Replacement of the essential Dictyostelium Arp2 gene by its Entamoeba homologue using parasexual genetics

<p>Abstract</p> <p>Background</p> <p>Cell motility is an essential feature of the pathogenesis and morbidity of amoebiasis caused by <it>Entamoeba histolytica</it>. As motility depends on cytoskeletal organisation and regulation, a study of the molecular components involved is key to a better understanding of amoebic pathogenesis. However, little is known about the physiological roles, interactions and regulation of the proteins of the <it>Entamoeba </it>cytoskeleton.</p> <p>Results</p> <p>We have established a genetic strategy that uses parasexual genetics to allow essential <it>Dictyostelium discoideum </it>genes to be manipulated and replaced with modified or tagged homologues. Our results show that actin related protein 2 (Arp2) is essential for survival, but that the <it>Dictyostelium </it>protein can be complemented by <it>E. histolytica </it>Arp2, despite the presence of an insertion of 16 amino acids in an otherwise highly conserved protein. Replacement of endogenous Arp2 with <it>myc</it>-tagged <it>Entamoeba </it>or <it>Dictyostelium </it>Arp2 has no obvious effects on growth and the protein incorporates effectively into the Arp2/3 complex.</p> <p>Conclusion</p> <p>We have established an effective two-step method for replacing genes that are required for survival. Our protocol will allow such genes to be studied far more easily, and also allows an unambiguous demonstration that particular genes are truly essential. In addition, cells in which the <it>Dictyostelium </it>Arp2 has been replaced by the <it>Entamoeba </it>protein are potential targets for drug screens.</p

Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase). Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity

The proteome of human brain microdialysate

BACKGROUND: Cerebral microdialysis has been established as a monitoring tool in neurocritically ill patients suffering from severe stroke. The technique allows to sample small molecules in the brain tissue for subsequent biochemical analysis. In this study, we investigated the proteomic profile of human cerebral microdialysate and if the identified proteins might be useful predictors for disease characteristics in stroke for tissue at risk in the contralateral hemisphere. We analysed cerebral protein expression in microdialysate from three stroke patients sampled from the hemisphere contralateral to the lesion. Using a proteomic approach based on two-dimensional gel electrophoresis and subsequent mass spectrometry, we created a protein map for the global protein expression pattern of human microdialyste. RESULTS: We found an average of 158 ± 24 (N = 18) protein spots in the human cerebral microdialysate and could identify 95 spots, representing 27 individual proteins. Most of these have been detected in human cerebrospinal fluid before, but 10 additional proteins mainly of cerebral intracellular origin were identified exclusively in the microdialysate. CONCLUSIONS: The 10 proteins found exclusively in human cerebral microdialysate, but not in cerebrospinal fluid, indicate the possibility to monitor the progression of the disease towards deterioration. The correlation of protein composition in the human cerebral microdialysate with the patients' clinical condition and results of cerebral imaging may be a useful approach to future applications for neurological stroke diagnosis, prognosis, and treatment

Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease

Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence

Role of multiparametric magnetic resonance imaging in early detection of prostate cancer.

UNLABELLED: Most prostate cancers (PC) are currently found on the basis of an elevated PSA, although this biomarker has only moderate accuracy. Histological confirmation is traditionally obtained by random transrectal ultrasound guided biopsy, but this approach may underestimate PC. It is generally accepted that a clinically significant PC requires treatment, but in case of an non-significant PC, deferment of treatment and inclusion in an active surveillance program is a valid option. The implementation of multiparametric magnetic resonance imaging (mpMRI) into a screening program may reduce the risk of overdetection of non-significant PC and improve the early detection of clinically significant PC. A mpMRI consists of T2-weighted images supplemented with diffusion-weighted imaging, dynamic contrast enhanced imaging, and/or magnetic resonance spectroscopic imaging and is preferably performed and reported according to the uniform quality standards of the Prostate Imaging Reporting and Data System (PIRADS). International guidelines currently recommend mpMRI in patients with persistently rising PSA and previous negative biopsies, but mpMRI may also be used before first biopsy to improve the biopsy yield by targeting suspicious lesions or to assist in the selection of low-risk patients in whom consideration could be given for surveillance. TEACHING POINTS: ? MpMRI may be used to detect or exclude significant prostate cancer. ? MpMRI can guide targeted rebiopsy in patients with previous negative biopsies. ? In patients with negative mpMRI consideration could be given for surveillance. ? MpMRI may add valuable information for the optimal treatment selection

Late Quaternary sedimentation off the western Sahara

Late Quaternary sediments on the West African continental margin between 24°N and 15°N were studied with RV METEOR (1971) and VALDIVIA (1975). Cores on the shelf were taken with a 6-m-vibrocorer, in deeper water mainly with a 12 m Kastenlot corer. During Holocene, and up to the present time, more or less and climatic conditions north of the Senegal River area reduced terrigenous supply. Therefore, the biogenic-carbonate content exceeds about 50%. Wüstenquarz numbers (red + yellow quartz : white quartz x 100) are high (20 to mmore than 200), indicating eolian input. The Senegal River supplied fine grained, green colored, terrigenous material with some plant debris. During Würm, the Mediterranean climatic zone with winter rains was shifted more than 5° to the South and was reaching Banc d'Arguin (at about 20°N). Therefore, the terrigenous supply was increased in this northern part and consequently the carbonate content and the Wüstenquartz numbers dropped below 50% and 10, respectively. The arid zone was also shifted to the south; as a consequence, the Senegal River did not reach the sea, eolian supply diluted the biogenic carbonates, and increased Wüstenquartz numbers to more than 200. Eolian dunes covered parts of the shelf. Ratios of radiolarians/plankonic foraminifera and planktonic/benthonic organisms and sedimentation rates of organic carbon indicate stronger upwelling in the northern region. Turbidity currents were more frequent, eroding as much as a third of the material supplied ba pelagic sedimentation

The Weddell Gyre, Southern Ocean: present knowledge and future challenges

The Weddell Gyre (WG) is one of the main oceanographic features of the Southern Ocean south of the Antarctic Circumpolar Current which plays an influential role in global ocean circulation as well as gas exchange with the atmosphere. We review the state‐of‐the art knowledge concerning the WG from an interdisciplinary perspective, uncovering critical aspects needed to understand this system's role in shaping the future evolution of oceanic heat and carbon uptake over the next decades. The main limitations in our knowledge are related to the conditions in this extreme and remote environment, where the polar night, very low air temperatures and presence of sea ice year‐round hamper field and remotely sensed measurements. We highlight the importance of winter and under‐ice conditions in the southern WG, the role that new technology will play to overcome present‐day sampling limitations, the importance of the WG connectivity to the low‐latitude oceans and atmosphere, and the expected intensification of the WG circulation as the westerly winds intensify. Greater international cooperation is needed to define key sampling locations that can be visited by any research vessel in the region. Existing transects sampled since the 1980s along the Prime Meridian and along an East‐West section at ~62°S should be maintained with regularity to provide answers to the relevant questions. This approach will provide long‐term data to determine trends and will improve representation of processes for regional, Antarctic‐wide and global modeling efforts – thereby enhancing predictions of the WG in global ocean circulation and climate