Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.     

The burden of zoonoses in Paraguay: A systematic review

IntroductionUnderestimation of zoonoses is exacerbated in low and middle-income countries due mainly to inequalities with serious consequences in healthcare. This is difficult to gauge and reduce the impact of those diseases. Our study focuses on Paraguay, where the livestock industry is one of the major components of the country’s economy. Therefore, the rationale of this study was to develop a case study in Paraguay to estimate the dual impact of zoonotic diseases on both the human health and animal health sector and thus determine the societal burden of such diseases.Methodology/Principal findingsWe conducted a systemic review (including a meta-analysis) to assess the burden of zoonoses in Paraguay, including official reports and grey literature of disease incidence and prevalence. We estimated the Disability Adjusted Life Years (DALYs) and Zoonosis Disability Adjusted Life Years (zDALYs) to measure the difference between the current health status and the desired health situation of animals and the Paraguayan population based on 50 zoonotic diseases suggested by the WHO (World Health Organization), OIE (World Organization for Animal Health) and the National Health in Paraguay. The total DALYs represent 19,384 (95% CI: from 15,805 to 29,733), and zDALYs, 62,178 (95% CI: from 48,696 to 77,188). According to the results, the priority pathogens for DALYs are E. coli, Trypanosoma cruzi, Leishmania spp, and Toxoplasma gondii. When we include the additional animal health burden, the most important pathogens are Brucella spp, E. coli, Trypanosoma cruzi, and Fasciola hepatica for zDALYs.Conclusion/SignificanceThis is the first study to integrate DALYs and zDALYs with important clues related to the health status of Paraguay. Through DALYs and zDALYs, our perspective becomes more complete because we consider not only human health but also animal health. This is important for setting priorities in disease control, especially in a society where livestock contribute significantly to the economy and to human well-being.     

Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions

Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.     

Pig melatonin receptor 1a (MTNR1A) genotype is associated with seasonal variation of sow litter size

The duration of nocturnal melatonin secretion reaches its minimum in summer, a physiological event that is likely related with the diminished sow fertility and delayed puberty typically observed in this season. Melatonin exerts its function by binding two different receptors named as MTNR1A and MTNR1B. Interestingly, the MTNR1A gene is located on a chromosome SSC17 region where QTL for prolificacy traits have been detected in previous studies. In this work, we have found a synonymous T162C polymorphism at exon 2 of the pig MTNR1A gene. An association analysis between this polymorphism and sow prolificacy in an Iberian ×  Meishan intercross was performed. The utilization of four statistical models of increasing complexity demonstrated that the MTNR1A gene has both additive and dominant effects on total number of born piglets (TNB) and number of piglets born alive (NBA). Additive effects were significant in summer (TNB, P < 0.01; NBA, P < 0.001), whereas dominant effects reached significance both in fall (TNB, P < 0.01; NBA, P < 0.05) and in winter (TNB, P < 0.001; NBA, P < 0.05). The seasonal variation observed for MTNR1A additive and dominant effects might be produced by the influence of photoperiod on the pattern and duration of melatonin secretion. These results illustrate that the complex interaction between genotype and environment can be an important source of phenotypic variation of reproductive traits.     

A Novel Role for hGas7b in Microtubular Maintenance: POSSIBLE IMPLICATION IN TAU-ASSOCIATED PATHOLOGY IN ALZHEIMER DISEASE*

Here, we report a novel role for hGas7b (human growth arrest specific protein 7b) in the regulation of microtubules. Using a bioinformatic approach, we studied the actin-binding protein hGas7b with a structural similarity to the WW domain of a peptidyl prolyl cis/trans isomerase, Pin1, that facilitates microtubule assembly. Thus, we have demonstrated that hGas7b binds Tau at the WW motif and that the hGas7b/Tau protein complex interacts with the microtubules, promoting tubulin polymerization. Tau, in turn, contributes to protein stability of hGas7b. Furthermore, we observed decreased levels of hGas7b in the brains from patients with Alzheimer disease. These results suggest an important role for hGas7b in microtubular maintenance and possible implication in Alzheimer disease.     

Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.     

Mapping the distribution of conformational information throughout a protein sequence

The three-dimensional structure of protein is encoded in the sequence, but many amino acid residues carry no essential conformational information, and the identity of those that are structure-determining is elusive. By circular permutation and terminal deletion, we produced and purified 25 Bacillus licheniformis beta-lactamase (ESBL) variants that lack 5-21 contiguous residues each, and collectively have 82% of the sequence and 92% of the non-local atom-atom contacts eliminated. Circular dichroism and size-exclusion chromatography showed that most of the variants form conformationally heterogeneous mixtures, but by measuring catalytic constants, we found that all populate, to a greater or lesser extent, conformations with the essential features of the native fold. This suggests that no segment of the ESBL sequence is essential to the structure as a whole, which is congruent with the notion that local information and modular organization can impart most of the tertiary fold specificity and cooperativity.     

Differential gene expression in ovaries of pregnant pigs with high and low prolificacy levels and identification of candidate genes for litter size

Previous results from a genome scan in an F(2) Iberian × Meishan pig intercross showed several chromosome regions associated with litter size traits in this species. In order to identify candidate genes underlying these quantitative trait loci (QTL), we performed an ovary gene expression analysis during the sow's pregnancy. F(2) sows were ranked by their estimated breeding values for prolificacy: six sows with the highest estimated breeding value (EBV) (i.e., high prolificacy) and six sows with the lowest EBV (low prolificacy) were selected. Samples were hybridized using an Affymetrix GeneChip porcine genome array. Statistical analysis with a mixed model approach identified 221 differentially expressed probes, representing 189 genes. These genes were functionally annotated in order to identify genetic pathways overrepresented in this list. Among the functional groups most represented was, in first position, immune system response activation against external stimulus. The second group consisted of integrated genes that regulate maternal homeostasis by complement and coagulation cascades. A third group was involved in lipid and fatty acid enzymes of metabolic processes, which participate in the steroidogenesis pathway. In order to identify powerful candidate genes for prolificacy, the second approach of this study was to merge microarray data with the QTL positional information affecting litter size, previously detected in the same experimental cross. As a result, we have identified 27 differentially expressed genes colocalizing with QTL for litter size traits, which fulfill the biological, positional, and functional criteria.