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Abstract The anaerobic oxidation of alkanes is a microbial process that mitigates the flux of hydrocarbon seeps into the oceans. In marine archaea, the process depends on sulphate-reducing bacterial partners to exhaust electrons, and it is generally assumed that the archaeal CO2-forming enzymes (CO dehydrogenase and formylmethanofuran dehydrogenase) are coupled to ferredoxin reduction. Here, we study the molecular basis of the CO2-generating steps of anaerobic ethane oxidation by characterising native enzymes of the thermophile Candidatus Ethanoperedens thermophilum obtained from microbial enrichment. We perform biochemical assays and solve crystal structures of the CO dehydrogenase and formylmethanofuran dehydrogenase complexes, showing that both enzymes deliver electrons to the F420 cofactor. Both multi-metalloenzyme harbour electronic bridges connecting CO and formylmethanofuran oxidation centres to a bound flavin-dependent F420 reductase. Accordingly, both systems exhibit robust coupled F420-reductase activities, which are not detected in the cell extract of related methanogens and anaerobic methane oxidisers. Based on the crystal structures, enzymatic activities, and metagenome mining, we propose a model in which the catabolic oxidising steps would wire electron delivery to F420 in this organism. Via this specific adaptation, the indirect electron transfer from reduced F420 to the sulphate-reducing partner would fuel energy conservation and represent the driving force of ethanotrophy.

Abstract Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50ʹ-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

Abstract Background Transcription bridges genetic information and phenotypes. Here, we evaluated how changes in transcriptional regulation enable maize (Zea mays), a crop originally domesticated in the tropics, to adapt to temperate environments. Result We generated 572 unique RNA-seq datasets from the roots of 340 maize genotypes. Genes involved in core processes such as cell division, chromosome organization and cytoskeleton organization showed lower heritability of gene expression, while genes involved in anti-oxidation activity exhibited higher expression heritability. An expression genome-wide association study (eGWAS) identified 19,602 expression quantitative trait loci (eQTLs) associated with the expression of 11,444 genes. A GWAS for alternative splicing identified 49,897 splicing QTLs (sQTLs) for 7614 genes. Genes harboring both cis-eQTLs and cis-sQTLs in linkage disequilibrium were disproportionately likely to encode transcription factors or were annotated as responding to one or more stresses. Independent component analysis of gene expression data identified loci regulating co-expression modules involved in oxidation reduction, response to water deprivation, plastid biogenesis, protein biogenesis, and plant-pathogen interaction. Several genes involved in cell proliferation, flower development, DNA replication, and gene silencing showed lower gene expression variation explained by genetic factors between temperate and tropical maize lines. A GWAS of 27 previously published phenotypes identified several candidate genes overlapping with genomic intervals showing signatures of selection during adaptation to temperate environments. Conclusion Our results illustrate how maize transcriptional regulatory networks enable changes in transcriptional regulation to adapt to temperate regions.

We previously demonstrated a beneficial effect of high-dose vitamin D in pregnancy on offspring bone and dental health. Here, we investigated the effect of maternal dietary patterns during pregnancy on the risk of bone fractures, bone mineralization and enamel defects until age 6 years in the offspring. Further, the influence of diet on the effect of high-dose vitamin D was analyzed in the COPSAC2010 mother–child cohort including 623 mother–child pairs. A weighted network analysis on FFQs revealed three specific maternal dietary patterns that associated (Bonferroni p < 0.05) with both offspring bone and dental health. The effect of prenatal high-dose (2800 IU/day) vs. standard-dose (400 IU/day) vitamin D on offspring bone mineral content (adjusted mean difference (aMD): 33.29 g, 95% CI: 14.48–52.09, p < 0.001), bone mineral density (aMD: 0.02 g/cm2 (0.01–0.04), p < 0.001), fracture risk (adjusted incidence rate ratio: 0.36 (0.16–0.84), p = 0.02), and enamel defects in primary (adjusted odds ratio (aOR): 0.13 (0.03–0.58), p < 0.01) and permanent molars (aOR: 0.25; (0.10–0.63), p < 0.01) was most pronounced when mothers had lower intake of fruit, vegetables, meat, eggs, sweets, whole grain, offal and fish. This study suggests that prenatal dietary patterns influence offspring bone and dental development, and should be considered in order to obtain the full benefits of vitamin D to enhance personalized supplementation strategy.

Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from Desulfovibrio species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from Methanococcales and many Bacteria corroborates this similarity. Indeed, Methanococcales HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in M. thermolithotrophicus, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.

Crystallization is the limiting step in X-ray structure determination of biological macromolecules. As crystallization experiments can be largely automatized, the diversity of precipitant solutions is often the determinant factor to obtain crystals of high quality. Here, we introduce a 96-well screening kit of crystallization conditions, centered on three ethoxylate-based organic polymers as precipitants and various additional compounds to promote crystal formation. This crystallization screen was tested on various non-standard proteins from bacteria and archaea. Structure determination succeeded for seven out of thirteen targets based on crystals that frequently diffracted to a higher resolution than those obtained with commercially available screening kits. Crystallization hits were rarely similar among the three ethoxylate-based organic polymers and, in comparison, with already available crystallization screens. Hence, the presented crystallization screen is an efficient tool to complement other screens and increase the likelihood of growing crystals suitable for X-ray structure determination.

Abstract The hopeful outcomes from 30 years of research in BH3-mimetics have indeed served a number of solid paradigms for targeting intermediates from the apoptosis pathway in a variety of diseased states. Not only have such rational approaches in drug design yielded several key therapeutics, such outputs have also offered insights into the integrated mechanistic aspects of basic and clinical research at the genetics level for the future. In no other area of medical research have the effects of such work been felt, than in cancer research, through targeting the BAX-Bcl-2 protein-protein interactions. With these promising outputs in mind, several mimetics, and their potential therapeutic applications, have also been developed for several other pathological conditions, such as cardiovascular disease and tissue fibrosis, thus highlighting the universal importance of the intrinsic arm of the apoptosis pathway and its input to general tissue homeostasis. Considering such recent developments, and in a field that has generated so much scientific interest, we take stock of how the broadening area of BH3-mimetics has developed and diversified, with a focus on their uses in single and combined cancer treatment regimens and recently explored therapeutic delivery methods that may aid the development of future therapeutics of this nature.

Ambrose Rukewe,1,2 Linea Nanyalo-Nashima,2 Nicola Olivier2 1University of Namibia, School of Medicine, Surgery & Anesthesiology Department, Hage Geingob Campus, Windhoek, Namibia; 2Department of Anaesthesia, Windhoek Central & Katutura State Hospitals, Windhoek, NamibiaCorrespondence: Ambrose RukeweUniversity of Namibia, School of Medicine, Surgery & Anesthesiology Department, Hage Geingob Campus, Windhoek, NamibiaTel +264-81-484-0892Email ambyrukewe@gmail.comAbstract: A recent inferior ST-elevation myocardial infarction and percutaneous coronary intervention in an elderly female patient scheduled for bilateral lower extremity operations simultaneously represent significant risks for re-infarction and mortality. Our index patient required an above-knee amputation of the left leg to prevent infection/progressing gangrene as well as application of a back-slab for the conservative management of a fractured right femur. We employed spinal injection of ultra-low-dose 0.5% isobaric bupivacaine 4 mg with morphine 75 mcg plus fentanyl 10 mcg which provided adequate anesthesia for radical amputation, effective postoperative analgesia and good hemodynamic stability.Keywords: recent myocardial infarction, percutaneous coronary intervention, geriatric, bilateral lower extremity operations, ultra-low-dose, isobaric bupivacaine

Domestication of CO2-fixation became a worldwide priority enhanced by the will to convert this greenhouse gas into fuels and valuable chemicals. Because of its high stability, CO2-activation/fixation represents a true challenge for chemists. Autotrophic microbial communities, however, perform these reactions under standard temperature and pressure. Recent discoveries shine light on autotrophic acetogenic bacteria and hydrogenotrophic methanogens, as these anaerobes use a particularly efficient CO2-capture system to fulfill their carbon and energy needs. While other autotrophs assimilate CO2 via carboxylation followed by a reduction, acetogens and methanogens do the opposite. They first generate formate and CO by CO2-reduction, which are subsequently fixed to funnel the carbon toward their central metabolism. Yet their CO2-reduction pathways, with acetate or methane as end-products, constrain them to thrive at the “thermodynamic limits of Life”. Despite this energy restriction acetogens and methanogens are growing at unexpected fast rates. To overcome the thermodynamic barrier of CO2-reduction they apply different ingenious chemical tricks such as the use of flavin-based electron-bifurcation or coupled reactions. This mini-review summarizes the current knowledge gathered on the CO2-fixation strategies among acetogens. While extensive biochemical characterization of the acetogenic formate-generating machineries has been done, there is no structural data available. Based on their shared mechanistic similarities, we apply the structural information obtained from hydrogenotrophic methanogens to highlight common features, as well as the specific differences of their CO2-fixation systems. We discuss the consequences of their CO2-reduction strategies on the evolution of Life, their wide distribution and their impact in biotechnological applications.

Ischemic cerebrovascular disease (CVD) is a relatively common condition in dogs but infrequent in cats, with acute or peracute onset of non-progressive neurological signs. Cerebellar artery infarction appears to be very uncommon in cats, with only two cases reported affecting the rostral cerebellar artery (RCA). This study aims to report for the first time the neurological signs, magnetic resonance imaging (MRI) findings and outcome in three cats diagnosed with presumed caudal cerebellar artery (CCA) infarction. Unique presentation of vestibular signs associated with CCA in three cats and similarities between dogs and humans are discussed.

The fatigue properties of ultrathin protective coatings on silicon thin films were investigated. The cohesive and delamination fatigue properties of 22 nm-thick atomic-layered-deposited (ALD) titania were characterized and compared to that of 25 nm-thick alumina. Both coatings were deposited at 200 °C. The fatigue rates are comparable at 30 °C, 50% relative humidity (RH) while they are one order of magnitude larger for alumina compared to titania at 80 °C, 90% RH. The improved fatigue performance is believed to be related to the improved stability of the ALD titania coating with water compared to ALD alumina, which may in part be related to the fact that ALD titania is crystalline, while ALD alumina is amorphous. Static fatigue crack nucleation and propagation was not observed. The underlying fatigue mechanism is different from previously documented mechanisms, such as stress corrosion cracking, and appears to result from the presence of compressive stresses and a rough coating–substrate interface.

The anaerobic oxidation of alkanes is a microbial process that mitigates the flux of hydrocarbon seeps into the oceans. In marine archaea, the process depends on sulphate-reducing bacterial partners to exhaust electrons, and it is generally assumed that the archaeal CO2-forming enzymes (CO dehydrogenase and formylmethanofuran dehydrogenase) are coupled to ferredoxin reduction. Here, we study the molecular basis of the CO2-generating steps of anaerobic ethane oxidation by characterising native enzymes of the thermophile Candidatus Ethanoperedens thermophilum obtained from microbial enrichment. We perform biochemical assays and solve crystal structures of the CO dehydrogenase and formylmethanofuran dehydrogenase complexes, showing that both enzymes deliver electrons to the F420 cofactor. Both multi-metalloenzyme harbour electronic bridges connecting CO and formylmethanofuran oxidation centres to a bound flavin-dependent F420 reductase. Accordingly, both systems exhibit robust coupled F420-reductase activities, which are not detected in the cell extract of related methanogens and anaerobic methane oxidisers. Based on the crystal structures, enzymatic activities, and metagenome mining, we propose a model in which the catabolic oxidising steps would wire electron delivery to F420 in this organism. Via this specific adaptation, the indirect electron transfer from reduced F420 to the sulphate-reducing partner would fuel energy conservation and represent the driving force of ethanotrophy. It is generally assumed that CO2 production is coupled to ferredoxin reduction during anaerobic alkane oxidation in archaea. Here, Lemaire et al. use biochemical and structural techniques to study native enzymes purified from an ethane-oxidising enrichment culture, showing that the enzymes deliver electrons to the F420 cofactor rather than to ferredoxin. [ABSTRACT FROM AUTHOR]

Although bulk silicon is not susceptible to fatigue, micron-scale silicon is. Several mechanisms have been proposed to explain this surprising behavior although the issue remains contentious. Here we describe published fatigue results for micron-scale thin siliconfilms and find that in general they display similar trends, in that lower cyclic stresses result in larger number of cycles to failure in stress-lifetime data. We further show that one of two classes of mechanisms is invariably proposed to explain the phenomenon. The first class attributes fatigue to a surface effect caused by subcritical (stable) cracking in the silicon-oxide layer, e.g., reaction-layer fatigue; the second class proposes that subcritical cracking in the silicon itself is the cause of fatigue in Si films. It is our contention that results to date from single and poly crystalline silicon fatigue studies provide no convincing experimentalevidence to support subcritical cracking in the silicon. Conversely, the reaction-layer mechanism is consistent with existing experimental results, and moreover provides a rational explanation for the marked difference in fatigue behavior of bulk and micron-scale silicon.