Comparative Mouse Genomics Centers Consortium: the Mouse Genotype Database

The Comparative Mouse Genomics Centers Consortium (CMGCC) is a branch of the Environmental Genome Project sponsored by the National Institute of Environmental Health Sciences (NIEHS) focusing upon the identification of human single nucleotide polymorphisms (SNPs) that may confer disease susceptibility within the human population. The goal of the CMGCC (http://www.niehs.nih.gov/cmgcc/) is to make genetic mouse models for human SNPs within cell cycle control, DNA replication and DNA repair genes that may be associated with human pathologies. In order to facilitate information sharing and analysis within the consortium a set of informatics resources have been generated to support the mouse model development efforts. The primary entry point for information about the mouse models developed by the consortium is through the CMGCC Genotype Database (http://mrages.niehs.nih.gov/genotype/), which maintains both a consortium specific and public access display of the available and developing mouse models.     

Histone Sequence Database: new histone fold family members.

Searches of the major public protein databases with core and linker chicken and human histone sequences have resulted in the compilation of an annotated set of histone protein sequences. In addition, new database searches with two distinct motif search algorithms have identified several members of the histone fold family, including human DRAP1 and yeast CSE4. Database resources include information on conflicts between similar sequence entries in different source databases, multiple sequence alignments, links to the Entrez integrated information retrieval system, structures for histone and histone fold proteins, and the ability to visualize structural data through Cn3D. The database currently contains >1000 protein sequences, which are searchable by protein type, accession number, organism name, or any other free text appearing in the definition line of the entry. All sequences and alignments in this database are available through the World Wide Web at http://www.nhgri.nih. gov/DIR/GTB/HISTONES or http://www.ncbi.nlm.nih. gov/Baxevani/HISTONES     

CDD: a curated Entrez database of conserved domain alignments

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.     

Human Telomere Length Correlates to the Size of the Associated Chromosome Arm

The majority of human telomere length studies have focused on the overall length of telomeres within a cell. In fact, very few studies have examined telomere length for individual chromosome arms. The objective of this study was to examine the relationship between chromosome arm size and the relative length of the associated telomere. Quantitative Fluorescence In Situ Hybridization (Q-FISH) was used to measure the relative telomere length of each chromosome arm in metaphases from cultured lymphocytes of 17 individuals. A statistically significant positive correlation (r = 0.6) was found between telomere length and the size of the associated chromosome arm, which was estimated based on megabase pair measurements from http://www.ncbi.nlm.nih.gov/projects/mapview/.     

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) ². Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor ^5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at ˜20 Å resolution ^9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH (http://biowulf.nih.gov). This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.Download video file.^{(47M, mov)}     

Histone Sequence Database: sequences, structures, post-translational modifications and genetic loci.

The Histone Sequence Database is an annotated and searchable collection of all available histone and histone fold sequences and structures. Particular emphasis has been placed on documenting conflicts between similar sequence entries from a number of source databases, conflicts that are not necessarily documented in the source databases themselves. New additions to the database include compilations of post-translational modifications for each of the core and linker histones, as well as genomic information in the form of map loci for the human histone gene complement, with the genetic loci linked to Online Mendelian Inheritance in Man (OMIM). The database is freely accessible through the World Wide Web at either http://genome.nhgri.nih.gov/histones/ or http://www.ncbi.nlm.nih. gov/Baxevani/HISTONES     

A Novel Cholinergic Action of Alcohol and the Development of Tolerance to That Effect in Caenorhabditis elegans

The Prp43 DExD/H-box protein is required for progression of the biochemically distinct pre-messenger RNA and ribosomal RNA (rRNA) maturation pathways. In Saccharomyces cerevisiae, the Spp382/Ntr1, Sqs1/Pfa1, and Pxr1/Gno1 proteins are implicated as cofactors necessary for Prp43 helicase activation during spliceosome dissociation (Spp382) and rRNA processing (Sqs1 and Pxr1). While otherwise dissimilar in primary sequence, these Prp43-binding proteins each contain a short glycine-rich G-patch motif required for function and thought to act in protein or nucleic acid recognition. Here yeast two-hybrid, domain-swap, and site-directed mutagenesis approaches are used to investigate G-patch domain activity and portability. Our results reveal that the Spp382, Sqs1, and Pxr1 G-patches differ in Prp43 two-hybrid response and in the ability to reconstitute the Spp382 and Pxr1 RNA processing factors. G-patch protein reconstitution did not correlate with the apparent strength of the Prp43 two-hybrid response, suggesting that this domain has function beyond that of a Prp43 tether. Indeed, while critical for Pxr1 activity, the Pxr1 G-patch appears to contribute little to the yeast two-hybrid interaction. Conversely, deletion of the primary Prp43 binding site within Pxr1 (amino acids 102–149) does not impede rRNA processing but affects small nucleolar RNA (snoRNA) biogenesis, resulting in the accumulation of slightly extended forms of select snoRNAs, a phenotype unexpectedly shared by the prp43 loss-of-function mutant. These and related observations reveal differences in how the Spp382, Sqs1, and Pxr1 proteins interact with Prp43 and provide evidence linking G-patch identity with pathway-specific DExD/H-box helicase activity.     

Integrating Constitutive Gene Expression and Chemoactivity: Mining the NCI60 Anticancer Screen

Studies into the genetic origins of tumor cell chemoactivity pose significant challenges to bioinformatic mining efforts. Connections between measures of gene expression and chemoactivity have the potential to identify clinical biomarkers of compound response, cellular pathways important to efficacy and potential toxicities; all vital to anticancer drug development. An investigation has been conducted that jointly explores tumor-cell constitutive NCI60 gene expression profiles and small-molecule NCI60 growth inhibition chemoactivity profiles, viewed from novel applications of self-organizing maps (SOMs) and pathway-centric analyses of gene expressions, to identify subsets of over- and under-expressed pathway genes that discriminate chemo-sensitive and chemo-insensitive tumor cell types. Linear Discriminant Analysis (LDA) is used to quantify the accuracy of discriminating genes to predict tumor cell chemoactivity. LDA results find 15% higher prediction accuracies, using ∼30% fewer genes, for pathway-derived discriminating genes when compared to genes derived using conventional gene expression-chemoactivity correlations. The proposed pathway-centric data mining procedure was used to derive discriminating genes for ten well-known compounds. Discriminating genes were further evaluated using gene set enrichment analysis (GSEA) to reveal a cellular genetic landscape, comprised of small numbers of key over and under expressed on- and off-target pathway genes, as important for a compound’s tumor cell chemoactivity. Literature-based validations are provided as support for chemo-important pathways derived from this procedure. Qualitatively similar results are found when using gene expression measurements derived from different microarray platforms. The data used in this analysis is available at http://pubchem.ncbi.nlm.nih.gov/and http://www.ncbi.nlm.nih.gov/projects/geo ({"type":"entrez-geo","attrs":{"text":"GPL96","term_id":"96"}}GPL96, {"type":"entrez-geo","attrs":{"text":"GSE32474","term_id":"32474"}}GSE32474).     

BIRS – Bioterrorism Information Retrieval System

Bioterrorism is the intended use of pathogenic strains of microbes to widen terror in a population. There is a definite need to promote research for development of vaccines, therapeutics and diagnostic methods as a part of preparedness to any bioterror attack in the future. BIRS is an open-access database of collective information on the organisms related to bioterrorism. The architecture of database utilizes the current open-source technology viz PHP ver 5.3.19, MySQL and IIS server under windows platform for database designing. Database stores information on literature, generic- information and unique pathways of about 10 microorganisms involved in bioterrorism. This may serve as a collective repository to accelerate the drug discovery and vaccines designing process against such bioterrorist agents (microbes). The available data has been validated from various online resources and literature mining in order to provide the user with a comprehensive information system.

Availability

The database is freely available at http://www.bioterrorism.biowaves.org     

Should Informed Consent for Cancer Treatment Include a Discussion about Hospital Outcome Disparities?

Background to the debate: Several studies have found disparities in the outcome of medical procedures across different hospitals—better outcomes have been associated with higher procedure volume. An Institute of Medicine workshop found such a “volume–outcome relationship” for two types of cancer surgery: resection of the pancreas and esophagus (http://www.iom.edu/?id=31508). This debate examines whether physicians have an ethical obligation to inform patients of hospital outcome disparities for these cancers.     

Brothers in arms

The horrific injuries and difficult working conditions faced by military medical personnel have forced the military to fund biomedical research to treat soldiers; those new technologies and techniques contribute significantly to civilian medicine.War is the father of all things, Heraclitus believed. The military''s demand for better weapons and transportation, as well as tools for communication, detection and surveillance has driven technological progress during the past 150 years or so, producing countless civilian applications as a fallout. The military has invested heavily into high-energy physics, materials science, navigation systems and cryptology. Similarly, military-funded biomedical research encompasses the whole range from basic to applied research programmes (Fig 1), and the portion of military-funded research in the biological and medical fields is now considerable.Open in a separate windowFigure 11944 advertisement for Diebold Inc. (Ohio, USA) in support of blood donations for soldiers wounded in the Second World War. The military has traditionally been one of the greatest proponents of active research on synthetic blood production, blood substitutes and oxygen therapeutics for treating battlefield casualties. One recent approach in this direction is The Defense Advanced Research Projects Agency''s (DARPA''s) Blood Pharming programme, which plans to use human haematopoietic stem cells—such as those obtained from umbilical cord blood—as a “starting material to develop an automated, fieldable cell culture and packaging system capable of producing transfusable amounts of universal donor red blood cells” (http://www.darpa.mil/Our_Work/DSO/Programs/Blood_Pharming.aspx).War has always driven medical advances. From ancient Roman to modern times, treating the wounds of war has yielded surgical innovations that have been adopted by mainstream medicine. For example, the terrible effect of modern artillery on soldiers in the First World War was a major impetus for plastic surgery. Similarly, microbiology has benefited from war and military research: from antiseptics to prevent and cure gangrene to the massive production of penicillin during the Second World War, as well as more basic research into a wide range of pathogens, militaries worldwide have long been enthusiastic sponsors of microbiology research. Nowadays, military-funded research on pathogens uses state-of-the-art genotyping methods to study outbreaks and the spread of infection and seeks new ways to combat antibiotic resistance that afflicts both combatants and civilians.…military-funded biomedical research encompasses the whole range from basic to applied research programmes…The US Military Infectious Diseases Research Program (MIDRP) is particularly active in vaccine development to protect soldiers, especially those deployed overseas. Its website notes that: “Since the passing of the 1962 Kefauver–Harris Drug Amendment, which added the FDA requirement for proof of efficacy in addition to proof of safety for human products, there have been 28 innovative vaccines licenced in the US, including 13 vaccines currently designated for paediatric use. These 28 innovative vaccine products targeted new microorganisms, utilized new technology, or consisted of novel combinations of vaccines. Of these 28, the US military played a significant role in the development of seven licenced vaccines” (https://midrp.amedd.army.mil/). These successes include tetravalent meningococcal vaccine and oral typhoid vaccine, while current research is looking into the development of vaccines against malaria, dengue fever and hepatitis E.Similarly, the US Military HIV Research Program (MHRP) is working on the development of a global HIV-1 vaccine (http://www.hivresearch.org). MHRP scientists were behind the RV144 vaccine study in Thailand—the largest ever HIV vaccine study conducted in humans—that demonstrated that the vaccine was capable of eliciting modest and transient protection against the virus [1]. In the wake of the cautious optimism raised by the trial, subsequent research is providing insights into the workings of RV144 and is opening new doors for vaccine designers to strengthen the vaccine. In a recent study, researchers isolated four monoclonal antibodies induced by the RV144 vaccine and directed at a particular region of the HIV virus envelope associated with reduced infection, the variable region 2. They found that these antibodies recognized HIV-1-infected CD4(+) T cells and tagged them for attack by the immune system [2].In response to the medical problems military personnel are suffering in Iraq and Afghanistan, a recent clinical trial funded by the US Department of the Army demonstrated the efficacy of the aminoglycoside antibiotic paromomycin—either with or without gentamicin—for the topical treatment of cutaneous leishmaniasis, the most common form of infection by Leishmania parasites. Cutaneous leishmaniasis—which is endemic in Iraq and Afghanistan and rather frequent among soldiers deployed there—is transmitted to humans through the bite of infected sandflies: it causes skin ulcers and sores and can cause serious disability and social prejudice [3]. Topical treatments would offer advantages over therapies that require the systemic administration of antiparasitic drugs. The study—a phase 3 trial—was conducted in Tunisia and enrolled some 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis. Patients, all aged between 5 and 65, received topical applications of a cream containing either 15% paromomycin with 0.5% gentamicin, 15% paromomycin alone or the control cream, which contained no antibiotic. The combination of paromomycin and gentamicin cured cutaneous leishmaniasis with an efficacy of 81%, compared with 82% for paromomycin alone and just 58% for control—the skin sores of cutaneous leishmaniasis often heal on their own. Researchers reported no adverse reactions to paronomycin-containing creams. Because the combination therapy with gentamicin is probably effective against a larger range of Leishmania parasitic species and strains causing the disease, it could become a first-line treatment for cutaneous leishmaniasis on a global scale the authors concluded [3].…military-funded research on pathogens uses state-of-the-art genotyping methods to study outbreaks and the spread of infectionNot surprisingly, trauma and regenerative and reconstructive medicine are other large areas of research in which military influence is prevalent. The treatment of wounds, shock and the rehabilitation of major trauma patients are the very essence of medical aid on the battlefield (Figs 2, ​,3).3). “Our experience of military conflict, in particular the medicine required to deal with severe injuries, has led to significant improvements in trauma medicine. Through advances in the prevention of blood loss and the treatment of coagulopathy for example, patients are now surviving injuries that 5–10 years ago would have been fatal,” said Professor Janet Lord, who leads a team investigating the inflammatory response in injured soldiers at the National Institute for Health Research Surgical Reconstruction and Microbiology Research Centre (NIHR SRMRC) in Birmingham, UK (http://www.srmrc.nihr.ac.uk/).Open in a separate windowFigure 2Medical services in Britain, 1917. Making an artificial leg for a wounded serviceman at Roehampton Hospital in Surrey. This image is from The First World War Poetry Digital Archive, University of Oxford (www.oucs.ox.ac.uk/ww1lit). Copyright: The Imperial War Museum.Open in a separate windowFigure 3US soldiers use the fireman''s carry to move a simulated casualty to safety during a hyper-realistic training environment, known as trauma lanes, as part of the final phase of the Combat Lifesaver Course given by medics from Headquarters Support Company, Headquarters and Headquarters Battalion, 25th Inf. Div., USD-C, at Camp Liberty, Iraq, March 2011. Credit: US Army, photo by Sgt Jennifer Sardam.NIHR SRMRC integrates basic researchers at Birmingham University with clinicians and surgeons at the Royal Centre for Defence Medicine and University Hospital Birmingham to improve the treatment of traumatic injury in both military and civilian patients. As Lord explained, the centre has two trauma-related themes. The first is looking at, “[t]he acute response to injury, which analyses the kinetics and nature of the inflammatory response to tissue damage and is developing novel therapies to ensure the body responds appropriately to injury and does not stay in a hyper-inflamed state. The latter is a particular issue with older patients whose chance of recovery from trauma is significantly lower than younger patients,” she said. The second theme is, “[n]eurotrauma and regeneration, which studies traumatic brain injury, trying to develop better ways to detect this early to prevent poor outcomes if it goes undiagnosed,” Lord said.Kevlar helmets and body armour have saved the lives of many soldiers, but they do not protect much the face and eyes, and in general against blasts to the head. Because human retinas and brains show little potential for regeneration, patients with face and eye injuries often suffer from loss of vision and other consequences for the rest of their lives. However, a new stem cell and regenerative approach for the treatment of retinal injury and blindness is on the horizon. “Recent progress in stem cell research has begun to emerge on the possible exploitation of stem cell-based strategies to repair the damaged CNS (central nervous system). In particular, research from our laboratory and others have demonstrated that Müller cells—dormant stem-like cells found throughout the retina—can serve as a source of retinal progenitor cells to regenerate photoreceptors as well as all other types of retinal neurons,” explained Dong Feng Chen at the Schepens Eye Research Institute, Massachusetts Eye and Ear of the Harvard Medical School in Boston (Massachusetts, USA). In collaboration with the US Department of Defence, the Schepens Institute is steering the Military Vision Research Program, “to develop new ways to save the vision of soldiers injured on today''s battlefield and to push the frontier of vision technologies forward” (http://www.schepens.harvard.edu).“My laboratory has shown that adult human and mouse Müller cells can not only regenerate retina-specific neurons, but can also do so following induction by a single small molecule compound, alpha-aminoadipate,” Chen explained. She said that alpha-aminoadipate causes isolated Müller glial cells in culture to loose their glial phenotype, express progenitor cell markers and divide. Injection of alpha-aminoadipate into the subretinal space of adult mice in vivo induces mature Müller glia to de-differentiate and generate new retinal neurons and photoreceptor cells [4]. “Our current effort seeks to further elucidate the molecular pathways underlying the regenerative behaviour of Muller cells and to achieve functional regeneration of the damaged retina with small molecule compounds,” Chen said. “As the retina has long served as a model of the CNS, and Müller cells share commonalities with astroglial lineage cells in the brain and spinal cord, the results of this study can potentially be broadened to future development of treatment strategies for other neurodegenerative diseases, such as brain and spinal cord trauma, or Alzheimer and Parkinson disease.”The treatment of wounds, shock and the rehabilitation of major trauma patients are the very essence of medical aid on the battlefieldBrain injuries account for a large percentage of the wounds sustained by soldiers. The Defense Advanced Research Projects Agency (DARPA), an agency of the US Department of Defense, recently awarded US$6 million to a team of researchers to develop nanotechnology therapies for the treatment of traumatic brain injury and associated infections. The researchers are trying to develop nanoparticles carrying small interfering RNA (siRNA) molecules to reach and treat drug-resistant bacteria and inflammatory cells in the brain. Protecting the siRNA within a nanocomplex covered with specific tissue homing and cell-penetrating peptides will make it possible to deliver the therapeutics to infected cells beyond the blood–brain barrier—which normally makes it difficult to get antibiotics to the brain. The project has been funded within the framework of DARPA''s In Vivo Nanoplatforms programme that “seeks to develop new classes of adaptable nanoparticles for persistent, distributed, unobtrusive physiologic and environmental sensing as well as the treatment of physiologic abnormalities, illness and infectious disease” (www.darpa.mil).“The DARPA funding agency often uses the term ‘DARPA-hard'' to refer to problems that are extremely tough to solve. What makes this a DARPA-hard problem is the fact that it is so difficult to deliver therapeutics to the brain. This is an underserved area of research,” explained team leader Michael Sailor, from the University of California San Diego, in a press release (http://ucsdnews.ucsd.edu/pressrelease/darpa_awards_6_million_to_develop_nanotech_therapies_for_traumatic_brain_in).In the near future, DARPA, whose budget is set for a 1.8% increase to US$2.9 billion next year, will focus on another important project dealing with the CNS. The BRAIN Initiative—short for Brain Research through Advancing Innovative Neurotechnologies—is a new research effort whose proponents intend will “revolutionize our understanding of the human mind and uncover new ways to treat, prevent, and cure brain disorders like Alzheimer''s, schizophrenia, autism, epilepsy and traumatic brain injury” (www.whitehouse.gov). Out of a total US$110 million investment, DARPA will obtain US$50 million to work on understanding the dynamic functions of the brain and demonstrating breakthrough applications based on these insights (Fig 4). In addition to exploring new research areas, this money will be directed towards ongoing projects of typical—although not exclusive—military interest that involve enhancing or recovering brain functions, such as the development of brain-interfaced prosthetics and uncovering the mechanisms underlying neural reorganization and plasticity to accelerate injury recovery.Open in a separate windowFigure 4The BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative infographic. A complete version can be downloaded at http://www.whitehouse.gov/infographics/brain-initiative.“[T]here is this enormous mystery waiting to be unlocked, and the BRAIN Initiative will change that by giving scientists the tools they need to get a dynamic picture of the brain in action and better understand how we think and how we learn and how we remember. And that knowledge could be—will be—transformative,” said US President Obama, presenting the initiative (http://www.whitehouse.gov/the-press-office/2013/04/02/remarks-president-brain-initiative-and-american-innovation).“The President''s initiative reinforces the significance of understanding how the brain records, processes, uses, stores and retrieves vast quantities of information. This kind of knowledge of brain function could inspire the design of a new generation of information processing systems; lead to insights into brain injury and recovery mechanisms; and enable new diagnostics, therapies and devices to repair traumatic injury,” explained DARPA Director Arati Prabhakar in a press release (http://www.darpa.mil/NewsEvents/Releases/2013/04/02.aspx).But BRAIN is also stirring up some controversy. Some scientists fear that this kind of ‘big and bold'' science, with a rigid top-down approach and vaguely defined objectives, will drain resources from smaller projects in fundamental biology [5]. Others ask whether the BRAIN project investment will really generate the huge return hinted at in Obama''s speech during the initiative''s launch, or whether a substantial amount of hype about the potential outcomes was used to sell the project (http://ksj.mit.edu/tracker/2013/04/obamas-brain-initiative-and-alleged-140).As these examples show, the most important player in military-funded biomedical research is the USA, with the UK following at a distance. But other countries with huge defence budgets are gearing up, although with less visibility. In July 2011, for instance, India and Kyrgyzstan opened the joint Mountain Biomedical Research Centre at the Kyrgyz capital Bishkek, to carry out research into the mechanisms of short- and long-term high-altitude adaptation. The institute will use molecular biological approaches to identify markers for screening people for high-altitude resistance and susceptibility to high-altitude sickness, and development of other mountain maladies. On the Indian side, the scientists involved in the new research centre belong to the Defence Institute of Physiology and Applied Sciences, and the money came from India''s defence budget.As mankind seems unlikely to give up on armed conflicts anytime soon, war-torn human bodies will still need to be cured and wounds healed. Whether the original impetus for military-funded biomedical research is noble or not, it nonetheless fuels considerable innovation leading to important medical discoveries that ultimately benefit all.     

Carbohydrate Metabolism Differences between Subgroup A1 and B2 Strains of Bacillus anthracis as Assessed by Comparative Genomics and Functional Genetics

In France, Bacillus anthracis subgroup B2 strains do not metabolize starch or glycogen but can use gluconate, whereas subgroup A1 strains show the inverse pattern. Functional genetic analysis revealed that mutations in the amyS and gntK genes encoding an alpha-amylase and a gluconate kinase, respectively, were responsible for these phenotypes.Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, aerobic soil bacterium. Multilocus variable-number tandem repeat analysis of a collection of French isolates shows that the main groups of B. anthracis groups A (subgroup A1) and B (subgroup B2) described worldwide are represented (1, 2). Subgroup B2 isolates are the most common isolates in France and are found particularly in southern mountain regions, but they are extremely rare elsewhere in the world. Biochemical characterization of French isolates indicates that subgroup A1 and B2 strains have different carbohydrate utilization patterns (P. Vaissaire, A. Fouet, K. L. Smith, C. Keys, C. Le Doujet, P. Sylvestre, M. Levy, P. Keim, and M. Mock, presented at the 5th International Conference on Anthrax and 3rd International Workshop on the Molecular Biology of Bacillus cereus, B. anthracis and B. thuringiensis, 30 March to 3 April 2003, Nice, France). French subgroup A1 strains metabolize starch and glycogen but not gluconate, and the inverse is true for subgroup B2 strains. The genomes of several B. anthracis strains are available on the NCBI website (http://www.ncbi.nlm.nih.gov/), and two of these strains, Ames and CNEVA, are representative of groups A and B, respectively. We compared the genomic sequences of Ames and CNEVA to identify mutations that may affect metabolic activities involved in the phenotypic differences.The Kegg pathway database (http://www.genome.jp/kegg/pathway.html) was used to select enzyme activities involved in the metabolic pathways for starch, glycogen, and gluconate. BLAST analysis of the corresponding open reading frame in the Ames (subgroup A3) and CNEVA (subgroup B2) genomes was then used to identify the selected genes that were interrupted or mutated. The functions and localizations of these open reading frames were then investigated with the Pfam (http://pfam.sanger.ac.uk/), CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), SMART (http://smart.embl-heidelberg.de/), SignalP (http://www.cbs.dtu.dk/services/SignalP/), and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) search programs. A single-base deletion in the amyS gene (BA3551) encoding an alpha-amylase linked to starch and glycogen metabolism was found in the CNEVA genome. The wild-type AmyS protein contains 513 amino acids, and its predicted molecular mass is 58.4 kDa. In subgroup B2, there is a frameshift due to deletion of an adenosine in the 7th position of the nucleotide sequence that leads to a premature stop codon in the 13th position. In the Ames genome, a single-base substitution was found in the gntK gene (BA0162) encoding a gluconate kinase linked to gluconate metabolism. The predicted wild-type GntK protein contains 511 amino acids, and its predicted molecular mass is 56.7 kDa. The mutation identified is a cytosine-to-adenosine substitution at position 530 of the nucleotide sequence that leads to a premature stop codon at amino acid position 176. We confirmed the presence of these two mutations in the other B. anthracis subgroup genomes accessible in the NCBI unfinished microbial genome database and sequenced 12 isolates with various genotypes belonging to subgroups A1 and B2 (6 isolates in each subgroup) originating from outbreaks that occurred in different regions of France over the last 15 years. These analyses revealed that the deletion in amyS is restricted to strains belonging to group B subgroups, whereas the substitution in gntK is restricted to strains belonging to group A subgroups. The mutations identified in amyS and gntK both result in premature stop codons that lead to a loss of the enzymatic activities and may thus account for the observed phenotypic differences between subgroup A1 and B2 strains. We therefore focused on these two genes and used French strains 9602R and RA3R belonging to subgroups A1 and B2, respectively, for further analysis.     

Comparative Analysis of Pdf-Mediated Circadian Behaviors Between Drosophila melanogaster and D. virilis

PAR proteins (partitioning defective) are major regulators of cell polarity and asymmetric cell division. One of the par genes, par-1, encodes a Ser/Thr kinase that is conserved from yeast to mammals. In Caenorhabditis elegans, par-1 governs asymmetric cell division by ensuring the polar distribution of cell fate determinants. However the precise mechanisms by which PAR-1 regulates asymmetric cell division in C. elegans remain to be elucidated. We performed a genomewide RNAi screen and identified six genes that specifically suppress the embryonic lethal phenotype associated with mutations in par-1. One of these suppressors is mpk-1, the C. elegans homolog of the conserved mitogen activated protein (MAP) kinase ERK. Loss of function of mpk-1 restored embryonic viability, asynchronous cell divisions, the asymmetric distribution of cell fate specification markers, and the distribution of PAR-1 protein in par-1 mutant embryos, indicating that this genetic interaction is functionally relevant for embryonic development. Furthermore, disrupting the function of other components of the MAPK signaling pathway resulted in suppression of par-1 embryonic lethality. Our data therefore indicates that MAP kinase signaling antagonizes PAR-1 signaling during early C. elegans embryonic polarization.ASYMMETRIC cell division, a process in which a mother cell divides in two different daughter cells, is a fundamental mechanism to achieve cell diversity during development. We use the early embryo of Caenorhabditis elegans as a model system to study asymmetric cell division. The C. elegans one-cell embryo divides asymmetrically along its anteroposterior axis, generating two cells of different sizes and fates: the larger anterior daughter cell will generate somatic tissues while the smaller posterior daughter cell will generate the germline (Sulston et al. 1983).A group of proteins called PAR proteins (partitioning defective) is required for asymmetric cell division in C. elegans (Kemphues et al. 1988). Depletion of any of the seven par genes (par-1 to -6 and pkc-3) leads to defects in asymmetric cell division and embryonic lethality (Kemphues et al. 1988; Kirby et al. 1990; Tabuse et al. 1998; Hung and Kemphues 1999; Hao et al. 2006). PAR-3 and PAR-6 are conserved proteins that contain PDZ-domains and form a complex with PKC-3 (Etemad-Moghadam et al. 1995; Izumi et al. 1998; Tabuse et al. 1998; Hung and Kemphues 1999). This complex becomes restricted to the anterior cortex of the embryo in response to spatially defined actomyosin contractions occurring in the embryo upon fertilization (Goldstein and Hird 1996; Munro et al. 2004). The posterior cortex of the embryo that becomes devoid of the anterior PAR proteins is occupied by the RING protein PAR-2 and the Ser/Thr kinase PAR-1 (Guo and Kemphues 1995; Boyd et al. 1996; Cuenca et al. 2003). Once polarized, the anterior and posterior PAR proteins mutually exclude each other from their respective cortices (Etemad-Moghadam et al. 1995; Boyd et al. 1996; Cuenca et al. 2003; Hao et al. 2006). Loss of function of the gene par-1, as opposed to loss of most other par genes, results in embryos that exhibit only subtle effects on the polarized cortical domains occupied by the other PAR proteins (Cuenca et al. 2003). However defects in this gene are associated with a more symmetric division in size, an aberrant distribution of cell fate specification markers, altered cell fates of the daughter cells of the embryo, and ultimately embryonic lethality (Kemphues et al. 1988; Guo and Kemphues 1995).PAR-1 controls asymmetric cell division and cell fate specification by regulating the localization of the two highly similar CCCH-type zinc-finger proteins MEX-5 and MEX-6 (referred to as MEX-5/6). MEX-5 and MEX-6 are 70% identical in their amino acid sequence and fulfill partially redundant functions in the embryo (Schubert et al. 2000). In wild-type animals, endogenous MEX-5 and GFP fusions of MEX-6 localize primarily to the anterior of the embryo while both proteins are evenly distributed in par-1 mutant embryos (Schubert et al. 2000; Cuenca et al. 2003). This suggests that in wild-type animals, PAR-1 acts in part by restricting MEX-5 and MEX-6 to the anterior of the embryo. The precise mechanism of this regulation is not known, but an elegant study performed for MEX-5 indicates that differential protein mobility in the anterior and posterior cytoplasm of the one-cell embryo contributes to this asymmetry (Tenlen et al. 2008). While increased mobility in the posterior of the one-cell embryo correlates with a par-1- and par-4-dependent phosphorylation on MEX-5, the kinase directly phosphorylating MEX-5 remains to be identified (Tenlen et al. 2008).Some of the phenotypes associated with loss of par-1 function are dependent on the function of mex-5 and mex-6. First, loss of function of par-1 leads to a decreased stability and aberrant localization of the posterior cell fate specification marker PIE-1, a protein that is usually inherited by the posterior daughter cell in wild-type animals and ensures the correct specification of the germline (Mello et al. 1996; Seydoux et al. 1996). This decreased stability is dependent on mex-5/6 function as PIE-1 levels are restored, albeit with symmetrical distribution, in mex-6(RNAi); mex-5(RNAi); par-1(b274) embryos (Schubert et al. 2000; Cuenca et al. 2003; Derenzo et al. 2003). Second, embryos lacking par-1 function exhibit decreased amounts of P granules in the one-cell embryo, while these markers are present in mex-6(pk440); mex-5(zu199); par-1(RNAi) embryos of comparable age (Cheeks et al. 2004). Third, in par-1(RNAi) one-cell embryos the posterior cortical domain occupied by the polarity protein PAR-2 is extended anteriorly, when compared to wild-type embryos (Cuenca et al. 2003). This anterior extension is rescued in embryos deficient for both par-1 and mex-5/6 (Cuenca et al. 2003). Taken together, these results indicate that par-1 acts in the embryo—at least in part—by regulating the localization and/or activity of the proteins MEX-5 and MEX-6. However, it remains unclear whether other proteins can modulate PAR-1 function to affect MEX-5/6 activity.To gain insight into the mechanisms of par-1 function in the embryo, we sought to identify genes that act together with par-1 during embryonic development. We performed an RNAi-based screen for genetic interactors of the temperature-sensitive allele par-1(zu310), using the embryonic lethal phenotype of this mutant as a readout. This method has proven successful in previous screens to identify genes involved in early embryonic processes (Labbé et al. 2006; O''Rourke et al. 2007). We were able to identify six genes that, upon disruption of their function, suppress the embryonic lethal phenotype of par-1 mutant embryos. One of these genes is mpk-1, the C. elegans homolog of the highly conserved MAP kinase ERK. Closer analysis subsequently showed that reduction of function of mpk-1 not only increases viability of par-1 mutant embryos, but also reverts several polarity phenotypes associated with loss of function of par-1. Our data indicate that mpk-1 antagonizes par-1 activity to regulate polarization and asymmetric cell divisions in the early embryo.