Detection of a MicroRNA Signal in an In Vivo Expression Set of mRNAs

Background. microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of a miRNA to the target mRNA. As a result, translation of the target mRNA is inhibited and the mRNA may be destabilized. The inhibitory effects of miRNAs have been linked to diverse cellular processes including malignant proliferation, apoptosis, development, differentiation, and metabolic processes. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. Methodology/Principal Findings. RNA was extracted from 12 human primary brain tumor biopsies. These samples were used to determine genome-wide mRN

High-throughput profiling of caenorhabditis elegans starvation-responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6-20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans

Chromosome conformation signatures define predictive markers of inadequate response to methotrexate in early rheumatoid arthritis

The authors would like to thank members of OBD Reference Facility Benjamin Foulkes, Chloe Bird, Emily Corfeld and Matthew Salter for expedient processing of clinical samples on the EpiSwitch™ platform and Magdalena Jeznach and Willem Westra for help with preparation of the manuscript. The study employed samples from the SERA Biobank used with permission and approval of the SERA Approval Group. We gratefully acknowledge the invaluable contribution of the clinicians and operating team in SERA. We would also like to thank Prof. Raju Kucherlapati (Harvard Medical School), and Prof. Jane Mellor (Oxford Univ.), Prof. John O’Shea (National Institute of Health) and Prof. John Isaacs (New Castle Univ.) for their independent and critical review of our study. A list of Scottish Early Rheumatoid Arthritis (SERA) inception cohort investigators is provided in Additional fle 1: Additional Note. Funding This work was funded by Oxford BioDynamics.Peer reviewedPublisher PD

Measuring network's entropy in ADHD: A new approach to investigate neuropsychiatric disorders

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The application of graph analysis methods to the topological organization of brain connectivity has been a useful tool in the characterization of brain related disorders. However, the availability of tools, which enable researchers to investigate functional brain networks, is still a major challenge. Most of the studies evaluating brain images are based on centrality and segregation measurements of complex networks. In this study, we applied the concept of graph spectral entropy (GSE) to quantify the complexity in the organization of brain networks. In addition, to enhance interpretability, we also combined graph spectral clustering to investigate the topological organization of sub-network's modules. We illustrate the usefulness of the proposed approach by comparing brain networks between attention deficit hyperactivity disorder (ADHD) patients and the brain networks of typical developing (TD) controls. The main findings highlighted that GSE involving sub-networks comprising the areas mostly bilateral pre and post central cortex, superior temporal gyrus, and inferior frontal gyri were statistically different (p-value = 0.002) between ADHD patients and TO controls. In the same conditions, the other conventional graph descriptors (betweenness centrality, clustering coefficient, and shortest path length) commonly used to identify connectivity abnormalities did not show statistical significant difference. We conclude that analysis of topological organization of brain sub-networks based on GSE can identify networks between brain regions previously unobserved to be in association with ADHD. (C) 2013 Elsevier Inc. All rights reserved.774451Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Pew Latin American FellowshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Structural characterization of maize SIRK1 kinase domain reveals an unusual architecture of the activation segment

Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK), have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK), the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1) is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists

Structural analysis of inhibitor binding to CAMKK1 identifies features necessary for design of specific inhibitors

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities

Development of 2-(4-pyridyl)-benzimidazoles as PKN2 chemical tools to probe cancer

Kinases are signalling proteins which have proven to be successful targets for the treatment of a variety of diseases, predominantly in cancers. However, only a small proportion of kinases (<20%) have been investigated for their therapeutic viability, likely due to the lack of available chemical tools across the kinome. In this work we describe initial efforts in the development of a selective chemical tool for protein kinase N2 (PKN2), a relatively unexplored kinase of interest in several types of cancer. The most successful compound, 5, has a measured IC<sub>50</sub> of 0.064 μM against PKN2, with ca. 17-fold selectivity over close homologue, PKN1

The c-terminal domains SnRK2 box and ABA box have a role in sugarcane SnRK2s auto-activation and activity

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s—known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function

The c-terminal domains SnRK2 box and ABA box have a role in sugarcane SnRK2s auto-activation and activity

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s—known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function

Discovery of a potent dual SLK/STK10 inhibitor based on a maleimide scaffold

SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound <b>31</b> as the most potent and selective inhibitor of SLK and STK10 yet reported