Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Evaluation of organic insecticides for management of spotted‐wing drosophila (Drosophila suzukii) in berry crops

Spotted‐wing drosophila, Drosophila suzukii (Matsumura), is an invasive pest affecting fruit production in many regions of the world. Insecticides are the primary tactic for controlling D. suzukii in organic as well as conventional production systems. Organic growers have a greater challenge because fewer insecticides are approved for use in organic agriculture. The most effective organically approved product is spinosad, but alternatives are needed because of label restrictions limiting the number of applications per year, toxicity to beneficial arthropods and the risk of developing resistance. We evaluated several organically approved insecticides against D. suzukii in laboratory assays and field trials conducted on organic blueberry and raspberry farms. Spinosad was consistently the most effective insecticide, but a few other insecticides such as azadirachtin + pyrethrins, Chromobacterium subtsugae and sabadilla alkaloids showed moderate activity. None of the treatments had long residual activity. Mortality started to decline by 3 days after treatment, and by 5 days after application, the treatments were not different from the controls. These products may be useful in rotation programmes, necessary for reducing reliance on spinosad and mitigating resistance. Cultural and biological control approaches are needed in fruit production for D. suzukii management, but insecticides will likely continue to be the dominant management tactic while these other approaches are being optimized and adopted.     

Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus

The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1(H) and HA-1(R), with a single amino acid difference. Whereas the immunogenicity of the HA-1(R) allele has not yet been shown, the nonameric HA-1(H) peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1(H) or HA-1(R) associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1(H/R) peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1(H/R) peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVL(H)/(R)DDL and decameric KECVL(H)/(R)DDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1(H) and HA-1(R) peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1(R) peptide did not induce CTLs that recognize the natural HLA-B60/HA-1(R) ligand. In contrast, dendritic cells pulsed with the nonameric HA-1(H) peptide induced IFN-gamma-secreting T cells specific for the natural HLA-B60/HA-1(H) ligand in three HLA-B60(+) HA-1(RR) individuals, demonstrating the immunogenicity of the HLA-B60/HA-1(H) ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.     

Polygyny in the tree swallow Tachycineta bicolor: a result of the cost of searching for an unmated male

The costs imposed by parental care duties on an individual's future survival and reproduction generate conflicts because parents should attempt to minimize their investment in the present brood, and exploit the parental care of the other parent. This conflict is likely to contribute to cases of both polygamy and desertion. Here, we study the costs of polygyny in the tree swallow Tachycineta bicolor , using observations on 52 nests that were attended by polygynous males over 14 y. Both females mated to polygynous males paid reproductive costs at several stages of the nesting cycle. Clutches laid by social mates of polygynous versus monogamous males did not differ in size. However, initial brood sizes for polygynously mated females were lower because a higher proportion of their eggs failed to hatch. Likewise, fledging success was lower and nest predation rates were higher, perhaps reflecting the direct or indirect effects of reduced male attention. These results demonstrate that females pay a productivity cost when breeding with reduced male parental care. In contrast, polygynous males fledge on average more young than monogamous ones and clearly benefit from the association. We suggest that a mate-search cost is leading to the few cases of polygamous males: although females are likely evaluating males for their prospective dedication to the breeding attempt, in a short-lived bird with a short breeding season, the cost to females of searching for a more dedicated male is the risk of not breeding at all.     

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.     

O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase

Irreversible inhibition by molecular oxygen (O(2)) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H(2)) production. Modification by O(2) of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe(H)) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O(2) exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O(2) reactions. The first phase (τ(1) ≤ 4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe(H)), and oxidation of one iron ion. The second phase (τ(2) ≈ 15 s) causes a ～50% decrease of the number of ～2.7-? Fe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ(3) ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe(H(+)) leading to the formation of a reactive oxygen species-like superoxide (O(2)(-)), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S](+) unit, leading to 3) complete O(2)-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O(2) tolerance in [FeFe]-hydrogenase.     

Eight new polymorphic microsatellite DNA markers for Sakhalin taimen Parahucho perryi

We developed eight polymorphic microsatellite markers for the endangered salmonid Parahucho perryi from genomic libraries enriched for (GACA)n and (GATA)n repeat sequences. Emphasis was placed on developing highly polymorphic, perfect repeats that could be scored with confidence. Six tetra- and two di-nucleotide loci were screened in 49 individuals from two populations on Sakhalin Island, Russia. Allelic variation was high with eight to 23 alleles per locus and expected heterozygosity ranging from 0.48 to 0.93. These highly variable markers should prove useful in evaluating inbreeding, gene flow and population structure in Sakhalin taimen throughout its range.     

Peripheral metabolic responses to prolonged weight reduction that promote rapid, efficient regain in obesity-prone rats

Weight regain after weight loss is the most significant impediment to long-term weight reduction. We have developed a rodent paradigm that models the process of regain after weight loss, and we have employed both prospective and cross-sectional analyses to characterize the compensatory adaptations to weight reduction that may contribute to the propensity to regain lost weight. Obese rats were fed an energy-restricted (50-60% kcal) low-fat diet that reduced body weight by 14%. This reduced weight was maintained for up to 16 wk with limited provisions of the low-fat diet. Intake restriction was then removed, and the rats were followed for 56 days as they relapsed to the obese state. Prolonged weight reduction was accompanied by 1) a persistent energy gap resulting from an increased drive to eat and a reduced expenditure of energy, 2) a higher caloric efficiency of regain that may be linked with suppressed lipid utilization early in the relapse process, 3) preferential lipid accumulation in adipose tissue accompanied by adipocyte hyperplasia, and 4) humoral adiposity signals that underestimate the level of peripheral adiposity and likely influence the neural pathways controlling energy balance. Taken together, long-term weight reduction in this rodent paradigm is accompanied by a number of interrelated compensatory adjustments in the periphery that work together to promote rapid and efficient weight regain. These metabolic adjustments to weight reduction are discussed in the context of a homeostatic feedback system that controls body weight.