Successful examples of the application of novel iterative trainable algorithms to guide rational mutation strategies for enzyme engineering: From prediction to lab testing to algorithm retraining

Both natural mutations occurring in a homologous enzyme family and mutations engineered in a given protein can have a tremendous impact in the activity and binding behavior of the enzyme towards substrates or other molecules. Binding and catalytic properties can be modified by rationally mutating selected amino acids in a protein. For instance, new specificity properties can be engineered into existing enzymes, which can be applied to the rational design of mutations to alter its catalysis. Although this approach has been largely used, the modifications introduced in the target protein have not been exempt of deleterious effects on protein function, binding or physicochemical properties. Much finer tuned modifications should be designed in order to alter the desired catalytic or binding properties of a protein and simultaneously not affecting other protein properties or functions. These engineered mutations usually require a thorough knowledge of the relevant structure-function relationships in the protein molecule. If no precise structure-function information is available for a protein, the amount of possible amino acid mutations to be tested precludes a direct search. Furthermore, in many cases a directed evolution strategy cannot be successfully used to achieve the desired results due to the unavailability of suitable screening tests. In the last years, we have developed new and powerful in silico methodologies to automatically propose, test and redesign mutagenesis strategies for a target protein, based only on evolutionarily conserved physicochemical properties of amino acids in a protein family where the target protein belongs, and on structural properties, including calculation of vibrational entropies, if available, with no need of explicit structure-function relationships. This methodology identifies amino acid positions that are putatively responsible for function, specificity, stability or binding interactions in a family of proteins and calculates amino acid propensity and distributions at each position. Not only conserved amino acid positions in a protein family can be labelled as functionally relevant, but also non-conserved amino acid positions can be identified to have a meaningful functional effect, and even amino acid substitutions that are unobserved in nature. These results can be used to predict if a given mutation can have a functional implication and which mutation is most likely to be functionally silent for a protein. Through several rounds of mutation suggestions, laboratory testing of the mutants and feedback of results to retrain the algorithms, our methodology can be used to rapidly and automatically discard any irrelevant mutation and guide the research focus toward functionally significant mutations. In this work, we will show how we have successfully used our publicly available methods to guide mutant design in enzyme engineering applied to xylanases (producing an improved octuple mutant in a single mutagenesis round), proteases, glucanases, ubiquitin ligases and other enzymes, to alter protein function, stability or thermodynamic properties independently of their catalytic properties in vitro and in vivo. We will also show how the predictions of these methods have been employed to shift chromatographic elution profiles of xylanases and ferritin nanocages for better purification without affecting their activity and to obtain ferritin variants with better properties to be used in nanotechnological applications, including modifications to the external and internal surface of the protein to change its interaction properties, improve its recombinant production, alter the characteristics of nanoparticles within or change its organic molecule carrier capacity. Finally, we will show how a similar approach has been integrated in an artificial intelligence classification scheme to identify somatic mutations in the human VHL gene that are related to renal clear-cell cancer and to predict the clinical outcome and prognosis of pVHL mutation and malfunction in humans, based on specific disruption of interactions with VHL binding partners. Clearly, our techniques show promising performance as a valuable and powerful bioinformatics tool to aid in the computer-aided design of engineered enzyme variants and in the understanding of function-structure, binding and affinity relationships in enzymes and other proteins

AAV gene therapy for alcoholism: Inhibition of mitochondrial aldehyde dehydrogenase enzyme expression in hepatoma cells

The process by which ethanol is metabolized in the liver occurs in two steps. The first step depends on the enzyme alcohol dehydrogenase (ADH) and the second step is catalyzed by the enzyme Aldehyde dehydrogenase (ALDH2). Some individuals of the Asian population who carry a mutation in the Aldehyde dehydrogenase gene (ALDH2*2) have a diminished capacity to metabolize acetaldehyde, producing strong effects including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2*2 by genetic technologies could result in a similar phenotype. We utilized scAAV2 vectors encoding ALDH2 shRNA to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. In the present study, we have shown that scAAV2 vectors encoding a single ALDH2 shRNA are effective in decreasing mitochondrial Aldehyde dehydrogenase expression in HEK-293/ALDH2, HepG2 and VL-17A HepG2 cell lines and increases acetaldehyde levels. Human cell lines 293 and HepG2 were transduced with scAAV2/shRNA showing a reduction in ALDH2 RNA and protein expression with the two viral concentrations assayed (1x104 and 1x105 vg/cell) at two different time points. In both cell lines ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 48% and 90%, respectively, five days post infection. ALDH2 silencing was evaluated in the VL-17A HepG2 cell line, which exhibits hepatocyte-like characteristics in response to ethanol. Cells were transduced with 1x105 vg/cell and ALDH2 expression was evaluated at the third day p.i. by Real Time RT-PCR and Western blot, showing an expression reduction of 40% as compared with the scramble control, in both analysis. As functional assay cells were incubated with ethanol (10, 25 and 100 mM) and acetaldehyde accumulation was measured by gas chromatography. Samples treated with the scAAV2/shRNA virus showed an increase of acetaldehyde levels of 50, 30 and 40% for each ethanol concentration assayed. Previously we have demonstrated a 50% decrease in ethanol consumption over 35 days with a similar gene therapy treatment of alcoholic mice that also inhibited the AlDH2 gene. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors

Can a community of practice enhance a palliative approach for people drawing close to death with dementia?

This action research study was conducted to trial a strategy intended to support a consistent, high-quality, palliative approach for people with dementia drawing close to death-the implementation of a community of practice. Professionals from community/residential care and hospitals formed this community of practice, which took on the role of an action research group. The group was supported to identify and address practice problems. Four action plans were implemented; outcomes from two are reported. When actioning the plan 'providing education and information for the staff', the staff's ratings of sessions and resources were positive but impacts upon knowledge, views, or confidence were small. When actioning 'supporting families', families providing care in non-hospital settings received information about severe dementia from suitably prepared staff, plus contact details to access support. Family feedback was primarily positive. Reference to additional practice change frameworks and inclusion of specialist palliative care professionals are recommendations for future initiatives; also focusing on targeted, achievable goals over longer timeframes

Evaluation of Restaurant Menus to Determine the Availability of Healthy Food Options and Guide Community Transformation Grant Activities in Massachusetts

INTRODUCTION. The availability of healthy menu options in restaurants is an important factor in the prevention of obesity. The Mass in Motion Initiative and two Community Transformation Grant (CTG) projects are conducting statewide longitudinal surveys to determine the availability of healthy food in restaurants in the state of Massachusetts. METHODS. The Community Nutrition Environment Evaluation Data System-Restaurant (C-NEEDS-R) was developed for food environment surveillance. C-NEEDS-R takes into account seasonal and geographic variations in food supplies, cultural relevance, and USDA dietary recommendations. Between summer 2012 and winter 2013, 506 restaurants in 36 Massachusetts towns and cities were surveyed and analyzed. Through menu and site evaluation, the availability of healthy entrees was examined for each restaurant, and the total number of healthy entrees as well as the percent of healthy entrees was calculated for each restaurant. For each municipality, the average number and average percentage of healthy entrees for restaurants within the community was also calculated. RESULTS. The surveyed restaurants had average 3.2 healthy entrees on the menu, accounting for 13.4% of the total number of entrees available. The percentage of healthy options varied widely by restaurant and restaurant type, ranging from 0 to 84%, and only 15 of the 506 surveyed restaurants ( DISCUSSION. As noted, menu evaluation demonstrated that the large majority of the surveyed restaurants had few healthy entrees, indicating a need to increase availability of healthy options. Analysis of restaurant- and community-level variations in availability is useful for CTG programs to formulate and prioritize interventions. Future longitudinal surveys of food stores in the intervention and control communities will help evaluate the effectiveness of CTG interventions

Cutting the Gordian knot : early and complete amino acid sequence confirmation of class II lasso peptides by HCD fragmentation

SAJ would like to thank the University of Aberdeen for an Elphinstone Scholarship. CC-A thanks CONICYT PFCHA/DOCTORADO BECAS CHILE/2016 (#21160585) fellowship and CONICYT Basal Centre Grant for the Centre for Biotechnology and Bioengineering, CeBiB (FB0001). JFC also thanks CONICYT for a National PhD Scholarship (#21110356) and a Visiting Student Scholarship.Peer reviewedPostprin

New genus-specific primers for PCR identification of Rubrobacter strains

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines.

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo