A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Δthr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Δthr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.     

Toll-Like Receptor Family Polymorphisms Are Associated with Primary Renal Diseases but Not with Renal Outcomes Following Kidney Transplantation

Toll-like receptors (TLRs) play a crucial role in innate- and adaptive immunity. The TLR pathways were shown to play key functional roles in experimental acute and chronic kidney injury, including the allo-immune response after experimental renal transplantation. Data about the precise impact of TLRs and their negative regulators on human renal transplant outcomes however are limited and contradictory. We studied twelve non-synonymous single nucleotide polymorphisms (SNPs) of which eleven in TLR1-8 and one in SIGIRR in a final cohort comprising 1116 matching donors and recipients. TLR3 p.Leu412Phe and SIGIRR p.Gln312Arg significantly deviated from Hardy-Weinberg equilibrium and were excluded. The frequency distribution of the minor alleles of the remaining 10 TLR variants were compared between patients with end-stage renal disease (recipients) and controls (kidney donors) in a case-control study. Secondly, the associations between the minor allele frequency of the TLR variants and delayed graft function, biopsy-proven acute rejection and death-censored graft failure after transplantation were investigated with Cox regression. Carrier frequencies of the minor alleles of TLR1 p.His305Leu (OR = 4.79, 95% CI = 2.35–9.75, P = 0.0002), TLR1 p.Asn248Ser (OR = 1.26, 95% CI = 1.07–1.47, P = 0.04) and TLR8 p.Met1Val (OR = 1.37, 95% CI = 1.14–1.64, P = 0.008) were significantly higher in patients with ESRD, with little specificity for the underlying renal disease entity (adjusted for age, gender and donor-recipient relatedness). The minor allele frequency of none of the TLR variants significantly associated with the surrogate and definite outcomes, even when multivariable models were created that could account for TLR gene redundancy. In conclusion, genetic variants in TLR genes were associated with the prevalence of ESRD but not renal transplant outcomes. Therefore, our data suggests that specific TLR signaling routes might play a role in the final common pathway of primary renal injury. A role for TLR signaling in the context of renal transplantation is probably limited.     

Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2K^k and H-2D^d MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2–5)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.     

Engineered vascular beds provide key signals to pancreatic hormone-producing cells

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.     

Apyrases (nucleoside triphosphate-diphosphohydrolases) play a key role in growth control in Arabidopsis

Expression of two Arabidopsis (Arabidopsis thaliana) apyrase (nucleoside triphosphate-diphosphohydrolase) genes with high similarity, APY1 and APY2, was analyzed during seedling development and under different light treatments using beta-glucuronidase fusion constructs with the promoters of both genes. As evaluated by beta-glucuronidase staining and independently confirmed by other methods, the highest expression of both apyrases was in rapidly growing tissues and/or tissues that accumulate high auxin levels. Red-light treatment of etiolated seedlings suppressed the protein and message level of both apyrases at least as rapidly as it inhibited hypocotyl growth. Adult apy1 and apy2 single mutants had near-normal growth, but apy1apy2 double-knockout plants were dwarf, due primarily to reduced cell elongation. Pollen tubes and etiolated hypocotyls overexpressing an apyrase had faster growth rates than wild-type plants. Growing pollen tubes released ATP into the growth medium and suppression of apyrase activity by antiapyrase antibodies or by inhibitors simultaneously increased medium ATP levels and inhibited pollen tube growth. These results imply that APY1 and APY2, like their homologs in animals, act to reduce the concentration of extracellular nucleotides, and that this function is important for the regulation of growth in Arabidopsis.     

Biallelic genome modification in F0 Xenopus tropicalis embryos using the CRISPR/Cas system

Gene inactivation is an important tool for correlation of phenotypic and genomic data, allowing researchers to infer normal gene function based on the phenotype when the gene is impaired. New and better approaches are needed to overcome the shortfalls of existing methods for any significant acceleration of scientific progress. We have adapted the CRISPR/Cas system for use in Xenopus tropicalis and report on the efficient creation of mutations in the gene encoding the enzyme tyrosinase, which is responsible for oculocutaneous albinism. Biallelic mutation of this gene was detected in the F₀ generation, suggesting targeting efficiencies similar to that of TALENs. We also find that off‐target mutagenesis seems to be negligible, and therefore, CRISPR/Cas may be a useful system for creating genome modifications in this important model organism. genesis 51:827–834. © 2013 Wiley Periodicals, Inc.     

Non hemolytic short peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents

Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt to find the pharmacophore of short antimicrobial peptidomimetics through systematic tuning of hydrophobic and hydrophilic patterns, we have identified a set of short histidine-derived antimicrobial peptides (SAMPs) with potent and broad-spectrum activity. A combination of high antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), without hemolytic activity and proteolytic stability makes these molecules promising candidates for novel antimicrobial therapeutics.