Search

Glioblastomas (GBM) are largely incurable as they diffusely infiltrate adjacent brain tissues and are difficult to diagnose at early stages. Biomarkers derived from serum, which can be obtained by minimally invasive procedures, may help in early diagnosis, prognosis and treatment monitoring. To develop a serum cytokine signature, we profiled 48 cytokines in sera derived from normal healthy individuals (n = 26) and different grades of glioma patients (n = 194). We divided the normal and grade IV glioma/GBM serum samples randomly into equal sized training and test sets. In the training set, the Prediction Analysis for Microarrays (PAM) identified a panel of 18 cytokines that could discriminate GBM sera from normal sera with maximum accuracy (95.40%) and minimum error (4.60%). The 18-cytokine signature obtained in the training set discriminated GBM sera from normal sera in the test set as well (accuracy 96.55%; error 3.45%). Interestingly, the 18-cytokine signature also differentiated grade II/Diffuse Astrocytoma (DA) and grade III/Anaplastic Astrocytoma (AA) sera from normal sera very efficiently (DA vs. normal-accuracy 96.00%, error 4.00%; AA vs. normal-accuracy 95.83%, error 4.17%). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using 18 cytokines resulted in the enrichment of two pathways, cytokine-cytokine receptor interaction and JAK-STAT pathways with high significance. Thus our study identified an 18-cytokine signature for distinguishing glioma sera from normal healthy individual sera and also demonstrated the importance of their differential abundance in glioma biology.

Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested.

The Neanthes acuminata species complex (Polychaeta) are cosmopolitan in distribution. Neanthesarenaceodentata , Southern California member of the N . acuminata complex, has been widely used as toxicological test animal in the marine environment. Method of reproduction is unique in this polychaete complex. Same sexes fight and opposite sexes lie side by side until egg laying. Females lose about 75% of their weight and die after laying eggs. The male, capable of reproducing up to nine times, fertilizes the eggs and incubates the embryos for 3-4 weeks. The objective of this study was to determine if there is any set of proteins that influences this unique pattern of reproduction. Gel-based two-dimensional electrophoresis (2-DE) and gel-free quantitative proteomics methods were used to identify differential protein expression patterns before and after spawning in both male and female N . arenaceodentata . Males showed a higher degree of similarity in protein expression patterns but females showed large changes in phosphoproteme before and after spawning. There was a decrease (about 70%) in the number of detected phosphoproteins in spent females. The proteins involved in muscular development, cell signaling, structure and integrity, and translation were differentially expressed. This study provides proteomic insights of the male and female worms that may serve as a foundation for better understanding of unusual reproductive patterns in polychaete worms.

Gli3 is a transcriptional regulator of Hedgehog (Hh) signaling that functions as a repressor (Gli3(R)) or activator (Gli3(A)) depending upon cellular context. Previously, we have shown that Gli3(R) is required for the formation of mammary placodes #3 and #5. Here, we report that this early loss of Gli3 results in abnormal patterning of two critical regulators: Bmp4 and Tbx3, within the presumptive mammary rudiment (MR) #3 zone. We also show that Gli3 loss leads to failure to maintain mammary mesenchyme specification and loss of epithelial Wnt signaling, which impairs the later development of remaining MRs: MR#2 showed profound evagination and ectopic hairs formed within the presumptive areola; MR#4 showed mild invagination defects and males showed inappropriate retention of mammary buds in Gli3(xt/xt) mice. Importantly, mice genetically manipulated to misactivate Hh signaling displayed the same phenotypic spectrum demonstrating that the repressor function of Gli3(R) is essential during multiple stages of mammary development. In contrast, positive Hh signaling occurs during nipple development in a mesenchymal cuff around the lactiferous duct and in muscle cells of the nipple sphincter. Collectively, these data show that repression of Hh signaling by Gli3(R) is critical for early placodal patterning and later mammary mesenchyme specification whereas positive Hh signaling occurs during nipple development.

Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×10(5) M(-1), -7.175 Kcal M(-1) for coumarin derivative (CD) enamide; 0.837±0.01×10(5) M(-1), -6.685 Kcal M(-1) for coumarin derivative (CD) enoate, and 0.606±0.01×10(5) M(-1), -6.49 Kcal M(-1) for coumarin derivative methylprop (CDM) enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

Robot-aided gait therapy offers a promising approach towards improving gait function in individuals with neurological disorders such as stroke or spinal cord injury. However, incorporation of appropriate control strategies is essential for actively engaging the patient in the therapeutic process. Although several control algorithms (such as assist-as-needed and error augmentation) have been proposed to improve active patient participation, we hypothesize that the therapeutic benefits of these control algorithms can be greatly enhanced if combined with a motor learning task to facilitate neural reorganization and motor recovery. Here, we describe an active robotic training approach (patient-cooperative robotic gait training combined with a motor learning task) using the Lokomat and pilot-tested whether this approach can enhance active patient participation during training. Six neurologically intact adults and three chronic stroke survivors participated in this pilot feasibility study. Participants walked in a Lokomat while simultaneously performing a foot target-tracking task that necessitated greater hip and knee flexion during the swing phase of the gait. We computed the changes in tracking error as a measure of motor performance and changes in muscle activation as a measure of active subject participation. Repeated practice of the motor-learning task resulted in significant reductions in target-tracking error in all subjects. Muscle activation was also significantly higher during active robotic training compared to simply walking in the robot. The data from stroke participants also showed a trend similar to neurologically intact participants. These findings provide a proof-of-concept demonstration that combining robotic gait training with a motor learning task enhances active participation.

The study of uterine leiomyomata (fibroids) provides a unique opportunity to investigate the physiological and molecular determinants of hormone dependent tumor growth and spontaneous tumor regression. We conducted a longitudinal clinical study of premenopausal women with leiomyoma that showed significantly different growth rates between white and black women depending on their age. Growth rates for leiomyoma were on average much higher from older black women than for older white women, and we now report gene expression pattern differences in tumors from these two groups of study participants. Total RNA from 52 leiomyoma and 8 myometrial samples were analyzed using Affymetrix Gene Chip expression arrays. Gene expression data was first compared between all leiomyoma and normal myometrium and then between leiomyoma from older black women (age 35 or older) and from older white women. Genes that were found significant in pairwise comparisons were further analyzed for canonical pathways, networks and biological functions using the Ingenuity Pathway Analysis (IPA) software. Whereas our comparison of leiomyoma to myometrium produced a very large list of genes highly similar to numerous previous studies, distinct sets of genes and signaling pathways were identified in comparisons of older black and white women whose tumors were likely to be growing and non-growing, respectively. Key among these were genes associated with regulation of apoptosis. To our knowledge, this is the first study to compare two groups of tumors that are likely to have different growth rates in order to reveal molecular signals likely to be influential in tumor growth.

Background As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures. Methodology/Principal Findings Here we demonstrate that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, we show that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis. Conclusions/Significance Collectively, our analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation.

BACKGROUND: Iron is an essential micronutrient for all organisms because it is a component of enzyme cofactors that catalyze redox reactions in fundamental metabolic processes. Even though iron is abundant on earth, it is often present in the insoluble ferric [Fe (III)] state, leaving many surface environments Fe-limited. The haploid green alga Chlamydomonas reinhardtii is used as a model organism for studying eukaryotic photosynthesis. This study explores structural and functional changes in PSI-LHCI supercomplexes under Fe deficiency as the eukaryotic photosynthetic apparatus adapts to Fe deficiency. RESULTS: 77K emission spectra and sucrose density gradient data show that PSI and LHCI subunits are affected under iron deficiency conditions. The visible circular dichroism (CD) spectra associated with strongly-coupled chlorophyll dimers increases in intensity. The change in CD signals of pigments originates from the modification of interactions between pigment molecules. Evidence from sucrose gradients and non-denaturing (green) gels indicates that PSI-LHCI levels were reduced after cells were grown for 72 h in Fe-deficient medium. Ultrafast fluorescence spectroscopy suggests that red-shifted pigments in the PSI-LHCI antenna were lost during Fe stress. Further, denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI subunits PsaC and PsaD decreased, while PsaE was completely absent after Fe stress. The light harvesting complexes were also susceptible to iron deficiency, with Lhca1 and Lhca9 showing the most dramatic decreases. These changes in the number and composition of PSI-LHCI supercomplexes may be caused by reactive oxygen species, which increase under Fe deficiency conditions. CONCLUSIONS: Fe deficiency induces rapid reduction of the levels of photosynthetic pigments due to a decrease in chlorophyll synthesis. Chlorophyll is important not only as a light-harvesting pigment, but also has a structural role, particularly in the pigment-rich LHCI subunits. The reduced level of chlorophyll molecules inhibits the formation of large PSI-LHCI supercomplexes, further decreasing the photosynthetic efficiency.

The polychaete Neanthes arenaceodentata- is cosmopolitan in distribution-, has been used as a laboratory test animal. Life history of this species has several unique features; the female dies after spawning and the male incubates the fertilized eggs through the 21-segmented stage. The larvae leave the tube and commence feeding. Changes in protein abundance and phosphorylation were examined during early development of N. arenaceodentata. A gel-based approach and gel-free enrichment of phosphopeptides coupled with mass spectrometry were used to identify proteins and phosphopeptides in fertilized ova and larval stages. Patterns of proteins and phosphoproteins changed from fertilized ova to larval stages. Twelve proteins occurred in phosphorylated form and nine as stage specific proteins. Cytoskeletal proteins have exhibited differential phosphorylation from ova to larval stages; whereas, other proteins exhibited stage-specific phosphorylation patterns. Ten phosphopeptides were identified that showed phosphorylation sites on serine or threonine residues. Sixty percent of the identified proteins were related to structural reorganization and others with protein synthesis, stress response and attachment. The abundance and distribution of two cytoskeleton proteins were examined further by 2-DE Western blot analysis. This is the first report on changes in protein expression and phosphorylation sites at Thr/Ser in early development of N. arenaceodentata. The 2-DE proteome maps and identified phosphoproteins contributes toward understanding the state of fertilized ova and early larval stages and serves as a basis for further studies on proteomics changes under different developmental conditions in this and other polychaete species.

Butenolide [5-octylfuran-2(5H)-one] is a recently discovered and very promising anti-marine-fouling compound. In this study, the acute toxicity of butenolide was assessed in several non-target organisms, including micro algae, crustaceans, and fish. Results were compared with previously reported results on the effective concentrations used on fouling (target) organisms. According to OECD's guideline, the predicted no effect concentration (PNEC) was 0.168 µg l(-1), which was among one of the highest in representative new biocides. Mechanistically, the phenotype of butenolide-treated Danio rerio (zebrafish) embryos was similar to the phenotype of the pro-caspase-3 over-expression mutant with pericardial edema, small eyes, small brains, and increased numbers of apoptotic cells in the bodies of zebrafish embryos. Butenolide also induced apoptosis in HeLa cells, with the activation of c-Jun N-terminal kinases (JNK), Bcl-2 family proteins, and caspases and proteasomes/lysosomes involved in this process. This is the first detailed toxicity and toxicology study on this antifouling compound.

Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.

Background Dendritic morphology largely determines patterns of synaptic connectivity and electrochemical properties of a neuron. Neurons display a myriad diversity of dendritic geometries which serve as a basis for functional classification. Several types of molecules have recently been identified which regulate dendrite morphology by acting at the levels of transcriptional regulation, direct interactions with the cytoskeleton and organelles, and cell surface interactions. Although there has been substantial progress in understanding the molecular mechanisms of dendrite morphogenesis, the specification of class-specific dendritic arbors remains largely unexplained. Furthermore, the presence of numerous regulators suggests that they must work in concert. However, presently, few genetic pathways regulating dendrite development have been defined. Methodology/Principal Findings The Drosophila gene turtle belongs to an evolutionarily conserved class of immunoglobulin superfamily members found in the nervous systems of diverse organisms. We demonstrate that Turtle is differentially expressed in Drosophila da neurons. Moreover, MARCM analyses reveal Turtle acts cell autonomously to exert class specific effects on dendritic growth and/or branching in da neuron subclasses. Using transgenic overexpression of different Turtle isoforms, we find context-dependent, isoform-specific effects on mediating dendritic branching in class II, III and IV da neurons. Finally, we demonstrate via chromatin immunoprecipitation, qPCR, and immunohistochemistry analyses that Turtle expression is positively regulated by the Cut homeodomain transcription factor and via genetic interaction studies that Turtle is downstream effector of Cut-mediated regulation of da neuron dendrite morphology. Conclusions/Significance Our findings reveal that Turtle proteins differentially regulate the acquisition of class-specific dendrite morphologies. In addition, we have established a transcriptional regulatory interaction between Cut and Turtle, representing a novel pathway for mediating class specific dendrite development.

Background Neurons are one of the most structurally and functionally diverse cell types found in nature, owing in large part to their unique class specific dendritic architectures. Dendrites, being highly specialized in receiving and processing neuronal signals, play a key role in the formation of functional neural circuits. Hence, in order to understand the emergence and assembly of a complex nervous system, it is critical to understand the molecular mechanisms that direct class specific dendritogenesis. Methodology/Principal Findings We have used the Drosophila dendritic arborization (da) neurons to gain systems-level insight into dendritogenesis by a comparative study of the morphologically distinct Class-I (C-I) and Class-IV (C-IV) da neurons. We have used a combination of cell-type specific transcriptional expression profiling coupled to a targeted and systematic in vivo RNAi functional validation screen. Our comparative transcriptomic analyses have revealed a large number of differentially enriched/depleted gene-sets between C-I and C-IV neurons, including a broad range of molecular factors and biological processes such as proteolytic and metabolic pathways. Further, using this data, we have identified and validated the role of 37 transcription factors in regulating class specific dendrite development using in vivo class-specific RNAi knockdowns followed by rigorous and quantitative neurometric analysis. Conclusions/Significance This study reports the first global gene-expression profiles from purified Drosophila C-I and C-IV da neurons. We also report the first large-scale semi-automated reconstruction of over 4,900 da neurons, which were used to quantitatively validate the RNAi screen phenotypes. Overall, these analyses shed global and unbiased novel insights into the molecular differences that underlie the morphological diversity of distinct neuronal cell-types. Furthermore, our class-specific gene expression datasets should prove a valuable community resource in guiding further investigations designed to explore the molecular mechanisms underlying class specific neuronal patterning.

Background: Recent research on glioblastoma (GBM) has focused on deducing gene signatures predicting prognosis. The present study evaluated the mRNA expression of selected genes and correlated with outcome to arrive at a prognostic gene signature. Methods: Patients with GBM (n = 123) were prospectively recruited, treated with a uniform protocol and followed up. Expression of 175 genes in GBM tissue was determined using qRT-PCR. A supervised principal component analysis followed by derivation of gene signature was performed. Independent validation of the signature was done using TCGA data. Gene Ontology and KEGG pathway analysis was carried out among patients from TCGA cohort. Results: A 14 gene signature was identified that predicted outcome in GBM. A weighted gene (WG) score was found to be an independent predictor of survival in multivariate analysis in the present cohort (HR = 2.507; B = 0.919; p < 0.001) and in TCGA cohort. Risk stratification by standardized WG score classified patients into low and high risk predicting survival both in our cohort (p =

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer's disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer's disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.