we elucidate the structural and functional differences between PRKX and PKA, as well as the possible roles of PRKX in development and related diseases. Finally, we propose future studies that could lead to important discoveries of more PRKX functions and the underlying mechanisms involved. This article is protected by copyright. All rights reserved

Description: Several key transcription factors regulate cell growth, survival, and differentiation during neural crest and melanoblast development in the embryo, and these same pathways may be reactivated in tumors arising from the progenitors of these cells. The transcription factors PAX3 and FOXD3 have essential roles in melanoblasts and melanoma. In this study, we define a regulatory pathway where FOXD3 promotes the expression of PAX3. Both factors are expressed in melanoma cells and there is a positive correlation between the transcript levels of PAX3 and FOXD3. The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. FOXD3 binds to both of these motifs in vitro but only one of these sites is preferentially utilized in melanoma cells. Overexpression of FOXD3 upregulates PAX3 levels while inhibition of FOXD3 function does not alter PAX3 protein levels, supporting that FOXD3 is sufficient but not necessary to drive PAX3 expression in melanoma cells. Here, we identify a molecular pathway where FOXD3 upregulates PAX3 expression and therefore contributes to melanoma progression. This article is protected by copyright. All rights reserved

Description: Metabolic networks are significantly altered in neoplastic cells. This altered metabolic program leads to increased glycolysis and lipogenesis and decreased dependence on oxidative phosphorylation and oxygen consumption. Despite their limited mitochondrial respiration, cancer cells, nonetheless, derive sufficient energy from alternative carbon sources and metabolic pathways to maintain cell proliferation. They do so, in part, by utilizing fatty acids, amino acids, ketone bodies and acetate, in addition to glucose. The alternative pathways used in the metabolism of these carbon sources provide opportunities for therapeutic manipulation. Acetate, in particular, has garnered increased attention in the context of cancer as both an epigenetic regulator of posttranslational protein modification, and as a carbon source for cancer cell biomass accumulation. However, to date, the data have not provided a clear understanding of the precise roles that protein acetylation and acetate oxidation play in carcinogenesis, cancer progression or treatment. This review highlights some of the major issues, discrepancies and opportunities associated with the manipulation of acetate metabolism and acetylation-based signaling in cancer development and treatment. This article is protected by copyright. All rights reserved

Description: Adipogenesis comprises a complex network of signaling pathways and transcriptional cascades; the GSK3β-C/EBPβ- srebf1a axis is a critical signaling pathway at early stages leading to the expression of PPARγ2, the master regulator of adipose differentiation. Previous work has demonstrated that retinoic acid inhibits adipogenesis affecting different signaling pathways. Here, we evaluated the anti-adipogenic effect of retinoic acid on the adipogenic transcriptional cascade, and the expression of adipogenic genes cebpb , srebf1a , srebf1c , pparg2 , and cebpa . Our results demonstrate that retinoic acid blocks adipose differentiation during commitment, returning cells to an apparent non-committed state, since they have to be newly induced to adipose conversion after the retinoid is removed from the culture medium. Retinoic acid down regulates the expression of the adipogenic genes, srebf1a, srebf1c , pparg2 , and cebpa . Retinoic acid did not down regulate the expression of cebpb , but it inhibited C/EBPβ phosphorylation at Thr188, a critical step for the progression of the adipogenic program. We also found that RA inhibition of adipogenesis did not increase the expression of dlk1 , the gene encoding for Pref1, a well-known anti-adipogenic transcription factor. This article is protected by copyright. All rights reserved

Description: ABSTRACT Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized, however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF + MVs) and that platelet-associated TF enhances thrombus formation at sites of vascular damage. Here we investigate the mechanisms implied in the interactions of TF + MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF + MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3-kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF + MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb-IIIa (GPIIb-IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF + MVs. Ultrastructural techniques revealed that uptake of TF + MVs was efficiently prevented by anti-CD36 and SERT inhibitor, but only moderately interfered by GPIIb-IIIa blockade. We conclude that internalization of TF + MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb-IIIa), involves the scavenger receptor CD36, SERT and engages PI3-Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF + MVs with platelets and possibly downregulate their prothrombotic phenotype. This article is protected by copyright. All rights reserved

Description: Human pancreatic and prostate cancers metastasize along nerve axons during perineural invasion. The extracellular matrix laminin class of proteins is an abundant component of both myelinated and non-myelinated nerves. Analysis of human pancreatic and prostate tissue revealed both perineural and endoneural invasion with Schwann cells surrounded or disrupted by tumor, respectively. Tumor and nerve cell co-culture conditions were used to determine if myelinating or non-myelinating Schwann cell (S16 and S16Y, respectively) phenotype was equally likely to promote integrin-dependent cancer cell invasion and migration on laminin. Conditioned medium from S16 cells increased tumor cell (DU145, PC3, and CFPAC1) invasion into laminin approximately 1.3 to 2.0 fold compared to fetal bovine serum (FBS) treated cells. Integrin function (e.g., ITGA6p formation) increased up to 1.5 fold in prostate (DU145, PC3, RWPE-1) and pancreatic (CFPAC1) cells, and invasion was dependent on ITGA6p formation and ITGB1 as determined by function-blocking antibodies. In contrast, conditioned medium isolated from S16Y cells (non-myelinating phenotype) decreased constitutive levels of ITGA6p in the tumor cells by 50% compared to untreated cells and decreased ITGA6p formation 3.0 fold compared to S16 treated cells. Flow cytometry and western blot analysis revealed loss of ITGA6p formation as reversible and independent of overall loss of ITGA6 expression. These results suggest that the myelinating phenotype of Schwann cells within the tumor microenvironment increased integrin-dependent tumor invasion on laminin. This article is protected by copyright. All rights reserved

Description: Profilin (Pfn1) regulates cytoskeletal reorganization and migration, but its role in osteoblasts is not known. BMPs (bone morphogenetic proteins) aree a multifunctional cytokine involved in osteoblastic differentiation and promote bone regeneration and repair. Although several molecules are known to modulate BMP signaling, mechanisms that determine the levels of BMP action in osteoblastic function are still incompletely understood. We therefore examine the expression of Pfn1 in osteoblasts and its role in BMP-induced differentiation in osteoblasts. In osteoblastic MC3T3-E1(MC) cells, Pfn1 mRNA is expressed constitutively and its expression levels are declined during the culture in a time dependent manner in contrast to the increase in alkaline phosphatase activity revealing that Pfn1 expression is down regulated along with differentiation. To test the effects of osteoblastic differentiation on Pfn1expression further, MC cells are treated with BMP. BMP treatment suppresses the levels of Pfn1 mRNA. This suppressive effect of BMP is time dependent and further down regulation of Pfn1 mRNA levels is observed when the BMP treatment is continued for a longer period of time. Pfn1mRNA knock down (KD) by siRNAs enhances BMP-induced increase in alkaline phosphatase (Alp) activity in MC cells. To analyze the regulatory mechanism, Alp mRNA levels are examined and Pfn1 KD enhances the BMP-induced increase in the levels of Alp mRNA expression. Furthermore, Pfn1 KD enhances BMP-induced transcriptional expression of luciferase reporter activity via BMP response element in osteoblasts. These data indicate that Pfn1 is a novel target of BMP and suppresses BMP-induced differentiation of osteoblasts at least in part via transcriptional event. This article is protected by copyright. All rights reserved

Description: One of the major features of neurodegenerative disease is the selective vulnerability of different neuronal populations that are affected in a progressive and often stereotyped manner. Despite the susceptible neuronal population varies between diseases, oxidative stress is implicated as the major pathogenic process in all of them. Natural Extract of Castanea sativa Mill . bark (ENC), recently characterized in its phenolic composition, acts as antioxidant and cardioprotective agent. Its neuroprotettive properties, however, have never been investigated. The aim of this study was to assess neuroprotection of ENC in in vitro models of oxidative-stress-mediate injury. Human neuroblastoma SH-SY5Y cells treated with glutamate (50 mM for 24h) or hydrogen peroxide (25 µM for 1h followed by 24 with medium) were used. The results showed that the addition of ENC (1-50 µg/ml) to cell medium before the neuronal damage provided neuroprotection in both experimental models used, while its addition after the injury was ineffective. In conclusion, the present results suggest that ENC could be a valuable support as dietary supplement, combining beneficial preventive neuroprotettive effects with a high antioxidant activity. This article is protected by copyright. All rights reserved

Description: PKR-like ER-resident kinase (PERK) phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α) under endoplasmic reticulum (ER) stress; this results in repression of general translation and induction of specific gene expression, such as activating transcription factor 4 (ATF4). We previously showed that, upon ER stress, transducin (beta)-like 2 (TBL2) was an ER-localized transmembrane protein and interacted with PERK and that TBL2 was involved in ATF4 expression and cell survival. Here, we show that TBL2 is able to associate with ATF4 mRNA and regulate its translation. The RNA-immunoprecipitation analysis using several TBL2 deletion mutants revealed that the WD40 domain was essential for association with ATF4 mRNA. Importantly, suppression of TBL2 by knockdown or overexpression of the TBL2 mutant with a defective WD40 domain diminished ATF4 induction at the translational level. Thus, our findings indicate that, under ER stress, TBL2 participates in ATF4 translation through its association with the mRNA. This article is protected by copyright. All rights reserved

Description: Toll-like receptor 2 (TLR2)-mediated signaling cascades and gene regulation are mainly involved in diseases such as immunity and inflammation. In this study microarray analysis was performed using bone marrow-derived macrophages (BMDM) and Raw 264.7 cells to identify novel proteins involved in the TLR2-mediated cellular response. We found that pleckstrin homology-like domain family, member 1 (PHLDA1) is a novel gene up-regulated by TLR2 stimulation and determined the unique signaling pathway for its expression. Treatment with TLR2 agonist Pam 3 CSK 4 increased mRNA, protein, and fluorescence staining of PHLDA1. Induction of PHLDA1 by TLR2 stimulation disappeared from TLR2 KO mice-derived BMDM. Among janus kinase (JAK) family members, JAK2 was involved in TLR2-stimulated PHLDA1 expression. Signal transducer and activator of transcription 3 (STAT3) also participated in PHLDA1 expression downstream of the JAK2. Interestingly, ERK1/2 was an intermediate between JAK2 and STAT3. In silico analysis revealed the presence of highly conserved γ-activated sites within mouse PHLDA1 promoter and confirmed the JAK2-STAT3 pathway is important to Pam 3 CSK 4 -induced PHLDA1 transcription. These findings suggest that the JAK2-ERK1/2-STAT3 pathway is an important signaling pathway for PHLDA1 expression and that these proteins may play a critical role in eliciting TLR2-mediated immune and inflammatory response. This article is protected by copyright. All rights reserved

Description: Autophagy is a catabolic cellular mechanism involving lysosomal degradation of unwanted cellular components. Interaction between Beclin-1 and Bcl-2 proteins is known to play a critical role in the initiation of autophagy. We report that malignantly transformed lung epithelial cells are resistant to autophagy, and express lower basal levels of autophagic proteins Beclin-1 and LC3-II as compared to non-tumorigenic cells. Additionally, increased levels of nitric oxide (NO) and Bcl-2 were observed in transformed cells. NO was found to negatively regulate autophagy initiation and autophagic flux by nitrosylating Bcl-2 and stabilizing its interaction with Beclin-1, resulting in inhibition of Beclin-1 activity. An increase in the apoptotic initiator caspase-9 and the apoptosis and autophagy-associated kinase p38/MAPK in both cell types indicated possible autophagy-apoptosis crosstalk. Pre-treatments with ABT-737 (Bcl-2 inhibitor) and aminoguanidine (NO inhibitor), and transfection with a non-nitrosylable Bcl-2 cysteine double-mutant plasmid resulted in increased autophagic flux (LC3-II/p62 upregulation) corresponding with decreased S -nitrocysteine expression, thus corroborating the regulatory role of Bcl-2 S -nitrosylation in autophagy. In conclusion, our study reveals a novel mechanism of autophagy resistance via post-translational modification of Bcl-2 protein by NO, which may be critical in driving cellular tumorigenesis. This article is protected by copyright. All rights reserved

Description: Accurately recovering the hippocampal shapes against rough and noisy segmentations is as challenging as achieving good anatomical correspondence between the individual shapes. To address these issues, we propose a mesh-to-volume registration approach, characterized by a progressive model deformation. Our model implements flexible weighting scheme for model rigidity under a multi-level neighborhood for vertex connectivity. This method induces a large-to-small scale deformation of a template surface to build the pairwise correspondence by minimizing geometric distortion while robustly restoring the individuals' shape characteristics. We evaluated the proposed method's 1) accuracy and robustness in smooth surface reconstruction, 2) sensitivity in detecting significant shape differences between healthy control and disease groups (mild cognitive impairment and Alzheimer's disease), 3) robustness in constructing the anatomical correspondence between individual shape models, and 4) applicability in identifying subtle shape changes in relation to cognitive abilities in a healthy population. We compared the performance of the proposed method with other well-known methods—SPHARM-PDM, ShapeWorks and LDDMM volume registration with template injection—using various metrics of shape similarity, surface roughness, volume, and shape deformity. The experimental results showed that the proposed method generated smooth surfaces with less volume differences and better shape similarity to input volumes than others. The statistical analyses with clinical variables also showed that it was sensitive in detecting subtle shape changes of hippocampus.

Description: We propose a conditional random field (CRF) based classifier for segmentation of small enhanced pathologies. Specifically, we develop a temporal hierarchical adaptive texture CRF (THAT-CRF) and apply it to the challenging problem of gad enhancing lesion segmentation in brain MRI of patients with multiple sclerosis. In this context, the presence of many nonlesion enhancements (such as blood vessels) renders the problem more difficult. In addition to voxel-wise features, the framework exploits multiple higher order textures to discriminate the true lesional enhancements from the pool of other enhancements. Since lesional enhancements show more variation over time as compared to the nonlesional ones, we incorporate temporal texture analysis in order to study the textures of enhanced candidates over time. The parameters of the THAT-CRF model are learned based on 2380 scans from a multi-center clinical trial. The effect of different components of the model is extensively evaluated on 120 scans from a separate multi-center clinical trial. The incorporation of the temporal textures results in a general decrease of the false discovery rate. Specifically, THAT-CRF achieves overall sensitivity of 95% along with false discovery rate of 20% and average false positive count of 0.5 lesions per scan. The sensitivity of the temporal method to the trained time interval is further investigated on five different intervals of 69 patients. Moreover, superior performance is achieved by the reviewed labelings of our model compared to the fully manual labeling when applied to the context of separating different treatment arms in a real clinical trial.

Description: We present a novel general-purpose compression method for tomographic images, termed 3D adaptive sparse representation based compression (3D-ASRC). In this paper, we focus on applications of 3D-ASRC for the compression of ophthalmic 3D optical coherence tomography (OCT) images. The 3D-ASRC algorithm exploits correlations among adjacent OCT images to improve compression performance, yet is sensitive to preserving their differences. Due to the inherent denoising mechanism of the sparsity based 3D-ASRC, the quality of the compressed images are often better than the raw images they are based on. Experiments on clinical-grade retinal OCT images demonstrate the superiority of the proposed 3D-ASRC over other well-known compression methods.

Description: Accurate segmentation is usually crucial in transrectal ultrasound (TRUS) image based prostate diagnosis; however, it is always hampered by heavy speckles. Contrary to the traditional view that speckles are adverse to segmentation, we exploit intrinsic properties induced by speckles to facilitate the task, based on the observations that sizes and orientations of speckles provide salient cues to determine the prostate boundary. Since the speckle orientation changes in accordance with a statistical prior rule, rotation-invariant texture feature is extracted along the orientations revealed by the rule. To address the problem of feature changes due to different speckle sizes, TRUS images are split into several arc-like strips. In each strip, every individual feature vector is sparsely represented, and representation residuals are obtained. The residuals, along with the spatial coherence inherited from biological tissues, are combined to segment the prostate preliminarily via graph cuts. After that, the segmentation is fine-tuned by a novel level sets model, which integrates 1) the prostate shape prior, 2) dark-to-light intensity transition near the prostate boundary, and 3) the texture feature just obtained. The proposed method is validated on two 2-D image datasets obtained from two different sonographic imaging systems, with the mean absolute distance on the mid gland images only $1.06pm 0.53~{hbox {mm}}$ and $1.25pm 0.77~{hbox {mm}}$ , respectively. The method is also extended to segment apex and base images, producing competitive results over the state of the art.

Description: Although the mechanism which regulates transcription in the 5'-UTR of the mu opioid receptor (MOR) gene (OPRM1) in lymphocytes has been well studied, a question remains as to whether there is post-transcriptional regulation of MOR gene OPRM1 gene in lymphocytes. In this study, we describe both the role played by miRNAs and the impact of SIVmac239 infection on post-transcriptional regulation of MOR gene OPRM1 gene in CEM x174 cells. Our results show that miR-16 is able to bind the target site in the range of 8699-8719 nt from the stop coden in MOR gene MOR-1 mRNA 3'-UTR and suppress the expression of MOR OPRM1 gene. Mutation of this target site reduces the effect of miR-16. Morphine (1 µM) inhibites the expression of miR-16, and this effect is reversed by the antagonist naloxone. Thus, morphine may up-regulate MOR receptor level by both stimulating MOR OPRM1 gene transcription and stabilizing its mRNA. SIVmac239 infection results in an apparent elevation of miR-16 and gradual reduction of MOR OPRM1 gene expression. The inverse correlation of elevated miR-16 and reduced MOR OPRM1 gene expression under viral loading confirmed the effect of SIVmac239 on post-transcriptional regulation of MOR OPRM1 gene in lymphocytes. We conclude that miR-16 is a primary factor in post-transcriptional regulation of MOR OPRM1 gene. SIVmac239 upregulates miR-16 levels and consequently suppresses MOR OPRM1 gene expression. This finding will be helpful for full understanding of the regulatory mechanism of MOR OPRM1 gene in lymphocytes, as well as the synergistic mechanism of HIV infection and morphine addiction in the pathogenesis of AIDS.

Description: Structural stability of Oldenlandia affinis cyclotide, kalata B1 of native (1NB1) and two mutants 2F2I ([P20D, V21K] kB1) and 2F2J ([W19K, P20N, V21K] kB1) was investigated. Single model analysis showed high number of intra-molecular interactions followed by more proportion of beta sheet contents in [P20D, V21K] kB1 as compared to that of native and the other mutant of kalata B1. Further, the modern conformational sampling approach, an alternate to classical molecular dynamics was introduced, which revealed that the [P20D, V21K] kB1 was identified as structurally stable one, substantiated by various structural events viz., root mean square deviation, root mean square fluctuation and angular deviation by Ramachandran plot. Moreover, the statistically validated contours of polar surface area, hydrogen bond distribution and the distance of disulfide bridges also supported the priority of [P20D, V21K] kB1 with respect to stability. From this work, it is proposed that the [P20D, V21K] kB1 (2F2I) could be the best template for scaffolding peptide based drug design.

Description: Inhibition of metabolic features which distinguish cancer cells from their non-malignant counterparts is a promising approach to cancer treatment. Energy support for drug extrusion in multidrug resistance (MDR) is a potential target for metabolic inhibition. Two major sources of ATP-based metabolic energy are partial (glycolysis) and complete (mitochondrial oxidative phosphorylation) oxidation of metabolic fuels. In cancer cells, the balance between them tends to be shifted towards glycolysis; this shift is considered to be characteristic of the cancer metabolic phenotype. Numerous earlier studies, conducted with cells cultured in a monolayer (2-D model), suggested inhibition of glycolytic ATP production as an efficient tool to suppress MDR in cancer cells. Yet, more recent work challenged the appropriateness of the 2-D model for such studies and suggested that a more clinically relevant approach would utilize a more advanced cellular model such as a 3-D model. Here we show that the transition from the 2-D model (cultured monolayer) to a 3-D model (cultured spheroids) introduces essential changes into the concept of energetic suppression of MDR. The 3-D cell organization leads to the formation of a discrete cell subpopulation (not formed in the 2-D model) with elevated MDR transport capacity. This subpopulation has a specific metabolic phenotype (mixed glycolytic/oxidative MDR support) different from that of cells cultured in the 2-D model. Finally, the shift to the oxidative phenotype becomes greater when the spheroids are grown under conditions of lactic acidosis that are typical for solid tumors. The potential clinical significance of these findings is discussed.

Description: We investigated the effects of exogenous sodium pyruvate (SP) on adipocyte differentiation, lipid accumulation, and the mRNA expression levels of adipogenesis-related genes in 3T3-L1 pre-adipocytes. Differentiation of pre-adipocytes was induced by MDI (3-isobutyl-1-methylxanthine: IBMX, dexamethasone: DEX, and insulin), in the presence or absence of SP. Adipogenesis was stimulated by SP in a concentration-dependent manner. SP also induced the expression of genes encoding aP2, GLUT4, and adiponectin, but had no effect on cell proliferation. Exogenous glucose did not promote adipogenesis or lipid accumulation. 2-deoxy-D-glucose inhibited adipogenesis initiated by MDI, but failed to influence the effects of SP on adipogenesis, whereas 3-bromopyruvate inhibited adipogenesis regardless of whether SP was present. The pro-adipogenic properties of SP were limited to the early events of adipogenesis. To determine whether SP mimics the adipogenic action of dexamethasone or insulin, we examined the effects of SP on adipogenesis with combinations of IBMX, DEX, and insulin. SP did not improve incomplete lipid accumulation observed in cells grown under IBMX-, DEX-, or insulin-free conditions. Insulin-stimulated ERK1/2 phosphorylation was diminished by SP, while phosphorylation of Akt was increased, correlating with increased glucose uptake in response to insulin. We also observed that SP stimulated immediate early expression of C/EBPβ and C/EBPδ. The PPARγ antagonist GW9662 inhibited adipogenesis. Our findings highlight the adipogenic function of exogenous SP by stimulating early events of adipogenesis.

Description: Diabetic nephropathy is characterized by inordinate secretion of extracellular matrix (ECM) proteins from mesangial cells (MCs), which is tightly associated with excessive activation of TGF-β signaling. The forkhead transcription factor O1 (FoxO1) protects mesangial cells from hyperglycemia-induced oxidative stress, which may be involved in ameliorating the redundant secretion of ECM proteins under high glucose conditions. Here we reported that high glucose elevated the level of p-Akt to attenuate endogenous FoxO1 bioactivities in MCs, accompanied with decreases in the mRNA expressions of catalase (CAT) and superoxide dismutase 2 (SOD2). Meanwhile, the expression of major ECM proteins-FN and Col I- increased under high glucose condition, in consistent with the activation of TGF-β/Smad signaling. By contrast, overexpression of nucleus-localized FoxO1 (insensitive to Akt phosphorylation) directly up-regulated the expressions of anti-oxidative enzymes, accompanied with inactivation of TGF-β/Smad3 pathway, as well as decreases of extracellular matrix proteins. Moreover, similar to those MCs overexpressed of nucleus-localized FoxO1 in high glucose conditions, MCs with down-regulation of FoxO1 by small interference-RNA under normal glucose conditions showed increased FN level and activated TGF-β/Smad3 pathway. Our findings link the anti-oxidative activity of FoxO1 and the TGF-β-induced secretion of ECM proteins, indicating the novel role of FoxO1 in protecting MCs under high glucose conditions.

Description: The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18kDa Fgf2 -IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18kD isoform of FGF2 (18kDa Fgf2/ LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type 2 collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion. This article is protected by copyright. All rights reserved

Description: TCERG1 was characterized previously as a repressor of the transcription factor C/EBPα through a mechanism that involved relocalization of TCERG1 from nuclear speckles to pericentromeric regions. The inhibitory activity as well as the relocalization activity has been demonstrated to lie in the amino terminal half of the protein, which contains several discrete motifs including an imperfect glutamine-alanine (QA) repeat. In the present study, we showed that deletion of this domain completely abrogated the ability of TCERG1 to inhibit the growth arrest activity of C/EBPα. Moreover, the QA repeat deletion mutant of TCERG1 lost the ability to be relocalized from nuclear speckles to pericentromeric regions, and caused an increase in the average size of individual speckles. We also showed that deletion of the QA repeat abrogated the complex formation between TCERG1 and C/EBPα. Examination of mutants with varying numbers of QA repeats indicated that a minimal number of repeats are required for inhibitory activity as well as relocalization ability. These data contribute to our overall understanding of how TCERG1 can have gene-specific effects in addition to its more general roles in coordinating transcription elongation and splicing. This article is protected by copyright. All rights reserved

Description: G-protein-coupled receptor 30 (GPR30) is an estrogen receptor that initiates several rapid, non-genomic signaling events triggered by E2. GPR30 has recently been identified in C2C12 cells; however, little is known about the intracelular distribution and its role in C2C12 myoblasts and myotubes. By western blotting and immunohistochemistry, we evidenced expression of GPR30. While in C2C12 myoblasts the receptor was present in nucleus, mitochondria and endoplasmic reticulum, in C2C12 myotubes it was additionally found in cytoplasm. Using trypan blue uptake assay to determine cellular death and fluorescent microscopy to evaluate picnotic nuclei and mitochondrial distribution, we demonstated that treatment of C2C12 myoblasts with G1 (GPR30 agonist) did not protect the cells against apoptosis induced by H 2 O 2 as E2. However, when G15 (GPR30 antagonist) was used, E2 could not prevent the damage caused by the oxidative stress. Further, some of the molecular mechanisms involved were investigated by wertern blot assays. Thus, E2 was able to induce AKT phosphorylation in apoptotic conditions and ERK phosphorylation in proliferating C2C12 cells but not when the cultures were incubated with G15. Additionally, using G15 antagonist we have found that GPR30 participates in the myogenin expression and creatine kinase activity stimulated by E2 in the first steps of C2C12 differentiation. Althogether these findings provide evidences showing that GPR30 is expressed in diverse intracellular compartments in undifferentiated and differentiated C2C12 cells and mediates E2 actions. This article is protected by copyright. All rights reserved

Description: Chd5 is an essential factor for neuronal differentiation and spermatogenesis, and known as a tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion, and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function. This article is protected by copyright. All rights reserved

Description: Previous studies have shown that promyelocytic leukemia zinc finger (PLZF), chemokine (C-X-C motif) receptor 4 (CXCR4) and mir146a were associated with the self-renewal of mouse spermatogonial stem cells (SSCs); however, there is little information on their effects on the fate of livestock SSCs. Here, we have identified a regulatory pathway in dairy goat mGSCs, involving PLZF, mir146a and the SDF-1 receptor CXCR4. PLZF overexpression downregulated mir146a and simultaneously upregulated the expression of CXCR4 protein, whereas PLZF knockdown (siPLZF) induced the specifically opposite effects. The in vitro assays demonstrated that PLZF specifically interacts with and suppresses the mir146a promoter, and mir146a targets CXCR4 to impede its translation. The levels of ERK1/2 phosphorylation in the mGSCs overexpressed CXCR4 and PLZF were upregulated, respectively, whereas mir146a expression was decreased and CXCR4 protein was increased. Mir146a overexpression and siPLZF impaired mGSC proliferation and differentiation, however, Mir146a knockdown induced the opposite effects. The effects of PLZF and mir146a were mediated regulation by mir146a and CXCR4, respectively. Overexpression of CXCR4 or addition of CXCL12 in cultures of dairy goat mGSCs resulted in the upregulation of their signaling, and the phosphorylation of ERK1/2 was increased. Collectively, these findings indicate that PLZF is an important transcription factor in the regulation of the expression of CXCR4 to promote dairy goat mGSC proliferation by targeting mir146a. This article is protected by copyright. All rights reserved

Description: Bacterial lipopolysaccharide (LPS) is the most important contributing factor in pathogenesis of bacterial infection in male accessory glands; and it has shown to inhibit testicular steroidogenesis and induce apoptosis. The present study demonstrates that LPS causes mitochondrial dysfunction via suppression of sirtuin 4 (SIRT4); which in turn affects Leydig cell function by modulating steroidogenesis and apoptosis. LC-540 Leydig cells treated with LPS (10µg/ml) showed impaired steroidogenesis and increased cellular apoptosis. The mRNA and protein expression of SIRT4 were decreased in LPS treated cells when compared to controls. The obtained data suggest that the c-Jun N-terminal kinase (JNK) activation suppresses SIRT4 expression in LPS treated Leydig cells. Furthermore, the overexpression of SIRT4 prevented LPS induced impaired steroidogenesis and cellular apoptosis by improving mitochondrial function. These findings provide valuable information that SIRT4 regulates LPS mediated Leydig cell dysfunction. This article is protected by copyright. All rights reserved

Description: Ketamine, a dissociative anesthetic, is misused and abused worldwide as an illegal recreational drug. In addition to its neuropathic toxicity, ketamine abuse has numerous effects, including renal failure; however, the underlying mechanism is poorly understood. The process called epithelial phenotypic changes (EPCs) causes the loss of cell-cell adhesion and cell polarity in renal diseases, as well as the acquisition of migratory and invasive properties. Madin-Darby canine kidney cells, an in vitro cell model, were subjected to experimental manipulation to investigate whether ketamine could promote EPCs. Our data showed that ketamine dramatically decreased transepithelial electrical resistance and increased paracellular permeability and junction disruption, which were coupled to decreased levels of apical junctional proteins (ZO-1, Occludin and E-cadherin). Consistent with the downregulation of epithelial markers, the mesenchymal markers N-cadherin, Fibronectin and Vimentin were markedly upregulated following ketamine stimulation. Of the E-cadherin repressor complexes tested, the mRNA levels of Snail, Slug, Twist, and ZEB1 were elevated. Moreover, ketamine significantly enhanced migration and invasion. Ketamine-mediated changes were at least partly caused by the inhibition of GSK-3β activity through Ser-9 phosphorylation by the PI3K/Akt pathway. Inhibiting PI3K/Akt with LY294002 reactivated GSK-3β and suppressed ketamine-enhanced permeability, EPCs and motility. These findings were recapitulated by the inactivation of GSK-3β using the inhibitor 3F8. Taken together, these results provide evidence that ketamine induces renal distal tubular EPCs through the downregulation of several junction proteins, the upregulation of mesenchymal markers, the activation of Akt, and the inactivation of GSK-3β. This article is protected by copyright. All rights reserved

Description: CCN2/connective tissue growth factor (CTGF) is a multifunctional molecule that promotes harmonized development and regeneration of cartilage through its matricellular interaction with a variety of extracellular biomolecules. Thus, deficiency in CCN2 supply profoundly affects a variety of cellular activities including basic metabolism. A previous study showed that the expression of a number of ribosomal protein genes was markedly enhanced in Ccn2 -null chondrocytes. Therefore, in this study, we analyzed the impact of CCN2 on amino acid and protein metabolism in chondrocytes. Comparative metabolome analysis of the amino acids in Ccn2 -null and wild type mouse chondrocytes revealed stable decreases in the cellular levels of all of the essential amino acids. Unexpectedly, uptake of such amino acids was rather enhanced in Ccn2 -null chondrocytes, and the addition of exogenous CCN2 to human chondrocytic cells resulted in decreased amino acid uptake. However, as expected, amino acid consumption by protein synthesis was also accelerated in Ccn2 -null chondrocytes. Furthermore, we newly found that expression of 2 genes encoding 2 glycolytic enzymes, as well as the previously reported Eno 1 gene, was repressed in those cells. Considering the impaired glycolysis and retained mitochondrial membrane potential in Ccn2 -null chondrocytes, these findings suggest that Ccn2 deficiency induces amino acid shortage in chondrocytes by accelerated amino acid consumption through protein synthesis and acquisition of aerobic energy. Interestingly, CCN2 was found to capture such free amino acids in vitro . Under physiological conditions, CCN2 may be regulating the levels of free amino acids in the extracellular matrix of cartilage. This article is protected by copyright. All rights reserved

Description: ABSTRACT Some cord blood banks freeze entire pieces of UC (mixed cord, MC) which after post-thaw yields mixed heterogeneous populations of mesenchymal stem cells (MSCs) from all its microanatomical compartments. Freezing of such entire tissues results in sub-optimal post-thaw cell recovery because of poor cryoprotectant diffusion and intracellular ice-formation, heat and water transport issues and damage to intercellular junctions. To develop a simple method of harvesting pure homogeneous MSCs for cord blood banks we compared the post-thaw behavior of three groups of frozen UC tissues (i) freshly harvested WJ without cell separation, (ii) MSCs isolated from WJ (WJSC) and (iii) MC. WJ and WJSC produced high post-thaw cell survival rates (93.52 ± 6.12% to 90.83 ± 4.51%) and epithelioid monolayers within 24h in primary culture whereas post-thaw MC explants showed slow growth with mixed epithelioid and fibroblastic cell outgrowths after several days. Viability and proliferation rates of post-thawed WJ and hWJSC were significantly greater than MC. Post-thaw WJ and WJSC produced significantly greater CD24 + and CD108 + fluorescence intensities and significantly lower CD40 + contaminants. Post-thaw WJ and WJSC produced significantly lesser annexin-V-positive and sub-G1 cells and greater degrees of osteogenic and chondrogenic differentiation compared to MC. qRT-PCR analysis of post-thaw MC showed significant decreases in anti-apoptotic gene expression (SURVIVIN, BCL2) and increases in pro-apoptotic (BAX) and cell cycle regulator genes (P53, P21, ROCK 1) compared to WJ and WJSC. We conclude that freezing of fresh WJ is a simple and reliable method of generating large numbers of clinically utilizable MSCs for cell-based therapies. This article is protected by copyright. All rights reserved

Description: The Wnt ligands are a family of secreted signaling proteins which play key roles in a number of cellular processes under physiological and pathological conditions. Wnts bind to their membrane receptors and initiate a signaling cascade which leads to the nuclear localization and transcriptional activity of β-catenin. The development of purified recombinant Wnt ligands has greatly aided in our understanding of Wnt signaling and its functions in development and disease. In the current study, we identified non-Wnt related signaling activities which were present in commercially available preparations of recombinant Wnt3a. Specifically, we found that treatment of cultured fibroblasts with recombinant Wnt3a induced immediate activation of TGF-β and BMP signaling and this activity appeared to be independent of the Wnt ligand itself. Therefore, while purified recombinant Wnt ligands continue to be a useful tool for studying this signaling pathway, one must exercise a degree of caution when analyzing the results of experiments that utilize purified recombinant Wnt ligands. This article is protected by copyright. All rights reserved

Description: CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1β was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1β promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis. This article is protected by copyright. All rights reserved

Description: ABSTRACT Immune responses are outcomes of complex molecular machinery which occur inside the cells. Unravelling the cellular mechanisms induced by immune stimulating molecules such as glycans and determining their structure-function relationship are therefore important factors to be assessed. With this viewpoint, the present study identifies the functional receptor binding unit of a well characterized heteroglycan and also delineates the cellular and molecular processes that are induced upon heteroglycan binding to specific cell surface receptors in immune cells. The heteroglycan was acid hydrolysed and it was revealed that 10-30 kDa fractions served as the functional receptor binding unit of the molecule. Increasing the size of 10-30 kDa heteroglycan showed prominent immune activity. The whole soluble heteroglycan was also conjugated with hyperbranched dendrimers so as to generate a particulate form of the molecule. Dectin-1 and TLR2 were identified as the major receptors in macrophages that bind to particulate as well as soluble form of the heteroglycan and subsequently caused downstream signaling molecules such as NF-κβ and MAPK to get activated. High levels of 1L-1β and IL-10 mRNA were observed in particulate heteroglycan treated macrophages, signifying that increasing the size and availability of the heteroglycan to its specific receptors is pertinent to its biological functioning. Upregulated expression of PKC and iNOS were also noted in particulate heteroglycan treated RAW 264.7 cells than the soluble forms. Taken together, our results indicate that biological functions of immunomodulatory heteroglycan are dependent on their size and molecular weight. This article is protected by copyright. All rights reserved

Description: Polyphenols are a class of natural compounds whose potential as antioxidant, anti-inflammatory and anti-angiogenesis has been reported in many pathological conditions. Red raspberry extract, rich in polyphenols, has been reported to exert anti-inflammatory effects and prevent cell proliferation in distinct animal models. However, the signalling pathways involved remain unknown. Herein, we used human microvascular endothelial cells (HMVECs) to determine the influence of red raspberry phenolic compound extract concentrations, ranging from 10-250 µg gallic acid equivalents (GAE)/mL, on endothelium viability (MTS assay), proliferation (BrdU incorporation), migration (injury assay) and capillary-like structures formation (Matrigel assay). Protein expression in cell lysates was determined by Western blot analysis. We showed that red raspberry extracts reduced cell viability (GI 50  = 87,64 ± 6,59 µg GAE/mL) and proliferation in a dose-dependent manner. A significant abrogation of cells ability to migrate to injured areas, even at low concentrations, was observed by injury assay. Cell assembly into capillary-like structures on Matrigel also decreased in a dose dependent-manner for higher extract concentrations, as well as the number of branching points per unit of area. Protein expression analysis showed a dose-dependent decrease in Phospho-VEGFR2 expression, implying abrogation of VEGF signalling activity. We also showed for the first time that red raspberry phenolic compounds induce the rearrangement of filamentous actin cytoskeleton, with an isotropy increase found for higher testing concentrations. Taken together, our findings corroborate the anti-angiogenic potential of red raspberry phenolic compounds and provide new insights into their mode of action upon endothelium. This article is protected by copyright. All rights reserved

Description: Evodiamine (Evo), extracted from the Chinese herbal medicine Evodia rutaecarpa, has cytotoxic effects on different types of human cancer cells. However, its effects on drug resistance and their molecular mechanism and therapeutic target in colorectal cancer are not well understood. In the present study, we observed that Evo inhibited cell growth and induced apoptosis in adose-and time-dependent mannner in HCT-116/L-OHP cells. Moreover, Evo treatment reduced Rhodamine 123 accumulation and ATPase activity in HCT-116/L-OHP cells, indicating that Evo decreased the efflux function in HCT-116/L-OHP cells. Interestingly, phosphorylation of NF-κB pathway, particularly p50/p65, was also inhibited by Evo treatment. Furthermore the effect of Evo in reversing drug resistance and suppressing phosphorylation of NF-κB pathway were attenuated after treatment with the NF-κB activator (LPS). Additionally, Evo inhibited the tumor growth in a colorectal MDR cancer xenograft model and down regulated p-NF-κB level in vivo . Our study provided the first direct evidence that Evo can attenuate multidrug resistance by blocking p-NF-κB signaling pathway in human colorectal cancer. Evo could be a potential candidate for cancer chemotherapy. This article is protected by copyright. All rights reserved

Description: Regulation of placental nutrient transport significantly affects fetal development and may modify intrauterine growth restriction (IUGR) and fetal programming. We hypothesized that placental nutrient transporters are differentially affected both by utero-placental insufficiency and prenatal surgical stress. Pregnant rats underwent bilateral uterine artery and vein ligation (LIG), sham operation (SOP) or no operation (controls, C) on gestational day E19. Placentas were obtained by caesarean section 4 h (LIG, n = 20 placentas; SOP, n = 24; C, n = 12), 24 h (LIG, n = 28; SOP, n = 20; C, n = 12) and 72 h (LIG, n = 20; SOP, n = 20; C, n = 24) after surgery. Gene and protein expression of placental nutrient transporters for fatty acids (h-FABP, CD36), amino acids (SNAT1, SNAT2) and glucose (GLUT-1, Connexin 26) were examined by qRT-PCR, western blot and immunohistochemistry. Interestingly, the mean protein expression of h-FABP was doubled in placentas of LIG and SOP animals 4, 24 (SOP significant) and 72 h (SOP significant) after surgery. CD36 protein was significantly increased in LIG after 72 h. SNAT1 and SNAT2 protein and gene expressions were significantly reduced in LIG and SOP after 24 h. Further significantly reduced proteins were GLUT-1 in LIG (4 h, 72 h) and SOP (24 h), and Connexin 26 in LIG (72 h). In conclusion, placental nutrient transporters are differentially affected both by reduced blood flow and stress, probably modifying the already disturbed intrauterine milieu and contributing to IUGR and fetal programming. Increased fatty acid transport capacity may affect energy metabolism and could be a compensatory reaction with positive effects on brain development. This article is protected by copyright. All rights reserved

Description: Previously, we reported that brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration by inducing periodontal ligament cell proliferation in vivo . In addition, the down growth of gingival epithelial cells, which comprises a major obstacle to the regeneration was not observed. However, the underlying molecular mechanism is still unclear. Therefore, this study aimed to investigate the effect of BDNF on cell proliferation and apoptosis in human periodontal ligament cells (HPL cells) and human gingival epithelial cells (OBA9 cells) and to explore the molecular mechanism in vitro . HPL cells dominantly expressed a BDNF receptor, TrkB, and BDNF increased cell proliferation and ERK phosphorylation. However, its proliferative effect was diminished by a MEK1/2 inhibitor (U0126) and TrkB siRNA transfection. Otherwise, OBA9 cells showed a higher expression level of p75, which is a pan-neurotrophin receptor, than that of HPL cells. BDNF facilitated not cell proliferation but cell apoptosis and JNK phosphorylation in OBA9 cells. A JNK inhibitor (SP600125) and p75 siRNA transfection attenuated the BDNF-induced cell apoptosis. Moreover, OBA9 cells pretreated with SP600125 or p75 siRNA showed cell proliferation by BDNF stimulation, though it was reduced by U0126 and TrkB siRNA. Interestingly, overexpression of p75 in HPL cells up-regulated cell apoptosis and JNK phosphorylation by BDNF treatment. These results indicated that TrkB-ERK signaling regulates BDNF-induced cell proliferation, whereas p75-JNK signaling plays roles in cell apoptotic and cytostatic effect of BDNF. Overall, BDNF activates periodontal ligament cells proliferation and inhibits the gingival epithelial cells growth via the distinct pathway. This article is protected by copyright. All rights reserved

Description: DMSO is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF-β activity. Here we show that DMSO enhances TGF-β activity by ∼3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF-β-stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF-β receptors (TβR-I and/or TβR-II) by ∼3-4-fold without altering their cellular levels as determined by 125 I-labeled TGF-β-cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TβR-II (but not TβR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TβR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TβR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TβR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TβR-II-HA-containing vesicles from the cytoplasm and co-localization of TβR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF-β activity presumably by inducing fusion of cytoplasmic vesicles (containing TβR-II) and the plasma membrane, resulting in increased localization of TβR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. This article is protected by copyright. All rights reserved

Description: The recently discovered MCPIP1 (monocyte chemoattractant protein-induced protein 1), a multidomain protein encoded by the MCPIP1 ( ZC3H12A) gene, has been described as a new differentiation factor, a ribonuclease, and a deubiquitination-supporting factor. However, its role in cancer is poorly recognized. Our recent analysis of microarrays data showed a lack of expression of the MCPIP1 transcript in primary neuroblastoma – the most common extracranial solid tumour in children. Additionally, enforced expression of the MCPIP1 gene in BE(2)-C cells caused a significant decrease in neuroblastoma proliferation and viability. Aim of the present study was to further investigate the role of MCPIP1 in neuroblastoma, using expression DNA microarrays and microRNA microarrays. Transient transfections of BE(2)-C cells were used for overexpression of either wild type of MCPIP1 (MCPIP1-wt), or its RN-ase defective mutant (MCPIP1-ΔPIN). We have analyzed changes of transcriptome and next, we have used qRT-PCR to verify mRNA levels of selected genes responding to MCPIP1 overexpression. Additionally, protein levels were determined for some of the selected genes. The choline transporter, CTL1, encoded by the SLC44A1 gene, was significantly repressed at the specific mRNA and protein levels and most importantly this translated into a decreased choline transport in MCPIP1-overexpressing cells. Then, we have found microRNA-3613-3p as the mostly altered in the pools of cells over-expressing the wild type MCPIP1. Next, we analyzed the predicted targets of the miR-3613-3p and validated them using qRT-PCR and western blot. These results indicate that the expression of miR-3613-3p might be regulated by MCPIP1 by cleavage of its precursor form. This article is protected by copyright. All rights reserved

Description: ABSTRACT Transplantation of hepatocytes is a promising therapy for end-stage liver disease, but the availability of functional cells currently precludes its clinical application. We now report a simple transient reprogramming approach to convert fibroblasts into hepatic-like cells. Human skin fibroblasts were treated with fish egg extracts to become the transiently-remodeled cells (TRCs). After infected with retroviral EGFP, they were directly injected into the fetal monkey liver, where they underwent in situ differentiation in the hepatic niche. The hepatic-like cells were functional as shown by the synthesis of hepatic markers in vivo , including albumin, cytokeratin-18, and hepatic serum antigen. Similarly, when implanted in the mouse liver, the TRCs were differentiated into hepatic-like cells that synthesize albumin and CK18 and became completely integrated into the liver parenchyma. The potency of TRCs was mechanistically related to the activation of several signal pathways, which reactivate endogenous genes related to cell potency. This study demonstrates the feasibility of a simple and inexpensive epigenetic remodeling approach to convert human fibroblasts into therapeutic hepatic-like cells for the treatment of end-stage liver disease. This article is protected by copyright. All rights reserved

Description: Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time PCa radio-resistance as a consequence of elevated expression of Hepatoma Up-Regulated Protein (HURP). In PCa cells, HURP expression suppresses γ- irradiation- induced apoptosis. γ- irradiation-induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ- irradiation- induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ- irradiation- induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio-resistance of PCa is thought to be the consequence of HURP-mediated destabilization of p53 and ATM proteins that are essential for γ-irradiation-induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F-1 in response to the elevated expression of HURP. This article is protected by copyright. All rights reserved

Description: The endo/lysosomal system in cells provides membranous platforms to assemble specific signaling complexes and to terminate signal transduction, thus, is essential for physiological signaling. Endocytic organelles can significantly extend signaling of activated cell surface receptors, and may additionally provide distinct locations for the generation of specific signaling outputs. Failures of regulation at different levels of endocytosis, recycling, degradation as well as aberrations in specific endo/lysosomal signaling pathways, such as mTORC1, might lead to different diseases including cancer. Therefore, a better understanding of spatio-temporal compartmentalization of sub-cellular signaling might provide an opportunity to interfere with aberrant signal transduction in pathological processes by novel combinatorial therapeutic approaches. This article is protected by copyright. All rights reserved

Description: Several gap junction connexins have been shown to be essential for appropriate placental development and function. It is known that the expression and distribution of connexins change in response to environmental oxygen levels. The placenta develops under various oxygen levels, beginning at a low oxygen tension of approximately 2% and increasing to a tension of 8% after the onset of the uteroplacental circulation. Moreover, it has been shown that during preeclampsia placentas are subjected to chronic hypoxia. Therefore, we investigated oxygen sensitivity of placental connexins 43 and 46. Using the trophoblast cell line Jar we demonstrated that the expression of connexin43 increased during acute hypoxia but decreased during chronic hypoxia. Chronic hypoxia resulted in the translocation of connexin43 from the membrane to the cytoplasm and in a reduction in its communication properties. In contrast, the expression of connexin46 was down-regulated during chronic hypoxia and was translocated from perinuclear areas to the cell membrane. Hypoxia-inducible factor (HIF) knockdown showed that the translocation of connexin43 but not that of connexin46 was HIF-2α dependent and was mediated by phosphoinositide 3-kinase. The upregulation of connexin43 in combination with the down-regulation of connexin46 was confirmed in placental explants cultivated under low oxygen and in placentas with early-onset preeclampsia. Taken together, in Jar cells placental connexins 43 and 46 are regulated during periods of low oxygen in opposite manners. The oxygen sensing of connexins in the trophoblast may play a role in physiological and pathophysiological oxygen conditions and thus may contribute to preeclampsia. This article is protected by copyright. All rights reserved

Description: Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/β-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/β-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3β inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/β-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect β-catenin mRNA expression levels, but reduced β-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted β-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with β-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/β-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/β-catenin signaling by enhancing proteasomal degradation of β-catenin. This article is protected by copyright. All rights reserved

Description: Patient-specific human induced-pluripotent stem cells (hiPSCs) represent important cell sources to treat patients with acquired blood disorders. To realize the therapeutic potential of hiPSCs, it is crucial to understand signals that direct hiPSC differentiation to a hematopoietic lineage fate. Our previous study demonstrated that CD34 + CD31 + cells derived from human pluripotent stem cells (hPSCs) contain progenitors that give rise to hematopoietic cells and endothelial cells. Here, we established a serum-free and feeder-free system to induce the differentiation of hPSC-derived CD34 + CD31 + progenitor cells to erythroid cells. We show that extracellular matrix (ECM) proteins promote the differentiation of CD34 + CD31 + progenitor cells into CD235a + erythroid cells through CD41 + CD235a + megakaryocyte-erythroid progenitors (MEP). Erythropoietin (EPO) is a predominant factor for CD34 + CD31 + progenitor differentiation to erythroid cells, whereas transforming growth factor beta (TGF-β) inhibits the development of CD34 + CD31 + progenitor cells. Apoptosis of progenitor cells is induced by TGF-β in early erythroid differentiation. Suppression of TGF-β signaling by SB431542 at early stage of CD34 + CD31 + progenitor differentiation induces the erythroid cell generation. Together, these findings suggest that TGF-β suppression and EPO stimulation promote erythropoiesis of CD34 + CD31 + progenitor cells derived from hPSCs. This article is protected by copyright. All rights reserved

Description: The microRNA-155 (miR155) regulates various functions of cells. Dysfunction or injury of endothelial cells (ECs) plays an important role in the pathogenesis of various vascular diseases. In this study, we investigated the role and potential mechanisms of miR155 in human brain microvessel endothelial cells (HBMECs) under physiological and pathological conditions. We detected the effects of miR155 silencing on ROS production, NO generation, apoptosis and functions of HBMECs at basal and in response to oxidized low density lipoprotein (ox-LDL). Western blot and q-PCR were used for analyzing the gene expression of epidermal growth factor receptor (EGFR)/ extracellular regulated protein kinases (ERK)/ p38 mitogen-activated protein kinase (p38 MAPK), phosphatidylinositol-3-kinase (PI3K) and serine/threonine kinase(Akt), activated caspase-3 and intercellular adhesion molecule-1 (ICAM-1). Results showed that under both basal and challenge situations: 1) Silencing of miR155 decreased apoptosis and reactive oxygen species (ROS) production of HBMECs, whereas, promoted nitric oxide (NO) generation. 2) Silencing of miR155 increased the proliferation, migration and tube formation ability of HBMECs, while decreased cell adhesion ability. 3) Gene expression analyses showed that EGFR/ ERK/ p38 MAPK and PI3K/Akt were increased and that activated caspase-3 and ICAM-1 mRNA were decreased after knockdown of miR155. In conclusion, knockdown of miR155 could modulate ROS production, NO generation, apoptosis and function of HBMECs via regulating diverse gene expression, such as caspase-3, ICAM-1 and EGFR/ERK/p38 MAPK and PI3K/Akt pathways. This article is protected by copyright. All rights reserved

Description: This study was designed to investigate the influence of mesenchymal stem cells (MSCs) on osteoblast (OB) differentiation. Rat bone marrow MSCs were cultured either in growth medium that maintained a MSC phenotype or in osteogenic medium that induced differentiation into OBs. Then, cells were grown in two different culture conditions: indirect co-culture of MSCs and OBs and OBs cultured in MSC-conditioned medium. As a control culture condition, OBs were grown in osteogenic medium without the influence of MSCs. We evaluated cell proliferation, the gene expression of key bone markers, alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) expression, and extracellular matrix mineralization. The results showed that, regardless of whether OBs were indirectly co-cultured with MSCs or cultured in MSC-conditioned medium, MSCs repressed OB differentiation, as evidenced by the downregulation of all evaluated bone marker genes, decreased ALP activity, inhibition of BSP protein expression, and reduced extracellular matrix mineralization. Taken together, these results indicate that despite the key role of both MSCs and OBs in the osteogenic process, the repressive effect of MSCs on OB differentiation in an osteogenic environment may represent a barrier to the strategy of using them together in cell-based therapies to induce bone repair. This article is protected by copyright. All rights reserved

Description: Osteoarthritis (OA) is a widespread chronic joint disease characterized by articular cartilage destruction and accompanied by pain and disability. In this study, we found that the expression of Insulin-like Growth Factor II (IGF-II) was reduced in articular cartilage in human OA patients as well as in the murine experimental OA model of destabilization of the medial meniscus (DMM). In primary human articular chondrocytes, ectopic expression of lentiviral IGF-II inhibited pro-inflammatory cytokine IL-1β-induced NF-κB activation as well as catabolic gene expression. Interestingly, IGF-II did not significantly alter the phosphorylation states of ERK1/2 or Akt, which are kinases typically activated by IGF-I. Instead, it induced the activity of phospholipase C (PLC) and a PLC inhibitor blocked the inhibitory activity of IGF-II against IL-1β, suggesting that this activity is mediated through PLC. Furthermore, IGF-II increased cartilage matrix levels and decreased MMP13 protein expression in explanted human OA cartilage cultures in vitro . In the in vivo DMM model, intraarticular injection of lentiviral IGF-II led to enhanced cartilage matrix levels and decreased MMP13 protein expression, as well as reduced osteophyte formation and subchondral bone sclerosis. Therefore, our results suggest that IGF-II can promote cartilage integrity and halt knee joint destruction in OA. This article is protected by copyright. All rights reserved

Description: ABSTRACT Adipose tissue-derived mesenchymal stem cells (Ad-MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet-rich plasma (PRP) on Ad-MSC and adipocyte function. PRP increased Ad-MSC viability, proliferation rate and G1- S cell cycle progression, by at least 7-, 2-, and 2.2-fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad-MSC growth. PRP also accelerated cell migration by at least 1.5-fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR-γ and AP-2 mRNAs, while it increased leptin production by 3.5-fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)-6, IL-8, IL-10, Interferon-γ and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad-MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro-angiogenic factors, which may facilitate tissue regeneration processes. This article is protected by copyright. All rights reserved

Description: Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit μ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death, however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 μM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies. This article is protected by copyright. All rights reserved

Description: Mesenchymal stem cells (MSCs) are population of adult stem cells and attractive candidates for cartilage repair due to their chondrogenic potential. Purinergic compounds (purinergic receptors and ecto-enzymes metabolizing nucleotides), together with nucleotides/nucleosides present in the extracellular environment, are known to play a key role in controlling the stem cells biological potential to proliferate and differentiate. Despite the available literature pointing to the importance of purinergic signalling in controlling the fate of mesenchymal stem cells, the research results linking nucleotides and ecto-nucleotidases with MSCs chondrogenic differentiation are indigent. Therefore, the aim of presented study was the characterization of the ecto-nucleotides hydrolysis profile and ecto-enzymes expression in human umbilical cord-derived mesenchymal stem cells and chondrogenically induced MSCs. We described substantial changes of ecto-nucleotides metabolism and ecto-enzymes expression profiles resulting from chondrogenic differentiation of human umbilical cord-derived mesenchymal stem cells. The increased rate of ADP hydrolysis, measured by ecto-nucleotidases activity, plays a pivotal role in the regulation of cartilage formation and resorption. Despite the increased level of NTPDase1 and NTPDase3 mRNA expression in chondrogenically induced MSCs, their activity toward3 remains quite low. Supported by the literature data, we hypothesize that structure-function relationships in chondrogenic lineage dictate the direction of nucleotides metabolism. In early neocartilage tissue, the beneficial role of ATP in improving biomechanical properties of cartilage, does not necessitate the high rate of enzymatic ATP degradation. This article is protected by copyright. All rights reserved

Description: Autophagy is a cellular process to recycle nutrients and has been implicated in cancer treatment. Oral squamous cell carcinoma (OSCC) is the most common oral cancer which ranks 3% of cancers in men and 2% in women. In this study, immunohistochemical staining of OSCC tumor specimens from human subjects and an athymic mouse model demonstrated high levels of autophagy markers LC3-II and ATG5 expression. Further, we identified high levels LC3-II expression in OSCC tumor cell lines (SCC-1, SCC-12 & SCC-14a) compared to normal human epithelial (RWPE-1) cells. OSCC cells express high levels of RANK ligand (RANKL); however a functional role in autophagy is unknown. Interestingly, RANKL stimulation significantly increased autophagosome related gene expressions such as LC3, ATG5, BECN1 and PI3KC3 mRNA expression in OSCC cells. Further, western blot analysis of total cell lysates demonstrated a dose-dependent increase in LC3-II and ATG5 expression in RANKL stimulated cells. In addition, RANKL increased expression of LC3-I and LC3-II, essential for autophagosome formation. Confocal microscopy analysis of LC3-II and localization with lysosome further confirms autophagosome formation in response to RANKL treatment in OSCC cells. Collectively, our results indicate a novel function of RANKL to induce autophagosome formation, and could be a potential therapeutic target to control OSCC tumor progression. This article is protected by copyright. All rights reserved

Description: The aspartate-histidine-histidine-cysteine (DHHC) protein family shares a 50-amino acid cysteine-rich domain with a conserved DHHC signature motif. DHHC proteins play a critical role in several biological processes. Several DHHC family members have been implicated in neuronal differentiation and synaptic plasticity. And disruptions to their function can lead to disease in the nervous system. Here, we investigate the role of Zdhhc15b, a DHHC family member, in neurodevelopment in zebrafish. Whole-mount in situ hybridization (WISH) revealed that zdhhc15b , an ortholog to human ZDHHC15, is abundant in zebrafish ( Danio rerio ) forebrain, especially in the diencephalon. Downregulation of zdhhc15b resulted in a smaller diencephalon and a reduction in mature dopaminergic neurons (DA neurons). In the meanshile, mutant zdhhc15b zebrafish was associated with poor learning behavior as detected by T-maze testing. The expression of zdhhc15b was upregulated during DA neuronal differentiation whereas knock-down of zdhhc15b diminished DA neuronal differentiation. Tyrosine hydroxylase (TH) immunofluorescence of cultured DA neurons in vitro also showed that DA neurons were immature following zdhhc15b knock-down. Consistent with the decreased number of DA neurons following knock-down of zdhhc15b , the expression of fate determination-related transcription factors such as nurr1, foxA2 , and lmx1a were also reduced in morphant zebrafish. Our results reveal that zdhhc15b controls DA neuronal fate decisions by regulating differentiation but not progenitor cell proliferation or DA neuronal survival. This article is protected by copyright. All rights reserved

Description: Photodynamic therapy (PDT) is a non-thermal technique for inducing tumour damage following administration of a light-activated photosensitizing drug (PS). In a previous work we found that PDT induces cytoskeleton changes in HB4a-Ras cells (human mammary breast carcinoma HB4a cells transfected with the RAS oncogene). In the present work we have studied the migratory and invasive features and the expression of proteins related to these processes on HB4a-Ras cells after 3 successive cycles of PDT using different PSs: 5-aminolevulinic acid (ALA), Verteporfin (Verte), m -tetrahydroxyphenylchlorin ( m -THPC) and Merocyanine 540 (MC). A slight (1.25- to -2 fold) degree of resistance was acquired in cell populations subjected to the three successive PDT treatments. However, complete cell killing was achieved after a light dose increase. Regardless of the PS employed, all the PDT-treated populations had shorter stress fibres than the untreated control HB4a-Ras cells, and the number of dorsal stress fibres was decreased in the PDT-treated populations. E-Cadherin distribution, which was already aberrant in HB4a-Ras cells, became even more diffuse in the PDT-treated populations, though its expression was increased in some of them. The strong migratory and invasive ability of HB4a-Ras cells in vitro was impaired in all the PDT-treated populations, with a behaviour that was similar to the parental non-tumoral HB4a cells. MMP-2 and MMP-9 metalloproteinase activities were also impaired in the PDT-treated populations. The evidence presented herein suggests that the cells surviving PDT would be less metastatic than the initial population. These findings encourage the use of PDT in combination with other treatments such as intraoperative or post-surgery therapeutic procedures. This article is protected by copyright. All rights reserved

Description: ABSTRACT Mechanotransduction is a key process by which cells perceive extracellular mechanical cues / intercellular physical interactions and transform them into intracellular biochemical signals. This physiological process is crucial during bone development and bone remodeling throughout childhood and adult life, whereas several aberrations during this process have emerged as a distinct pathogenic molecular entity in bone maladies and tumor formation. The present review focuses on recent advances regarding the mechanobiology of osteosarcoma, the most common type of bone cancer. Special emphasis is given on the mechano-responsive signal transduction pathways underlying osteosarcoma pathology and on specific mechanosensitive molecules engaged in osteosarcoma development. This article is protected by copyright. All rights reserved

Description: ABSTRACT Protandim and 6-gingerol, two potent nutraceuticals, have been shown to decrease free radicals production through enhancing endogenous antioxidant enzymes. In this study, we evaluated the effects of these products on the expression of different factors involved in osteoarthritis (OA) process. Human OA chondrocytes were treated with 1 ng/ml IL-1β in the presence or absence of protandim (0-10 μg/ml) or 6-gingerol (0-10 μM). OA was induced surgically in mice by destabilization of the medial meniscus (DMM). The animals were treated weekly with an intraarticular injection of 10 μl of vehicle or protandim (10 μg/ml) for 8 weeks. Sham-operated mice served as controls. In vitro , we demonstrated that protandim and 6-gingerol preserve cell viability and mitochondrial metabolism and prevented 4-hydroxynonenal (HNE)-induced cell mortality. They activated Nrf2 transcription factor, abolished IL-1β-induced NO, PGE 2 , MMP-13, and HNE production as well as IL-β − induced GSTA4-4 down-regulation. Nrf2 overexpression reduced IL-1β-induced HNE and MMP-13 as well as IL-1β-induced GSTA4-4 down-regulation. Nrf2 knockdown following siRNA transfection abolished protandim protection against oxidative stress and catabolism. The activation of MAPK and NF-κB by IL-1β was not affected by 6-gingerol. In vivo , we observed that Nrf2 and GSTA4-4 expression was significantly lower in OA cartilage from humans and mice compared to normal controls. Interestingly, protandim administration reduced OA score in DMM mice. Altogether, our data indicate that protandim and 6-gingerol are essential in preserving cartilage and abolishing a number of factors known to be involved in OA pathogenesis. This article is protected by copyright. All rights reserved

Description: ABSTRACT Epithelial morphogenesis in the mammary gland proceeds as a consequence of complex cell behaviors including apoptotic cell death and epithelial-mesenchymal transition (EMT); the extracellular matrix (ECM) protein laminin is crucially involved. Syntaxins mediate intracellular vesicular fusion, yet certain plasmalemmal members have been shown to possess latent extracellular functions. In this study, the extracellular subpopulation of syntaxin-4, extruded in response to the induction of differentiation or apoptosis in mammary epithelial cells, was detected. Using a tetracycline-repressive transcriptional system and clonal mammary epithelial cells, SCp2, we found that the expression of cell surface syntaxin-4 elicits EMT-like cell behaviors. Intriguingly, these cells did not up-regulate key transcription factors associated with the canonical EMT such as snail, slug , or twist , and repressed translation of E-cadherin. Concurrently, the cells completely evaded the cellular aggregation/rounding triggered by a potent EMT blocker laminin-111. We found that the recombinant form of syntaxin-4 not only bound to laminin but also latched onto the glycosaminoglycan (GAG) side chains of syndecan-1, a laminin receptor that mediates epithelial morphogenesis. Thus, temporal extracellular extrusion of syntaxin-4 emerged as a novel regulatory element for laminin-induced mammary epithelial cell behaviors. This article is protected by copyright. All rights reserved

Description: Glial cell line-derived neurotrophic factor (GDNF) is known to mediate multiple biological activities such as promotion of cell motility and proliferation, and morphogenesis. However, little is known about its effects on periodontal ligament (PDL) cells. Recently, we reported that GDNF expression is increased in wounded rat PDL tissue and human PDL cells (HPDLCs) treated with proinflammatory cytokines. Here, we investigated the associated expression of GDNF and the proinflammatory cytokine interleukin-1 beta (IL-1β) in wounded PDL tissue, and whether HPDLCs secrete GDNF which affects neurocytic differentiation. Rat PDL cells near the wounded area showed intense immunoreactions against an anti-GDNF antibody, where immunoreactivity was also increased against an anti-IL-1β antibody. Compared with untreated cells, HPDLCs treated with IL-1β or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1β-treated HPDLCs (IL-1β-CM) increased neurite outgrowth of PC12 rat adrenal pheochromocytoma cells. The expression levels of two neural regeneration-associated genes, growth-associated protein-43 (Gap-43) and small proline-rich repeat protein 1A (Sprr1A), were also upregulated in IL-1β-CM-treated PC12 cells. These stimulatory effects of IL-1β-CM were significantly inhibited by a neutralizing antibody against GDNF. In addition, U0126, a MEK inhibitor, inhibited GDNF-induced neurite outgrowth of PC12 cells. These findings suggest that an increase of GDNF in wounded PDL tissue might play an important role in neural regeneration probably via the MEK/ERK signaling pathway. This article is protected by copyright. All rights reserved

Description: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder that has recently seen serious increase in the number of affected subjects. In the last decade, neuroimaging has been shown to be a useful tool to understand AD and its prodromal stage, amnestic mild cognitive impairment (MCI). The majority of AD/MCI studies have focused on disease diagnosis, by formulating the problem as classification with a binary outcome of AD/MCI or healthy controls. There have recently emerged studies that associate image scans with continuous clinical scores that are expected to contain richer information than a binary outcome. However, very few studies aim at modeling multiple clinical scores simultaneously, even though it is commonly conceived that multivariate outcomes provide correlated and complementary information about the disease pathology. In this article, we propose a sparse multi-response tensor regression method to model multiple outcomes jointly as well as to model multiple voxels of an image jointly. The proposed method is particularly useful to both infer clinical scores and thus disease diagnosis, and to identify brain subregions that are highly relevant to the disease outcomes. We conducted experiments on the Alzheimer's Disease Neuroimaging Initiative (ADNI) dataset, and showed that the proposed method enhances the performance and clearly outperforms the competing solutions.

Description: A plethora of techniques for cardiac deformation imaging with 3D ultrasound, typically referred to as 3D speckle tracking techniques, are available from academia and industry. Although the benefits of single methods over alternative ones have been reported in separate publications, the intrinsic differences in the data and definitions used makes it hard to compare the relative performance of different solutions. To address this issue, we have recently proposed a framework to simulate realistic 3D echocardiographic recordings and used it to generate a common set of ground-truth data for 3D speckle tracking algorithms, which was made available online. The aim of this study was therefore to use the newly developed database to contrast non-commercial speckle tracking solutions from research groups with leading expertise in the field. The five techniques involved cover the most representative families of existing approaches, namely block-matching, radio-frequency tracking, optical flow and elastic image registration. The techniques were contrasted in terms of tracking and strain accuracy. The feasibility of the obtained strain measurements to diagnose pathology was also tested for ischemia and dyssynchrony.

Description: Ultra-high field MRI is an area of great interest for clinical research and basic science due to the increased signal-to-noise, spatial resolution and magnetic-susceptibility-based contrast. However, the fact that the electromagnetic wavelength in tissue is comparable to the relevant body dimensions means that the uniformity of the excitation field is much poorer than at lower field strengths. In addition to techniques such as transmit arrays, one simple but effective method to counteract this effect is to use high permittivity “pads”. Very high permittivities enable thinner, flexible pads to be used, but the limiting factor is wavelength effects within the pads themselves, which can lead to image artifacts. So far, all studies have used simple continuous rectangular/circular pad geometries. In this work we investigate how the wavelength effects can be partially mitigated utilizing shaped pad with holes. Several arrangements have been simulated, including low order pre-fractal geometries, which maintain the overall coverage of the pad, but can provide better image homogeneity in the region of interest or higher sensitivity depending on the setup. Experimental data in the form of in vivo human images at 7T were acquired to validate the simulation results.

Description: In this paper, we consider combined TV denoising and diffusion tensor fitting in DTI using the affine-invariant Riemannian metric on the space of diffusion tensors. Instead of first fitting the diffusion tensors, and then denoising them, we define a suitable TV type energy functional which incorporates the measured DWIs (using an inverse problem setup) and which measures the nearness of neighboring tensors in the manifold. To approach this functional, we propose generalized forward- backward splitting algorithms which combine an explicit and several implicit steps performed on a decomposition of the functional. We validate the performance of the derived algorithms on synthetic and real DTI data. In particular, we work on real 3D data. To our knowledge, the present paper describes the first approach to TV regularization in a combined manifold and inverse problem setup.

Description: We present a generic method for automatic detection of abnormal regions in medical images as deviations from a normative data base. The algorithm decomposes an image, or more broadly a function defined on the image grid, into the superposition of a normal part and a residual term. A statistical model is constructed with regional sparse learning to represent normative anatomical variations among a reference population (e.g., healthy controls), in conjunction with a Markov random field regularization that ensures mutual consistency of the regional learning among partially overlapping image blocks. The decomposition is performed in a principled way so that the normal part fits well with the learned normative model, while the residual term absorbs pathological patterns, which may then be detected through a statistical significance test. The decomposition is applied to multiple image features from an individual scan, detecting abnormalities using both intensity and shape information. We form an iterative scheme that interleaves abnormality detection with deformable registration, gradually improving robustness of the spatial normalization and precision of the detection. The algorithm is evaluated with simulated images and clinical data of brain lesions, and is shown to achieve robust deformable registration and localize pathological regions simultaneously. The algorithm is also applied on images from Alzheimer’s disease patients to demonstrate the generality of the method.

Description: Staining and scanning of tissue samples for microscopic examination is fraught with undesirable color variations arising from differences in raw materials and manufacturing techniques of stain vendors, staining protocols of labs, and color responses of digital scanners. When comparing tissue samples, color normalization and stain separation of the tissue images can be helpful for both pathologists and software. Techniques that are used for natural images fail to utilize structural properties of stained tissue samples and produce undesirable color distortions. The stain concentration cannot be negative. Tissue samples are stained with only a few stains and most tissue regions are characterized by at most one effective stain. We model these physical phenomena that define the tissue structure by first decomposing images in an unsupervised manner into stain density maps that are sparse and non-negative. For a given image, we combine its stain density maps with stain color basis of a pathologist-preferred target image, thus altering only its color while preserving its structure described by the maps. Stain density correlation with ground truth and preference by pathologists were higher for images normalized using our method when compared to other alternatives. We also propose a computationally faster extension of this technique for large whole-slide images that selects an appropriate patch sample instead of using the entire image to compute the stain color basis.

Description: Prospective authors are requested to submit new, unpublished manuscripts for inclusion in the upcoming event described in this call for papers.

Description: We present a methodology to recover the geometrical calibration of conventional X-ray settings with the help of an ordinary video camera and visible fiducials that are present in the scene. After calibration, equivalent points of interest can be easily identifiable with the help of the epipolar geometry. The same procedure also allows the measurement of real anatomic lengths and angles and obtains accurate 3D locations from image points. Our approach completely eliminates the need for X-ray-opaque reference marks (and necessary supporting frames) which can sometimes be invasive for the patient, occlude the radiographic picture, and end up projected outside the imaging sensor area in oblique protocols. Two possible frameworks are envisioned: a spatially shifting X-ray anode around the patient/object and a moving patient that moves/rotates while the imaging system remains fixed. As a proof of concept, experiences with a device under test (DUT), an anthropomorphic phantom and a real brachytherapy session have been carried out. The results show that it is possible to identify common points with a proper level of accuracy and retrieve three-dimensional locations, lengths and shapes with a millimetric level of precision. The presented approach is simple and compatible with both current and legacy widespread diagnostic X-ray imaging deployments and it can represent a good and inexpensive alternative to other radiological modalities like CT.

Description: Herpes simplex viruses can cause uncommon systemic complications as acute liver failure or urinary tract dysfunctions. Diphenyl diselenide, (PhSe) 2 , a classical studied organic selenium compound, has a novel antiviral action against HSV-2 infection and well-known antioxidant and anti-inflammatory properties. This study aimed to investigate if (PhSe) 2 reduces oxidative stress and systemic toxicity caused by HSV-2 infection in mice. Adult BALB/c mice were pre-treated with (PhSe) 2 (5 mg kg −1 /day, intragastric, i.g.) during 5 days; at day 6 mice were infected with HSV-2 (10 µl-10 5 PFU/ml −1 ) and post-treated with (PhSe) 2 for more 5 days. At day 11, they were killed and samples of liver and kidney were obtained to determine: reactive species (RS); malondialdehyde (MDA) and non-protein thiols (NPSH) levels; the activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). The activities of adenosine deaminase (ADA), Na + /K + -ATPase (liver and kidney); alanine aminotransferase (ALT), aspartate aminotransferase (AST) and the levels of urea (plasma) were determined as markers of hepatic and renal toxicity. The results revealed that (PhSe) 2 treatment was effective against the increase of renal and hepatic oxidative stress in infected mice and also normalized hepatic and renal ADA activity. It recovered the activity of Na + /K + - and was not effective against the increase in urea levels in infected mice. Different from (PhSe) 2 , acyclovir (positive control), caused an increase in ADA activity and a decrease in hepatic CAT activity. Considering the interest of alternative therapies to treat HSV-2 infections and secondary complications, (PhSe) 2 become a notable candidate. This article is protected by copyright. All rights reserved

Description: Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. This article is protected by copyright. All rights reserved

Description: Apolipoprotein A-I (ApoA-I) is a key component of High Density Lipoproteins which possess anti-atherosclerotic and anti-inflammatory properties. Insulin is a crucial mediator of the glucose and lipid metabolism that has been implicated in atherosclerotic and inflammatory processes. Important mediators of insulin signaling such as Liver X Receptors (LXRs) and Forkhead Box A2 (FOXA2) are known to regulate apoA-I expression in liver. Forkhead Box O1 (FOXO1) is a well-known target of insulin signaling and a key mediator of oxidative stress response. Low doses of insulin were shown to activate apoA-I expression in human hepatoma HepG2 cells. However, the detailed mechanisms for these processes are still unknown. We studied the possible involvement of FOXO1, FOXA2, LXRα and LXRβ transcription factors in the insulin-mediated regulation of apoA-I expression. Treatment of HepG2 cells with high doses of insulin (48 hours, 100 nM) suppresses apoA-I gene expression. siRNAs against FOXO1, FOXA2, LXRβ or LXRα abrogated this effect. FOXO1 forms a complex with LXRβ and insulin treatment impairs FOXO1/LXRβ complex binding to hepatic enhancer and triggers its nuclear export. Insulin as well as LXR ligand TO901317 enhance the interaction between FOXA2, LXRα and hepatic enhancer. These data suggest that high doses of insulin downregulate apoA-I gene expression in HepG2 cells through redistribution of FOXO1/LXRβ complex, FOXA2 and LXRα on hepatic enhancer of apoA-I gene. This article is protected by copyright. All rights reserved

Description: ABSTRACT Chronic inflammation and metabolic reprogramming have been proposed as hallmarks of cancer development. Currently, many of the functional clues between these two phenomena are studied under the integrative view of functional stroma-epithelia interaction. It has been proposed that stromal cells, due to their abundance and avidity for glucose, are able to modify the metabolic behavior of an entire solid tumor”. In the present study, using a mammary stromal cell line derived from healthy tissue subjected to long-term culture in low (5 mM) or high (25 mM) glucose, we found that the hyperglycemic condition favors the establishment of a pro-inflammatory and pro-oxidant environment characterized by the induction of the COX-2/PGE2 axis. In this condition, epithelial migration was stimulated. Moreover, we also found that stromal-derived PGE2, acting as a stimulator of IL-1 epithelial expression was one of the factors that promote the acquisition of motile properties by epithelial cells and the maintenance of a COX-2/PGE2-dependent inflammatory condition. Overall, our work provides experimental evidence that glucose stimulates a tumor inflammatory environment that, as a result of a functional cross-talk between stroma and epithelia, may be responsible for tumor progression. This article is protected by copyright. All rights reserved

Description: The neural crest is a population of cells in the vertebrate embryo that gives rise to a wide range of tissues and cell types, including components of the peripheral nervous system and the craniofacial skeleton as well as melanocytes and the adrenal medulla. Aberrations in neural crest development can lead to numerous diseases, including cancers such as melanoma and neuroblastoma. Cancer stem cells (CSCs) have been identified in these neural crest-derived tumors, and these CSCs demonstrate resistance to treatment and are likely key contributors to disease relapse. Patients with neural crest-derived tumors often have poor outcomes due to frequent relapses, likely due to the continued presence of residual treatment-resistant CSCs, and therapies directed against these CSCs are likely to improve patient outcomes. CSCs share many of the same genetic and biologic features of primordial neural crest cells, and therefore a better understanding of neural crest development will likely lead to the development of effective therapies directed against these CSCs. Signaling through STAT3 has been shown to be required for neural crest development, and granulocyte colony stimulating factor (GCSF)-mediated activation of STAT3 has been shown to play a role in the pathogenesis of neural crest-derived tumors. Expression of the cell surface marker CD114 (the receptor for GCSF) has been identified as a potential marker for CSCs in neural crest-derived tumors, suggesting that CD114 expression and function may contribute to disease relapse and poor patient outcomes. In this review we review the processes of neural crest development and tumorigenesis and we discuss the previously identified markers for CSC subpopulations identified in neural crest tumors and their role in neural crest tumor biology. We also discuss the potential for CD114 and downstream intracellular signaling pathways as potential targets for CSC-directed therapy. This article is protected by copyright. All rights reserved

Description: Human topoisomerase I is partitioned between the nucleolus and the nucleoplasm in the interphase cells. Under unstressed conditions it is concentrated in the first compartment but nucleolar concentration of the full length protein is lost after inactivation of relaxation activity. Due to the above, subnuclear localization of topoisomerase I is linked with DNA relaxation activity of topoisomerase I. Looking for other factors responsible for subnuclear distribution of topoisomerase I, we studied here localization of the fluorescently tagged fragments and point mutants of topoisomerase I in HeLa cells. We found that two regions of topoisomerase I, the N-terminal and the linker domains, were critical for subnuclear localization of the enzyme. The linker domain and the distal region of the N-terminal domain directed topoisomerase I to the nucleolus, whereas the remaining region of the N-terminal domain was responsible for the nucleoplasmic localization. The effects exhibited by the regions which contributed to nuclear distribution of topoisomerase I were independent of DNA relaxation activity. Localization mutations in both domains complemented one another giving the wild type phenotype for the double mutant. These results suggest a two-stage model of regulation of partitioning of topoisomerase I between the nucleolus and the nucleoplasm. The first stage is a net of interactions provided by the N-terminal and the linker domains. The other stage, accessible only if the first net is balanced, is driven by DNA relaxation activity. This article is protected by copyright. All rights reserved

Description: ABSTRACT ZC3H12D belongs to a recently discovered family of proteins containing four members of which the most studied and best described is the RNase ZC3H12A (MCPIP1/Regnase-1). ZC3H12A is a crucial negative regulator of inflammation. It accelerates the turnover of transcripts of a spectrum of proinflammatory cytokines, as well as its own mRNA. The biological role of ZC3H12D is less clear, although it was shown that this member of ZC3H12 family is also involved in the regulation of inflammation. Here, we show that ZC3H12A and ZC3H12D recognize a set of common target mRNAs encoding proteins that play important roles in the course of the inflammation. Similarly to ZC3H12A, ZC3H12D participates in the 3'UTR-dependent regulation of the turnover of mRNAs encoding interleukin-6 (IL-6), tumor necrosis factor (TNF) and immediate early response 3 gene ( IER3 ). The ZC3H12A mRNA is also among the identified ZC3H12D targets. Using the combination of immunofluorescence with single molecule RNA fluorescence in situ hybridization (smRNA FISH) we have shown that ZC3H12D protein interacts with the ZC3H12A transcript. The direct binding of these two molecules in vivo was further confirmed by RNA immunoprecipitation. Simultaneously, overexpression of ZC3H12D increases the turnover rate of transcripts containing ZC3H12A 3'UTR. Using reporter gene assays we have confirmed that the Asp95 residue present in the NYN/PIN-like domain is crucial for ZC3H12D biological activity. We have also revealed that ZC3H12D recognizes the same structural elements present in the 3'UTRs of the investigated transcripts, as ZC3H12A. This article is protected by copyright. All rights reserved

Description: Fiber based fluorescence lifetime imaging has shown great potential for intraoperative diagnosis and guidance of surgical procedures. Here we describe a novel method addressing a significant challenge for the practical implementation of this technique, i.e., the real-time display of the quantified biochemical or functional tissue properties superimposed on the interrogated area. Specifically, an aiming beam (450 nm) generated by a continuous-wave laser beam was merged with the pulsed fluorescence excitation light in a single delivery/collection fiber and then imaged and segmented using a color-based algorithm. We demonstrate that this approach enables continuous delineation of the interrogated location and dynamic augmentation of the acquired frames with the corresponding fluorescence decay parameters. The method was evaluated on a fluorescence phantom and fresh tissue samples. Current results demonstrate that 34 frames per second can be achieved for augmenting videos of 640 $times $ 512 pixels resolution. Also we show that the spatial resolution of the fluorescence lifetime map depends on the tissue optical properties, the scanning speed, and the frame rate. The dice similarity coefficient between the fluorescence phantom and the reconstructed maps was estimated to be as high as 93%. The reported method could become a valuable tool for augmenting the surgeon's field of view with diagnostic information derived from the analysis of fluorescence lifetime data in real-time using handheld, automated, or endoscopic scanning systems. Current method provides also a means for maintaining the tissue light exposure within safety limits. This study provides a framework for using an aiming beam with other point spectroscopy applications.

Description: Simultaneous Multi-Slice (SMS) magnetic resonance imaging (MRI) is a rapidly evolving technique for increasing imaging speed. Controlled aliasing techniques utilize periodic undersampling patterns to help mitigate the loss in signal-to-noise ratio (SNR) in SMS MRI. To evaluate the performance of different undersampling patterns, a quantitative description of the image SNR loss is needed. Additionally, eddy current effects in echo planar imaging (EPI) lead to slice-specific Nyquist ghosting artifacts. These artifacts cannot be accurately corrected for each individual slice before or after slice-unaliasing. In this work, we propose a hybrid-space sensitivity encoding (SENSE) reconstruction framework for SMS MRI by adopting a three-dimensional representation of the SMS acquisition. Analytical SNR loss maps are derived for SMS acquisitions with arbitrary phase encoding undersampling patterns. Moreover, we propose a matrix-decoding correction method that corrects the slice-specific Nyquist ghosting artifacts in SMS EPI acquisitions. Brain images demonstrate that the proposed hybrid-space SENSE reconstruction generates images with comparable quality to commonly used split-slice-generalized autocalibrating partially parallel acquisition reconstruction. The analytical SNR loss maps agree with those calculated by a Monte Carlo based method, but require less computation time for high quality maps. The analytical maps enable a fair comparison between the performances of coherent and incoherent SMS undersampling patterns. Phantom and brain SMS EPI images show that the matrix-decoding method performs better than the single-slice and slice-averaged Nyquist ghosting correction methods under the hybrid-space SENSE reconstruction framework.

Description: Photoacoustic (PA) images utilize pulsed lasers and ultrasound transducers to visualize targets with higher optical absorption than the surrounding medium. However, they are susceptible to acoustic clutter and background noise artifacts that obfuscate biomedical structures of interest. We investigated three spatial-angular compounding methods to improve PA image quality for biomedical applications, implemented by combining multiple images acquired as an ultrasound probe was rotated about the elevational axis with the laser beam and target fixed. Compounding with conventional averaging was based on the pose information of each PA image, while compounding with weighted and selective averaging utilized both the pose and image content information. Weighted-average compounding enhanced PA images with the least distortion of signal size, particularly when there were large (i.e., 2.5 mm and 7 $^{circ}$ ) perturbations from the initial probe position. Selective-average compounding offered the best improvement in image quality with up 181, 1665, and 1568 times higher contrast, CNR, and SNR, respectively, compared to the mean values of individual PA images. The three presented spatial compounding methods have promising potential to enhance image quality in multiple photoacoustic applications.

Description: Analysis of cranial nerve systems, such as the anterior visual pathway (AVP), from MRI sequences is challenging due to their thin long architecture, structural variations along the path, and low contrast with adjacent anatomic structures. Segmentation of a pathologic AVP (e.g., with low-grade gliomas) poses additional challenges. In this work, we propose a fully automated partitioned shape model segmentation mechanism for AVP steered by multiple MRI sequences and deep learning features. Employing deep learning feature representation, this framework presents a joint partitioned statistical shape model able to deal with healthy and pathological AVP. The deep learning assistance is particularly useful in the poor contrast regions, such as optic tracts and pathological areas. Our main contributions are: 1) a fast and robust shape localization method using conditional space deep learning, 2) a volumetric multiscale curvelet transform-based intensity normalization method for robust statistical model, and 3) optimally partitioned statistical shape and appearance models based on regional shape variations for greater local flexibility. Our method was evaluated on MRI sequences obtained from 165 pediatric subjects. A mean Dice similarity coefficient of 0.779 was obtained for the segmentation of the entire AVP (optic nerve only $=0.791$ ) using the leave-one-out validation. Results demonstrated that the proposed localized shape and sparse appearance-based learning approach significantly outperforms current state-of-the-art segmentation approaches and is as robust as the manual segmentation.

Description: Analytical (closed-form) inversion schemes have been the standard approach for image reconstruction in optoacoustic tomography due to their fast reconstruction abilities and low memory requirements. Yet, the need for quantitative imaging and artifact reduction has led to the development of more accurate inversion approaches, which rely on accurate forward modeling of the optoacoustic wave generation and propagation. In this way, multiple experimental factors can be incorporated, such as the exact detection geometry, spatio-temporal response of the transducers, and acoustic heterogeneities. The model-based inversion commonly results in very large sparse matrix formulations that require computationally extensive and memory demanding regularization schemes for image reconstruction, hindering their effective implementation in real-time imaging applications. Herein, we introduce a new discretization procedure for efficient model-based reconstructions in two-dimensional optoacoustic tomography that allows for parallel implementation on a graphics processing unit (GPU) with a relatively low numerical complexity. By on-the-fly calculation of the model matrix in each iteration of the inversion procedure, the new approach results in imaging frame rates exceeding 10 Hz, thus enabling real-time image rendering using the model-based approach.

Description: This paper introduces a statistical estimation framework for magnetic resonance (MR) fingerprinting, a recently proposed quantitative imaging paradigm. Within this framework, we present a maximum likelihood (ML) formalism to estimate multiple MR tissue parameter maps directly from highly undersampled, noisy ${bf k}$ -space data. A novel algorithm, based on variable splitting, the alternating direction method of multipliers, and the variable projection method, is developed to solve the resulting optimization problem. Representative results from both simulations and in vivo experiments demonstrate that the proposed approach yields significantly improved accuracy in parameter estimation, compared to the conventional MR fingerprinting reconstruction. Moreover, the proposed framework provides new theoretical insights into the conventional approach. We show analytically that the conventional approach is an approximation to the ML reconstruction; more precisely, it is exactly equivalent to the first iteration of the proposed algorithm for the ML reconstruction, provided that a gridding reconstruction is used as an initialization.

Description: We propose a unified Bayesian framework to detect both hyper- and hypo-active communities within whole-brain fMRI data. Specifically, our model identifies dense subgraphs that exhibit population-level differences in functional synchrony between a control and clinical group. We derive a variational EM algorithm to solve for the latent posterior distributions and parameter estimates, which subsequently inform us about the afflicted network topology. We demonstrate that our method provides valuable insights into the neural mechanisms underlying social dysfunction in autism, as verified by the Neurosynth meta-analytic database. In contrast, both univariate testing and community detection via recursive edge elimination fail to identify stable functional communities associated with the disorder.

Description: A recent entry into the rapidly evolving field of integrated PET/MR scanners is presented in this paper: a whole body hybrid PET/MR system (SIGNA PET/MR, GE Healthcare) capable of simultaneous acquisition of both time-of-flight (TOF) PET and high resolution MR data. The PET ring was integrated into an existing 3T MR system resulting in a (patient) bore opening of 60 cm diameter, with a 25 cm axial FOV. PET performance was evaluated both on the standalone PET ring and on the same detector integrated into the MR system, to assess the level of mutual interference between both subsystems. In both configurations we obtained detector performance data. PET detector performance was not significantly affected by integration into the MR system. The global energy resolution was within 2% (10.3% versus 10.5%), and the system coincidence time resolution showed a maximum change of 〈 3% (385 ps versus 394 ps) when measured outside MR and during simultaneous PET/MRI acquisitions, respectively. To evaluate PET image quality and resolution, the NEMA IQ phantom was acquired with MR idle and with MR active. Impact of PET on MR IQ was assessed by comparing SNR with PET acquisition on and off. B0 and B1 homogeneities were acquired before and after the integration of the PET ring inside the magnet. In vivo brain and whole body head-to-thighs data were acquired to demonstrate clinical image quality.

Description: The performance of photoacoustic microscopy (PAM) degrades due to the turbidity of the skull that introduces attenuation and distortion of both laser and stimulated ultrasound. In this manuscript, we demonstrated that a newly developed skull optical clearing solution (SOCS) could enhance not only the transmittance of light, but also that of ultrasound in the skull in vitro. Thus the photoacoustic signal was effectively elevated, and the relative strength of the artifacts induced by the skull could be suppressed. Furthermore in vivo studies demonstrated that SOCS could drastically enhance the performance of photoacoustic microscopy for cerebral microvasculature imaging.

Description: Cryo-balloon catheters have attracted an increasing amount of interest in the medical community as they can reduce patient risk during left atrial pulmonary vein ablation procedures. As cryo-balloon catheters are not equipped with electrodes, they cannot be localized automatically by electro-anatomical mapping systems. As a consequence, X-ray fluoroscopy has remained an important means for guidance during the procedure. Most recently, image guidance methods for fluoroscopy-based procedures have been proposed, but they provide only limited support for cryo-balloon catheters and require significant user interaction. To improve this situation, we propose a novel method for automatic cryo-balloon catheter detection in fluoroscopic images by detecting the cryo-balloon catheter's built-in X-ray marker. Our approach is based on a blob detection algorithm to find possible X-ray marker candidates. Several of these candidates are then excluded using prior knowledge. For the remaining candidates, several catheter specific features are introduced. They are processed using a machine learning approach to arrive at the final X-ray marker position. Our method was evaluated on 75 biplane fluoroscopy images from 40 patients, from two sites, acquired with a biplane angiography system. The method yielded a success rate of 99.0% in plane A and 90.6% in plane B, respectively. The detection achieved an accuracy of $1.00~{rm mm}pm 0.82~{rm mm}$ in plane A and $1.13~{rm mm}pm 0.24~{rm mm}$ in plane B. The localization in 3-D was associated with an average error of $0.36~{rm mm}pm 0.86~{rm mm}$ .

Description: Segmentation of the wrist bones in CT images has been frequently used in different clinical applications including arthritis evaluation, bone age assessment and image-guided interventions. The major challenges include non-uniformity and spongy textures of the bone tissue as well as narrow inter-bone spaces. In this work, we propose an automatic wrist bone segmentation technique for CT images based on a statistical model that captures the shape and pose variations of the wrist joint across 60 example wrists at nine different wrist positions. To establish the correspondences across the training shapes at neutral positions, the wrist bone surfaces are jointly aligned using a group-wise registration framework based on a Gaussian Mixture Model. Principal component analysis is then used to determine the major modes of shape variations. The variations in poses not only across the population but also across different wrist positions are incorporated in two pose models. An intra-subject pose model is developed by utilizing the similarity transforms at all wrist positions across the population. Further, an inter-subject pose model is used to model the pose variations across different wrist positions. For segmentation of the wrist bones in CT images, the developed model is registered to the edge point cloud extracted from the CT volume through an expectation maximization based probabilistic approach. Residual registration errors are corrected by application of a non-rigid registration technique. We validate the proposed segmentation method by registering the wrist model to a total of 66 unseen CT volumes of average voxel size of 0.38 mm. We report a mean surface distance error of 0.33 mm and a mean Jaccard index of 0.86.

Description: ABSTRACT The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has driven efforts to develop Hh pathway inhibitors as anti-cancer agents. The majority of these agents antagonize Smoothened (Smo), a plasma membrane-associated signal transducer molecule. However, several such Smo antagonists have failed in clinical trials to benefit patients with cancers that arise from aberrant Hh signaling (which often bypasses Smo). In this study, we report that a naturally occurring oxysterol, 20α, 22( R )-dihydroxycholesterol (Oxy16), a known metabolite in the biosynthesis of steroid hormones, strongly inhibits Hh signaling induced in C3H10T1/2 embryonic fibroblasts and NIH3T3-E1 fibroblasts through a mechanism that is independent of liver X receptor (LXR) activation. We demonstrate that Oxy16 inhibits Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells, indicating that its inhibitory effect on Hh signaling is epistatic to Sufu. We further demonstrate that Oxy16 inhibits Gli1 transcriptional activity in NIH3T3-E1 cells overexpressing Gli1 and a Gli-dependent reporter construct. Altogether, data presented here suggest that Oxy16 may be a suitable starting point for the development of new drugs that inhibit Hh signaling downstream of Smo. By targeting aberrant Hh signaling, such novel Hh pathway inhibitors could significantly broaden the range of clinical applications compared to existing Smo antagonists. Furthermore, the present study adds a new facet to the spectrum of Hh pathway modulation that naturally occurring oxysterol derivatives are capable of, ranging from allosteric activation of the pathway via Smo binding to inhibition of the pathway downstream of Smo. This article is protected by copyright. All rights reserved

Description: Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic humans and animals. We recently reported that targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non-obese mice. To elucidate the mechanism, we examined human proximal tubule cells (hPTC) and C57BL/6 mice fed with high-fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower protein level of IR in HFD compare to normal chow diet (NCD). Furthermore, a blunted rise in p-AKT 308 levels in the kidney cortex of HFD mice was observed in response to acute insulin (0.75 IU/kg body weight, i.p) relative to NCD n = 8/group, p 〈 0.05). Moreover, we found significantly higher transcript levels of phosphoenolpyruvate carboxykinase (PEPCK, a key gluconeogenic enzyme) in the kidney cortex from HFD, relative to mice on NCD. The higher level of PEPCK in HFD was confirmed by immunoblotting. However, no significant differences were observed in cortical glucose-6-phosphatase (G6Pase) or fructose-1,6, bisphosphosphatase (FBPase) enzyme transcript levels. Furthermore, we demonstrated insulin inhibited glucose production in hPTC treated with cyclic AMP and dexamethasone (cAMP/DEXA) to stimulate gluconeogenesis. Transcript levels of the gluconeogenic enzyme PEPCK were significantly increased in cAMP/DEXA-stimulated hPTC cells (n = 3, p 〈 0.05), and insulin attenuated this upregulation Furthermore, the effect of insulin on cAMP/DEXA–induced gluconeogenesis and PEPCK induction was significantly attenuated in IR (siRNA) silenced hPTC (n = 3, p 〈 0.05). Overall the above data indicate a direct role for IR expression as a determinant of PT-gluconeogenesis. Thus reduced insulin signaling of the proximal tubule may contribute to hyperglycemia in the metabolic syndrome via elevated gluconeogenesis. This article is protected by copyright. All rights reserved

Description: ABSTRACT Osteoblasts are essential for maintaining skeletal architecture and modulating bone microenvironment homeostasis. From numerous associated investigations, the BMP-2 pathway has been well-defined as a vital positive modulator of bone homeostasis. Gremlin2 (Grem2) is a bone morphogenetic protein (BMP) antagonists. However, the effect of Grem2 on the BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs) remains ambiguous. This study aimed to analyze the procedure in vitro and in vivo. The differentiation of hBMSCs was assessed by determining the expression levels of several osteoblastic genes, as well as the enzymatic activity and calcification of alkaline phosphatase. We found that Grem2 expression was upregulated by BMP-2 within the range of 0–1 µg/mL, and significant increases were evident at 48, 72, and 96 h after BMP-2 treatment. Si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs, whereas over-expression of Grem2 had the opposite trend. The result was confirmed using a defective femur model. We also discovered that the BMP-2/Smad/Runx2 pathway played an important role in the process. This study showed that si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs via the BMP-2/Smad/Runx2 pathway. This article is protected by copyright. All rights reserved

Description: The expression of Caudal -related homeobox transcription factor 2 (CDX2) is impaired by tumor necrosis factor-α (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) in ulcerative colitis (UC). Laminin subunit γ2 (LAMC2) is an epithelial basement membrane protein implicated in cell migration, proliferation, differentiation as well as tumor invasion and intestinal inflammation, and its expression is enhanced by TNF-α in a NF-κB-dependent regulation of the recently identified LAMC2 enhancer. The aim was to determine whether CDX2 is involved in the basal regulation of LAMC2 in epithelial cells and to assess the influence of inflammation. Transcriptional regulation of LAMC2 was examined by reporter gene assays, overexpression, and shRNA-mediated knock-down of CDX2 . CDX2-DNA interactions were assessed by chromatin immunoprecipitation on Caco-2 cells without or with TNF-α as well as in purified colonic human epithelial cells. Immunohistochemical staining and quantitative reverse-transcription polymerase chain reaction analyses were used to measure the expression of CDX2 and LAMC2 in colonic biopsies from healthy controls and patients with UC. These data indicate that CDX2 directly regulates LAMC2 gene expression through interaction with elements in the LAMC2 promoter region. We further revealed an inverse effect of inflammation on CDX2 and LAMC2. The data presented provide a novel insight into how CDX2 is implicated in the transcriptional regulation of LAMC2 in intestinal epithelial cells, a function that is impaired during mucosal inflammation where a high level of TNF-α is present. This article is protected by copyright. All rights reserved

Description: Human multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next-generation sequencer to investigate the changes in gene expression during the osteoblastic differentiation of hMSCs. The hMSCs used in this study possessed both multipotency and self-renewal ability. Whole-transcriptome analysis revealed that the expression of zinc finger and BTB domain containing 16 (ZBTB16) was significantly increased during the osteoblastogenesis of hMSCs. ZBTB16 mRNA and protein expression was enhanced by culturing the hMSCs with OIM. Small interfering RNA (siRNA)-mediated gene silencing of ZBTB16 decreased the activity of alkaline phosphatase (ALP); the expression of osteogenic genes, such as osteocalcin (OCN) and bone sialoprotein (BSP); and the mineralized nodule formation induced by OIM. siRNA-mediated gene silencing of Osterix (Osx), which is known as an essential regulator of osteoblastic differentiation, markedly downregulated the expression of ZBTB16. In addition, chromatin immunoprecipitation (ChIP) assays showed that Osx associated with the ZBTB16 promoter region containing the GC-rich canonical Sp1 sequence, which is the specific Osx binding site. These findings suggest that ZBTB16 acts as a downstream transcriptional regulator of Osx and can be useful as a late marker of osteoblastic differentiation. This article is protected by copyright. All rights reserved

Description: Phototherapy is the most common therapy used for severe jaundice. There is increasing evidence that phototherapy can directly affect the expression and function of cell surface receptors including adhesion molecules, cytokines, and growth factor receptors. In this study, the effect of two infantile phototherapy regimens, including single and intensive phototherapy was investigated on biological features of circulation endothelial progenitor cells (cEPCs) as well as on serum secretion of two important chemotactic cytokines, SDF-1 and VEGF. Sixty infants diagnosed with severe hyperbilirubinemia and exposed to phototherapy were enrolled in this study. cEPCs were isolated before and after phototherapy and then migratory, proliferative, tubulogenic, and functional properties of these cells were analyzed. Our results revealed that intensive phototherapy markedly increased the release of EPCs into the circulation, and augmented the serum concentrations of both SDF-1 and VEGF cytokines. Cell proliferation, tubulogenic, and migratory properties of cEPCs isolated and expanded from infants with intensive phototherapy were significantly improved. cEPCs from infants with intensive phototherapy also showed greater levels of acetylated low-density lipoprotein and lectin binding. Overall, our results showed that the intensive phototherapy regimen can mobilize functional EPCs into the circulation through up-regulation of serum levels of VEGF and SDF-1, indicating phototherapy as an effective modality for improvement of stem cell mobilization in the therapeutic regenerative medicine. This article is protected by copyright. All rights reserved

Description: ABSTRACT The mammalian hairless (Hr) protein plays critical roles in skin and brain tissues, but how it interacts with DNA and partner protein is only now being defined. Our initial tests of four consensus response elements, revealed that rat Hr can specifically bind to a consensus p53 response element (p53RE), 5'-AGACATGCCTAGACATGCCT-3', but not to response elements for NF-κB, TCF4 or Sp1. We then employed ChIP assays which verified that human HR binds to a p53RE of the GADD45A gene in both HEK293 (embryonic kidney) and U87 (glioblastoma) cells. Further, HR was shown to interact directly with the p53 protein in a co-immunoprecipitation assay. Cotransfections with p53RE reporter gene constructs revealed that rat Hr can boost p53-mediated transactivation of a reporter gene linked to the GADD45A p53RE, but blunts p53-mediated transactivation when the reporter gene is linked to a p21 promoter fragment containing a p53RE, with implications for the regulation of these two cell cycle control genes. Finally, our investigations of HR phosphorylation revealed that rat Hr is a substrate for PKC, but not PKA, and that human HR is phosphorylated in intact U87 cells at Ser-416, located in a highly conserved region which partially fulfills the criteria of a PKC site. We propose that mammalian Hr is a phosphoprotein which can exert cross-talk with the p53 pathway with important implications for the regulation of cell proliferation and differentiation in tissues such as skin and brain where Hr is highly expressed. This article is protected by copyright. All rights reserved

Description: The majority of advanced breast cancers have genetic alterations that are potentially targetable with drugs. Through initiatives such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), data can be mined to provide context for next-generation sequencing (NGS) results in the landscape of advanced breast cancer. Therapies for targets other than estrogen receptor alpha (ER) and HER2, such as cyclin-dependent kinases CDK4 and CDK6, were recently approved based on efficacy in patient subpopulations, but no predictive biomarkers have been found, leaving clinicians to continue a trial-and-error approach with each patient. Next-generation sequencing identifies potentially actionable alterations in genes thought to be drivers in the cancerous process including phosphatidylinositol 3-kinase (PI3K), AKT, fibroblast growth factor receptors (FGFRs), and mutant HER2. Epigenetically-directed and immunologic therapies have also shown promise for the treatment of breast cancer via histone deacetylases (HDAC) 1 and 3, programmed T cell death 1 (PD-1), and programmed T cell death ligand 1 (PD-L1). Identifying biomarkers to predict primary resistance in breast cancer will ultimately affect clinical decisions regarding adjuvant therapy in the first-line setting. However, the bulk of medical decision-making is currently made in the secondary resistance setting. Herein, we review the clinical potential of PI3K, AKT, FGFRs, mutant HER2, HDAC1/3, PD-1, and PD-L1 as therapeutic targets in breast cancer, focusing on the rationale for therapeutic development and the status of clinical testing. This article is protected by copyright. All rights reserved

Description: Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer related deaths among women worldwide. The purpose of this study is to evaluate the cytotoxic effects and possible molecular mechanisms of the antiproliferative properties of the antiangiogenic 1-aryl-3-[3-(thieno[3,2- b ]pyridin-7-ylthio)phenyl]ureas 1a - e , prepared earlier by us, on two human breast cancer cell lines of distinct histological types: hormone-dependent MCF-7 (ER positive), and hormone independent MDA-MB-231 (ER/PR/HER2 negative), this latter being the most aggressive and difficult to treat. Our findings clearly demonstrated that compounds 1a - e suppress breast cancer cell survival, proliferation, migration and colony formation at very low concentrations, not showing cytotoxicity in normal human mammary cells (MCF-10A). TUNEL assay demonstrated that compounds 1a - e induced apoptosis in MDA-MB-231, but not in MCF-7 at the concentrations tested. PI3K/Akt and MAPK/Erk cell signaling pathways were investigated using Western blot analysis, revealing that these compounds decrease their activity in both breast cancer cell lines. Compounds 1b (R 2  = F) 1c (R 2  = Me) and 1e (R 1  = Cl, R 2  = CF 3 ) were the most effective particularly in MDA-MB-231 cells. Overall, 1c and 1e compounds are the most promising antitumor compounds. These findings, together with the antiangiogenic activity previously described by us, render these compounds a relevant breakthrough for cancer therapy. This article is protected by copyright. All rights reserved

Description: ABSTRACT Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of µ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor µ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. This article is protected by copyright. All rights reserved