Genetic and biochemical consequences of thymidylate stress

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.     

Molecular analysis of the interaction of LCMV with its cellular receptor [alpha]-dystroglycan

alpha-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a beta-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to alpha-DG and addressed the role of alpha-DG-associated host-derived proteins in virus infection. We found that the COOH-terminal region of alpha-DG's first globular domain and the NH2-terminal region of the mucin-related structures of alpha-DG together form the binding site for LCMV. The virus-alpha-DG binding unlike ECM alpha-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on alpha-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to alpha-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to alpha-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to alpha-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express alpha-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.     

Prehospital Thrombolysis: A Manual from Berlin

In acute ischemic stroke, time from symptom onset to intervention is a decisive prognostic factor. In order to reduce this time, prehospital thrombolysis at the emergency site would be preferable. However, apart from neurological expertise and laboratory investigations a computed tomography (CT) scan is necessary to exclude hemorrhagic stroke prior to thrombolysis. Therefore, a specialized ambulance equipped with a CT scanner and point-of-care laboratory was designed and constructed. Further, a new stroke identifying interview algorithm was developed and implemented in the Berlin emergency medical services. Since February 2011 the identification of suspected stroke in the dispatch center of the Berlin Fire Brigade prompts the deployment of this ambulance, a stroke emergency mobile (STEMO). On arrival, a neurologist, experienced in stroke care and with additional training in emergency medicine, takes a neurological examination. If stroke is suspected a CT scan excludes intracranial hemorrhage. The CT-scans are telemetrically transmitted to the neuroradiologist on-call. If coagulation status of the patient is normal and patient''s medical history reveals no contraindication, prehospital thrombolysis is applied according to current guidelines (intravenous recombinant tissue plasminogen activator, iv rtPA, alteplase, Actilyse).Thereafter patients are transported to the nearest hospital with a certified stroke unit for further treatment and assessment of strokeaetiology. After a pilot-phase, weeks were randomized into blocks either with or without STEMO care. Primary end-point of this study is time from alarm to the initiation of thrombolysis. We hypothesized that alarm-to-treatment time can be reduced by at least 20 min compared to regular care.     

A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover

A detailed understanding of the mechanisms underlying the capacity of a virus to break the species barrier is crucial for pathogen surveillance and control. New World (NW) mammarenaviruses constitute a diverse group of rodent-borne pathogens that includes several causative agents of severe viral hemorrhagic fever in humans. The ability of the NW mammarenaviral attachment glycoprotein (GP) to utilize human transferrin receptor 1 (hTfR1) as a primary entry receptor plays a key role in dictating zoonotic potential. The recent isolation of Tacaribe and lymphocytic choriominingitis mammarenaviruses from host-seeking ticks provided evidence for the presence of mammarenaviruses in arthropods, which are established vectors for numerous other viral pathogens. Here, using next generation sequencing to search for other mammarenaviruses in ticks, we identified a novel replication-competent strain of the NW mammarenavirus Tamiami (TAMV-FL), which we found capable of utilizing hTfR1 to enter mammalian cells. During isolation through serial passaging in mammalian immunocompetent cells, the quasispecies of TAMV-FL acquired and enriched mutations leading to the amino acid changes N151K and D156N, within GP. Cell entry studies revealed that both substitutions, N151K and D156N, increased dependence of the virus on hTfR1 and binding to heparan sulfate proteoglycans. Moreover, we show that the substituted residues likely map to the sterically constrained trimeric axis of GP, and facilitate viral fusion at a lower pH, resulting in viral egress from later endosomal compartments. In summary, we identify and characterize a naturally occurring TAMV strain (TAMV-FL) within ticks that is able to utilize hTfR1. The TAMV-FL significantly diverged from previous TAMV isolates, demonstrating that TAMV quasispecies exhibit striking genetic plasticity that may facilitate zoonotic spillover and rapid adaptation to new hosts.     

Maxillofacial reconstruction with prefabricated osseous free flaps: a 3-year experience with 24 patients

Between January of 1998 and May of 2002, 25 prefabricated osseous free flaps (23 fibula and two iliac crest flaps) were transferred in 24 patients to repair maxillary (six flaps) or mandibular (eight flaps) defects after tumor resection, severe maxillary (four flaps) or mandibular (one flap) atrophy (Cawood VI), maxillary (one flap) or mandibular (three flaps) defects after gunshot injury, and maxillary (two flaps) defects after traffic accidents. Prefabrication included insertion of dental implants, positioned with a drilling template in a preplanned position, and split-thickness grafting. Drilling template construction was based on the prosthetic planning. The template determined the position of the implants and the site and angulation of osteotomies, if necessary. The mean delay between prefabrication and flap transfer was 6 weeks (range, 4 to 8 weeks). While the flap was harvested, a bar construction with overdentures was mounted onto the implants. The overdentures were used as an occlusal key for exact three-dimensional positioning of the graft within the defect. The bar construction also helped to stabilize the horseshoe shape of the graft. The follow-up period ranged from 2 months to 4 years (mean, 21 months), during which time two total and three partial flap losses occurred. One total loss was due to thrombosis of the flap veins during the delay period, whereas the other total loss was caused by spasm of the peroneal artery. Two partial losses were due to oversegmentation of the flaps with necrosis of the distal fragment, whereas one partial loss was caused by disruption of the vessel from the distal part. Of the 90 implants that were inserted into the prefabricated flaps during the study period, 10 were lost in conjunction with flap failure; of the remaining 80 implants, four were lost during the observation period, for a success rate of 95 percent. Flap prefabrication based on prosthetic planning offers a powerful tool for various reconstructive problems in the maxillofacial area. Although it involves a two-stage procedure, the time for complete rehabilitation is shorter than with conventional procedures.     

Evaluation of the Duopath Legionella Lateral Flow Assay for Identification of Legionella pneumophila and Legionella Species Culture Isolates

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries’ regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.     

Active transport of proteins into the nucleus

Nuclear proteins are actively and posttranslationally transported across the nuclear envelope. This transport is a highly selective process that can be divided into two steps, receptor-binding followed by translocation through the nuclear envelope. Receptor-binding is mediated by nuclear localization signals that have been identified in many nuclear proteins. Translocation is energy-dependent and occurs through the nuclear pore complex.     

Proteins and peroxidase in callus and suspension cultures of apple : a study using ultrathin-layer isoelectric focusing, sensitive silver staining of proteins, and peroxidase isozyme visualization

Different methods for the isolation of soluble proteins were applied to cell cultures of three apple cultivars (Malus sylvestris Mill.), best results being obtained with a rapid technique based on freezing and thawing. Ultrathin-layer isoelectric focusing followed by an improved silver staining method has shown that proteins from apple callus cultures consist of some 60 to 80 zones, with isoelectric points mainly between pH 4 to 7. Depending on protein content, adequate silver staining is achieved with 50 to 500 cells. Protein patterns of callus cultures allowed clear discrimination of cultivars. Protein and peroxidase isozyme patterns in cell saps of suspension cultures show striking differences during the growth cycle, whereas the protein patterns from the nutrient media were constant over the entire cultivation period and closely resembled the patterns of stationary phase and callus cells.