Involvement of Tetraspanin C189 in Cell-to-Cell Spreading of the Dengue Virus in C6/36 Cells

by Chao-Fu Yang, Cheng-Hsun Tu, Yin-Ping Lo, Chih-Chieh Cheng, Wei-June Chen

Dengue virus (DENV) is naturally transmitted by mosquitoes to humans, infecting cells of both hosts. Unlike in mammalian cells, DENV usually does not cause extremely deleterious effects on cells of mosquitoes. Despite this, clustered progeny virions were found to form infection foci in a high density cell culture. It is thus interesting to know how the virus spreads among cells in tissues such as the midgut within live mosquitoes. This report demonstrates that cell-to-cell spread is one way for DENV to infect neighboring cells without depending on the “release and entry” mode. In the meantime, a membrane-bound vacuole incorporating tetraspanin C189 was formed in response to DENV infection in the C6/36 cell and was subsequently transported along with the contained virus from one cell to another. Knockdown of C189 in DENV-infected C6/36 cells is shown herein to reduce cell-to-cell transmission of the virus, which may be recovered by co-transfection with a C189-expressing vector in DENV-infected C6/36 cells. Moreover, cell-to-cell transmission usually occurred at the site where the donor cell directly contacts the recipient cell. It suggested that C189 is crucially involved in the intercellular spread of progeny viral particles between mosquito cells. This novel finding presumably accounts for the rapid and efficient infection of DENV after its initial replication within tissues of the mosquito.
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Functional Activity Limitation and Quality of Life of Leprosy Cases in an Endemic Area in Northeastern Brazil

by Victor S. Santos, Laudice S. Oliveira, Fabrícia D. N. Castro, Vanessa T. Gois-Santos, Ligia M. D. Lemos, Maria do C. O. Ribeiro, Luis E. Cuevas, Ricardo Q. Gurgel

Background

Few studies have evaluated the association between quality of life (QoL) and functional activity limitations (FAL) of leprosy patients as determined by the Screening of Activity Limitation and Safety Awareness scale (SALSA).

Aim

To identify the association between FALs and the QoL of patients during and post leprosy treatment.

Materials and Methods

Cross-sectional survey of 104 patients with leprosy followed in specialist reference centres in Sergipe, Brazil, between June and October 2014. QoL was evaluated using the World Health Organization-QoL-BREF (WHOQoL-BREF) questionnaire. The SALSA scale was used to measure FALs.

Results

Low SALSA scores were present in 76% of patients. QoL scores were lower for the physical and environmental domains, with median (interquartile range (IQR)) scores of 53.6 (32.1–67.9) and 53.1 (46.9–64.8), respectively. There was a statistical association between increasing SALSA scores and lower QoL as measured by the WHOQoL-BREF.

Conclusion

Functional limitations are associated with lower QoL in leprosy patients, especially in the physical and environmental WHOQoL-BREF domains.


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Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA

by Andreas Gallmetzer, Lucia Silvestrini, Thorsten Schinko, Bernd Gesslbauer, Peter Hortschansky, Christoph Dattenböck, María Isabel Muro-Pastor, Andreas Kungl, Axel A. Brakhage, Claudio Scazzocchio, Joseph Strauss

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the NiRD, NES exposure, and relocation of the inactive NirA to the cytosol.
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A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5′ RNA-seq

by Sergey Y. Druzhinin, Ngat T. Tran, Kyle S. Skalenko, Seth R. Goldman, Jared G. Knoblauch, Simon L. Dove, Bryce E. Nickels

Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, “primer-dependent initiation,” (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5′ ends (5′ RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T−1A+1 or G−1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.
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TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis

by Lei Gao, Dantong Li, Ke Ma, Wenjuan Zhang, Tao Xu, Cong Fu, Changbin Jing, Xiaoe Jia, Shuang Wu, Xin Sun, Mei Dong, Min Deng, Yi Chen, Wenge Zhu, Jinrong Peng, Fengyi Wan, Yi Zhou, Leonard I. Zon, Weijun Pan

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.
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Nmf9 Encodes a Highly Conserved Protein Important to Neurological Function in Mice and Flies

by Shuxiao Zhang, Kevin D. Ross, Glen A. Seidner, Michael R. Gorman, Tiffany H. Poon, Xiaobo Wang, Elizabeth M. Keithley, Patricia N. Lee, Mark Q. Martindale, William J. Joiner, Bruce A. Hamilton

Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.
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Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer

by Kim Clarke, Thomas Daubon, Nil Turan, Fabienne Soulet, Maihafizah Mohd Zahari, Katie R. Ryan, Sarah Durant, Shan He, John Herbert, John Ankers, John K. Heath, Rolf Bjerkvig, Roy Bicknell, Neil A. Hotchin, Andreas Bikfalvi, Francesco Falciani

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.
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