Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

The Functional Potential of Microbial Communities in Hydraulic Fracturing Source Water and Produced Water from Natural Gas Extraction Characterized by Metagenomic Sequencing

Microbial activity in produced water from hydraulic fracturing operations can lead to undesired environmental impacts and increase gas production costs. However, the metabolic profile of these microbial communities is not well understood. Here, for the first time, we present results from a shotgun metagenome of microbial communities in both hydraulic fracturing source water and wastewater produced by hydraulic fracturing. Taxonomic analyses showed an increase in anaerobic/facultative anaerobic classes related to Clostridia, Gammaproteobacteria, Bacteroidia and Epsilonproteobacteria in produced water as compared to predominantly aerobic Alphaproteobacteria in the fracturing source water. The metabolic profile revealed a relative increase in genes responsible for carbohydrate metabolism, respiration, sporulation and dormancy, iron acquisition and metabolism, stress response and sulfur metabolism in the produced water samples. These results suggest that microbial communities in produced water have an increased genetic ability to handle stress, which has significant implications for produced water management, such as disinfection.     

Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system

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PfHRP2-PfHRP3 diversity among Kenyan isolates and comparative evaluation of PfHRP2/pLDH malaria RDT with microscopy and nested PCR methodologies

Rapid diagnostic tests (RDT) are valuable tools that support prudent and timely use of antimalarial drugs, particularly if reliable microscopy is not available. However, the performance and reliability of these tests vary between and within geographical regions. The present study evaluated the performance of routine malaria RDT in Kenyan febrile patients in Busia County, Kenya. A cross sectional study design was employed to recruit febrile patients attending health facilities between August and November 2016. A total of 192 febrile patients who were slide positive and negative were evaluated for their infection status by nested PCR and RDTs (PfHRP2/pLDH). In addition, P. falciparum diversity of the histidine-rich proteins 2 and 3, that influences the RDT test results were determined. All individuals were P. falciparum positive. Among the investigated 192 febrile patients, 76 (40%) were positive by microscopy, 101 (53%) by RDTs and 80 (42%) were PCR positive. The performance of the CareStart? HRP2/pLDH (pf) RDTs was better than microscopy (Sensitivity 94%; Specificity 75%) and Nucleic acid testing (sensitivity 95%, specificity 77%) with high negative predictive values, indicating the suitability of the RDT in routine practice. Specific pfhrp2/pfhrp3 deletions shown to associate with RDT false negativity was not observed. However, high genetic diversity among pfhrp2 gene was observed. Eleven new PfHRP2 and nine PfHRP3 repeats were observed. False positivity by microscopy and under reporting of infections may thus be a barrier in malaria control and elimination programs. The HRP2/pLDH(Pf) based RDT yet demonstrate to be an effective tool for malaria surveillance program.     

Linkage analysis and identification of quantitative trait loci associated with freeze tolerance and turf quality traits in St. Augustinegrass

St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is a warm-season turfgrass commonly grown in the southern USA. In this study, the first linkage map for all nine haploid chromosomes of the species was constructed for cultivar ‘Raleigh’ and cultivar ‘Seville’ using a pseudo-F₂ mapping strategy. A total of 160 simple sequence repeat markers were mapped to nine linkage groups (LGs) covering a total distance of 1176.24 cM. To demonstrate the usefulness of the map, quantitative trait loci (QTL) were mapped controlling field winter survival, laboratory-based freeze tolerance, and turf quality traits. Multiple genomic regions associated with these traits were identified. Moreover, overlapping QTL were found for winterkill and spring green up on LG 3 (99.21 cM); turf quality, turf density, and leaf texture on LG 3 (68.57–69.50 cM); and surviving green tissue and regrowth on LGs 1 (38.31 cM), 3 (77.70 cM), 6 (49.51 cM), and 9 (34.20 cM). Additional regions, where QTL identified in both field and laboratory-based/controlled environment freeze testing co-located, provided strong support that these regions are good candidates for true gene locations. These results present the first complete linkage map produced for St. Augustinegrass, providing a template for further genetic mapping. Additionally, markers linked to the QTL identified may be useful to breeders for transferring these traits into new breeding lines and cultivars.     

Natural products as sources of new fungicides (V): Design and synthesis of acetophenone derivatives against phytopathogenic fungi in vitro and in vivo

A series of acetophenone derivatives (10a–10i, 11, 12a–12g, 13a–13g, 14a–14d and 15a–15l) were designed, synthesized and evaluated for antifungal activities in vitro and in vivo. The antifungal activities of 53 compounds were tested against several plant pathogens, and their structure–activity relationship was summarized. Compounds 10a–10f displayed better antifungal effects than two reference fungicides. Interestingly, the most potent compound 10d exhibited antifungal properties against Cytospora sp., Botrytis cinerea, Magnaporthe grisea, with IC₅₀ values of 6.0–22.6?µg/mL, especially Cytospora sp. (IC₅₀?=?6.0?µg/mL). In the in vivo antifungal assays, 10d displayed the significant protective efficacy of 55.3% to Botrytis cinerea and 73.1% to Cytospora sp. The findings indicated that 10d may act as a potential pesticide lead compound that merits further investigation.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Does governance play a role in the distribution of invasive alien species?

Invasive alien species (IAS) constitute a major threat to global biological diversity. In order to control their spread, a detailed understanding of the factors influencing their distribution is essential. Although international trade is regarded as a major force structuring spatial patterns of IAS, the role of other social factors remains unclear. Despite studies highlighting the importance of strong governance in slowing drivers of biodiversity loss such as logging, deforestation, and agricultural intensification, no study has yet analyzed its contribution to the issue of IAS. Using estimates of governance quality and comprehensive spatiotemporal IAS data, we performed multiple linear regressions to investigate the effect of governance quality upon the distribution of species listed under “100 of the worst” IAS in 38 Eurasian countries as defined by DASIE. Our model suggested that for countries with higher GDP, stronger governance was associated with a greater number of the worst IAS; in contrast, for the lowest GDP countries under analysis, stronger governance was associated with fewer of these IAS. We elucidate how the quality of governance within a country has implications for trade, tourism, transport, legislation, and economic development, all of which influence the spread of IAS. While our findings support the common assumption that strengthening governance benefits conservation interventions in countries of smaller economy, we find that this effect is not universal. Stronger governance alone cannot adequately address the problem of IAS, and targeted action is required in relatively high‐GDP countries in order to stem the influx of IAS associated with high volumes of trade.