Engineering the Relatively Conserved Residues in Active Site Architecture of Thermophilic Chitinase SsChi18A Enhanced Catalytic Activity.

Chitinase plays a vital role in the efficient biotransformation of the chitin substrate. This study aimed to modify and elucidate the contribution of the relatively conserved residues in the active site architecture of a thermophilic chitinase SsChi18A from Streptomyces sp. F-3 in processive catalysis. The enzymatic activity on colloidal chitin increased to 151%, 135%, and 129% in variants Y286W, E287A, and K186A compared with the wild type (WT). Also, the apparent processive parameter G2/G1 was lower in the variants compared to the WT, indicating the essential role of Tyr-286, Glu-287, and Lys-186 in processive catalysis. Additionally, the enzymatic activity on the crystalline chitin of F48W and double mutants F48W/Y209F and F48W/Y286W increased by 35%, 16%, and 36% compared with that for WT. Molecular dynamics simulations revealed that the driving force of processive catalysis might be related to the changes in interaction energy. This study provided a rational design strategy targeting relatively conserved residues to enhance the catalytic activity of GH18 processive chitinases.

Unraveling the metabolic potential of biocontrol fungi through omics data: a key to enhancing large-scaleapplication strategies.

Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of insignificant effects in large-scale applications. Therefore, an in-depth exploration of the metabolic potential of biocontrol fungi based on big omics data is crucial for a comprehensive and systematic understanding of the specific modes of action operated by various biocontrol fungi. This article analyzes the preferences for extracellular carbon and nitrogen source degradation, secondary metabolites (nonribosomal peptides, polyketide synthases) and their product characteristics and the conversion relationship between extracellular primary metabolism and intracellular secondary metabolism for eight different filamentous fungi with characteristics appropriate for the biological control of bacterial pathogens and phytopathogenic nematodes. Further clarification is provided that Paecilomyces lilacinus, encoding a large number of hydrolase enzymes capable of degrading pathogen protection barrier, can be directly applied in the field as a predatory biocontrol fungus, whereas Trichoderma, as an antibiosis-active biocontrol control fungus, can form dominant strains on preferred substrates and produce a large number of secondary metabolites to achieve antibacterial effects. By clarifying the levels of biological control achievable by different biocontrol fungi, we provide a theoretical foundation for their application to cropping habitats.

Stage-specific dual function: EZH2 regulates human erythropoiesis by eliciting histone and non-histone methylation.

Enhancer of zeste homolog 2 (EZH2) is the lysine methyltransferase of polycomb repressive complex 2 (PRC2) that catalyzes H3K27 tri-methylation. Aberrant expression and loss-of-function mutations of EZH2 have been demonstrated to be tightly associated with the pathogenesis of various myeloid malignancies characterized by ineffective erythropoiesis, such as myelodysplastic syndrome (MDS). However, the function and mechanism of EZH2 in human erythropoiesis still remains largely unknown. Here, we demonstrated that EZH2 regulates human erythropoiesis in a stage-specific, dual-function manner by catalyzing histone and non-histone methylation. During the early erythropoiesis, EZH2 deficiency caused cell cycle arrest in the G1 phase, which impaired cell growth and differentiation. Chromatin immunoprecipitation sequencing and RNA sequencing discovered that EZH2 knockdown caused a reduction of H3K27me3 and upregulation of cell cycle proteindependent kinase inhibitors. In contrast, EZH2 deficiency led to the generation of abnormal nuclear cells and impaired enucleation during the terminal erythropoiesis. Interestingly, EZH2 deficiency downregulated the methylation of HSP70 by directly interacting with HSP70. RNA-sequencing analysis revealed that the expression of AURKB was significantly downregulated in response to EZH2 deficiency. Furthermore, treatment with an AURKB inhibitor and small hairpin RNAmediated AURKB knockdown also led to nuclear malformation and decreased enucleation efficiency. These findings strongly suggest that EZH2 regulates terminal erythropoiesis through a HSP70 methylation-AURKB axis. Our findings have implications for improved understanding of ineffective erythropoiesis with EZH2 dysfunction.

Data-driven strategies for the computational design of enzyme thermal stability: trends, perspectives, and prospects.

Thermal stability is one of the most important properties of enzymes, which sustains life and determines the potential for the industrial application of biocatalysts. Although traditional methods such as directed evolution and classical rational design contribute greatly to this field, the enormous sequence space of proteins implies costly and arduous experiments. The development of enzyme engineering focuses on automated and efficient strategies because of the breakthrough of high-throughput DNA sequencing and machine learning models. In this review, we propose a data-driven architecture for enzyme thermostability engineering and summarize some widely adopted datasets, as well as machine learning-driven approaches for designing the thermal stability of enzymes. In addition, we present a series of existing challenges while applying machine learning in enzyme thermostability design, such as the data dilemma, model training, and use of the proposed models. Additionally, a few promising directions for enhancing the performance of the models are discussed. We anticipate that the efficient incorporation of machine learning can provide more insights and solutions for the design of enzyme thermostability in the coming years.

Expectations about motion direction affect perception and anticipatory smooth pursuit differently.

Smooth pursuit eye movements and visual motion perception rely on the integration of current sensory signals with past experience. Experience shapes our expectation of current visual events and can drive eye movement responses made in anticipation of a target, such as anticipatory pursuit. Previous research revealed consistent effects of expectation on anticipatory pursuit-eye movements follow the expected target direction or speed-and contrasting effects on motion perception, but most studies considered either eye movement or perceptual responses. The current study directly compared effects of direction expectation on perception and anticipatory pursuit within the same direction discrimination task to investigate whether both types of responses are affected similarly or differently. Observers (n = 10) viewed high-coherence random-dot kinematograms (RDKs) moving rightward and leftward with a probability of 50%, 70%, or 90% in a given block of trials to build up an expectation of motion direction. They were asked to judge motion direction of interleaved low-coherence RDKs (0%-15%). Perceptual judgements were compared with changes in anticipatory pursuit eye movements as a function of probability. Results show that anticipatory pursuit velocity scaled with probability and followed direction expectation (attraction bias), whereas perceptual judgments were biased opposite to direction expectation (repulsion bias). Control experiments suggest that the repulsion bias in perception was not caused by retinal slip induced by anticipatory pursuit, or by motion adaptation. We conclude that direction expectation can be processed differently for perception and anticipatory pursuit.NEW & NOTEWORTHY We show that expectations about motion direction that are based on long-term trial history affect perception and anticipatory pursuit differently. Whereas anticipatory pursuit direction was coherent with the expected motion direction (attraction bias), perception was biased opposite to the expected direction (repulsion bias). These opposite biases potentially reveal different ways in which perception and action utilize prior information and support the idea of different information processing for perception and pursuit.

A highly efficient protein degradation system in Bacillus sp. CN2: a functional-degradomics study.

A novel protease-producing Bacillus sp. CN2 isolated from chicken manure composts exhibited a relatively high proteolytic specific activity. The strain CN2 degradome consisted of at least 149 proteases and homolog candidates, which were distributed into 4 aspartic, 30 cysteine, 55 metallo, 56 serine, and 4 threonine proteases. Extracellular proteolytic activity was almost completely inhibited by PMSF (phenylmethylsulfonyl fluoride) rather than o-P, E-64, or pepstatin A, suggesting that strain CN2 primarily secreted serine protease. More importantly, analysis of the extracellular proteome of strain CN2 revealed the presence of a highly efficient protein degradation system. Three serine proteases of the S8 family with different active site architectures firstly fragmented protein substrates which were then degraded to smaller peptides by a M4 metalloendopeptidase that prefers to degrade hydrophobic peptides and by a S13 carboxypeptidase. Those enzymes acted synergistically to degrade intact substrate proteins outside the cell. Furthermore, highly expressed sequence-specific intracellular aminopeptidases from multiple families (M20, M29, and M42) accurately degraded peptides into oligopeptides or amino acids, thus realizing the rapid acquisition and utilization of nitrogen sources. In this paper, a systematic study of the functional-degradome provided a new perspective for understanding the complexity of the protease hydrolysis system of Bacillus, and laid a solid foundation for further studying the precise degradation of proteins with the cooperative action of different family proteases. KEY POINTS: • Bacillus sp. CN2 has relatively high proteolytic specific activity. • Bacillus sp. CN2 harbors a highly efficient protein degradation system. • The site-specific endopeptidases were secreted extracellular, while the sequence-specific aminopeptidases played a role in the cell.

TET2 deficiency leads to stem cell factor-dependent clonal expansion of dysfunctional erythroid progenitors.

Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)-dependent hyperproliferation and impaired differentiation of human colony-forming unit-erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors "marker CFU-E" cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis.

The contribution of specific subsites to catalytic activities in active site architecture of a GH11 xylanase.

BACKGROUND: Xylanase with high specific activity plays a crucial role in hemicellulose biodegradation and has important industrial application. The amino acids located in the active site determine the enzyme biological characterization. In this study, structure bioinformatics analysis and alanine screening experiments were performed to explore the roles of amino acids at each subsite of the GH11 xylanase active site. RESULTS: There are highly conserved amino acids at - 2 to + 1 subsites, and the network of the interactions is concentrated near the catalytic sites (E86, E178). However, the amino acids at relatively distal subsites, especially at the + 2 and + 3 subsites, are few but diverse. Alanine substitution of amino acids in the active site architecture exerted different impacts on catalytic efficiency. Interestingly, mutants Y180A at the + 2 subsite and Y96A at the + 3 subsite had reduced enzymatic activities by almost 95%, which indicate that these two aromatic residues are necessary for the catalysis of substrates in addition to the highly conserved residues at the - 2 and + 1 subsites. Moreover, in these two subsites, aromatic amino acids with different side-chain properties also affected enzyme activity. The mutants Y180W and Y96W showed 6.2% and 12.8% increase in specific activities by comparison with wild-type enzyme at 50 °C, respectively. CONCLUSION: We elucidated the interaction between amino acids and substrates in the active site, which will aid understanding of the protein-ligand interaction in enzyme engineering. KEY POINTS: • Xylanase of GH11 family is a good industrial candidate. • The roles of residues at each subsite of GH11 xylanase active site are explored. • The two aromatic residues at the + 2 and + 3 subsites are necessary for the catalysis. • Y180W and Y96W increased the enzymatic activity by 6.2% and 12.8% at low temperature.

On the relation between anticipatory ocular torsion and anticipatory smooth pursuit.

Humans and other animals move their eyes in anticipation to compensate for sensorimotor delays. Such anticipatory eye movements can be driven by the expectation of a future visual object or event. Here we investigate whether such anticipatory responses extend to ocular torsion, the eyes' rotation about the line of sight. We recorded three-dimensional eye position in head-fixed healthy human adults who tracked a rotating dot pattern moving horizontally across a computer screen. This kind of stimulus triggers smooth pursuit with a horizontal and torsional component. In three experiments, we elicited expectation of stimulus rotation by repeatedly showing the same rotation (Experiment 1), or by using different types of higher-level symbolic cues indicating the rotation of the upcoming target (Experiments 2 and 3). Across all experiments, results reveal reliable anticipatory horizontal smooth pursuit. However, anticipatory torsion was only elicited by stimulus repetition, but not by symbolic cues. In summary, torsion can be made in anticipation of an upcoming visual event only when low-level motion signals are accumulated by repetition. Higher-level cognitive mechanisms related to a symbolic cue reliably evoke anticipatory pursuit but did not modulate torsion. These findings indicate that anticipatory torsion and anticipatory pursuit are at least partly decoupled and might be controlled separately.

Crowding and Binding: Not All Feature Dimensions Behave in the Same Way.

Humans often fail to identify a target because of nearby flankers. The nature and stages at which this crowding occurs are unclear, and whether crowding operates via a common mechanism across visual dimensions is unknown. Using a dual-estimation report (N = 42), we quantitatively assessed the processing of features alone and in conjunction with another feature both within and between dimensions. Under crowding, observers misreported colors and orientations (i.e., reported a flanker value instead of the target's value) but averaged the target's and flankers' spatial frequencies (SFs). Interestingly, whereas orientation and color errors were independent, orientation and SF errors were interdependent. These qualitative differences of errors across dimensions revealed a tight link between crowding and feature binding, which is contingent on the type of feature dimension. These results and a computational model suggest that crowding and misbinding are due to pooling across a joint coding of orientations and SFs but not of colors.

Ocular torsion is related to perceived motion-induced position shifts.

Ocular torsion (i.e., rotations of the eye about the line of sight) can be induced by visual rotational motion. It remains unclear whether and how such visually induced torsion is related to perception. By using the flash-grab effect, an illusory position shift of a briefly flashed stationary target superimposed on a rotating pattern, we examined the relationship between torsion and perception. In two experiments, 25 observers reported the perceived location of a flash while their three-dimensional eye movements were recorded. In Experiment 1, the flash coincided with a direction reversal of a large, centrally displayed, rotating grating. The grating triggered visually induced torsion in the direction of stimulus rotation. The magnitude of torsional eye rotation correlated with the illusory perceptual position shift. To test whether torsion caused the illusion, in Experiment 2, the flash was superimposed on two peripheral gratings rotating in opposite directions. Even though torsion was eliminated, the illusory position shift persisted. Despite the lack of a causal relationship, the torsion-perception correlations indicate a close link between both systems, either through similar visual-input processing or a boost of visual rotational signal strength via oculomotor feedback.

Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.

XynB from Aspergillus niger ATCC1015 (AnXynB) is a mesophilic glycoside hydrolase (GH) family 11 xylanase which holds great potentials in a wide variety of industrial applications. In the present study, the catalytic activity and stability of AnXynB were improved by a combination of computational and experimental approaches. Virtual mutation and molecular dynamics simulations indicated that the introduction of Glu and Asn altered the interaction network at the - 3 subsite. Interestingly, the double mutant S41N/T43E displayed 72% increase in catalytic activity when compared to the wild type (WT). In addition, it also showed a better thermostability than the WT enzyme. Kinetic determination of the T43E and S41N/T43E mutants suggested that the higher xylanase activity is probably due to the increasing binding affinity of enzyme and substrate. Consequently, the enzyme activity and thermostability of AnXynB was both increased by selective site-directed mutagenesis at the - 3 subsite of its active site architecture which provides a good example for a successfully engineered enzyme for potential industrial application. Moreover, the molecular evolution approach adopted in this study led to the design of a library of sequences that captures a meaningful functional diversity in a limited number of protein variants.

Latent variable mixture models to test for differential item functioning: a population-based analysis.

BACKGROUND: Comparisons of population health status using self-report measures such as the SF-36 rest on the assumption that the measured items have a common interpretation across sub-groups. However, self-report measures may be sensitive to differential item functioning (DIF), which occurs when sub-groups with the same underlying health status have a different probability of item response. This study tested for DIF on the SF-36 physical functioning (PF) and mental health (MH) sub-scales in population-based data using latent variable mixture models (LVMMs). METHODS: Data were from the Canadian Multicentre Osteoporosis Study (CaMos), a prospective national cohort study. LVMMs were applied to the ten PF and five MH SF-36 items. A standard two-parameter graded response model with one latent class was compared to multi-class LVMMs. Multivariable logistic regression models with pseudo-class random draws characterized the latent classes on demographic and health variables. RESULTS: The CaMos cohort consisted of 9423 respondents. A three-class LVMM fit the PF sub-scale, with class proportions of 0.59, 0.24, and 0.17. For the MH sub-scale, a two-class model fit the data, with class proportions of 0.69 and 0.31. For PF items, the probabilities of reporting greater limitations were consistently higher in classes 2 and 3 than class 1. For MH items, respondents in class 2 reported more health problems than in class 1. Differences in item thresholds and factor loadings between one-class and multi-class models were observed for both sub-scales. Demographic and health variables were associated with class membership. CONCLUSIONS: This study revealed DIF in population-based SF-36 data; the results suggest that PF and MH sub-scale scores may not be comparable across sub-groups defined by demographic and health status variables, although effects were frequently small to moderate in size. Evaluation of DIF should be a routine step when analysing population-based self-report data to ensure valid comparisons amongst sub-groups.

The pervasive effect of youth self-report of hunger on depression over 6 years of follow up.

PURPOSE: We used longitudinal data to clarify the association between self-report of hunger and subsequent depression risk among youth and young adults, accounting for other risk factors. METHODS: Youth self-report of ever experiencing hunger data were collected from cycles 4-6 of the National Longitudinal Survey of Children and Youth cohort of Canadian youth 16 years and older (n = 4139). Data on depressive symptoms (CES-D 12) were collected over three cycles (2004-2009, cycles 6-8). We used multivariable regression based on generalized estimating equations (GEE) to examine prior youth hunger on later depression risk, adjusting for time-stable, time-varying, and lagged variables (e.g., depressive symptoms in previous cycle), thereby clarifying the temporal relationship. RESULTS: The prevalence of youth hunger experience and depression risk reached 5.9 and 15.0%, respectively. The adjusted odds ratio of depression for participants reporting hunger was 2.31 (95% CI 1.54, 3.46) and changed little [2.17 (95% CI 1.29, 3.67)] after accounting for previous CES-D 12 scores, suggesting a temporal relationship in which hunger contributes to depression risk. Unlike never-hungry youth, depression in ever-hungry youth remained comparatively elevated over time. CONCLUSIONS: Our models support an independent and temporal relationship between youth self-report of hunger and depression in adolescence and young adulthood.

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence-assisted carbohydrate electrophoresis.

The structure of xylan, which has a 1,4-linked ß-xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence-assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA(2) Xyl3 and MeGlcA(2) Xyl4 , respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family-specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and ß-xylosidase degraded MeGlcA(2) Xyl4 into xylose and MeGlcA(2) Xyl3 but further degradation of MeGlcA(2) Xyl3 required additional enzymes. Synergy between xylanases and ß-xylosidase was also found to significantly accelerate the conversion of XOS into xylose.

On power idealization filter topologies of lattice implication algebras.

The aim of this paper is to introduce power idealization filter topologies with respect to filter topologies and power ideals of lattice implication algebras, and to investigate some properties of power idealization filter topological spaces and their quotient spaces.

Complete genome sequence of Bacillus cereus A01.

Bacillus cereus, a class of facultative aerobic gram-positive bacteria, is frequently isolated from soil, growing plants, and the intestinal tract of insects and mammals. Here, we report the complete genome sequence of B. cereus A01, whose total genome length is 6,097,808 bp, with a GC content of 34.92%.

Complete genome sequence of Bifidobacterium animalis B01.

Bifidobacterium, a class of strictly anaerobic Gram-positive bacteria, existed mainly in the gut of humans and many mammals. Here, we report the complete genome sequence of Bifidobacterium animalis B01, whose genome length is 1,935,423 bp with a GC content of 60.49%.

Complete genome sequence of Lacticaseibacillus paracasei A02.

Lacticaseibacillus paracasei, a facultative anaerobic gram-positive bacterium, is commonly found in the gut of humans and animals, as well as in dairy products and plant ferments. Here, we report the complete genome sequence of L. paracasei A02, whose total genome length is 3,038,472 bp, with a GC content of 46.41%.

Health-Related Quality of Life in Relation to Health Behaviour Patterns among Canadian Children.

Poor health behaviours in childhood, including sedentary behaviour, low physical activity levels, inadequate sleep, and unhealthy diet, are established risk factors for both chronic diseases and mental illness. Scant studies have examined the importance of such health behaviour patterns for health-related quality of life (HRQoL). This study aimed to examine the association of health behaviour patterns with HRQoL among Canadian children. Data from 2866 grade five students were collected through a provincially representative school-based survey of the 2014 Raising Healthy Eating and Active Living Kids in Alberta study. Latent class analysis was used to identify health behaviour patterns based on 11 lifestyle behaviours: sedentary behaviour (using a computer, playing video games, watching TV), physical activity (with and without a coach), sleep (bedtime on weekdays and weekends), and diet (fruit and vegetables intake, grain products, milk and alternatives, meat and alternatives). Multivariable multilevel logistic regression was applied to examine the associations of health behaviour patterns with HRQoL. Three groupings with distinct health behaviour patterns were identified: the first grouping (55%) is characterized by relatively healthy levels of sedentary behaviour, physical activity, and sleep, but a less healthy diet ("activity-focused" group). The second grouping (24%) is characterized by a relatively healthy diet, but moderately healthy levels of sedentary behaviour, physical activity, and sleep ("diet-focused" group). The third grouping (21%) is characterized by mostly unhealthy behaviours ("not health-focused" group). Students in the third and second groupings ("not health-focused" and "diet-focused") were more likely to report lower HRQoL relative to students in the first grouping ("activity-focused"). The findings suggest that health promotion strategies may be more effective when considering the patterns of health behaviours as distinct targets in the efforts to improve HRQoL. Future research should include prospective observational and intervention studies to further elucidate the relationship between health behaviour patterns and HRQoL among children.