Kinetic Characterization of 100 Glycoside Hydrolase Mutants Enables the Discovery of Structural Features Correlated with Kinetic Constants

The use of computational modeling algorithms to guide the design of novel enzyme catalysts is a rapidly growing field. Force-field based methods have now been used to engineer both enzyme specificity and activity. However, the proportion of designed mutants with the intended function is often less than ten percent. One potential reason for this is that current force-field based approaches are trained on indirect measures of function rather than direct correlation to experimentally-determined functional effects of mutations. We hypothesize that this is partially due to the lack of data sets for which a large panel of enzyme variants has been produced, purified, and kinetically characterized. Here we report the k_cat and K_M values of 100 purified mutants of a glycoside hydrolase enzyme. We demonstrate the utility of this data set by using machine learning to train a new algorithm that enables prediction of each kinetic parameter based on readily-modeled structural features. The generated dataset and analyses carried out in this study not only provide insight into how this enzyme functions, they also provide a clear path forward for the improvement of computational enzyme redesign algorithms.     

Proteomic approaches for cancer epigenetics research

Introduction: Epigenetic dysregulation drives or supports numerous human cancers. The chromatin landscape in cancer cells is often marked by abnormal histone post-translational modification (PTM) patterns and by aberrant assembly and recruitment of protein complexes to specific genomic loci. Mass spectrometry-based proteomic analyses can support the discovery and characterization of both phenomena.

Areas covered: We broadly divide this literature into two parts: ‘modification-centric’ analyses that link histone PTMs to cancer biology; and ‘complex-centric’ analyses that examine protein–protein interactions that occur de novo as a result of oncogenic mutations. We also discuss proteomic studies of oncohistones. We highlight relevant examples, discuss limitations, and speculate about forthcoming innovations regarding each application.

Expert commentary: ‘Modification-centric’ analyses have been used to further understanding of cancer’s histone code and to identify associated therapeutic vulnerabilities. ‘Complex-centric’ analyses have likewise revealed insights into mechanisms of oncogenesis and suggested potential therapeutic targets, particularly in MLL-associated leukemia. Proteomic experiments have also supported some of the pioneering studies of oncohistone-mediated tumorigenesis. Additional applications of proteomics that may benefit cancer epigenetics research include middle-down and top-down histone PTM analysis, chromatin reader profiling, and genomic locus-specific protein identification. In the coming years, proteomic approaches will remain powerful ways to interrogate the biology of cancer.     

Selection of favorable alleles of genes controlling flowering and senescence improves malt barley quality

Molecular Breeding - Maize amylose is a type of high value-added starch used for medical, food, and chemical applications. Mutations in the starch branching enzyme (SBEIIb), with recessive ae...     

Community development by forest understory plants after prolonged burial by tephra

Several processes bury plants, but sediment can also be subsequently removed, often by delayed erosion. Thus, the ability to survive multiple years of burial and to respond when released are important to vegetation changes and population dynamics. We experimentally evaluated the effects of delayed removal of tephra (aerially transported volcanic ejecta) in an old-growth forest understory near Mount St. Helens, using 1-m² plots assigned to three treatments: tephra removed 4 months after deposition (50 plots), tephra removed 28 months after deposition (the delayed erosion treatment, 50 plots), and undisturbed, natural tephra (100 plots). Prior to tephra removal, species density, cover, shoot density, and shoot size in the delayed erosion treatment were all similar to values in natural plots and significantly less than values in plots cleared initially, indicating that 24 months of additional burial adversely affected understory plants. However, all attributes eventually approached pre-eruption values for shrubs and herbs, indicating that erosion greatly facilitated vegetation recovery. Responses varied substantially among species and growth forms. Overall, our experimental results indicate that some plants of most species can respond effectively after release from burial of at least three growing seasons. In addition, the delay of erosion retards ecosystem recovery relative to early erosion, facilitates recovery relative to no erosion, and modifies the trajectory of post-disturbance vegetation change.

     

A Matched-Filter-Based Algorithm for Subcellular Classification of T-System in Cardiac Tissues

In mammalian ventricular cardiomyocytes, invaginations of the surface membrane form the transverse tubular system (T-system), which consists of transverse tubules (TTs) that align with sarcomeres and Z-lines as well as longitudinal tubules (LTs) that are present between Z-lines in some species. In many cardiac disease etiologies, the T-system is perturbed, which is believed to promote spatially heterogeneous, dyssynchronous Ca²⁺ release and inefficient contraction. In general, T-system characterization approaches have been directed primarily at isolated cells and do not detect subcellular T-system heterogeneity. Here, we present MatchedMyo, a matched-filter-based algorithm for subcellular T-system characterization in isolated cardiomyocytes and millimeter-scale myocardial sections. The algorithm utilizes “filters” representative of TTs, LTs, and T-system absence. Application of the algorithm to cardiomyocytes isolated from rat disease models of myocardial infarction (MI), dilated cardiomyopathy induced via aortic banding, and sham surgery confirmed and quantified heterogeneous T-system structure and remodeling. Cardiomyocytes from post-MI hearts exhibited increasing T-system disarray as proximity to the infarct increased. We found significant (p < 0.05, Welch’s t-test) increases in LT density within cardiomyocytes proximal to the infarct (12 ± 3%, data reported as mean ± SD, n = 3) versus sham (4 ± 2%, n = 5), but not distal to the infarct (7 ± 1%, n = 3). The algorithm also detected decreases in TTs within 5° of the myocyte minor axis for isolated aortic banding (36 ± 9%, n = 3) and MI cardiomyocytes located intermediate (37 $\pm$ 4%, n = 3) and proximal (34 ± 4%, n = 3) to the infarct versus sham (57 ± 12%, n = 5). Application of bootstrapping to rabbit MI tissue revealed distal sections comprised 18.9 ± 1.0% TTs, whereas proximal sections comprised 10.1 ± 0.8% TTs (p < 0.05), a 46.6% decrease. The matched-filter approach therefore provides a robust and scalable technique for T-system characterization from isolated cells through millimeter-scale myocardial sections.     

Soil Solution Chemistry and Element Fluxes in Three European Heathlands and Their Responses to Warming and Drought

Soil water chemistry and element budgets were studied at three northwestern European Calluna vulgaris heathland sites in Denmark (DK), The Netherlands (NL), and Wales (UK). Responses to experimental nighttime warming and early summer drought were followed during a two-year period. Soil solution chemistry measured below the organic soil layer and below the rooting zone and water fluxes estimated with hydrological models were combined to calculate element budgets. Remarkably high N leaching was observed at the NL heath with 18 and 6.4 kg N ha^–1 year^–1 of NO₃–N and NH₄–N leached from the control plots, respectively, indicating that this site is nitrogen saturated. Increased soil temperature of +0.5°C in the heated plots almost doubled the concentrations and losses of NO₃–N and DON at this site. Temperature also increased mobilization of N in the O horizon at the UK and DK heaths in the first year, but, because of high retention of N in the vegetation or mineral soil, there were no significant effects of warming on seepage water NO₃–N and NH₄–N. Retention of P was high at all three sites. In several cases, drought increased concentrations of elements momentarily, but element fluxes decreased because of a lower flux of water. Seepage water DOC and DON was highly significantly correlated at the UK site where losses of N were low, whereas losses of C and N were uncoupled at the NL site where atmospheric N input was greatest. Based on N budgets, calculations of the net change in the C sink or source strength in response to warming suggest no change or an increase in the C sink strength during these early years.     

The cortical control of movement revisited

Recently, we found that electrical stimulation of motor cortex caused monkeys to make coordinated, complex movements. These evoked movements were arranged across the cortex in a map of spatial locations to which the hand moved. We suggest that some of the subdivisions previously described within primary motor and premotor cortex may represent different types of actions that monkeys tend to make in different regions of space. According to this view, primary and premotor cortex may fit together into a larger map of manual space.