Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes

Summary Molecular cloning of DNA fragments between 1.5 and 8kb from BamHI, EcoRI, HindIII, SalI, or Sau3A digests permitted the isolation of structural genes coding for TEM-1, ROB-1, OXA-1, OXA-3, OXA-4, OXA-5, PSE-1, PSE-2, PSE-3, PSE-4, CARB-3, CARB-4, AER-1, and LCR-1 -lactamases. Ampicillin-resistant clones were selected and it was confirmed that they contained the respective -lactamase genes by isoelectric focusing. Detailed physical maps of 14 different recombinant plasmids were constructed using 8 restriction endonucleases. Plasmid deletions and lacZ fusions were used to localize the -lactamase structural genes. DNA probes were constructed for the TEM01, ROB-1, OXA-1, and PSE-1 genes. Under conditions of high stringency, hybridization was observed between the genes for TEM-1 and TEM-2 or TLE-1, OXA-1 and OXA-4, and PSE-1 and PSE-4 or CARB-3, while the ROB-1 gene probe showed no cross-hybridization. Such bla gene probes should facilitate studies of -lactamase molecular epidemiology.     

Coevolution is linked with phenotypic diversification but not speciation in avian brood parasites

Coevolution is often invoked as an engine of biological diversity. Avian brood parasites and their hosts provide one of the best-known examples of coevolution. Brood parasites lay their eggs in the nests of other species, selecting for host defences and reciprocal counteradaptations in parasites. In theory, this arms race should promote increased rates of speciation and phenotypic evolution. Here, we use recently developed methods to test whether the three largest avian brood parasitic lineages show changes in rates of phenotypic diversity and speciation relative to non-parasitic lineages. Our results challenge the accepted paradigm, and show that there is little consistent evidence that lineages of brood parasites have higher speciation or extinction rates than non-parasitic species. However, we provide the first evidence that the evolution of brood parasitic behaviour may affect rates of evolution in morphological traits associated with parasitism. Specifically, egg size and the colour and pattern of plumage have evolved up to nine times faster in parasitic than in non-parasitic cuckoos. Moreover, cuckoo clades of parasitic species that are sympatric (and share similar host genera) exhibit higher rates of phenotypic evolution. This supports the idea that competition for hosts may be linked to the high phenotypic diversity found in parasitic cuckoos.     

Status and prospects for renewable energy using wood pellets from the southeastern United States

The ongoing debate about costs and benefits of wood‐pellet based bioenergy production in the southeastern United States (SE USA) requires an understanding of the science and context influencing market decisions associated with its sustainability. Production of pellets has garnered much attention as US exports have grown from negligible amounts in the early 2000s to 4.6 million metric tonnes in 2015. Currently, 98% of these pellet exports are shipped to Europe to displace coal in power plants. We ask, ‘How is the production of wood pellets in the SE USA affecting forest systems and the ecosystem services they provide?’ To address this question, we review current forest conditions and the status of the wood products industry, how pellet production affects ecosystem services and biodiversity, and what methods are in place to monitor changes and protect vulnerable systems. Scientific studies provide evidence that wood pellets in the SE USA are a fraction of total forestry operations and can be produced while maintaining or improving forest ecosystem services. Ecosystem services are protected by the requirement to utilize loggers trained to apply scientifically based best management practices in planning and implementing harvest for the export market. Bioenergy markets supplement incomes to private rural landholders and provide an incentive for forest management practices that simultaneously benefit water quality and wildlife and reduce risk of fire and insect outbreaks. Bioenergy also increases the value of forest land to landowners, thereby decreasing likelihood of conversion to nonforest uses. Monitoring and evaluation are essential to verify that regulations and good practices are achieving goals and to enable timely responses if problems arise. Conducting rigorous research to understand how conditions change in response to management choices requires baseline data, monitoring, and appropriate reference scenarios. Long‐term monitoring data on forest conditions should be publicly accessible and utilized to inform adaptive management.     

Enhancement of Histological Detail Using Metanil Yellow as Counterstain in Periodic Acid Schiff's Hematoxylin Staining of Glycol Methacrylate Tissue Sections

Histological detail in sections from tissues embedded in glycol methacrylate was improved by counterstaining PAS/iron-hematoxylin stained sections with a dilute solution of metanil yellow. The addition of the counterstain increases contrast in tissue sections and highlights PAS-positive entities. The staining protocol provides sharp definition of tissue morphology, differentiates cell types and other tissue components and does not produce background staining.     

Detection of genes essential in specific niches by signature-tagged mutagenesis

Variations of the signature-tagged mutagenesis (STM) technique are now possible and the method can be applied to most pathogens that have an STM-selectable phenotype in a host system. STM screening of 15,040 mutants from 11 bacterial species identified 323 in vivo attenuated mutants. As a genome-scanning tool, STM will yield information about genes with unknown functions as well as information crucial for understanding microbial pathogenesis.     

Suppression of VLDL secretion by cultured hepatocytes incubated with chylomicron remnants enriched in n−3 polyunsaturated fatty acids is regulated by hepatic nuclear factor-4α

Dietary n? 3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4α (HNF-4α) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n? 3 PUFA) oil were incubated with cultured rat hepatocytes (24 h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4α, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4α protein and mRNA levels were significantly decreased by CRLPs enriched in n? 3 PUFA, but not SFA, and the expression of mRNA for HNF-4α target genes, including HNF-1α, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n? 3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n? 3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4α.     

Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (～5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had～50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems.