Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Inflammatory cytokines and metabolic responses to high-intensity intermittent training: effect of the exercise intensity

To examine the effects of two high-intensity intermittent training (HIIT) programs of varying intensities (100% vs. 110% of maximal aerobic velocity [MAV]) on metabolic, hormonal and inflammatory markers in young men. Thirty-seven active male volunteers were randomly assigned into: HIIT experimental groups (100% MAV [EG₁₀₀, n = 9] and 110% MAV [EG₁₁₀, n = 9]) and a control groups (CG₁₀₀, n = 9 and CG₁₁₀, n = 9). Particpants performed high intesity intermittent exercise test (HIIE) at 100% or 110% MAV. Venous blood samples were obtained before, at the end of HIIE and at 15 min of recovery, and before and after 8 weeks of HIIT programs. After training, Glucose was lower (p < 0.01) in EG₁₀₀ (d = 0.72) and EG₁₁₀ (d = 1.20) at the end of HIIE, and at 15 min recovery only in EG₁₁₀ (d = 0.95). After training, Insulin and Cortisol were lower than before training in EG₁₀₀ and EG₁₁₀ at the end of HIIE (p < 0.001). After HIIT, IL-6 deceased (p < 0.001) in EG₁₀₀ (d = 1.43) and EG₁₁₀ (d = 1.56) at rest, at the end of HIIE (d = 1.03; d = 1.75, respectively) and at 15 min of recovery (d = 0.88;d = 1.7, respectively). This decrease was more robust (p < 0.05) in EG₁₁₀ compared to EG₁₀₀. After HIIT, TNF-α deceased (p < 0.001) in EG₁₀₀ (d = 1.43) and EG₁₁₀ (d = 0.60) at rest, at the end of HIIE (0.71 < d < 0.98) and at 15 min of recovery (0.70 < d < 2.78). HIIT with 110% MAV is more effective in young males on the improvements of some metabolic (Glucose), hormonal (Cortisol) and inflammatory (IL-6) markers at rest, at the end of HIIE and 15 min of recovery than training at 100 % MAV.     

First laboratory confirmation and sequencing of Zaire ebolavirus in Uganda following two independent introductions of cases from the 10th Ebola Outbreak in the Democratic Republic of the Congo,June 2019

Uganda established a domestic Viral Hemorrhagic Fever (VHF) testing capacity in 2010 in response to the increasing occurrence of filovirus outbreaks. In July 2018, the neighboring Democratic Republic of Congo (DRC) experienced its 10^th Ebola Virus Disease (EVD) outbreak and for the duration of the outbreak, the Ugandan Ministry of Health (MOH) initiated a national EVD preparedness stance. Almost one year later, on 10^th June 2019, three family members who had contracted EVD in the DRC crossed into Uganda to seek medical treatment.Samples were collected from all the suspected cases using internationally established biosafety protocols and submitted for VHF diagnostic testing at Uganda Virus Research Institute. All samples were initially tested by RT-PCR for ebolaviruses, marburgviruses, Rift Valley fever (RVF) virus and Crimean-Congo hemorrhagic fever (CCHF) virus. Four people were identified as being positive for Zaire ebolavirus, marking the first report of Zaire ebolavirus in Uganda. In-country Next Generation Sequencing (NGS) and phylogenetic analysis was performed for the first time in Uganda, confirming the outbreak as imported from DRC at two different time point from different clades. This rapid response by the MoH, UVRI and partners led to the control of the outbreak and prevention of secondary virus transmission.     

Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-β-glucanases that efficiently hydrolyse cellulosic substrates

The hydrolysis of cellulose into fermentable sugars is a costly and rate-limiting step in the production of biofuels from renewable feedstocks. Developing new cellulase systems capable of increased cellulose hydrolysis rates would reduce biofuel production costs. With this in mind, we screened 55 fungal endoglucanases for their abilities to be expressed at high levels by Aspergillus niger and to hydrolyze amorphous cellulose at rates significantly greater than that obtained with TrCel5A, one of the major endoglucanases in the Trichoderma reesei cellulase system. This screen identified three endoglucanases, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A and Sporotrichum thermophile StCel5A. We determined that A. niger expressed the three endoglucanases at relatively high levels (≥0.3 g/l) and that the hydrolysis rate of ApCel5A and StCel5A with carboxymethylcellulose 4M as substrate was five and two times greater than the T. reesei Cel5A. The ApCel5A, GtCel12A and StCel5A enzymes also demonstrated significant synergy with Cel7A/CbhI, the major exoglucanase in the T. reesei cellulase system. The three endoglucanases characterized in this study are, therefore, promising candidate endoglucanases for developing new cellulase systems with increased rates of cellulose saccharification.     

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.     

Genetic variation in catechol-<Emphasis Type="Italic">O</Emphasis>-methyltransferase (<Emphasis Type="Italic">COMT</Emphasis>) and obesity in the prostate,lung, colorectal,and ovarian (PLCO) cancer screening trial

Catechol-O-methyltransferase (COMT) is an important modulator in the catabolism of extraneural dopamine, which plays an important role in drug reward mechanisms. It is hypothesized that genetic variations in the COMT gene, which can result in a three to fourfold difference in COMT enzyme activity, may be associated with several reward-motivated behaviors. The aim of our study was to examine the relationship between COMT polymorphisms with smoking, obesity and alcohol. Three single nucleotide polymorphisms (SNPs) in COMT were genotyped in 2,371 participants selected randomly from the screening arm of the PLCO Cancer Screening Trial after stratifying by sex, age, and smoking status. Smoking, obesity, and alcohol consumption were assessed by questionnaire. SNP and haplotype associations were estimated using odds ratios (ORs) and 95% confidence intervals (CIs) derived from conditional logistic regression models, adjusted for race/ethnicity. The COMT Ex4-76C > G (Leu136Leu) polymorphism was statistically significantly associated with individuals who had >30% increases in BMI from ages 20 to 50 years, compared to those with 0–5% increase in BMI (0–5%) over the same age period: (CC is referent; OR_CG = 1.42, OR_GG = 1.46, P _trend = 0.06). By sex, the increased risk was further pronounced among females (OR_CG = 1.50, OR_GG = 2.10, P _trend = 0.03). Consistent with our analyses of single polymorphisms, individuals whose BMI increased >30% from ages 20 to 50 years were more likely than individuals with 0–5% increases in BMI to possess COMT haplotypes [COMT Ex3-104C > T–COMT Ex4-76 C > G–COMT Ex4-12 A > G] that included the variant allele for COMT Ex4-76 C > G: C-G-G (T-C-A is referent: OR_C-G-G = 1.33, 95% CI 1.01–1.77) and C-G-A (OR_C-G-A = 1.79, 95% CI 0.72–4.49). We observed no association between any of the COMT polymorphisms with smoking behavior or alcohol intake. The COMT Ex4-76C > G (Leu136Leu) polymorphism appears to play a role in large increases in BMI. The null association with smoking and alcohol and the pronounced association with increasing BMI among women further implicates COMT’s role in estrogen metabolism as a potentially culpable pathway. Our results support a need for comprehensive evaluation of COMT variations and their functional relevance as COMT may be an important molecular target to evaluate for new treatments regarding obesity.     

Parallel production and verification of protein products using a novel high-throughput screening method

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.     

Climate‐ and successional‐related changes in functional composition of European forests are strongly driven by tree mortality

Intense droughts combined with increased temperatures are one of the major threats to forest persistence in the 21st century. Despite the direct impact of climate change on forest growth and shifts in species abundance, the effect of altered demography on changes in the composition of functional traits is not well known. We sought to (1) quantify the recent changes in functional composition of European forests; (2) identify the relative importance of climate change, mean climate and forest development for changes in functional composition; and (3) analyse the roles of tree mortality and growth underlying any functional changes in different forest types. We quantified changes in functional composition from the 1980s to the 2000s across Europe by two dimensions of functional trait variation: the first dimension was mainly related to changes in leaf mass per area and wood density (partially related to the trait differences between angiosperms and gymnosperms), and the second dimension was related to changes in maximum tree height. Our results indicate that climate change and mean climatic effects strongly interacted with forest development and it was not possible to completely disentangle their effects. Where recent climate change was not too extreme, the patterns of functional change generally followed the expected patterns under secondary succession (e.g. towards late‐successional short‐statured hardwoods in Mediterranean forests and taller gymnosperms in boreal forests) and latitudinal gradients (e.g. larger proportion of gymnosperm‐like strategies at low water availability in forests formerly dominated by broad‐leaved deciduous species). Recent climate change generally favoured the dominance of angiosperm‐like related traits under increased temperature and intense droughts. Our results show functional composition changes over relatively short time scales in European forests. These changes are largely determined by tree mortality, which should be further investigated and modelled to adequately predict the impacts of climate change on forest function.