Evolution of novel reassortant A/H3N2 influenza viruses in North American swine and humans, 2009-2011

Novel H3N2 influenza viruses (H3N2v) containing seven genome segments from swine lineage triple-reassortant H3N2 viruses and a 2009 pandemic H1N1 (H1N1pdm09) matrix protein segment (pM) were isolated from 12 humans in the United States between August and December 2011. To understand the evolution of these novel H3N2 viruses in swine and humans, we undertook a phylogenetic analysis of 674 M sequences and 388 HA and NA sequences from influenza viruses isolated from North American swine during 2009-2011, as well as HA, NA, and M sequences from eight H3N2v viruses isolated from humans. We identified 34 swine influenza viruses (termed rH3N2p) with the same combination of H3, N2, and pM segments as the H3N2v viruses isolated from humans. Notably, these rH3N2p viruses were generated in swine via reassortment events between H3N2 viruses and the pM segment approximately 4 to 10 times since 2009. The pM segment has also reassorted with multiple distinct lineages of H1 virus, especially H1δ viruses. Importantly, the N2 segment of all H3N2v viruses isolated from humans is derived from a genetically distinct N2 lineage that has circulated in swine since being acquired by reassortment with seasonal human H3N2 viruses in 2001-2002, rather than from the N2 that is associated with the 1998 H3N2 swine lineage. The identification of this N2 variant may have implications for influenza vaccine design and the potential pandemic threat of H3N2v to human age groups with differing levels of prior exposure and immunity.     

BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells

Background aimsTo date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts.MethodsWe describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively.ResultsTransgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin.ConclusionsIt appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.     

Ingestion of spinosad bait GF-120 and resulting impact on adult Chrysoperla carnea (Neuroptera: Chrysopidae)

The spinosad bait spray GF-120 Naturalyte F® (Dow AgroSciences LLC, Indianapolis, IN, USA) is widely used in commercial olives in California. Because of concern about its non-target effects on beneficial insects, we studied its impact on feeding behavior, mortality, and reproductive parameters of adult green lacewings, Chrysoperla carnea (Stephens), under laboratory conditions. Male and female feeding rate and quantity, and subsequent mortality, were compared over a 5-day period following a 24-h exposure to honey, GF-120, or the bait component (without spinosad) in two-choice and no-choice assays. All treatments were ingested equally when offered alone. Spinosad did not impart repellency or preference to GF-120 relative to the bait. Honey was preferred in choice tests with GF-120 or bait. Significant mortality occurred when GF-120 was offered alone, but was not significant when it was offered in choice with honey or bait. In a separate assay of female longevity and reproduction, mortality rate as a function of time was similar in all treatments, except for some significantly earlier mortality due to GF-120 compared with honey. GF-120 ingestion significantly reduced fecundity compared with bait during the first week after treatment. Egg hatch was not significantly affected. The preference of adult C. carnea for food should ameliorate the potential negative effect of GF-120 on adult C. carnea mortality in the field. The laboratory study finds that GF-120 is a potentially safer choice for C. carnea than an aqueous suspension of spinosad, and provides no new evidence for definitive risk to field populations.     

Maintenance and thermal stabilization of NADH dehydrogenase-2 conformation upon elimination of its C-terminal region

Development of an artificial enzyme with activity and structure comparable to that of natural enzymes is an important goal in biological chemistry. Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein, belonging to a group of enzymes with scarce structural information. By eliminating the C-terminal region of NDH-2, a water soluble version with significant enzymatic activity was previously obtained. Here, NDH-2 structural features were established, in comparison to those of the truncated version. Far-UV circular dichroism, Fourier transform infrared spectroscopy and limited proteolysis analysis showed that the overall structure of both proteins was similar at 30 °C. Experimental data agree with the predicted NDH-2 structure (PDB: 1OZK). The absence of C-terminal region stabilized in ∼5–10 °C the truncated protein conformation. However, truncation impaired enzymatic activity at low temperatures, probably due to the weak interaction of the mutant protein with FAD cofactor.     

Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to “sense” pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.     

Quantitative trait loci regulating sugar moiety of acylsugars in tomato

Acylsugars are broad-spectrum insect resistance sugar esters produced at very high levels by some accessions of the wild tomato, Solanum pennellii. Transferring acylsugar production from S. pennellii LA716 to cultivated tomato through traditional breeding developed the benchmark acylsugar breeding line CU071026. The base moiety of acylsugars (sucrose vs. glucose) can vary among S. pennellii accessions. Additionally the accession S. pennellii LA716 produces almost exclusively acylglucoses, but the breeding line CU071026 derived from S. pennellii LA716 produces exclusively acylsucroses. This study uses a BC1F1 and a BC1F2 population derived from the cross CU071026 × (CU071026 × S. pennellii LA716) to identify and confirm the action of three quantitative trait loci (QTL) on chromosomes 3, 4, and 11. The QTL on chromosomes 3 and 11 are both required for acylglucose production, while addition of the chromosome 4 QTL affects the level of acylglucose produced in the presence of the QTL on chromosomes 3 and 11. A three-way interaction between these acylglucose QTL was confirmed with a post hoc ANOVA. Identification of these three QTL provides a blueprint for breeding to shift acylsucrose production to acylglucose production in tomato breeding lines. The implications of these QTL and two additional QTL affecting total acylsugar level in the BC1F2 are discussed.     

Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii. Received April 16, 1998; accepted June 30, 1998.