The role of STATs in lung carcinogenesis: an emerging target for novel therapeutics.

PubMed

Karamouzis, Michalis V; Konstantinopoulos, Panagiotis A; Papavassiliou, Athanasios G

2007-05-01

The signal transducer and activator of transcription (STAT) proteins are a family of latent cytoplasmic transcription factors, which form dimers when activated by cytokine receptors, tyrosine kinase growth factor receptors as well as non-receptor tyrosine kinases. Dimeric STATs translocate to the nucleus, where they bind to specific DNA-response elements in the promoters of target genes, thereby inducing unique gene expression programs often in association with other transcription regulatory proteins. The functional consequence of different STAT proteins activation varies, as their target genes play diverse roles in normal cellular/tissue functions, including growth, apoptosis, differentiation and angiogenesis. Certain activated STATs have been implicated in human carcinogenesis, albeit only few studies have focused into their role in lung tumours. Converging evidence unravels their molecular interplays and complex multipartite regulation, rendering some of them appealing targets for lung cancer treatment with new developing strategies.

A targeted IL-15 fusion protein with potent anti-tumor activity

PubMed Central

Chen, Siqi; Huang, Qiang; Liu, Jiayu; Xing, Jieyu; Zhang, Ning; Liu, Yawei; Wang, Zhong; Li, Qing

2015-01-01

IL-15 has been actively investigated for its potential in tumor immunotherapy. To enhance the anti-tumor activity of IL-15, the novel PFC-1 construct was designed, which comprises the following 3 parts: (1) IL-15Rα fused with IL-15 to enhance IL-15 activity, (2) an Fc fragment to increase protein half-life, and (3) an integrin-targeting RGD peptide to enhance tumor targeting. PFC-1 showed tumor cell targeting without compromising IL-15 activity. PFC-1 also had potent anti-tumor activities in xenograft models, suggesting the potential application of this multi-functional fusion protein in tumor therapy. PMID:26176990

The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

PubMed Central

Kucher, Volodymyr; Li, Emily Y.; Conforti, Laura; Zahedi, Kamyar A.

2012-01-01

The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane. PMID:22442137

Functional imaging reveals rapid reorganization of cortical activity after parietal inactivation in monkeys

PubMed Central

Wilke, Melanie; Kagan, Igor; Andersen, Richard A.

2012-01-01

Impairments of spatial awareness and decision making occur frequently as a consequence of parietal lesions. Here we used event-related functional MRI (fMRI) in monkeys to investigate rapid reorganization of spatial networks during reversible pharmacological inactivation of the lateral intraparietal area (LIP), which plays a role in the selection of eye movement targets. We measured fMRI activity in control and inactivation sessions while monkeys performed memory saccades to either instructed or autonomously chosen spatial locations. Inactivation caused a reduction of contralesional choices. Inactivation effects on fMRI activity were anatomically and functionally specific and mainly consisted of: (i) activity reduction in the upper bank of the superior temporal sulcus (temporal parietal occipital area) for single contralesional targets, especially in the inactivated hemisphere; and (ii) activity increase accompanying contralesional choices between bilateral targets in several frontal and parieto-temporal areas in both hemispheres. There was no overactivation for ipsilesional targets or choices in the intact hemisphere. Task-specific effects of LIP inactivation on blood oxygen level-dependent activity in the temporal parietal occipital area underline the importance of the superior temporal sulcus for spatial processing. Furthermore, our results agree only partially with the influential interhemispheric competition model of spatial neglect and suggest an additional component of interhemispheric cooperation in the compensation of neglect deficits. PMID:22562793

PPAR Ligands Function as Suppressors That Target Biological Actions of HMGB1

PubMed Central

Chen, Tianhui

2016-01-01

High mobility group box 1 (HMGB1), which has become one of the most intriguing molecules in inflammatory disorders and cancers and with which ligand-activated peroxisome proliferator-activated receptors (PPARs) are highly associated, is considered as a therapeutic target. Of particular interest is the fact that certain PPAR ligands have demonstrated their potent anti-inflammatory activities and potential anticancer effects. In this review article we summarize recent experimental evidence that PPAR ligands function as suppressors that target biological actions of HMGB1, including intracellular expression, receptor signaling cascades, and extracellular secretion of HMGB1 in cell lines and/or animal models. We also propose the possible mechanisms underlying PPAR involvement in inflammatory disorders and discuss the future therapeutic value of PPAR ligands targeting HMGB1 molecule for cancer prevention and treatment. PMID:27563308

Dual role of Brg chromatin remodeling factor in Sonic hedgehog signaling during neural development.

PubMed

Zhan, Xiaoming; Shi, Xuanming; Zhang, Zilai; Chen, Yu; Wu, Jiang I

2011-08-02

Sonic hedgehog (Shh) signaling plays diverse roles during animal development and adult tissue homeostasis through differential regulation of Gli family transcription factors. Dysregulated Shh signaling activities have been linked to birth defects and tumorigenesis. Here we report that Brg, an ATP-dependent chromatin remodeling factor, has dual functions in regulating Shh target gene expression. Using a Brg conditional deletion in Shh-responding neural progenitors and fibroblasts, we demonstrate that Brg is required both for repression of the basal expression and for the activation of signal-induced transcription of Shh target genes. In developing telencephalons deficient for Brg, Shh target genes were derepressed, whereas Brg-deleted cerebellar granule neuron precursors failed to respond to Shh to increase their proliferation. The repressor function of Brg was mediated through Gli3 and both the repressor and activator functions of Brg appeared to be independent of its ATPase activity. Furthermore, Brg facilitates Gli coactivator histone deacetylase (HDAC) binding to the regulatory regions of Shh target genes, providing a possible mechanism for its positive role in Shh signaling. Our results thus reveal that a complex chromatin regulation mechanism underlies the precise transcription outcomes of Shh signaling and its diverse roles during development.

A strategy for actualization of active targeting nanomedicine practically functioning in a living body.

PubMed

Lee, Kyoung Jin; Shin, Seol Hwa; Lee, Jae Hee; Ju, Eun Jin; Park, Yun-Yong; Hwang, Jung Jin; Suh, Young-Ah; Hong, Seung-Mo; Jang, Se Jin; Lee, Jung Shin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

2017-10-01

Designing nanocarriers with active targeting has been increasingly emphasized as for an ideal delivery mechanism of anti-cancer therapeutic agents, but the actualization has been constrained by lack of reliable strategy ultimately applicable. Here, we designed and verified a strategy to achieve active targeting nanomedicine that works in a living body, utilizing animal models bearing a patient's tumor tissue and subjected to the same treatments that would be used in the clinic. The concept for this strategy was that a novel peptide probe and its counterpart protein, which responded to a therapy, were identified, and then the inherent ability of the peptide to target the designated tumor protein was used for active targeting in vivo. An initial dose of ionizing radiation was locally delivered to the gastric cancer (GC) tumor of a patient-derived xenograft mouse model, and phage-displayed peptide library was intravenously injected. The peptides tightly bound to the tumor were recovered, and the counterpart protein was subsequently identified. Peptide-conjugated liposomal drug showed dramatically improved therapeutic efficacy and possibility of diagnostic imaging with radiation. These results strongly suggested the potential of our strategy to achieve in vivo functional active targeting and to be applied clinically for human cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Temporal Dynamics of Activation of Thematic and Functional Knowledge During Conceptual Processing of Manipulable Artifacts

PubMed Central

Kalénine, Solène; Mirman, Daniel; Middleton, Erica L.; Buxbaum, Laurel J.

2012-01-01

The current research aimed at specifying the activation time course of different types of semantic information during object conceptual processing and the effect of context on this time course. We distinguished between thematic and functional knowledge and the specificity of functional similarity. Two experiments were conducted with healthy older adults using eye tracking in a word-to-picture matching task. The time course of gaze fixations was used to assess activation of distractor objects during the identification of manipulable artifact targets (e.g., broom). Distractors were (a) thematically related (e.g., dustpan), (b) related by a specific function (e.g., vacuum cleaner), or (c) related by a general function (e.g., sponge). Growth curve analyses were used to assess competition effects when target words were presented in isolation (Experiment 1) and embedded in contextual sentences of different generality levels (Experiment 2). In the absence of context, there was earlier and shorter lasting activation of thematically related as compared to functionally related objects. The time course difference was more pronounced for general functions than specific functions. When contexts were provided, functional similarities that were congruent with context generality level increased in salience with earlier activation of those objects. Context had little impact on thematic activation time course. These data demonstrate that processing a single manipulable artifact concept implicitly activates thematic and functional knowledge with different time courses and that context speeds activation of context-congruent functional similarity. PMID:22449134

Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug-target sites.

PubMed

Singh, Shailza; Mandlik, Vineetha; Shinde, Sonali

2015-03-01

GPI12 represents an important enzyme in the GPI biosynthetic pathway of several parasites like 'Leishmania'. GPI activity is generally regulated through either the hindrance in GPI complex assembly formation or the modulation of the lipophosphoglycan (LPG) flux to either reduce or enhance the pathogenicity in an organism. Of the various GPI molecules known, GPI12 is an important enzyme in the GPI biosynthetic pathway which can be exploited as a target due to the substrate specificity difference in parasites and humans. In the present study, the functional importance of the co-evolving residues of the GPI12 protein of Leishmania has been highlighted using the GPI proteins belonging to the GlcNAC-deacetylase family. Exploring the active site of the GPI12 protein and designing inhibitors against the functional residues provide ways and means to change the efficiency of deacetylation activity of the enzyme. The activity of de-N-acetylase is low in the absence of metal ions like zinc. Hence we designed eight small molecules in order to modulate the activity of GPI12. Compound 8 was found to be an appropriate choice to target the agonist (GPI12) active site thereby targeting the residues which were essential in the Zn binding and chelation activity. Inhibition of these sites offered a strong constraint to block the protein activity and in turn GPI biosynthesis.

Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system.

PubMed

Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

2016-01-27

The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

EBF factors drive expression of multiple classes of target genes governing neuronal development.

PubMed

Green, Yangsook S; Vetter, Monica L

2011-04-30

Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

Genomewide Analysis of Aryl Hydrocarbon Receptor Binding Targets Reveals an Extensive Array of Gene Clusters that Control Morphogenetic and Developmental Programs

PubMed Central

Sartor, Maureen A.; Schnekenburger, Michael; Marlowe, Jennifer L.; Reichard, John F.; Wang, Ying; Fan, Yunxia; Ma, Ci; Karyala, Saikumar; Halbleib, Danielle; Liu, Xiangdong; Medvedovic, Mario; Puga, Alvaro

2009-01-01

Background The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand. Objectives We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand. Methods The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts. Results We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes. Conclusions The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury. PMID:19654925

Genome-wide STAT3 binding analysis after histone deacetylase inhibition reveals novel target genes in dendritic cells

PubMed Central

Sun, Yaping; Iyer, Matthew; McEachin, Richard; Zhao, Meng; Wu, Yi-Mi; Cao, Xuhong; Oravecz-Wilson, Katherine; Zajac, Cynthia; Mathewson, Nathan; Wu, Shin-Rong Julia; Rossi, Corinne; Toubai, Tomomi; Qin, Zhaohui S.; Chinnaiya, Arul M.; Reddy, Pavan

2016-01-01

STAT3 is a master transcriptional regulator that plays an important role in the induction of both immune activation and immune tolerance in dendritic cells (DCs). The transcriptional targets of STAT3 in promoting DC activation are becoming increasingly understood; however, the mechanisms underpinning its role in causing DC suppression remain largely unknown. To determine the functional gene targets of STAT3, we compared the genome-wide binding of STAT3 using ChIP-seq coupled with gene expression microarrays to determine STAT3-dependent gene regulation in DCs after histone deacetylase (HDAC) inhibition. HDAC inhibition boosted the ability of STAT3 to bind to distinct DNA targets and regulate gene expression. Among the top 500 STAT3 binding sites, the frequency of canonical motifs was significantly higher than that of non-canonical motifs. Functional analysis revealed that after treatment with an HDAC inhibitor, the upregulated STAT3 target genes were those that were primarily the negative regulators of pro-inflammatory cytokines and those in the IL-10 signaling pathway. The downregulated STAT3-dependent targets were those involved in immune effector processes and antigen processing/presentation. The expression and functional relevance of these genes were validated. Specifically, functional studies confirmed that the upregulation of IL-10Ra by STAT3 contributed to the suppressive function of DCs following HDAC inhibition. PMID:27866206

STAT3 or USF2 Contributes to HIF Target Gene Specificity

PubMed Central

Pawlus, Matthew R.; Wang, Liyi; Murakami, Aya; Dai, Guanhai; Hu, Cheng-Jun

2013-01-01

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. PMID:23991099

Return-to-Duty Toolkit: Assessments and Tasks for Determining Military Functional Performance Following Neurosensory Injury

DTIC Science & Technology

2017-09-29

the warfighter to engage in aerobic activity such as running in place or push-ups until 65–85% of the target heart rate is reached (the target heart...85% of the target heart rate is reached (the target heart rate is 220 minus age). Options for activity include but are not limited to running in...Time • Pursuit Tracking • Running Memory CPT • Simple Reaction Time • Sleep Scale • Spatial Processing – Sequential and Simultaneous • Manikin

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

PubMed

Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

2014-09-01

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival.

PubMed

Lin, Zhiqiang; Zhou, Pingzhu; von Gise, Alexander; Gu, Fei; Ma, Qing; Chen, Jinghai; Guo, Haidong; van Gorp, Pim R R; Wang, Da-Zhi; Pu, William T

2015-01-02

Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence-specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus-mediated Pik3cb expression. Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival. © 2014 American Heart Association, Inc.

Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.

PubMed

Peretti, Marta; Angelini, Marina; Savalli, Nicoletta; Florio, Tullio; Yuspa, Stuart H; Mazzanti, Michele

2015-10-01

In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers." Copyright © 2015 Elsevier B.V. All rights reserved.

Methods to enable the design of bioactive small molecules targeting RNA

PubMed Central

Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

2014-01-01

RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

Methods to enable the design of bioactive small molecules targeting RNA.

PubMed

Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

2014-02-21

RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

Attentional priorities and access to short-term memory: parietal interactions.

PubMed

Gillebert, Céline R; Dyrholm, Mads; Vangkilde, Signe; Kyllingsbæk, Søren; Peeters, Ronald; Vandenberghe, Rik

2012-09-01

The intraparietal sulcus (IPS) has been implicated in selective attention as well as visual short-term memory (VSTM). To contrast mechanisms of target selection, distracter filtering, and access to VSTM, we combined behavioral testing, computational modeling and functional magnetic resonance imaging. Sixteen healthy subjects participated in a change detection task in which we manipulated both target and distracter set sizes. We directly compared the IPS response as a function of the number of targets and distracters in the display and in VSTM. When distracters were not present, the posterior and middle segments of IPS showed the predicted asymptotic activity increase with an increasing target set size. When distracters were added to a single target, activity also increased as predicted. However, the addition of distracters to multiple targets suppressed both middle and posterior IPS activities, thereby displaying a significant interaction between the two factors. The interaction between target and distracter set size in IPS could not be accounted for by a simple explanation in terms of number of items accessing VSTM. Instead, it led us to a model where items accessing VSTM receive differential weights depending on their behavioral relevance, and secondly, a suppressive effect originates during the selection phase when multiple targets and multiple distracters are simultaneously present. The reverse interaction between target and distracter set size was significant in the right temporoparietal junction (TPJ), where activity was highest for a single target compared to any other condition. Our study reconciles the role of middle IPS in attentional selection and biased competition with its role in VSTM access. Copyright © 2012 Elsevier Inc. All rights reserved.

Application of chemical biology in target identification and drug discovery.

PubMed

Zhu, Yue; Xiao, Ting; Lei, Saifei; Zhou, Fulai; Wang, Ming-Wei

2015-09-01

Drug discovery and development is vital to the well-being of mankind and sustainability of the pharmaceutical industry. Using chemical biology approaches to discover drug leads has become a widely accepted path partially because of the completion of the Human Genome Project. Chemical biology mainly solves biological problems through searching previously unknown targets for pharmacologically active small molecules or finding ligands for well-defined drug targets. It is a powerful tool to study how these small molecules interact with their respective targets, as well as their roles in signal transduction, molecular recognition and cell functions. There have been an increasing number of new therapeutic targets being identified and subsequently validated as a result of advances in functional genomics, which in turn led to the discovery of numerous active small molecules via a variety of high-throughput screening initiatives. In this review, we highlight some applications of chemical biology in the context of drug discovery.

A Synthetic DNA-Binding Domain Guides Distinct Chromatin-Modifying Small Molecules to Activate an Identical Gene Network.

PubMed

Han, Le; Pandian, Ganesh N; Chandran, Anandhakumar; Sato, Shinsuke; Taniguchi, Junichi; Kashiwazaki, Gengo; Sawatani, Yoshito; Hashiya, Kaori; Bando, Toshikazu; Xu, Yufang; Qian, Xuhong; Sugiyama, Hiroshi

2015-07-20

Synthetic dual-function ligands targeting specific DNA sequences and histone-modifying enzymes were applied to achieve regulatory control over multi-gene networks in living cells. Unlike the broad array of targeting small molecules for histone deacetylases (HDACs), few modulators are known for histone acetyltransferases (HATs), which play a central role in transcriptional control. As a novel chemical approach to induce selective HAT-regulated genes, we conjugated a DNA-binding domain (DBD) "I" to N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB), an artificial HAT activator. In vitro enzyme activity assays and microarray studies were used to demonstrate that distinct functional small molecules could be transformed to have identical bioactivity when conjugated with a targeting DBD. This proof-of-concept synthetic strategy validates the switchable functions of HDACs and HATs in gene regulation and provides a molecular basis for developing versatile bioactive ligands. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syeda, Farisa; Fagan, Rebecca L.; Wean, Matthew

Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenicmore » DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.« less

Targeting the p53 signaling pathway in cancer therapy - The promises, challenges, and perils

PubMed Central

Stegh, Alexander H.

2012-01-01

Introduction Research over the past three decades has identified p53 as a multifunctional transcription factor, which regulates the expression of >2,500 target genes. p53 impacts myriad, highly diverse cellular processes, including the maintenance of genomic stability and fidelity, metabolism, longevity, and represents one of the most important and extensively studied tumor suppressors. Activated by various stresses, foremost genotoxic damage, hypoxia, heat shock and oncogenic assault, p53 blocks cancer progression by provoking transient or permanent growth arrest, by enabling DNA repair or by advancing cellular death programs. This potent and versatile anti-cancer activity profile, together with genomic and mutational analyses documenting inactivation of p53 in more than 50% of human cancers, motivated drug development efforts to (re-) activate p53 in established tumors. Areas covered In this review the complexities of p53 signaling in cancer are summarized. Current strategies and challenges to restore p53’s tumor suppressive function in established tumors, i.e. adenoviral gene transfer and small molecules to activate p53, to inactivate p53 inhibitors and to restore wild type function of p53 mutant proteins are discussed. Expert opinion It is indubitable that p53 represents an attractive target for the development of anti-cancer therapies. Whether p53 is ‘druggable’, however, remains an area of active research and discussion, as p53 has pro-survival functions and chronic p53 activation accelerates aging, which may compromise the long-term homeostasis of an organism. Thus, the complex biology and dual functions of p53 in cancer prevention and age-related cellular responses pose significant challenges on the development of p53-targeting cancer therapies. PMID:22239435

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation

PubMed Central

Kozhuharova, Ana; Sharma, Harshita; Ohyama, Takako; Fasolo, Francesca; Yamazaki, Toshio; Cotella, Diego; Santoro, Claudio; Zucchelli, Silvia; Gustincich, Stefano; Carninci, Piero

2018-01-01

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest. PMID:29414979

Comparing sixteen scoring functions for predicting biological activities of ligands for protein targets.

PubMed

Xu, Weijun; Lucke, Andrew J; Fairlie, David P

2015-04-01

Accurately predicting relative binding affinities and biological potencies for ligands that interact with proteins remains a significant challenge for computational chemists. Most evaluations of docking and scoring algorithms have focused on enhancing ligand affinity for a protein by optimizing docking poses and enrichment factors during virtual screening. However, there is still relatively limited information on the accuracy of commercially available docking and scoring software programs for correctly predicting binding affinities and biological activities of structurally related inhibitors of different enzyme classes. Presented here is a comparative evaluation of eight molecular docking programs (Autodock Vina, Fitted, FlexX, Fred, Glide, GOLD, LibDock, MolDock) using sixteen docking and scoring functions to predict the rank-order activity of different ligand series for six pharmacologically important protein and enzyme targets (Factor Xa, Cdk2 kinase, Aurora A kinase, COX-2, pla2g2a, β Estrogen receptor). Use of Fitted gave an excellent correlation (Pearson 0.86, Spearman 0.91) between predicted and experimental binding only for Cdk2 kinase inhibitors. FlexX and GOLDScore produced good correlations (Pearson>0.6) for hydrophilic targets such as Factor Xa, Cdk2 kinase and Aurora A kinase. By contrast, pla2g2a and COX-2 emerged as difficult targets for scoring functions to predict ligand activities. Although possessing a high hydrophobicity in its binding site, β Estrogen receptor produced reasonable correlations using LibDock (Pearson 0.75, Spearman 0.68). These findings can assist medicinal chemists to better match scoring functions with ligand-target systems for hit-to-lead optimization using computer-aided drug design approaches. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of motivation on reward and attentional networks: an fMRI study.

PubMed

Ivanov, Iliyan; Liu, Xun; Clerkin, Suzanne; Schulz, Kurt; Friston, Karl; Newcorn, Jeffrey H; Fan, Jin

2012-11-01

Existing evidence suggests that reward and attentional networks function in concert and that activation in one system influences the other in a reciprocal fashion; however, the nature of these influences remains poorly understood. We therefore developed a three-component task to assess the interaction effects of reward anticipation and conflict resolution on the behavioral performance and the activation of brain reward and attentional systems. Sixteen healthy adult volunteers aged 21-45 years were scanned with functional magnetic resonance imaging (fMRI) while performing the task. A two-way repeated measures analysis of variance (ANOVA) with cue (reward vs. non-reward) and target (congruent vs. incongruent) as within-subjects factors was used to test for main and interaction effects. Neural responses to anticipation, conflict, and reward outcomes were tested. Behaviorally there were main effects of both reward cue and target congruency on reaction time. Neuroimaging results showed that reward anticipation and expected reward outcomes activated components of the attentional networks, including the inferior parietal and occipital cortices, whereas surprising non-rewards activated the frontoinsular cortex bilaterally and deactivated the ventral striatum. In turn, conflict activated a broad network associated with cognitive control and motor functions. Interaction effects showed decreased activity in the thalamus, anterior cingulated gyrus, and middle frontal gyrus bilaterally when difficult conflict trials (e.g., incongruent targets) were preceded by reward cues; in contrast, the ventral striatum and orbitofrontal cortex showed greater activation during congruent targets preceded by reward cues. These results suggest that reward anticipation is associated with lower activation in attentional networks, possibly due to increased processing efficiency, whereas more difficult, conflict trials are associated with lower activity in regions of the reward system, possibly because such trials are experienced as less rewarding.

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL).

PubMed

Woyach, Jennifer A; Bojnik, Engin; Ruppert, Amy S; Stefanovski, Matthew R; Goettl, Virginia M; Smucker, Kelly A; Smith, Lisa L; Dubovsky, Jason A; Towns, William H; MacMurray, Jessica; Harrington, Bonnie K; Davis, Melanie E; Gobessi, Stefania; Laurenti, Luca; Chang, Betty Y; Buggy, Joseph J; Efremov, Dimitar G; Byrd, John C; Johnson, Amy J

2014-02-20

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Enhanced anti-tumoral activity of methotrexate-human serum albumin conjugated nanoparticles by targeting with Luteinizing Hormone-Releasing Hormone (LHRH) peptide.

PubMed

Taheri, Azade; Dinarvand, Rassoul; Atyabi, Fatemeh; Ahadi, Fatemeh; Nouri, Farank Salman; Ghahremani, Mohammad Hossein; Ostad, Seyed Nasser; Borougeni, Atefeh Taheri; Mansoori, Pooria

2011-01-01

Active targeting could increase the efficacy of anticancer drugs. Methotrexate-human serum albumin (MTX-HSA) conjugates, functionalized by luteinizing hormone-releasing hormone (LHRH) as targeting moieties, with the aim of specifically targeting the cancer cells, were prepared. Owing to the high expression of LHRH receptors in many cancer cells as compared to normal cells, LHRH was used as the targeting ligand in this study. LHRH was conjugated to MTX-HSA nanoparticles via a cross-linker. Three types of LHRH targeted nanoparticles with a mean particle size between 120-138 nm were prepared. The cytotoxicity of LHRH targeted and non-targeted nanoparticles were determined on the LHRH positive and negative cell lines. The internalization of the targeted and non-targeted nanoparticles in LHRH receptor positive and negative cells was investigated using flow cytometry analysis and fluorescence microscopy. The cytotoxicity of the LHRH targeted nanoparticles on the LHRH receptor positive cells were significantly more than non-targeted nanoparticles. LHRH targeted nanoparticles were also internalized by LHRH receptor positive cells significantly more than non-targeted nanoparticles. There were no significant differences between the uptake of targeted and non-targeted nanoparticles to the LHRH receptor negative cells. The active targeting procedure using LHRH targeted MTX-HSA nanoparticles could increase the anti-tumoral activity of MTX.

ZFP36 RNA-binding proteins restrain T-cell activation and anti-viral immunity.

PubMed

Moore, Michael J; Blachere, Nathalie E; Fak, John J; Park, Christopher Y; Sawicka, Kirsty; Parveen, Salina; Zucker-Scharff, Ilana; Moltedo, Bruno; Rudensky, Alexander Y; Darnell, Robert B

2018-05-31

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in mouse T cells, revealing unanticipated actions in regulating T cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through novel AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T cell activation kinetics cell autonomously, by attenuating activation marker expression, limiting T cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T cell responses to acute viral infection and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T cell expansion and effector functions, and suggest ZFP36 inhibition as a strategy to enhance immune-based therapies. © 2018, Moore et al.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

The prokaryotic FAD synthetase family: a potential drug target.

PubMed

Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

2013-01-01

Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

Targeting PCSK9 for therapeutic gains: Have we addressed all the concerns?

PubMed

Banerjee, Yajnavalka; Santos, Raul D; Al-Rasadi, Khalid; Rizzo, Manfredi

2016-05-01

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) regulates the expression of low-density lipoprotein (LDL)-receptors, through reducing their recycling by binding to the receptor along with LDL and targeting it for lysosomal destruction. PCSK9 also enhances the degradation of very-low-density-lipoprotein receptor (VLDLR) and lipoprotein receptor-related protein 1 (LRP-1) in a LDL-receptor independent manner. This role in lipid homeostasis presents PCSK9 as an attractive target for the therapeutic management of familial hypercholesterolemia as well as other refractory dyslipidaemias. However, PCSK9 mediates multifarious functions independent of its role in lipid homeostasis, which can be grouped under "pleiotropic functions" of the protein. This includes PCSK9's role in: trafficking of epithelial sodium channel; hepatic regeneration; pancreatic integrity and glucose homeostasis; antiviral activity; antimalarial activity; regulation of different cell signalling pathways; cortical neural differentiation; neuronal apoptosis and Alzheimer's disease. The question that needs to be investigated in depth is "How will the pleotropic functions of PCSK9, be affected by the therapeutic intervention of the protease's LDL-receptor lowering activity?" In this review, we appraise the different lipid lowering strategies targeting PCSK9 in light of the protein's different pleiotropic functions. Additionally, we delineate the key areas that require further examination, to ensure the long-term safety of the above lipid-lowering strategies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Brain activation of semantic category-based grouping in multiple identity tracking task

PubMed Central

Wei, Liuqing; Lyu, Chuang; Hu, Siyuan; Li, Zhen

2017-01-01

Using Multiple Identity Tracking task and the functional magnetic resonance imaging (fMRI) technology, the present study aimed to isolate and visualize the functional anatomy of neural systems involved in the semantic category-based grouping process. Three experiment conditions were selected and compared: the category-based targets grouping (TG) condition, the targets-distractors grouping (TDG) condition and the homogenous condition. In the TG condition, observers could utilize the categorical distinction between targets and distractors, to construct a uniform presentation of targets, that is, to form a group of the targets to facilitate tracking. In the TDG condition, half the targets and half the distractors belonged to the same category. Observers had to inhibit the grouping of targets and distractors in one category to complete tracking. In the homogenous condition, where targets and distractors consisted of the same objects, no grouping could be formed. The “TG-Homogenous” contrast (p<0.01) revealed the activation of the left fusiform and the pars triangularis of inferior frontal gyrus (IFG). The “TG-TDG” contrast only revealed the activation of the left anterior cingulate gyrus (ACC). The fusiform and IFG pars triangularis might participate in the representation of semantic knowledge, IFG pars triangularis might relate intensely with the classification of semantic categories. The ACC might be responsible for the initiation and maintenance of grouping representation. PMID:28505166

Cramer-Rao Lower Bound Evaluation for Linear Frequency Modulation Based Active Radar Networks Operating in a Rice Fading Environment.

PubMed

Shi, Chenguang; Salous, Sana; Wang, Fei; Zhou, Jianjiang

2016-12-06

This paper investigates the joint target parameter (delay and Doppler) estimation performance of linear frequency modulation (LFM)-based radar networks in a Rice fading environment. The active radar networks are composed of multiple radar transmitters and multichannel receivers placed on moving platforms. First, the log-likelihood function of the received signal for a Rician target is derived, where the received signal scattered off the target comprises of dominant scatterer (DS) component and weak isotropic scatterers (WIS) components. Then, the analytically closed-form expressions of the Cramer-Rao lower bounds (CRLBs) on the Cartesian coordinates of target position and velocity are calculated, which can be adopted as a performance metric to access the target parameter estimation accuracy for LFM-based radar network systems in a Rice fading environment. It is found that the cumulative Fisher information matrix (FIM) is a linear combination of both DS component and WIS components, and it also demonstrates that the joint CRLB is a function of signal-to-noise ratio (SNR), target's radar cross section (RCS) and transmitted waveform parameters, as well as the relative geometry between the target and the radar network architectures. Finally, numerical results are provided to indicate that the joint target parameter estimation performance of active radar networks can be significantly improved with the exploitation of DS component.

Doxycycline and its quaternary ammonium derivative for adjuvant therapies of chondrosarcoma.

PubMed

Miladi, Imen; Vivier, Magali; Dauplat, Marie-Mélanie; Chatard, Morgane; Besse, Sophie; Vidal, Aurélien; Chassain, Karine; Jean, Betty; Forestier, Christiane; Chezal, Jean-Michel; Rédini, Francoise; Degoul, Francoise; Miot-Noirault, Elisabeth

2017-09-01

This study was conducted during the development of innovative treatment targeting the microenvironment of chondrosarcoma. In this context, MMP inhibitors were conjugated with a quaternary ammonium (QA) function as a targeting ligand to proteoglycans of chondrosarcoma extracellular matrix. Here we report the proof of concept of this strategy applied to the MMP13 inhibitor, doxycycline (Dox). A quaternary ammonium derivative of the MMP13 inhibitor doxycycline (QA-Dox) was synthesized, and its anticancer activity was evaluated in the Swarm rat chondrosarcoma (SRC) model compared with the parent drug doxycycline, in vitro and in vivo. In vivo, dox and QA-Dox efficiency was assessed at equimolar doses according to a q4dx4 schedule by monitoring tumour volume by MRI and PG-targeted scintigraphy. Molecular mechanism (MMP13 expression, proteoglycan level) and histology studies were performed on tumours. The link of QA targeting function to Dox maintained the MMP13 inhibitory activity in vitro. Interestingly, the bacteriostatic activity was lost. SRC cells incubated with both drugs were blocked in S and G2 M phases. Tumour growth inhibition (confirmed by histology) was observed for both Dox and QA-Dox. Undesirable blood effects (leukocyte decrease) were reduced when Dox was targeted to tumour tissue using the QA function. In the SRC model, the MMP13 inhibitor Dox and its QA derivative are promising as adjuvant therapies for chondrosarcoma management.

Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

PubMed Central

Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

2014-01-01

The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

EMSA Analysis of DNA Binding By Rgg Proteins

PubMed Central

LaSarre, Breah; Federle, Michael J.

2016-01-01

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004

EMSA Analysis of DNA Binding By Rgg Proteins.

PubMed

LaSarre, Breah; Federle, Michael J

2013-08-20

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

Can the ICF osteoarthritis core set represent a future clinical tool in measuring functioning in persons with osteoarthritis undergoing hip and knee joint replacement?

PubMed

Alviar, Maria Jenelyn; Olver, John; Pallant, Julie F; Brand, Caroline; de Steiger, Richard; Pirpiris, Marinis; Bucknill, Andrew; Khan, Fary

2012-11-01

To determine the dimensionality, reliability, model fit, adequacy of the qualifier levels, response patterns across different factors, and targeting of the International Classification of Functioning, Disability and Health (ICF) osteoarthritis core set categories in people with osteoarthritis undergoing hip and knee arthroplasty. The osteoarthritis core set was rated in 316 persons with osteoarthritis who were either in the pre-operative or within one year post-operative stage. Rasch analyses were performed using the RUMM 2030 program. Twelve of the 13 body functions categories and 13 of the 19 activity and participation categories had good model fit. The qualifiers displayed disordered thresholds necessitating rescoring. There was uneven spread of ICF categories across the full range of the patients' scores indicating off--targeting. Subtest analysis of the reduced ICF categories of body functions and activity and participation showed that the two components could be integrated to form one measure. The results suggest that it is possible to measure functioning using a unidimensional construct based on ICF osteoarthritis core set categories of body functions and activity and participation in this population. However, omission of some categories and reduction in qualifier levels are necessary. Further studies are needed to determine whether better targeting is achieved, particularly during the pre-operative and during the sub-acute care period.

Properties of Native High-Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery Vehicles.

PubMed

McMahon, Kaylin M; Plebanek, Michael P; Thaxton, C Shad

2016-11-15

Efficient systemic administration of therapeutic short interfering RNA (siRNA) is challenging. High-density lipoproteins (HDL) are natural in vivo RNA delivery vehicles. Specifically, native HDLs: 1) Load single-stranded RNA; 2) Are anionic, which requires charge reconciliation between the RNA and HDL, and 3) Actively target scavenger receptor type B-1 (SR-B1) to deliver RNA. Emphasizing these particular parameters, we employed templated lipoprotein particles (TLP), mimics of spherical HDLs, and self-assembled them with single-stranded complements of, presumably, any highly unmodified siRNA duplex pair after formulation with a cationic lipid. Resulting siRNA templated lipoprotein particles (siRNA-TLP) are anionic and tunable with regard to RNA assembly and function. Data demonstrate that the siRNA-TLPs actively target SR-B1 to potently reduce androgen receptor (AR) and enhancer of zeste homolog 2 (EZH2) proteins in multiple cancer cell lines. Systemic administration of siRNA-TLPs demonstrated no off-target toxicity and significantly reduced the growth of prostate cancer xenografts. Thus, native HDLs inspired the synthesis of a hybrid siRNA delivery vehicle that can modularly load single-stranded RNA complements after charge reconciliation with a cationic lipid, and that function due to active targeting of SR-B1.

Surface functionality affects the biodistribution and microglia-targeting of intra-amniotically delivered dendrimers.

PubMed

Zhang, Fan; Nance, Elizabeth; Zhang, Zhi; Jasty, Venkatasai; Kambhampati, Siva P; Mishra, Manoj K; Burd, Irina; Romero, Roberto; Kannan, Sujatha; Kannan, Rangaramanujam M

2016-09-10

Cerebral Palsy (CP) is a chronic childhood disorder with limited therapeutic options. Maternal intrauterine inflammation/infection is a major risk factor in the pathogenesis of CP. In pre-clinical models, dendrimer-based therapies are viable in postnatal period, attenuating inflammation and improving motor function in vivo. However, treatment to the mother, in the prenatal period, may provide the possibility of preventing/resolving inflammation at early stages. Towards this goal, we used a maternal intrauterine inflammation-induced rabbit model of CP to study fetal-maternal transport and neuroinflammation targeting of intra-amniotically administrated dendrimers with neutral/anionic surface functionality. Our study suggested both hydroxyl-terminated 'neutral' (D-OH) and carboxyl-terminated 'anionic' (D-COOH) Polyamidoamine (PAMAM) dendrimers were absorbed by fetuses and demonstrated bi-directional transport between fetuses and mother. D-OH was more effective in crossing the fetal blood-brain barrier, and targeting activated microglia. The cell-specific targeting was associated with the extent of microglia activation. This study demonstrated intra-amniotically administered hydroxyl PAMAM dendrimers could be an effective drug delivery vehicle for targeting fetal inflammation and preventing subsequent neurologic injury associated with chorioamnionitis. Copyright © 2016 Elsevier B.V. All rights reserved.

Surface functionality affects the biodistribution and microglia-targeting of intra-amniotically delivered dendrimers☆

PubMed Central

Zhang, Fan; Nance, Elizabeth; Zhang, Zhi; Jasty, Venkatasai; Kambhampati, Siva P.; Mishra, Manoj K.; Burd, Irina; Romero, Roberto; Kannan, Sujatha; Kannan, Rangaramanujam M.

2017-01-01

Cerebral Palsy (CP) is a chronic childhood disorder with limited therapeutic options. Maternal intrauterine inflammation/infection is a major risk factor in the pathogenesis of CP. In pre-clinical models, dendrimer-based therapies are viable in postnatal period, attenuating inflammation and improving motor function in vivo. However, treatment to the mother, in the prenatal period, may provide the possibility of preventing/resolving inflammation at early stages. Towards this goal, we used a maternal intrauterine inflammation-induced rabbit model of CP to study fetal-maternal transport and neuroinflammation targeting of intra-amniotically administrated dendrimers with neutral/anionic surface functionality. Our study suggested both hydroxyl-terminated ‘neutral’ (D-OH) and carboxyl-terminated ‘anionic’ (D-COOH) Polyamidoamine (PAMAM) dendrimers were absorbed by fetuses and demonstrated bi-directional transport between fetuses and mother. D-OH was more effective in crossing the fetal blood-brain barrier, and targeting activated microglia. The cell-specific targeting was associated with the extent of microglia activation. This study demonstrated intra-amniotically administered hydroxyl PAMAM dendrimers could be an effective drug delivery vehicle for targeting fetal inflammation and preventing subsequent neurologic injury associated with chorioamnionitis. PMID:27378700

SHP-2 expression negatively regulates NK cell function1,2

PubMed Central

Purdy, Amanda K.; Campbell, Kerry S.

2009-01-01

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)4 is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human natural killer (NK) cells upon recruitment to killer cell Ig-like receptors (KIR)4. To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN-γ production, whereas cells overexpressing wild type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center (MTOC)4 polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts MTOC-based cytoskeletal rearrangement and granule release. PMID:19915046

Enhanced Anti-Tumoral Activity of Methotrexate-Human Serum Albumin Conjugated Nanoparticles by Targeting with Luteinizing Hormone-Releasing Hormone (LHRH) Peptide

PubMed Central

Taheri, Azade; Dinarvand, Rassoul; Atyabi, Fatemeh; Ahadi, Fatemeh; Nouri, Farank Salman; Ghahremani, Mohammad Hossein; Ostad, Seyed Nasser; Borougeni, Atefeh Taheri; Mansoori, Pooria

2011-01-01

Active targeting could increase the efficacy of anticancer drugs. Methotrexate-human serum albumin (MTX-HSA) conjugates, functionalized by luteinizing hormone-releasing hormone (LHRH) as targeting moieties, with the aim of specifically targeting the cancer cells, were prepared. Owing to the high expression of LHRH receptors in many cancer cells as compared to normal cells, LHRH was used as the targeting ligand in this study. LHRH was conjugated to MTX-HSA nanoparticles via a cross-linker. Three types of LHRH targeted nanoparticles with a mean particle size between 120–138 nm were prepared. The cytotoxicity of LHRH targeted and non-targeted nanoparticles were determined on the LHRH positive and negative cell lines. The internalization of the targeted and non-targeted nanoparticles in LHRH receptor positive and negative cells was investigated using flow cytometry analysis and fluorescence microscopy. The cytotoxicity of the LHRH targeted nanoparticles on the LHRH receptor positive cells were significantly more than non-targeted nanoparticles. LHRH targeted nanoparticles were also internalized by LHRH receptor positive cells significantly more than non-targeted nanoparticles. There were no significant differences between the uptake of targeted and non-targeted nanoparticles to the LHRH receptor negative cells. The active targeting procedure using LHRH targeted MTX-HSA nanoparticles could increase the anti-tumoral activity of MTX. PMID:21845098

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Targeted polypeptide degradation

DOEpatents

Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

2008-05-13

This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

TS-Chemscore, a Target-Specific Scoring Function, Significantly Improves the Performance of Scoring in Virtual Screening.

PubMed

Wang, Wen-Jing; Huang, Qi; Zou, Jun; Li, Lin-Li; Yang, Sheng-Yong

2015-07-01

Most of the scoring functions currently used in structure-based drug design belong to 'universal' scoring functions, which often give a poor correlation between the calculated scores and experimental binding affinities. In this investigation, we proposed a simple strategy to construct target-specific scoring functions based on known 'universal' scoring functions. This strategy was applied to Chemscore, a widely used empirical scoring function, which led to a new scoring function, termed TS-Chemscore. TS-Chemscore was validated on 14 protein targets, which cover a wide range of biological target categories. The results showed that TS-Chemscore significantly improved the correlation between the calculated scores and experimental binding affinities compared with the original Chemscore. TS-Chemscore was then applied in virtual screening to retrieve novel JAK3 and YopH inhibitors. Top 30 compounds for each target were selected for experimental validation. Six active compounds for JAK3 and four for YopH were obtained. These compounds were out of the lists of top 30 compounds sorted by Chemscore. Collectively, TS-Chemscore established in this study showed a better performance in virtual screening than its counterpart Chemscore. © 2014 John Wiley & Sons A/S.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

PubMed

Brown, David A; Di Cerbo, Vincenzo; Feldmann, Angelika; Ahn, Jaewoo; Ito, Shinsuke; Blackledge, Neil P; Nakayama, Manabu; McClellan, Michael; Dimitrova, Emilia; Turberfield, Anne H; Long, Hannah K; King, Hamish W; Kriaucionis, Skirmantas; Schermelleh, Lothar; Kutateladze, Tatiana G; Koseki, Haruhiko; Klose, Robert J

2017-09-05

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Real-time functional imaging for monitoring miR-133 during myogenic differentiation.

PubMed

Kato, Yoshio; Miyaki, Shigeru; Yokoyama, Shigetoshi; Omori, Shin; Inoue, Atsushi; Horiuchi, Machiko; Asahara, Hiroshi

2009-11-01

MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3' untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events.

Delayed pituitary adenylate cyclase-activating polypeptide delivery after brain stroke improves functional recovery by inducing m2 microglia/macrophage polarization.

PubMed

Brifault, Coralie; Gras, Marjorie; Liot, Donovan; May, Victor; Vaudry, David; Wurtz, Olivier

2015-02-01

Until now, except thrombolysis, the therapeutical strategies targeting the acute phase of cerebral ischemia have been proven ineffective, and no approach is available to attenuate the delayed cell death mechanisms and the resulting functional deficits in the late phase. Then, we investigated whether a targeted and delayed delivery of pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide known to exert neuroprotective activities, may dampen delayed pathophysiological processes improving functional recovery. Three days after permanent focal ischemia, PACAP-producing stem cells were transplanted intracerebro ventricularly in nonimmunosuppressed mice. At 7 and 14 days post ischemia, the effects of this stem cell-based targeted delivery of PACAP on functional recovery, volume lesions, and inflammatory processes were analyzed. The delivery of PACAP in the vicinity of the infarct zone 3 days post stroke promotes fast, stable, and efficient functional recovery. This was correlated with a modulation of the postischemic inflammatory response. Transcriptomic and Ingenuity Pathway Analysis-based bioinformatic analyses identified several gene networks, functions, and key transcriptional factors, such as nuclear factor-κB, C/EBP-β, and Notch/RBP-J as PACAP's potential targets. Such PACAP-dependent immunomodulation was further confirmed by morphometric and phenotypic analyses of microglial cells showing increased number of Arginase-1(+) cells in mice treated with PACAP-expressing cells specifically, demonstrating the redirection of the microglial response toward a neuroprotective M2 phenotype. Our results demonstrated that immunomodulatory strategies capable of redirecting the microglial response toward a neuroprotective M2 phenotype in the late phase of brain ischemia could represent attractive options for stroke treatment in a new and unexploited therapeutical window. © 2014 American Heart Association, Inc.

Brain activation underlying threat detection to targets of different races.

PubMed

Senholzi, Keith B; Depue, Brendan E; Correll, Joshua; Banich, Marie T; Ito, Tiffany A

2015-01-01

The current study examined blood oxygen level-dependent signal underlying racial differences in threat detection. During functional magnetic resonance imaging, participants determined whether pictures of Black or White individuals held weapons. They were instructed to make shoot responses when the picture showed armed individuals but don't shoot responses to unarmed individuals, with the cost of not shooting armed individuals being greater than that of shooting unarmed individuals. Participants were faster to shoot armed Blacks than Whites, but faster in making don't shoot responses to unarmed Whites than Blacks. Brain activity differed to armed versus unarmed targets depending on target race, suggesting different mechanisms underlying threat versus safety decisions. Anterior cingulate cortex was preferentially engaged for unarmed Whites than Blacks. Parietal and visual cortical regions exhibited greater activity for armed Blacks than Whites. Seed-based functional connectivity of the amygdala revealed greater coherence with parietal and visual cortices for armed Blacks than Whites. Furthermore, greater implicit Black-danger associations were associated with increased amygdala activation to armed Blacks, compared to armed Whites. Our results suggest that different neural mechanisms may underlie racial differences in responses to armed versus unarmed targets.

Rethinking the role of the rTPJ in attention and social cognition in light of the opposing domains hypothesis: findings from an ALE-based meta-analysis and resting-state functional connectivity

PubMed Central

Kubit, Benjamin; Jack, Anthony I.

2013-01-01

The right temporo-parietal junction (rTPJ) has been associated with two apparently disparate functional roles: in attention and in social cognition. According to one account, the rTPJ initiates a “circuit-breaking” signal that interrupts ongoing attentional processes, effectively reorienting attention. It is argued this primary function of the rTPJ has been extended beyond attention, through a process of evolutionarily cooption, to play a role in social cognition. We propose an alternative account, according to which the capacity for social cognition depends on a network which is both distinct from and in tension with brain areas involved in focused attention and target detection: the default mode network (DMN). Theory characterizing the rTPJ based on the area's purported role in reorienting may be falsely guided by the co-occurrence of two distinct effects in contiguous regions: activation of the supramarginal gyrus (SMG), associated with its functional role in target detection; and the transient release, during spatial reorienting, of suppression of the angular gyrus (AG) associated with focused attention. Findings based on meta-analysis and resting functional connectivity are presented which support this alternative account. We find distinct regions, possessing anti-correlated patterns of resting connectivity, associated with social reasoning (AG) and target detection (SMG) at the rTPJ. The locus for reorienting was spatially intermediate between the AG and SMG and showed a pattern of connectivity with similarities to social reasoning and target detection seeds. These findings highlight a general methodological concern for brain imaging. Given evidence that certain tasks not only activate some areas but also suppress activity in other areas, it is suggested that researchers need to distinguish two distinct putative mechanisms, either of which may produce an increase in activity in a brain area: functional engagement in the task vs. release of suppression. PMID:23847497

P21-activated kinase in inflammatory and cardiovascular disease.

PubMed

Taglieri, Domenico M; Ushio-Fukai, Masuko; Monasky, Michelle M

2014-09-01

P-21 activated kinases, or PAKs, are serine-threonine kinases that serve a role in diverse biological functions and organ system diseases. Although PAK signaling has been the focus of many investigations, still our understanding of the role of PAK in inflammation is incomplete. This review consolidates what is known about PAK1 across several cell types, highlighting the role of PAK1 and PAK2 in inflammation in relation to NADPH oxidase activation. This review explores the physiological functions of PAK during inflammation, the role of PAK in several organ diseases with an emphasis on cardiovascular disease, and the PAK signaling pathway, including activators and targets of PAK. Also, we discuss PAK1 as a pharmacological anti-inflammatory target, explore the potentials and the limitations of the current pharmacological tools to regulate PAK1 activity during inflammation, and provide indications for future research. We conclude that a vast amount of evidence supports the idea that PAK is a central molecule in inflammatory signaling, thus making PAK1 itself a promising prospective pharmacological target. Copyright © 2014 Elsevier Inc. All rights reserved.

Targeted brain activation using an MR-compatible wrist torque measurement device and isometric motor tasks during functional magnetic resonance imaging.

PubMed

Vlaar, Martijn P; Mugge, Winfred; Groot, Paul F C; Sharifi, Sarvi; Bour, Lo J; van der Helm, Frans C T; van Rootselaar, Anne-Fleur; Schouten, Alfred C

2016-07-01

Dedicated pairs of isometric wrist flexion tasks, with and without visual feedback of the exerted torque, were designed to target activation of the CBL and BG in healthy subjects during functional magnetic resonance imaging (fMRI). Selective activation of the cerebellum (CBL) and basal ganglia (BG), often implicated in movement disorders such as tremor and dystonia, may help identify pathological changes and expedite diagnosis. A prototyped MR-compatible wrist torque measurement device, free of magnetic and conductive materials, allowed safe execution of tasks during fMRI without causing artifacts. A significant increase of activity in CBL and BG was found in healthy volunteers during a constant torque task with visual feedback compared to a constant torque task without visual feedback. This study shows that specific pairs of motor tasks using MR-compatible equipment at the wrist allow for targeted activation of CBL and BG, paving a new way for research into the pathophysiology of movement disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Neuropilin-1-targeted gold nanoparticles enhance therapeutic efficacy of platinum(IV) drug for prostate cancer treatment.

PubMed

Kumar, Anil; Huo, Shuaidong; Zhang, Xu; Liu, Juan; Tan, Aaron; Li, Shengliang; Jin, Shubin; Xue, Xiangdong; Zhao, YuanYuan; Ji, Tianjiao; Han, Lu; Liu, Hong; Zhang, XiaoNing; Zhang, Jinchao; Zou, Guozhang; Wang, Tianyou; Tang, Suoqin; Liang, Xing-Jie

2014-05-27

Platinum-based anticancer drugs such as cisplatin, oxaliplatin, and carboplatin are some of the most potent chemotherapeutic agents but have limited applications due to severe dose-limiting side effects and a tendency for cancer cells to rapidly develop resistance. The therapeutic index can be improved through use of nanocarrier systems to target cancer cells efficiently. We developed a unique strategy to deliver a platinum(IV) drug to prostate cancer cells by constructing glutathione-stabilized (Au@GSH) gold nanoparticles. Glutathione (GSH) has well-known antioxidant properties, which lead to cancer regression. Here, we exploit the advantages of both the antioxidant properties and high surface-area-to-volume ratio of Au@GSH NPs to demonstrate their potential for delivery of a platinum(IV) drug by targeting the neuropilin-1 receptor (Nrp-1). A lethal dose of a platinum(IV) drug functionalized with the Nrp-1-targeting peptide (CRGDK) was delivered specifically to prostate cancer cells in vitro. Targeted peptide ensures specific binding to the Nrp-1 receptor, leading to enhanced cellular uptake level and cell toxicity. The nanocarriers were themselves nontoxic, but exhibited high cytotoxicity and increased efficacy when functionalized with the targeting peptide and drug. The uptake of drug-loaded nanocarriers is dependent on the interaction with Nrp-1 in cell lines expressing high (PC-3) and low (DU-145) levels of Nrp-1, as confirmed through inductively coupled plasma mass spectrometry and confocal microscopy. The nanocarriers have effective anticancer activity, through upregulation of nuclear factor kappa-B (NF-κB) protein (p50 and p65) expression and activation of NF-κB-DNA-binding activity. Our preliminary investigations with platinum(IV)-functionalized gold nanoparticles along with a targeting peptide hold significant promise for future cancer treatment.

The Quest for Targets Executing MYC-Dependent Cell Transformation.

PubMed

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired.

The Quest for Targets Executing MYC-Dependent Cell Transformation

PubMed Central

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired. PMID:27313991

Mitochondrial pyruvate carrier regulates autophagy, inflammation, and neurodegeneration in experimental models of Parkinson's disease.

PubMed

Ghosh, Anamitra; Tyson, Trevor; George, Sonia; Hildebrandt, Erin N; Steiner, Jennifer A; Madaj, Zachary; Schulz, Emily; Machiela, Emily; McDonald, William G; Escobar Galvis, Martha L; Kordower, Jeffrey H; Van Raamsdonk, Jeremy M; Colca, Jerry R; Brundin, Patrik

2016-12-07

Mitochondrial and autophagic dysfunction as well as neuroinflammation are involved in the pathophysiology of Parkinson's disease (PD). We hypothesized that targeting the mitochondrial pyruvate carrier (MPC), a key controller of cellular metabolism that influences mTOR (mammalian target of rapamycin) activation, might attenuate neurodegeneration of nigral dopaminergic neurons in animal models of PD. To test this, we used MSDC-0160, a compound that specifically targets MPC, to reduce its activity. MSDC-0160 protected against 1-methyl-4-phenylpyridinium (MPP + ) insult in murine and cultured human midbrain dopamine neurons and in an α-synuclein-based Caenorhabditis elegans model. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, MSDC-0160 improved locomotor behavior, increased survival of nigral dopaminergic neurons, boosted striatal dopamine levels, and reduced neuroinflammation. Long-term targeting of MPC preserved motor function, rescued the nigrostriatal pathway, and reduced neuroinflammation in the slowly progressive Engrailed1 (En1 +/- ) genetic mouse model of PD. Targeting MPC in multiple models resulted in modulation of mitochondrial function and mTOR signaling, with normalization of autophagy and a reduction in glial cell activation. Our work demonstrates that changes in metabolic signaling resulting from targeting MPC were neuroprotective and anti-inflammatory in several PD models, suggesting that MPC may be a useful therapeutic target in PD. Copyright © 2016, American Association for the Advancement of Science.

Cramer-Rao Lower Bound Evaluation for Linear Frequency Modulation Based Active Radar Networks Operating in a Rice Fading Environment

PubMed Central

Shi, Chenguang; Salous, Sana; Wang, Fei; Zhou, Jianjiang

2016-01-01

This paper investigates the joint target parameter (delay and Doppler) estimation performance of linear frequency modulation (LFM)-based radar networks in a Rice fading environment. The active radar networks are composed of multiple radar transmitters and multichannel receivers placed on moving platforms. First, the log-likelihood function of the received signal for a Rician target is derived, where the received signal scattered off the target comprises of dominant scatterer (DS) component and weak isotropic scatterers (WIS) components. Then, the analytically closed-form expressions of the Cramer-Rao lower bounds (CRLBs) on the Cartesian coordinates of target position and velocity are calculated, which can be adopted as a performance metric to access the target parameter estimation accuracy for LFM-based radar network systems in a Rice fading environment. It is found that the cumulative Fisher information matrix (FIM) is a linear combination of both DS component and WIS components, and it also demonstrates that the joint CRLB is a function of signal-to-noise ratio (SNR), target’s radar cross section (RCS) and transmitted waveform parameters, as well as the relative geometry between the target and the radar network architectures. Finally, numerical results are provided to indicate that the joint target parameter estimation performance of active radar networks can be significantly improved with the exploitation of DS component. PMID:27929433

Membrane nanotubes facilitate long-distance interactions between natural killer cells and target cells

PubMed Central

Chauveau, Anne; Aucher, Anne; Eissmann, Philipp; Vivier, Eric; Davis, Daniel M.

2010-01-01

Membrane nanotubes are membranous tethers that physically link cell bodies over long distances. Here, we present evidence that nanotubes allow human natural killer (NK) cells to interact functionally with target cells over long distances. Nanotubes were formed when NK cells contacted target cells and moved apart. The frequency of nanotube formation was dependent on the number of receptor/ligand interactions and increased on NK cell activation. Most importantly, NK cell nanotubes contained a submicron scale junction where proteins accumulated, including DAP10, the signaling adaptor that associates with the activating receptor NKG2D, and MHC class I chain-related protein A (MICA), a cognate ligand for NKG2D, as occurs at close intercellular synapses between NK cells and target cells. Quantitative live-cell fluorescence imaging suggested that MICA accumulated at small nanotube synapses in sufficient numbers to trigger cell activation. In addition, tyrosine-phosphorylated proteins and Vav-1 accumulated at such junctions. Functionally, nanotubes could aid the lysis of distant target cells either directly or by moving target cells along the nanotube path into close contact for lysis via a conventional immune synapse. Target cells moving along the nanotube path were commonly polarized such that their uropods faced the direction of movement. This is the opposite polarization than for normal cell migration, implying that nanotubes can specifically drive target cell movement. Finally, target cells that remained connected to an NK cell by a nanotube were frequently lysed, whereas removing the nanotube using a micromanipulator reduced lysis of these target cells. PMID:20212116

Sequential CD4-Coreceptor Interactions in Human Immunodeficiency Virus Type 1 Env Function: Soluble CD4 Activates Env for Coreceptor-Dependent Fusion and Reveals Blocking Activities of Antibodies against Cryptic Conserved Epitopes on gp120

PubMed Central

Salzwedel, Karl; Smith, Erica D.; Dey, Barna; Berger, Edward A.

2000-01-01

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems. PMID:10590121

Affective context interferes with cognitive control in unipolar depression: An fMRI investigation

PubMed Central

Dichter, Gabriel S.; Felder, Jennifer N.; Smoski, Moria J.

2009-01-01

Background Unipolar major depressive disorder (MDD) is characterized by aberrant amygdala responses to sad stimuli and poor cognitive control, but the interactive effects of these impairments are poorly understood. Aim To evaluate brain activation in MDD in response to cognitive control stimuli embedded within sad and neutral contexts. Method Fourteen adults with MDD and fifteen matched controls participated in a mixed block/event-related functional magnetic resonance imaging (fMRI) task that presented oddball target stimuli embedded within blocks of sad or neutral images. Results Target events activated similar prefrontal brain regions in both groups. However, responses to target events embedded within blocks of emotional images revealed a clear group dissociation. During neutral blocks, the control group demonstrated greater activation to targets in the midfrontal gyrus and anterior cingulate relative to the MDD group, replicating previous findings of prefrontal hypo-activation in MDD samples to cognitive control stimuli. However, during sad blocks, the MDD group demonstrated greater activation in a number of prefrontal regions, including the mid-, inferior, and orbito-frontal gyri and the anterior cingulate, suggesting that relatively more prefrontal brain activation was required to disengage from the sad images to respond to the target events. Limitations A larger sample size would have provided greater statistical power, and more standardized stimuli would have increased external validity. Conclusions This double dissociation of prefrontal responses to target events embedded within neutral and sad context suggests that MDD impacts not only responses to affective events, but extends to other cognitive processes carried out in the context of affective engagement. This implies that emotional reactivity to sad events in MDD may impact functioning more broadly than previously understood. PMID:18706701

Unravelling ``off-target'' effects of redox-active polymers and polymer multilayered capsules in prostate cancer cells

NASA Astrophysics Data System (ADS)

Beretta, Giovanni L.; Folini, Marco; Cavalieri, Francesca; Yan, Yan; Fresch, Enrico; Kaliappan, Subramanian; Hasenöhrl, Christoph; Richardson, Joseph J.; Tinelli, Stella; Fery, Andreas; Caruso, Frank; Zaffaroni, Nadia

2015-03-01

Redox-active polymers and carriers are oxidizing nanoagents that can potentially trigger intracellular off-target effects. In the present study, we investigated the occurrence of off-target effects in prostate cancer cells following exposure to redox-active polymer and thin multilayer capsules with different chemical properties. We show that, depending on the intracellular antioxidant capacity, thiol-functionalized poly(methacrylic acid), PMASH triggers cell defense responses/perturbations that result in off-target effects (i.e., induction of autophagy and down-regulation of survivin). Importantly, the conversion of the carboxyl groups of PMASH into the neutral amides of poly(hydroxypropylmetacrylamide) (pHPMASH) nullified the off-target effects and cytotoxicity in tested cell lines. This suggests that the simultaneous action of carboxyl and disulfide groups in PMASH polymer or capsules may play a role in mediating the intracellular off-target effects. Our work provides evidence that the rational design of redox-active carriers for therapeutic-related application should be guided by a careful investigation on potential disturbance of the cellular machineries related to the carrier association.Redox-active polymers and carriers are oxidizing nanoagents that can potentially trigger intracellular off-target effects. In the present study, we investigated the occurrence of off-target effects in prostate cancer cells following exposure to redox-active polymer and thin multilayer capsules with different chemical properties. We show that, depending on the intracellular antioxidant capacity, thiol-functionalized poly(methacrylic acid), PMASH triggers cell defense responses/perturbations that result in off-target effects (i.e., induction of autophagy and down-regulation of survivin). Importantly, the conversion of the carboxyl groups of PMASH into the neutral amides of poly(hydroxypropylmetacrylamide) (pHPMASH) nullified the off-target effects and cytotoxicity in tested cell lines. This suggests that the simultaneous action of carboxyl and disulfide groups in PMASH polymer or capsules may play a role in mediating the intracellular off-target effects. Our work provides evidence that the rational design of redox-active carriers for therapeutic-related application should be guided by a careful investigation on potential disturbance of the cellular machineries related to the carrier association. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07240e

How activation, entanglement, and searching a semantic network contribute to event memory.

PubMed

Nelson, Douglas L; Kitto, Kirsty; Galea, David; McEvoy, Cathy L; Bruza, Peter D

2013-08-01

Free-association norms indicate that words are organized into semantic/associative neighborhoods within a larger network of words and links that bind the net together. We present evidence indicating that memory for a recent word event can depend on implicitly and simultaneously activating related words in its neighborhood. Processing a word during encoding primes its network representation as a function of the density of the links in its neighborhood. Such priming increases recall and recognition and can have long-lasting effects when the word is processed in working memory. Evidence for this phenomenon is reviewed in extralist-cuing, primed free-association, intralist-cuing, and single-item recognition tasks. The findings also show that when a related word is presented in order to cue the recall of a studied word, the cue activates the target in an array of related words that distract and reduce the probability of the target's selection. The activation of the semantic network produces priming benefits during encoding, and search costs during retrieval. In extralist cuing, recall is a negative function of cue-to-distractor strength, and a positive function of neighborhood density, cue-to-target strength, and target-to-cue strength. We show how these four measures derived from the network can be combined and used to predict memory performance. These measures play different roles in different tasks, indicating that the contribution of the semantic network varies with the context provided by the task. Finally, we evaluate spreading-activation and quantum-like entanglement explanations for the priming effects produced by neighborhood density.

Functional validation of the oncogenic cooperativity and targeting potential of tuberous sclerosis mutation in medulloblastoma using a MYC-amplified model cell line.

PubMed

Henderson, Jacob J; Wagner, Jacob P; Hofmann, Nicolle E; Eide, Christopher A; Cho, Yoon-Jae; Druker, Brian J; Davare, Monika A

2017-10-01

Medulloblastoma is the most common malignant brain tumor of childhood. To identify targetable vulnerabilities, we employed inhibitor screening that revealed mTOR inhibitor hypersensitivity in the MYC-overexpressing medulloblastoma cell line, D341. Concomitant exome sequencing unveiled an uncharacterized missense mutation, TSC2 A415V , in these cells. We biochemically demonstrate that the TSC2 A415V mutation is functionally deleterious, leading to shortened half-life and proteasome-mediated protein degradation. These data suggest that MYC cooperates with activated kinase pathways, enabling pharmacologic intervention in these treatment refractory tumors. We propose that identification of activated kinase pathways may allow for tailoring targeted therapy to improve survival and treatment-related morbidity in medulloblastoma. © 2017 Wiley Periodicals, Inc.

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB

PubMed Central

2017-01-01

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure–activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc1 oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis. PMID:29035551

AMPK-mediated regulation of neuronal metabolism and function in brain diseases.

PubMed

Liu, Yu-Ju; Chern, Yijuang

2015-01-01

The AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a key energy sensor in a wide variety of tissues. This kinase has been a major drug target for metabolic diseases (e.g., type 2 diabetes) and cancers. For example, metformin (an activator of AMPK) is a first-line diabetes drug that protects against cancers. Abnormal regulation of AMPK has been implicated in several brain diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and stroke. Given the emerging importance of neurodegenerative diseases in our aging societies, this review features the recent studies that have delineated the functions of AMPK in brain diseases and discusses their potential clinical implications or roles as drug targets in brain diseases.

Dissociable prefrontal brain systems for attention and emotion

NASA Astrophysics Data System (ADS)

Yamasaki, Hiroshi; Labar, Kevin S.; McCarthy, Gregory

2002-08-01

The prefrontal cortex has been implicated in a variety of attentional, executive, and mnemonic mental operations, yet its functional organization is still highly debated. The present study used functional MRI to determine whether attentional and emotional functions are segregated into dissociable prefrontal networks in the human brain. Subjects discriminated infrequent and irregularly presented attentional targets (circles) from frequent standards (squares) while novel distracting scenes, parametrically varied for emotional arousal, were intermittently presented. Targets differentially activated middle frontal gyrus, posterior parietal cortex, and posterior cingulate gyrus. Novel distracters activated inferior frontal gyrus, amygdala, and fusiform gyrus, with significantly stronger activation evoked by the emotional scenes. The anterior cingulate gyrus was the only brain region with equivalent responses to attentional and emotional stimuli. These results show that attentional and emotional functions are segregated into parallel dorsal and ventral streams that extend into prefrontal cortex and are integrated in the anterior cingulate. These findings may have implications for understanding the neural dynamics underlying emotional distractibility on attentional tasks in affective disorders. novelty | prefrontal cortex | amygdala | cingulate gyrus

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Preparation of stimulus responsive multiple emulsions by membrane emulsification using con a as biochemical sensor.

PubMed

Piacentini, Emma; Drioli, Enrico; Giorno, Lidietta

2011-04-01

In this work, a novel strategy for the controlled fabrication of biomolecular stimulus responsive water-in-oil-in-water (W/O/W) multiple emulsion using the membrane emulsification process was investigated. The emulsions interface was functionalized with a biomolecule able to function as a receptor for a target compound. The interaction between the biomolecular receptor and target stimulus activated the release of bioactive molecules contained within the structured emulsion. A glucose sensitive emulsion was investigated as a model study case. Concanavalin A (Con A) was used as the biomolecular glucose sensor. Various physicochemical strategies for stimulus responsive materials formulation are available in literature, but the preparation of biomolecule-responsive emulsions has been explored for the first time in this paper. The development of novel drug delivery systems requires advanced and highly precise techniques to obtain their particular properties and targeting requirements. The present study has proven the flexibility and suitability of membrane emulsification for the preparation of stable and functional multiple emulsions containing Con A as interfacial biomolecular receptor able to activate the release of a bioactive molecule as a consequence of interaction with the glucose target molecule. The influence of emulsion interfacial composition and membrane emulsification operating conditions on droplets stability and functional properties have been investigated. The release of the bioactive molecule as a function of glucose stimulus and its concentration has been demonstrated. Copyright © 2010 Wiley Periodicals, Inc.

Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

NASA Astrophysics Data System (ADS)

Shah, Dhiral Ashwin

Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. The same concept of naturally occurring protein-protein interactions can also be implemented to selectively bring intracellular protein targets in close proximity to proteasomal degradation machinery in cells and effect their depletion from the cellular compartments. This approach will be able to not only target entire pool of proteins to ubiquitination-mediated degradation, but also to specific sub-pools of posttranslationally modified proteins in the cell, provided peptides having distinct binding affinities are identified for posttranslational modifications. This system can then be tested for intracellular protein delivery using nanoparticle carriers to identify roles of different posttranslational modifications on the protein's activity. In future work, we propose to develop a cellular detection system, based on GFP complementation, which can be used to evaluate the efficiency of different protein delivery carriers to internalize proteins into the cell cytosol. We envision the application of nanoscale materials as intracellular protein delivery vehicles to target diverse cell signaling pathways at the posttranslational level, and subsequent metabolic manipulation, which may have interesting therapeutic properties and can potentially target stem cell fate.

Activator protein 1 promotes gemcitabine-induced apoptosis in pancreatic cancer by upregulating its downstream target Bim.

PubMed

Ren, Xiaoxia; Zhao, Wenjing; Du, Yongxing; Zhang, Taiping; You, Lei; Zhao, Yupei

2016-12-01

Gemcitabine is a commonly used chemotherapy drug in pancreatic cancer. The function of activator protein 1 (AP-1) is cell-specific, and its function depends on the expression of other complex members. In the present study, we added gemcitabine to the media of Panc-1 and SW1990 cells at clinically achieved concentrations (10 µM). Compared with constitutive c-Fos expression, c-Jun expression increased in a dose-dependent manner upon gemcitabine treatment. c-Jun overexpression increased gemcitabine-induced apoptosis through Bim activation, while cell apoptosis and Bim expression decreased following c-Jun knockdown. Furthermore, gemcitabine-induced apoptosis and Bim levels decreased when c-Jun phosphorylation was blocked by SP600125. Our findings suggest that c-Jun, which is a member of the AP-1 complex, functions in gemcitabine-induced apoptosis by regulating its downstream target Bim in pancreatic cancer cells.

Subcortical roles in lexical task processing: Inferences from thalamic and subthalamic event-related potentials.

PubMed

Tiedt, Hannes O; Ehlen, Felicitas; Krugel, Lea K; Horn, Andreas; Kühn, Andrea A; Klostermann, Fabian

2017-01-01

Subcortical functions for language capacities are poorly defined, but may be investigated in the context of deep brain stimulation. Here, we studied event-related potentials recorded from electrodes in the subthalamic nucleus (STN) and the thalamic ventral intermediate nucleus (VIM) together with surface-EEG. Participants completed a lexical decision task (LDT), which required the differentiation of acoustically presented words from pseudo-words by button press. Target stimuli were preceded by prime-words. In recordings from VIM, a slow potential shift apparent at the lower electrode contacts persisted during target stimulus presentation (equally for words and pseudo-words). In contrast, recordings from STN electrodes showed a short local activation on prime-words but not target-stimuli. In both depth-recording regions, further components related to contralateral motor responses to target words were evident. On scalp level, mid-central activations on (pseudo)lexical stimuli were obtained, in line with the expression of N400 potentials. The prolonged activity recorded from VIM, exclusively accompanying the relevant LDT phase, is in line with the idea of thalamic "selective engagement" for supporting the realization of the behavioral focus demanded by the task. In contrast, the phasic prime related activity rather indicates "procedural" STN functions, for example, for trial sequencing or readiness inhibition of prepared target reactions. Hum Brain Mapp 38:370-383, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Task difficulty modulates brain activation in the emotional oddball task.

PubMed

Siciliano, Rachel E; Madden, David J; Tallman, Catherine W; Boylan, Maria A; Kirste, Imke; Monge, Zachary A; Packard, Lauren E; Potter, Guy G; Wang, Lihong

2017-06-01

Previous functional magnetic resonance imaging (fMRI) studies have reported that task-irrelevant, emotionally salient events can disrupt target discrimination, particularly when attentional demands are low, while others demonstrate alterations in the distracting effects of emotion in behavior and neural activation in the context of attention-demanding tasks. We used fMRI, in conjunction with an emotional oddball task, at different levels of target discrimination difficulty, to investigate the effects of emotional distractors on the detection of subsequent targets. In addition, we distinguished different behavioral components of target detection representing decisional, nondecisional, and response criterion processes. Results indicated that increasing target discrimination difficulty led to increased time required for both the decisional and nondecisional components of the detection response, as well as to increased target-related neural activation in frontoparietal regions. The emotional distractors were associated with activation in ventral occipital and frontal regions and dorsal frontal regions, but this activation was attenuated with increased difficulty. Emotional distraction did not alter the behavioral measures of target detection, but did lead to increased target-related frontoparietal activation for targets following emotional images as compared to those following neutral images. This latter effect varied with target discrimination difficulty, with an increased influence of the emotional distractors on subsequent target-related frontoparietal activation in the more difficult discrimination condition. This influence of emotional distraction was in addition associated specifically with the decisional component of target detection. These findings indicate that emotion-cognition interactions, in the emotional oddball task, vary depending on the difficulty of the target discrimination and the associated limitations on processing resources. Copyright © 2017 Elsevier B.V. All rights reserved.

Robust aptamer-polydopamine-functionalized M-PLGA-TPGS nanoparticles for targeted delivery of docetaxel and enhanced cervical cancer therapy.

PubMed

Xu, Guojun; Yu, Xinghua; Zhang, Jinxie; Sheng, Yingchao; Liu, Gan; Tao, Wei; Mei, Lin

2016-01-01

One limitation of current biodegradable polymeric nanoparticles (NPs) is the contradiction between functional modification and maintaining formerly excellent bioproperties with simple procedures. Here, we reported a robust aptamer-polydopamine-functionalized mannitol-functionalized poly(lactide-co-glycolide) (M-PLGA)-D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) nanoformulation (Apt-pD-NPs) for the delivery of docetaxel (DTX) with enhanced cervical cancer therapy effects. The novel DTX-loaded Apt-pD-NPs possess satisfactory advantages: 1) increased drug loading content and encapsulation efficiency induced by star-shaped copolymer M-PLGA-TPGS; 2) significant active targeting effect caused by conjugated AS1411 aptamers; and 3) excellent long-term compatibility by incorporation of TPGS. Therefore, with simple preparation procedures and excellent bioproperties, the new functionalized Apt-pD-NPs could maximally increase the local effective drug concentration on tumor sites, achieving enhanced treatment effectiveness and minimizing side effects. In a word, the robust DTX-loaded Apt-pD-NPs could be used as potential nanotherapeutics for cervical cancer treatment, and the aptamer-polydopamine modification strategy could be a promising method for active targeting of cancer therapy with simple procedures.

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

PubMed

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

UnPAKing the class differences among p21-activated kinases.

PubMed

Eswaran, Jeyanthy; Soundararajan, Meera; Kumar, Rakesh; Knapp, Stefan

2008-08-01

The p21-activated kinases (PAKs) are signal transducers, central to many vital cellular processes, including cell morphology, motility, survival, gene transcription and hormone signalling. The mammalian PAK family contains six serine/threonine kinases divided into two subgroups, group I (PAK 1-3) and group II (PAK4-6), based on their domain architecture and regulation. PAKs functioning as dynamic signalling nodes present themselves as attractive therapeutic targets in tumours, neurological diseases and infection. The recent findings across all PAKs, including newly reported structures, shed light on the cellular functions of PAKs, highlighting molecular mechanisms of activation, catalysis and substrate specificity. We believe that a comprehensive understanding of the entire PAK family is essential for developing strategies towards PAK-targeted therapeutics.

Trace amine-associated receptor 1: a promising target for the treatment of psychostimulant addiction

PubMed Central

Jing, Li; Li, Jun-Xu

2015-01-01

Abuse of and addiction to psychostimulants remains a challenging clinical issue, yet no effective pharmacotherapy is available. Trace amine associated receptor 1 (TAAR 1) is increasingly recognized as a novel drug target that participates in the modulation of drug abuse. This review analyzed existing preclinical evidence from electrophysiological, biochemical to behavioral aspects regarding the functional interactions between TAAR 1 and dopaminergic system. TAAR 1 knockout mice demonstrate increased sensitivity to dopaminergic activation while TAAR 1 agonists reduce the neurochemical effects of cocaine and amphetamines, attenuate abuse- and addiction-related behavioral effects of cocaine and methamphetamine. It is concluded that TAAR 1 activation functionally modulate the dopaminergic activity and TAAR 1 agonists appear to be promising pharmacotherapies against psychostimulant addiction. PMID:26092759

Altered prefrontal function with aging: insights into age-associated performance decline.

PubMed

Solbakk, Anne-Kristin; Fuhrmann Alpert, Galit; Furst, Ansgar J; Hale, Laura A; Oga, Tatsuhide; Chetty, Sundari; Pickard, Natasha; Knight, Robert T

2008-09-26

We examined the effects of aging on visuo-spatial attention. Participants performed a bi-field visual selective attention task consisting of infrequent target and task-irrelevant novel stimuli randomly embedded among repeated standards in either attended or unattended visual fields. Blood oxygenation level dependent (BOLD) responses to the different classes of stimuli were measured using functional magnetic resonance imaging. The older group had slower reaction times to targets, and committed more false alarms but had comparable detection accuracy to young controls. Attended target and novel stimuli activated comparable widely distributed attention networks, including anterior and posterior association cortex, in both groups. The older group had reduced spatial extent of activation in several regions, including prefrontal, basal ganglia, and visual processing areas. In particular, the anterior cingulate and superior frontal gyrus showed more restricted activation in older compared with young adults across all attentional conditions and stimulus categories. The spatial extent of activations correlated with task performance in both age groups, but the regional pattern of association between hemodynamic responses and behavior differed between the groups. Whereas the young subjects relied on posterior regions, the older subjects engaged frontal areas. The results indicate that aging alters the functioning of neural networks subserving visual attention, and that these changes are related to cognitive performance.

Musashi Protein-directed Translational Activation of Target mRNAs Is Mediated by the Poly(A) Polymerase, Germ Line Development Defective-2*

PubMed Central

Cragle, Chad; MacNicol, Angus M.

2014-01-01

The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation. PMID:24644291

Expectations Regarding Aging, Physical Activity, and Physical Function in Older Adults

PubMed Central

Breda, Aili I.; Watts, Amber S.

2017-01-01

Objective: The present study examined how expectations regarding aging (ERA) influence physical activity participation and physical function. Method: We surveyed 148 older adults about their ERA (ERA-38), health-promoting lifestyles (HPLP-II), and self-rated health (RAND-36). We tested the mediating effect of physical activity on the relationships between ERA and physical function. Results: Positive expectations were associated with more engagement in physical activity (B = 0.016, p < .05) and better physical function (B = 0.521, p < .01). Physical activity mediated the relationship between ERA and physical function (B = 5.890, p < .01, indirect effect 0.092, CI = [0.015, 0.239]). Discussion: ERA play an important role in adoption of physically active lifestyles in older adults and may influence health outcomes, such as physical function. Future research should evaluate whether attempts to increase physical activity are more successful when modifications to ERA are also targeted. PMID:28491915

Zn(II)-curc targets p53 in thyroid cancer cells.

PubMed

Garufi, Alessia; D'Orazi, Valerio; Crispini, Alessandra; D'Orazi, Gabriella

2015-10-01

TP53 mutation is a common event in many cancers, including thyroid carcinoma. Defective p53 activity promotes cancer resistance to therapies and a more malignant phenotype, acquiring oncogenic functions. Rescuing the function of mutant p53 (mutp53) protein is an attractive anticancer therapeutic strategy. Zn(II)-curc is a novel small molecule that has been shown to target mutp53 protein in several cancer cells, but its effect in thyroid cancer cells remains unclear. Here, we investigated whether Zn(II)-curc could affect p53 in thyroid cancer cells with both p53 mutation (R273H) and wild-type p53. Zn(II)-curc induced mutp53H273 downregulation and reactivation of wild-type functions, such as binding to canonical target promoters and target gene transactivation. This latter effect was similar to that induced by PRIMA-1. In addition, Zn(II)-curc triggered p53 target gene expression in wild-type p53-carrying cells. In combination treatments, Zn(II)-curc enhanced the antitumor activity of chemotherapeutic drugs, in both mutant and wild-type-carrying cancer cells. Taken together, our data indicate that Zn(II)-curc promotes the reactivation of p53 in thyroid cancer cells, providing in vitro evidence for a potential therapeutic approach in thyroid cancers.

Activation in mesolimbic and visuospatial neural circuits elicited by smoking cues: evidence from functional magnetic resonance imaging.

PubMed

Due, Deborah L; Huettel, Scott A; Hall, Warren G; Rubin, David C

2002-06-01

The authors sought to increase understanding of the brain mechanisms involved in cigarette addiction by identifying neural substrates modulated by visual smoking cues in nicotine-deprived smokers. Event-related functional magnetic resonance imaging (fMRI) was used to detect brain activation after exposure to smoking-related images in a group of nicotine-deprived smokers and a nonsmoking comparison group. Subjects viewed a pseudo-random sequence of smoking images, neutral nonsmoking images, and rare targets (photographs of animals). Subjects pressed a button whenever a rare target appeared. In smokers, the fMRI signal was greater after exposure to smoking-related images than after exposure to neutral images in mesolimbic dopamine reward circuits known to be activated by addictive drugs (right posterior amygdala, posterior hippocampus, ventral tegmental area, and medial thalamus) as well as in areas related to visuospatial attention (bilateral prefrontal and parietal cortex and right fusiform gyrus). In nonsmokers, no significant differences in fMRI signal following exposure to smoking-related and neutral images were detected. In most regions studied, both subject groups showed greater activation following presentation of rare target images than after exposure to neutral images. In nicotine-deprived smokers, both reward and attention circuits were activated by exposure to smoking-related images. Smoking cues are processed like rare targets in that they activate attentional regions. These cues are also processed like addictive drugs in that they activate mesolimbic reward regions.

Regulatory link between DNA methylation and active demethylation in Arabidopsis

PubMed Central

Lei, Mingguang; Zhang, Huiming; Julian, Russell; Tang, Kai; Xie, Shaojun; Zhu, Jian-Kang

2015-01-01

De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression. PMID:25733903

Repetition suppression of faces is modulated by emotion

NASA Astrophysics Data System (ADS)

Ishai, Alumit; Pessoa, Luiz; Bikle, Philip C.; Ungerleider, Leslie G.

2004-06-01

Single-unit recordings and functional brain imaging studies have shown reduced neural responses to repeated stimuli in the visual cortex. By using event-related functional MRI, we compared the activation evoked by repetitions of neutral and fearful faces, which were either task relevant (targets) or irrelevant (distracters). We found that within the inferior occipital gyri, lateral fusiform gyri, superior temporal sulci, amygdala, and the inferior frontal gyri/insula, targets evoked stronger responses than distracters and their repetition was associated with significantly reduced responses. Repetition suppression, as manifested by the difference in response amplitude between the first and third repetitions of a target, was stronger for fearful than neutral faces. Distracter faces, regardless of their repetition or valence, evoked negligible activation, indicating top-down attenuation of behaviorally irrelevant stimuli. Our findings demonstrate a three-way interaction between emotional valence, repetition, and task relevance and suggest that repetition suppression is influenced by high-level cognitive processes in the human brain. face perception | functional MRI

The potential of targeting Ras proteins in lung cancer.

PubMed

McCormick, Frank

2015-04-01

The Ras pathway is a major driver in lung adenocarcinoma: over 75% of all cases harbor mutations that activate this pathway. While spectacular clinical successes have been achieved by targeting activated receptor tyrosine kinases in this pathway, little, if any, significant progress has been achieved targeting Ras proteins themselves or cancers driven by oncogenic Ras mutants. New approaches to drug discovery, new insights into Ras function, new ways of attacking undruggable proteins through RNA interference and new ways of harnessing the immune system could change this landscape in the relatively near future.

Using "Functional" Target Coordinates of the Subthalamic Nucleus to Assess the Indirect and Direct Methods of the Preoperative Planning: Do the Anatomical and Functional Targets Coincide?

PubMed

Rabie, Ahmed; Verhagen Metman, Leo; Slavin, Konstantin V

2016-12-21

To answer the question of whether the anatomical center of the subthalamic nucleus (STN), as calculated indirectly from stereotactic atlases or by direct visualization on magnetic resonance imaging (MRI), corresponds to the best functional target. Since the neighboring red nucleus (RN) is well visualized on MRI, we studied the relationships of the final target to its different borders. We analyzed the data of 23 PD patients (46 targets) who underwent bilateral frame-based STN deep brain stimulation (DBS) procedure with microelectrode recording guidance. We calculated coordinates of the active contact on DBS electrode on postoperative MRI, which we referred to as the final "functional/optimal" target. The coordinates calculated by the atlas-based "indirect" and "direct" methods, as well as the coordinates of the different RN borders were compared to these final coordinates. The mean ± SD of the final target coordinates was 11.7 ± 1.5 mm lateral (X), 2.4 ± 1.5 mm posterior (Y), and 6.1 ± 1.7 mm inferior to the mid-commissural point (Z). No significant differences were found between the "indirect" X, Z coordinates and those of the final targets. The "indirect" Y coordinate was significantly posterior to Y of the final target, with mean difference of 0.6 mm ( p = 0.014). No significant differences were found between the "direct" X, Y, and Z coordinates and those of the final targets. The functional STN target is located in direct proximity to its anatomical center. During preoperative targeting, we recommend using the "direct" method, and taking into consideration the relationships of the final target to the mid-commissural point (MCP) and the different RN borders.

Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets

PubMed Central

Stadler, Michael; Artiles, Karen; Pak, Julia; Fire, Andrew

2012-01-01

miRNAs are post-transcriptional regulators of gene activity that reduce protein accumulation from target mRNAs. Elucidating precise molecular effects that animal miRNAs have on target transcripts has proven complex, with varied evidence indicating that miRNA regulation may produce different molecular outcomes in different species, systems, and/or physiological conditions. Here we use high-throughput ribosome profiling to analyze detailed translational parameters for five well-studied targets of miRNAs that regulate C. elegans developmental timing. For two targets of the miRNA lin-4 (lin-14 and lin-28), functional down-regulation was associated with decreases in both overall mRNA abundance and ribosome loading; however, these changes were of substantially smaller magnitude than corresponding changes observed in protein abundance. For three functional targets of the let-7 miRNA family for which down-regulation is critical in temporal progression of the animal (daf-12, hbl-1, and lin-41), we observed only modest changes in mRNA abundance and ribosome loading. lin-41 provides a striking example in that populations of ribosome-protected fragments from this gene remained essentially unchanged during the L3–L4 time interval when lin-41 activity is substantially down-regulated by let-7. Spectra of ribosomal positions were also examined for the five lin-4 and let-7 target mRNAs as a function of developmental time, with no indication of miRNA-induced ribosomal drop-off or significant pauses in translation. These data are consistent with models in which physiological regulation by this set of C. elegans miRNAs derives from combinatorial effects including suppressed recruitment/activation of translational machinery, compromised stability of target messages, and post- or peri-translational effects on lifetimes of polypeptide products. PMID:22855835

Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study.

PubMed

Simon, Gabriel M; Cravatt, Benjamin F

2010-04-09

Genome sequencing projects have uncovered thousands of uncharacterized enzymes in eukaryotic and prokaryotic organisms. Deciphering the physiological functions of enzymes requires tools to profile and perturb their activities in native biological systems. Activity-based protein profiling has emerged as a powerful chemoproteomic strategy to achieve these objectives through the use of chemical probes that target large swaths of enzymes that share active-site features. Here, we review activity-based protein profiling and its implementation to annotate the enzymatic proteome, with particular attention given to probes that target serine hydrolases, a diverse superfamily of enzymes replete with many uncharacterized members.

Physical and functional interactions between Drosophila TRAF2 and Pelle kinase contribute to Dorsal activation.

PubMed

Shen, B; Liu, H; Skolnik, E Y; Manley, J L

2001-07-17

Signaling through the Toll receptor is required for dorsal/ventral polarity in Drosophila embryos, and also plays an evolutionarily conserved role in the immune response. Upon ligand binding, Toll appears to multimerize and activate the associated kinase, Pelle. However, the immediate downstream targets of Pelle have not been identified. Here we show that Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2), a homologue of human TRAF6, physically and functionally interacts with Pelle, and is phosphorylated by Pelle in vitro. Importantly, dTRAF2 and Pelle cooperate to activate Dorsal synergistically in cotransfected Schneider cells. Deletion of the C-terminal TRAF domain of dTRAF2 enhances Dorsal activation, perhaps reflecting the much stronger interaction of the mutant protein with phosphorylated, active Pelle. Taken together, our results indicate that Pelle and dTRAF2 physically and functionally interact, and that the TRAF domain acts as a regulator of this interaction. dTRAF2 thus appears to be a downstream target of Pelle. We discuss these results in the context of Toll signaling in flies and mammals.

Neuroimaging somatosensory perception and masking.

PubMed

Meador, Kimford J; Revill, Kathleen Pirog; Epstein, Charles M; Sathian, K; Loring, David W; Rorden, Chris

2017-01-08

The specific cortical and subcortical regions involved in conscious perception and masking are uncertain. This study sought to identify brain areas involved in conscious perception of somatosensory stimuli during a masking task using functional magnetic resonance (fMRI) to contrast perceived vs. non-perceived targets. Electrical trains were delivered to the right index finger for targets and to the left index finger for masks. Target intensities were adjusted to compensate for threshold drift. Sham target trials were given in ~10% of the trials, and target stimuli without masks were delivered in one of the five runs (68 trials/run). When healthy dextral adult volunteers (n=15) perceived right hand targets, greater left- than right-cerebral activations were seen with similar patterns across the parietal cortex, thalamus, insula, claustrum, and midbrain. When targets were not perceived, left/right cerebral activations were similar overall. Directly comparing perceived vs. non-perceived stimuli with similar intensities in the masking task revealed predominate activations contralateral to masks. In contrast, activations were greater contralateral to perceived targets if no masks were given or if masks were given but target stimulus intensities were greater for perceived than non-perceived targets. The novel aspects of this study include: 1) imaging of cortical and subcortical activations in healthy humans related to somatosensory perception during a masking task, 2) activations in the human thalamus and midbrain related to perception of stimuli compared to matched non-perceived stimuli, and 3) similar left/right cerebral activation patterns across cortical, thalamic and midbrain structures suggesting interactions across all three levels during conscious perception in humans. Copyright © 2016 Elsevier Ltd. All rights reserved.

Helping Eve overcome ADAM: G-quadruplexes in the ADAM-15 promoter as new molecular targets for breast cancer therapeutics.

PubMed

Brown, Robert V; Gaerig, Vanessa C; Simmons, Taesha; Brooks, Tracy A

2013-12-05

ADAM-15, with known zymogen, secretase, and disintegrin activities, is a catalytically active member of the ADAM family normally expressed in early embryonic development and aberrantly expressed in various cancers, including breast, prostate and lung. ADAM-15 promotes extracellular shedding of E-cadherin, a soluble ligand for the HER2/neu receptor, leading to activation, increased motility, and proliferation. Targeted downregulation of both ADAM-15 and HER2/neu function synergistically kills breast cancer cells, but to date there are no therapeutic options for decreasing ADAM-15 function or expression. In this vein, we have examined a unique string of guanine-rich DNA within the critical core promoter of ADAM-15. This region of DNA consists of seven contiguous runs of three or more consecutive guanines, which, under superhelical stress, can relax from duplex DNA to form an intrastrand secondary G-quadruplex (G4) structure. Using biophysical and biological techniques, we have examined the G4 formation within the entire and various truncated regions of the ADAM-15 promoter, and demonstrate strong intrastrand G4 formation serving to function as a biological silencer element. Characterization of the predominant G4 species formed within the ADAM-15 promoter will allow for specific drug targeting and stabilization, and the further development of novel, targeted therapeutics.

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

PubMed

Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

2017-03-17

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

Structural and functional analyses reveal the contributions of the C- and N-lobes of Argonaute protein to selectivity of RNA target cleavage.

PubMed

Dayeh, Daniel M; Kruithoff, Bradley C; Nakanishi, Kotaro

2018-04-27

Some gene transcripts have cellular functions as regulatory noncoding RNAs. For example, ∼23-nucleotide (nt)-long siRNAs are loaded into Argonaute proteins. The resultant ribonucleoprotein assembly, the RNA-induced silencing complex (RISC), cleaves RNAs that are extensively base-paired with the loaded siRNA. To date, base complementarity is recognized as the major determinant of specific target cleavage (or slicing), but little is known about how Argonaute inspects base pairing before cleavage. A hallmark of Argonaute proteins is their bilobal structure, but despite the significance of this structure for curtailing slicing activity against mismatched targets, the molecular mechanism remains elusive. Here, our structural and functional studies of a bilobed yeast Argonaute protein and its isolated catalytic C-terminal lobe (C-lobe) revealed that the C-lobe alone retains almost all properties of bilobed Argonaute: siRNA-duplex loading, passenger cleavage/ejection, and siRNA-dependent RNA cleavage. A 2.1 Å-resolution crystal structure revealed that the catalytic C-lobe mirrors the bilobed Argonaute in terms of guide-RNA recognition and that all requirements for transitioning to the catalytically active conformation reside in the C-lobe. Nevertheless, we found that in the absence of the N-terminal lobe (N-lobe), target RNAs are scanned for complementarity only at positions 5-14 on a 23-nt guide RNA before endonucleolytic cleavage, thereby allowing for some off-target cleavage. Of note, acquisition of an N-lobe expanded the range of the guide RNA strand used for inspecting target complementarity to positions 2-23. These findings offer clues to the evolution of the bilobal structure of catalytically active Argonaute proteins. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Source-based neurofeedback methods using EEG recordings: training altered brain activity in a functional brain source derived from blind source separation

PubMed Central

White, David J.; Congedo, Marco; Ciorciari, Joseph

2014-01-01

A developing literature explores the use of neurofeedback in the treatment of a range of clinical conditions, particularly ADHD and epilepsy, whilst neurofeedback also provides an experimental tool for studying the functional significance of endogenous brain activity. A critical component of any neurofeedback method is the underlying physiological signal which forms the basis for the feedback. While the past decade has seen the emergence of fMRI-based protocols training spatially confined BOLD activity, traditional neurofeedback has utilized a small number of electrode sites on the scalp. As scalp EEG at a given electrode site reflects a linear mixture of activity from multiple brain sources and artifacts, efforts to successfully acquire some level of control over the signal may be confounded by these extraneous sources. Further, in the event of successful training, these traditional neurofeedback methods are likely influencing multiple brain regions and processes. The present work describes the use of source-based signal processing methods in EEG neurofeedback. The feasibility and potential utility of such methods were explored in an experiment training increased theta oscillatory activity in a source derived from Blind Source Separation (BSS) of EEG data obtained during completion of a complex cognitive task (spatial navigation). Learned increases in theta activity were observed in two of the four participants to complete 20 sessions of neurofeedback targeting this individually defined functional brain source. Source-based EEG neurofeedback methods using BSS may offer important advantages over traditional neurofeedback, by targeting the desired physiological signal in a more functionally and spatially specific manner. Having provided preliminary evidence of the feasibility of these methods, future work may study a range of clinically and experimentally relevant brain processes where individual brain sources may be targeted by source-based EEG neurofeedback. PMID:25374520

Abnormality in catalase import into peroxisomes leads to severe neurological disorder

PubMed Central

Sheikh, Faruk G.; Pahan, Kalipada; Khan, Mushfiquddin; Barbosa, Ernest; Singh, Inderjit

1998-01-01

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H2O2 or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the in situ levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H2O2 toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease. PMID:9501198

Small interfering RNA targeting focal adhesion kinase prevents cardiac dysfunction in endotoxemia.

PubMed

Guido, Maria C; Clemente, Carolina F; Moretti, Ana I; Barbeiro, Hermes V; Debbas, Victor; Caldini, Elia G; Franchini, Kleber G; Soriano, Francisco G

2012-01-01

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

The transcriptional PPARβ/δ network in human macrophages defines a unique agonist-induced activation state

PubMed Central

Adhikary, Till; Wortmann, Annika; Schumann, Tim; Finkernagel, Florian; Lieber, Sonja; Roth, Katrin; Toth, Philipp M.; Diederich, Wibke E.; Nist, Andrea; Stiewe, Thorsten; Kleinesudeik, Lara; Reinartz, Silke; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-01-01

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor with established metabolic functions, whereas its anti-inflammatory function is poorly understood. To address this issue, we determined the global PPARβ/δ-regulated signaling network in human monocyte-derived macrophages. Besides cell type-independent, canonical target genes with metabolic and immune regulatory functions we identified a large number of inflammation-associated NFκB and STAT1 target genes that are repressed by agonists. Accordingly, PPARβ/δ agonists inhibited the expression of multiple pro-inflammatory mediators and induced an anti-inflammatory, IL-4-like morphological phenotype. Surprisingly, bioinformatic analyses also identified immune stimulatory effects. Consistent with this prediction, PPARβ/δ agonists enhanced macrophage survival under hypoxic stress and stimulated CD8+ T cell activation, concomitantly with the repression of immune suppressive target genes and their encoded products CD274 (PD-1 ligand), CD32B (inhibitory Fcγ receptor IIB) and indoleamine 2,3-dioxygenase 1 (IDO-1), as well as a diminished release of the immune suppressive IDO-1 metabolite kynurenine. Comparison with published data revealed a significant overlap of the PPARβ/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines. Our findings indicate that PPARβ/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/δ in immune regulation. PMID:25934804

A novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da

2009-08-01

The pursuit of efficient and highly targeting-selective transporters is an active topic in cancer-targeting therapy. In this study, a novel cancer-targeting transporter with integrin αvβ3 monoclonal antibody functionalized single-walled carbon nanotubes (SWCNTs) was developed to investigate cancer cell targeting in vitro. SWCNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). PL-PEG functionalized SWCNTs were then conjugated with fluorescein isothiocyanate (FITC) labeled integrin αvβ3 monoclonal antibody to construct SWCNT-integrin αvβ3 monoclonal antibody system (denoted as SWCNT-PEG-mAb). In vitro study revealed that the system had a high efficiency in cancer cell targeting in integrin αvβ3 positive U87MG cells. Moreover, the SWCNT-PEG-mAb is stable in physiological media, and can be readily transported into U87MG cells via integrin αvβ3-mediated endocytosis in cell. In summary, the integrin αvβ3 monoclonal antibody labeled SWCNT is a potential carrier-candidate for cancer-imaging and drug-delivering in cancer-targeting therapy.

Targeting of TAM Receptors Ameliorates Fibrotic Mechanisms in Idiopathic Pulmonary Fibrosis.

PubMed

Espindola, Milena S; Habiel, David M; Narayanan, Rohan; Jones, Isabelle; Coelho, Ana L; Murray, Lynne A; Jiang, Dianhua; Noble, Paul W; Hogaboam, Cory M

2018-06-01

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling, which progressively abolishes lung function in an RTK (receptor tyrosine kinase)-dependent manner. Gas6 (growth arrest-specific 6) ligand, Tyro3 (TYRO3 protein tyrosine kinase 3), and Axl (anexelekto) RTK expression and activity are increased in IPF. To determine if targeting these RTK pathways would inhibit fibroblast activation and the development of pulmonary fibrosis. Quantitative genomic, proteomic, and functional analyses were used to determine Gas6/TAM (Tyro3, Axl, and Mertk [MER proto-oncogene, tyrosine kinase]) RTK expression and activation in tissues and fibroblasts from normal and IPF lungs. The profibrotic impact of these RTK pathways were also examined in bleomycin-induced pulmonary fibrosis and in SCID/Bg mice that developed pulmonary fibrosis after the intravenous administration of primary IPF fibroblasts. Gas6, Axl, and Tyro3 were increased in both rapidly and slowly progressive IPF compared with normal lung samples and fibroblasts. Targeting these pathways with either specific antibodies directed at Gas6 or Axl, or with small-molecule TAM inhibitors indicated that the small molecule-mediated targeting approach was more efficacious in both in vitro and in vivo studies. Specifically, the TAM receptor inhibitor R428 (also known as BGB324) significantly inhibited the synthetic, migratory, and proliferative properties of IPF fibroblasts compared with the other Gas6/TAM receptor targeting agents. Finally, loss of Gas6 expression decreased lung fibrotic responses to bleomycin and treatment with R428 inhibited pulmonary fibrosis in humanized SCID/Bg mice. Gas6/TAM receptor activity contributes to the activation of pulmonary fibroblasts in IPF, suggesting that targeting this RTK pathway might be an effective antifibrotic strategy in this disease.

Presenilin-1 affects trafficking and processing of βAPP and is targeted in a complex with nicastrin to the plasma membrane

PubMed Central

Kaether, Christoph; Lammich, Sven; Edbauer, Dieter; Ertl, Michaela; Rietdorf, Jens; Capell, Anja; Steiner, Harald; Haass, Christian

2002-01-01

Amyloid β-peptide (Aβ) is generated by the consecutive cleavages of β- and γ-secretase. The intramembraneous γ-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with γ-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Aβ production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the γ-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with γ-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by γ-secretase inhibitors results in delayed reinternalization of the β-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in γ-secretase processing and in trafficking. PMID:12147673

Targeting Self-Binding Peptides as a Novel Strategy To Regulate Protein Activity and Function: A Case Study on the Proto-oncogene Tyrosine Protein Kinase c-Src.

PubMed

Bai, Zhengya; Hou, Shasha; Zhang, Shilei; Li, Zhongyan; Zhou, Peng

2017-04-24

Previously, we have reported a new biomolecular phenomenon spanning between protein folding and binding, termed as self-binding peptides (SBPs), where a short peptide segment in monomeric protein functions as a molecular switch by dynamically binding to/unbinding from its cognate domain in the monomer (Yang et al. J. Chem. Inf. 2015, 55, 329-342). Here, we attempt to raise the SBP as a new class of druggable targets to regulate the biological activity and function of proteins. A case study was performed on the proto-oncogene nonreceptor tyrosine kinase, c-Src, which contains two SBPs that bind separately to SH3 and SH2 domains of the kinase. State-of-the-art molecular dynamics (MD) simulations and post binding energetics analysis revealed that disrupting the kinase-intramolecular interactions of SH3 and SH2 domains with their cognate SBP ligands can result in totally different effects on the structural dynamics of c-Src kinase architecture; targeting the SH2 domain unlocks the autoinhibitory form of the kinase-this is very similar to the pTyr527 dephosphorylation that functionally activates the kinase, whereas targeting the SH3 domain can only release the domain from the tightly packed kinase but has a moderate effect on the kinase activity. Subsequently, based on the cognate SBP sequence we computationally designed a number of SH2-binding phosphopeptides using a motif grafting strategy. Fluorescence polarization (FP) assay observed that most of the designed phosphopeptides have higher binding affinity to SH2 domain as compared to the native SBP segment (K d = 53 nM). Kinase assay identified a typical dose-response relationship of phosphopeptides against kinase activation, substantiating that disruption of SH2-SBP interaction can mimic c-Src dephosphorylation and activate the kinase. Two rationally designed phosphopeptides, namely EPQpYEEIEN and EPQpYEELEN, were determined as strong binders of SH2 domain (K d = 8.3 and 15 nM, respectively) and potent activators of c-Src kinase (EC 50 = 3.2 and 41 μM, respectively).

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

PubMed Central

Arango, Daniel; Morohashi, Kengo; Yilmaz, Alper; Kuramochi, Kouji; Parihar, Arti; Brahimaj, Bledi; Grotewold, Erich; Doseff, Andrea I.

2013-01-01

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits. PMID:23697369

The transcription factor p53: Not a repressor, solely an activator

PubMed Central

Fischer, Martin; Steiner, Lydia; Engeland, Kurt

2014-01-01

The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway. PMID:25486564

Morphological and functional effects of graphene on the synthesis of uranium carbide for isotopes production targets.

PubMed

Biasetto, L; Corradetti, S; Carturan, S; Eloirdi, R; Amador-Celdran, P; Staicu, D; Blanco, O Dieste; Andrighetto, A

2018-05-29

The development of tailored targets for the production of radioactive isotopes represents an active field in nuclear research. Radioactive beams find applications in nuclear medicine, in astrophysics, matter physics and materials science. In this work, we study the use of graphene both as carbon source for UO 2 carbothermal reduction to produce UC x targets, and also as functional properties booster. At fixed composition, the UC x target grain size, porosity and thermal conductivity represent the three main points that affect the target production efficiency. UC x was synthesized using both graphite and graphene as the source of carbon and the target properties in terms of composition, grain size, porosity, thermal diffusivity and thermal conductivity were studied. The main output of this work is related to the remarkable enhancement achieved in thermal conductivity, which can profitably improve thermal dissipation during operational stages of UC x targets.

Ephrin-B3 coordinates timed axon targeting and amygdala spinogenesis for innate fear behaviour

PubMed Central

Zhu, Xiao-Na; Liu, Xian-Dong; Sun, Suya; Zhuang, Hanyi; Yang, Jing-Yu; Henkemeyer, Mark; Xu, Nan-Jie

2016-01-01

Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period. PMID:27008987

Ephrin-B3 coordinates timed axon targeting and amygdala spinogenesis for innate fear behaviour.

PubMed

Zhu, Xiao-Na; Liu, Xian-Dong; Sun, Suya; Zhuang, Hanyi; Yang, Jing-Yu; Henkemeyer, Mark; Xu, Nan-Jie

2016-03-24

Innate emotion response to environmental stimuli is a fundamental brain function that is controlled by specific neural circuits. Dysfunction of early emotional circuits may lead to neurodevelopmental disorders such as autism and schizophrenia. However, how the functional circuits are formed to prime initial emotional behaviours remain elusive. We reveal here using gene-targeted mutations an essential role for ephrin-B3 ligand-like activity in the development of innate fear in the neonatal brain. We further demonstrate that ephrin-B3 controls axon targeting and coordinates spinogenesis and neuronal activity within the amygdala. The morphological and behavioural abnormalities in ephrin-B3 mutant mice are rescued by conditional knock-in of wild-type ephrin-B3 during the critical period when axon targeting and fear responses are initiated. Our results thus define a key axonal molecule that participates in the wiring of amygdala circuits and helps bring about fear emotion during the important adolescence period.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew

The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence ofmore » replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.« less

Active colloids as mobile microelectrodes for unified label-free selective cargo transport.

PubMed

Boymelgreen, Alicia M; Balli, Tov; Miloh, Touvia; Yossifon, Gilad

2018-02-22

Utilization of active colloids to transport both biological and inorganic cargo has been widely examined in the context of applications ranging from targeted drug delivery to sample analysis. In general, carriers are customized to load one specific target via a mechanism distinct from that driving the transport. Here we unify these tasks and extend loading capabilities to include on-demand selection of multiple nano/micro-sized targets without the need for pre-labelling or surface functionalization. An externally applied electric field is singularly used to drive the active cargo carrier and transform it into a mobile floating electrode that can attract (trap) or repel specific targets from its surface by dielectrophoresis, enabling dynamic control of target selection, loading and rate of transport via the electric field parameters. In the future, dynamic selectivity could be combined with directed motion to develop building blocks for bottom-up fabrication in applications such as additive manufacturing and soft robotics.

Cytotoxic T cells use mechanical force to potentiate target cell killing

PubMed Central

Basu, Roshni; Whitlock, Benjamin M.; Husson, Julien; Le Floc’h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C.; Huse, Morgan

2016-01-01

SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. PMID:26924577

TALE-mediated modulation of transcriptional enhancers in vivo.

PubMed

Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.

PubMed

Rodríguez-Escudero, Isabel; Oliver, María D; Andrés-Pons, Amparo; Molina, María; Cid, Víctor J; Pulido, Rafael

2011-11-01

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

Reorganization of functional brain networks mediates the improvement of cognitive performance following real-time neurofeedback training of working memory.

PubMed

Zhang, Gaoyan; Yao, Li; Shen, Jiahui; Yang, Yihong; Zhao, Xiaojie

2015-05-01

Working memory (WM) is essential for individuals' cognitive functions. Neuroimaging studies indicated that WM fundamentally relied on a frontoparietal working memory network (WMN) and a cinguloparietal default mode network (DMN). Behavioral training studies demonstrated that the two networks can be modulated by WM training. Different from the behavioral training, our recent study used a real-time functional MRI (rtfMRI)-based neurofeedback method to conduct WM training, demonstrating that WM performance can be significantly improved after successfully upregulating the activity of the target region of interest (ROI) in the left dorsolateral prefrontal cortex (Zhang et al., [2013]: PloS One 8:e73735); however, the neural substrate of rtfMRI-based WM training remains unclear. In this work, we assessed the intranetwork and internetwork connectivity changes of WMN and DMN during the training, and their correlations with the change of brain activity in the target ROI as well as with the improvement of post-training behavior. Our analysis revealed an "ROI-network-behavior" correlation relationship underlying the rtfMRI training. Further mediation analysis indicated that the reorganization of functional brain networks mediated the effect of self-regulation of the target brain activity on the improvement of cognitive performance following the neurofeedback training. The results of this study enhance our understanding of the neural basis of real-time neurofeedback and suggest a new direction to improve WM performance by regulating the functional connectivity in the WM related networks. © 2014 Wiley Periodicals, Inc.

Visual target modulation of functional connectivity networks revealed by self-organizing group ICA.

PubMed

van de Ven, Vincent; Bledowski, Christoph; Prvulovic, David; Goebel, Rainer; Formisano, Elia; Di Salle, Francesco; Linden, David E J; Esposito, Fabrizio

2008-12-01

We applied a data-driven analysis based on self-organizing group independent component analysis (sogICA) to fMRI data from a three-stimulus visual oddball task. SogICA is particularly suited to the investigation of the underlying functional connectivity and does not rely on a predefined model of the experiment, which overcomes some of the limitations of hypothesis-driven analysis. Unlike most previous applications of ICA in functional imaging, our approach allows the analysis of the data at the group level, which is of particular interest in high order cognitive studies. SogICA is based on the hierarchical clustering of spatially similar independent components, derived from single subject decompositions. We identified four main clusters of components, centered on the posterior cingulate, bilateral insula, bilateral prefrontal cortex, and right posterior parietal and prefrontal cortex, consistently across all participants. Post hoc comparison of time courses revealed that insula, prefrontal cortex and right fronto-parietal components showed higher activity for targets than for distractors. Activation for distractors was higher in the posterior cingulate cortex, where deactivation was observed for targets. While our results conform to previous neuroimaging studies, they also complement conventional results by showing functional connectivity networks with unique contributions to the task that were consistent across subjects. SogICA can thus be used to probe functional networks of active cognitive tasks at the group-level and can provide additional insights to generate new hypotheses for further study. Copyright 2007 Wiley-Liss, Inc.

Molecular pathways: targeting p21-activated kinase 1 signaling in cancer--opportunities, challenges, and limitations.

PubMed

Eswaran, Jeyanthy; Li, Da-Qiang; Shah, Anil; Kumar, Rakesh

2012-07-15

The evolution of cancer cells involves deregulation of highly regulated fundamental pathways that are central to normal cellular architecture and functions. p21-activated kinase 1 (PAK1) was initially identified as a downstream effector of the GTPases Rac and Cdc42. Subsequent studies uncovered a variety of new functions for this kinase in growth factor and steroid receptor signaling, cytoskeleton remodeling, cell survival, oncogenic transformation, and gene transcription, largely through systematic discovery of its direct, physiologically relevant substrates. PAK1 is widely upregulated in several human cancers, such as hormone-dependent cancer, and is intimately linked to tumor progression and therapeutic resistance. These exciting developments combined with the kinase-independent role of PAK1-centered phenotypic signaling in cancer cells elevated PAK1 as an attractive drug target. Structural and biochemical studies revealed the precise mechanism of PAK1 activation, offering the possibility to develop PAK1-targeted cancer therapeutic approaches. In addition, emerging reports suggest the potential of PAK1 and its specific phosphorylated substrates as cancer prognostic markers. Here, we summarize recent findings about the PAK1 molecular pathways in human cancer and discuss the current status of PAK1-targeted anticancer therapies.

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

PubMed

Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

1997-09-15

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Bactobolin resistance is conferred by mutations in the L2 ribosomal protein.

PubMed

Chandler, Josephine R; Truong, Thao T; Silva, Patricia M; Seyedsayamdost, Mohammad R; Carr, Gavin; Radey, Matthew; Jacobs, Michael A; Sims, Elizabeth H; Clardy, Jon; Greenberg, E Peter

2012-12-18

Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins interact with a novel antibiotic target.

Relationships between structure, in vivo function and long-range axonal target of cortical pyramidal tract neurons.

PubMed

Rojas-Piloni, Gerardo; Guest, Jason M; Egger, Robert; Johnson, Andrew S; Sakmann, Bert; Oberlaender, Marcel

2017-10-11

Pyramidal tract neurons (PTs) represent the major output cell type of the neocortex. To investigate principles of how the results of cortical processing are broadcasted to different downstream targets thus requires experimental approaches, which provide access to the in vivo electrophysiology of PTs, whose subcortical target regions are identified. On the example of rat barrel cortex (vS1), we illustrate that retrograde tracer injections into multiple subcortical structures allow identifying the long-range axonal targets of individual in vivo recorded PTs. Here we report that soma depth and dendritic path lengths within each cortical layer of vS1, as well as spiking patterns during both periods of ongoing activity and during sensory stimulation, reflect the respective subcortical target regions of PTs. We show that these cellular properties result in a structure-function parameter space that allows predicting a PT's subcortical target region, without the need to inject multiple retrograde tracers.The major output cell type of the neocortex - pyramidal tract neurons (PTs) - send axonal projections to various subcortical areas. Here the authors combined in vivo recordings, retrograde tracings, and reconstructions of PTs in rat somatosensory cortex to show that PT structure and activity can predict specific subcortical targets.

Mechanism of Mediator recruitment by tandem Gcn4 activation domains and three Gal11 activator-binding domains.

PubMed

Herbig, Eric; Warfield, Linda; Fish, Lisa; Fishburn, James; Knutson, Bruce A; Moorefield, Beth; Pacheco, Derek; Hahn, Steven

2010-05-01

Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.

Ideal resuscitation pressure for uncontrolled hemorrhagic shock in different ages and sexes of rats

PubMed Central

2013-01-01

Introduction Our previous studies demonstrated that 50-60 mmHg mean arterial blood pressure was the ideal target hypotension for uncontrolled hemorrhagic shock during the active hemorrhage in sexually mature rats. The ideal target resuscitation pressure for immature and older rats has not been determined. Methods To elucidate this issue, using uncontrolled hemorrhagic-shock rats of different ages and sexes (6 weeks, 14 weeks and 1.5 years representing pre-adult, adult and older rats, respectively), the resuscitation effects of different target pressures (40, 50, 60, 70 and 80 mmHg) on uncontrolled hemorrhagic shock during active hemorrhage and the age and sex differences were observed. Results Different target resuscitation pressures had different resuscitation outcomes for the same age and sex of rats. The optimal target resuscitation pressures for 6-week-old, 14-week-old and 1.5-year-old rats were 40 to 50 mmHg, 50 to 60 mmHg and 70 mmHg respectively. Ideal target resuscitation pressures were significantly superior to other resuscitation pressures in improving the hemodynamics, blood perfusion, organ function and animal survival of uncontrolled hemorrhagic-shock rats (P < 0.01). For same target resuscitation pressures, the beneficial effect on hemorrhagic shock had a significant age difference (P < 0.01) but no sex difference (P > 0.05). Different resuscitation pressures had no effect on coagulation function. Conclusion Hemorrhagic-shock rats at different ages have different target resuscitation pressures during active hemorrhage. The ideal target resuscitation hypotension for 6-week-old, 14-week-old and 1.5-year-old rats was 40 to 50 mmHg, 50 to 60 mmHg and 70 mmHg, respectively. Their resuscitation effects have significant age difference but had no sex difference. PMID:24020401

The Pseudomonas aeruginosa AmrZ C-terminal domain mediates tetramerization and is required for its activator and repressor functions

PubMed Central

Xu, Binjie; Ju, Yue; Soukup, Randal J.; Ramsey, Deborah M.; Fishel, Richard; Wysocki, Vicki H.; Wozniak, Daniel J.

2015-01-01

Summary Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher-order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets. PMID:26549743

Bacterial virulence effectors and their activities.

PubMed

Hann, Dagmar R; Gimenez-Ibanez, Selena; Rathjen, John P

2010-08-01

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes. Copyright 2010 Elsevier Ltd. All rights reserved.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

PubMed Central

Ismail, Hanafy M.; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

2016-01-01

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

Detecting microRNAs of high influence on protein functional interaction networks: a prostate cancer case study

PubMed Central

2012-01-01

Background The use of biological molecular network information for diagnostic and prognostic purposes and elucidation of molecular disease mechanism is a key objective in systems biomedicine. The network of regulatory miRNA-target and functional protein interactions is a rich source of information to elucidate the function and the prognostic value of miRNAs in cancer. The objective of this study is to identify miRNAs that have high influence on target protein complexes in prostate cancer as a case study. This could provide biomarkers or therapeutic targets relevant for prostate cancer treatment. Results Our findings demonstrate that a miRNA’s functional role can be explained by its target protein connectivity within a physical and functional interaction network. To detect miRNAs with high influence on target protein modules, we integrated miRNA and mRNA expression profiles with a sequence based miRNA-target network and human functional and physical protein interactions (FPI). miRNAs with high influence on target protein complexes play a role in prostate cancer progression and are promising diagnostic or prognostic biomarkers. We uncovered several miRNA-regulated protein modules which were enriched in focal adhesion and prostate cancer genes. Several miRNAs such as miR-96, miR-182, and miR-143 demonstrated high influence on their target protein complexes and could explain most of the gene expression changes in our analyzed prostate cancer data set. Conclusions We describe a novel method to identify active miRNA-target modules relevant to prostate cancer progression and outcome. miRNAs with high influence on protein networks are valuable biomarkers that can be used in clinical investigations for prostate cancer treatment. PMID:22929553

Surfactant protein-A nanobody-conjugated liposomes loaded with methylprednisolone increase lung-targeting specificity and therapeutic effect for acute lung injury.

PubMed

Li, Nan; Weng, Dong; Wang, Shan-Mei; Zhang, Yuan; Chen, Shan-Shan; Yin, Zhao-Fang; Zhai, Jiali; Scoble, Judy; Williams, Charlotte C; Chen, Tao; Qiu, Hui; Wu, Qin; Zhao, Meng-Meng; Lu, Li-Qin; Mulet, Xavier; Li, Hui-Ping

2017-11-01

The advent of nanomedicine requires novel delivery vehicles to actively target their site of action. Here, we demonstrate the development of lung-targeting drug-loaded liposomes and their efficacy, specificity and safety. Our study focuses on glucocorticoids methylprednisolone (MPS), a commonly used drug to treat lung injuries. The steroidal molecule was loaded into functionalized nano-sterically stabilized unilamellar liposomes (NSSLs). Targeting functionality was performed through conjugation of surfactant protein A (SPANb) nanobodies to form MPS-NSSLs-SPANb. MPS-NSSLs-SPANb exhibited good size distribution, morphology, and encapsulation efficiency. Animal experiments demonstrated the high specificity of MPS-NSSLs-SPANb to the lung. Treatment with MPS-NSSLs-SPANb reduced the levels of TNF-α, IL-8, and TGF-β1 in rat bronchoalveolar lavage fluid and the expression of NK-κB in the lung tissues, thereby alleviating lung injuries and increasing rat survival. The nanobody functionalized nanoparticles demonstrate superior performance to treat lung injury when compared to that of antibody functionalized systems.

Bounded Empathy: Neural Responses to Outgroup Targets’ (Mis)fortunes

PubMed Central

Cikara, Mina; Fiske, Susan T.

2013-01-01

The current study investigates whether mere stereotypes are sufficient to modulate empathic responses to other people’s (mis)fortunes, how these modulations manifest in the brain, and whether affective and neural responses relate to endorsing harm against different outgroup targets. Participants feel least bad when misfortunes befall envied targets, and worst when misfortunes befall pitied targets, as compared to ingroup targets. Participants are also least willing to endorse harming pitied targets, despite pitied targets being outgroup members. However, those participants who exhibit increased activation in functionally-defined insula/MFG when viewing pity targets experience positive events not only report feeling worse about those events, but also more willing to harm pity targets in a tradeoff scenario. Similarly, increased activation in anatomically-defined bilateral anterior insula, in response to positive events, predicts increased willingness to harm envy targets, but decreased willingness to harm ingroup targets, above and beyond self-reported affect in response to the events. Stereotypes’ specific content, and not just outgroup membership, modulates empathic responses and related behavioral consequences including harm. PMID:21671744

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

PubMed

Green, Yangsook Song; Vetter, Monica L

2011-10-01

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

An integrated miRNA functional screening and target validation method for organ morphogenesis.

PubMed

Rebustini, Ivan T; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A; Maas, Richard L

2016-03-16

The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

E-box-independent regulation of transcription and differentiation by MYC.

PubMed

Uribesalgo, Iris; Buschbeck, Marcus; Gutiérrez, Arantxa; Teichmann, Sophia; Demajo, Santiago; Kuebler, Bernd; Nomdedéu, Josep F; Martín-Caballero, Juan; Roma, Guglielmo; Benitah, Salvador Aznar; Di Croce, Luciano

2011-10-23

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.

Garp as a therapeutic target for modulation of T regulatory cell function.

PubMed

Shevach, Ethan M

2017-02-01

Foxp3 + T regulatory cells (Tregs) play critical roles in immune homeostasis primarily by suppressing many aspects of the immune response. Tregs uniquely express GARP on their cell surface and GARP functions as a delivery system for latent TGF-β. As Treg-derived TGF-β may mediate the suppressive functions of Tregs, GARP may represent a target to inhibit Treg suppression in cancer or augment suppression in autoimmunity. Areas covered: This article will focus on 1) the role of Treg-derived TGF-β in the suppressive activity of Treg, 2) the cellular and molecular regulation of expression of GARP on mouse and human Tregs, 3) the role of integrins in the activation of latent-TGF-β/GARP complex, 4) an overview of our present understanding of the function of the latent-TGF-β/GARP complex. Expert opinion: Two approaches are outlined for targeting the L-TGF-β1/GARP complex for therapeutic purposes. Tregs play a major role in suppressive effector T cell responses to tumors and TGF-β1 may be a major contributor to this process. One approach is to specifically block the production of active TGF-β1 from Tregs as an adjunct to tumor immunotherapy. The second approach in autoimmunity is to selectively enhance the production of TGF-β by Tregs at sites of chronic inflammation.

Connectivity-based neurofeedback: Dynamic causal modeling for real-time fMRI☆

PubMed Central

Koush, Yury; Rosa, Maria Joao; Robineau, Fabien; Heinen, Klaartje; W. Rieger, Sebastian; Weiskopf, Nikolaus; Vuilleumier, Patrik; Van De Ville, Dimitri; Scharnowski, Frank

2013-01-01

Neurofeedback based on real-time fMRI is an emerging technique that can be used to train voluntary control of brain activity. Such brain training has been shown to lead to behavioral effects that are specific to the functional role of the targeted brain area. However, real-time fMRI-based neurofeedback so far was limited to mainly training localized brain activity within a region of interest. Here, we overcome this limitation by presenting near real-time dynamic causal modeling in order to provide feedback information based on connectivity between brain areas rather than activity within a single brain area. Using a visual–spatial attention paradigm, we show that participants can voluntarily control a feedback signal that is based on the Bayesian model comparison between two predefined model alternatives, i.e. the connectivity between left visual cortex and left parietal cortex vs. the connectivity between right visual cortex and right parietal cortex. Our new approach thus allows for training voluntary control over specific functional brain networks. Because most mental functions and most neurological disorders are associated with network activity rather than with activity in a single brain region, this novel approach is an important methodological innovation in order to more directly target functionally relevant brain networks. PMID:23668967

Flowcytometry – A rapid tool to correlate functional activities of human peripheral blood lymphocytes with their corresponding phenotypes after in vitro stimulation.

PubMed Central

Nirmala, R; Narayanan, PR

2002-01-01

Background While dealing with mixed in vitro lymphocyte cultures one is faced with the problem of relative contributions of different populations to the activity being studied. This is especially true in the controversy relating to the contributions of lymphocyte sub-populations to the Lymphokine Activated Killer (LAK) phenomenon. Flowcytometry can be used to highlight relative contributions of lymphocyte subpopulations towards LAK activity without resorting to difficult purification strategies. We set up long-term in vitro lymphocyte cultures, stimulated them with cytokines IL-2/IL-12, recorded their phenotypic changes and cytotoxic activity against U-937 tumor targets. Results The results indicated that natural killer cells (NK) constituted the predominant proliferating cell population in the cytokine stimulatedcultures. Flowcytometric evidence revealed that CD56+ T cells contributed little to LAK activity against U937 target cells as compared to cells with NK phenotype which were predominantly responsible for spontaneous killing of the tumor targets. The two cytokines, IL-2 and IL-12, had an additive effect on cell proliferation and spontaneous cytotoxicity. Conclusion Flowcytometry can be used to rapidly delineate phenotypic changes in immune cells after stimulation and simultaneously correlate them with corresponding functional activity. This approach may find application as a initial screening tool for studying different types of cells in mixed cultures and their respective activities under stimulatory / inhibitory conditions. PMID:12165101

Auditory Cortical Plasticity Drives Training-Induced Cognitive Changes in Schizophrenia

PubMed Central

Dale, Corby L.; Brown, Ethan G.; Fisher, Melissa; Herman, Alexander B.; Dowling, Anne F.; Hinkley, Leighton B.; Subramaniam, Karuna; Nagarajan, Srikantan S.; Vinogradov, Sophia

2016-01-01

Schizophrenia is characterized by dysfunction in basic auditory processing, as well as higher-order operations of verbal learning and executive functions. We investigated whether targeted cognitive training of auditory processing improves neural responses to speech stimuli, and how these changes relate to higher-order cognitive functions. Patients with schizophrenia performed an auditory syllable identification task during magnetoencephalography before and after 50 hours of either targeted cognitive training or a computer games control. Healthy comparison subjects were assessed at baseline and after a 10 week no-contact interval. Prior to training, patients (N = 34) showed reduced M100 response in primary auditory cortex relative to healthy participants (N = 13). At reassessment, only the targeted cognitive training patient group (N = 18) exhibited increased M100 responses. Additionally, this group showed increased induced high gamma band activity within left dorsolateral prefrontal cortex immediately after stimulus presentation, and later in bilateral temporal cortices. Training-related changes in neural activity correlated with changes in executive function scores but not verbal learning and memory. These data suggest that computerized cognitive training that targets auditory and verbal learning operations enhances both sensory responses in auditory cortex as well as engagement of prefrontal regions, as indexed during an auditory processing task with low demands on working memory. This neural circuit enhancement is in turn associated with better executive function but not verbal memory. PMID:26152668

Advances in the management of rheumatoid arthritis.

PubMed

Dale, James

2015-08-01

Modern early rheumatoid arthritis strategies are usually based upon a number of important overarching principles: 1. early diagnosis facilitates early commencement of disease modifying anti-rheumatic therapy; 2. early commencement of treatment reduces the long-term risk of erosive damage and functional decline; 3. composite disease activity measures should be used to quantify global rheumatoid arthritis disease activity; and 4. therapy should be intensified until a predefined disease activity target has been achieved. A substantial minority of rheumatoid arthritis patients (approximately 40%) will experience an adequate response to methotrexate monotherapy; however, the remainder may require disease modifying anti-rheumatic combination therapy, and/or biologic therapy, to achieve disease activity targets. Importantly, short term trials of methotrexate monotherapy do not appear to disadvantage outcomes provided treatment continues to be intensified if disease activity targets are not achieved. © The Author(s) 2015.

Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import

PubMed Central

Banci, Lucia; Bertini, Ivano; Cefaro, Chiara; Cenacchi, Lucia; Ciofi-Baffoni, Simone; Felli, Isabella Caterina; Gallo, Angelo; Gonnelli, Leonardo; Luchinat, Enrico; Sideris, Dionisia; Tokatlidis, Kostas

2010-01-01

Several proteins of the mitochondrial intermembrane space are targeted by internal targeting signals. A class of such proteins with α-helical hairpin structure bridged by two intramolecular disulfides is trapped by a Mia40-dependent oxidative process. Here, we describe the oxidative folding mechanism underpinning this process by an exhaustive structural characterization of the protein in all stages and as a complex with Mia40. Two consecutive induced folding steps are at the basis of the protein-trapping process. In the first one, Mia40 functions as a molecular chaperone assisting α-helical folding of the internal targeting signal of the substrate. Subsequently, in a Mia40-independent manner, folding of the second substrate helix is induced by the folded targeting signal functioning as a folding scaffold. The Mia40-induced folding pathway provides a proof of principle for the general concept that internal targeting signals may operate as a folding nucleus upon compartment-specific activation. PMID:21059946

Omega-3 Fatty Acids Modulate TRPV4 Function through Plasma Membrane Remodeling.

PubMed

Caires, Rebeca; Sierra-Valdez, Francisco J; Millet, Jonathan R M; Herwig, Joshua D; Roan, Esra; Vásquez, Valeria; Cordero-Morales, Julio F

2017-10-03

Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoid's epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Functional and genetic screening of acute myeloid leukemia associated with mediastinal germ cell tumor identifies MEK inhibitor as an active clinical agent.

PubMed

Leonard, Jessica T; Raess, Philipp W; Dunlap, Jennifer; Hayes-Lattin, Brandon; Tyner, Jeffrey W; Traer, Elie

2016-03-31

Hematologic malignancies arising in the setting of established germ cell tumors have been previously described and have a dismal prognosis. Identification of targetable mutations and pathway dysregulation through massively parallel sequencing and functional assays provides new approaches to disease management. Herein, we report the case of a 23-year-old male who was diagnosed with a mediastinal germ cell tumor and subsequent acute myeloid leukemia. A shared clonal origin was demonstrated through identification of identical NRAS and TP53 somatic mutations in both malignancies. The patient's leukemia was refractory to standard therapies with short interval relapse. Functional assays demonstrated the patient's blasts to be sensitive to the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, correlating with the activating NRAS mutation. The patient experienced a sustained partial remission while on trametinib therapy but ultimately suffered relapse of the germ cell tumor. The leukemic clone remained stable and sensitive to trametinib at that time. This case highlights the potential power of combining genetic sequencing and in vitro functional assays with targeted therapies in the treatment of rare diseases.

Targeting Alertness to Improve Cognition in Older Adults: A Preliminary Report of Benefits in Executive Function and Skill Acquisition

PubMed Central

Van Vleet, Thomas M.; DeGutis, Joseph M.; Merzenich, Michael M.; Simpson, Gregory V.; Zomet, Ativ; Dabit, Sawsan

2016-01-01

Efficient self-regulation of alertness declines with age exacerbating normal declines in performance across multiple cognitive domains, including learning and skill acquisition. Previous cognitive intervention studies have shown that it is possible to enhance alertness in patients with acquired brain injury and marked attention impairments, and that this benefit generalizes to improvements in more global cognitive functions. In the current preliminary studies, we sought to test whether this approach, that targets both tonic (over a period of minutes) and phasic (moment-to-moment) alertness, can improve key executive functioning declines in older adults, and enhance the rate of skill acquisition. The results of both experiments 1 and 2 demonstrate that, compared to active control training, alertness training significantly enhanced performance in several validated executive function measures. In experiment 2, alertness training significantly improved skill acquisition compared to active control training in a well-characterized speed of processing task, with the largest benefits shown in the most challenging speed of processing blocks. The results of the current study suggest that targeting intrinsic alertness in cognitive training provides a novel approach to improve executive functions in older adults and may be a useful adjunct treatment to enhance benefits gained in other clinically validated treatments. PMID:27372902

Metformin targets multiple signaling pathways in cancer.

PubMed

Lei, Yong; Yi, Yanhua; Liu, Yang; Liu, Xia; Keller, Evan T; Qian, Chao-Nan; Zhang, Jian; Lu, Yi

2017-01-26

Metformin, an inexpensive and well-tolerated oral agent commonly used in the first-line treatment of type 2 diabetes, has become the focus of intense research as a candidate anticancer agent. Here, we discuss the potential of metformin in cancer therapeutics, particularly its functions in multiple signaling pathways, including AMP-activated protein kinase, mammalian target of rapamycin, insulin-like growth factor, c-Jun N-terminal kinase/mitogen-activated protein kinase (p38 MAPK), human epidermal growth factor receptor-2, and nuclear factor kappaB pathways. In addition, cutting-edge targeting of cancer stem cells by metformin is summarized.

Directed intermittent search for a hidden target on a dendritic tree

NASA Astrophysics Data System (ADS)

Newby, Jay M.; Bressloff, Paul C.

2009-08-01

Motivated by experimental observations of active (motor-driven) intracellular transport in neuronal dendrites, we analyze a stochastic model of directed intermittent search on a tree network. A particle injected from the cell body or soma into the primary branch of the dendritic tree randomly switches between a stationary search phase and a mobile nonsearch phase that is biased in the forward direction. A (synaptic) target is presented somewhere within the tree, which the particle can locate if it is within a certain range and in the searching phase. We approximate the moment generating function using Green’s function methods. The moment generating function is then used to compute the hitting probability and conditional mean first passage time to the target. We show that in contrast to a previously explored finite interval case, there is a range of parameters for which a bidirectional search strategy is more efficient than a unidirectional one in finding the target.

The Integration of a Family Systems Approach for Understanding Youth Obesity, Physical Activity, and Dietary Programs

PubMed Central

Wilson, Dawn K.; St. George, Sara M.; Lawman, Hannah; Segal, Michelle; Fairchild, Amanda

2012-01-01

Rates of overweight in youth have reached epidemic proportions and are associated with adverse health outcomes. Family-based programs have been widely used to treat overweight in youth. However, few programs incorporate a theoretical framework for studying a family systems approach in relation to youth health behavior change. Therefore, this review provides a family systems theory framework for evaluating family-level variables in weight loss, physical activity, and dietary approaches in youth. Studies were reviewed and effect sizes were calculated for interventions that manipulated the family system, including components that targeted parenting styles, parenting skills, or family functioning, or which had novel approaches for including the family. Twenty-one weight loss interventions were identified, and 25 interventions related to physical activity and/or diet were identified. Overall, family-based treatment programs that incorporated training for authoritative parenting styles, parenting skills, or child management, and family functioning had positive effects on youth weight loss. Programs to improve physical activity and dietary behaviors that targeted the family system also demonstrated improvements in youth health behaviors; however, direct effects of parent-targeted programming is not clear. Both treatment and prevention programs would benefit from evaluating family functioning and parenting styles as possible mediators of intervention outcomes. Recommendations are provided to guide the development of future family-based obesity prevention and treatment programs for youth. PMID:20689989

Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide.

PubMed

Biscaglia, Francesca; Rajendran, Senthilkumar; Conflitti, Paolo; Benna, Clara; Sommaggio, Roberta; Litti, Lucio; Mocellin, Simone; Bocchinfuso, Gianfranco; Rosato, Antonio; Palleschi, Antonio; Nitti, Donato; Gobbo, Marina; Meneghetti, Moreno

2017-12-01

Plasmonic nanostructures show important properties for biotechnological applications, but they have to be guided on the target for exploiting their potentialities. Antibodies are the natural molecules for targeting. However, their possible adverse immunogenic activity and their cost have suggested finding other valid substitutes. Small molecules like peptides can be an alternative source of targeting agents, even if, as single molecules, their binding affinity is usually not very good. GE11 is a small dodecapeptide with specific binding to the epidermal growth factor receptor (EGFR) and low immunogenicity. The present work shows that thousands of polyethylene glycol (PEG) chains modified with lysines and functionalized with GE11 on clusters of naked gold nanoparticles, obtained by laser ablation in water, achieves a better targeting activity than that recorded with nanoparticles decorated with the specific anti-EGFR antibody Cetuximab (C225). The insertion of the cationic spacer between the polymeric part of the ligand and the targeting peptide allows for a proper presentation of GE11 on the surface of the nanosystems. Surface enhanced resonance Raman scattering signals of the plasmonic gold nanoparticles are used for quantifying the targeting activity. Molecular dynamic calculations suggest that subtle differences in the exposition of the peptide on the PEG sea are important for the targeting activity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Common and distinct neural targets of treatment: changing brain function in substance addiction.

PubMed

Konova, Anna B; Moeller, Scott J; Goldstein, Rita Z

2013-12-01

Neuroimaging offers an opportunity to examine the neurobiological effects of therapeutic interventions for human drug addiction. Using activation likelihood estimation, the aim of the current meta-analysis was to quantitatively summarize functional neuroimaging studies of pharmacological and cognitive-based interventions for drug addiction, with an emphasis on their common and distinct neural targets. More exploratory analyses also contrasted subgroups of studies based on specific study and sample characteristics. The ventral striatum, a region implicated in reward, motivation, and craving, and the inferior frontal gyrus and orbitofrontal cortex, regions involved in inhibitory control and goal-directed behavior, were identified as common targets of pharmacological and cognitive-based interventions; these regions were observed when the analysis was limited to only studies that used established or efficacious interventions, and across imaging paradigms and types of addictions. Consistent with theoretical models, cognitive-based interventions were additionally more likely to activate the anterior cingulate cortex, middle frontal gyrus, and precuneus, implicated in self-referential processing, cognitive control, and attention. These results suggest that therapeutic interventions for addiction may target the brain structures that are altered across addictions and identify potential neurobiological mechanisms by which the tandem use of pharmacological and cognitive-based interventions may yield synergistic or complementary effects. These findings could inform the selection of novel functional targets in future treatment development for this difficult-to-treat disorder. Copyright © 2013 Elsevier Ltd. All rights reserved.

Common and distinct neural targets of treatment: changing brain function in substance addiction

PubMed Central

Konova, Anna B.; Moeller, Scott J.; Goldstein, Rita Z.

2013-01-01

Neuroimaging offers an opportunity to examine the neurobiological effects of therapeutic interventions for human drug addiction. Using activation likelihood estimation, the aim of the current meta-analysis was to quantitatively summarize functional neuroimaging studies of pharmacological and cognitive-based interventions for drug addiction, with an emphasis on their common and distinct neural targets. More exploratory analyses also contrasted subgroups of studies based on specific study and sample characteristics. The ventral striatum, a region implicated in reward, motivation, and craving, and the inferior frontal gyrus and orbitofrontal cortex, regions involved in inhibitory control goal-directed behavior, were identified as common targets of pharmacological and cognitive-based interventions; these regions were observed when the analysis was limited to only studies that used established or efficacious interventions, and across imaging paradigms and types of addictions. Consistent with theoretical models, cognitive-based interventions were additionally more likely to activate the anterior cingulate cortex, middle frontal gyrus, and precuneus, implicated in self-referential processing, cognitive control, and attention. These results suggest that therapeutic interventions for addiction may target the brain structures that are altered across addictions and identify potential neurobiological mechanisms by which the tandem use of pharmacological and cognitive-based interventions may yield synergistic or complementary effects. These findings could inform the selection of novel functional targets in future treatment development for this difficult-to-treat disorder. PMID:24140399

Lmx1b-targeted cis-regulatory modules involved in limb dorsalization.

PubMed

Haro, Endika; Watson, Billy A; Feenstra, Jennifer M; Tegeler, Luke; Pira, Charmaine U; Mohan, Subburaman; Oberg, Kerby C

2017-06-01

Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% ( n =617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential cis -regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related Gdf5 that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with Lmx1b that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization. © 2017. Published by The Company of Biologists Ltd.

Simulation of cosmic irradiation conditions in thick target arrangements

NASA Technical Reports Server (NTRS)

Theis, S.; Englert, P.; Reedy, R. C.; Arnold, J. R.

1986-01-01

One approach to simulate 2-pi irradiation conditions of planetary surfaces which has been widely applied in the past are bombardments of so called thick targets. A very large thick target was exposed recently to 2.1 GeV protons at the Bevatron-Bevalac in Berkeley. In a 100x100x180 cm steel-surrounded granodiorite target radioactive medium and high energy spallation products of the incident primary and of secondary particles were analyzed along the beam axis down to depths of 140 g/cm(2) in targets such as Cu, Ni, Co, Fe, T, Si, SiO2 and Al. Activities of these nuclides were exclusively determined via instrumental gamma-ray spectroscopy. Relative yields of neutron capture and spallation products induced in Co and Cu targets during the thick target bombardment are shown as a function of depth. The majority of the medium energy products such as Co-58 from Co targets exhibit a maximum at shallow depths of 40-60 g/cm(2) and then decrease exponentially. In a comparable 600 MeV proton bombarded thick target such a slight maximum for medium energy products was not observed. Rather, Co-58 activities in Co decreased steadily with the highest activity at the surface. The activities of the n-capture product Co-60 increase steadily starting at the surface. This indicates the rapidly growing flux of low energy neutrons within the target.

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

PubMed

Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

2013-02-28

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

PubMed Central

Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.

2011-01-01

Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377

Heart failure—potential new targets for therapy

PubMed Central

Nabeebaccus, Adam; Zheng, Sean; Shah, Ajay M.

2016-01-01

Abstract Introduction/background Heart failure is a major cause of cardiovascular morbidity and mortality. This review covers current heart failure treatment guidelines, emerging therapies that are undergoing clinical trial, and potential new therapeutic targets arising from basic science advances. Sources of data A non-systematic search of MEDLINE was carried out. International guidelines and relevant reviews were searched for additional articles. Areas of agreement Angiotensin-converting enzyme inhibitors and beta-blockers are first line treatments for chronic heart failure with reduced left ventricular function. Areas of controversy Treatment strategies to improve mortality in heart failure with preserved left ventricular function are unclear. Growing points Many novel therapies are being tested for clinical efficacy in heart failure, including those that target natriuretic peptides and myosin activators. A large number of completely novel targets are also emerging from laboratory-based research. Better understanding of pathophysiological mechanisms driving heart failure in different settings (e.g. hypertension, post-myocardial infarction, metabolic dysfunction) may allow for targeted therapies. Areas timely for developing research Therapeutic targets directed towards modifying the extracellular environment, angiogenesis, cell viability, contractile function and microRNA-based therapies. PMID:27365454

Decoding Target Distance and Saccade Amplitude from Population Activity in the Macaque Lateral Intraparietal Area (LIP)

PubMed Central

Bremmer, Frank; Kaminiarz, Andre; Klingenhoefer, Steffen; Churan, Jan

2016-01-01

Primates perform saccadic eye movements in order to bring the image of an interesting target onto the fovea. Compared to stationary targets, saccades toward moving targets are computationally more demanding since the oculomotor system must use speed and direction information about the target as well as knowledge about its own processing latency to program an adequate, predictive saccade vector. In monkeys, different brain regions have been implicated in the control of voluntary saccades, among them the lateral intraparietal area (LIP). Here we asked, if activity in area LIP reflects the distance between fovea and saccade target, or the amplitude of an upcoming saccade, or both. We recorded single unit activity in area LIP of two macaque monkeys. First, we determined for each neuron its preferred saccade direction. Then, monkeys performed visually guided saccades along the preferred direction toward either stationary or moving targets in pseudo-randomized order. LIP population activity allowed to decode both, the distance between fovea and saccade target as well as the size of an upcoming saccade. Previous work has shown comparable results for saccade direction (Graf and Andersen, 2014a,b). Hence, LIP population activity allows to predict any two-dimensional saccade vector. Functional equivalents of macaque area LIP have been identified in humans. Accordingly, our results provide further support for the concept of activity from area LIP as neural basis for the control of an oculomotor brain-machine interface. PMID:27630547

In vivo targeting and imaging of tumor vasculature with radiolabeled, antibody-conjugated nanographene.

PubMed

Hong, Hao; Yang, Kai; Zhang, Yin; Engle, Jonathan W; Feng, Liangzhu; Yang, Yunan; Nayak, Tapas R; Goel, Shreya; Bean, Jero; Theuer, Charles P; Barnhart, Todd E; Liu, Zhuang; Cai, Weibo

2012-03-27

Herein we demonstrate that nanographene can be specifically directed to the tumor neovasculature in vivo through targeting of CD105 (i.e., endoglin), a vascular marker for tumor angiogenesis. The covalently functionalized nanographene oxide (GO) exhibited excellent stability and target specificity. Pharmacokinetics and tumor targeting efficacy of the GO conjugates were investigated with serial noninvasive positron emission tomography imaging and biodistribution studies, which were validated by in vitro, in vivo, and ex vivo experiments. The incorporation of an active targeting ligand (TRC105, a monoclonal antibody that binds to CD105) led to significantly improved tumor uptake of functionalized GO, which was specific for the neovasculature with little extravasation, warranting future investigation of these GO conjugates for cancer-targeted drug delivery and/or photothermal therapy to enhance therapeutic efficacy. Since poor extravasation is a major hurdle for nanomaterial-based tumor targeting in vivo, this study also establishes CD105 as a promising vascular target for future cancer nanomedicine. © 2012 American Chemical Society

Septins - active GTPases or just GTP-binding proteins?

PubMed

Abbey, Megha; Gaestel, Matthias; Menon, Manoj B

2018-05-10

Septins are conserved cytoskeletal proteins with unique filament forming capabilities and roles in cytokinesis and cell morphogenesis. Septins undergo hetero-oligomerization and assemble into higher order structures including filaments, rings and cages. Hetero- and homotypic interactions of septin isoforms involve alternating GTPase (G)-domain interfaces and those mediated by N- and C-terminal extensions. While most septins bind GTP, display weak GTP-hydrolysis activity and incorporate guanine nucleotides in their interaction interfaces, studies using GTPase-inactivating mutations have failed to conclusively establish a crucial role for GTPase activity in mediating septin functions. In this mini-review, we will critically assess the role of GTP-binding and -hydrolysis on septin assembly and function. The relevance of G-domain activity will also be discussed in the context of human septin mutations as well as the development of specific small-molecules targeting septin polymerization. As structural determinants of septin oligomer interfaces, G-domains are attractive targets for ligand-based inhibition of septin assembly. Whether such an intervention can predictably alter septin function is a major question for future research. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

The G Protein α Chaperone Ric-8 as a Potential Therapeutic Target

PubMed Central

Papasergi, Makaía M.; Patel, Bharti R.

2015-01-01

Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein–coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein–protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies. PMID:25319541

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

PubMed Central

Zhang, Jiping; Valianou, Matthildi; Simmons, Heidi; Robinson, Matthew K.; Lee, Hyung-Ok; Mullins, Stefanie R.; Marasco, Wayne A.; Adams, Gregory P.; Weiner, Louis M.; Cheng, Jonathan D.

2013-01-01

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.-O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. PMID:23104982

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

PubMed Central

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

2016-01-01

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores.

PubMed

Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S; Peters, Jan-Michael; Ellenberg, Jan

2016-03-14

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. © 2016 Isokane et al.

Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

PubMed

Ta, H T; Prabhu, S; Leitner, E; Jia, F; von Elverfeldt, D; Jackson, Katherine E; Heidt, T; Nair, A K N; Pearce, H; von Zur Muhlen, C; Wang, X; Peter, K; Hagemeyer, C E

2011-08-05

Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

Long-term outcomes of treat-to-target strategy in established rheumatoid arthritis: a daily practice prospective cohort study.

PubMed

de Andrade, Nicole Pamplona Bueno; da Silva Chakr, Rafael Mendonça; Xavier, Ricardo Machado; Viecceli, Daniela; Correa, Ricardo Henrique Bilycz; de Oliveira Filho, Cilomar Martins; Brenol, Claiton Viegas

2017-06-01

To examine disease activity and physical function after implementation of treat-to-target (T2T) strategy in patients with established rheumatoid arthritis (RA) over a long-term period. Patients with RA were started on a T2T strategy in 2005 and followed through 2014. Patients were seen every 3-4 months until remission/low disease activity was achieved and every 6 months thereafter. Disease activity was measured by the DAS28 and CDAI, and physical function by the HAQ-DI. Results were presented as all observed data, without imputation for missing values. Changes in disease activity and physical function were evaluated by generalized estimating equations (GEE). Two hundred and twenty-nine patients were included, with a mean (SD) disease duration of 10.6 (7.4) years. Significant improvements were seen in both composite scores during the follow-up period, as demonstrated by DAS28 (β coefficient = 0.19; 95% CI = 0.16-0.21; p < 0.01) and by CDAI (β coefficient = 1.59; 95% CI = 1.84-1.34; p < 0.01). Physical function also improved, as demonstrated by HAQ-DI (β coefficient = 0.03; 95% CI = 0.02-0.04; p < 0.01). Biological therapy was associated with improvement in disease activity and in physical function. Leflunomide was only associated with improvement in physical function. Clinically meaningful reductions of DAS28, CDAI and HAQ-DI were observed in patients with established rheumatoid arthritis from 2005 to 2014. Implementation of new therapeutic options, in the scenario of T2T strategy, was associated with improvement in disease activity and physical function.

Trajectory of change in pain, depression, and physical functioning after physical activity adoption in fibromyalgia.

PubMed

Steiner, Jennifer L; Bigatti, Silvia M; Ang, Dennis C

2015-07-01

Fibromyalgia is associated with widespread pain, depression, and declines in physical functioning. The purpose of this study was to examine the trajectory of these symptoms over time related to physical activity adoption and maintenance via motivational interviewing versus education, to increase physical activity. There were no treatment group differences; we divided the sample (n = 184) based on changes in physical activity. Repeated measures analyses demonstrated differential patterns in depression, pain, and physical functioning at 24 and 36 weeks. Findings suggest increased physical activity may serve as a multiple-target intervention that provides moderate to large, long-lasting benefits for individuals with fibromyalgia. © The Author(s) 2013.

The simulation study on optical target laser active detection performance

NASA Astrophysics Data System (ADS)

Li, Ying-chun; Hou, Zhao-fei; Fan, Youchen

2014-12-01

According to the working principle of laser active detection system, the paper establishes the optical target laser active detection simulation system, carry out the simulation study on the detection process and detection performance of the system. For instance, the performance model such as the laser emitting, the laser propagation in the atmosphere, the reflection of optical target, the receiver detection system, the signal processing and recognition. We focus on the analysis and modeling the relationship between the laser emitting angle and defocus amount and "cat eye" effect echo laser in the reflection of optical target. Further, in the paper some performance index such as operating range, SNR and the probability of the system have been simulated. The parameters including laser emitting parameters, the reflection of the optical target and the laser propagation in the atmosphere which make a great influence on the performance of the optical target laser active detection system. Finally, using the object-oriented software design methods, the laser active detection system with the opening type, complete function and operating platform, realizes the process simulation that the detection system detect and recognize the optical target, complete the performance simulation of each subsystem, and generate the data report and the graph. It can make the laser active detection system performance models more intuitive because of the visible simulation process. The simulation data obtained from the system provide a reference to adjust the structure of the system parameters. And it provides theoretical and technical support for the top level design of the optical target laser active detection system and performance index optimization.

Identification, visualization, and sorting of translationally active microbial consortia from deep-sea methane seeps

NASA Astrophysics Data System (ADS)

Hatzenpichler, R.; Connon, S. A.; Goudeau, D.; Malmstrom, R.; Woyke, T.; Orphan, V. J.

2015-12-01

Within the past few years, great progress has been made in tapping the genomes of individual cells separated from environmental samples. Unfortunately, however, most often these efforts have been target blind, as they did not pre-select for taxa of interest or focus on metabolically active cells that could be considered key species of the system at the time. This problem is particularly pronounced in low-turnover systems such as deep sea sediments. In an effort to tap the genetic potential hidden within functionally active cells, we have recently developed an approach for the in situ fluorescent tracking of protein synthesis in uncultured cells via bioorthogonal non-canonical amino acid-tagging (BONCAT). This technique depends on the incorporation of synthetic amino acids that carry chemically modifiable tags into newly made proteins, which later can be visualized via click chemistry-mediated fluorescence-labeling. BONCAT is thus able to specifically target proteins that have been expressed in reaction to an experimental condition. We are particularly interested in using BONCAT to understand the functional potential of slow-growing syntrophic consortia of anaerobic methanotrophic archaea and sulfate-reducing bacteria which together catalyze the anaerobic oxidation of methane (AOM) in marine methane seeps. In order to specifically target consortia that are active under varying environmental regimes, we are studying different subpopulations of these inter-domain consortia via a combination of BONCAT with rRNA-targeted FISH. We then couple the BONCAT-enabled staining of active consortia with their separation from inactive members of the community via fluorescence-activated cell-sorting (FACS) and metagenomic sequencing of individual consortia. Using this approach, we were able to identify previously unrecognized AOM-partnerships. By comparing the mini-metagenomes obtained from individual consortia with each other we are starting to gain a more hollistic understanding of the genetic similarities and niche-determining characteristics of a range of functional and taxonomic clades of AOM-consortia.

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.

PubMed

Morgens, David W; Wainberg, Michael; Boyle, Evan A; Ursu, Oana; Araya, Carlos L; Tsui, C Kimberly; Haney, Michael S; Hess, Gaelen T; Han, Kyuho; Jeng, Edwin E; Li, Amy; Snyder, Michael P; Greenleaf, William J; Kundaje, Anshul; Bassik, Michael C

2017-05-05

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

PubMed Central

Morgens, David W.; Wainberg, Michael; Boyle, Evan A.; Ursu, Oana; Araya, Carlos L.; Tsui, C. Kimberly; Haney, Michael S.; Hess, Gaelen T.; Han, Kyuho; Jeng, Edwin E.; Li, Amy; Snyder, Michael P.; Greenleaf, William J.; Kundaje, Anshul; Bassik, Michael C.

2017-01-01

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens. PMID:28474669

A COL11A1-correlated pan-cancer gene signature of activated fibroblasts for the prioritization of therapeutic targets

PubMed Central

Jia, Dongyu; Liu, Zhenqiu; Deng, Nan; Tan, Tuan Zea; Huang, Ruby Yun-Ju; Taylor-Harding, Barbie; Cheon, Dong-Joo; Lawrenson, Kate; Wiedemeyer, Wolf R.; Walts, Ann E.; Karlan, Beth Y.; Orsulic, Sandra

2016-01-01

Although cancer-associated fibroblasts (CAFs) are viewed as a promising therapeutic target, the design of rational therapy has been hampered by two key obstacles. First, attempts to ablate CAFs have resulted in significant toxicity because currently used biomarkers cannot effectively distinguish activated CAFs from non-cancer associated fibroblasts and mesenchymal progenitor cells. Second, it is unclear whether CAFs in different organs have different molecular and functional properties that necessitate organ-specific therapeutic designs. Our analyses uncovered COL11A1 as a highly specific biomarker of activated CAFs. Using COL11A1 as a ‘seed’, we identified co-expressed genes in 13 types of primary carcinoma in The Cancer Genome Atlas. We demonstrated that a molecular signature of activated CAFs is conserved in epithelial cancers regardless of organ site and transforming events within cancer cells, suggesting that targeting fibroblast activation should be effective in multiple cancers. We prioritized several potential pan-cancer therapeutic targets that are likely to have high specificity for activated CAFs and minimal toxicity in normal tissues. PMID:27609069

Pore-forming activity of pestivirus p7 in a minimal model system supports genus-specific viroporin function

USDA-ARS?s Scientific Manuscript database

Viroporins are small integral membrane proteins functional in viral assembly and egress by promoting permeabilization. Blocking of viroporin function therefore constitutes a target for antiviral development. Classical swine fever virus (CSFV) protein p7 has been recently regarded as a class II viro...

Consequences of inhibiting amyloid precursor protein processing enzymes on synaptic function and plasticity.

PubMed

Wang, Hui; Megill, Andrea; He, Kaiwen; Kirkwood, Alfredo; Lee, Hey-Kyoung

2012-01-01

Alzheimer's disease (AD) is a neurodegenerative disease, one of whose major pathological hallmarks is the accumulation of amyloid plaques comprised of aggregated β-amyloid (Aβ) peptides. It is now recognized that soluble Aβ oligomers may lead to synaptic dysfunctions early in AD pathology preceding plaque deposition. Aβ is produced by a sequential cleavage of amyloid precursor protein (APP) by the activity of β- and γ-secretases, which have been identified as major candidate therapeutic targets of AD. This paper focuses on how Aβ alters synaptic function and the functional consequences of inhibiting the activity of the two secretases responsible for Aβ generation. Abnormalities in synaptic function resulting from the absence or inhibition of the Aβ-producing enzymes suggest that Aβ itself may have normal physiological functions which are disrupted by abnormal accumulation of Aβ during AD pathology. This interpretation suggests that AD therapeutics targeting the β- and γ-secretases should be developed to restore normal levels of Aβ or combined with measures to circumvent the associated synaptic dysfunction(s) in order to have minimal impact on normal synaptic function.

TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy

PubMed Central

Grada, Zakaria; Hegde, Meenakshi; Byrd, Tiara; Shaffer, Donald R; Ghazi, Alexia; Brawley, Vita S; Corder, Amanda; Schönfeld, Kurt; Koch, Joachim; Dotti, Gianpietro; Heslop, Helen E; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Ahmed, Nabil

2013-01-01

Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor—the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment. PMID:23839099

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

PubMed

Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

2017-07-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Targeted, activity-dependent spinal stimulation produces long-lasting motor recovery in chronic cervical spinal cord injury

PubMed Central

McPherson, Jacob G.; Miller, Robert R.; Perlmutter, Steve I.

2015-01-01

Use-dependent movement therapies can lead to partial recovery of motor function after neurological injury. We attempted to improve recovery by developing a neuroprosthetic intervention that enhances movement therapy by directing spike timing-dependent plasticity in spared motor pathways. Using a recurrent neural–computer interface in rats with a cervical contusion of the spinal cord, we synchronized intraspinal microstimulation below the injury with the arrival of functionally related volitional motor commands signaled by muscle activity in the impaired forelimb. Stimulation was delivered during physical retraining of a forelimb behavior and throughout the day for 3 mo. Rats receiving this targeted, activity-dependent spinal stimulation (TADSS) exhibited markedly enhanced recovery compared with animals receiving targeted but open-loop spinal stimulation and rats receiving physical retraining alone. On a forelimb reach and grasp task, TADSS animals recovered 63% of their preinjury ability, more than two times the performance level achieved by the other therapy groups. Therapeutic gains were maintained for 3 additional wk without stimulation. The results suggest that activity-dependent spinal stimulation can induce neural plasticity that improves behavioral recovery after spinal cord injury. PMID:26371306

Targeting AMPK Signaling as a Neuroprotective Strategy in Parkinson's Disease.

PubMed

Curry, Daniel W; Stutz, Bernardo; Andrews, Zane B; Elsworth, John D

2018-03-26

Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is characterized by the accumulation of intracellular α-synuclein aggregates and the degeneration of nigrostriatal dopaminergic neurons. While no treatment strategy has been proven to slow or halt the progression of the disease, there is mounting evidence from preclinical PD models that activation of 5'-AMP-activated protein kinase (AMPK) may have broad neuroprotective effects. Numerous dietary supplements and pharmaceuticals (e.g., metformin) that increase AMPK activity are available for use in humans, but clinical studies of their effects in PD patients are limited. AMPK is an evolutionarily conserved serine/threonine kinase that is activated by falling energy levels and functions to restore cellular energy balance. However, in response to certain cellular stressors, AMPK activation may exacerbate neuronal atrophy and cell death. This review describes the regulation and functions of AMPK, evaluates the controversies in the field, and assesses the potential of targeting AMPK signaling as a neuroprotective treatment for PD.

Systematic Review of the Effectiveness of Occupational Therapy–Related Interventions for People With Parkinson’s Disease

PubMed Central

Foster, Erin R.; Bedekar, Mayuri

2014-01-01

We describe the results of a systematic review of the literature on occupational therapy–related interventions for people with Parkinson’s disease (PD). Three broad categories of intervention emerged: (1) exercise or physical activity; (2) environmental cues, stimuli, and objects; and (3) self-management and cognitive–behavioral strategies. Moderate to strong evidence exists for task-specific benefits of targeted physical activity training on motor performance, postural stability, and balance. Low to moderate evidence indicates that more complex, multimodal activity training supports improvement in functional movement activities. The evidence is moderate that the use of external supports during functional mobility or other movement activities has positive effects on motor control. In addition, moderate evidence is available that individualized interventions focused on promoting participant wellness initiatives and personal control by means of cognitive–behavioral strategies can improve targeted areas of quality of life. The implications for practice, education, and research are discussed. PMID:24367954

Transcription factor activity of estrogen receptor α activation upon nonylphenol or bisphenol A treatment enhances the in vitro proliferation, invasion, and migration of neuroblastoma cells

PubMed Central

Ma, Hongda; Yao, Yao; Wang, Changli; Zhang, Liyu; Cheng, Long; Wang, Yiren; Wang, Tao; Liang, Erguang; Jia, Hui; Ye, Qinong; Hou, Mingxiao; Feng, Fan

2016-01-01

Many kinds of endocrine-disrupting chemicals (EDCs), for example, the environmental estrogens bisphenol A and nonylphenol, may regulate the activity of estrogen receptor α (ERα) and therefore induce potential disruption of normal endocrine function. However, the involvement of EDCs in human cancers, especially in endocrine-related cancer neuroblastoma regulation, is not very clear. In this work, results showed that upon bisphenol A or nonylphenol treatment, the transcription factor activity of ERα was significantly increased in neuroblastoma cell line SH-SY5Y. Bisphenol A and nonylphenol could enhance ERα activity via recruiting it to the target gene promoter. Furthermore, treatment of bisphenol A and nonylphenol enhanced the in vitro proliferation, invasion, and migration ability of neuroblastoma cells. By investigating the role of EDC-induced ERα upregulation, our data extend the understanding of the function of EDCs and further suggest that ERα might be a potential therapeutic target in human neuroblastoma treatment. PMID:27366082

Quantitative regulation of histone variant H2A.Z during cell cycle by ubiquitin proteasome system and SUMO-targeted ubiquitin ligases.

PubMed

Takahashi, Daisuke; Orihara, Yuki; Kitagawa, Saho; Kusakabe, Masayuki; Shintani, Takahiro; Oma, Yukako; Harata, Masahiko

2017-08-01

Quantitative control of histones and histone variants during cell cycle is relevant to their epigenetic functions. We found that the level of yeast histone variant H2A.Z in the G2/M-phase is actively kept low by the ubiquitin proteasome system and SUMO-targeted ubiquitin ligases. Overexpression of H2A.Z induced defects in mitotic progression, suggesting functional importance of this quantitative control.

[Supercomputer investigation of the protein-ligand system low-energy minima].

PubMed

Oferkin, I V; Sulimov, A V; Katkova, E V; Kutov, D K; Grigoriev, F V; Kondakova, O A; Sulimov, V B

2015-01-01

The accuracy of the protein-ligand binding energy calculations and ligand positioning is strongly influenced by the choice of the docking target function. This work demonstrates the evaluation of the five different target functions used in docking: functions based on MMFF94 force field and functions based on PM7 quantum-chemical method accounting or without accounting the implicit solvent model (PCM, COSMO or SGB). For these purposes the ligand positions corresponding to the minima of the target function and the experimentally known ligand positions in the protein active site (crystal ligand positions) were compared. Each function was examined on the same test-set of 16 protein-ligand complexes. The new parallelized docking program FLM based on Monte Carlo search algorithm was developed to perform the comprehensive low-energy minima search and to calculate the protein-ligand binding energy. This study demonstrates that the docking target function based on the MMFF94 force field can be used to detect the crystal or near crystal positions of the ligand by the finding the low-energy local minima spectrum of the target function. The importance of solvent accounting in the docking process for the accurate ligand positioning is also shown. The accuracy of the ligand positioning as well as the correlation between the calculated and experimentally determined protein-ligand binding energies are improved when the MMFF94 force field is substituted by the new PM7 method with implicit solvent accounting.

The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids.

PubMed Central

El-Robh, Mohamed Samir; Busby, Stephen J W

2002-01-01

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters. PMID:12350222

Social hierarchy modulates neural responses of empathy for pain.

PubMed

Feng, Chunliang; Li, Zhihao; Feng, Xue; Wang, Lili; Tian, Tengxiang; Luo, Yue-Jia

2016-03-01

Recent evidence indicates that empathic responses to others' pain are modulated by various situational and individual factors. However, few studies have examined how empathy and underlying brain functions are modulated by social hierarchies, which permeate human society with an enormous impact on social behavior and cognition. In this study, social hierarchies were established based on incidental skill in a perceptual task in which all participants were mediumly ranked. Afterwards, participants were scanned with functional magnetic resonance imaging while watching inferior-status or superior-status targets receiving painful or non-painful stimulation. The results revealed that painful stimulation applied to inferior-status targets induced higher activations in the anterior insula (AI) and anterior medial cingulate cortex (aMCC), whereas these empathic brain activations were significantly attenuated in response to superior-status targets' pain. Further, this neural empathic bias to inferior-status targets was accompanied by stronger functional couplings of AI with brain regions important in emotional processing (i.e. thalamus) and cognitive control (i.e. middle frontal gyrus). Our findings indicate that emotional sharing with others' pain is shaped by relative positions in a social hierarchy such that underlying empathic neural responses are biased toward inferior-status compared with superior-status individuals. © The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Insights into the structural/conformational requirements of cytotoxic oxadiazoles as potential chemotherapeutic target binding agents

NASA Astrophysics Data System (ADS)

Alikhani, Radin; Razzaghi-Asl, Nima; Ramazani, Ali; Hosseinzadeh, Zahra

2018-07-01

A few novel previously synthesized 2,5-disubstituted 1,3,4-oxadiazoles with cytotoxic activity (1-17) were subjected to combined docking/quantum mechanical studies against chemotherapeutic targets. Selected macromolecular targets were those that were previously known to be inhibited by 1,3,4-oxadiazoles. Within this work, favorable binding modes/affinities of the oxadiazoles toward validated cancer targets were elucidated. Some oxadiazole structures exhibited ΔGbs comparable to or stronger than crystallographic ligands that were previously demonstrated to inhibit such targets. On the basis of obtained results, a general structure activity/binding relationship (SAR/SBR) was developed and a few 2,5-disubstituted 1,3,4-oxadiazole structures were proposed and virtually validated as potential cytotoxic candidates. To get more insight into structure binding relationship of candidate molecules within best correlated targets, docked conformation of the best in silico in vitro correlated oxadiazole structure was analyzed in terms of intermolecular binding energy components by functional B3LYP in association with split valence basis set using polarization functions (Def2-SVP). We believe that such modeling studies may be complementary to our previous results on the synthesis and cytotoxicity assessment of novel 1,3,4-oxadiazole derivatives through extending the scope of privileged structures toward designing new potential anti-tumor compounds.

Extracts Obtained from Pterocarpus angolensis DC and Ziziphus mucronata Exhibit Antiplasmodial Activity and Inhibit Heat Shock Protein 70 (Hsp70) Function.

PubMed

Zininga, Tawanda; Anokwuru, Chinedu P; Sigidi, Muendi T; Tshisikhawe, Milingoni P; Ramaite, Isaiah I D; Traoré, Afsatou N; Hoppe, Heinrich; Shonhai, Addmore; Potgieter, Natasha

2017-07-28

Malaria parasites are increasingly becoming resistant to currently used antimalarial therapies, therefore there is an urgent need to expand the arsenal of alternative antimalarial drugs. In addition, it is also important to identify novel antimalarial drug targets. In the current study, extracts of two plants, Pterocarpus angolensis and Ziziphus mucronata were obtained and their antimalarial functions were investigated. Furthermore, we explored the capability of the extracts to inhibit Plasmodium falciparum heat shock protein 70 (Hsp70) function. Heat shock protein 70 (Hsp70) are molecular chaperones whose function is to facilitate protein folding. Plasmodium falciparum the main agent of malaria, expresses two cytosol-localized Hsp70s: PfHsp70-1 and PfHsp70-z. The PfHsp70-z has been reported to be essential for parasite survival, while inhibition of PfHsp70-1 function leads to parasite death. Hence both PfHsp70-1 and PfHsp70-z are potential antimalarial drug targets. Extracts of P. angolensis and Z. mucronata inhibited the basal ATPase and chaperone functions of the two parasite Hsp70s. Furthermore, fractions of P. angolensis and Z. mucronata inhibited P. falciparum 3D7 parasite growth in vitro. The extracts obtained in the current study exhibited antiplasmodial activity as they killed P. falciparum parasites maintained in vitro. In addition, the findings further suggest that some of the compounds in P. angolensis and Z. mucronata may target parasite Hsp70 function.

Multi-functional liposomes showing radiofrequency-triggered release and magnetic resonance imaging for tumor multi-mechanism therapy.

PubMed

Du, Bin; Han, Shuping; Li, Hongyan; Zhao, Feifei; Su, Xiangjie; Cao, Xiaohui; Zhang, Zhenzhong

2015-03-12

Recently, nanoplatforms with multiple functions, such as tumor-targeting drug carriers, MRI, optical imaging, thermal therapy etc., have become popular in the field of cancer research. The present study reports a novel multi-functional liposome for cancer theranostics. A dual targeted drug delivery with radiofrequency-triggered drug release and imaging based on the magnetic field influence was used advantageously for tumor multi-mechanism therapy. In this system, the surface of fullerene (C60) was decorated with iron oxide nanoparticles, and PEGylation formed a hybrid nanosystem (C60-Fe3O4-PEG2000). Thermosensitive liposomes (dipalmitoylphosphatidylcholine, DPPC) with DSPE-PEG2000-folate wrapped up the hybrid nanosystem and docetaxel (DTX), which were designed to combine features of biological and physical (magnetic) drug targeting for fullerene radiofrequency-triggered drug release. The magnetic liposomes not only served as powerful tumor diagnostic magnetic resonance imaging (MRI) contrast agents, but also as powerful agents for photothermal ablation of tumors. Furthermore, a remarkable thermal therapy combined chemotherapy multi-functional liposome nanoplatform converted radiofrequency energy into thermal energy to release drugs from thermosensitive liposomes, which was also observed during both in vitro and in vivo treatment. The multi-functional liposomes also could selectively kill cancer cells in highly localized regions via their excellent active tumor targeting and magnetic targeted abilities.

Targeting TGFβ Signaling in Subchondral Bone and Articular Cartilage Homeostasis

PubMed Central

Zhen, Gehau; Cao, Xu

2014-01-01

Osteoarthritis (OA) is the most common degenerative joint disease, and there is no disease-modifying therapy for OA currently available. Targeting of articular cartilage alone may not be sufficient to halt this disease progression. Articular cartilage and subchondral bone act as a functional unit. Increasing evidence indicates that transforming growth factor β (TGFβ) plays a crucial role in maintaining homeostasis of both articular cartilage and subchondral bone. Activation of extracellular matrix latent TGFβ at the appropriate time and location is the prerequisite for its function. Aberrant activation of TGFβ in the subchondral bone in response to abnormal mechanical loading environment induces formation of osteroid islets at onset of osteoarthritis. As a result, alteration of subchondral bone structure changes the stress distribution on the articular cartilage and leads to its degeneration. Thus, inhibition of TGFβ activity in the subchondral bone may provide a new avenue of treatment for OA. In this review, we will respectively discuss the role of TGFβ in homeostasis of articular cartilage and subchondral bone as a novel target for OA therapy. PMID:24745631

A Novel Function of the Fe65 Neuronal Adaptor in Estrogen Receptor Action in Breast Cancer Cells*

PubMed Central

Sun, Yuefeng; Kasiappan, Ravi; Tang, Jinfu; Webb, Panida L.; Quarni, Waise; Zhang, Xiaohong; Bai, Wenlong

2014-01-01

Fe65 is a multidomain adaptor with established functions in neuronal cells and neurodegeneration diseases. It binds to the C terminus of the Aβ amyloid precursor protein and is involved in regulating gene transcription. The present studies show that Fe65 is expressed in breast cancer (BCa) cells and acts as an ERα transcriptional coregulator that is recruited by 17β-estradiol to the promoters of estrogen target genes. Deletion analyses mapped the ERα binding domain to the phosphotyrosine binding domain 2 (PTB2). Ectopic Fe65 increased the transcriptional activity of the ERα in a PTB2-dependent manner in reporter assays. Fe65 knockdown decreased, whereas its stable expression increased the transcriptional activity of endogenous ERα in BCa cells and the ability of estrogens to stimulate target gene expression, ERα, and coactivator recruitment to target gene promoters and cell growth. Furthermore, Fe65 expression decreased the antagonistic activity of tamoxifen (TAM), suggesting a role for Fe65 in TAM resistance. Overall, the studies define a novel role for the neuronal adaptor in estrogen actions in BCa cells. PMID:24619425

Peroxisome proliferator-activated receptors for hypertension

PubMed Central

Usuda, Daisuke; Kanda, Tsugiyasu

2014-01-01

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes (α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin II receptor blockers, should be studied. This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases. PMID:25228953

Modulation of empathy in the left ventrolateral prefrontal cortex facilitates altruistic behavior: An fNIRS study.

PubMed

Himichi, Toshiyuki; Nomura, Michio

2015-06-01

Previous studies suggest that the ventrolateral prefrontal cortex (VLPFC) is involved in modulating empathy. However, it is unclear whether VLPFC activation while an individual empathizes with others is related to subsequent altruistic behavior. In the present study, participants observed two people playing a card game while empathizing with one of them (target person); PFC activation during the task was measured using functional near-infrared spectroscopy (fNIRS). After this task, participants distributed money to the target person. Results showed that activation in the left VLPFC during empathizing with the target person, who lost a small amount of money, was positively correlated with the amount of money distributed; this activation mediated a relationship between norm of restitution toward helping and the amount of distributed money. These results suggest that activation in the left VLPFC during empathizing with others experiencing negative circumstances is associated with altruistic behavior.

Dynamic visual acuity using "far" and "near" targets

NASA Technical Reports Server (NTRS)

Peters, Brian T.; Bloomberg, Jacob J.

2005-01-01

CONCLUSIONS: DVA may be useful for assessing the functional consequences of an impaired gaze stabilization mechanism or for testing the effectiveness of a rehabilitation paradigm. Because target distance influences the relative contributions of canal and otolith inputs, the ability to measure DVA at near and far viewing distances may also lead to tests that will independently assess canal and otolith function. OBJECTIVE: To present and test a methodology that uses dynamic visual acuity (DVA) to assess the efficacy of compensatory gaze mechanisms during a functionally relevant activity that differentially measures canal and otolith function. MATERIAL AND METHODS: The effect of treadmill walking at a velocity of 1.79 m/s on subjects' visual acuity was assessed at each of two viewing distances. A custom-written threshold determination program was used to display Landolt C optotypes on a laptop computer screen during a "far" (4 m) target condition and on a micro-display for a "near" (50 cm) target condition. The walking acuity scores for each target distance were normalized by subtracting a corresponding acuity measure obtained while standing still on the treadmill belt. RESULTS: As predicted by subjective reports of relative target motion, the decrease in visual acuity was significantly greater (p < 0.00001) for the near compared to the far condition.

Rubisco Activase Activity Assays

USDA-ARS?s Scientific Manuscript database

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase functions as a mechano-chemical motor protein using the energy from ATP hydrolysis to contort the structure of its target protein, Rubisco. This action modulates the activation state of Rubisco by removing tightly-bound inhibitory s...

Gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice

PubMed Central

Fang, Yu; Chen, Hao; Hu, Yuhui; Djukic, Zorka; Tevebaugh, Whitney; Shaheen, Nicholas J.; Orlando, Roy C.; Hu, Jianguo

2013-01-01

The barrier function of the esophageal epithelium is a major defense against gastroesophageal reflux disease. Previous studies have shown that reflux damage is reflected in a decrease in transepithelial electrical resistance associated with tight junction alterations in the esophageal epithelium. To develop novel therapies, it is critical to understand the molecular mechanisms whereby contact with a refluxate impairs esophageal barrier function. In this study, surgical models of duodenal and mixed reflux were developed in mice. Mouse esophageal epithelium was analyzed by gene microarray. Gene set enrichment analysis showed upregulation of inflammation-related gene sets and the NF-κB pathway due to reflux. Significance analysis of microarrays revealed upregulation of NF-κB target genes. Overexpression of NF-κB subunits (p50 and p65) and NF-κB target genes (matrix metalloproteinases-3 and -9, IL-1β, IL-6, and IL-8) confirmed activation of the NF-κB pathway in the esophageal epithelium. In addition, real-time PCR, Western blotting, and immunohistochemical staining also showed downregulation and mislocalization of claudins-1 and -4. In a second animal experiment, treatment with an NF-κB inhibitor, BAY 11-7085 (20 mg·kg−1·day−1 ip for 10 days), counteracted the effects of duodenal and mixed reflux on epithelial resistance and NF-κB-regulated cytokines. We conclude that gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice and that targeting the NF-κB pathway may strengthen esophageal barrier function against reflux. PMID:23639809

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

PubMed

Basu, Roshni; Whitlock, Benjamin M; Husson, Julien; Le Floc'h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C; Huse, Morgan

2016-03-24

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of novel targets for PGC-1{alpha} and histone deacetylase inhibitors in neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cowell, Rita M.; Department of Neurology, University of Michigan, Ann Arbor, MI 48109; Talati, Pratik

2009-02-06

Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1{alpha} express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1{alpha} in neuronal cells and whether there are ways to pharmacologically target PGC-1{alpha} in neurons. Here, PGC-1{alpha} overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3more » activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1{alpha} and glucose transporter 4 (GLUT4). These results suggest that PGC-1{alpha} regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1{alpha} and GLUT4 in HD and other neurological disorders.« less

NF-κB Signaling Pathway and its Potential as a Target for Therapy in Lymphoid Neoplasms

PubMed Central

Yu, Li; Li, Ling; Medeiros, L. Jeffrey; Young, Ken H.

2016-01-01

The NF-κB pathway, a critical regulator of apoptosis, plays a key role in many normal cellular functions. Genetic alterations and other mechanisms leading to constitutive activation of the NF-κB pathway contribute to cancer development, progression and therapy resistance by activation of downstream anti-apoptotic pathways, unfavorable microenvironment interactions, and gene dysregulation. Not surprisingly, given its importance to normal and cancer cell function, the NF-κB pathway has emerged as a target for therapy. In the review, we present the physiologic role of the NF-κB pathway and recent advances in better understanding of the pathologic roles of the NF-κB pathway in major types of lymphoid neoplasms. We also provide an update of clinical trials that use NF-κB pathway inhibitors. These trials are exploring the clinical efficiency of combining NF-κB pathway inhibitors with various agents that target diverse mechanisms of action with the goal being to optimize novel therapeutic opportunities for targeting oncogenic pathways to eradicate cancer cells. PMID:27773462

[Anti-epidermal growth factor receptor treatment: a new paradigm for conducting therapeutic trials].

PubMed

Marty, Michel; Bedairia, Naima; Armand, Jean-Pierre

2003-11-01

Agents which modify biological properties of tumour tissue can target many tenths of functions over- or underexpressed in human tumours. In general these agents are cytostatic rather than cytotoxic and will affect only that fraction of human tumours where the target plays and important and unique role for the viability of the tumour tissue. Alternatively it is expected that acute toxicity will not be observed at active dose-time exposure; rather subacute or chronic toxicity can be observed with these agents. Clinical studies will have to follow the following rules: characterisation of the pharmacological target and of its functional role on tumour tissue; definition of an optimal biological dose rather than a maximum tolerated dose; importance of validated pharmacodynamic endpoints; importance and thus need for early studies of combination regimens. It is still too early to define general guidelines for the study of these different therapeutic families. Nevertheless, studies already conducted with agents interfering with EGF mediated signalization have already permitted preliminary indications on pharmacodynamics, target assessment, level of activity and conduct of clinical trials with combination regimens.

Immobilized magnetic beads-based multi-target affinity selection coupled with HPLC-MS for screening active compounds from traditional Chinese medicine and natural products.

PubMed

Chen, Yaqi; Chen, Zhui; Wang, Yi

2015-01-01

Screening and identifying active compounds from traditional Chinese medicine (TCM) and other natural products plays an important role in drug discovery. Here, we describe a magnetic beads-based multi-target affinity selection-mass spectrometry approach for screening bioactive compounds from natural products. Key steps and parameters including activation of magnetic beads, enzyme/protein immobilization, characterization of functional magnetic beads, screening and identifying active compounds from a complex mixture by LC/MS, are illustrated. The proposed approach is rapid and efficient in screening and identification of bioactive compounds from complex natural products.

Matched and Mismatched Metabolic Fuels in Lymphocyte Function

PubMed Central

Caro-Maldonado, Alfredo; Gerriets, Valerie A.; Rathmell, Jeffrey C.

2012-01-01

Immunological function requires metabolic support to suit the needs of lymphocytes at a variety of distinct differentiation and activation states. It is now evident that the signaling pathways that drive lymphocyte survival and activity can directly control cellular metabolism. This linkage provides a mechanism by which activation and specific signaling pathways provide a supply of appropriate and required nutrients to support cell functions in a pro-active supply rather than consumption-based metabolic model. In this way, the metabolism and fuel choices of lymphocytes are guided to specifically match the anticipated needs. If the fuel choice or metabolic pathways of lymphocytes are dysregulated, however, metabolic checkpoints can become activated to disrupt immunological function. These changes are now shown in several immunological diseases and may open new opportunities to selectively enhance or suppress specific immune functions through targeting of glucose, lipid, or amino acid metabolism. PMID:23290889

Identifying transcription factor functions and targets by phenotypic activation

PubMed Central

Chua, Gordon; Morris, Quaid D.; Sopko, Richelle; Robinson, Mark D.; Ryan, Owen; Chan, Esther T.; Frey, Brendan J.; Andrews, Brenda J.; Boone, Charles; Hughes, Timothy R.

2006-01-01

Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology. PMID:16880382

CRTC2 is required for β-cell function and proliferation.

PubMed

Eberhard, Chandra E; Fu, Accalia; Reeks, Courtney; Screaton, Robert A

2013-07-01

Previous work in insulinoma cell lines has established that calcineurin plays a critical role in the activation of cAMP-responsive element binding protein (Creb), a key transcription factor required for β-cell function and survival, by dephosphorylating the Creb coactivator Creb-regulated transcription coactivator (Crtc)2 at 2 regulatory sites, Ser171 and Ser275. Here, we report that Crtc2 is essential both for glucose-stimulated insulin secretion and cell survival in the β-cell. Endogenous Crtc2 activation is achieved via increasing glucose levels to the physiological feeding range, indicating that Crtc2 is a sensor that couples ambient glucose concentrations to Creb activity in the β-cell. Immunosuppressant drugs such as cyclosporin A and tacrolimus that target the protein phosphatase calcineurin are commonly administered after organ transplantation. Chronic use is associated with reduced insulin secretion and new onset diabetes, suggestive of pancreatic β-cell dysfunction. Importantly, we show that overexpression of a Crtc2 mutant rendered constitutively active by introduction of nonphosphorylatable alanine residues at Ser171 and Ser275 permits Creb target gene activation under conditions when calcineurin is inhibited. Taken together, these data suggest that promoting Crtc2-Creb activity is required for β-cell function and proliferation and promoting this pathway could ameliorate symptoms of new onset diabetes after transplantation.

4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

PubMed

Winter, Matthew J; Windell, Dylan; Metz, Jeremy; Matthews, Peter; Pinion, Joe; Brown, Jonathan T; Hetheridge, Malcolm J; Ball, Jonathan S; Owen, Stewart F; Redfern, Will S; Moger, Julian; Randall, Andrew D; Tyler, Charles R

2017-07-26

Functional neuroimaging, using genetically-encoded Ca 2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies

PubMed Central

Colombo, Miriam; Fiandra, Luisa; Alessio, Giulia; Mazzucchelli, Serena; Nebuloni, Manuela; De Palma, Clara; Kantner, Karsten; Pelaz, Beatriz; Rotem, Rany; Corsi, Fabio; Parak, Wolfgang J.; Prosperi, Davide

2016-01-01

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect. PMID:27991503

Anticancer molecules targeting fibroblast growth factor receptors.

PubMed

Liang, Guang; Liu, Zhiguo; Wu, Jianzhang; Cai, Yuepiao; Li, Xiaokun

2012-10-01

The fibroblast growth factor receptor (FGFR) family includes four highly conserved receptor tyrosine kinases: FGFR1-4. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. These FGFR cascades play crucial roles in tumor cell proliferation, angiogenesis, migration, and survival. The combination of knockdown studies and pharmaceutical inhibition in preclinical models demonstrates that FGFRs are attractive targets for therapeutic intervention in cancer. Multiple FGFR inhibitors with various structural skeletons have been designed, synthesized, and evaluated. Reviews on FGFRs have recently focused on FGFR signaling, pathophysiology, and functions in cancer or other diseases. In this article, we review recent advances in structure-activity relationships (SAR) of FGFR inhibitors, as well as the FGFR-targeting drug design strategies currently employed in targeting deregulated FGFRs by antibodies and small molecule inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dorso-Lateral Frontal Cortex of the Ferret Encodes Perceptual Difficulty during Visual Discrimination

PubMed Central

Zhou, Zhe Charles; Yu, Chunxiu; Sellers, Kristin K.; Fröhlich, Flavio

2016-01-01

Visual discrimination requires sensory processing followed by a perceptual decision. Despite a growing understanding of visual areas in this behavior, it is unclear what role top-down signals from prefrontal cortex play, in particular as a function of perceptual difficulty. To address this gap, we investigated how neurons in dorso-lateral frontal cortex (dl-FC) of freely-moving ferrets encode task variables in a two-alternative forced choice visual discrimination task with high- and low-contrast visual input. About two-thirds of all recorded neurons in dl-FC were modulated by at least one of the two task variables, task difficulty and target location. More neurons in dl-FC preferred the hard trials; no such preference bias was found for target location. In individual neurons, this preference for specific task types was limited to brief epochs. Finally, optogenetic stimulation confirmed the functional role of the activity in dl-FC before target touch; suppression of activity in pyramidal neurons with the ArchT silencing opsin resulted in a decrease in reaction time to touch the target but not to retrieve reward. In conclusion, dl-FC activity is differentially recruited for high perceptual difficulty in the freely-moving ferret and the resulting signal may provide top-down behavioral inhibition. PMID:27025995

Dorso-Lateral Frontal Cortex of the Ferret Encodes Perceptual Difficulty during Visual Discrimination.

PubMed

Zhou, Zhe Charles; Yu, Chunxiu; Sellers, Kristin K; Fröhlich, Flavio

2016-03-30

Visual discrimination requires sensory processing followed by a perceptual decision. Despite a growing understanding of visual areas in this behavior, it is unclear what role top-down signals from prefrontal cortex play, in particular as a function of perceptual difficulty. To address this gap, we investigated how neurons in dorso-lateral frontal cortex (dl-FC) of freely-moving ferrets encode task variables in a two-alternative forced choice visual discrimination task with high- and low-contrast visual input. About two-thirds of all recorded neurons in dl-FC were modulated by at least one of the two task variables, task difficulty and target location. More neurons in dl-FC preferred the hard trials; no such preference bias was found for target location. In individual neurons, this preference for specific task types was limited to brief epochs. Finally, optogenetic stimulation confirmed the functional role of the activity in dl-FC before target touch; suppression of activity in pyramidal neurons with the ArchT silencing opsin resulted in a decrease in reaction time to touch the target but not to retrieve reward. In conclusion, dl-FC activity is differentially recruited for high perceptual difficulty in the freely-moving ferret and the resulting signal may provide top-down behavioral inhibition.

Effects of Δ9-tetrahydrocannabinol administration on human encoding and recall memory function: a pharmacological FMRI study.

PubMed

Bossong, Matthijs G; Jager, Gerry; van Hell, Hendrika H; Zuurman, Lineke; Jansma, J Martijn; Mehta, Mitul A; van Gerven, Joop M A; Kahn, René S; Ramsey, Nick F

2012-03-01

Deficits in memory function are an incapacitating aspect of various psychiatric and neurological disorders. Animal studies have recently provided strong evidence for involvement of the endocannabinoid (eCB) system in memory function. Neuropsychological studies in humans have shown less convincing evidence but suggest that administration of cannabinoid substances affects encoding rather than recall of information. In this study, we examined the effects of perturbation of the eCB system on memory function during both encoding and recall. We performed a pharmacological MRI study with a placebo-controlled, crossover design, investigating the effects of Δ9-tetrahydrocannabinol (THC) inhalation on associative memory-related brain function in 13 healthy volunteers. Performance and brain activation during associative memory were assessed using a pictorial memory task, consisting of separate encoding and recall conditions. Administration of THC caused reductions in activity during encoding in the right insula, the right inferior frontal gyrus, and the left middle occipital gyrus and a network-wide increase in activity during recall, which was most prominent in bilateral cuneus and precuneus. THC administration did not affect task performance, but while during placebo recall activity significantly explained variance in performance, this effect disappeared after THC. These findings suggest eCB involvement in encoding of pictorial information. Increased precuneus activity could reflect impaired recall function, but the absence of THC effects on task performance suggests a compensatory mechanism. These results further emphasize the eCB system as a potential novel target for treatment of memory disorders and a promising target for development of new therapies to reduce memory deficits in humans.

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo

PubMed Central

Yallowitz, Alisha R.; Gong, Ke-Qin; Swinehart, Ilea T.; Nelson, Lisa T.; Wellik, Deneen M.

2009-01-01

Summary Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity is poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins. PMID:19716816

A biomimetic colorimetric logic gate system based on multi-functional peptide-mediated gold nanoparticle assembly

NASA Astrophysics Data System (ADS)

Li, Yong; Li, Wang; He, Kai-Yu; Li, Pei; Huang, Yan; Nie, Zhou; Yao, Shou-Zhuo

2016-04-01

In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation.In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation. Electronic supplementary information (ESI) available: Additional figures (Tables S1-S3 and Fig. S1-S6). See DOI: 10.1039/c6nr01072e

Target mimics: an embedded layer of microRNA-involved gene regulatory networks in plants.

PubMed

Meng, Yijun; Shao, Chaogang; Wang, Huizhong; Jin, Yongfeng

2012-05-21

MicroRNAs (miRNAs) play an essential role in gene regulation in plants. At the same time, the expression of miRNA genes is also tightly controlled. Recently, a novel mechanism called "target mimicry" was discovered, providing another layer for modulating miRNA activities. However, except for the artificial target mimics manipulated for functional studies on certain miRNA genes, only one example, IPS1 (Induced by Phosphate Starvation 1)-miR399 was experimentally confirmed in planta. To date, few analyses for comprehensive identification of natural target mimics have been performed in plants. Thus, limited evidences are available to provide detailed information for interrogating the questionable issue whether target mimicry was widespread in planta, and implicated in certain biological processes. In this study, genome-wide computational prediction of endogenous miRNA mimics was performed in Arabidopsis and rice, and dozens of target mimics were identified. In contrast to a recent report, the densities of target mimic sites were found to be much higher within the untranslated regions (UTRs) when compared to those within the coding sequences (CDSs) in both plants. Some novel sequence characteristics were observed for the miRNAs that were potentially regulated by the target mimics. GO (Gene Ontology) term enrichment analysis revealed some functional insights into the predicted mimics. After degradome sequencing data-based identification of miRNA targets, the regulatory networks constituted by target mimics, miRNAs and their downstream targets were constructed, and some intriguing subnetworks were further exploited. These results together suggest that target mimicry may be widely implicated in regulating miRNA activities in planta, and we hope this study could expand the current understanding of miRNA-involved regulatory networks.

Rhodium(II) proximity-labeling identifies a novel target site on STAT3 for inhibitors with potent anti-leukemia activity

PubMed Central

Minus, Matthew B.; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M.; Long, Xin; Krueger, Michael; Stevens, Alexandra; Kolosov, Mikhail I.; Sison, Edward Allen R.; Ball, Zachary T.

2015-01-01

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. PMID:26480340

Interaction of p53 with prolyl isomerases: Healthy and unhealthy relationships.

PubMed

Mantovani, Fiamma; Zannini, Alessandro; Rustighi, Alessandra; Del Sal, Giannino

2015-10-01

The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Promising landscape for regulating macrophage polarization: epigenetic viewpoint

PubMed Central

Chen, Lu; Zhang, Wen; Xu, Zhenyu; Zuo, Jian; Jiang, Hui; Luan, Jiajie

2017-01-01

Macrophages are critical myeloid cells with the hallmark of phenotypic heterogeneity and functional plasticity. Macrophages phenotypes are commonly described as classically-activated M1 and alternatively-activated M2 macrophages which play an essential role in the tissues homeostasis and diseases pathogenesis. Alternations of macrophage polarization and function states require precise regulation of target-gene expression. Emerging data demonstrate that epigenetic mechanisms and transcriptional factors are becoming increasingly appreciated in the orchestration of macrophage polarization in response to local environmental signals. This review is to focus on the advanced concepts of epigenetics changes involved with the macrophage polarization, including microRNAs, DNA methylation and histone modification, which are responsible for the altered cellular signaling and signature genes expression during M1 or M2 polarization. Eventually, the persistent investigation and understanding of epigenetic mechanisms in tissue macrophage polarization and function will enhance the potential to develop novel therapeutic targets for various diseases. PMID:28915705

Myeloid derived suppressor cells-An overview of combat strategies to increase immunotherapy efficacy.

PubMed

Draghiciu, Oana; Lubbers, Joyce; Nijman, Hans W; Daemen, Toos

2015-01-01

Myeloid-derived suppressor cells (MDSCs) contribute to tumor-mediated immune escape and negatively correlate with overall survival of cancer patients. Nowadays, a variety of methods to target MDSCs are being investigated. Based on the intervention stage of MDSCs, namely development, expansion and activation, function and turnover, these methods can be divided into: (I) prevention or differentiation to mature cells, (II) blockade of MDSC expansion and activation, (III) inhibition of MDSC suppressive activity or (IV) depletion of intratumoral MDSCs. This review describes effective mono- or multimodal-therapies that target MDSCs for the benefit of cancer treatment.

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth

PubMed Central

Saha, Nayanendu; Eissman, Moritz F.; Xu, Kai; Llerena, Carmen; Kusebauch, Ulrike; Ding, Bi-Sen; Cao, Zhongwei; Rafii, Shahin; Ernst, Matthias; Scott, Andrew M.; Nikolov, Dimitar B.; Lackmann, Martin

2016-01-01

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance. PMID:27503072

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth.

PubMed

Atapattu, Lakmali; Saha, Nayanendu; Chheang, Chanly; Eissman, Moritz F; Xu, Kai; Vail, Mary E; Hii, Linda; Llerena, Carmen; Liu, Zhanqi; Horvay, Katja; Abud, Helen E; Kusebauch, Ulrike; Moritz, Robert L; Ding, Bi-Sen; Cao, Zhongwei; Rafii, Shahin; Ernst, Matthias; Scott, Andrew M; Nikolov, Dimitar B; Lackmann, Martin; Janes, Peter W

2016-08-22

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance. © 2016 Atapattu et al.

219. Changes in Functional Networks Underlying Social Cognition Following Cognitive Training in Individuals at Risk for Psychosis

PubMed Central

Haut, Kristen; Saxena, Abhishek; Yin, Hong; Carol, Emily; Dodell-Feder, David; Lincoln, Sarah Hope; Tully, Laura; Keshavan, Matcheri; Seidman, Larry J.; Nahum, Mor; Hooker, Christine

2017-01-01

Abstract Background: Deficits in social cognition are prominent features of schizophrenia that play a large role in functional impairments and disability. Performance deficits in these domains are associated with altered activity in functional networks, including those that support social cognitive abilities such as emotion recognition. These social cognitive deficits and alterations in neural networks are present prior to the onset of frank psychotic symptoms and thus present a potential target for intervention in early phases of the illness, including in individuals at clinical high risk (CHR) for psychosis. This study assessed changes in social cognitive functional networks following targeted cognitive training (TCT) in CHR individuals. Methods: 14 CHR subjects (7 male, mean age = 21.9) showing attenuated psychotic symptoms as assessed by the SIPS were included in the study. Subjects underwent a clinical evaluation and a functional MRI session prior to and subsequent to completing 40 hours (8 weeks) of targeted cognitive and social cognitive training using Lumosity and SocialVille. 14 matched healthy control (HC) subjects also underwent a single fMRI session as a comparison group for functional activity. Resting state fMRI was acquired as well as fMRI during performance of an emotion recognition task. Group level differences in BOLD activity between HC and CHR group before TCT, and CHR group before and after TCT were computed. Changes in social cognitive network functional connectivity at rest and during task performance was evaluated using seed-based connectivity analyses and psychophysiological interaction (PPI). Results: Prior to training, CHR individuals demonstrated hyperactivity in the amygdala, posterior cingulate, and superior temporal sulcus (STS) during emotion recognition, suggesting inefficient processing. This hyperactivity normalized somewhat after training, with CHR individuals showing less hyperactivity in the amygdala in response to emotional faces. In addition, training was associated with increased connectivity in emotion processing networks, including greater STS-medial prefrontal connectivity and normalization of amygdala connectivity patterns. Conclusion: These results suggest that targeted cognitive training produced improvements in emotion recognition and may be effective in altering functional network connectivity in networks associated with psychosis risk. TCT may be a useful tool for early intervention in individuals at risk for psychotic disorders to address behaviors that impact functional outcome.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

The Putzig-NURF nucleosome remodeling complex is required for ecdysone receptor signaling and innate immunity in Drosophila melanogaster.

PubMed

Kugler, Sabrina J; Gehring, Eva-Maria; Wallkamm, Veronika; Krüger, Victoria; Nagel, Anja C

2011-05-01

Putzig (Pzg) was originally identified as being an integral component of the TRF2/DREF complex in Drosophila melanogaster, thereby regulating the transcriptional activation of replication-related genes. In a DREF-independent manner, Pzg was shown to mediate Notch target gene activation. This function of Pzg entails an association with the nucleosome remodeling factor complex NURF, which directly binds the ecdysone receptor EcR and coregulates targets of the EcR via the NURF-specific subunit Nurf-301. In contrast, Nurf-301 acts as a negative regulator of JAK/STAT signaling. Here, we provide evidence to show that Pzg is fundamental for these functions of NURF, apart from the regulation of Notch signaling activity. A jump-out mutagenesis provided us with a pzg null mutant displaying early larval lethality, defects in growth, and molting accompanied by aberrant feeding behavior. We show that Pzg is associated with EcR in vivo and required for the transcriptional induction of EcR target genes, whereas reduced ecdysteroid levels imply a NURF-independent function of Pzg. Moreover, pzg interferes with JAK/STAT-signaling activity by acting as a corepressor of Ken. Lamellocyte differentiation was consistently affected in a JAK/STAT mutant background and the expression level of defense response genes was elevated in pzg mutants, leading to the formation of melanotic tumors. Our results suggest that Pzg acts as an important partner of NURF in the regulation of EcR and JAK/STAT signaling.

NF-E2 p45 Is Important for Establishing Normal Function of Platelets

PubMed Central

Fujita, Rie; Takayama-Tsujimoto, Mariko; Satoh, Hironori; Gutiérrez, Laura; Aburatani, Hiroyuki; Fujii, Satoshi; Sarai, Akinori; Bresnick, Emery H.

2013-01-01

NF-E2 is a heterodimeric transcription factor consisting of p45 and small Maf subunits. Since p45−/− mice display severe thrombocytopenia, p45 is recognized as a critical regulator of platelet production from megakaryocytes. To identify direct p45 target genes in megakaryocytes, we used chromatin immunoprecipitation (ChIP) sequencing to analyze the genome-wide chromatin occupancy of p45 in primary megakaryocytes. p45 target gene candidates obtained from the analysis are implicated in the production and function of platelets. Two of these genes, Selp and Myl9, were verified as direct p45 targets through multiple approaches. Since P-selectin, encoded by Selp, plays a critical role in platelet function during thrombogenesis, we tested whether p45 determines the intrinsic reactivity and potency of platelets generated from megakaryocytes. Mice expressing a hypomorphic p45 mutant instead of wild-type p45 in megakaryocytes (p45−/−:ΔNTD-Tg mice) displayed platelet hypofunction accompanied by mild thrombocytopenia. Furthermore, lung metastasis of melanoma cells, which requires platelet activation, was repressed in p45−/−:ΔNTD-Tg mice compared to control mice, validating the impaired function of platelets produced from p45−/−:ΔNTD-Tg megakaryocytes. By activating genes in megakaryocytes that mediate platelet production and function, p45 determines the quantity and quality of platelets. PMID:23648484

Study of the temperature dependent immuno-reaction kinetics for the bio-functionalized magnetic nanoparticle assay of bio-markers of colorectal cancer

NASA Astrophysics Data System (ADS)

Yang, S. Y.; Chang, J. F.; Chen, T. C.; Yang, C. C.; Ho, C. S.

2014-01-01

By conjugating antibodies on magnetic nanoparticles, target antigens can be quantitatively detected by measuring the magnetic signals of the magnetic nanoparticles due to their association with target antigens. This method of detection is called magnetically labeled immunoassay. The assay characteristics of magnetically labeled immunoassay have been reported widely. However, the immuno-reaction kinetics of magnetically labeled immunoassay has not been studied. In this work, the reaction rates between magnetic nanoparticles and target antigens are measured at various temperatures. It is found that the temperature dependent reaction rate obeys Arrhenius's equation, which shows the collision frequency and activation energy for the immuno-reaction between antibody-functionalized magnetic nanoparticles and target antigens. The carcinoembryonic antigen, which is a regular blood bio-marker for in-vitro diagnosis of colorectal cancer, is used as a target antigen for the example.

Discovery of novel drug targets and their functions using phenotypic screening of natural products.

PubMed

Chang, Junghwa; Kwon, Ho Jeong

2016-03-01

Natural products are valuable resources that provide a variety of bioactive compounds and natural pharmacophores in modern drug discovery. Discovery of biologically active natural products and unraveling their target proteins to understand their mode of action have always been critical hurdles for their development into clinical drugs. For effective discovery and development of bioactive natural products into novel therapeutic drugs, comprehensive screening and identification of target proteins are indispensable. In this review, a systematic approach to understanding the mode of action of natural products isolated using phenotypic screening involving chemical proteomics-based target identification is introduced. This review highlights three natural products recently discovered via phenotypic screening, namely glucopiericidin A, ecumicin, and terpestacin, as representative case studies to revisit the pivotal role of natural products as powerful tools in discovering the novel functions and druggability of targets in biological systems and pathological diseases of interest.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

PubMed

Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

2012-03-01

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

Functional Biomimetic Architectures

NASA Astrophysics Data System (ADS)

Levine, Paul M.

N-substituted glycine oligomers, or 'peptoids,' are a class of sequence--specific foldamers composed of tertiary amide linkages, engendering proteolytic stability and enhanced cellular permeability. Peptoids are notable for their facile synthesis, sequence diversity, and ability to fold into distinct secondary structures. In an effort to establish new functional peptoid architectures, we utilize the copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction to generate peptidomimetic assemblies bearing bioactive ligands that specifically target and modulate Androgen Receptor (AR) activity, a major therapeutic target for prostate cancer. Additionally, we explore chemical ligation protocols to generate semi-synthetic hybrid biomacromolecules capable of exhibiting novel structures and functions not accessible to fully biosynthesized proteins.

N-acetylgalactosamine-functionalized dendrimers as hepatic cancer cell-targeted carriers.

PubMed

Medina, Scott H; Tekumalla, Venkatesh; Chevliakov, Maxim V; Shewach, Donna S; Ensminger, William D; El-Sayed, Mohamed E H

2011-06-01

There is an urgent need for novel polymeric carriers that can selectively deliver a large dose of chemotherapeutic agents into hepatic cancer cells to achieve high therapeutic activity with minimal systemic side effects. PAMAM dendrimers are characterized by a unique branching architecture and a large number of chemical surface groups suitable for coupling of chemotherapeutic agents. In this article, we report the coupling of N-acetylgalactosamine (NAcGal) to generation 5 (G5) of poly(amidoamine) (PAMAM-NH₂) dendrimers via peptide and thiourea linkages to prepare NAcGal-targeted carriers used for targeted delivery of chemotherapeutic agents into hepatic cancer cells. We describe the uptake of NAcGal-targeted and non-targeted G5 dendrimers into hepatic cancer cells (HepG2) as a function of G5 concentration and incubation time. We examine the contribution of the asialoglycoprotein receptor (ASGPR) to the internalization of NAcGal-targeted dendrimers into hepatic cancer cells through a competitive inhibition assay. Our results show that uptake of NAcGal-targeted G5 dendrimers into hepatic cancer cells occurs via ASGPR-mediated endocytosis. Internalization of these targeted carriers increased with the increase in G5 concentration and incubation time following Michaelis-Menten kinetics characteristic of receptor-mediated endocytosis. These results collectively indicate that G5-NAcGal conjugates function as targeted carriers for selective delivery of chemotherapeutic agents into hepatic cancer cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Anyalysis of Msx1 and Msx2 Transactivation Function in the Context of the Heat Shock 70 (Hspa1b) Gene Promoter

PubMed Central

Zhuang, Fengfeng; Nguyen, Manuel P.; Shuler, Charles; Liu, Yi-Hsin

2009-01-01

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent trans-activation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and Heat shock factors may play a contributing role. PMID:19338779

Connectivity-based parcellation of human cingulate cortex and its relation to functional specialization.

PubMed

Beckmann, Matthias; Johansen-Berg, Heidi; Rushworth, Matthew F S

2009-01-28

Whole-brain neuroimaging studies have demonstrated regional variations in function within human cingulate cortex. At the same time, regional variations in cingulate anatomical connections have been found in animal models. It has, however, been difficult to estimate the relationship between connectivity and function throughout the whole cingulate cortex within the human brain. In this study, magnetic resonance diffusion tractography was used to investigate cingulate probabilistic connectivity in the human brain with two approaches. First, an algorithm was used to search for regional variations in the probabilistic connectivity profiles of all cingulate cortex voxels with the whole of the rest of the brain. Nine subregions with distinctive connectivity profiles were identified. It was possible to characterize several distinct areas in the dorsal cingulate sulcal region. Several distinct regions were also found in subgenual and perigenual cortex. Second, the probabilities of connection between cingulate cortex and 11 predefined target regions of interest were calculated. Cingulate voxels with a high probability of connection with the different targets formed separate clusters within cingulate cortex. Distinct connectivity fingerprints characterized the likelihood of connections between the extracingulate target regions and the nine cingulate subregions. Last, a meta-analysis of 171 functional studies reporting cingulate activation was performed. Seven different cognitive conditions were selected and peak activation coordinates were plotted to create maps of functional localization within the cingulate cortex. Regional functional specialization was found to be related to regional differences in probabilistic anatomical connectivity.

Estradiol targets T cell signaling pathways in human systemic lupus.

PubMed

Walters, Emily; Rider, Virginia; Abdou, Nabih I; Greenwell, Cindy; Svojanovsky, Stan; Smith, Peter; Kimler, Bruce F

2009-12-01

The major risk factor for developing systemic lupus erythematosus (SLE) is being female. The present study utilized gene profiles of activated T cells from females with SLE and healthy controls to identify signaling pathways uniquely regulated by estradiol that could contribute to SLE pathogenesis. Selected downstream pathway genes (+/- estradiol) were measured by real time polymerase chain amplification. Estradiol uniquely upregulated six pathways in SLE T cells that control T cell function including interferon-alpha signaling. Measurement of interferon-alpha pathway target gene expression revealed significant differences (p= 0.043) in DRIP150 (+/- estradiol) in SLE T cell samples while IFIT1 expression was bimodal and correlated moderately (r= 0.55) with disease activity. The results indicate that estradiol alters signaling pathways in activated SLE T cells that control T cell function. Differential expression of transcriptional coactivators could influence estrogen-dependent gene regulation in T cell signaling and contribute to SLE onset and disease pathogenesis.

Immunoconjugates and long circulating systems: Origins, current state of the art and future directions☆

PubMed Central

Koshkaryev, Alexander; Sawant, Rupa; Deshpande, Madhura; Torchilin, Vladimir

2012-01-01

Significant progress has been made recently in the area of immunoconjugated drugs and drug delivery systems (DDS). The immuno-modification of either the drug or DDS has proven to be a very promising approach that has significantly improved the targeted accumulation in pathological sites while decreasing its undesirable side effects in healthy tissues. The arrangement for both prolonged life in the circulation and specific target recognition represents another potent strategy in the development of immuno-targeted systems. The longevity of immuno-targeted DDS such as immunoliposomes and immunomicelles improves their targetability even in the presence of the additional passive accumulation in areas with a compromised vasculature. The added use of the immuno-targeted systems takes advantage of the specific microenvironment of pathological sites including lowered pH, increased temperature, and variation in the enzymatic activity. “Smart” stimulus-responsive systems combine different valuable functionalities including PEG-protection, targeting antibody, cell-penetration, and stimulus-sensitive functions. In this review we examined the evolution, current status and future directions in the area of therapeutical immunoconjugates and long-circulating immuno-targeted DDS. PMID:22964425

Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules

PubMed Central

Bakajsova, Diana; Samarel, Allen M.

2011-01-01

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC. PMID:21289057

Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

PubMed Central

Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

2014-01-01

Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

Targeting mechanisms of high voltage-activated Ca2+ channels.

PubMed

Herlitze, Stefan; Xie, Mian; Han, Jing; Hümmer, Alexander; Melnik-Martinez, Katya V; Moreno, Rosa L; Mark, Melanie D

2003-12-01

Functional voltage-dependent Ca2+ channel complexes are assembled by three to four subunits: alpha1, beta, alpha2delta subunits (C. Leveque et al., 1994, J. Biol Chem. 269, 6306-6312; M. W. McEnery et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099) and at least in muscle cells also y subunits (B. M. Curtis and W. A. Catterall, 1984, Biochemistry 23, 2113-2118). Ca2+ channels mediate the voltage-dependent Ca2+ influx in subcellular compartments, triggering such diverse processes as neurotransmitter release, dendritic action potentials, excitation-contraction, and excitation-transcription coupling. The targeting of biophysically defined Ca2+ channel complexes to the correct subcellular structures is, thus, critical to proper cell and physiological functioning. Despite their importance, surprisingly little is known about the targeting mechanisms by which Ca2+ channel complexes are transported to their site of function. Here we summarize what we know about the targeting of Ca2+ channel complexes through the cell to the plasma membrane and subcellular structures.

Dendritic cell targeted vaccines: Recent progresses and challenges

PubMed Central

Chen, Pengfei; Liu, Xinsheng; Sun, Yuefeng; Zhou, Peng; Wang, Yonglu; Zhang, Yongguang

2016-01-01

ABSTRACT Dendritic cells (DCs) are known to be a set of morphology, structure and function of heterogeneous professional antigen presenting cells (APCs), as well as the strongest functional antigen presenting cells, which can absorb, process and present antigens. As the key regulators of innate and adaptive immune responses, DCs are at the center of the immune system and capable of interacting with both B cells and T cells, thereby manipulating the humoral and cellular immune responses. DCs provide an essential link between the innate and adaptive immunity, and the strong immune activation function of DCs and their properties of natural adjuvants, make them a valuable target for antigen delivery. Targeting antigens to DC-specific endocytic receptors in combination with the relevant antibodies or ligands along with immunostimulatory adjuvants has been recently recognized as a promising strategy for designing an effective vaccine that elicits a strong and durable T cell response against intracellular pathogens and cancer. This opinion article provides a brief summary of the rationales, superiorities and challenges of existing DC-targeting approaches. PMID:26513200

Blockade of Treg Cell Differentiation and Function by the Interleukin-21-Mechanistic Target of Rapamycin Axis Via Suppression of Autophagy in Patients With Systemic Lupus Erythematosus.

PubMed

Kato, Hiroshi; Perl, Andras

2018-03-01

The mechanistic target of rapamycin (mTOR) has become a therapeutic target in systemic lupus erythematosus (SLE). In T cells, mTOR plays a central role in lineage specification, including development of regulatory cells (Treg cells). This study sought to investigate whether mTOR is activated within Treg cells and whether this contributes to the depletion and dysfunction of Treg cells in patients with SLE. Activities of mTOR complexes 1 (mTORC1) and 2 (mTORC2) were examined by quantifying phosphorylation of translation initiation factor 4E-binding protein 1, S6 kinase, and Akt in SLE patients relative to age- and sex-matched female healthy control subjects. Polarization of Treg cells from naive CD4+ T cells was assessed in the presence of interleukin-6 (IL-6), IL-17, and IL-21. The suppressor function of sorted CD4+CD25+ Treg cells was measured by determining their impact on the proliferation of autologous CD4+CD25- responder T cells. Treg cell expression of FoxP3, GATA-3, and CTLA-4 was monitored by flow cytometry. Autophagy was assessed using immunoblotting of light chain 3 lipidation. The effect of mTOR blockade was evaluated by testing the impact of rapamycin treatment on Treg cell function. SLE Treg cells exhibited increased activities of mTORC1 and mTORC2, whereas autophagy, the expression of GATA-3 and CTLA-4, and the suppressor function of Treg cells were diminished. IL-21, but not IL-6 or IL-17, blocked the development of Treg cells. IL-21 stimulated mTORC1 and mTORC2, and it abrogated the autophagy, differentiation, and function of Treg cells. Moreover, IL-21 constrained the expression of GATA-3 and CTLA-4 selectively in Treg cells. In turn, blockade of mTORC1 by 3-day rapamycin treatment enhanced transforming growth factor β production, while dual blockade of mTORC1 and mTORC2 by 4-week rapamycin treatment induced autophagy, restored the expression of GATA-3 and CTLA-4, and corrected Treg cell function. IL-21-driven mTOR activation is a pharmacologically targetable checkpoint of the deficient autophagy that underlies Treg cell dysfunction in SLE. © 2017, American College of Rheumatology.

Structure-based design of bacterial nitric oxide synthase inhibitors

DOE PAGES

Holden, Jeffrey K.; Kang, Soosung; Hollingsworth, Scott A.; ...

2014-12-18

Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial. Here wemore » present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Lastly, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.« less

MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.

PubMed

Cowan, Colleen; Muraleedharan, Chithra K; O'Donnell, James J; Singh, Pawan K; Lum, Hazel; Kumar, Ashok; Xu, Shunbin

2014-07-01

Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions. Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

The Throw-and-Catch Model of Human Gait: Evidence from Coupling of Pre-Step Postural Activity and Step Location

PubMed Central

Bancroft, Matthew J.; Day, Brian L.

2016-01-01

Postural activity normally precedes the lift of a foot from the ground when taking a step, but its function is unclear. The throw-and-catch hypothesis of human gait proposes that the pre-step activity is organized to generate momentum for the body to fall ballistically along a specific trajectory during the step. The trajectory is appropriate for the stepping foot to land at its intended location while at the same time being optimally placed to catch the body and regain balance. The hypothesis therefore predicts a strong coupling between the pre-step activity and step location. Here we examine this coupling when stepping to visually-presented targets at different locations. Ten healthy, young subjects were instructed to step as accurately as possible onto targets placed in five locations that required either different step directions or different step lengths. In 75% of trials, the target location remained constant throughout the step. In the remaining 25% of trials, the intended step location was changed by making the target jump to a new location 96 ms ± 43 ms after initiation of the pre-step activity, long before foot lift. As predicted by the throw-and-catch hypothesis, when the target location remained constant, the pre-step activity led to body momentum at foot lift that was coupled to the intended step location. When the target location jumped, the pre-step activity was adjusted (median latency 223 ms) and prolonged (on average by 69 ms), which altered the body’s momentum at foot lift according to where the target had moved. We conclude that whenever possible the coupling between the pre-step activity and the step location is maintained. This provides further support for the throw-and-catch hypothesis of human gait. PMID:28066208

The Throw-and-Catch Model of Human Gait: Evidence from Coupling of Pre-Step Postural Activity and Step Location.

PubMed

Bancroft, Matthew J; Day, Brian L

2016-01-01

Postural activity normally precedes the lift of a foot from the ground when taking a step, but its function is unclear. The throw-and-catch hypothesis of human gait proposes that the pre-step activity is organized to generate momentum for the body to fall ballistically along a specific trajectory during the step. The trajectory is appropriate for the stepping foot to land at its intended location while at the same time being optimally placed to catch the body and regain balance. The hypothesis therefore predicts a strong coupling between the pre-step activity and step location. Here we examine this coupling when stepping to visually-presented targets at different locations. Ten healthy, young subjects were instructed to step as accurately as possible onto targets placed in five locations that required either different step directions or different step lengths. In 75% of trials, the target location remained constant throughout the step. In the remaining 25% of trials, the intended step location was changed by making the target jump to a new location 96 ms ± 43 ms after initiation of the pre-step activity, long before foot lift. As predicted by the throw-and-catch hypothesis, when the target location remained constant, the pre-step activity led to body momentum at foot lift that was coupled to the intended step location. When the target location jumped, the pre-step activity was adjusted (median latency 223 ms) and prolonged (on average by 69 ms), which altered the body's momentum at foot lift according to where the target had moved. We conclude that whenever possible the coupling between the pre-step activity and the step location is maintained. This provides further support for the throw-and-catch hypothesis of human gait.

Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

PubMed Central

2014-01-01

Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

Microarray-based determination of anti-inflammatory genes targeted by 6-(methylsulfinyl)hexyl isothiocyanate in macrophages.

PubMed

Chen, Jihua; Uto, Takuhiro; Tanigawa, Shunsuke; Yamada-Kato, Tomeo; Fujii, Makoto; Hou, DE-Xing

2010-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as 'defense, inflammatory response, cytokine activities and receptor activities' and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function.

Molecular Determinants of Magnolol Targeting Both RXRα and PPARγ

PubMed Central

Chen, Lili; Chen, Jing; Hu, Lihong; Jiang, Hualiang; Shen, Xu

2011-01-01

Nuclear receptors retinoic X receptor α (RXRα) and peroxisome proliferator activated receptor γ (PPARγ) function potently in metabolic diseases, and are both important targets for anti-diabetic drugs. Coactivation of RXRα and PPARγ is believed to synergize their effects on glucose and lipid metabolism. Here we identify the natural product magnolol as a dual agonist targeting both RXRα and PPARγ. Magnolol was previously reported to enhance adipocyte differentiation and glucose uptake, ameliorate blood glucose level and prevent development of diabetic nephropathy. Although magnolol can bind and activate both of these two nuclear receptors, the transactivation assays indicate that magnolol exhibits biased agonism on the transcription of PPAR-response element (PPRE) mediated by RXRα:PPARγ heterodimer, instead of RXR-response element (RXRE) mediated by RXRα:RXRα homodimer. To further elucidate the molecular basis for magnolol agonism, we determine both the co-crystal structures of RXRα and PPARγ ligand-binding domains (LBDs) with magnolol. Structural analyses reveal that magnolol adopts its two 5-allyl-2-hydroxyphenyl moieties occupying the acidic and hydrophobic cavities of RXRα L-shaped ligand-binding pocket, respectively. While, two magnolol molecules cooperatively accommodate into PPARγ Y-shaped ligand-binding pocket. Based on these two complex structures, the key interactions for magnolol activating RXRα and PPARγ are determined. As the first report on the dual agonist targeting RXRα and PPARγ with receptor-ligand complex structures, our results are thus expected to help inspect the potential pharmacological mechanism for magnolol functions, and supply useful hits for nuclear receptor multi-target ligand design. PMID:22140563

Neural correlates of the spatial and expectancy components of endogenous and stimulus-driven orienting of attention in the Posner task.

PubMed

Doricchi, Fabrizio; Macci, Enrica; Silvetti, Massimo; Macaluso, Emiliano

2010-07-01

Voluntary orienting of visual attention is conventionally measured in tasks with predictive central cues followed by frequent valid targets at the cued location and by infrequent invalid targets at the uncued location. This implies that invalid targets entail both spatial reorienting of attention and breaching of the expected spatial congruency between cues and targets. Here, we used event-related functional magnetic resonance imaging (fMRI) to separate the neural correlates of the spatial and expectancy components of both endogenous orienting and stimulus-driven reorienting of attention. We found that during endogenous orienting with predictive cues, there was a significant deactivation of the right Temporal-Parietal Junction (TPJ). We also discovered that the lack of an equivalent deactivation with nonpredictive cues was matched to drop in attentional costs and preservation of attentional benefits. The right TPJ showed equivalent responses to invalid targets following predictive and nonpredictive cues. On the contrary, infrequent-unexpected invalid targets following predictive cues specifically activated the right Middle and Inferior Frontal Gyrus (MFG-IFG). Additional comparisons with spatially neutral trials demonstrated that, independently of cue predictiveness, valid targets activate the left TPJ, whereas invalid targets activate both the left and right TPJs. These findings show that the selective right TPJ activation that is found in the comparison between invalid and valid trials results from the reciprocal cancelling of the different activations that in the left TPJ are related to the processing of valid and invalid targets. We propose that left and right TPJs provide "matching and mismatching to attentional template" signals. These signals enable reorienting of attention and play a crucial role in the updating of the statistical contingency between cues and targets.

Echo scintillation Index affected by cat-eye target's caliber with Cassegrain lens

NASA Astrophysics Data System (ADS)

Shan, Cong-miao; Sun, Hua-yan; Zhao, Yan-zhong; Zheng, Yong-hui

2015-10-01

The optical aperture of cat-eye target has the aperture averaging effect to the active detecting laser of active laser detection system, which can be used to identify optical targets. The echo scintillation characteristics of the transmission-type lens target have been studied in previous work. Discussing the differences of the echo scintillation characteristics between the transmission-type lens target and Cassegrain lens target can be helpful to targets classified. In this paper, the echo scintillation characteristics of Cat-eye target's caliber with Cassegrain lens has been discussed . By using the flashing theory of spherical wave in the weak atmospheric turbulence, the annular aperture filter function and the Kolmogorov power spectrum, the analytic expression of the scintillation index of the cat-eye target echo of the horizontal path two-way transmission was given when the light is normal incidence. Then the impact of turbulence inner and outer scale to the echo scintillation index and the analytic expression of the echo scintillation index at the receiving aperture were presented using the modified Hill spectrum and the modified Von Karman spectrum. Echo scintillation index shows the tendency of decreasing with the target aperture increases and different ratios of the inner and outer aperture diameter show the different echo scintillation index curves. This conclusion has a certain significance for target recognition in the active laser detection system that can largely determine the target type by largely determining the scope of the cat-eye target which depending on echo scintillation index.

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice.

PubMed

Le Henaff, Carole; Mansouri, Rafik; Modrowski, Dominique; Zarka, Mylène; Geoffroy, Valérie; Marty, Caroline; Tarantino, Nadine; Laplantine, Emmanuel; Marie, Pierre J

2015-07-17

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice*

PubMed Central

Le Henaff, Carole; Mansouri, Rafik; Modrowski, Dominique; Zarka, Mylène; Geoffroy, Valérie; Marty, Caroline; Tarantino, Nadine; Laplantine, Emmanuel; Marie, Pierre J.

2015-01-01

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis. PMID:26060255

Cell Death and Cancer Therapy: Don't Forget to Kill the Cancer Cell!

PubMed

Letai, Anthony

2015-11-15

In our current age of targeted therapies, there is understandably considerable attention paid to the specific molecular targets of pharmaceutical intervention. For a targeted drug to work, it must bind to a target selectively and impair its function. Monitoring biomarkers of the impaired target function can provide vital in vivo pharmacodynamic information. Moreover, genetic changes to the target are often the source of resistance to targeted agents. However, for the treatment of cancer, it is necessary that the therapy not only provide efficient binding and inhibition of the target, but also that this intervention reliably kills the cancer cell. In this CCR Focus section, four articles make the connection between therapies that target T-cell activation, autophagy, IAP proteins, and BCL-2 and the commitment of cancer cells to cell death. Before addressing those exciting classes of targeted therapies, however, an overview is provided to discuss cell death induced by what is arguably still the most successful set of drugs in the history of medical oncology, conventional chemotherapy. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy." ©2015 American Association for Cancer Research.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

PubMed Central

Planas, Delphine; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R. M.

2017-01-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation. PMID:28727807

GLI activation by atypical protein kinase C ι/λ regulates the growth of basal cell carcinomas.

PubMed

Atwood, Scott X; Li, Mischa; Lee, Alex; Tang, Jean Y; Oro, Anthony E

2013-02-28

Growth of basal cell carcinomas (BCCs) requires high levels of hedgehog (HH) signalling through the transcription factor GLI. Although inhibitors of membrane protein smoothened (SMO) effectively suppress HH signalling, early tumour resistance illustrates the need for additional downstream targets for therapy. Here we identify atypical protein kinase C ι/λ (aPKC-ι/λ) as a novel GLI regulator in mammals. aPKC-ι/λ and its polarity signalling partners co-localize at the centrosome and form a complex with missing-in-metastasis (MIM), a scaffolding protein that potentiates HH signalling. Genetic or pharmacological loss of aPKC-ι/λ function blocks HH signalling and proliferation of BCC cells. Prkci is a HH target gene that forms a positive feedback loop with GLI and exists at increased levels in BCCs. Genome-wide transcriptional profiling shows that aPKC-ι/λ and SMO control the expression of similar genes in tumour cells. aPKC-ι/λ functions downstream of SMO to phosphorylate and activate GLI1, resulting in maximal DNA binding and transcriptional activation. Activated aPKC-ι/λ is upregulated in SMO-inhibitor-resistant tumours and targeting aPKC-ι/λ suppresses signalling and growth of resistant BCC cell lines. These results demonstrate that aPKC-ι/λ is critical for HH-dependent processes and implicates aPKC-ι/λ as a new, tumour-selective therapeutic target for the treatment of SMO-inhibitor-resistant cancers.

Elevated microRNA-34a in obesity reduces NAD+ levels and SIRT1 activity by directly targeting NAMPT.

PubMed

Choi, Sung-E; Fu, Ting; Seok, Sunmi; Kim, Dong-Hyun; Yu, Eunkyung; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Kemper, Byron; Kemper, Jongsook Kim

2013-12-01

SIRT1 is an NAD(+)-dependent deacetylase that is implicated in prevention of many age-related diseases including metabolic disorders. As SIRT1 deacetylase activity is dependent on NAD(+) levels and the development of compounds that directly activate SIRT1 has been controversial, indirectly activating SIRT1 through enhancing NAD(+) bioavailability has received increasing attention. NAD(+) levels are reduced in obesity and the aged, but the underlying mechanisms remain unclear. We recently showed that hepatic microRNA-34a (miR-34a), which is elevated in obesity, directly targets and decreases SIRT1 expression. Here, we further show that miR-34a reduces NAD(+) levels and SIRT1 activity by targeting NAMPT, the rate-limiting enzyme for NAD(+) biosynthesis. A functional binding site for miR-34a is present in the 3' UTR of NAMPT mRNA. Hepatic overexpression of miR-34a reduced NAMPT/NAD(+) levels, increased acetylation of the SIRT1 target transcriptional regulators, PGC-1α, SREBP-1c, FXR, and NF-κB, and resulted in obesity-mimetic outcomes. The decreased NAMPT/NAD(+) levels were independent of miR-34a effects on SIRT1 levels as they were also observed in SIRT1 liver-specific knockout mice. Further, the miR-34a-mediated decreases were reversed by treatment with the NAD(+) intermediate, nicotinamide mononucleotide. Conversely, antagonism of miR-34a in diet-induced obese mice restored NAMPT/NAD(+) levels and alleviated steatosis, inflammation, and glucose intolerance. Anti-miR-34a-mediated increases in NAD(+) levels were attenuated when NAMPT was downregulated. Our findings reveal a novel function of miR-34a in reducing both SIRT1 expression and activity in obesity. The miR-34a/NAMPT axis presents a potential target for treating obesity- and aging-related diseases involving SIRT1 dysfunction like steatosis and type 2 diabetes. © 2013 the Anatomical Society and John Wiley & Sons Ltd.

Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.

PubMed

McLellan, Lauren; Forder, Cassie; Cranston, Aaron; Harrigan, Jeanine; Jacq, Xavier

2016-01-01

The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors.

Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos.

PubMed

Hunter, C P; Kenyon, C

1996-10-18

The early asymmetric cleavages of Caenorhabditis elegans embryos produce blastomeres with distinct developmental potentials. Here, we show that the caudal-like homeodomain protein PAL-1 is required to specify the somatic identity of one posterior blastomere in the 4 cell embryo. We find that pal-1 activity is sequentially restricted to this blastomere. First, at the 4 cell stage, it is translated only in the two posterior blastomeres. Then, its function is restricted to one of these blastomeres. This second targeting step is dependent on the activities of the posteriorly localized SKN-1 and asymmetrically segregated PIE-1 proteins. We propose that the segregation of PIE-1, combined with the temporal decay of SKN-1, targets pal-1 activity to this posterior lineage, thus coupling the regulation of this conserved posterior patterning gene to asymmetric cell cleavages.

Development of an aptamer-ampicillin conjugate for treating biofilms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lijuan, Cheng; Department of Biochemistry and Molecular Biology, Hunan University of Chinese Medicine, Changsha, Hunan, 410208; Xing, Yan

Biofilm formation involves the development of extracellular matrix and initially depends on adherence and tropism by flagellar movement. With the widespread development of antibiotic resistance and tolerance of biofilms, there is a growing need for novel anti-infective strategies. No currently approved medications specifically target biofilms. Aptamers are single-stranded nucleic acid molecules that may bind to their targets with high affinity and affect the target functions. We developed a bifunctional conjugate by linking an aptamer targeting bacterial flagella with ampicillin. We investigated its influence on biofilm prevention and dissolution by ultraviolet–visible spectrophotometry, inverted microscopy, and atomic force microscopy. This conjugate hadmore » distinctive antibacterial activity. Notably, the conjugate was more active than either component, and thus had a synergistic effect against biofilms.« less

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

PubMed

Parker, Lindsay M; Damanhuri, Hanafi A; Fletcher, Sophie P S; Goodchild, Ann K

2015-04-16

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate. Copyright © 2015 Elsevier B.V. All rights reserved.

Computational Models of Neuronal Biophysics and the Characterization of Potential Neuropharmacological Targets

PubMed Central

Ferrante, Michele; Blackwell, Kim T.; Migliore, Michele; Ascoli, Giorgio A.

2012-01-01

The identification and characterization of potential pharmacological targets in neurology and psychiatry is a fundamental problem at the intersection between medicinal chemistry and the neurosciences. Exciting new techniques in proteomics and genomics have fostered rapid progress, opening numerous questions as to the functional consequences of ligand binding at the systems level. Psycho- and neuro-active drugs typically work in nerve cells by affecting one or more aspects of electrophysiological activity. Thus, an integrated understanding of neuropharmacological agents requires bridging the gap between their molecular mechanisms and the biophysical determinants of neuronal function. Computational neuroscience and bioinformatics can play a major role in this functional connection. Robust quantitative models exist describing all major active membrane properties under endogenous and exogenous chemical control. These include voltage-dependent ionic channels (sodium, potassium, calcium, etc.), synaptic receptor channels (e.g. glutamatergic, GABAergic, cholinergic), and G protein coupled signaling pathways (protein kinases, phosphatases, and other enzymatic cascades). This brief review of neuromolecular medicine from the computational perspective provides compelling examples of how simulations can elucidate, explain, and predict the effect of chemical agonists, antagonists, and modulators in the nervous system. PMID:18855673

The nucleolus as a fundamental regulator of the p53 response and a new target for cancer therapy.

PubMed

Woods, Simone J; Hannan, Katherine M; Pearson, Richard B; Hannan, Ross D

2015-07-01

Recent studies have highlighted the fundamental role that key oncogenes such as MYC, RAS and PI3K occupy in driving RNA Polymerase I transcription in the nucleolus. In addition to maintaining essential levels of protein synthesis, hyperactivated ribosome biogenesis and nucleolar function plays a central role in suppressing p53 activation in response to oncogenic stress. Consequently, disruption of ribosome biogenesis by agents such as the small molecule inhibitor of RNA Polymerase I transcription, CX-5461, has shown unexpected, potent, and selective effects in killing tumour cells via disruption of nucleolar function leading to activation of p53, independent of DNA damage. This review will explore the mechanism of DNA damage-independent activation of p53 via the nucleolar surveillance pathway and how this can be utilised to design novel cancer therapies. Non-genotoxic targeting of nucleolar function may provide a new paradigm for treatment of a broad range of oncogene-driven malignancies with improved therapeutic windows. This article is part of a Special Issue entitled: Translation and Cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Pro-tumorigenic roles of fibroblast activation protein in cancer: back to the basics.

PubMed

Puré, Ellen; Blomberg, Rachel

2018-05-03

Fibroblast activation protein (FAP) is a cell-surface serine protease that acts on various hormones and extracellular matrix components. FAP is highly upregulated in a wide variety of cancers, and is often used as a marker for pro-tumorigenic stroma. It has also been proposed as a molecular target of cancer therapies, and, especially in recent years, a great deal of research has gone into design and testing of diverse FAP-targeted treatments. Yet despite this growing field of research, our knowledge of FAP's basic biology and functional roles in various cancers has lagged behind its use as a tumor-stromal marker. In this review, we summarize and analyze recent advances in understanding the functions of FAP in cancer, most notably its prognostic value in various tumor types, cellular effects on various cell types, and potential as a therapeutic target. We highlight outstanding questions in the field, the answers to which could shape preclinical and clinical studies of FAP.

Dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy

NASA Astrophysics Data System (ADS)

Zhao, Xu; Yang, Cheng-Xiong; Chen, Li-Gong; Yan, Xiu-Ping

2017-05-01

The integrated functions of diagnostics and therapeutics make theranostics great potential for personalized medicine. Stimulus-responsive therapy allows spatial control of therapeutic effect only in the site of interest, and offers promising opportunities for imaging-guided precision therapy. However, the imaging strategies in previous stimulus-responsive therapies are `always on' or irreversible `turn on' modality, resulting in poor signal-to-noise ratios or even `false positive' results. Here we show the design of dual-stimuli-responsive and reversibly activatable nanoprobe for precision tumour-targeting and fluorescence-guided photothermal therapy. We fabricate the nanoprobe from asymmetric cyanine and glycosyl-functionalized gold nanorods (AuNRs) with matrix metalloproteinases (MMPs)-specific peptide as a linker to achieve MMPs/pH synergistic and pH reversible activation. The unique activation and glycosyl targetibility makes the nanoprobe bright only in tumour sites with negligible background, while AuNRs and asymmetric cyanine give synergistic photothermal effect. This work paves the way to designing efficient nanoprobes for precision theranostics.

Under-reactive but easily distracted: An fMRI investigation of attentional capture in autism spectrum disorder.

PubMed

Keehn, Brandon; Nair, Aarti; Lincoln, Alan J; Townsend, Jeanne; Müller, Ralph-Axel

2016-02-01

For individuals with autism spectrum disorder (ASD), salient behaviorally-relevant information often fails to capture attention, while subtle behaviorally-irrelevant details commonly induce a state of distraction. The present study used functional magnetic resonance imaging (fMRI) to investigate the neurocognitive networks underlying attentional capture in sixteen high-functioning children and adolescents with ASD and twenty-one typically developing (TD) individuals. Participants completed a rapid serial visual presentation paradigm designed to investigate activation of attentional networks to behaviorally-relevant targets and contingent attention capture by task-irrelevant distractors. In individuals with ASD, target stimuli failed to trigger bottom-up activation of the ventral attentional network and the cerebellum. Additionally, the ASD group showed no differences in behavior or occipital activation associated with contingent attentional capture. Rather, results suggest that to-be-ignored distractors that shared either task-relevant or irrelevant features captured attention in ASD. Results indicate that individuals with ASD may be under-reactive to behaviorally-relevant stimuli, unable to filter irrelevant information, and that both top-down and bottom-up attention networks function atypically in ASD. Lastly, deficits in target-related processing were associated with autism symptomatology, providing further support for the hypothesis that non-social attentional processes and their neurofunctional underpinnings may play a significant role in the development of sociocommunicative impairments in ASD. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Novel treatment strategies for smooth muscle disorders: Targeting Kv7 potassium channels.

PubMed

Haick, Jennifer M; Byron, Kenneth L

2016-09-01

Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body. Published by Elsevier Inc.

Processing of targets in smooth or apparent motion along the vertical in the human brain: an fMRI study.

PubMed

Maffei, Vincenzo; Macaluso, Emiliano; Indovina, Iole; Orban, Guy; Lacquaniti, Francesco

2010-01-01

Neural substrates for processing constant speed visual motion have been extensively studied. Less is known about the brain activity patterns when the target speed changes continuously, for instance under the influence of gravity. Using functional MRI (fMRI), here we compared brain responses to accelerating/decelerating targets with the responses to constant speed targets. The target could move along the vertical under gravity (1g), under reversed gravity (-1g), or at constant speed (0g). In the first experiment, subjects observed targets moving in smooth motion and responded to a GO signal delivered at a random time after target arrival. As expected, we found that the timing of the motor responses did not depend significantly on the specific motion law. Therefore brain activity in the contrast between different motion laws was not related to motor timing responses. Average BOLD signals were significantly greater for 1g targets than either 0g or -1g targets in a distributed network including bilateral insulae, left lingual gyrus, and brain stem. Moreover, in these regions, the mean activity decreased monotonically from 1g to 0g and to -1g. In the second experiment, subjects intercepted 1g, 0g, and -1g targets either in smooth motion (RM) or in long-range apparent motion (LAM). We found that the sites in the right insula and left lingual gyrus, which were selectively engaged by 1g targets in the first experiment, were also significantly more active during 1g trials than during -1g trials both in RM and LAM. The activity in 0g trials was again intermediate between that in 1g trials and that in -1g trials. Therefore in these regions the global activity modulation with the law of vertical motion appears to hold for both RM and LAM. Instead, a region in the inferior parietal lobule showed a preference for visual gravitational motion only in LAM but not RM.

Supporting Executive Functions during Children's Preliteracy Learning with the Computer

ERIC Educational Resources Information Center

Van de Sande, E.; Segers, E.; Verhoeven, L.

2016-01-01

The present study examined how embedded activities to support executive functions helped children to benefit from a computer intervention that targeted preliteracy skills. Three intervention groups were compared on their preliteracy gains in a randomized controlled trial design: an experimental group that worked with software to stimulate early…

An innovative pre-targeting strategy for tumor cell specific imaging and therapy

NASA Astrophysics Data System (ADS)

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-08-01

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the ``biotin-avidin'' interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments. Electronic supplementary information (ESI) available: Experimental details, peptide structures, molecular weights, and additional data. See DOI: 10.1039/c5nr03862f

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

PubMed Central

Bruntz, Ronald C.; Lindsley, Craig W.

2014-01-01

Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein–coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. PMID:25244928

Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer.

PubMed

Bruntz, Ronald C; Lindsley, Craig W; Brown, H Alex

2014-10-01

Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein-coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Social hierarchy modulates neural responses of empathy for pain

PubMed Central

Feng, Chunliang; Li, Zhihao; Feng, Xue; Wang, Lili; Tian, Tengxiang

2016-01-01

Recent evidence indicates that empathic responses to others’ pain are modulated by various situational and individual factors. However, few studies have examined how empathy and underlying brain functions are modulated by social hierarchies, which permeate human society with an enormous impact on social behavior and cognition. In this study, social hierarchies were established based on incidental skill in a perceptual task in which all participants were mediumly ranked. Afterwards, participants were scanned with functional magnetic resonance imaging while watching inferior-status or superior-status targets receiving painful or non-painful stimulation. The results revealed that painful stimulation applied to inferior-status targets induced higher activations in the anterior insula (AI) and anterior medial cingulate cortex (aMCC), whereas these empathic brain activations were significantly attenuated in response to superior-status targets’ pain. Further, this neural empathic bias to inferior-status targets was accompanied by stronger functional couplings of AI with brain regions important in emotional processing (i.e. thalamus) and cognitive control (i.e. middle frontal gyrus). Our findings indicate that emotional sharing with others’ pain is shaped by relative positions in a social hierarchy such that underlying empathic neural responses are biased toward inferior-status compared with superior-status individuals. PMID:26516169

Evaluation of the biological activity of the molluscicidal fraction of Solanum sisymbriifolium against non target organisms.

PubMed

Bagalwa, Jean-Jacques M; Voutquenne-Nazabadioko, Laurence; Sayagh, Charlotte; Bashwira, Augustin S

2010-10-01

The evaluation of the biocidal activity of the fruit of Solanum sisymbriifolium involving non target organisms such as aquatic insects, fish and snails lead to the isolation of the steroidal alkaloids, solamargine and β-solamarine, from the active fractions. The fractions A3 and C, with biological activity against fish, snail and aquatic insect and larvae, are able to affect the good functioning of ecosystem found on alimentary chain. The fraction B seems to be less toxic to fish and aquatic insect and larvae. The fraction B could thus be used as molluscicide in the future. Copyright © 2010 Elsevier B.V. All rights reserved.

Targeting the RAS oncogene

PubMed Central

Takashima, Asami

2013-01-01

Introduction The Ras proteins (K-Ras, N-Ras, H-Ras) are GTPases that function as molecular switches for a variety of critical cellular activities and their function is tightly and temporally regulated in normal cells. Oncogenic mutations in the RAS genes, which create constitutively-active Ras proteins, can result in uncontrolled proliferation or survival in tumor cells. Areas covered The paper discusses three therapeutic approaches targeting the Ras pathway in cancer: 1) Ras itself, 2) Ras downstream pathways, and 3) synthetic lethality. The most adopted approach is targeting Ras downstream signaling, and specifically the PI3K-AKT-mTOR and Raf-MEK pathways, as they are frequently major oncogenic drivers in cancers with high Ras signaling. Although direct targeting of Ras has not been successful clinically, newer approaches being investigated in preclinical studies, such as RNA interference-based and synthetic lethal approaches, promise great potential for clinical application. Expert opinion The challenges of current and emerging therapeutics include the lack of “tumor specificity” and their limitation to those cancers which are “dependent” upon aberrant Ras signaling for survival. While the newer approaches have the potential to overcome these limitations, they also highlight the importance of robust preclinical studies and bidirectional translational research for successful clinical development of Ras-related targeted therapies. PMID:23360111

"We're Not Just Sitting on the Periphery": A Staff Perspective of Physical Activity in Older Adults with Schizophrenia

ERIC Educational Resources Information Center

Leutwyler, Heather; Hubbard, Erin M.; Jeste, Dilip V.; Vinogradov, Sophia

2013-01-01

Targeted physical activity interventions to improve the poor physical function of older adults with schizophrenia are necessary but currently not available. Given disordered thought processes and institutionalization, it is likely that older adults with schizophrenia have unique barriers and facilitators to physical activity. It is necessary to…

Drug sensitivity profiling identifies potential therapies for lymphoproliferative disorders with overactive JAK/STAT3 signaling

PubMed Central

Kuusanmäki, Heikki; Dufva, Olli; Parri, Elina; van Adrichem, Arjan J.; Rajala, Hanna; Majumder, Muntasir M.; Yadav, Bhagwan; Parsons, Alun; Chan, Wing C.; Wennerberg, Krister; Mustjoki, Satu; Heckman, Caroline A.

2017-01-01

Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3. PMID:29228628

Molecular Targets for Antiepileptic Drug Development

PubMed Central

Meldrum, Brian S.; Rogawski, Michael A.

2007-01-01

Summary This review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the α subunits of voltage-gated Na+ channels and also T-type voltage-gated Ca2+ channels) or influence GABA-mediated inhibition. Recently, α2–δ voltage-gated Ca2+ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na+ channels and GABAA receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca2+ channel subunits and auxiliary proteins, A- or M-type voltage-gated K+ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABAB and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated current Ih; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na+ channels underlying persistent Na+ currents or GABAA receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies. PMID:17199015

Targeting RhoA/Rho kinase and p21-activated kinase signaling to prevent cancer development and progression.

PubMed

Chang, Yu-Wen E; Bean, Ronald R; Jakobi, Rolf

2009-06-01

Elevated RhoA/Rho kinase and p21-activated kinase signaling have been shown to promote cancer development and metastasis and have drawn much attention as potential targets of anti-cancer therapy. Elevated RhoA and Rho kinase activity promote cancer cell invasion and eventually lead to metastasis by disrupting E-cadherin-mediated adherens junctions and degradation of the extracellular matrix. Elevated p21-activated kinase activity promotes invasion by stimulating cell motility but also promotes cancer cell survival and growth. In this review we describe normal functions of RhoA/Rho kinase and p21-activated kinase signaling, mechanisms that lead to constitutive activation of RhoA/Rho kinase and p21-activated kinase pathways, and processes by which constitutive RhoA/Rho kinase and p21-activated kinase activity promote cancer development and progression to more aggressive and metastatic phenotypes. In addition, we summarize relevant patents on RhoA/Rho kinase and p21-activated kinase as targets of anti-cancer therapy and discuss the clinical potential of different approaches to modulate RhoA/Rho kinase and p21-activated kinase signaling.

Interaction of Herbal Compounds with Biological Targets: A Case Study with Berberine

PubMed Central

Chen, Xiao-Wu; Di, Yuan Ming; Zhang, Jian; Zhou, Zhi-Wei; Li, Chun Guang; Zhou, Shu-Feng

2012-01-01

Berberine is one of the main alkaloids found in the Chinese herb Huang lian (Rhizoma Coptidis), which has been reported to have multiple pharmacological activities. This study aimed to analyze the molecular targets of berberine based on literature data followed by a pathway analysis using the PANTHER program. PANTHER analysis of berberine targets showed that the most classes of molecular functions include receptor binding, kinase activity, protein binding, transcription activity, DNA binding, and kinase regulator activity. Based on the biological process classification of in vitro berberine targets, those targets related to signal transduction, intracellular signalling cascade, cell surface receptor-linked signal transduction, cell motion, cell cycle control, immunity system process, and protein metabolic process are most frequently involved. In addition, berberine was found to interact with a mixture of biological pathways, such as Alzheimer's disease-presenilin and -secretase pathways, angiogenesis, apoptosis signalling pathway, FAS signalling pathway, Hungtington disease, inflammation mediated by chemokine and cytokine signalling pathways, interleukin signalling pathway, and p53 pathways. We also explored the possible mechanism of action for the anti-diabetic effect of berberine. Further studies are warranted to elucidate the mechanisms of action of berberine using systems biology approach. PMID:23213296

Role of co-regulators in metabolic and transcriptional actions of thyroid hormone.

PubMed

Astapova, Inna

2016-04-01

Thyroid hormone (TH) controls a wide range of physiological processes through TH receptor (TR) isoforms. Classically, TRs are proposed to function as tri-iodothyronine (T3)-dependent transcription factors: on positively regulated target genes, unliganded TRs mediate transcriptional repression through recruitment of co-repressor complexes, while T3 binding leads to dismissal of co-repressors and recruitment of co-activators to activate transcription. Co-repressors and co-activators were proposed to play opposite roles in the regulation of negative T3 target genes and hypothalamic-pituitary-thyroid axis, but exact mechanisms of the negative regulation by TH have remained elusive. Important insights into the roles of co-repressors and co-activators in different physiological processes have been obtained using animal models with disrupted co-regulator function. At the same time, recent studies interrogating genome-wide TR binding have generated compelling new data regarding effects of T3, local chromatin structure, and specific response element configuration on TR recruitment and function leading to the proposal of new models of transcriptional regulation by TRs. This review discusses data obtained in various mouse models with manipulated function of nuclear receptor co-repressor (NCoR or NCOR1) and silencing mediator of retinoic acid receptor and thyroid hormone receptor (SMRT or NCOR2), and family of steroid receptor co-activators (SRCs also known as NCOAs) in the context of TH action, as well as insights into the function of co-regulators that may emerge from the genome-wide TR recruitment analysis. © 2016 Society for Endocrinology.

Modulation of Astrocyte Activity by Cannabidiol, a Nonpsychoactive Cannabinoid

PubMed Central

Kozela, Ewa; Juknat, Ana; Vogel, Zvi

2017-01-01

The astrocytes have gained in recent decades an enormous interest as a potential target for neurotherapies, due to their essential and pleiotropic roles in brain physiology and pathology. Their precise regulation is still far from understood, although several candidate molecules/systems arise as promising targets for astrocyte-mediated neuroregulation and/or neuroprotection. The cannabinoid system and its ligands have been shown to interact and affect activities of astrocytes. Cannabidiol (CBD) is the main non-psychotomimetic cannabinoid derived from Cannabis. CBD is devoid of direct CB1 and CB2 receptor activity, but exerts a number of important effects in the brain. Here, we attempt to sum up the current findings on the effects of CBD on astrocyte activity, and in this way on central nervous system (CNS) functions, across various tested models and neuropathologies. The collected data shows that increased astrocyte activity is suppressed in the presence of CBD in models of ischemia, Alzheimer-like and Multiple-Sclerosis-like neurodegenerations, sciatic nerve injury, epilepsy, and schizophrenia. Moreover, CBD has been shown to decrease proinflammatory functions and signaling in astrocytes. PMID:28788104

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure–Activity Relationship and Target Identification Studies

PubMed Central

2017-01-01

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell–wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure–activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties. PMID:29148755

Modulation of Astrocyte Activity by Cannabidiol, a Nonpsychoactive Cannabinoid.

PubMed

Kozela, Ewa; Juknat, Ana; Vogel, Zvi

2017-07-31

The astrocytes have gained in recent decades an enormous interest as a potential target for neurotherapies, due to their essential and pleiotropic roles in brain physiology and pathology. Their precise regulation is still far from understood, although several candidate molecules/systems arise as promising targets for astrocyte-mediated neuroregulation and/or neuroprotection. The cannabinoid system and its ligands have been shown to interact and affect activities of astrocytes. Cannabidiol (CBD) is the main non-psychotomimetic cannabinoid derived from Cannabis . CBD is devoid of direct CB1 and CB2 receptor activity, but exerts a number of important effects in the brain. Here, we attempt to sum up the current findings on the effects of CBD on astrocyte activity, and in this way on central nervous system (CNS) functions, across various tested models and neuropathologies. The collected data shows that increased astrocyte activity is suppressed in the presence of CBD in models of ischemia, Alzheimer-like and Multiple-Sclerosis-like neurodegenerations, sciatic nerve injury, epilepsy, and schizophrenia. Moreover, CBD has been shown to decrease proinflammatory functions and signaling in astrocytes.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

PubMed

Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

2015-10-26

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18*

PubMed Central

Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

2017-01-01

Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. PMID:28087594

Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

PubMed

Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

PubMed Central

Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

Brain activation in parietal area during manipulation with a surgical robot simulator.

PubMed

Miura, Satoshi; Kobayashi, Yo; Kawamura, Kazuya; Nakashima, Yasutaka; Fujie, Masakatsu G

2015-06-01

we present an evaluation method to qualify the embodiment caused by the physical difference between master-slave surgical robots by measuring the activation of the intraparietal sulcus in the user's brain activity during surgical robot manipulation. We show the change of embodiment based on the change of the optical axis-to-target view angle in the surgical simulator to change the manipulator's appearance in the monitor in terms of hand-eye coordination. The objective is to explore the change of brain activation according to the change of the optical axis-to-target view angle. In the experiments, we used a functional near-infrared spectroscopic topography (f-NIRS) brain imaging device to measure the brain activity of the seven subjects while they moved the hand controller to insert a curved needle into a target using the manipulator in a surgical simulator. The experiment was carried out several times with a variety of optical axis-to-target view angles. Some participants showed a significant peak (P value = 0.037, F-number = 2.841) when the optical axis-to-target view angle was 75°. The positional relationship between the manipulators and endoscope at 75° would be the closest to the human physical relationship between the hands and eyes.

Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

PubMed

Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

2010-11-01

Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

Role of protein kinase C in the TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells

PubMed Central

Abraha, Abraham B.; Rana, Krupa; Whalen, Margaret M.

2010-01-01

Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposures of NK cells to tributyltin (TBT) greatly diminish their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in the NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. The TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in the inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposures. TBT caused a 2–3 fold activation of PKC at concentrations ranging from 50–300 nM (16–98 ng/mL), indicating that activation of PKC occurs in response to TBT exposures. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that in NK cells where PKC activation was blocked there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including the activation of p44/42 by TBT in NK cells. PMID:20390410

Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity.

PubMed

Ding, Baoyue; Wu, Xin; Fan, Wei; Wu, Zhaoyong; Gao, Jing; Zhang, Wei; Ma, Lulu; Xiang, Wang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2011-01-01

The increased incidence of malignant melanoma in recent decades, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Novel therapeutic strategies, such as immunotherapy and targeted therapy, are urgently needed for melanoma. In this study, a new active targeting drug delivery system was constructed to combine chemotherapy and active specific immunotherapy. The chemotherapeutic drug, dacarbazine (DTIC), that induces apoptosis through the intrinsic pathway which typically responds to severe DNA damage, was used as a model drug to prepare DTIC-loaded polylactic acid (PLA) nanoparticles (DTIC-NPs), which were covalently conjugated to a highly specific targeting functional TRAIL-receptor 2 (DR5) monoclonal antibody (mAb) that can contribute directly to cancer cell apoptosis or growth inhibition through the extrinsic pathway. Our in vitro experiments demonstrated that DTIC-PLA-DR5 mAb nanoparticles (DTIC-NPs-DR5 mAb) are an active targeting drug delivery system which can specifically target DR5-overexpressing malignant melanoma cells and become efficiently internalized. Most strikingly, compared with conventional DTIC-NPs, DTIC-NPs-DR5 mAb showed significantly enhanced cytotoxicity and increased cell apoptosis in DR5-positive malignant melanoma cells. The DTIC-NPs-DR5 mAb described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to DR5-overexpressing metastatic melanoma.

More complete gene silencing by fewer siRNAs: transparent optimized design and biophysical signature

PubMed Central

Ladunga, Istvan

2007-01-01

Highly accurate knockdown functional analyses based on RNA interference (RNAi) require the possible most complete hydrolysis of the targeted mRNA while avoiding the degradation of untargeted genes (off-target effects). This in turn requires significant improvements to target selection for two reasons. First, the average silencing activity of randomly selected siRNAs is as low as 62%. Second, applying more than five different siRNAs may lead to saturation of the RNA-induced silencing complex (RISC) and to the degradation of untargeted genes. Therefore, selecting a small number of highly active siRNAs is critical for maximizing knockdown and minimizing off-target effects. To satisfy these needs, a publicly available and transparent machine learning tool is presented that ranks all possible siRNAs for each targeted gene. Support vector machines (SVMs) with polynomial kernels and constrained optimization models select and utilize the most predictive effective combinations from 572 sequence, thermodynamic, accessibility and self-hairpin features over 2200 published siRNAs. This tool reaches an accuracy of 92.3% in cross-validation experiments. We fully present the underlying biophysical signature that involves free energy, accessibility and dinucleotide characteristics. We show that while complete silencing is possible at certain structured target sites, accessibility information improves the prediction of the 90% active siRNA target sites. Fast siRNA activity predictions can be performed on our web server at . PMID:17169992

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness

PubMed Central

Brimer, Nicole

2017-01-01

Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A), by binding to an LXXLL peptide (ELTLQELLGEE) displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS) on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs) and cetacean (porpoises and dolphins) hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution. PMID:29281732

Physical Activity, Functional Ability, and Obesity in Older Adults: A Gender Difference.

PubMed

Gretebeck, Kimberlee A; Sabatini, LeAnn M; Black, David R; Gretebeck, Randall J

2017-09-01

Disability, institutionalization, and loss of independence may be directly caused or exacerbated by physical inactivity and obesity. The purpose of the current cross-sectional survey was to explore the impact of gender and obesity on functional ability tasks, physical activity, and psychosocial factors in older adults. Participants comprised 964 University retirees (55% female, mean age = 75.3 years, SD = 6.7 years) with a mean body mass index (BMI) of 26.1 kg/m 2 (SD = 4.7 kg/m 2 ). Results revealed significant gender and BMI interaction effects. Women were less active than men and obese women were most functionally impaired, particularly in activities that target lower extremity function, regardless of weight status. These findings suggest that physical activity interventions for older adults should focus on exercises that improve functional ability and are tailored to meet individual needs while considering weight and gender. Type, intensity, frequency, and duration of exercises should be individualized to limit injuries and improve functional ability and physical activity adherence. [Journal of Gerontological Nursing, 43(9), 38-46.]. Copyright 2017, SLACK Incorporated.

Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation.

PubMed

Bellissimo, Teresa; Masciarelli, Silvia; Poser, Elena; Genovese, Ilaria; Del Rio, Alberto; Colotti, Gianni; Fazi, Francesco

2017-01-01

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain.

PubMed

Heier, Christoph; Kien, Benedikt; Huang, Feifei; Eichmann, Thomas O; Xie, Hao; Zechner, Rudolf; Chang, Ping-An

2017-11-17

Mammalian patatin-like phospholipase domain-containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure-function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

PubMed Central

Thakur, Shweta; Sarkar, Bibekananda; Cholia, Ravi P; Gautam, Nandini; Dhiman, Monisha; Mantha, Anil K

2014-01-01

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1α, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a ‘hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed. PMID:25033834

Mitochondrial function as a therapeutic target in heart failure

PubMed Central

Brown, David A.; Perry, Justin B.; Allen, Mitchell E.; Sabbah, Hani N.; Stauffer, Brian L.; Shaikh, Saame Raza; Cleland, John G. F.; Colucci, Wilson S.; Butler, Javed; Voors, Adriaan A.; Anker, Stefan D.; Pitt, Bertram; Pieske, Burkert; Filippatos, Gerasimos; Greene, Stephen J.; Gheorghiade, Mihai

2017-01-01

Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria. PMID:28004807

Reducing RBM20 activity improves diastolic dysfunction and cardiac atrophy.

PubMed

Hinze, Florian; Dieterich, Christoph; Radke, Michael H; Granzier, Henk; Gotthardt, Michael

2016-12-01

Impaired diastolic filling is a main contributor to heart failure with preserved ejection fraction (HFpEF), a syndrome with increasing prevalence and no treatment. Both collagen and the giant sarcomeric protein titin determine diastolic function. Since titin's elastic properties can be adjusted physiologically, we evaluated titin-based stiffness as a therapeutic target. We adjusted RBM20-dependent cardiac isoform expression in the titin N2B knockout mouse with increased ventricular stiffness. A ~50 % reduction of RBM20 activity does not only maintain cardiac filling in diastole but also ameliorates cardiac atrophy and thus improves cardiac function in the N2B-deficient heart. Reduced RBM20 activity partially normalized gene expression related to muscle development and fatty acid metabolism. The adaptation of cardiac growth was related to hypertrophy signaling via four-and-a-half lim-domain proteins (FHLs) that translate mechanical input into hypertrophy signals. We provide a novel link between cardiac isoform expression and trophic signaling via FHLs and suggest cardiac splicing as a therapeutic target in diastolic dysfunction. Increasing the length of titin isoforms improves ventricular filling in heart disease. FHL proteins are regulated via RBM20 and adapt cardiac growth. RBM20 is a therapeutic target in diastolic dysfunction.

Understanding the origin of non-immune cell-mediated weakness in the idiopathic inflammatory myopathies - potential role of ER stress pathways.

PubMed

Lightfoot, Adam P; Nagaraju, Kanneboyina; McArdle, Anne; Cooper, Robert G

2015-11-01

Discussion of endoplasmic reticulum (ER) stress pathway activation in idiopathic inflammatory myopathies (IIM), and downstream mechanisms causative of muscle weakness. In IIM, ER stress is an important pathogenic process, but how it causes muscle dysfunction is unknown. We discuss relevant pathways modified in response to ER stress in IIM: reactive oxygen species (ROS) generation and mitochondrial dysfunction, and muscle cytokine (myokine) generation. First, ER stress pathway activation can induce changes in mitochondrial bioenergetics and ROS production. ROS can oxidize cellular components, causing muscle contractile dysfunction and energy deficits. Novel compounds targeting ROS generation and/or mitochondrial dysfunction can improve muscle function in several myopathologies. Second, recent research has demonstrated that skeletal muscle produces multiple myokines. It is suggested that these play a role in causing muscle weakness. Myokines are capable of immune cell recruitment, thus contributing to perturbed muscle function. A characterization of myokines in IIM would clarify their pathogenic role, and so identify new therapeutic targets. ER stress pathway activation is clearly of etiological relevance in IIM. Research to better understand mechanisms of weakness downstream of ER stress is now required, and which may discover new therapeutic targets for nonimmune cell-mediated weakness.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity

PubMed Central

2016-01-01

SUMMARY Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. PMID:27784797

Surface engineered magnetic nanoparticles for specific immunotargeting of cadherin expressing cells

NASA Astrophysics Data System (ADS)

Moros, Maria; Delhaes, Flavien; Puertas, Sara; Saez, Berta; de la Fuente, Jesús M.; Grazú, Valeria; Feracci, Helene

2016-02-01

In spite of historic advances in cancer biology and recent development of sophisticated chemotherapeutics, the outlook for patients with advanced cancer is still grim. In this sense nanoparticles (NPs), through their unique physical properties, enable the development of new approaches for cancer diagnosis and treatment. Thus far the most used active targeting scheme involves NPs functionalization with antibodies specific to molecules overexpressed on cancer cell’s surface. Therefore, such active targeting relies on differences in NPs uptake kinetics rates between tumor and healthy cells. Many cancers of epithelial origin are associated with the inappropriate expression of non-epithelial cadherins (e.g. N-, P-, -11) with concomitant loss of E-cadherin. Such phenomenon named cadherin switching favors tumor development and metastasis via interactions of tumor cells with stromal components. That is why we optimized the oriented functionalization of fluorescently labelled magnetic NPs with a novel antibody specific for the extracellular domain of cadherin-11. The obtained Ab-NPs exhibited high specificity when incubated with two cell lines used as models of tumor and healthy cells. Thus, cadherin switching offers a great opportunity for the development of active targeting strategies aimed to improve the early detection and treatment of cancer.

LIN-12/Notch signaling instructs postsynaptic muscle arm development by regulating UNC-40/DCC and MADD-2 in Caenorhabditis elegans

PubMed Central

Li, Pengpeng; Collins, Kevin M; Koelle, Michael R; Shen, Kang

2013-01-01

The diverse cell types and the precise synaptic connectivity between them are the cardinal features of the nervous system. Little is known about how cell fate diversification is linked to synaptic target choices. Here we investigate how presynaptic neurons select one type of muscles, vm2, as a synaptic target and form synapses on its dendritic spine-like muscle arms. We found that the Notch-Delta pathway was required to distinguish target from non-target muscles. APX-1/Delta acts in surrounding cells including the non-target vm1 to activate LIN-12/Notch in the target vm2. LIN-12 functions cell-autonomously to up-regulate the expression of UNC-40/DCC and MADD-2 in vm2, which in turn function together to promote muscle arm formation and guidance. Ectopic expression of UNC-40/DCC in non-target vm1 muscle is sufficient to induce muscle arm extension from these cells. Therefore, the LIN-12/Notch signaling specifies target selection by selectively up-regulating guidance molecules and forming muscle arms in target cells. DOI: http://dx.doi.org/10.7554/eLife.00378.001 PMID:23539368

Pharmacological enhancement of calcium-activated potassium channel function reduces the effects of repeated stress on fear memory

PubMed Central

Atchley, Derek; Hankosky, Emily R.; Gasparotto, Kaylyn; Rosenkranz, J. Amiel

2012-01-01

Repeated stress impacts emotion, and can induce mood and anxiety disorders. These disorders are characterized by imbalance of emotional responses. The amygdala is fundamental in expression of emotion, and is hyperactive in many patients with mood or anxiety disorders. Stress also leads to hyperactivity of the amygdala in humans. In rodent studies, repeated stress causes hyperactivity of the amygdala, and increases fear conditioning behavior that is mediated by the basolateral amygdala (BLA). Calcium-activated potassium (KCa) channels regulate BLA neuronal activity, and evidence suggests reduced small conductance KCa (SK) channel function in male rats exposed to repeated stress. Pharmacological enhancement of SK channels reverses the BLA neuronal hyperexcitability caused by repeated stress. However, it is not known if pharmacological targeting of SK channels can repair the effects of repeated stress on amygdala-dependent behaviors. The purpose of this study was to test whether enhancement of SK channel function reverses the effects of repeated restraint on BLA-dependent auditory fear conditioning. We found that repeated restraint stress increased the expression of cued conditioned fear in male rats. However, 1-EBIO (1 or 10 mg/kg) or CyPPA (5 mg/kg) administered 30 minutes prior to testing of fear expression brought conditioned freezing to control levels, with little impact on fear expression in control handled rats. These results demonstrate that enhancement of SK channel function can reduce the abnormalities of BLA-dependent fear memory caused by repeated stress. Furthermore, this indicates that pharmacological targeting of SK channels may provide a novel target for alleviation of psychiatric symptoms associated with amygdala hyperactivity. PMID:22487247

The contributions of cerebro-cerebellar circuitry to executive verbal working memory.

PubMed

Marvel, Cherie L; Desmond, John E

2010-01-01

Contributions of cerebro-cerebellar function to executive verbal working memory were examined using event-related functional magnetic resonance imaging (fMRI) while 16 subjects completed two versions of the Sternberg task. In both versions subjects were presented with two or six target letters during the encoding phase, which were held in memory during the maintenance phase. A single probe letter was presented during the retrieval phase. In the "match condition", subjects decided whether the probe matched the target letters. In the "executive condition", subjects created a new probe by counting two alphabetical letters forward (e.g., f-->h) and decided whether the new probe matched the target letters. Neural activity during the match and executive conditions was compared during each phase of the task. There were four main findings. First, cerebro-cerebellar activity increased as a function of executive load. Second, the dorsal cerebellar dentate co-activated with the supplementary motor area (SMA) during encoding. This likely represented the formation of an articulatory (motor) trajectory. Third, the ventral cerebellar dentate co-activated with anterior prefrontal regions Brodmann Area (BA) 9/46 and the pre-SMA during retrieval. This likely represented the manipulation of information and formation of a response. A functional dissociation between the dorsal "motor" dentate and "cognitive" ventral dentate agrees with neuroanatomical tract tracing studies that have demonstrated separate neural pathways involving each region of the dentate: the dorsal dentate projects to frontal motor areas (including the SMA), and the ventral dentate projects to frontal cognitive areas (including BA 9/46 and the pre-SMA). Finally, activity during the maintenance phase in BA 9, anterior insula, pre-SMA and ventral dentate predicted subsequent accuracy of response to the probe during the retrieval phase. This finding underscored the significant contribution of the pre-SMA/ventral dentate pathway--observed several seconds prior to any motor response to the probe--to executive verbal working memory. Copyright (c) 2009 Elsevier Srl. All rights reserved.

The Contributions of Cerebro-Cerebellar Circuitry to Executive Verbal Working Memory

PubMed Central

Marvel, Cherie L.; Desmond, John E.

2009-01-01

Contributions of cerebro-cerebellar function to executive verbal working memory were examined using event-related functional magnetic resonance imaging (fMRI) while 16 subjects completed two versions of the Sternberg task. In both versions subjects were presented with two or six target letters during the encoding phase, which were held in memory during the maintenance phase. A single probe letter was presented during the retrieval phase. In the “match condition”, subjects decided whether the probe matched the target letters. In the “executive condition”, subjects created a new probe by counting two alphabetical letters forward (e.g., f → h) and decided whether the new probe matched the target letters. Neural activity during the match and executive conditions was compared during each phase of the task. There were four main findings. First, cerebro-cerebellar activity increased as a function of executive load. Second, the dorsal cerebellar dentate co-activated with the supplementary motor area (SMA) during encoding. This likely represented the formation of an articulatory (motor) trajectory. Third, the ventral cerebellar dentate co-activated with anterior prefrontal regions BA 9/46 and the pre-SMA during retrieval. This likely represented the manipulation of information and formation of a response. A functional dissociation between the dorsal “motor” dentate and “cognitive” ventral dentate agrees with neuroanatomical tract tracing studies that have demonstrated separate neural pathways involving each region of the dentate: the dorsal dentate projects to frontal motor areas (including the SMA), and the ventral dentate projects to frontal cognitive areas (including BA 9/46 and the pre-SMA). Finally, activity during the maintenance phase in BA 9, anterior insula, pre-SMA and ventral dentate predicted subsequent accuracy of response to the probe during the retrieval phase. This finding underscored the significant contribution of the pre-SMA/ventral dentate pathway – observed several seconds prior to any motor response to the probe -- to executive verbal working memory. PMID:19811779

Connexin-Dependent Neuroglial Networking as a New Therapeutic Target.

PubMed

Charvériat, Mathieu; Naus, Christian C; Leybaert, Luc; Sáez, Juan C; Giaume, Christian

2017-01-01

Astrocytes and neurons dynamically interact during physiological processes, and it is now widely accepted that they are both organized in plastic and tightly regulated networks. Astrocytes are connected through connexin-based gap junction channels, with brain region specificities, and those networks modulate neuronal activities, such as those involved in sleep-wake cycle, cognitive, or sensory functions. Additionally, astrocyte domains have been involved in neurogenesis and neuronal differentiation during development; they participate in the "tripartite synapse" with both pre-synaptic and post-synaptic neurons by tuning down or up neuronal activities through the control of neuronal synaptic strength. Connexin-based hemichannels are also involved in those regulations of neuronal activities, however, this feature will not be considered in the present review. Furthermore, neuronal processes, transmitting electrical signals to chemical synapses, stringently control astroglial connexin expression, and channel functions. Long-range energy trafficking toward neurons through connexin-coupled astrocytes and plasticity of those networks are hence largely dependent on neuronal activity. Such reciprocal interactions between neurons and astrocyte networks involve neurotransmitters, cytokines, endogenous lipids, and peptides released by neurons but also other brain cell types, including microglial and endothelial cells. Over the past 10 years, knowledge about neuroglial interactions has widened and now includes effects of CNS-targeting drugs such as antidepressants, antipsychotics, psychostimulants, or sedatives drugs as potential modulators of connexin function and thus astrocyte networking activity. In physiological situations, neuroglial networking is consequently resulting from a two-way interaction between astrocyte gap junction-mediated networks and those made by neurons. As both cell types are modulated by CNS drugs we postulate that neuroglial networking may emerge as new therapeutic targets in neurological and psychiatric disorders.

p21-activated kinases in cancer.

PubMed

Kumar, Rakesh; Gururaj, Anupama E; Barnes, Christopher J

2006-06-01

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of cancers. p21-activated kinases (Paks) are serine/threonine kinases that function as downstream nodes for various oncogenic signalling pathways. Paks are well-known regulators of cytoskeletal remodelling and cell motility, but have recently also been shown to promote cell proliferation, regulate apoptosis and accelerate mitotic abnormalities, which results in tumour formation and cell invasiveness. Alterations in Pak expression have been detected in human tumours, which makes them an attractive new therapeutic target.

Individual muscle control using an exoskeleton robot for muscle function testing.

PubMed

Ueda, Jun; Ming, Ding; Krishnamoorthy, Vijaya; Shinohara, Minoru; Ogasawara, Tsukasa

2010-08-01

Healthy individuals modulate muscle activation patterns according to their intended movement and external environment. Persons with neurological disorders (e.g., stroke and spinal cord injury), however, have problems in movement control due primarily to their inability to modulate their muscle activation pattern in an appropriate manner. A functionality test at the level of individual muscles that investigates the activity of a muscle of interest on various motor tasks may enable muscle-level force grading. To date there is no extant work that focuses on the application of exoskeleton robots to induce specific muscle activation in a systematic manner. This paper proposes a new method, named "individual muscle-force control" using a wearable robot (an exoskeleton robot, or a power-assisting device) to obtain a wider variety of muscle activity data than standard motor tasks, e.g., pushing a handle by hand. A computational algorithm systematically computes control commands to a wearable robot so that a desired muscle activation pattern for target muscle forces is induced. It also computes an adequate amount and direction of a force that a subject needs to exert against a handle by his/her hand. This individual muscle control method enables users (e.g., therapists) to efficiently conduct neuromuscular function tests on target muscles by arbitrarily inducing muscle activation patterns. This paper presents a basic concept, mathematical formulation, and solution of the individual muscle-force control and its implementation to a muscle control system with an exoskeleton-type robot for upper extremity. Simulation and experimental results in healthy individuals justify the use of an exoskeleton robot for future muscle function testing in terms of the variety of muscle activity data.

New Pharmacotherapy Targeting Cognitive Dysfunction of Schizophrenia via Modulation of GABA Neuronal Function

PubMed Central

Jeon, Won Je; Sumiyoshi, Tomiki; Kurachi, Masayoshi

2015-01-01

Schizophrenia is considered a neurodevelopmental and neurodegenerative disorder. Cognitive impairment is a core symptom in patients with the illness, and has been suggested a major predictor of functional outcomes. Reduction of parvalbumin (PV)-positive γ-aminobutyric acid (GABA) interneurons has been associated with the pathophysiology of schizophrenia, in view of the link between the abnormality of GABA neurons and cognitive impairments of the disease. It is assumed that an imbalance of excitatory and inhibitory (E-I) activity induced by low activity of glutamatergic projections and PV-positive GABA interneurons in the prefrontal cortex resulted in sustained neural firing and gamma oscillation, leading to impaired cognitive function. Therefore, it is important to develop novel pharmacotherapy targeting GABA neurons and their activities. Clinical evidence suggests serotonin (5-HT) 1A receptor agonist improves cognitive disturbances of schizophrenia, consistent with results from preclinical studies, through mechanism that corrects E-I imbalance via the suppression of GABA neural function. On the other hand, T-817MA, a novel neurotrophic agent, ameliorated loss of PV-positive GABA neurons in the medial prefrontal cortex and reduction of gamma-band activity, as well as cognitive dysfunction in animal model of schizophrenia. In conclusion, a pharmacotherapy to alleviate abnormalities in GABA neurons through 5-HT1A agonists and T-817MA is expected to prevent the onset and/or progression of schizophrenia. PMID:26630957

New Pharmacotherapy Targeting Cognitive Dysfunction of Schizophrenia via Modulation of GABA Neuronal Function.

PubMed

Uehara, Takashi; Sumiyoshi, Tomiki; Kurachi, Masayoshi

2015-01-01

Schizophrenia is considered a neurodevelopmental and neurodegenerative disorder. Cognitive impairment is a core symptom in patients with the illness, and has been suggested a major predictor of functional outcomes. Reduction of parvalbumin (PV)-positive γ-aminobutyric acid (GABA) interneurons has been associated with the pathophysiology of schizophrenia, in view of the link between the abnormality of GABA neurons and cognitive impairments of the disease. It is assumed that an imbalance of excitatory and inhibitory (E-I) activity induced by low activity of glutamatergic projections and PV-positive GABA interneurons in the prefrontal cortex resulted in sustained neural firing and gamma oscillation, leading to impaired cognitive function. Therefore, it is important to develop novel pharmacotherapy targeting GABA neurons and their activities. Clinical evidence suggests serotonin (5-HT) 1A receptor agonist improves cognitive disturbances of schizophrenia, consistent with results from preclinical studies, through mechanism that corrects E-I imbalance via the suppression of GABA neural function. On the other hand, T-817MA, a novel neurotrophic agent, ameliorated loss of PV-positive GABA neurons in the medial prefrontal cortex and reduction of gamma-band activity, as well as cognitive dysfunction in animal model of schizophrenia. In conclusion, a pharmacotherapy to alleviate abnormalities in GABA neurons through 5-HT1A agonists and T-817MA is expected to prevent the onset and/or progression of schizophrenia.

New target for inhibition of bacterial RNA polymerase: 'switch region'.

PubMed

Srivastava, Aashish; Talaue, Meliza; Liu, Shuang; Degen, David; Ebright, Richard Y; Sineva, Elena; Chakraborty, Anirban; Druzhinin, Sergey Y; Chatterjee, Sujoy; Mukhopadhyay, Jayanta; Ebright, Yon W; Zozula, Alex; Shen, Juan; Sengupta, Sonali; Niedfeldt, Rui Rong; Xin, Cai; Kaneko, Takushi; Irschik, Herbert; Jansen, Rolf; Donadio, Stefano; Connell, Nancy; Ebright, Richard H

2011-10-01

A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery. Copyright © 2011 Elsevier Ltd. All rights reserved.

A cortical-spinal prosthesis for targeted limb movement in paralyzed primate avatars

PubMed Central

Shanechi, Maryam M.; Hu, Rollin C.; Williams, Ziv M.

2014-01-01

Motor paralysis is among the most disabling aspects of injury to the central nervous system. Here we develop and test a target-based cortical-spinal neural prosthesis that employs neural activity recorded from pre-motor neurons to control limb movements in functionally paralyzed primate avatars. Given the complexity by which muscle contractions are naturally controlled, we approach the problem of eliciting goal-directed limb movement in paralyzed animals by focusing on the intended targets of movement rather than their intermediate trajectories. We then match this information in real-time with spinal cord and muscle stimulation parameters that produce free planar limb movements to those intended target locations. We demonstrate that both the decoded activities of pre-motor populations and their adaptive responses can be used, after brief training, to effectively direct an avatar’s limb to distinct targets variably displayed on a screen. These findings advance the future possibility of reconstituting targeted limb movement in paralyzed subjects. PMID:24549394

Gain-of-Function Alleles in Caenorhabditis elegans Nuclear Hormone Receptor nhr-49 Are Functionally Distinct

PubMed Central

Lee, Kayoung; Goh, Grace Ying Shyen; Wong, Marcus Andrew; Klassen, Tara Leah

2016-01-01

Nuclear hormone receptors (NHRs) are transcription factors that regulate numerous physiological and developmental processes and represent important drug targets. NHR-49, an ortholog of Hepatocyte Nuclear Factor 4 (HNF4), has emerged as a key regulator of lipid metabolism and life span in the nematode worm Caenorhabditis elegans. However, many aspects of NHR-49 function remain poorly understood, including whether and how it regulates individual sets of target genes and whether its activity is modulated by a ligand. A recent study identified three gain-of-function (gof) missense mutations in nhr-49 (nhr-49(et7), nhr-49(et8), and nhr-49(et13), respectively). These substitutions all affect the ligand-binding domain (LBD), which is critical for ligand binding and protein interactions. Thus, these alleles provide an opportunity to test how three specific residues contribute to NHR-49 dependent gene regulation. We used computational and molecular methods to delineate how these mutations alter NHR-49 activity. We find that despite originating from a screen favoring the activation of specific NHR-49 targets, all three gof alleles cause broad upregulation of NHR-49 regulated genes. Interestingly, nhr-49(et7) and nhr-49(et8) exclusively affect nhr-49 dependent activation, whereas the nhr-49(et13) surprisingly affects both nhr-49 mediated activation and repression, implicating the affected residue as dually important. We also observed phenotypic non-equivalence of these alleles, as they unexpectedly caused a long, short, and normal life span, respectively. Mechanistically, the gof substitutions altered neither protein interactions with the repressive partner NHR-66 and the coactivator MDT-15 nor the subcellular localization or expression of NHR-49. However, in silico structural modeling revealed that NHR-49 likely interacts with small molecule ligands and that the missense mutations might alter ligand binding, providing a possible explanation for increased NHR-49 activity. In sum, our findings indicate that the three nhr-49 gof alleles are non-equivalent, and highlight the conserved V411 residue affected by et13 as critical for gene activation and repression alike. PMID:27618178

SIRT3 Is a Mitochondrial Tumor Suppressor and Genetic Loss Results in a Murine Model for ER/PR-Positive Mammary Tumors Connecting Metabolism and Carcinogenesis

DTIC Science & Technology

2013-09-01

deacetylation targets and determine if these targets are regulated by extracellular stimuli known to activate sirtuin function (e.g., resveratrol ...Determine if exposure to resveratrol or overexpression of a MnSOD gene will prevent increases in ROS in MEFs and/or decrease the development of mammary

Neural mechanisms tracking popularity in real-world social networks.

PubMed

Zerubavel, Noam; Bearman, Peter S; Weber, Jochen; Ochsner, Kevin N

2015-12-08

Differences in popularity are a key aspect of status in virtually all human groups and shape social interactions within them. Little is known, however, about how we track and neurally represent others' popularity. We addressed this question in two real-world social networks using sociometric methods to quantify popularity. Each group member (perceiver) viewed faces of every other group member (target) while whole-brain functional MRI data were collected. Independent functional localizer tasks were used to identify brain systems supporting affective valuation (ventromedial prefrontal cortex, ventral striatum, amygdala) and social cognition (dorsomedial prefrontal cortex, precuneus, temporoparietal junction), respectively. During the face-viewing task, activity in both types of neural systems tracked targets' sociometric popularity, even when controlling for potential confounds. The target popularity-social cognition system relationship was mediated by valuation system activity, suggesting that observing popular individuals elicits value signals that facilitate understanding their mental states. The target popularity-valuation system relationship was strongest for popular perceivers, suggesting enhanced sensitivity to differences among other group members' popularity. Popular group members also demonstrated greater interpersonal sensitivity by more accurately predicting how their own personalities were perceived by other individuals in the social network. These data offer insights into the mechanisms by which status guides social behavior.

Small molecules targeting heterotrimeric G proteins.

PubMed

Ayoub, Mohammed Akli

2018-05-05

G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30-40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the "hot" areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Kupffer Cell Metabolism and Function

PubMed Central

Nguyen-Lefebvre, Anh Thu; Horuzsko, Anatolij

2015-01-01

Kupffer cells are resident liver macrophages and play a critical role in maintaining liver functions. Under physiological conditions, they are the first innate immune cells and protect the liver from bacterial infections. Under pathological conditions, they are activated by different components and can differentiate into M1-like (classical) or M2-like (alternative) macrophages. The metabolism of classical or alternative activated Kupffer cells will determine their functions in liver damage. Special functions and metabolism of Kupffer cells suggest that they are an attractive target for therapy of liver inflammation and related diseases, including cancer and infectious diseases. Here we review the different types of Kupffer cells and their metabolism and functions in physiological and pathological conditions. PMID:26937490

PROBLEM OF FORMING IN A MAN-OPERATOR A HABIT OF TRACKING A MOVING TARGET,

DTIC Science & Technology

Cybernetics stimulated the large-scale use of the method of functional analogy which makes it possible to compare technical and human activity systems...interesting and highly efficient human activity because of the psychological control factor involved in its operation. The human tracking system is

Design and Testing of Bi-functional, P-loop Targeted MDM2 Inhibitors

DTIC Science & Technology

2008-03-01

Nucleotide Binding Activity of the Mdm2 RING Domain Christina Priest, Masha Poyurovsky, Brent Stockwell and Carol Prives Department of Biological Sciences...Deconstructing nucleotide binding activity of the Mdm2 RING domain Christina Priest, Carol Prives* and Masha V. Poyurovsky Department of Biological Sciences

Authentic Text Types and Corresponding Activities: A List for the Foreign Language Instructor.

ERIC Educational Resources Information Center

Geltrich-Ludgate, Brigitta; Tovar, Deanna

1987-01-01

The study of authentic texts gives students the opportunity to become functional in target-language situations. An exhaustive alphabetical list of authentic text types and corresponding learning activities are presented. Text types include: advertisements, bank materials, bills and receipts, calendars, captions, comics, commercials, decals, forms,…

Sounds Activate Visual Cortex and Improve Visual Discrimination

PubMed Central

Störmer, Viola S.; Martinez, Antigona; McDonald, John J.; Hillyard, Steven A.

2014-01-01

A recent study in humans (McDonald et al., 2013) found that peripheral, task-irrelevant sounds activated contralateral visual cortex automatically as revealed by an auditory-evoked contralateral occipital positivity (ACOP) recorded from the scalp. The present study investigated the functional significance of this cross-modal activation of visual cortex, in particular whether the sound-evoked ACOP is predictive of improved perceptual processing of a subsequent visual target. A trial-by-trial analysis showed that the ACOP amplitude was markedly larger preceding correct than incorrect pattern discriminations of visual targets that were colocalized with the preceding sound. Dipole modeling of the scalp topography of the ACOP localized its neural generators to the ventrolateral extrastriate visual cortex. These results provide direct evidence that the cross-modal activation of contralateral visual cortex by a spatially nonpredictive but salient sound facilitates the discriminative processing of a subsequent visual target event at the location of the sound. Recordings of event-related potentials to the targets support the hypothesis that the ACOP is a neural consequence of the automatic orienting of visual attention to the location of the sound. PMID:25031419

Synthesis of galactosyl compounds for targeted gene delivery.

PubMed

Ren, T; Zhang, G; Liu, D

2001-11-01

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

PubMed

Safavi-Sohi, Reihaneh; Maghari, Shokoofeh; Raoufi, Mohammad; Jalali, Seyed Amir; Hajipour, Mohammad J; Ghassempour, Alireza; Mahmoudi, Morteza

2016-09-07

Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications.

Search for α-Cluster Structure in Exotic Nuclei with the Prototype Active-Target Time-Projection Chamber

NASA Astrophysics Data System (ADS)

Fritsch, A.; Ayyad, Y.; Bazin, D.; Beceiro-Novo, S.; Bradt, J.; Carpenter, L.; Cortesi, M.; Mittig, W.; Suzuki, D.; Ahn, T.; Kolata, J. J.; Becchetti, F. D.; Howard, A. M.

2016-03-01

Some exotic nuclei appear to exhibit α-cluster structure. While various theoretical models currently describe such clustering, more experimental data are needed to constrain model predictions. The Prototype Active-Target Time-Projection Chamber (PAT-TPC) has low-energy thresholds for charged-particle decay and a high luminosity due to its thick gaseous active target volume, making it well-suited to search for low-energy α-cluster reactions. Radioactive-ion beams produced by the TwinSol facility at the University of Notre Dame were delivered to the PAT-TPC to study nuclei including 14C and 14O via α-resonant scattering. Differential cross sections and excitation functions were measured. Preliminary results from our recent experiments will be presented. This work is supported by the U.S. National Science Foundation.

Tyrosine kinase inhibitors and mesenchymal stromal cells: effects on self-renewal, commitment and functions

PubMed Central

Borriello, Adriana; Caldarelli, Ilaria; Bencivenga, Debora; Stampone, Emanuela; Perrotta, Silverio; Oliva, Adriana; Ragione, Fulvio Della

2017-01-01

The hope of selectively targeting cancer cells by therapy and eradicating definitively malignancies is based on the identification of pathways or metabolisms that clearly distinguish “normal” from “transformed” phenotypes. Some tyrosine kinase activities, specifically unregulated and potently activated in malignant cells, might represent important targets of therapy. Consequently, tyrosine kinase inhibitors (TKIs) might be thought as the “vanguard” of molecularly targeted therapy for human neoplasias. Imatinib and the successive generations of inhibitors of Bcr-Abl1 kinase, represent the major successful examples of TKI use in cancer treatment. Other tyrosine kinases have been selected as targets of therapy, but the efficacy of their inhibition, although evident, is less definite. Two major negative effects exist in this therapeutic strategy and are linked to the specificity of the drugs and to the role of the targeted kinase in non-malignant cells. In this review, we will discuss the data available on the TKIs effects on the metabolism and functions of mesenchymal stromal cells (MSCs). MSCs are widely distributed in human tissues and play key physiological roles; nevertheless, they might be responsible for important pathologies. At present, bone marrow (BM) MSCs have been studied in greater detail, for both embryological origins and functions. The available data are evocative of an unexpected degree of complexity and heterogeneity of BM-MSCs. It is conceivable that this grade of intricacy occurs also in MSCs of other organs. Therefore, in perspective, the negative effects of TKIs on MSCs might represent a critical problem in long-term cancer therapies based on such inhibitors. PMID:27750212

Differential Effects of HNF-1α Mutations Associated with Familial Young-Onset Diabetes on Target Gene Regulation

PubMed Central

Galán, Maria; García-Herrero, Carmen-Maria; Azriel, Sharona; Gargallo, Manuel; Durán, Maria; Gorgojo, Juan-Jose; Andía, Victor-Manuel; Navas, Maria-Angeles

2011-01-01

Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype. PMID:21170474

Dorsal premotor cortex: neural correlates of reach target decisions based on a color-location matching rule and conflicting sensory evidence

PubMed Central

Coallier, Émilie; Michelet, Thomas

2015-01-01

We recorded single-neuron activity in dorsal premotor (PMd) and primary motor cortex (M1) of two monkeys in a reach-target selection task. The monkeys chose between two color-coded potential targets by determining which target's color matched the predominant color of a multicolored checkerboard-like Decision Cue (DC). Different DCs contained differing numbers of colored squares matching each target. The DCs provided evidence about the correct target ranging from unambiguous (one color only) to very ambiguous and conflicting (nearly equal number of squares of each color). Differences in choice behavior (reach response times and success rates as a function of DC ambiguity) of the monkeys suggested that each applied a different strategy for using the target-choice evidence in the DCs. Nevertheless, the appearance of the DCs evoked a transient coactivation of PMd neurons preferring both potential targets in both monkeys. Reach response time depended both on how long it took activity to increase in neurons that preferred the chosen target and on how long it took to suppress the activity of neurons that preferred the rejected target, in both correct-choice and error-choice trials. These results indicate that PMd neurons in this task are not activated exclusively by a signal proportional to the net color bias of the DCs. They are instead initially modulated by the conflicting evidence supporting both response choices; final target selection may result from a competition between representations of the alternative choices. The results also indicate a temporal overlap between action selection and action initiation processes in PMd and M1. PMID:25787952

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

PubMed Central

Drube, Sebastian; Beyer, Mandy; Rothe, Mandy; Rabenhorst, Anja; Göpfert, Christiane; Meininger, Isabel; Diamanti, Michaela A.; Stegner, David; Häfner, Norman; Böttcher, Martin; Reinecke, Kirstin; Herdegen, Thomas; Greten, Florian R.; Nieswandt, Bernhard; Hartmann, Karin; Krämer, Oliver H.; Kamradt, Thomas

2015-01-01

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca2+-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term “subthreshold IKK activation”. This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33. We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo. Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure. PMID:25749030

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy.

PubMed

Yang, Chul-Su; Kim, Jwa-Jin; Lee, Hye-Mi; Jin, Hyo Sun; Lee, Sang-Hee; Park, Ji-Hoon; Kim, Soung Jung; Kim, Jin-Man; Han, Yong-Mahn; Lee, Myung-Shik; Kweon, Gi Ryang; Shong, Minho; Jo, Eun-Kyeong

2014-05-01

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), β (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism.

Calculation and application of activity discriminants in lead optimization.

PubMed

Luo, Xincai; Krumrine, Jennifer R; Shenvi, Ashok B; Pierson, M Edward; Bernstein, Peter R

2010-11-01

We present a technique for computing activity discriminants of in vitro (pharmacological, DMPK, and safety) assays and the application to the prediction of in vitro activities of proposed synthetic targets during the lead optimization phase of drug discovery projects. This technique emulates how medicinal chemists perform SAR analysis and activity prediction. The activity discriminants that are functions of 6 commonly used medicinal chemistry descriptors can be interpreted easily by medicinal chemists. Further, visualization with Spotfire allows medicinal chemists to analyze how the query molecule is related to compounds tested previously, and to evaluate easily the relevance of the activity discriminants to the activities of the query molecule. Validation with all compounds synthesized and tested in AstraZeneca Wilmington since 2006 demonstrates that this approach is useful for prioritizing new synthetic targets for synthesis. Copyright © 2010 Elsevier Inc. All rights reserved.

Functional modular architecture underlying attentional control in aging.

PubMed

Monge, Zachary A; Geib, Benjamin R; Siciliano, Rachel E; Packard, Lauren E; Tallman, Catherine W; Madden, David J

2017-07-15

Previous research suggests that age-related differences in attention reflect the interaction of top-down and bottom-up processes, but the cognitive and neural mechanisms underlying this interaction remain an active area of research. Here, within a sample of community-dwelling adults 19-78 years of age, we used diffusion reaction time (RT) modeling and multivariate functional connectivity to investigate the behavioral components and whole-brain functional networks, respectively, underlying bottom-up and top-down attentional processes during conjunction visual search. During functional MRI scanning, participants completed a conjunction visual search task in which each display contained one item that was larger than the other items (i.e., a size singleton) but was not informative regarding target identity. This design allowed us to examine in the RT components and functional network measures the influence of (a) additional bottom-up guidance when the target served as the size singleton, relative to when the distractor served as the size singleton (i.e., size singleton effect) and (b) top-down processes during target detection (i.e., target detection effect; target present vs. absent trials). We found that the size singleton effect (i.e., increased bottom-up guidance) was associated with RT components related to decision and nondecision processes, but these effects did not vary with age. Also, a modularity analysis revealed that frontoparietal module connectivity was important for both the size singleton and target detection effects, but this module became central to the networks through different mechanisms for each effect. Lastly, participants 42 years of age and older, in service of the target detection effect, relied more on between-frontoparietal module connections. Our results further elucidate mechanisms through which frontoparietal regions support attentional control and how these mechanisms vary in relation to adult age. Copyright © 2017 Elsevier Inc. All rights reserved.

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

PubMed

Church, Jarrod E; Trieu, Jennifer; Chee, Annabel; Naim, Timur; Gehrig, Stefan M; Lamon, Séverine; Angelini, Corrado; Russell, Aaron P; Lynch, Gordon S

2014-04-01

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

Genome-wide Functional Analysis of CREB/Long-Term Memory-Dependent Transcription Reveals Distinct Basal and Memory Gene Expression Programs

PubMed Central

Lakhina, Vanisha; Arey, Rachel N.; Kaletsky, Rachel; Kauffman, Amanda; Stein, Geneva; Keyes, William; Xu, Daniel; Murphy, Coleen T.

2014-01-01

SUMMARY Induced CREB activity is a hallmark of long-term memory, but the full repertoire of CREB transcriptional targets required specifically for memory is not known in any system. To obtain a more complete picture of the mechanisms involved in memory, we combined memory training with genome-wide transcriptional analysis of C. elegans CREB mutants. This approach identified 757 significant CREB/memory-induced targets and confirmed the involvement of known memory genes from other organisms, but also suggested new mechanisms and novel components that may be conserved through mammals. CREB mediates distinct basal and memory transcriptional programs at least partially through spatial restriction of CREB activity: basal targets are regulated primarily in nonneuronal tissues, while memory targets are enriched for neuronal expression, emanating from CREB activity in AIM neurons. This suite of novel memory-associated genes will provide a platform for the discovery of orthologous mammalian long-term memory components. PMID:25611510

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE PAGES

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

2017-09-11

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Reciprocal Regulation of ERα and ERβ Stability and Activity by Diptoindonesin G.

PubMed

Zhao, Zibo; Wang, Lu; James, Taryn; Jung, Youngeun; Kim, Ikyon; Tan, Renxiang; Hoffmann, F Michael; Xu, Wei

2015-12-17

ERβ is regarded as a "tumor suppressor" in breast cancer due to its anti-proliferative effects. However, unlike ERα, ERβ has not been developed as a therapeutic target in breast cancer due to loss of ERβ in aggressive cancers. In a small-molecule library screen for ERβ stabilizers, we identified Diptoindonesin G (Dip G), which significantly increases ERβ protein stability while decreasing ERα protein levels. Dip G enhances the transcription and anti-proliferative activities of ERβ, while attenuating the transcription and proliferative effects of ERα. Further investigation revealed that instead of targeting ER, Dip G targets the CHIP E3 ubiquitin ligase shared by ERα and ERβ. Thus, Dip G is a dual-functional moiety that reciprocally controls ERα and ERβ protein stability and activities via an indirect mechanism. The ERβ stabilization effects of Dip G may enable the development of ERβ-targeted therapies for human breast cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nanotized PPARα Overexpression Targeted to Hypertrophied Myocardium Improves Cardiac Function by Attenuating the p53-GSK3β-Mediated Mitochondrial Death Pathway.

PubMed

Rana, Santanu; Datta, Ritwik; Chaudhuri, Ratul Datta; Chatterjee, Emeli; Chawla-Sarkar, Mamta; Sarkar, Sagartirtha

2018-05-09

Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy. Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo. Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium. PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac function, thereby, opening up a potential avenue for cardiac tissue engineering during hypertrophic cardiac pathophysiology.

Mediator kinase module and human tumorigenesis.

PubMed

Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

2015-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

Mediator kinase module and human tumorigenesis

PubMed Central

Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

2016-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Hydrophobic surface functionalization of Philippine natural zeolite for a targeted oil remediation application

NASA Astrophysics Data System (ADS)

Osonio, Airah P.; Olegario-Sanchez, Eleanor M.

2017-12-01

The objective of this study is to modify and compare the oil sorption capacity on the surface of natural zeolite (NZ) and functionalized natural zeolite (FNZ) and to compare with activated charcoal samples. The NZ samples were surface modified via esterification process and characterized using XRD, SEM, and IR spectroscopy. The NZ, FNZ and activated charcoal were then tested using ASTM method F726-12 to validate the oil sorption capacity and TGA was used for the oil selectivity of the adsorbents. The results indicate that FNZ has an improved oil/water adsorption capacity than NZ when functionalized with ester and has a comparable capacity with activated charcoal.

Regulation and function of mTOR signalling in T cell fate decision

PubMed Central

Chi, Hongbo

2012-01-01

The evolutionary conserved kinase mTOR couples cell growth and metabolism to environmental inputs in eukaryotes. T cells depend on mTOR signalling to integrate immune signals and metabolic cues for their proper maintenance and activation. Under steady-state conditions, mTOR is actively controlled by multiple inhibitory mechanisms, and this enforces normal T cell homeostasis. Antigen recognition by naïve CD4+ and CD8+ T cells triggers mTOR activation, which in turn programs their differentiation into functionally distinct lineages. This Review focuses on the signalling mechanisms of mTOR in T cell homeostatic and functional fates and therapeutic implications of targeting mTOR in T cells. PMID:22517423

The SYK tyrosine kinase: a crucial player in diverse biological functions

PubMed Central

Mócsai, Attila; Ruland, Jürgen; Tybulewicz, Victor L. J.

2016-01-01

Spleen tyrosine kinase (SYK) has been known to relay adaptive immune receptor signalling. However, recent reports indicate that SYK also mediates other, unexpectedly diverse biological functions including cellular adhesion, innate immune recognition, osteoclast maturation, platelet activation and vascular development. SYK is activated by C-type lectins and integrins, and activates novel targets including the CARD9/CARMA1–BCL10–MALT1 pathway and the NLRP3 inflammasome. Drosophila studies indicate evolutionary ancient origin of SYK-mediated signalling. Moreover, SYK has a crucial role in autoimmune diseases and haematological malignancies. This Review summarizes our current understanding of SYK functions and the translation of this knowledge for therapeutic purposes. PMID:20467426

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity

PubMed Central

Mittal, Nimisha; Muthuswami, Rohini

2017-01-01

Background Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani. Methodology Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors. Conclusion Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis. PMID:28493888

Structure-function analysis of RBP-J-interacting and tubulin-associated (RITA) reveals regions critical for repression of Notch target genes.

PubMed

Tabaja, Nassif; Yuan, Zhenyu; Oswald, Franz; Kovall, Rhett A

2017-06-23

The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance, and aberrant Notch signaling has been implicated in various cancers, congenital defects, and cardiovascular diseases. Notch signaling activates the expression of target genes, which are regulated by the transcription factor CSL (CBF1/RBP-J, Su(H), Lag-1). CSL interacts with both transcriptional corepressor and coactivator proteins, functioning as both a repressor and activator, respectively. Although Notch activation complexes are relatively well understood at the structural level, less is known about how CSL interacts with corepressors. Recently, a new RBP-J (mammalian CSL ortholog)-interacting protein termed RITA has been identified and shown to export RBP-J out of the nucleus, thereby leading to the down-regulation of Notch target gene expression. However, the molecular details of RBP-J/RITA interactions are unclear. Here, using a combination of biochemical/cellular, structural, and biophysical techniques, we demonstrate that endogenous RBP-J and RITA proteins interact in cells, map the binding regions necessary for RBP-J·RITA complex formation, and determine the X-ray structure of the RBP-J·RITA complex bound to DNA. To validate the structure and glean more insights into function, we tested structure-based RBP-J and RITA mutants with biochemical/cellular assays and isothermal titration calorimetry. Whereas our structural and biophysical studies demonstrate that RITA binds RBP-J similarly to the RAM (RBP-J-associated molecule) domain of Notch, our biochemical and cellular assays suggest that RITA interacts with additional regions in RBP-J. Taken together, these results provide molecular insights into the mechanism of RITA-mediated regulation of Notch signaling, contributing to our understanding of how CSL functions as a transcriptional repressor of Notch target genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A biomimetic colorimetric logic gate system based on multi-functional peptide-mediated gold nanoparticle assembly.

PubMed

Li, Yong; Li, Wang; He, Kai-Yu; Li, Pei; Huang, Yan; Nie, Zhou; Yao, Shou-Zhuo

2016-04-28

In natural biological systems, proteins exploit various functional peptide motifs to exert target response and activity switch, providing a functional and logic basis for complex cellular activities. Building biomimetic peptide-based bio-logic systems is highly intriguing but remains relatively unexplored due to limited logic recognition elements and complex signal outputs. In this proof-of-principle work, we attempted to address these problems by utilizing multi-functional peptide probes and the peptide-mediated nanoparticle assembly system. Here, the rationally designed peptide probes function as the dual-target responsive element specifically responsive to metal ions and enzymes as well as the mediator regulating the assembly of gold nanoparticles (AuNPs). Taking advantage of Zn2+ ions and chymotrypsin as the model inputs of metal ions and enzymes, respectively, we constructed the peptide logic system computed by the multi-functional peptide probes and outputted by the readable colour change of AuNPs. In this way, the representative binary basic logic gates (AND, OR, INHIBIT, NAND, IMPLICATION) have been achieved by delicately coding the peptide sequence, demonstrating the versatility of our logic system. Additionally, we demonstrated that the three-input combinational logic gate (INHIBIT-OR) could also be successfully integrated and applied as a multi-tasking biosensor for colorimetric detection of dual targets. This nanoparticle-based peptide logic system presents a valid strategy to illustrate peptide information processing and provides a practical platform for executing peptide computing or peptide-related multiplexing sensing, implying that the controllable nanomaterial assembly is a promising and potent methodology for the advancement of biomimetic bio-logic computation.

Randomized Clinical Trial Replication of a Psychosocial Treatment for Children with High-Functioning Autism Spectrum Disorders

ERIC Educational Resources Information Center

Thomeer, Marcus L.; Lopata, Christopher; Volker, Martin A.; Toomey, Jennifer A.; Lee, Gloria K.; Smerbeck, Audrey M.; Rodgers, Jonathan D.; McDonald, Christin A.; Smith, Rachael A.

2012-01-01

This replication randomized clinical trial examined the efficacy of a comprehensive psychosocial intervention for children aged 7 to 12 years with high-functioning autism spectrum disorders (HFASDs). Participants were randomly assigned to treatment or wait-list conditions. Treatment included instruction and therapeutic activities targeting social…

Effect of the Purinergic Inhibitor Oxidized ATP in a Model of Islet Allograft Rejection

PubMed Central

Vergani, Andrea; Fotino, Carmen; D’Addio, Francesca; Tezza, Sara; Podetta, Michele; Gatti, Francesca; Chin, Melissa; Bassi, Roberto; Molano, Ruth D.; Corradi, Domenico; Gatti, Rita; Ferrero, Maria E.; Secchi, Antonio; Grassi, Fabio; Ricordi, Camillo; Sayegh, Mohamed H.; Maffi, Paola; Pileggi, Antonello; Fiorina, Paolo

2013-01-01

The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2XRs) sense ATP released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2XRs during islet allograft rejection and to establish a novel anti-P2XRs strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet allograft-infiltrating cells, that only P2X7R is increasingly expressed during alloimmune response, and that P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP]) delays islet allograft rejection, reduces the frequency of Th1/Th17 cells, and induces hyporesponsiveness toward donor antigens. oATP-treated mice displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. Peripheral blood mononuclear cells obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R+CD4+ T cells compared with controls. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function. PMID:23315496

Nanomedicine Strategies to Target Tumor-Associated Macrophages

PubMed Central

Binnemars-Postma, Karin; Storm, Gert; Prakash, Jai

2017-01-01

In recent years, the influence of the tumor microenvironment (TME) on cancer progression has been better understood. Macrophages, one of the most important cell types in the TME, exist in different subtypes, each of which has a different function. While classically activated M1 macrophages are involved in inflammatory and malignant processes, activated M2 macrophages are more involved in the wound-healing processes occurring in tumors. Tumor-associated macrophages (TAM) display M2 macrophage characteristics and support tumor growth and metastasis by matrix remodeling, neo-angiogenesis, and suppressing local immunity. Due to their detrimental role in tumor growth and metastasis, selective targeting of TAM for the treatment of cancer may prove to be beneficial in the treatment of cancer. Due to the plastic nature of macrophages, their activities may be altered to inhibit tumor growth. In this review, we will discuss the therapeutic options for the modulation and targeting of TAM. Different therapeutic strategies to deplete, inhibit recruitment of, or re-educate TAM will be discussed. Current strategies for the targeting of TAM using nanomedicine are reviewed. Passive targeting using different nanoparticle systems is described. Since TAM display a number of upregulated surface proteins compared to non-TAM, specific targeting using targeting ligands coupled to nanoparticles is discussed in detail. PMID:28471401

Activity-based proteome profiling of potential cellular targets of Orlistat--an FDA-approved drug with anti-tumor activities.

PubMed

Yang, Peng-Yu; Liu, Kai; Ngai, Mun Hong; Lear, Martin J; Wenk, Markus R; Yao, Shao Q

2010-01-20

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

Functional interrogation of non-coding DNA through CRISPR genome editing

PubMed Central

Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.

2017-01-01

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. PMID:28288828

Targets Fishing and Identification of Calenduloside E as Hsp90AB1: Design, Synthesis, and Evaluation of Clickable Activity-Based Probe

PubMed Central

Wang, Shan; Tian, Yu; Zhang, Jing-Yi; Xu, Hui-Bo; Zhou, Ping; Wang, Min; Lu, Sen-Bao; Luo, Yun; Wang, Min; Sun, Gui-Bo; Xu, Xu-Dong; Sun, Xiao-Bo

2018-01-01

Calenduloside E (CE), a natural triterpenoid compound isolated from Aralia elata, can protect against ox-LDL-induced human umbilical vein endothelial cell (HUVEC) injury in our previous reports. However, the exact targets and mechanisms of CE remain elusive. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. Based on the previous studies of the structure-activity relationship (SAR), we introduced an alkyne moiety at the C-28 carboxylic group of CE, which kept the protective and anti-apoptosis activity. Via proteomic approach, one of the potential proteins bound to CE-P was identified as Hsp90AB1, and further verification was performed by pure recombinant Hsp90AB1 and competitive assay. These results demonstrated that CE could bind to Hsp90AB1. We also found that CE could reverse the Hsp90AB1 decrease after ox-LDL treatment. To make our results more convincing, we performed SPR analysis and the affinity kinetic assay showed that CE/CE-P could bind to Hsp90AB1 in a dose-dependent manner. Taken together, our research showed CE could probably bind to Hsp90AB1 to protect the cell injury, which might provide the basis for the further exploration of its cardiovascular protective mechanisms. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. PMID:29875664

Topographical Organization of Attentional, Social, and Memory Processes in the Human Temporoparietal Cortex123

PubMed Central

Webb, Taylor W.; Kelly, Yin T.; Graziano, Michael S. A.

2016-01-01

Abstract The temporoparietal junction (TPJ) is activated in association with a large range of functions, including social cognition, episodic memory retrieval, and attentional reorienting. An ongoing debate is whether the TPJ performs an overarching, domain-general computation, or whether functions reside in domain-specific subdivisions. We scanned subjects with fMRI during five tasks known to activate the TPJ, probing social, attentional, and memory functions, and used data-driven parcellation (independent component analysis) to isolate task-related functional processes in the bilateral TPJ. We found that one dorsal component in the right TPJ, which was connected with the frontoparietal control network, was activated in all of the tasks. Other TPJ subregions were specific for attentional reorienting, oddball target detection, or social attribution of belief. The TPJ components that participated in attentional reorienting and oddball target detection appeared spatially separated, but both were connected with the ventral attention network. The TPJ component that participated in the theory-of-mind task was part of the default-mode network. Further, we found that the BOLD response in the domain-general dorsal component had a longer latency than responses in the domain-specific components, suggesting an involvement in distinct, perhaps postperceptual, computations. These findings suggest that the TPJ performs both domain-general and domain-specific computations that reside within spatially distinct functional components. PMID:27280153

Progranulin promotes peripheral nerve regeneration and reinnervation: role of notch signaling.

PubMed

Altmann, Christine; Vasic, Verica; Hardt, Stefanie; Heidler, Juliana; Häussler, Annett; Wittig, Ilka; Schmidt, Mirko H H; Tegeder, Irmgard

2016-10-22

Peripheral nerve injury is a frequent cause of lasting motor deficits and chronic pain. Although peripheral nerves are capable of regrowth they often fail to re-innervate target tissues. Using newly generated transgenic mice with inducible neuronal progranulin overexpression we show that progranulin accelerates axonal regrowth, restoration of neuromuscular synapses and recovery of sensory and motor functions after injury of the sciatic nerve. Oppositely, progranulin deficient mice have long-lasting deficits in motor function tests after nerve injury due to enhanced losses of motor neurons and stronger microglia activation in the ventral horn of the spinal cord. Deep proteome and gene ontology (GO) enrichment analysis revealed that the proteins upregulated in progranulin overexpressing mice were involved in 'regulation of transcription' and 'response to insulin' (GO terms). Transcription factor prediction pointed to activation of Notch signaling and indeed, co-immunoprecipitation studies revealed that progranulin bound to the extracellular domain of Notch receptors, and this was functionally associated with higher expression of Notch target genes in the dorsal root ganglia of transgenic mice with neuronal progranulin overexpression. Functionally, these transgenic mice recovered normal gait and running, which was not achieved by controls and was stronger impaired in progranulin deficient mice. We infer that progranulin activates Notch signaling pathways, enhancing thereby the regenerative capacity of partially injured neurons, which leads to improved motor function recovery.

RNA interference can be used to disrupt gene function in tardigrades

PubMed Central

Tenlen, Jennifer R.; McCaskill, Shaina; Goldstein, Bob

2012-01-01

How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We show that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions, and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments. PMID:23187800

RNA interference can be used to disrupt gene function in tardigrades.

PubMed

Tenlen, Jennifer R; McCaskill, Shaina; Goldstein, Bob

2013-05-01

How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments.

Musculoskeletal Model-Guided, Customizable Selection of Shoulder and Elbow Muscles for a C5 SCI Neuroprosthesis

PubMed Central

Hincapie, Juan Gabriel; Blana, Dimitra; Chadwick, Edward K.; Kirsch, Robert F.

2010-01-01

Individuals with C5/C6 spinal cord injury (SCI) have a number of paralyzed muscles in their upper extremities that can be electrically activated in a coordinated manner to restore function. The selection of a practical subset of paralyzed muscles for stimulation depends on the specific condition of the individual, the functions targeted for restoration, and surgical considerations. This paper presents a musculoskeletal model-based approach for optimizing the muscle set used for functional electrical stimulation (FES) of the shoulder and elbow in this population. Experimentally recorded kinematics from able-bodied subjects served as inputs to a musculoskeletal model of the shoulder and elbow, which was modified to reflect the reduced muscle force capacities of an individual with C5 SCI but also the potential of using FES to activate paralyzed muscles. A large number of inverse dynamic simulations mimicking typical activities of daily living were performed that included 1) muscles with retained voluntary control and 2) many different combinations of stimulated paralyzed muscles. These results indicate that a muscle set consisting of the serratus anterior, infraspinatus and triceps would enable the greatest range of relevant movements. This set will become the initial target in a C5 SCI neuroprosthesis to restore shoulder and elbow function. PMID:18586604

Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway.

PubMed

Namba, Takushi; Chu, Kiki; Kodama, Rika; Byun, Sanguine; Yoon, Kyoung Wan; Hiraki, Masatsugu; Mandinova, Anna; Lee, Sam W

2015-08-21

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53.

Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

PubMed Central

Kodama, Rika; Byun, Sanguine; Yoon, Kyoung Wan; Hiraki, Masatsugu; Mandinova, Anna; Lee, Sam W.

2015-01-01

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53. PMID:26254280

RGD-functionalized ultrasmall iron oxide nanoparticles for targeted T1-weighted MR imaging of gliomas

NASA Astrophysics Data System (ADS)

Luo, Yu; Yang, Jia; Yan, Yu; Li, Jingchao; Shen, Mingwu; Zhang, Guixiang; Mignani, Serge; Shi, Xiangyang

2015-08-01

We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration range, and display targeting specificity to glioma cells overexpressing αvβ3 integrin in vitro. With the relatively high r1 relaxivity (r1 = 1.4 mM-1 s-1), the Fe3O4-PEG-RGD particles can be used as an efficient nanoprobe for targeted T1-weighted positive MR imaging of glioma cells in vitro and the xenografted tumor model in vivo via an active RGD-mediated targeting pathway. The developed RGD-functionalized Fe3O4 NPs may hold great promise to be used as a nanoprobe for targeted T1-weighted MR imaging of different αvβ3 integrin-overexpressing cancer cells or biological systems.We report a convenient approach to prepare ultrasmall Fe3O4 nanoparticles (NPs) functionalized with an arginylglycylaspartic acid (RGD) peptide for in vitro and in vivo magnetic resonance (MR) imaging of gliomas. In our work, stable sodium citrate-stabilized Fe3O4 NPs were prepared by a solvothermal route. Then, the carboxylated Fe3O4 NPs stabilized with sodium citrate were conjugated with polyethylene glycol (PEG)-linked RGD. The formed ultrasmall RGD-functionalized nanoprobe (Fe3O4-PEG-RGD) was fully characterized using different techniques. We show that these Fe3O4-PEG-RGD particles with a size of 2.7 nm are water-dispersible, stable, cytocompatible and hemocompatible in a given concentration range, and display targeting specificity to glioma cells overexpressing αvβ3 integrin in vitro. With the relatively high r1 relaxivity (r1 = 1.4 mM-1 s-1), the Fe3O4-PEG-RGD particles can be used as an efficient nanoprobe for targeted T1-weighted positive MR imaging of glioma cells in vitro and the xenografted tumor model in vivo via an active RGD-mediated targeting pathway. The developed RGD-functionalized Fe3O4 NPs may hold great promise to be used as a nanoprobe for targeted T1-weighted MR imaging of different αvβ3 integrin-overexpressing cancer cells or biological systems. Electronic supplementary information (ESI) available: Additional experimental results. See DOI: 10.1039/c5nr04003e

[Visualization and Functional Regulation of Live Cell Proteins Based on Labeling Probe Design].

PubMed

Mizukami, Shin; Kikuchi, Kazuya

2016-01-01

  There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

PubMed

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Functional Role of PPARs in Ruminants: Potential Targets for Fine-Tuning Metabolism during Growth and Lactation

PubMed Central

Chen, Shuowen; Khan, Muhammad J.; Loor, Juan J.

2013-01-01

Characterization and biological roles of the peroxisome proliferator-activated receptor (PPAR) isotypes are well known in monogastrics, but not in ruminants. However, a wealth of information has accumulated in little more than a decade on ruminant PPARs including isotype tissue distribution, response to synthetic and natural agonists, gene targets, and factors affecting their expression. Functional characterization demonstrated that, as in monogastrics, the PPAR isotypes control expression of genes involved in lipid metabolism, anti-inflammatory response, development, and growth. Contrary to mouse, however, the PPARγ gene network appears to controls milk fat synthesis in lactating ruminants. As in monogastrics, PPAR isotypes in ruminants are activated by long-chain fatty acids, therefore, making them ideal candidates for fine-tuning metabolism in this species via nutrients. In this regard, using information accumulated in ruminants and monogastrics, we propose a model of PPAR isotype-driven biological functions encompassing key tissues during the peripartal period in dairy cattle. PMID:23737762

Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation.

PubMed

Zhao, Likun; Zhang, Jiangyan; Xu, Huiming; Geng, Hao; Cheng, Yongqiang

2016-09-07

Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity.

The Human Natural Killer Cell Immune Synapse

NASA Astrophysics Data System (ADS)

Davis, Daniel M.; Chiu, Isaac; Fassett, Marlys; Cohen, George B.; Mandelboim, Ofer; Strominger, Jack L.

1999-12-01

Inhibitory killer Ig-like receptors (KIR) at the surface of natural killer (NK) cells induced clustering of HLA-C at the contacting surface of target cells. In this manner, inhibitory immune synapses were formed as human NK cells surveyed target cells. At target/NK cell synapses, HLA-C/KIR distributed into rings around central patches of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1, the opposite orientation to mature murine T cell-activating synapses. This organization of protein was stable for at least 20 min. Cells could support multiple synapses simultaneously, and clusters of HLA-C moved as NK cells crawled over target cells. Clustering required a divalent metal cation, explaining how metal chelators inhibit KIR function. Surprisingly, however, formation of inhibitory synapses was unaffected by ATP depletion and the cytoskeletal inhibitors, colchicine and cytochalsins B and D. Clearly, supramolecular organization within plasma membranes is critical for NK cell immunosurveillance.

Analysis of Msx1 and Msx2 transactivation function in the context of the heat shock 70 (Hspa1b) gene promoter.

PubMed

Zhuang, Fengfeng; Nguyen, Manuel P; Shuler, Charles; Liu, Yi-Hsin

2009-04-03

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent transactivation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and heat shock factors may play a contributing role.

CRISPR-Cas9 Structures and Mechanisms.

PubMed

Jiang, Fuguo; Doudna, Jennifer A

2017-05-22

Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

Range estimation of passive infrared targets through the atmosphere

NASA Astrophysics Data System (ADS)

Cho, Hoonkyung; Chun, Joohwan; Seo, Doochun; Choi, Seokweon

2013-04-01

Target range estimation is traditionally based on radar and active sonar systems in modern combat systems. However, jamming signals tremendously degrade the performance of such active sensor devices. We introduce a simple target range estimation method and the fundamental limits of the proposed method based on the atmosphere propagation model. Since passive infrared (IR) sensors measure IR signals radiating from objects in different wavelengths, this method has robustness against electromagnetic jamming. The measured target radiance of each wavelength at the IR sensor depends on the emissive properties of target material and various attenuation factors (i.e., the distance between sensor and target and atmosphere environment parameters). MODTRAN is a tool that models atmospheric propagation of electromagnetic radiation. Based on the results from MODTRAN and atmosphere propagation-based modeling, the target range can be estimated. To analyze the proposed method's performance statistically, we use maximum likelihood estimation (MLE) and evaluate the Cramer-Rao lower bound (CRLB) via the probability density function of measured radiance. We also compare CRLB and the variance of MLE using Monte-Carlo simulation.

Radiance and atmosphere propagation-based method for the target range estimation

NASA Astrophysics Data System (ADS)

Cho, Hoonkyung; Chun, Joohwan

2012-06-01

Target range estimation is traditionally based on radar and active sonar systems in modern combat system. However, the performance of such active sensor devices is degraded tremendously by jamming signal from the enemy. This paper proposes a simple range estimation method between the target and the sensor. Passive IR sensors measures infrared (IR) light radiance radiating from objects in dierent wavelength and this method shows robustness against electromagnetic jamming. The measured target radiance of each wavelength at the IR sensor depends on the emissive properties of target material and is attenuated by various factors, in particular the distance between the sensor and the target and atmosphere environment. MODTRAN is a tool that models atmospheric propagation of electromagnetic radiation. Based on the result from MODTRAN and measured radiance, the target range is estimated. To statistically analyze the performance of proposed method, we use maximum likelihood estimation (MLE) and evaluate the Cramer-Rao Lower Bound (CRLB) via the probability density function of measured radiance. And we also compare CRLB and the variance of and ML estimation using Monte-Carlo.

Glycyrrhetinic acid-functionalized mesoporous silica nanoparticles as hepatocellular carcinoma-targeted drug carrier.

PubMed

Lv, Yongjiu; Li, Jingjing; Chen, Huali; Bai, Yan; Zhang, Liangke

2017-01-01

In this study, a glycyrrhetinic acid-functionalized mesoporous silica nanoparticle (MSN-GA) was prepared for active tumor targeting. MSN-GA exhibited satisfactory loading capacity for insoluble drugs, uniform size distribution, and specific tumor cell targeting. Glycyrrhetinic acid, a hepatocellular carcinoma-targeting group, was covalently decorated on the surface of MSN via an amido bond. The successful synthesis of MSN-GA was validated by the results of Fourier transform infrared spectroscopy, transmission electron microscopy (TEM), and zeta potential measurement. TEM images revealed the spherical morphology and uniform size distribution of the naked MSN and MSN-GA. Curcumin (CUR), an insoluble model drug, was loaded into MSN-GA (denoted as MSN-GA-CUR) with a high-loading capacity (8.78%±1.24%). The results of the in vitro cellular experiment demonstrated that MSN-GA-CUR significantly enhanced cytotoxicity and cellular uptake toward hepatocellular carcinoma (HepG2) cells via a specific GA receptor-mediated endocytosis mechanism. The results of this study provide a promising nanoplatform for the targeting of hepatocellular carcinoma.

Validation studies of the site-directed docking program LibDock.

PubMed

Rao, Shashidhar N; Head, Martha S; Kulkarni, Amit; LaLonde, Judith M

2007-01-01

The performance of the site-features docking algorithm LibDock has been evaluated across eight GlaxoSmithKline targets as a follow-up to a broad validation study of docking and scoring software (Warren, G. L.; Andrews, W. C.; Capelli, A.; Clarke, B.; Lalonde, J.; Lambert, M. H.; Lindvall, M.; Nevins, N.; Semus, S. F.; Senger, S.; Tedesco, G.; Walls, I. D.; Woolven, J. M.; Peishoff, C. E.; Head, M. S. J. Med. Chem. 2006, 49, 5912-5931). Docking experiments were performed to assess both the accuracy in reproducing the binding mode of the ligand and the retrieval of active compounds in a virtual screening protocol using both the DJD (Diller, D. J.; Merz, K. M., Jr. Proteins 2001, 43, 113-124) and LigScore2 (Krammer, A. K.; Kirchoff, P. D.; Jiang, X.; Venkatachalam, C. M.; Waldman, M. J. Mol. Graphics Modell. 2005, 23, 395-407) scoring functions. This study was conducted using DJD scoring, and poses were rescored using all available scoring functions in the Accelrys LigandFit module, including LigScore2. For six out of eight targets at least 30% of the ligands were docked within a root-mean-square difference (RMSD) of 2.0 A for the crystallographic poses when the LigScore2 scoring function was used. LibDock retrieved at least 20% of active compounds in the top 10% of screened ligands for four of the eight targets in the virtual screening protocol. In both studies the LigScore2 scoring function enhanced the retrieval of crystallographic poses or active compounds in comparison with the results obtained using the DJD scoring function. The results for LibDock accuracy and ligand retrieval in virtual screening are compared to 10 other docking and scoring programs. These studies demonstrate the utility of the LigScore2 scoring function and that LibDock as a feature directed docking method performs as well as docking programs that use genetic/growing and Monte Carlo driven algorithms.

The left inferior parietal lobe represents stored hand-postures for object use and action prediction

PubMed Central

van Elk, Michiel

2014-01-01

Action semantics enables us to plan actions with objects and to predict others' object-directed actions as well. Previous studies have suggested that action semantics are represented in a fronto-parietal action network that has also been implicated to play a role in action observation. In the present fMRI study it was investigated how activity within this network changes as a function of the predictability of an action involving multiple objects and requiring the use of action semantics. Participants performed an action prediction task in which they were required to anticipate the use of a centrally presented object that could be moved to an associated target object (e.g., hammer—nail). The availability of actor information (i.e., presenting a hand grasping the central object) and the number of possible target objects (i.e., 0, 1, or 2 target objects) were independently manipulated, resulting in different levels of predictability. It was found that making an action prediction based on actor information resulted in an increased activation in the extrastriate body area (EBA) and the fronto-parietal action observation network (AON). Predicting actions involving a target object resulted in increased activation in the bilateral IPL and frontal motor areas. Within the AON, activity in the left inferior parietal lobe (IPL) and the left premotor cortex (PMC) increased as a function of the level of action predictability. Together these findings suggest that the left IPL represents stored hand-postures that can be used for planning object-directed actions and for predicting other's actions as well. PMID:24795681

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

PubMed Central

Johnson, Amanda N.; Weil, P. Anthony

2017-01-01

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways. PMID:28196871

Inhibition of Bacterial Rna Polymerase by Streptolydigin: Stabilization of A Straight-Bridge-Helix Active-Center Conformation

PubMed Central

Tuske, Steven; Sarafianos, Stefan G.; Wang, Xinyue; Hudson, Brian; Sineva, Elena; Mukhopadhyay, Jayanta; Birktoft, Jens J.; Leroy, Olivier; Ismail, Sajida; Clark, Arthur D.; Dharia, Chhaya; Napoli, Andrew; Laptenko, Oleg; Lee, Jookyung; Borukhov, Sergei; Ebright, Richard H.; Arnold, Eddy

2009-01-01

We define the target, mechanism, and structural basis of inhibition of bacterial RNA polymerase (RNAP) by the tetramic-acid antibiotic streptolydigin (Stl). Stl binds to a site adjacent to, but not overlapping, the RNAP active center and stabilizes an RNAP-active-center conformational state with a straight bridge helix. The results provide direct support for the proposals that alternative straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations exist, and that cycling between straight-bridge-helix and bent-bridge-helix RNAP-active-center conformations is required for RNAP function. The results set bounds on models for RNAP function and suggest strategies for design of novel antibacterial agents. PMID:16122422

Impaired modulation of attention and emotion in schizophrenia.

PubMed

Dichter, Gabriel S; Bellion, Carolyn; Casp, Michael; Belger, Aysenil

2010-05-01

Fronto-limbic interactions facilitate the generation of task-relevant responses while inhibiting interference from emotionally distracting information. Schizophrenia is associated with deficits in both executive attention and affective regulation. This study aims to elucidate the neural correlates of emotion-attention regulation and shifting in schizophrenia. We employed functional magnetic resonance imaging to probe the fronto-limbic regions in 16 adults with schizophrenia and 13 matched adults with no history of psychiatric illness. Subjects performed a forced-choice visual oddball task where they detected infrequent target circles embedded in a series of infrequent nontarget aversive and neutral pictures and frequent squares. In control participants, target events activated a dorsal frontoparietal network, whereas these regions were deactivated by aversive stimuli. Conversely, ventral frontolimbic brain regions were activated by aversive stimuli and deactivated by target events. In the patient group, regional hemodynamic timecourses revealed not only reduced activation to target and aversive events in dorsal executive and ventral limbic regions, respectively, but also reduced deactivation to target and aversive stimuli in ventral and dorsal regions, respectively, relative to the control group. Patients further showed reduced spatial extent of activation in the right inferior frontal gyrus during the target and aversive conditions. Activation of the anterior cingulate to aversive images was inversely related to severity of avolition and anhedonia symptoms in the schizophrenia group. These results suggest both frontal and limbic dysfunction in schizophrenia as well as aberrant reciprocal inhibitions between these regions during attention-emotion modulation in this disorder.

Modulation of Immune Cell Functions by the E3 Ligase Cbl-b

PubMed Central

Lutz-Nicoladoni, Christina; Wolf, Dominik; Sopper, Sieghart

2015-01-01

Maintenance of immunological tolerance is a critical hallmark of the immune system. Several signaling checkpoints necessary to balance activating and inhibitory input to immune cells have been described so far, among which the E3 ligase Cbl-b appears to be a central player. Cbl-b is expressed in all leukocyte subsets and regulates several signaling pathways in T cells, NK cells, B cells, and different types of myeloid cells. In most cases, Cbl-b negatively regulates activation signals through antigen or pattern recognition receptors and co-stimulatory molecules. In line with this function, cblb-deficient immune cells display lower activation thresholds and cblb knockout mice spontaneously develop autoimmunity and are highly susceptible to experimental autoimmunity. Interestingly, genetic association studies link CBLB-polymorphisms with autoimmunity also in humans. Vice versa, the increased activation potential of cblb-deficient cells renders them more potent to fight against malignancies or infections. Accordingly, several reports have shown that cblb knockout mice reject tumors, which mainly depends on cytotoxic T and NK cells. Thus, targeting Cbl-b may be an interesting strategy to enhance anti-cancer immunity. In this review, we summarize the findings on the molecular function of Cbl-b in different cell types and illustrate the potential of Cbl-b as target for immunomodulatory therapies. PMID:25815272

Role of Nicotinic and Muscarinic Receptors on Synaptic Plasticity and Neurological Diseases.

PubMed

Fuenzalida, Marco; Pérez, Miguel Ángel; Arias, Hugo R

2016-01-01

The cholinergic activity in the brain is fundamental for cognitive functions. The modulatory activity of the neurotransmitter acetylcholine (ACh) is mediated by activating a variety of nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (mAChR). Accumulating evidence indicates that both nAChR and mAChRs can modulate the release of several other neurotransmitters, modify the threshold of long-term plasticity, finally improving learning and memory processes. Importantly, the expression, distribution, and/or function of these systems are altered in several neurological diseases. The aim of this review is to discuss our current knowledge on cholinergic receptors and their regulating synaptic functions and neuronal network activities as well as their use as targets for the development of new and clinically useful cholinergic ligands. These new therapies involve the development of novel and more selective cholinergic agonists and allosteric modulators as well as selective cholinesterase inhibitors, which may improve cognitive and behavioral symptoms, and also provide neuroprotection in several brain diseases. The review will focus on two nAChR receptor subtypes found in the mammalian brain and the most commonly targeted in drug discovery programs for neuropsychiatric disorder, the ligands of α4β2 nAChR and α7 nAChRs.

MUC1-C activates EZH2 expression and function in human cancer cells.

PubMed

Rajabi, Hasan; Hiraki, Masayuki; Tagde, Ashujit; Alam, Maroof; Bouillez, Audrey; Christensen, Camilla L; Samur, Mehmet; Wong, Kwok-Kin; Kufe, Donald

2017-08-07

The EZH2 histone methyltransferase is a member of the polycomb repressive complex 2 (PRC2) that is highly expressed in diverse human cancers and is associated with a poor prognosis. MUC1-C is an oncoprotein that is similarly overexpressed in carcinomas and has been linked to epigenetic regulation. A role for MUC1-C in regulating EZH2 and histone methylation is not known. Here, we demonstrate that targeting MUC1-C in diverse human carcinoma cells downregulates EZH2 and other PRC2 components. MUC1-C activates (i) the EZH2 promoter through induction of the pRB→E2F pathway, and (ii) an NF-κB p65 driven enhancer in exon 1. We also show that MUC1-C binds directly to the EZH2 CXC region adjacent to the catalytic SET domain and associates with EZH2 on the CDH1 and BRCA1 promoters. In concert with these results, targeting MUC1-C downregulates EZH2 function as evidenced by (i) global and promoter-specific decreases in H3K27 trimethylation (H3K27me3), and (ii) activation of tumor suppressor genes, including BRCA1. These findings highlight a previously unreported role for MUC1-C in activating EZH2 expression and function in cancer cells.

Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines.

PubMed

Gulec, Cagri; Coban, Neslihan; Ozsait-Selcuk, Bilge; Sirma-Ekmekci, Sema; Yildirim, Ozlem; Erginel-Unaltuna, Nihan

2017-04-01

ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses. Copyright © 2017 Elsevier Inc. All rights reserved.

New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment?

PubMed

Coqueret, Olivier

2003-02-01

Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions for these proteins in the control of CDK activation, in the regulation of apoptosis and in transcriptional activation. This article discusses recent insights into these possible additional functions of p21 and p27.

Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets

PubMed Central

Akala, Hoseah M.; Macharia, Rosaline W.; Juma, Dennis W.; Cheruiyot, Agnes C.; Andagalu, Ben; Brown, Mathew L.; El-Shemy, Hany A.; Nyanjom, Steven G.

2017-01-01

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens. PMID:29088219

MicroRNA-212 activates hepatic stellate cells and promotes liver fibrosis via targeting SMAD7.

PubMed

Zhu, Jie; Zhang, Ziqiang; Zhang, Yitong; Li, Wenshuai; Zheng, Wanwei; Yu, Jianghong; Wang, Bangting; Chen, Lirong; Zhuo, Qin; Chen, Lin; Zhang, Jun; Liu, Jie

2018-01-29

There has been an increasing number of researches about microRNAs (miRNAs) in the progression of liver fibrosis from the point of their comprehensive functions in regulating the activation of hepatic stellate cells (HSCs). Among them, it has been reported that miR-212 is up-regulated in activated rat primary HSCs. However, its mechanism has not been determined yet. Here, we confirmed that the level of miR-212-3p was up-regulated in livers of carbon tetrachloride (CCl 4 )-treated mice compared with the normal control, which is a classical model of chronically damaged fibrotic liver. In vitro, we demonstrated that TGF-β, a master fibrogenic cytokine, could induce the level of miR-212. In turn, overexpression of miR-212 could induce the activation marker of HSC including α-smooth muscle actin (α-SMA) and collagens by activating TGF-β signaling pathway. Furthermore, SMAD7, a dominant suppressor of TGF-β pathway, was identified as a direct target of miR-212-3p. Our results indicate that miR-212-3p facilitates the activation of HSCs and TGF-β pathway by targeting SMAD7, highlighting that it can be served as a novel biomarker or therapeutic target for liver fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

PubMed Central

Chen, Hui; Kazemier, Hinke G; de Groote, Marloes L.; Ruiters, Marcel H. J.; Xu, Guo-Liang; Rots, Marianne G.

2014-01-01

Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs’ methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks. PMID:24194590

USAF (United States Air Force) Avionics Master Plan.

DTIC Science & Technology

1982-12-01

is updated annually by MAPG activities to reflect changes in emphasis resulting from new direction, threat developments , and other armament and...many different kinds of functional electronic subsystems, a building block approach to the development of new subsystems can be taken. This approach...technologies targeted for precision all weather weapon delivery. A new program will develop the capability to detect and locate ground moving targets not

In Search of a Cure for Proteostasis-Addicted Cancer: A AAA Target Revealed.

PubMed

Xia, Di; Ye, Yihong

2015-11-09

Tumorigenesis is often associated with an unbalanced protein homeostasis (proteostasis) network, which sensitizes cancer cells to drugs targeting protein quality control (PQC) regulators. In this issue of Cancer Cell, Anderson and colleagues investigated the anti-cancer activity of a new class of inhibitor against a multi-functional ATPase essential for proteostasis maintenance. Copyright © 2015 Elsevier Inc. All rights reserved.

SIRT3 Is a Mitochondrial Tumor Suppressor and Genetic Loss Results in a Murine Model for ER/PR-Positive Mammary Tumors Connecting Metabolism and Carcinogenesis

DTIC Science & Technology

2012-09-01

and determine if these targets are regulated by extracellular stimuli known to activate sirtuin function (e.g., resveratrol ). These targets will... resveratrol or overexpression of a MnSOD gene will prevent increases in ROS in MEFs and/or decrease the development of mammary tumors in Sirt3

CD44 functions in Wnt signaling by regulating LRP6 localization and activation

PubMed Central

Schmitt, M; Metzger, M; Gradl, D; Davidson, G; Orian-Rousseau, V

2015-01-01

Wnt reception at the membrane is complex and not fully understood. CD44 is a major Wnt target gene in the intestine and is essential for Wnt-induced tumor progression in colorectal cancer. Here we show that CD44 acts as a positive regulator of the Wnt receptor complex. Downregulation of CD44 expression decreases, whereas CD44 overexpression increases Wnt activity in a concentration-dependent manner. Epistasis experiments place CD44 function at the level of the Wnt receptor LRP6. Mechanistically, CD44 physically associates with LRP6 upon Wnt treatment and modulates LRP6 membrane localization. Moreover, CD44 regulates Wnt signaling in the developing brain of Xenopus laevis embryos as shown by a decreased expression of Wnt targets tcf-4 and en-2 in CD44 morphants. PMID:25301071

Characterization of the functional requirements of West Nile virus membrane fusion.

PubMed

Moesker, Bastiaan; Rodenhuis-Zybert, Izabela A; Meijerhof, Tjarko; Wilschut, Jan; Smit, Jolanda M

2010-02-01

Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the fluorophore, pyrene. Fusion of WNV with liposomes occurred on the timescale of seconds and was strictly dependent on mildly acidic pH. Optimal fusion kinetics were observed at pH 6.3, the threshold for fusion being pH 6.9. Preincubation of the virus alone at pH 6.3 resulted in a rapid loss of fusion capacity. WNV fusion activity is strongly promoted by the presence of cholesterol in the target membrane. Furthermore, we provide direct evidence that cleavage of prM to M is a requirement for fusion activity of WNV.

Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells.

PubMed

Mariscal, Ana M; Kakizawa, Shigeyuki; Hsu, Jonathan Y; Tanaka, Kazuki; González-González, Luis; Broto, Alicia; Querol, Enrique; Lluch-Senar, Maria; Piñero-Lambea, Carlos; Sun, Lijie; Weyman, Philip D; Wise, Kim S; Merryman, Chuck; Tse, Gavin; Moore, Adam J; Hutchison, Clyde A; Smith, Hamilton O; Tomita, Masaru; Venter, J Craig; Glass, John I; Piñol, Jaume; Suzuki, Yo

2018-05-22

Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

Cross-orientation suppression in human visual cortex

PubMed Central

Heeger, David J.

2011-01-01

Cross-orientation suppression was measured in human primary visual cortex (V1) to test the normalization model. Subjects viewed vertical target gratings (of varying contrasts) with or without a superimposed horizontal mask grating (fixed contrast). We used functional magnetic resonance imaging (fMRI) to measure the activity in each of several hypothetical channels (corresponding to subpopulations of neurons) with different orientation tunings and fit these orientation-selective responses with the normalization model. For the V1 channel maximally tuned to the target orientation, responses increased with target contrast but were suppressed when the horizontal mask was added, evident as a shift in the contrast gain of this channel's responses. For the channel maximally tuned to the mask orientation, a constant baseline response was evoked for all target contrasts when the mask was absent; responses decreased with increasing target contrast when the mask was present. The normalization model provided a good fit to the contrast-response functions with and without the mask. In a control experiment, the target and mask presentations were temporally interleaved, and we found no shift in contrast gain, i.e., no evidence for suppression. We conclude that the normalization model can explain cross-orientation suppression in human visual cortex. The approach adopted here can be applied broadly to infer, simultaneously, the responses of several subpopulations of neurons in the human brain that span particular stimulus or feature spaces, and characterize their interactions. In addition, it allows us to investigate how stimuli are represented by the inferred activity of entire neural populations. PMID:21775720

The impact of target site accessibility on the design of effective siRNAs.

PubMed

Tafer, Hakim; Ameres, Stefan L; Obernosterer, Gregor; Gebeshuber, Christoph A; Schroeder, Renée; Martinez, Javier; Hofacker, Ivo L

2008-05-01

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.

A new target ligand Ser-Glu for PEPT1-overexpressing cancer imaging.

PubMed

Dai, Tongcheng; Li, Na; Zhang, Lingzhi; Zhang, Yuanxing; Liu, Qin

2016-01-01

Nanoparticles functionalized with active target ligands have been widely used for tumor-specific diagnosis and therapy. The target ligands include antibodies, peptides, proteins, small molecules, and nucleic acid aptamers. Here, we utilize dipeptide Ser-Glu (DIP) as a new ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for pancreatic cancer target imaging. We demonstrate that in the first step, Ser-Glu-conjugated NPs (NPs-DIP) efficiently bind to AsPC-1 and in the following NPs-DIP are internalized into AsPC-1 in vitro. The peptide transporter 1 inhibition experiment reveals that the targeting effects mainly depend on the specific binding of DIP to peptide transporter 1, which is remarkably upregulated in pancreatic cancer cells compared with varied normal cells. Furthermore, NPs-DIP specifically accumulate in the site of pancreatic tumor xenograft and are further internalized into the tumor cells in vivo after intravenous administration, indicating that DIP successfully enhanced nanoparticles internalization efficacy into tumor cells in vivo. This work establishes Ser-Glu to be a new tumor-targeting ligand and provides a promising tool for future tumor diagnostic or therapeutic applications.

Functional genomics analysis of big data identifies novel peroxisome proliferator–activated receptor gamma target single nucleotide polymorphisms showing association with cardiometabolic outcomes

USDA-ARS?s Scientific Manuscript database

Background Cardiovascular disease and type 2 diabetes mellitus represent overlapping diseases where a large portion of the variation attributable to genetics remains unexplained. An important player in their pathogenesis is peroxisome proliferator–activated receptor gamma (PPARgamma) that is involve...

Cell Cycle Regulators Guide Mitochondrial Activity in Radiation-Induced Adaptive Response

PubMed Central

Alexandrou, Aris T.

2014-01-01

Abstract Significance: There are accruing concerns on potential genotoxic agents present in the environment including low-dose ionizing radiation (LDIR) that naturally exists on earth's surface and atmosphere and is frequently used in medical diagnosis and nuclear industry. Although its long-term health risk is being evaluated and remains controversial, LDIR is shown to induce temporary but significant adaptive responses in mammalian cells and animals. The mechanisms guiding the mitochondrial function in LDIR-induced adaptive response represent a unique communication between DNA damage and cellular metabolism. Elucidation of the LDIR-regulated mitochondrial activity may reveal new mechanisms adjusting cellular function to cope with hazardous environmental stress. Recent Advances: Key cell cycle regulators, including Cyclin D1/CDK4 and Cyclin B1/cyclin-dependent kinase 1 (CDK1) complexes, are actively involved in the regulation of mitochondrial functions via phosphorylation of their mitochondrial targets. Accumulating new evidence supports a concept that the Cyclin B1/CDK1 complex acts as a mediator in the cross talk between radiation-induced DNA damage and mitochondrial functions to coordinate cellular responses to low-level genotoxic stresses. Critical Issues: The LDIR-mediated mitochondrial activity via Cyclin B1/CDK1 regulation is an irreplaceable network that is able to harmonize vital cellular functions with adjusted mitochondrial metabolism to enhance cellular homeostasis. Future Directions: Further investigation of the coordinative mechanism that regulates mitochondrial activities in sublethal stress conditions, including LDIR, will reveal new insights of how cells cope with genotoxic injury and will be vital for future targeted therapeutic interventions that reduce environmental injury and cancer risk. Antioxid. Redox Signal. 20, 1463–1480. PMID:24180340

Mechanistic and structural basis of bioengineered bovine Cathelicidin-5 with optimized therapeutic activity

NASA Astrophysics Data System (ADS)

Sahoo, Bikash R.; Maruyama, Kenta; Edula, Jyotheeswara R.; Tougan, Takahiro; Lin, Yuxi; Lee, Young-Ho; Horii, Toshihiro; Fujiwara, Toshimichi

2017-03-01

Peptide-drug discovery using host-defense peptides becomes promising against antibiotic-resistant pathogens and cancer cells. Here, we customized the therapeutic activity of bovine cathelicidin-5 targeting to bacteria, protozoa, and tumor cells. The membrane dependent conformational adaptability and plasticity of cathelicidin-5 is revealed by biophysical analysis and atomistic simulations over 200 μs in thymocytes, leukemia, and E. coli cell-membranes. Our understanding of energy-dependent cathelicidin-5 intrusion in heterogeneous membranes aided in designing novel loss/gain-of-function analogues. In vitro findings identified leucine-zipper to phenylalanine substitution in cathelicidin-5 (1-18) significantly enhance the antimicrobial and anticancer activity with trivial hemolytic activity. Targeted mutants of cathelicidin-5 at kink region and N-terminal truncation revealed loss-of-function. We ensured the existence of a bimodal mechanism of peptide action (membranolytic and non-membranolytic) in vitro. The melanoma mouse model in vivo study further supports the in vitro findings. This is the first structural report on cathelicidin-5 and our findings revealed potent therapeutic application of designed cathelicidin-5 analogues.

Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity

PubMed Central

Ding, Baoyue; Wu, Xin; Fan, Wei; Wu, Zhaoyong; Gao, Jing; Zhang, Wei; Ma, Lulu; Xiang, Wang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2011-01-01

Background The increased incidence of malignant melanoma in recent decades, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Novel therapeutic strategies, such as immunotherapy and targeted therapy, are urgently needed for melanoma. In this study, a new active targeting drug delivery system was constructed to combine chemotherapy and active specific immunotherapy. Methods The chemotherapeutic drug, dacarbazine (DTIC), that induces apoptosis through the intrinsic pathway which typically responds to severe DNA damage, was used as a model drug to prepare DTIC-loaded polylactic acid (PLA) nanoparticles (DTIC-NPs), which were covalently conjugated to a highly specific targeting functional TRAIL-receptor 2 (DR5) monoclonal antibody (mAb) that can contribute directly to cancer cell apoptosis or growth inhibition through the extrinsic pathway. Results Our in vitro experiments demonstrated that DTIC-PLA-DR5 mAb nanoparticles (DTIC-NPs-DR5 mAb) are an active targeting drug delivery system which can specifically target DR5-overexpressing malignant melanoma cells and become efficiently internalized. Most strikingly, compared with conventional DTIC-NPs, DTIC-NPs-DR5 mAb showed significantly enhanced cytotoxicity and increased cell apoptosis in DR5-positive malignant melanoma cells. Conclusion The DTIC-NPs-DR5 mAb described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to DR5-overexpressing metastatic melanoma. PMID:21976975

Taiman acts as a coactivator of Yorkie in the Hippo pathway to promote tissue growth and intestinal regeneration.

PubMed

Wang, Chao; Yin, Meng-Xin; Wu, Wei; Dong, Liang; Wang, Shimin; Lu, Yi; Xu, Jinjin; Wu, Wenqing; Li, Sheng; Zhao, Yun; Zhang, Lei

2016-01-01

The Hippo signaling pathway regulates tissue growth and organ size through controlling cell growth, proliferation and apoptosis. During these processes, the coactivator Yorkie partners with the transcription factor Scalloped to mediate Hippo pathway-regulated cellular functions. Here, we demonstrate that Taiman facilitates the activity of Yorkie. First, Taiman overexpression upregulates Hippo pathway-responsive genes and induces tissue overgrowth. Second, the loss of tai downregulates the expression of Hippo pathway target genes and reduces organ size as well as tissue overgrowth caused by Yorkie overexpression. Furthermore, we provide evidence that Taiman binds to Yorkie and facilitates the activity of Yorkie-Scalloped to activate the transcription of several Hippo pathway target genes. Moreover, we found that the C-terminus of Taiman is indispensable for the function of Taiman in Hippo signaling. Finally, we demonstrate that Taiman is also required in intestinal stem cell proliferation. Our findings suggest Taiman is an essential coactivator of Yorkie.

Development of Novel Antisense Oligonucleotides for the Functional Regulation of RNA-Induced Silencing Complex (RISC) by Promoting the Release of microRNA from RISC.

PubMed

Ariyoshi, Jumpei; Momokawa, Daiki; Eimori, Nao; Kobori, Akio; Murakami, Akira; Yamayoshi, Asako

2015-12-16

MicroRNAs (miRNAs) are known to be important post-transcription regulators of gene expression. Aberrant miRNA expression is associated with pathological disease processes, including carcinogenesis. Therefore, miRNAs are considered significant therapeutic targets for cancer therapy. MiRNAs do not act alone, but exhibit their functions by forming RNA-induced silencing complex (RISC). Thus, the regulation of RISC activity is a promising approach for cancer therapy. MiRNA is a core component of RISC and is an essential to RISC for recognizing target mRNA. Thereby, it is expected that development of the method to promote the release of miRNA from RISC would be an effective approach for inhibition of RISC activity. In this study, we synthesized novel peptide-conjugated oligonucleotides (RINDA-as) to promote the release of miRNA from RISC. RINDA-as showed a high rate of miRNA release from RISC and high level of inhibitory effect on RISC activity.

Cell-mediated immunity in herpes simplex virus-infected mice: functional analysis of lymph node cells during periods of acute and latent infection, with reference to cytotoxic and memory cells.

PubMed

Nash, A A; Quartey-Papafio, R; Wildy, P

1980-08-01

The functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at lease up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.

A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation

PubMed Central

Shen, Yang; Zeng, Lin; Novosyadlyy, Ruslan; Forest, Amelie; Zhu, Aiping; Korytko, Andrew; Zhang, Haifan; Eastman, Scott W; Topper, Michael; Hindi, Sagit; Covino, Nicole; Persaud, Kris; Kang, Yun; Burtrum, Douglas; Surguladze, David; Prewett, Marie; Chintharlapalli, Sudhakar; Wroblewski, Victor J; Shen, Juqun; Balderes, Paul; Zhu, Zhenping; Snavely, Marshall; Ludwig, Dale L

2015-01-01

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions. PMID:26073904

Ionotropic glutamate receptors: regulation by G-protein-coupled receptors.

PubMed

Rojas, Asheebo; Dingledine, Raymond

2013-04-01

The function of many ion channels is under dynamic control by coincident activation of G-protein-coupled receptors (GPCRs), particularly those coupled to the Gαs and Gαq family members. Such regulation is typically dependent on the subunit composition of the ionotropic receptor or channel as well as the GPCR subtype and the cell-specific panoply of signaling pathways available. Because GPCRs and ion channels are so highly represented among targets of U.S. Food and Drug Administration-approved drugs, functional cross-talk between these drug target classes is likely to underlie many therapeutic and adverse effects of marketed drugs. GPCRs engage a myriad of signaling pathways that involve protein kinases A and C (PKC) and, through PKC and interaction with β-arrestin, Src kinase, and hence the mitogen-activated-protein-kinase cascades. We focus here on the control of ionotropic glutamate receptor function by GPCR signaling because this form of regulation can influence the strength of synaptic plasticity. The amino acid residues phosphorylated by specific kinases have been securely identified in many ionotropic glutamate (iGlu) receptor subunits, but which of these sites are GPCR targets is less well known even when the kinase has been identified. N-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and heteromeric kainate receptors are all downstream targets of GPCR signaling pathways. The details of GPCR-iGlu receptor cross-talk should inform a better understanding of how synaptic transmission is regulated and lead to new therapeutic strategies for neuropsychiatric disorders.

Molecular mechanisms of programmed cell death-1 dependent T cell suppression: relevance for immunotherapy

PubMed Central

Zuazo, Miren; Gato-Cañas, Maria; Llorente, Noelia; Ibañez-Vea, María; Arasanz, Hugo

2017-01-01

Programmed cell death-1 (PD1) has become a significant target for cancer immunotherapy. PD1 and its receptor programmed cell death 1 ligand 1 (PDL1) are key regulatory physiological immune checkpoints that maintain self-tolerance in the organism by regulating the degree of activation of T and B cells amongst other immune cell types. However, cancer cells take advantage of these immunosuppressive regulatory mechanisms to escape T and B cell-mediated immunity. PD1 engagement on T cells by PDL1 on the surface of cancer cells dramatically interferes with T cell activation and the acquisition of effector capacities. Interestingly, PD1-targeted therapies have demonstrated to be highly effective in rescuing T cell anti-tumor effector functions. Amongst these the use of anti-PD1/PDL1 monoclonal antibodies are particularly efficacious in human therapies. Furthermore, clinical findings with PD1/PDL1 blockers over several cancer types demonstrate clinical benefit. Despite the successful results, the molecular mechanisms by which PD1-targeted therapies rescue T cell functions still remain elusive. Therefore, it is a key issue to uncover the molecular pathways by which these therapies exert its function in T cells. A profound knowledge of PDL1/PD1 mechanisms will surely uncover a new array of targets susceptible of therapeutic intervention. Here, we provide an overview of the molecular events underlying PD1-dependent T cell suppression in cancer. PMID:29114543

"Failure-to-Identify" Hunting Incidents: A Resilience Engineering Approach.

PubMed

Bridges, Karl E; Corballis, Paul M; Hollnagel, Erik

2018-03-01

Objective The objective was to develop an understanding, using the Functional Resonance Analysis Method (FRAM), of the factors that could cause a deer hunter to misidentify their intended target. Background Hunting is a popular activity in many communities. However, hunters vary considerably based on training, experience, and expertise. Surprisingly, safety in hunting has not received much attention, especially failure-to-identify hunting incidents. These are incidents in which the hunter, after spotting and targeting their quarry, discharge their firearm only to discover they have been spotting and targeting another human, an inanimate object, or flora by mistake. The hunter must consider environment, target, time of day, weather, and many other factors-continuously evaluating whether the hunt should continue. To understand how these factors can relate to one another is fundamental to begin to understand how incidents happen. Method Workshops with highly experienced and active hunters led to the development of a FRAM model detailing the functions of a "Hunting FRAM." The model was evaluated for correctness based on confidential and anonymous near-miss event submissions by hunters. Results A FRAM model presenting the functions of a hunt was produced, evaluated, and accepted. Using the model, potential sources of incidents or other unintended outcomes were identified, which in turn helped to improve the model. Conclusion Utilizing principles of understanding and visualization tools of the FRAM, the findings create a foundation for safety improvements potentially through training or safety messages based on an increased understanding of the complexity of hunting.

Interventions for coordination of walking following stroke: systematic review.

PubMed

Hollands, Kristen L; Pelton, Trudy A; Tyson, Sarah F; Hollands, Mark A; van Vliet, Paulette M

2012-03-01

Impairments in gait coordination may be a factor in falls and mobility limitations after stroke. Therefore, rehabilitation targeting gait coordination may be an effective way to improve walking post-stroke. This review sought to examine current treatments that target impairments of gait coordination, the theoretical basis on which they are derived and the effects of such interventions. Few high quality RCTs with a low risk of bias specifically targeting and measuring restoration of coordinated gait were found. Consequently, we took a pragmatic approach to describing and quantifying the available evidence and included non-randomised study designs and limited the influence of heterogeneity in experimental design and control comparators by restricting meta-analyses to pre- and post-test comparisons of experimental interventions only. Results show that physiotherapy interventions significantly improved gait function and coordination. Interventions involving repetitive task-specific practice and/or auditory cueing appeared to be the most promising approaches to restore gait coordination. The fact that overall improvements in gait coordination coincided with increased walking speed lends support to the hypothesis that targeting gait coordination gait may be a way of improving overall walking ability post-stroke. However, establishing the mechanism for improved locomotor control requires a better understanding of the nature of both neuroplasticity and coordination deficits in functional tasks after stroke. Future research requires the measurement of impairment, activity and cortical activation in an effort to establish the mechanism by which functional gains are achieved. Copyright © 2011 Elsevier B.V. All rights reserved.

Targeted ablation of cardiac sympathetic neurons improves ventricular electrical remodelling in a canine model of chronic myocardial infarction.

PubMed

Xiong, Liang; Liu, Yu; Zhou, Mingmin; Wang, Guangji; Quan, Dajun; Shen, Caijie; Shuai, Wei; Kong, Bin; Huang, Congxin; Huang, He

2018-05-31

The purpose of this study was to evaluate the cardiac electrophysiologic effects of targeted ablation of cardiac sympathetic neurons (TACSN) in a canine model of chronic myocardial infarction (MI). Thirty-eight anaesthetized dogs were randomly assigned into the sham-operated, MI, and MI-TACSN groups, respectively. Myocardial infarction-targeted ablation of cardiac sympathetic neuron was induced by injecting cholera toxin B subunit-saporin compound in the left stellate ganglion (LSG). Five weeks after surgery, the cardiac function, heart rate variability (HRV), ventricular electrophysiological parameters, LSG function and neural activity, serum norepinephrine (NE), nerve growth factor (NGF), and brain natriuretic peptide (BNP) levels were measured. Cardiac sympathetic innervation was determined with immunofluorescence staining of growth associated protein-43 (GAP43) and tyrosine hydroxylase (TH). Compared with MI group, TACSN significantly improved HRV, attenuated LSG function and activity, prolonged corrected QT interval, decreased Tpeak-Tend interval, prolonged ventricular effective refractory period (ERP), and action potential duration (APD), decreased the slopes of APD restitution curves, suppressed the APD alternans, increased ventricular fibrillation threshold, and reduced serum NE, NGF, and BNP levels. Moreover, the densities of GAP43 and TH-positive nerve fibres in the infarcted border zone in the MI-TACSN group were lower than those in the MI group. Targeted ablation of cardiac sympathetic neuron attenuates sympathetic remodelling and improves ventricular electrical remodelling in the chronic phase of MI. These data suggest that TACSN may be a novel approach to treating ventricular arrhythmias.

Antagonistic Rgg regulators mediate quorum sensing via competitive DNA binding in Streptococcus pyogenes.

PubMed

Lasarre, Breah; Aggarwal, Chaitanya; Federle, Michael J

2013-01-02

Recent studies have established the fact that multiple members of the Rgg family of transcriptional regulators serve as key components of quorum sensing (QS) pathways that utilize peptides as intercellular signaling molecules. We previously described a novel QS system in Streptococcus pyogenes which utilizes two Rgg-family regulators (Rgg2 and Rgg3) that respond to neighboring signaling peptides (SHP2 and SHP3) to control gene expression and biofilm formation. We have shown that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes, and that SHPs function to activate the QS system. The mechanisms by which Rgg proteins regulate both QS-dependent and QS-independent processes remain poorly defined; thus, we sought to further elucidate how Rgg2 and Rgg3 mediate gene regulation. Here we provide evidence that S. pyogenes employs a unique mechanism of direct competition between the antagonistic, peptide-responsive proteins Rgg2 and Rgg3 for binding at target promoters. The highly conserved, shared binding sites for Rgg2 and Rgg3 are located proximal to the -35 nucleotide in the target promoters, and the direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP. These results suggest that exclusionary binding of target promoters by Rgg3 may prevent Rgg2 binding under SHP-limiting conditions, thereby preventing premature induction of the quorum sensing circuit. Rgg-family transcriptional regulators are widespread among low-G+C Gram-positive bacteria and in many cases contribute to bacterial physiology and virulence. Only recently was it discovered that several Rgg proteins function in cell-to-cell communication (quorum sensing [QS]) via direct interaction with signaling peptides. The mechanism(s) by which Rgg proteins mediate regulation is poorly understood, and further insight into Rgg function is anticipated to be of great importance for the understanding of both regulatory-network architecture and intercellular communication in Rgg-containing species. The results of this study on the Rgg2/3 QS circuit of S. pyogenes demonstrate that DNA binding of target promoters by the activator Rgg2 is directly inhibited by competitive binding by the repressor Rgg3, thereby preventing transcriptional activation of the target genes and premature induction of the QS circuit. This is a unique regulatory mechanism among Rgg proteins and other peptide-responsive QS regulators.

Pre-clinical study of drug combinations that reduce breast cancer burden due to aberrant mTOR and metabolism promoted by LKB1 loss

PubMed Central

Andrade-Vieira, Rafaela; Goguen, Donna; Bentley, Heidi A.; Bowen, Chris V.; Marignani, Paola A.

2014-01-01

Cancer therapies that simultaneously target activated mammalian target of rapamycin (mTOR) and cell metabolism are urgently needed. The goal of our study was to identify therapies that effectively inhibited both mTOR activity and cancer cell metabolism in primary tumors in vivo. Using our mouse model of spontaneous breast cancer promoted by loss of LKB1 expression in an ErbB2 activated model; referred to as LKB1−/−NIC mice, we evaluated the effect of novel therapies in vivo on primary tumors. Treatment of LKB1−/−NIC mice with AZD8055 and 2-DG mono-therapies significantly reduced mammary gland tumorigenesis by inhibiting mTOR pathways and glycolytic metabolism; however simultaneous inhibition of these pathways with AZD8055/2-DG combination was significantly more effective at reducing tumor volume and burden. At the molecular level, combination treatment inhibited mTORC1/mTORC2 activity, selectively inhibited mitochondria function and blocked MAPK pro-survival signaling responsible for the ERK-p90RSK feedback loop. Our findings suggest that loss of LKB1 expression be considered a marker for metabolic dysfunction given its role in regulating AMPK and mTOR function. Finally, the outcome of our pre-clinical study confirms therapies that simultaneously target mTORC1/mTORC2 and glycolytic metabolism in cancer produce the best therapeutic outcome for the treatment of patients harboring metabolically active HER2 positive breast cancers. PMID:25436981

Lentiviral-mediated targeted NF-kappaB blockade in dorsal spinal cord glia attenuates sciatic nerve injury-induced neuropathic pain in the rat.

PubMed

Meunier, Alice; Latrémolière, Alban; Dominguez, Elisa; Mauborgne, Annie; Philippe, Stéphanie; Hamon, Michel; Mallet, Jacques; Benoliel, Jean-Jacques; Pohl, Michel

2007-04-01

Neuropathic pain developing after peripheral nerve injury is associated with altered neuronal and glial cell functions in the spinal cord. Activated glia produces algogenic mediators, exacerbating pain. Among the different intracellular pathways possibly involved in the modified glial function, the nuclear factor kappaB (NF-kappaB) system is of particular interest, as numerous genes encoding inflammation- and pain-related molecules are controlled by this transcription factor. NF-kappaB is a pleiotropic factor also involved in central nervous system homeostasy. To study its role in chronic pain, it is thus essential to inhibit the NF-kappaB pathway selectively in activated spinal glial cells. Here, we show that when restricted to spinal cord and targeted to glial cells, lentiviral vector-mediated delivery of NF-kappaB super- repressor IkappaBalpha resulted in an inhibition of the NF-kappaB pathway activated in the rat spinal cord after sciatic nerve injury (chronic constriction injury, CCI). Concomitantly, IkappaBalpha overproduction prevented the enhanced expression of interleukin-6 and of inducible nitric oxide synthase associated with chronic constriction injury and resulted in prolonged antihyperalgesic and antiallodynic effects. These data show that targeted blockade of NF-kappaB activity in spinal glia efficiently alleviates pain behavior in CCI rats, demonstrating the active participation of the glial NF-kappaB pathway in the development of neuropathic pain after peripheral nerve injury.

AMP-guided tumour-specific nanoparticle delivery via adenosine A1 receptor.

PubMed

Dai, Tongcheng; Li, Na; Han, Fajun; Zhang, Hua; Zhang, Yuanxing; Liu, Qin

2016-03-01

Active targeting-ligands have been increasingly used to functionalize nanoparticles for tumour-specific clinical applications. Here we utilize nucleotide adenosine 5'-monophosphate (AMP) as a novel ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for tumour-targeted imaging. We demonstrate that AMP-conjugated NPs (NPs-AMP) efficiently bind to and are following internalized into colon cancer cell CW-2 and breast cancer cell MDA-MB-468 in vitro. RNA interference and inhibitor assays reveal that the targeting effects mainly rely on the specific binding of AMP to adenosine A1 receptor (A1R), which is greatly up-regulated in cancer cells than in matched normal cells. More importantly, NPs-AMP specifically accumulate in the tumour site of colon and breast tumour xenografts and are further internalized into the tumour cells in vivo via tail vein injection, confirming that the high in vitro specificity of AMP can be successfully translated into the in vivo efficacy. Furthermore, NPs-AMP exhibit an active tumour-targeting behaviour in various colon and breast cancer cells, which is positively related to the up-regulation level of A1R in cancer cells, suggesting that AMP potentially suits for more extensive A1R-overexpressing cancer models. This work establishes AMP to be a novel tumour-targeting ligand and provides a promising strategy for future diagnostic or therapeutic applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Emergence of gamma motor activity in an artificial neural network model of the corticospinal system.

PubMed

Grandjean, Bernard; Maier, Marc A

2017-02-01

Muscle spindle discharge during active movement is a function of mechanical and neural parameters. Muscle length changes (and their derivatives) represent its primary mechanical, fusimotor drive its neural component. However, neither the action nor the function of fusimotor and in particular of γ-drive, have been clearly established, since γ-motor activity during voluntary, non-locomotor movements remains largely unknown. Here, using a computational approach, we explored whether γ-drive emerges in an artificial neural network model of the corticospinal system linked to a biomechanical antagonist wrist simulator. The wrist simulator included length-sensitive and γ-drive-dependent type Ia and type II muscle spindle activity. Network activity and connectivity were derived by a gradient descent algorithm to generate reciprocal, known target α-motor unit activity during wrist flexion-extension (F/E) movements. Two tasks were simulated: an alternating F/E task and a slow F/E tracking task. Emergence of γ-motor activity in the alternating F/E network was a function of α-motor unit drive: if muscle afferent (together with supraspinal) input was required for driving α-motor units, then γ-drive emerged in the form of α-γ coactivation, as predicted by empirical studies. In the slow F/E tracking network, γ-drive emerged in the form of α-γ dissociation and provided critical, bidirectional muscle afferent activity to the cortical network, containing known bidirectional target units. The model thus demonstrates the complementary aspects of spindle output and hence γ-drive: i) muscle spindle activity as a driving force of α-motor unit activity, and ii) afferent activity providing continuous sensory information, both of which crucially depend on γ-drive.

Structure-Function Analysis of the Drosophila melanogaster Caudal Transcription Factor Provides Insights into Core Promoter-preferential Activation.

PubMed

Shir-Shapira, Hila; Sharabany, Julia; Filderman, Matan; Ideses, Diana; Ovadia-Shochat, Avital; Mannervik, Mattias; Juven-Gershon, Tamar

2015-07-10

Regulation of RNA polymerase II transcription is critical for the proper development, differentiation, and growth of an organism. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters encompass the RNA start site and consist of functional elements such as the TATA box, initiator, and downstream core promoter element (DPE), which confer specific properties to the core promoter. We have previously discovered that Drosophila Caudal, which is a master regulator of genes involved in development and differentiation, is a DPE-specific transcriptional activator. Here, we show that the mouse Caudal-related homeobox (Cdx) proteins (mCdx1, mCdx2, and mCdx4) are also preferential core promoter transcriptional activators. To elucidate the mechanism that enables Caudal to preferentially activate DPE transcription, we performed structure-function analysis. Using a systematic series of deletion mutants (all containing the intact DNA-binding homeodomain) we discovered that the C-terminal region of Caudal contributes to the preferential activation of the fushi tarazu (ftz) Caudal target gene. Furthermore, the region containing both the homeodomain and the C terminus of Caudal was sufficient to confer core promoter-preferential activation to the heterologous GAL4 DNA-binding domain. Importantly, we discovered that Drosophila CREB-binding protein (dCBP) is a co-activator for Caudal-regulated activation of ftz. Strikingly, dCBP conferred the ability to preferentially activate the DPE-dependent ftz reporter to mini-Caudal proteins that were unable to preferentially activate ftz transcription themselves. Taken together, it is the unique combination of dCBP and Caudal that enables the co-activation of ftz in a core promoter-preferential manner. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

PubMed

Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F; She, Qunxin

2017-02-28

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles.

PubMed

Molina-Sánchez, Maria D; García-Rodríguez, Fernando M; Toro, Nicolás

2016-01-01

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3' end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro . The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles

PubMed Central

Molina-Sánchez, Maria D.; García-Rodríguez, Fernando M.; Toro, Nicolás

2016-01-01

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods. PMID:27730127

Silk gland-specific proteinase inhibitor serpin16 from the Bombyx mori shows cysteine proteinase inhibitory activity.

PubMed

Guo, Peng-Chao; Dong, Zhaoming; Xiao, Li; Li, Tao; Zhang, Yan; He, Huawei; Xia, Qingyou; Zhao, Ping

2015-01-30

Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity. Copyright © 2014 Elsevier Inc. All rights reserved.

Small molecule promotes β-catenin citrullination and inhibits Wnt signaling in cancer.

PubMed

Qu, Yi; Olsen, Jan Roger; Yuan, Xing; Cheng, Phil F; Levesque, Mitchell P; Brokstad, Karl A; Hoffman, Paul S; Oyan, Anne Margrete; Zhang, Weidong; Kalland, Karl-Henning; Ke, Xisong

2018-01-01

Wnt (wingless)/β-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of β-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of β-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or β-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of β-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

Synthesis and Evaluation of the Anticancer and Trypanocidal Activities of Boronic Tyrphostins.

PubMed

de J Hiller, Noemi; A A E Silva, Nayane; Faria, Robson X; Souza, André Luís A; Resende, Jackson A L C; Borges Farias, André; Correia Romeiro, Nelilma; de Luna Martins, Daniela

2018-06-01

Molecules containing an (cyanovinyl)arene moiety are known as tyrphostins because of their ability to inhibit proteins from the tyrosine kinase family, an interesting target for the development of anticancer and trypanocidal drugs. In the present work, (E)-(cyanovinyl)benzeneboronic acids were synthesized by Knoevenagel condensations without the use of any catalysts in water through a simple protocol that completely avoided the use of organic solvents in the synthesis and workup process. The in vitro anticancer and trypanocidal activities of the synthesized boronic acids were also evaluated, and it was discovered that the introduction of the boronic acid functionality improved the activity of the boronic tyrphostins. In silico target fishing with the use of a chemogenomic approach suggested that tyrosine-phosphorylation-regulated kinase 1a (DYRK1A) was a potential target for some of the designed compounds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Search for α -Cluster Structure in Exotic Nuclei with the Prototype Active-Target Time-Projection Chamber

NASA Astrophysics Data System (ADS)

Fritsch, A.; Ayyad, Y.; Bazin, D.; Beceiro-Novo, S.; Bradt, J.; Carpenter, L.; Cortesi, M.; Mittig, W.; Suzuki, D.; Ahn, T.; Kolata, J. J.; Howard, A. M.; Becchetti, F. D.; Wolff, M.

Some exotic nuclei appear to exhibit α -cluster structure, which may impact nucleosynthesis reaction rates. While various theoretical models currently describe such clustering, more experimental data are needed to constrain model predictions. The Prototype Active-Target Time-Projection Chamber (PAT-TPC) has low-energy thresholds for charged-particle decay and a high detection efficiency due to its thick gaseous active target volume, making it well-suited to search for low-energy α -cluster reactions. Radioactive-ion beams produced by the TwinSol facility at the University of Notre Dame were delivered to the PAT-TPC to study 14C via α -resonant scattering. Differential cross sections and excitation functions were measured and show evidence of three-body exit channels. Additional data were measured with an updated Micromegas detector more sensitive to three-body decay. Preliminary results are presented.

RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes

PubMed Central

Rose, Nathan R; King, Hamish W; Blackledge, Neil P; Fursova, Nadezda A; Ember, Katherine JI; Fischer, Roman; Kessler, Benedikt M; Klose, Robert J

2016-01-01

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation. DOI: http://dx.doi.org/10.7554/eLife.18591.001 PMID:27705745

Microarray-based determination of anti-inflammatory genes targeted by 6-(methylsulfinyl)hexyl isothiocyanate in macrophages

PubMed Central

CHEN, JIHUA; UTO, TAKUHIRO; TANIGAWA, SHUNSUKE; YAMADA-KATO, TOMEO; FUJII, MAKOTO; HOU, DE-XING

2010-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as ‘defense, inflammatory response, cytokine activities and receptor activities’ and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function. PMID:23136589

Teaching refusal skills to sexually active adolescents.

PubMed

Warzak, W J; Page, T J

1990-06-01

Refusal skills training was extended to sexually active handicapped female adolescents who lacked an effective refusal strategy. Role-plays for assessment and training were developed using the who, what, when and where of situations which resulted in unwanted intercourse. Refusal skills were trained following the format of rationale, modeling, rehearsal, feedback, and reinforcement. Baseline rates of most target behaviors were quite low. High frequencies of target behaviors were observed as each behavior became the focus of training. Generalization across staff and time was also observed. The skillfulness and effectiveness of the subjects' refusal skills were judged to be improved as a function of training. One-year follow-up showed decreased sexual activity for each girl.

The inhibitory effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members on the activity of cellular microRNAs.

PubMed

Zhang, Hui

2010-01-01

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or APOBEC3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. However, the cellular function of APOBEC3G remains to be further clarified. It has been reported that APOBEC3s can restrict the mobility of endogenous retroviruses and LTR-retrotransposons, suggesting that they can maintain stability in host genomes. However, APOBEC3G is normally cytoplasmic. Further studies have demonstrated that it is associated with an RNase-sensitive high molecular mass (HMM) and located in processing bodies (P-bodies) of replicating T-cells, indicating that the major cellular function of APOBEC3G seems to be related to P-body-related RNA processing and metabolism. As the function of P-body is closely related to miRNA activity, APOBEC3G could affect the miRNA function. Recent studies have demonstrated that APOBEC3G and its family members counteract miRNA-mediated repression of protein translation. Further, APOBEC3G enhances the association of miRNA-targeted mRNA with polysomes, and facilitates the dissociation of miRNA-targeted mRNA from P-bodies. As such, APOBEC3G regulate the activity of cellular miRNAs. Whether this function is related to its potent antiviral activity remains to be further determined.

Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR).

PubMed

Ward, Richard A; Anderton, Mark J; Ashton, Susan; Bethel, Paul A; Box, Matthew; Butterworth, Sam; Colclough, Nicola; Chorley, Christopher G; Chuaqui, Claudio; Cross, Darren A E; Dakin, Les A; Debreczeni, Judit É; Eberlein, Cath; Finlay, M Raymond V; Hill, George B; Grist, Matthew; Klinowska, Teresa C M; Lane, Clare; Martin, Scott; Orme, Jonathon P; Smith, Peter; Wang, Fengjiang; Waring, Michael J

2013-09-12

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Nonlinear changes in brain activity during continuous word repetition: an event-related multiparametric functional MR imaging study.

PubMed

Hagenbeek, R E; Rombouts, S A R B; Veltman, D J; Van Strien, J W; Witter, M P; Scheltens, P; Barkhof, F

2007-10-01

Changes in brain activation as a function of continuous multiparametric word recognition have not been studied before by using functional MR imaging (fMRI), to our knowledge. Our aim was to identify linear changes in brain activation and, what is more interesting, nonlinear changes in brain activation as a function of extended word repetition. Fifteen healthy young right-handed individuals participated in this study. An event-related extended continuous word-recognition task with 30 target words was used to study the parametric effect of word recognition on brain activation. Word-recognition-related brain activation was studied as a function of 9 word repetitions. fMRI data were analyzed with a general linear model with regressors for linearly changing signal intensity and nonlinearly changing signal intensity, according to group average reaction time (RT) and individual RTs. A network generally associated with episodic memory recognition showed either constant or linearly decreasing brain activation as a function of word repetition. Furthermore, both anterior and posterior cingulate cortices and the left middle frontal gyrus followed the nonlinear curve of the group RT, whereas the anterior cingulate cortex was also associated with individual RT. Linear alteration in brain activation as a function of word repetition explained most changes in blood oxygen level-dependent signal intensity. Using a hierarchically orthogonalized model, we found evidence for nonlinear activation associated with both group and individual RTs.

Transient Receptor Potential Mucolipin 1 (TRPML1) and Two-pore Channels Are Functionally Independent Organellar Ion Channels*

PubMed Central

Yamaguchi, Soichiro; Jha, Archana; Li, Qin; Soyombo, Abigail A.; Dickinson, George D.; Churamani, Dev; Brailoiu, Eugen; Patel, Sandip; Muallem, Shmuel

2011-01-01

NAADP is a potent second messenger that mobilizes Ca2+ from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca2+ release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca2+-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca2+ influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca2+ signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca2+ signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca2+ oscillations in pancreatic acinar cells were identical in wild-type and TRPML1−/− cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP. PMID:21540176

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma

PubMed Central

Phelps, Michael P.; Bailey, Jenna N.; Vleeshouwer-Neumann, Terra

2016-01-01

Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity. PMID:27956629

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma.

PubMed

Phelps, Michael P; Bailey, Jenna N; Vleeshouwer-Neumann, Terra; Chen, Eleanor Y

2016-12-27

Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.

Control of vascular smooth muscle function by Src-family kinases and reactive oxygen species in health and disease

PubMed Central

MacKay, Charles E; Knock, Greg A

2015-01-01

Abstract Reactive oxygen species (ROS) are now recognised as second messenger molecules that regulate cellular function by reversibly oxidising specific amino acid residues of key target proteins. Amongst these are the Src-family kinases (SrcFKs), a multi-functional group of non-receptor tyrosine kinases highly expressed in vascular smooth muscle (VSM). In this review we examine the evidence supporting a role for ROS-induced SrcFK activity in normal VSM contractile function and in vascular remodelling in cardiovascular disease. VSM contractile responses to G-protein-coupled receptor stimulation, as well as hypoxia in pulmonary artery, are shown to be dependent on both ROS and SrcFK activity. Specific phosphorylation targets are identified amongst those that alter intracellular Ca2+ concentration, including transient receptor potential channels, voltage-gated Ca2+ channels and various types of K+ channels, as well as amongst those that regulate actin cytoskeleton dynamics and myosin phosphatase activity, including focal adhesion kinase, protein tyrosine kinase-2, Janus kinase, other focal adhesion-associated proteins, and Rho guanine nucleotide exchange factors. We also examine a growing weight of evidence in favour of a key role for SrcFKs in multiple pro-proliferative and anti-apoptotic signalling pathways relating to oxidative stress and vascular remodelling, with a particular focus on pulmonary hypertension, including growth-factor receptor transactivation and downstream signalling, hypoxia-inducible factors, positive feedback between SrcFK and STAT3 signalling and positive feedback between SrcFK and NADPH oxidase dependent ROS production. We also discuss evidence for and against the potential therapeutic targeting of SrcFKs in the treatment of pulmonary hypertension. PMID:25384773

The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

PubMed Central

Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

2013-01-01

BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

Factors associated with fear of falling in people with Parkinson’s disease

PubMed Central

2014-01-01

Background This study aimed to comprehensibly investigate potential contributing factors to fear of falling (FOF) among people with idiopathic Parkinson’s disease (PD). Methods The study included 104 people with PD. Mean (SD) age and PD-duration were 68 (9.4) and 5 (4.2) years, respectively, and the participants’ PD-symptoms were relatively mild. FOF (the dependent variable) was investigated with the Swedish version of the Falls Efficacy Scale, i.e. FES(S). The first multiple linear regression model replicated a previous study and independent variables targeted: walking difficulties in daily life; freezing of gait; dyskinesia; fatigue; need of help in daily activities; age; PD-duration; history of falls/near falls and pain. Model II included also the following clinically assessed variables: motor symptoms, cognitive functions, gait speed, dual-task difficulties and functional balance performance as well as reactive postural responses. Results Both regression models showed that the strongest contributing factor to FOF was walking difficulties, i.e. explaining 60% and 64% of the variance in FOF-scores, respectively. Other significant independent variables in both models were needing help from others in daily activities and fatigue. Functional balance was the only clinical variable contributing additional significant information to model I, increasing the explained variance from 66% to 73%. Conclusions The results imply that one should primarily target walking difficulties in daily life in order to reduce FOF in people mildly affected by PD. This finding applies even when considering a broad variety of aspects not previously considered in PD-studies targeting FOF. Functional balance performance, dependence in daily activities, and fatigue were also independently associated with FOF, but to a lesser extent. Longitudinal studies are warranted to gain an increased understanding of predictors of FOF in PD and who is at risk of developing a FOF. PMID:24456482

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation

USDA-ARS?s Scientific Manuscript database

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-t...

Open-Trial Pilot Study of a Comprehensive Outpatient Psychosocial Treatment for Children with High-Functioning Autism Spectrum Disorder

ERIC Educational Resources Information Center

Lopata, Christopher; Lipinski, Alanna M.; Thomeer, Marcus L.; Rodgers, Jonathan D.; Donnelly, James P.; McDonald, Christin A.; Volker, Martin A.

2017-01-01

This study examined the feasibility and initial outcomes of a comprehensive outpatient psychosocial treatment (MAXout) for children aged 7-12 years with high-functioning autism spectrum disorder. The 18-week treatment, two 90-minute sessions per week, included instruction and therapeutic activities targeting social/social communication skills,…

Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover

PubMed Central

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J.; Shi, Yang; Harper, J. Wade

2014-01-01

Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

PubMed

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J; Shi, Yang; Harper, J Wade

2013-10-10

Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

Nuclear Glycolytic Enzyme Enolase of Toxoplasma gondii Functions as a Transcriptional Regulator

PubMed Central

Mouveaux, Thomas; Oria, Gabrielle; Werkmeister, Elisabeth; Slomianny, Christian; Fox, Barbara A.; Bzik, David J.; Tomavo, Stanislas

2014-01-01

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii. PMID:25153525

Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics.

PubMed

Suzuki, Hiroshi I; Spengler, Ryan M; Grigelioniene, Giedre; Kobayashi, Tatsuya; Sharp, Phillip A

2018-05-01

RNA interference (RNAi) is a major, powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a crosstalk scenario, designated as crosstalk with endogenous RBPs' (ceRBP), in which small interfering RNAs or microRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of small interfering RNA (siRNA) off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, identified that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of microRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics.

Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

PubMed Central

Torres, Jaume; Surya, Wahyu; Li, Yan; Liu, Ding Xiang

2015-01-01

Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i) the envelope protein in coronaviruses and (ii) the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity. PMID:26053927

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Mitochondrial Targets for Pharmacological Intervention in Human Disease

PubMed Central

2015-01-01

Over the past several years, mitochondrial dysfunction has been linked to an increasing number of human illnesses, making mitochondrial proteins (MPs) an ever more appealing target for therapeutic intervention. With 20% of the mitochondrial proteome (312 of an estimated 1500 MPs) having known interactions with small molecules, MPs appear to be highly targetable. Yet, despite these targeted proteins functioning in a range of biological processes (including induction of apoptosis, calcium homeostasis, and metabolism), very few of the compounds targeting MPs find clinical use. Recent work has greatly expanded the number of proteins known to localize to the mitochondria and has generated a considerable increase in MP 3D structures available in public databases, allowing experimental screening and in silico prediction of mitochondrial drug targets on an unprecedented scale. Here, we summarize the current literature on clinically active drugs that target MPs, with a focus on how existing drug targets are distributed across biochemical pathways and organelle substructures. Also, we examine current strategies for mitochondrial drug discovery, focusing on genetic, proteomic, and chemogenomic assays, and relevant model systems. As cell models and screening techniques improve, MPs appear poised to emerge as relevant targets for a wide range of complex human diseases, an eventuality that can be expedited through systematic analysis of MP function. PMID:25367773

Functional characterisation of Arabidopsis SPL7 conserved protein domains suggests novel regulatory mechanisms in the Cu deficiency response.

PubMed

Garcia-Molina, Antoni; Xing, Shuping; Huijser, Peter

2014-08-30

The Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis. Cytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function. Here, we propose a revised model of SPL7 activation and regulation. According to our results, SPL7 would be initially located to endomembranes and activated during ER stress as a result of Cu deficiency. Furthermore, we added the SPL7 dimerization in the presence of Cu ions as an additional regulatory mechanism to modulate the Cu deficiency response.

Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion

PubMed Central

Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

2013-01-01

Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion. PMID:23337583

Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion.

PubMed

Zhou, Jing; Tan, Shi-Hao; Nicolas, Valérie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming

2013-04-01

Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

Nanodiamonds as Carriers for Address Delivery of Biologically Active Substances

NASA Astrophysics Data System (ADS)

Purtov, K. V.; Petunin, A. I.; Burov, A. E.; Puzyr, A. P.; Bondar, V. S.

2010-03-01

Surface of detonation nanodiamonds was functionalized for the covalent attachment of immunoglobulin, and simultaneously bovine serum albumin and Rabbit Anti-Mouse Antibody. The nanodiamond-IgGI125 and RAM-nanodiamond-BSAI125 complexes are stable in blood serum and the immobilized proteins retain their biological activity. It was shown that the RAM-nanodiamond-BSAI125 complex is able to bind to the target antigen immobilized on the Sepharose 6B matrix through antibody-antigen interaction. The idea can be extended to use nanodiamonds as carriers for delivery of bioactive substances (i.e., drugs) to various targets in vivo.

EphB4 promotes or suppresses Ras/MEK/ERK pathway in a context-dependent manner: Implications for EphB4 as a cancer target.

PubMed

Xiao, Zhan; Carrasco, Rosa; Kinneer, Krista; Sabol, Darrin; Jallal, Bahija; Coats, Steve; Tice, David A

2012-06-01

EphB4 is a member of the Eph receptor tyrosine kinase family shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4's function in cancer is much less clear. Here we illustrate both tumor promoting as well as suppressing roles of EphB4, by showing that its activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but activation of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with EphrinB2, its natural ligand, or an agonistic monoclonal antibody for EphB4. Correspondingly, EphB4 activation stimulated MCF7 growth while inhibiting HUVEC cell proliferation. The reason for these dramatic differences is due to functional coupling of EphB4 to different downstream effectors. Reduction of p120 RasGAP in HUVEC cells attenuated the inhibitory effect of EphB4 activation on the ERK pathway, whereas knockdown of PP2A in MCF7 cells attenuated EphB4 activation of the ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been demonstrated leading to activation of an oncogenic pathway. Our study illustrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the conflicting effects on cancer cell and endothelial cell compartments.

CREB at the Crossroads of Activity-Dependent Regulation of Nervous System Development and Function.

PubMed

Belgacem, Yesser H; Borodinsky, Laura N

2017-01-01

The central nervous system is a highly plastic network of cells that constantly adjusts its functions to environmental stimuli throughout life. Transcription-dependent mechanisms modify neuronal properties to respond to external stimuli regulating numerous developmental functions, such as cell survival and differentiation, and physiological functions such as learning, memory, and circadian rhythmicity. The discovery and cloning of the cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB) constituted a big step toward deciphering the molecular mechanisms underlying neuronal plasticity. CREB was first discovered in learning and memory studies as a crucial mediator of activity-dependent changes in target gene expression that in turn impose long-lasting modifications of the structure and function of neurons. In this chapter, we review the molecular and signaling mechanisms of neural activity-dependent recruitment of CREB and its cofactors. We discuss the crosstalk between signaling pathways that imprints diverse spatiotemporal patterns of CREB activation allowing for the integration of a wide variety of stimuli.

FAR and NEAR Target Dynamic Visual Acuity: A Functional Assessment of Canal and Otolith Performance

NASA Technical Reports Server (NTRS)

Peters, Brian T.; Brady, Rachel A.; Landsness, Eric C.; Black, F. Owen; Bloomberg, Jacob J.

2004-01-01

Upon their return to earth, astronauts experience the effects of vestibular adaptation to microgravity. The postflight changes in vestibular information processing can affect postural and locomotor stability and may lead to oscillopsia during activities of daily living. However, it is likely that time spent in microgravity affects canal and otolith function differently. As a result, the isolated rotational stimuli used in traditional tests of canal function may fail to identify vestibular deficits after spaceflight. Also, the functional consequences of deficits that are identified often remain unknown. In a gaze control task, the relative contributions of the canal and otolith organs are modulated with viewing distance. The ability to stabilize gaze during a perturbation, on visual targets placed at different distances from the head may therefore provide independent insight into the function of this systems. Our goal was to develop a functional measure of gaze control that can also offer independent information about the function of the canal and otolith organs.

WAVE3-NFκB Interplay Is Essential for the Survival and Invasion of Cancer Cells

PubMed Central

Davuluri, Gangarao; Augoff, Katarzyna; Schiemann, William P.; Plow, Edward F.; Sossey-Alaoui, Khalid

2014-01-01

The WAVE3 cytoskeletal protein promotes cancer invasion and metastasis. We have shown that the WAVE3-mediated activation of cancer cell invasion is due, in part, to its regulation of expression and activity of key metalloproteinases (MMPs), including MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potential linkage of WAVE3 to this pathway, which we sought to investigate. Mechanistically, we found that loss of WAVE3 in cancer cells leads to inhibition of NFκB signaling as a result of a decrease in the nuclear translocation of NFκB and therefore loss of activation of NFκB target genes. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity. Both pharmacologic and genetic manipulations of NFκB effector molecules show that the biological consequence of loss of WAVE3 function in the NFκB pathway result the inhibition of invadopodia formation and ECM degradation by cancer cells, and these changes are a consequence of decreased MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to apoptosis and cell death driven by TNFα, through the inhibition of the AKT pro-survival pathway. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation. Therefore, targeted therapeutic inhibition of WAVE3 will sensitize cancer cells to apoptosis and cell death, and suppress cancer invasion and metastasis. PMID:25329315

WAVE3-NFκB interplay is essential for the survival and invasion of cancer cells.

PubMed

Davuluri, Gangarao; Augoff, Katarzyna; Schiemann, William P; Plow, Edward F; Sossey-Alaoui, Khalid

2014-01-01

The WAVE3 cytoskeletal protein promotes cancer invasion and metastasis. We have shown that the WAVE3-mediated activation of cancer cell invasion is due, in part, to its regulation of expression and activity of key metalloproteinases (MMPs), including MMP9, which is centrally involved in invadopodia-mediated degradation of the extracellular matrix (ECM). MMP9 is also a major NFκB target gene, suggesting a potential linkage of WAVE3 to this pathway, which we sought to investigate. Mechanistically, we found that loss of WAVE3 in cancer cells leads to inhibition of NFκB signaling as a result of a decrease in the nuclear translocation of NFκB and therefore loss of activation of NFκB target genes. Conversely, overexpression of WAVE3 was sufficient to enhance NFκB activity. Both pharmacologic and genetic manipulations of NFκB effector molecules show that the biological consequence of loss of WAVE3 function in the NFκB pathway result the inhibition of invadopodia formation and ECM degradation by cancer cells, and these changes are a consequence of decreased MMP9 expression and activity. Loss of WAVE3 also sensitized cancer cells to apoptosis and cell death driven by TNFα, through the inhibition of the AKT pro-survival pathway. Our results identify a novel function of WAVE3 in NFκB signaling, where its activity is essential for the regulation of invadopodia and ECM degradation. Therefore, targeted therapeutic inhibition of WAVE3 will sensitize cancer cells to apoptosis and cell death, and suppress cancer invasion and metastasis.

Inhibition of human papillomavirus expression using DNAzymes.

PubMed

Benítez-Hess, María Luisa; Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis Marat

2011-01-01

Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects.

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity.

PubMed

Liang, Lunxi; Wang, Huanbin; Shi, Hubing; Li, Zhaoli; Yao, Han; Bu, Zhigao; Song, Ningning; Li, Chushu; Xiang, Dabin; Zhang, Yao; Wang, Jilin; Hu, Ye; Xu, Qi; Ma, Yanlei; Cheng, Zhongyi; Wang, Yingchao; Zhao, Shuliang; Qian, Jin; Chen, Yingxuan; Fang, Jing-Yuan; Xu, Jie

2018-06-21

Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule. Copyright © 2018 Elsevier Ltd. All rights reserved.

MicroRNA cluster miR-17-92 Cluster in Exosomes Enhance Neuroplasticity and Functional Recovery After Stroke in Rats.

PubMed

Xin, Hongqi; Katakowski, Mark; Wang, Fengjie; Qian, Jian-Yong; Liu, Xian Shuang; Ali, Meser M; Buller, Benjamin; Zhang, Zheng Gang; Chopp, Michael

2017-03-01

Multipotent mesenchymal stromal cell (MSC) harvested exosomes are hypothesized as the major paracrine effectors of MSCs. In vitro, the miR-17-92 cluster promotes oligodendrogenesis, neurogenesis, and axonal outgrowth. We, therefore, investigated whether the miR-17-92 cluster-enriched exosomes harvested from MSCs transfected with an miR-17-92 cluster plasmid enhance neurological recovery compared with control MSC-derived exosomes. Rats subjected to 2 hours of transient middle cerebral artery occlusion were intravenously administered miR-17-92 cluster-enriched exosomes, control MSC exosomes, or liposomes and were euthanized 28 days post-middle cerebral artery occlusion. Histochemistry, immunohistochemistry, and Golgi-Cox staining were used to assess dendritic, axonal, synaptic, and myelin remodeling. Expression of phosphatase and tensin homolog and activation of its downstream proteins, protein kinase B, mechanistic target of rapamycin, and glycogen synthase kinase 3β in the peri-infarct region were measured by means of Western blots. Compared with the liposome treatment, both exosome treatment groups exhibited significant improvement of functional recovery, but miR-17-92 cluster-enriched exosome treatment had significantly more robust effects on improvement of neurological function and enhancements of oligodendrogenesis, neurogenesis, and neurite remodeling/neuronal dendrite plasticity in the ischemic boundary zone (IBZ) than the control MSC exosome treatment. Moreover, miR-17-92 cluster-enriched exosome treatment substantially inhibited phosphatase and tensin homolog, a validated miR-17-92 cluster target gene, and subsequently increased the phosphorylation of phosphatase and tensin homolog downstream proteins, protein kinase B, mechanistic target of rapamycin, and glycogen synthase kinase 3β compared with control MSC exosome treatment. Our data suggest that treatment of stroke with tailored exosomes enriched with the miR-17-92 cluster increases neural plasticity and functional recovery after stroke, possibly via targeting phosphatase and tensin homolog to activate the PI3K/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3β signaling pathway. © 2017 American Heart Association, Inc.

Diacylglycerol kinase α inactivation is an integral component of the costimulatory pathway that amplifies TCR signals.

PubMed

Arranz-Nicolás, Javier; Ogando, Jesús; Soutar, Denise; Arcos-Pérez, Raquel; Meraviglia-Crivelli, Daniel; Mañes, Santos; Mérida, Isabel; Ávila-Flores, Antonia

2018-06-01

The arsenal of cancer therapies has evolved to target T lymphocytes and restore their capacity to destroy tumor cells. T cells rely on diacylglycerol (DAG) to carry out their functions. DAG availability and signaling are regulated by the enzymes diacylglycerol kinase (DGK) α and ζ, whose excess function drives T cells into hyporesponsive states. Targeting DGKα is a promising strategy for coping with cancer; its blockade could reinstate T-cell attack on tumors while limiting tumor growth, due to positive DGKα functions in several oncogenic pathways. Here, we made a side-by-side comparison of the effects of commercial pharmacological DGK inhibitors on T-cell responses with those promoted by DGKα and DGKζ genetic deletion or silencing. We show the specificity for DGKα of DGK inhibitors I and II and the structurally similar compound ritanserin. Inhibitor treatment promoted Ras/ERK (extracellular signal-regulated kinase) signaling and AP-1 (Activator protein-1) transcription, facilitated DGKα membrane localization, reduced the requirement for costimulation, and cooperated with enhanced activation following DGKζ silencing/deletion. DGKiII and ritanserin had similar effects on TCR proximal signaling, but ritanserin counteracted long-term T-cell activation, an effect that was potentiated in DGKα -/- cells. In contrast with enhanced activation triggered by pharmacological inhibition, DGKα silencing/genetic deletion led to impaired Lck (lymphocyte-specific protein tyrosine kinase) activation and limited costimulation responses. Our results demonstrate that pharmacological inhibition of DGKα downstream of the TCR provides a gain-of-function effect that amplifies the DAG-dependent signaling cascade, an ability that could be exploited therapeutically to reinvigorate T cells to attack tumors.

Aversive stimuli exacerbate defensive motor behaviour in motor conversion disorder.

PubMed

Blakemore, Rebekah L; Sinanaj, Indrit; Galli, Silvio; Aybek, Selma; Vuilleumier, Patrik

2016-12-01

Conversion disorder or functional neurological symptom disorder (FND) can affect the voluntary motor system, without an organic cause. Functional symptoms are thought to be generated unconsciously, arising from underlying psychological stressors. However, attempts to demonstrate a direct relationship between the limbic system and disrupted motor function in FND are lacking. We tested whether negative affect would exacerbate alterations of motor control and corresponding brain activations in individuals with FND. Ten patients and ten healthy controls produced an isometric precision-grip contraction at 10% of maximum force while either viewing visual feedback of their force output, or unpleasant or pleasant emotional images (without feedback). Force magnitude was continuously recorded together with change in brain activity using fMRI. For controls, force output decayed from the target level while viewing pleasant and unpleasant images. Patients however, maintained force at the target level without decay while viewing unpleasant images, indicating a pronounced effect of negative affect on force output in FND. This emotional modulation of force control was associated with different brain activation patterns between groups. Contrasting the unpleasant with the pleasant condition, controls showed increased activity in the inferior frontal cortex and pre-supplementary motor area, whereas patients had greater activity in the cerebellum (vermis), posterior cingulate cortex, and hippocampus. Engagement of a cerebellar-limbic network in patients is consistent with heightened processing of emotional salience, and supports the role of the cerebellum in freezing responses in the presence of aversive events. These data highlight a possible neural circuit through which psychological stressors elicit defensive behaviour and modulate motor function in FND. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sorafenib paradoxically activates the RAS/RAF/ERK pathway in polyclonal human NK cells during expansion and thereby enhances effector functions in a dose- and time-dependent manner.

PubMed

Lohmeyer, J; Nerreter, T; Dotterweich, J; Einsele, H; Seggewiss-Bernhardt, R

2018-03-24

Natural killer (NK) cells play a major role in host immunity against leukaemia and lymphoma. However, clinical trials applying NK cells have not been as efficient as hoped for. Patients treated with rapidly accelerated fibrosarcoma (RAF) inhibitors exhibit increased tumour infiltration by immune cells, suggesting that a combination of RAF inhibitors with immunotherapy might be beneficial. As mitogen-activated protein kinases (MAPKs) such as raf-1 proto-oncogene, serine/threonine kinase (CRAF) regulate NK cell functions, we performed an in-vitro investigation on the potential of clinically relevant short-acting tyrosine kinase inhibitors (TKIs) as potential adjuvants for NK cell therapy: NK cells from healthy human blood donors were thus treated with sorafenib, sunitinib or the pan-RAF inhibitor ZM336372 during ex-vivo expansion. Functional outcomes assessed after washout of the drugs included cytokine production, degranulation, cytotoxicity, apoptosis induction and signal transduction with/without target cell contact. Paradoxically, sorafenib enhanced NK cell effector functions in a time- and dose-dependent manner by raising the steady-state activation level. Of note, this did not lead to NK cell exhaustion, but enhanced activity against target cells such as K562 or Daudis mediated via the RAS/RAF/extracellular-regulated kinase (ERK) pathway, but not via protein kinase B (AKT). Our data will pave the path to develop a rationale for the considered use of RAF inhibitors such as sorafenib for pre-activation in NK cell-based adoptive immune therapy. © 2018 British Society for Immunology.

Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

PubMed

Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D

2017-04-07

Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.

Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

PubMed Central

Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D.

2017-01-01

Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification. PMID:28387220

Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

PubMed

Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

2015-01-14

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

Digestive proteolysis in the Colorado potato beetle, Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network.

PubMed

Srp, Jaroslav; Nussbaumerová, Martina; Horn, Martin; Mareš, Michael

2016-11-01

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease.

PubMed

Denadai-Souza, Alexandre; Bonnart, Chrystelle; Tapias, Núria Solà; Marcellin, Marlène; Gilmore, Brendan; Alric, Laurent; Bonnet, Delphine; Burlet-Schiltz, Odile; Hollenberg, Morley D; Vergnolle, Nathalie; Deraison, Céline

2018-05-18

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

Drug-Target Kinetics in Drug Discovery.

PubMed

Tonge, Peter J

2018-01-17

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.

Drug–Target Kinetics in Drug Discovery

PubMed Central

2017-01-01

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure–kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug–target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug–target kinetics into predictions of drug activity. PMID:28640596

2D and 3D similarity landscape analysis identifies PARP as a novel off-target for the drug Vatalanib.

PubMed

Gohlke, Bjoern-Oliver; Overkamp, Tim; Richter, Anja; Richter, Antje; Daniel, Peter T; Gillissen, Bernd; Preissner, Robert

2015-09-24

Searching for two-dimensional (2D) structural similarities is a useful tool to identify new active compounds in drug-discovery programs. However, as 2D similarity measures neglect important structural and functional features, similarity by 2D might be underestimated. In the present study, we used combined 2D and three-dimensional (3D) similarity comparisons to reveal possible new functions and/or side-effects of known bioactive compounds. We utilised more than 10,000 compounds from the SuperTarget database with known inhibition values for twelve different anti-cancer targets. We performed all-against-all comparisons resulting in 2D similarity landscapes. Among the regions with low 2D similarity scores are inhibitors of vascular endothelial growth factor receptor (VEGFR) and inhibitors of poly ADP-ribose polymerase (PARP). To demonstrate that 3D landscape comparison can identify similarities, which are untraceable in 2D similarity comparisons, we analysed this region in more detail. This 3D analysis showed the unexpected structural similarity between inhibitors of VEGFR and inhibitors of PARP. Among the VEGFR inhibitors that show similarities to PARP inhibitors was Vatalanib, an oral "multi-targeted" small molecule protein kinase inhibitor being studied in phase-III clinical trials in cancer therapy. An in silico docking simulation and an in vitro HT universal colorimetric PARP assay confirmed that the VEGFR inhibitor Vatalanib exhibits off-target activity as a PARP inhibitor, broadening its mode of action. In contrast to the 2D-similarity search, the 3D-similarity landscape comparison identifies new functions and side effects of the known VEGFR inhibitor Vatalanib.

PDK1/Akt/PDE4D axis identified as a target for asthma remedy synergistic with β2 AR agonists by a natural agent arctigenin.

PubMed

Fang, R; Cui, Q; Sun, J; Duan, X; Ma, X; Wang, W; Cheng, B; Liu, Y; Hou, Y; Bai, G

2015-12-01

Asthma is a heterogenetic disorder characterized by chronic inflammation with variable airflow obstruction and airway hyper-responsiveness. As the most potent and popular bronchodilators, β2 adrenergic receptor (β2 AR) agonists bind to the β2 ARs that are coupled via a stimulatory G protein to adenylyl cyclase, thereby improving cAMP accumulation and resulting in airway smooth muscle relaxation. We previously demonstrated arctigenin had a synergistic function with the β2 AR agonist, but the target for this remained elusive. Chemical proteomics capturing was used to enrich and uncover the target of arctigenin in human bronchial smooth muscle cells, and reverse docking and molecular dynamic stimulation were performed to evaluate the binding of arctigenin and its target. In vitro enzyme activities and protein levels were demonstrated with special kits and Western blotting. Finally, guinea pig tracheal muscle segregation and ex vivo function were analysed. Arctigenin bound to PDK1 with an ideal binding free energy -25.45 kcal/mol and inhibited PDK1 kinase activity without changing its protein level. Additionally, arctigenin reduced PKB/Akt-induced phosphorylation of PDE4D, which was first identified in this study. Attenuation of PDE4D resulted in cAMP accumulation in human bronchial smooth muscle. The inhibition of PDK1 showed a synergistic function with β2 AR agonists and relaxed the constriction of segregated guinea pig tracheal muscle. The PDK1/Akt/PDE4D axis serves as a novel asthma target, which may benefit airflow obstruction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dissociation in decision bias mechanism between probabilistic information and previous decision

PubMed Central

Kaneko, Yoshiyuki; Sakai, Katsuyuki

2015-01-01

Target detection performance is known to be influenced by events in the previous trials. It has not been clear, however, whether this bias effect is due to the previous sensory stimulus, motor response, or decision. Also it remains open whether or not the previous trial effect emerges via the same mechanism as the effect of knowledge about the target probability. In the present study, we asked normal human subjects to make a decision about the presence or absence of a visual target. We presented a pre-cue indicating the target probability before the stimulus, and also a decision-response mapping cue after the stimulus so as to tease apart the effect of decision from that of motor response. We found that the target detection performance was significantly affected by the probability cue in the current trial and also by the decision in the previous trial. While the information about the target probability modulated the decision criteria, the previous decision modulated the sensitivity to target-relevant sensory signals (d-prime). Using functional magnetic resonance imaging (fMRI), we also found that activation in the left intraparietal sulcus (IPS) was decreased when the probability cue indicated a high probability of the target. By contrast, activation in the right inferior frontal gyrus (IFG) was increased when the subjects made a target-present decision in the previous trial, but this change was observed specifically when the target was present in the current trial. Activation in these regions was associated with individual-difference in the decision computation parameters. We argue that the previous decision biases the target detection performance by modulating the processing of target-selective information, and this mechanism is distinct from modulation of decision criteria due to expectation of a target. PMID:25999844

Structure–function studies of STAR family Quaking proteins bound to their in vivo RNA target sites

PubMed Central

Teplova, Marianna; Hafner, Markus; Teplov, Dmitri; Essig, Katharina; Tuschl, Thomas; Patel, Dinshaw J.

2013-01-01

Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins. PMID:23630077

Immune function in arctic mammals: Natural killer (NK) cell-like activity in polar bear, muskox and reindeer.

PubMed

Desforges, Jean-Pierre; Jasperse, Lindsay; Jensen, Trine Hammer; Grøndahl, Carsten; Bertelsen, Mads F; Guise, Sylvain De; Sonne, Christian; Dietz, Rune; Levin, Milton

2018-01-01

Natural killer (NK) cells are a vital part of the rapid and non-specific immune defense against invading pathogens and tumor cells. This study evaluated NK cell-like activity by flow cytometry for the first time in three ecologically and culturally important Arctic mammal species: polar bear (Ursus maritimus), muskox (Ovibos moschatus) and reindeer (Rangifer tarandus). NK cell-like activity for all three species was most effective against the mouse lymphoma cell line YAC-1, compared to the human leukemia cell line K562; NK cell response displayed the characteristic increase in cytotoxic activity when the effector:target cell ratio increased. Comparing NK activity between fresh and cryopreserved mouse lymphocytes revealed little to no difference in function, highlighting the applicability of cryopreserving cells in field studies. The evaluation of this important innate immune function in Arctic mammals can contribute to future population health assessments, especially as pollution-induced suppression of immune function may increase infectious disease susceptibility. Copyright © 2017 Elsevier B.V. All rights reserved.

Biological and functional relevance of CASP predictions

PubMed Central

Liu, Tianyun; Ish‐Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D.

2017-01-01

Abstract Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo‐sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo‐sites), and Ten sites containing important motifs, loops, or key residues with important disease‐associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best‐ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand‐binding sites, most prediction methods have higher performance on apo‐sites than holo‐sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein‐protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein‐protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. PMID:28975675

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

PubMed

Karim, Zubair A; Vemana, Hari Priya; Alshbool, Fatima Z; Lin, Olivia A; Alshehri, Abdullah M; Javaherizadeh, Payam; Paez Espinosa, Enma V; Khasawneh, Fadi T

2015-03-01

Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies. © 2015 American Heart Association, Inc.

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression

PubMed Central

Garcia, Gloria R.; Goodale, Britton C.; Wiley, Michelle W.; La Du, Jane K.; Hendrix, David A.

2017-01-01

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway. PMID:28385905

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

PubMed

Garcia, Gloria R; Goodale, Britton C; Wiley, Michelle W; La Du, Jane K; Hendrix, David A; Tanguay, Robert L

2017-06-01

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA ( slincR ) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway. Copyright © 2017 by The Author(s).

Nitric Oxide Regulates Skeletal Muscle Fatigue, Fiber Type, Microtubule Organization, and Mitochondrial ATP Synthesis Efficiency Through cGMP-Dependent Mechanisms.

PubMed

Moon, Younghye; Balke, Jordan E; Madorma, Derik; Siegel, Michael P; Knowels, Gary; Brouckaert, Peter; Buys, Emmanuel S; Marcinek, David J; Percival, Justin M

2017-06-10

Skeletal muscle nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways are impaired in Duchenne and Becker muscular dystrophy partly because of reduced nNOSμ and soluble guanylate cyclase (GC) activity. However, GC function and the consequences of reduced GC activity in skeletal muscle are unknown. In this study, we explore the functions of GC and NO-cGMP signaling in skeletal muscle. GC1, but not GC2, expression was higher in oxidative than glycolytic muscles. GC1 was found in a complex with nNOSμ and targeted to nNOS compartments at the Golgi complex and neuromuscular junction. Baseline GC activity and GC agonist responsiveness was reduced in the absence of nNOS. Structural analyses revealed aberrant microtubule directionality in GC1 -/- muscle. Functional analyses of GC1 -/- muscles revealed reduced fatigue resistance and postexercise force recovery that were not due to shifts in type IIA-IIX fiber balance. Force deficits in GC1 -/- muscles were also not driven by defects in resting mitochondrial adenosine triphosphate (ATP) synthesis. However, increasing muscle cGMP with sildenafil decreased ATP synthesis efficiency and capacity, without impacting mitochondrial content or ultrastructure. GC may represent a new target for alleviating muscle fatigue and that NO-cGMP signaling may play important roles in muscle structure, contractility, and bioenergetics. These findings suggest that GC activity is nNOS dependent and that muscle-specific control of GC expression and differential GC targeting may facilitate NO-cGMP signaling diversity. They suggest that nNOS regulates muscle fiber type, microtubule organization, fatigability, and postexercise force recovery partly through GC1 and suggest that NO-cGMP pathways may modulate mitochondrial ATP synthesis efficiency. Antioxid. Redox Signal. 26, 966-985.

β2-Microglobulin-mediated signaling as a target for cancer therapy.

PubMed

Nomura, Takeo; Huang, Wen-Chin; Zhau, Haiyen E; Josson, Sajni; Mimata, Hiromitsu; Chung, Leland W K

2014-03-01

β2-microglobulin (β2-m) has become the focus of intense scrutiny since the discovery of its undesirable roles promoting osteomimicry and cancer progression. β2-m is a well-known housekeeping protein that forms complexes with the heavy chain of major histocompatibility complex class I molecules, which are heterodimeric cell surface proteins that present antigenic peptides to cytotoxic T cells. On recognition of foreign peptide antigens on cell surfaces, T cells actively bind and lyse antigen-presenting cancer cells. In addition to its roles in tumor immunity, β2-m has two different functions in cancer cells, either tumor promoting or tumor suppressing, in cancer cell context-dependent manner. Our studies have demonstrated that β2-m is involved extensively in the functional regulation of growth, survival, apoptosis, and even metastasis of cancer cells. We found that β2-m is a soluble growth factor and a pleiotropic signaling molecule which interacts with its receptor, hemochromatosis protein, to modulate epithelial-to-mesenchymal transition (EMT) through iron-responsive pathways. Specific antibodies against β2-m have remarkable tumoricidal activity in cancer, through β2-m action on iron flux, alterations of intracellular reactive oxygen species, DNA damage and repair enzyme activities, β-catenin activation and cadherin switching, and tumor responsiveness to hypoxia. These novel functions of β2-m and β2-m signaling may be common to several solid tumors including human lung, breast, renal, and prostate cancers. Our experimental results could lead to the development of a novel class of antibody-based pharmaceutical agents for cancer growth control. In this review, we briefly summarize the recent data regarding β2-m as a promising new cancer therapeutic target and discuss antagonizing this therapeutic target with antibody therapy for the treatment of localized and disseminated cancers.

β2-Microglobulin-mediated Signaling as a Target for Cancer Therapy

PubMed Central

Nomura, Takeo; Huang, Wen-Chin; Zhau, Haiyen E.; Josson, Sajni; Mimata, Hiromitsu; Kaur, Mandeep

2014-01-01

β2-microglobulin (β2-m) has become the focus of intense scrutiny since the discovery of its undesirable roles promoting osteomimicry and cancer progression. β2-m is a well-known housekeeping protein that forms complexes with the heavy chain of major histocompatibility complex class I molecules, which are heterodimeric cell surface proteins that present antigenic peptides to cytotoxic T cells. On recognition of foreign peptide antigens on cell surfaces, T cells actively bind and lyse antigen-presenting cancer cells. In addition to its roles in tumor immunity, β2-m has two different functions in cancer cells, either tumor promoting or tumor suppressing, in cancer cell context-dependent manner. Our studies have demonstrated that β2-m is involved extensively in the functional regulation of growth, survival, apoptosis, and even metastasis of cancer cells. We found that β2-m is a soluble growth factor and a pleiotropic signaling molecule which interacts with its receptor, hemochromatosis protein, to modulate epithelial-to-mesenchymal transition (EMT) through iron-responsive pathways. Specific antibodies against β2-m have remarkable tumoricidal activity in cancer, through β2-m action on iron flux, alterations of intracellular reactive oxygen species, DNA damage and repair enzyme activities, β-catenin activation and cadherin switching, and tumor responsiveness to hypoxia. These novel functions of β2-m and β2-m signaling may be common to several solid tumors including human lung, breast, renal, and prostate cancers. Our experimental results could lead to the development of a novel class of antibody-based pharmaceutical agents for cancer growth control. In this review, we briefly summarize the recent data regarding β2-m as a promising new cancer therapeutic target and discuss antagonizing this therapeutic target with antibody therapy for the treatment of localized and disseminated cancers. PMID:23848204

Method for multimodal analysis of independent source differences in schizophrenia: combining gray matter structural and auditory oddball functional data.

PubMed

Calhoun, V D; Adali, T; Giuliani, N R; Pekar, J J; Kiehl, K A; Pearlson, G D

2006-01-01

The acquisition of both structural MRI (sMRI) and functional MRI (fMRI) data for a given study is a very common practice. However, these data are typically examined in separate analyses, rather than in a combined model. We propose a novel methodology to perform independent component analysis across image modalities, specifically, gray matter images and fMRI activation images as well as a joint histogram visualization technique. Joint independent component analysis (jICA) is used to decompose a matrix with a given row consisting of an fMRI activation image resulting from auditory oddball target stimuli and an sMRI gray matter segmentation image, collected from the same individual. We analyzed data collected on a group of schizophrenia patients and healthy controls using the jICA approach. Spatially independent joint-components are estimated and resulting components were further analyzed only if they showed a significant difference between patients and controls. The main finding was that group differences in bilateral parietal and frontal as well as posterior temporal regions in gray matter were associated with bilateral temporal regions activated by the auditory oddball target stimuli. A finding of less patient gray matter and less hemodynamic activity for target detection in these bilateral anterior temporal lobe regions was consistent with previous work. An unexpected corollary to this finding was that, in the regions showing the largest group differences, gray matter concentrations were larger in patients vs. controls, suggesting that more gray matter may be related to less functional connectivity in the auditory oddball fMRI task. Hum Brain Mapp, 2005. (c) 2005 Wiley-Liss, Inc.

C1 domain-targeted isophthalates as protein kinase C modulators: structure-based design, structure-activity relationships and biological activities.

PubMed

Talman, Virpi; Provenzani, Riccardo; Boije af Gennäs, Gustav; Tuominen, Raimo K; Yli-Kauhaluoma, Jari

2014-12-01

Protein kinase C (PKC) is a serine/threonine kinase belonging to the AGC family. PKC isoenzymes are activated by phospholipid-derived second messengers, transmit their signal by phosphorylating specific substrates and play a pivotal role in the regulation of various cell functions, including metabolism, growth, differentiation and apoptosis. Therefore they represent an interesting molecular target for the treatment of several diseases, such as cancer and Alzheimer's disease. Adopting a structure-based approach on the crystal structure of the PKCδ C1B domain, our team has developed isophthalic acid derivatives that are able to modify PKC functions by binding to the C1 domain of the enzyme. Bis[3-(trifluoromethyl)benzyl] 5-(hydroxymethyl)isophthalate (HMI-1a3) and bis(1-ethylpentyl) 5-(hydroxymethyl)isophthalate (HMI-1b11) were selected from a set of compounds for further studies due to their high affinity for the C1 domains of PKCα and PKCδ. HMI-1a3 showed marked antiproliferative activity in HeLa cells whereas HMI-1b11 induced differentiation and supported neurite growth in SH-SY5Y cells. Our aim in the future is to improve the selectivity and potency of isophthalate derivatives, to clarify their mechanism of action in the cellular environment and to assess their efficacy in cell-based and in vivo disease models. HMI-1a3 has already been selected for a further project and redesigned to function as a probe immobilized on an affinity chromatography column. It will be used to identify cellular target proteins from cell lysates, providing new insights into the mechanism of action of HMI-1a3.

Functional interrogation of non-coding DNA through CRISPR genome editing.

PubMed

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

PubMed

Brockhoff, Marielle; Rion, Nathalie; Chojnowska, Kathrin; Wiktorowicz, Tatiana; Eickhorst, Christopher; Erne, Beat; Frank, Stephan; Angelini, Corrado; Furling, Denis; Rüegg, Markus A; Sinnreich, Michael; Castets, Perrine

2017-02-01

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

Hepatitis C virus p7 mediates membrane-to-membrane adhesion.

PubMed

Lee, Gi Young; Lee, Sora; Lee, Hye-Ra; Yoo, Young Do

2016-09-01

Viroporin p7 of the hepatitis C virus (HCV) acts as an ion channel for pH equilibration to stabilize HCV particles; most studies of p7 have focused on this role. However, pH equilibration by p7 via its ion channel activity does not fully explain the importance of p7 in HCV particle production. Indeed, several researchers have suggested p7 to have an unidentified ion channel-independent function. Here, we show that p7 has a novel role as a lipid raft adhesion factor, which is independent of its ion channel activity. We found that p7 targets not only the liquid-disordered (Ld) phase, but also the negatively-charged liquid-ordered (Lo) phase that can be represented as a lipid raft. p7 clusters at the phase boundary of the neutral Ld phase and the negatively-charged Lo phase. Interestingly, p7 targeting the Lo phase facilitates membrane-to-membrane adhesion, and this activity is not inhibited by p7 ion channel inhibitors. Our results demonstrated that HCV p7 has dual roles as a viroporin and as a lipid raft adhesion factor. This ion channel-independent function of p7 might be an attractive target for development of anti-HCV compounds. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

PubMed Central

Brockhoff, Marielle; Rion, Nathalie; Chojnowska, Kathrin; Wiktorowicz, Tatiana; Eickhorst, Christopher; Erne, Beat; Frank, Stephan; Angelini, Corrado; Rüegg, Markus A.; Sinnreich, Michael

2017-01-01

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3′-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease. PMID:28067669

Potential Regulators Driving the Transition in Nonalcoholic Fatty Liver Disease: a Stage-Based View.

PubMed

Lou, Yi; Chen, Yi-Dan; Sun, Fu-Rong; Shi, Jun-Ping; Song, Yu; Yang, Jin

2017-01-01

The incidence of nonalcoholic fatty liver disease (NAFLD), ranging from mild steatosis to hepatocellular injury and inflammation, increases with the rise of obesity. However, the implications of transcription factors network in progressive NAFLD remain to be determined. A co-regulatory network approach by combining gene expression and transcription influence was utilized to dissect transcriptional regulators in different NAFLD stages. In vivo, mice models of NAFLD were used to investigate whether dysregulated expression be undertaken by transcriptional regulators. Through constructing a large-scale co-regulatory network, sample-specific regulator activity was estimated. The combinations of active regulators that drive the progression of NAFLD were identified. Next, top regulators in each stage of NAFLD were determined, and the results were validated using the different experiments and bariatric surgical samples. In particular, Adipocyte enhancer-binding protein 1 (AEBP1) showed increased transcription activity in nonalcoholic steatohepatitis (NASH). Further characterization of the AEBP1 related transcription program defined its co-regulators, targeted genes, and functional organization. The dynamics of AEBP1 and its potential targets were verified in an animal model of NAFLD. This study identifies putative functions for several transcription factors in the pathogenesis of NAFLD and may thus point to potential targets for therapeutic interventions. © 2017 The Author(s) Published by S. Karger AG, Basel.

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

NASA Technical Reports Server (NTRS)

Love, J.; Scott, A. C.; Thompson, W. F.; Brown, C. S. (Principal Investigator)

2000-01-01

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.

YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity.

PubMed

Ma, Ka-Wai; Ma, Wenbo

2016-12-01

Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Estimating Fast Neural Input Using Anatomical and Functional Connectivity

PubMed Central

Eriksson, David

2016-01-01

In the last 20 years there has been an increased interest in estimating signals that are sent between neurons and brain areas. During this time many new methods have appeared for measuring those signals. Here we review a wide range of methods for which connected neurons can be identified anatomically, by tracing axons that run between the cells, or functionally, by detecting if the activity of two neurons are correlated with a short lag. The signals that are sent between the neurons are represented by the activity in the neurons that are connected to the target population or by the activity at the corresponding synapses. The different methods not only differ in the accuracy of the signal measurement but they also differ in the type of signal being measured. For example, unselective recording of all neurons in the source population encompasses more indirect pathways to the target population than if one selectively record from the neurons that project to the target population. Infact, this degree of selectivity is similar to that of optogenetic perturbations; one can perturb selectively or unselectively. Thus it becomes possible to match a given signal measurement method with a signal perturbation method, something that allows for an exact input control to any neuronal population. PMID:28066189

Engineering intracellular active transport systems as in vivo biomolecular tools.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bachand, George David; Carroll-Portillo, Amanda

2006-11-01

Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptionalmore » regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo applications. Further development could potentially enable selective capture of intracellular antigens, targeted delivery of therapeutic agents, or disruption of the transport systems and consequently the infection and pathogenesis cycle of biothreat agents.« less

Divergent synthesis and identification of the cellular targets of deoxyelephantopins

NASA Astrophysics Data System (ADS)

Lagoutte, Roman; Serba, Christelle; Abegg, Daniel; Hoch, Dominic G.; Adibekian, Alexander; Winssinger, Nicolas

2016-08-01

Herbal extracts containing sesquiterpene lactones have been extensively used in traditional medicine and are known to be rich in α,β-unsaturated functionalities that can covalently engage target proteins. Here we report synthetic methodologies to access analogues of deoxyelephantopin, a sesquiterpene lactone with anticancer properties. Using alkyne-tagged cellular probes and quantitative proteomics analysis, we identified several cellular targets of deoxyelephantopin. We further demonstrate that deoxyelephantopin antagonizes PPARγ activity in situ via covalent engagement of a cysteine residue in the zinc-finger motif of this nuclear receptor.

Auditory phonological priming in children and adults during word repetition

NASA Astrophysics Data System (ADS)

Cleary, Miranda; Schwartz, Richard G.

2004-05-01

Short-term auditory phonological priming effects involve changes in the speed with which words are processed by a listener as a function of recent exposure to other similar-sounding words. Activation of phonological/lexical representations appears to persist beyond the immediate offset of a word, influencing subsequent processing. Priming effects are commonly cited as demonstrating concurrent activation of word/phonological candidates during word identification. Phonological priming is controversial, the direction of effects (facilitating versus slowing) varying with the prime-target relationship. In adults, it has repeatedly been demonstrated, however, that hearing a prime word that rhymes with the following target word (ISI=50 ms) decreases the time necessary to initiate repetition of the target, relative to when the prime and target have no phonemic overlap. Activation of phonological representations in children has not typically been studied using this paradigm, auditory-word + picture-naming tasks being used instead. The present study employed an auditory phonological priming paradigm being developed for use with normal-hearing and hearing-impaired children. Initial results from normal-hearing adults replicate previous reports of faster naming times for targets following a rhyming prime word than for targets following a prime having no phonemes in common. Results from normal-hearing children will also be reported. [Work supported by NIH-NIDCD T32DC000039.

The Spatial Release of Cognitive Load in Cocktail Party Is Determined by the Relative Levels of the Talkers.

PubMed

Andéol, Guillaume; Suied, Clara; Scannella, Sébastien; Dehais, Frédéric

2017-06-01

In a multi-talker situation, spatial separation between talkers reduces cognitive processing load: this is the "spatial release of cognitive load". The present study investigated the role played by the relative levels of the talkers on this spatial release of cognitive load. During the experiment, participants had to report the speech emitted by a target talker in the presence of a concurrent masker talker. The spatial separation (0° and 120° angular distance in azimuth) and the relative levels of the talkers (adverse, intermediate, and favorable target-to-masker ratio) were manipulated. The cognitive load was assessed with a prefrontal functional near-infrared spectroscopy. Data from 14 young normal-hearing listeners revealed that the target-to-masker ratio had a direct impact on the spatial release of cognitive load. Spatial separation significantly reduced the prefrontal activity only for the intermediate target-to-masker ratio and had no effect on prefrontal activity for the favorable and the adverse target-to-masker ratios. Therefore, the relative levels of the talkers might be a key point to determine the spatial release of cognitive load and more specifically the prefrontal activity induced by spatial cues in multi-talker situations.

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

PubMed

Rockah-Shmuel, Liat; Tawfik, Dan S

2012-12-01

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

Nanoparticle-based B-cell targeting vaccines: Tailoring of humoral immune responses by functionalization with different TLR-ligands.

PubMed

Zilker, Claudia; Kozlova, Diana; Sokolova, Viktoriya; Yan, Huimin; Epple, Matthias; Überla, Klaus; Temchura, Vladimir

2017-01-01

Induction of an appropriate type of humoral immune response during vaccination is essential for protection against viral and bacterial infections. We recently observed that biodegradable calcium phosphate (CaP) nanoparticles coated with proteins efficiently targeted and activated naïve antigen-specific B-cells in vitro. We now compared different administration routes for CaP-nanoparticles and demonstrated that intramuscular immunization with such CaP-nanoparticles induced stronger immune responses than immunization with monovalent antigen. Additional functionalization of the CaP-nanoparticles with TRL-ligands allowed modulating the IgG subtype response and the level of mucosal IgA antibodies. CpG-containing CaP-nanoparticles were as immunogenic as a virus-like particle vaccine. Functionalization of CaP-nanoparticles with T-helper cell epitopes or CpG also allowed overcoming lack of T-cell help. Thus, our results indicate that CaP-nanoparticle-based B-cell targeting vaccines functionalized with TLR-ligands can serve as a versatile platform for efficient induction and modulation of humoral immune responses in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Two Isomeric C16 Oxo-Fatty Acids from the Diatom Chaetoceros karianus Show Dual Agonist Activity towards Human Peroxisome Proliferator-Activated Receptors (PPARs) α/γ

PubMed Central

Moldes-Anaya, Angel; Sæther, Thomas; Uhlig, Silvio; Nebb, Hilde I.; Larsen, Terje; Eilertsen, Hans C.; Paulsen, Steinar M.

2017-01-01

The peroxisome proliferator-activated receptors (PPARs) function as ligand-activated transcription factors that convert signals in the form of lipids to physiological responses through the activation of metabolic target genes. Due to their key roles in lipid and carbohydrate metabolism, the PPARs are important drug targets. However, for several of the PPAR drugs currently in use, adverse side effects have been reported. In an effort to identify compounds from marine organisms that may serve as molecular scaffolds for the development of novel and safer PPAR-targeting drugs, we performed a bioassay-guided screening of organic extracts made from organisms supplied by the Norwegian Biobank of Arctic Marine Organisms (Marbank). Among several interesting hits, we identified two poorly described isomeric oxo-fatty acids from the microalgae Chaetoceros karianus for which we provide the first evidence that they might display dual specificity towards human PPARα and PPARγ. Principal component analysis showed that C. karianus stood out from other Chaetoceros species, both with respect to the metabolic profile and the PPAR activity. The isolation of these compounds holds the potential of uncovering a PPAR pharmacophore with tunable activity and specificity. PMID:28587091