Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.DNA polymerases are essential for DNA replication and DNA repair. Based on sequence similarities and phylogenetic relationships, DNA polymerases are grouped into six different families (A, B, C, D, X, and Y) (17). In this study, we used a DNA polymerase I (polA) mutant of Escherichia coli as a host for the screening of metagenomic libraries. PolA belongs to family A and contains three different domains: a 5Ј-3Ј exonuclease domain at the N terminus, a central proofreading 3Ј-5Ј exonuclease domain, and a polymerase domain at the C terminus of the enzyme (11). These polymerases are employed as tools in molecular biology, including probe labeling, DNA sequencing, and mutagenic PCR (13). To improve their suitability for such applications, various family A DNA polymerases have been modified; e.g., the Klenow fragment of E. coli DNA polymerase I has been redesigned by the removal of the 5Ј-3Ј exonuclease domain (12). Nevertheless, expanding the known DNA polymerase sequence space and discovery of polymerases with novel properties are required for the development of novel or improved molecular methods and tools (13,20).Metagenomics based on direct isolation of DNA from environmental samples, generation of metagenomic libraries from the isolated DNA, and function-based screening of the constructed libraries has led to identification and characterization of a variety of novel biocatalysts, such as lipases, amylases, amidases, nitrilases, and oxidoreductases (for reviews, see references 6, 7, and 10). In particular, the use of host strains or mutants of host strains that require heterologous complementation for growth under selective conditions has proven to be an efficient strategy to screen complex metagenomic libraries. This approach has been applied to, e.g., the isolation of genes encoding Na ϩ /H ϩ antiporters (14), antibiotic resistance (18), or enzymes involved in poly-3-hydroxybutyrate metabolism (21).In this study, we employed the last-named strategy to recover functional genes encoding DNA polymerases. To our knowledge, this is the first report of identification of polymerases or other DNA-modifying enzymes by function-driven screening of metagenomes. For this purpose, we constructed small-insert and large-insert metagenomic libraries from DNA isolated from glacial ice. The employment of glacial ice samples for metagenomic library construction has not been reported by other researchers. The screening for the targeted genes was based on complementation of a cold-sensitive lethal mutation in the polA gene of E. coli (16).Sample collection and construction of metagenomic glacier ice libraries. G...

Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function-associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein-coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1-phosphate-directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4 + CD25 + FoxP3 + regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation.

Background: Valproic acid (VPA) has been approved for the treatment of seizure disorders. It is also commonly used in psychiatric disorders, such as schizophrenia spectrum disorders. With increasing administration, reports of intoxications are more frequently reported. The most common findings of VPA intoxication are central nervous system depression, respiratory depression, hypotension, metabolic acidosis, and elevated lactate, among others.Methods: We describe a case report of VPA intoxication with hemodiafiltration (HDF) as extracorporeal treatment (ECTR) for removal of VPA. This treatment modality has only rarely been reported in the current literature. In addition, we performed an updated systematic literature review (SLR) of additional cases on the topic ranging from December 1st, 2014 to April 20th, 2018. We searched MEDLINE and Web of Science for relevant references.Results: In the presented case, VPA intoxication occurred in a 46-year-old female patient after oral ingestion of 56 g of VPA. In addition to vasopressors and endotracheal intubation, we administered L-Carnitine (L-Car) and performed hemodiafiltration treatment. After intravenous therapy with L-Car and simultaneous HDF sessions, we observed full recovery without neurological sequelae. The SLR identified 8 additional articles reporting favorable outcomes with extracorporeal treatments in most cases.Conclusion: HDF and other extracorporeal procedures are safe and effective therapeutic options in patients with VPA intoxication. The choice of ECTR modality mainly depends on local experience and the setting. In the present case, ingestion of 56 g was successfully treated with HDF. These findings are in line with several other case reports describing positive outcomes. Extracorporeal treatment, including HDF, should be considered early in the management of VPA intoxication. Supporting evidence is emerging, but it is of limited quality.