Engineered, Scalable Production of Optically Pure l-Phenylalanines Using Phenylalanine Ammonia-Lyase from Arabidopsis thaliana.

An efficient preparative-scale synthetic procedure of l-phenylalanine derivatives has been developed using mutant variants of phenylalanine ammonia-lyase from Arabidopsis thaliana (AtPAL). After rigorous reaction engineering, the AtPAL-catalyzed hydroamination reaction of cinnamic acids provided several unnatural amino acids of high synthetic value, such as (S)-m- and (S)-p-methoxyphenylalanine; (S)-o- and (S)-m-methylphenylalanine; and (S)-o- and (S)-p-bromophenylalanine at preparative scale, significantly surpassing the catalytic efficiency in terms of conversions and yields of the previously reported PcPAL-based biotransformations. The AtPAL variants tolerated high substrate and product concentrations, representing an important extension of the PAL-toolbox, while the engineered biocatalytic procedures of improved E-factor and space-time yields fulfill the requirements of sustainable and green chemistry, providing facile access to valuable amino acid building blocks.

Low socioeconomic status, parental stress, depression, and the buffering role of network social capital in mothers.

BACKGROUND: Pathways underlying the stress-depression relationship in mothers, and the factors that buffer this relationship are not well understood. AIMS: Drawing from the Stress Process model, this study examines (1) if parental stress mediates the association between socioeconomic characteristics and depressive symptoms, and (2) if social support and network capital moderate these pathways. METHOD: Data came from 101 mothers from Montreal. Generalized structural equation models were conducted, with depressive symptoms (CES-D scores) as the outcome, socioeconomic stressors as independent variables, parental stress as the mediator, and social support and network social capital as moderators. RESULTS: Parental stress partially mediated the association between household income and depressive symptoms (indirect effect: ß = -0.09, Bootstrap SE = 0.03, 95% CI = -0.15 to -0.03 p = 0.00). Network diversity moderated the relationship between parental stress and depressive symptoms (ß = -0.25, 95% CI = -0.42 to -0.09, p = 0.00); at high levels of stress, mothers with high compared to low network diversity reported fewer symptoms. CONCLUSION: Findings highlight the role that socioeconomic factors play in influencing women's risk of depression and shaping the benefits that ensue from social resources. Addressing these factors requires interventions that target the social determinants of depression.

Robust Suppression of Lipopolysaccharide Deficiency in Acinetobacter baumannii by Growth in Minimal Medium.

Lipopolysaccharide (LPS) is normally considered to be essential for viability in Gram-negative bacteria but can be removed in Acinetobacter baumannii Mutant cells lacking this component of the outer membrane show growth and morphological defects. Here, we report that growth rates equivalent to the wild type can be achieved simply by propagation in minimal medium. The loss of LPS requires that cells rely on phospholipids for both leaflets of the outer membrane. We show that growth rate in the absence of LPS is not limited by nutrient availability but by the rate of outer membrane biogenesis. We hypothesize that because cells grow more slowly, outer membrane synthesis ceases to be rate limiting in minimal medium.IMPORTANCE Gram-negative bacteria are defined by their asymmetric outer membrane that consists of phospholipids on the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS is essential in all but a few Gram-negative species; the reason for this differential essentiality is not well understood. One species that can survive without LPS, Acinetobacter baumannii, shows characteristic growth and morphology phenotypes. We show that these phenotypes can be suppressed under conditions of slow growth and describe how LPS loss is connected to the growth defects. In addition to better defining the challenges A. baumannii cells face in the absence of LPS, we provide a new hypothesis that may explain the species-dependent conditional essentiality.

A cluster of residues in the lipopolysaccharide exporter that selects substrate variants for transport to the outer membrane.

Most Gram-negative bacteria assemble lipopolysaccharides (LPS) on their surface to form a permeability barrier against many antimicrobials. LPS is synthesized at the inner membrane and then transported to the outer leaflet of the outer membrane. Although the overall LPS structure is conserved, LPS molecules can differ in composition at the species and strain level. Some bacteria also regulate when to modify phosphates on LPS at the inner membrane in order to become resistant to cationic antimicrobial peptides. The multi-protein Lpt trans-envelope machine, which transports LPS from the inner to the outer membrane, must therefore handle a variety of substrates. The most poorly understood step in LPS transport is how the ATP-binding cassette LptB2 FG transporter extracts LPS from the inner membrane. Here, we define residue K34 in LptG as a site within the structural cavity of the Escherichia coli LptB2 FG transporter that interacts electrostatically with phosphates on unmodified LPS. Alterations to this residue cause transport defects that are suppressed by the activation of the BasSR two-component signaling system, which results in modifications to the LPS phosphates. We also show this residue is part of a larger site in LptG that differentially contributes to the transport of unmodified and modified LPS.

Decarboxylative and dehydrative coupling of dienoic acids and pentadienyl alcohols to form 1,3,6,8-tetraenes.

Dienoic acids and pentadienyl alcohols are coupled in a decarboxylative and dehydrative manner at ambient temperature using Pd(0) catalysis to generate 1,3,6,8-tetraenes. Contrary to related decarboxylative coupling reactions, an anion-stabilizing group is not required adjacent to the carboxyl group. Of mechanistic importance, it appears that both the diene of the acid and the diene of the alcohol are required for this reaction. To further understand this reaction, substitutions at every unique position of both coupling partners was examined and two potential mechanisms are presented.

Tracking emergency response actions during COVID-19 leads to development of an innovative public health evaluation tool.

SETTING: Early in the pandemic, KFL&A Public Health needed a way to capture, organize, and display COVID-19-related events to be accountable for and evaluate our actions. INTERVENTION: We used accessible software (Microsoft Office 365 suite, Microsoft PowerBI) to develop a data collection and visualization system. The Canadian Institute for Health Information (CIHI) developed a timeline and categorization approach for provincial and national COVID-related interventions, which was used to develop a regional version for local events using similar categories. We collected and displayed qualitative data alongside epidemiological data that allowed users to display different timelines of actions and outcomes and evaluate our response. OUTCOMES: In developing the timeline, we took stock of the information and data we wanted to collect, sort, and display locally. Next, we collected information on response actions, case and contact tracing, and staffing changes in a database that we displayed on a timeline. We included CIHI's data set to provide insight into pandemic response across all jurisdictions. IMPLICATIONS: Our timeline tool has many advantages for public health authorities beyond responding to a rapidly evolving emergency. By collecting information on events as they occur, decisions and actions are documented that may otherwise be overlooked. This enables decision-makers to visualize the impact of public health actions on health outcomes over time. The tool is completely customizable and scalable depending on the project scope and we plan to apply this method to other public health programming. Finally, we include lessons learned from quickly developing these tools in a real-time pandemic setting, both locally at KFL&A Public Health and nationally at CIHI.

RéSUMé: LIEU: Au début de la pandémie, le Bureau de santé de Kingston, Frontenac, Lennox et Addington (KFL&A) avait besoin d'un moyen de saisir, d'organiser et de présenter les événements liés à la COVID-19 pour pouvoir en rendre compte et évaluer ses actions. INTERVENTION: Nous avons utilisé des logiciels accessibles (Microsoft Office 365, Microsoft PowerBI) pour mettre au point un système de collecte et de visualisation de données. L'Institut canadien d'information sur la santé (ICIS) a créé un fil d'actualité et une approche de catégorisation pour les interventions provinciales et nationales liées à la COVID; nous avons créé une version régionale de ces outils pour présenter les événements locaux, en utilisant des catégories semblables. Nous avons collecté et affiché des données qualitatives en plus des données épidémiologiques, ce qui a permis aux utilisateurs d'afficher les mesures prises et leurs résultats sur différentes périodes et d'évaluer leurs interventions. RéSULTATS: Pour créer ce fil d'actualité, nous avons fait l'inventaire des informations et des données que nous voulions collecter, trier et présenter localement. Ensuite, nous avons réuni des informations sur les mesures d'intervention, les enquêtes sur les cas, la recherche de contacts et les changements de personnel dans une base de données, et nous les avons présentées dans un fil d'actualité. Nous avons inclus le jeu de données de l'ICIS pour apporter un éclairage sur la lutte contre la pandémie dans toutes les administrations du pays. CONSéQUENCES: Notre fil d'actualité présente de nombreux avantages pour les autorités de santé publique, en plus de la possibilité de réagir à une situation urgence qui évolue rapidement. En collectant des informations sur les événements à mesure qu'ils se produisent, il est possible de documenter des décisions et des mesures qui risquent d'être oubliées sinon. Cela permet aux décideurs de visualiser dans le temps l'effet des mesures de santé publique sur les résultats cliniques. L'outil est entièrement personnalisable et évolutif, selon la portée du projet, et nous avons l'intention de l'appliquer à d'autres programmes de santé publique. Enfin, nous présentons les leçons de la mise au point rapide d'un tel outil pendant une pandémie en temps réel, tant à l'échelle locale (à la Santé publique de KFL&A) qu'à l'échelle nationale (à l'ICIS).

Toolbox for the structure-guided evolution of ferulic acid decarboxylase (FDC).

The interest towards ferulic acid decarboxylase (FDC), piqued by the enzyme's unique 1,3-dipolar cycloaddition mechanism and its atypic prFMN cofactor, provided several applications of the FDC mediated decarboxylations, such as the synthesis of styrenes, or its diverse derivatives, including 1,3-butadiene and the enzymatic activation of C-H bonds through the reverse carboligation reactions. While rational design-based protein engineering was successfully employed for tailoring FDC towards diverse substrates of interest, the lack of high-throughput FDC-activity assay hinders its directed evolution-based protein engineering. Herein we report a toolbox, useful for the directed evolution based and/or structure-guided protein engineering of FDC, which was validated representatively on the well described FDC, originary from Saccharomyces cerevisiae (ScFDC). Accordingly, the developed fluorescent plate-assay allows in premiere the FDC-activity screens of a mutant library in a high-throughput manner. Moreover, using the plate-assay for the activity screens of a rationally designed 23-membered ScFDC variant library against a substrate panel comprising of 16, diversely substituted cinnamic acids, revealed several variants of improved activity. The superior catalytic properties of the hits revealed by the plate-assay, were also supported by the conversion values from their analytical scale biotransformations. The computational results further endorsed the experimental findings, showing inactive binding poses of several non-transformed substrate analogues within the active site of the wild-type ScFDC, but favorable ones within the catalytic site of the variants of improved activity. The results highlight several 'hot-spot' residues involved in substrate specificity modulation of FDC, such as I189, I330, F397, I398 or Q192, of which mutations to sterically less demanding residues increased the volume of the active site, thus facilitated proper binding and increased conversions of diverse non-natural substrates. Upon revealing which mutations improve the FDC activity towards specific substrate analogues, we also provide key for the rational substrate-tailoring of FDC.

Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases.

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

The production of L- and D-phenylalanines using engineered phenylalanine ammonia lyases from Petroselinum crispum.

The biocatalytic synthesis of L- and D-phenylalanine analogues of high synthetic value have been developed using as biocatalysts mutant variants of phenylalanine ammonia lyase from Petroselinum crispum (PcPAL), specifically tailored towards mono-substituted phenylalanine and cinnamic acid substrates. The catalytic performance of the engineered PcPAL variants was optimized within the ammonia elimination and ammonia addition reactions, focusing on the effect of substrate concentration, biocatalyst:substrate ratio, reaction buffer and reaction time, on the conversion and enantiomeric excess values. The optimal conditions provided an efficient preparative scale biocatalytic procedure of valuable phenylalanines, such as (S)-m-methoxyphenylalanine (Y = 40%, ee > 99%), (S)-p-bromophenylalanine (Y = 82%, ee > 99%), (S)-m-(trifluoromethyl)phenylalanine (Y = 26%, ee > 99%), (R)-p-methylphenylalanine, (Y = 49%, ee = 95%) and (R)-m-(trifluoromethyl)phenylalanine (Y = 34%, ee = 93%).

Exploring the substrate scope of ferulic acid decarboxylase (FDC1) from Saccharomyces cerevisiae.

Ferulic acid decarboxylase from Saccharomyces cerevisiae (ScFDC1) was described to possess a novel, prenylated flavin mononucleotide cofactor (prFMN) providing the first enzymatic 1,3-dipolar cycloaddition mechanism. The high tolerance of the enzyme towards several non-natural substrates, combined with its high quality, atomic resolution structure nominates FDC1 an ideal candidate as flexible biocatalyst for decarboxylation reactions leading to synthetically valuable styrenes. Herein the substrate scope of ScFDC1 is explored on substituted cinnamic acids bearing different functional groups (-OCH3, -CF3 or -Br) at all positions of the phenyl ring (o-, m-, p-), as well as on several biaryl and heteroaryl cinnamic acid analogues or derivatives with extended alkyl chain. It was found that E. coli whole cells expressing recombinant ScFDC1 could transform a large variety of substrates with high conversion, including several bulky aryl and heteroaryl cinnamic acid analogues, that characterize ScFDC1 as versatile and highly efficient biocatalyst. Computational studies revealed energetically favoured inactive binding positions and limited active site accessibility for bulky and non-linear substrates, such as 2-phenylthiazol-4-yl-, phenothiazine-2-yl- and 5-(4-bromophenyl)furan-2-yl) acrylic acids. In accordance with the computational predictions, site-directed mutagenesis of residue I330 provided variants with catalytic activity towards phenothiazine-2-yl acrylic acid and provides a basis for altering the substrate specificity of ScFDC1 by structure based rational design.

Tailored Mutants of Phenylalanine Ammonia-Lyase from Petroselinum crispum for the Synthesis of Bulky l- and d-Arylalanines.

Tailored mutants of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non-natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)-arylacrylates. The wild-type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well-studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4-methoxyphenyl)-, (napthalen-2-yl)-, ([1,1'-biphenyl]-4-yl)-, (4'-fluoro-[1,1'-biphenyl]-4-yl)-, and (5-phenylthiophene-2-yl)alanines.

Social interventions: An effective approach to reduce adult depression?

BACKGROUND: Social interventions that aim to facilitate bonds and interaction among individuals could reduce depression at a population level; yet, the scope and effectiveness of these interventions remain unclear. This systematic review classifies and reports on social interventions that have been implemented to target depression in adults. METHODS: Search terms related to 'intervention', 'depression', and 'social' were entered into databases, including: The Cochrane Database of Systematic Reviews, MEDLINE, Embase, PsycInfo, CINAHL, and TRoPHI. Inclusion criteria included: (1) depression was an intervention outcome, (2) depression was not attributable to concomitant illnesses or circumstances (e.g., chronic illness or exposure to natural disasters), (3) the intervention facilitated social interaction, (4) the intervention targeted adults (18-64), (5) the sample was community-based, (6) the study was available in English, and (7) within-group or between-group comparison group information was available. RESULTS: Of the 24 studies meeting the inclusion criteria, 17 reported reductions in depressive symptoms. Social interventions often incorporated multiple strategies to improve depressive symptoms, including: peer support (n=17), skill building (n=11), group-based activities (n =11), psycho-education (n =9), psychotherapy (n =5), exercise (n =5), and links to community resources (n=3). LIMITATIONS: Findings of this review may not be generalizable to specific population subgroups with depression, including those who have chronic illnesses or postpartum depression. CONCLUSIONS: Various types of social interventions can be effective in reducing adult depression. Social interventions can be tailored to diverse groups, are feasible in resource-scarce communities, and have the potential to reduce population-level depression due to their group formats.

Parental social capital and children's sleep disturbances.

OBJECTIVES: Sleep plays a critical role in the health and well-being of children. Individual and household factors, including parent's social connections, may impact children's sleep. Our study assessed the association between children's sleep disturbances and parent's social capital in a sample of Canadian households. DESIGN: Cross-sectional, observational study. SETTINGS AND PARTICIPANTS: Data came from 339 children and their parents who completed a telephone and follow-up survey in 2013 as part of the Canada Brain-to-Society study. Participants were parents (73.1% female) with children aged 6 to 12years residing in Montreal, Canada. MEASUREMENTS: Parental social capital was assessed using a position generator, and children's sleep disturbances were measured with the Children's Sleep Habits Questionnaire. Other household demographic and socioeconomic characteristics were measured, including parental and child age and sex, foreign-born status, and income. Linear regression was used to examine the association between parental social capital and children's sleep disturbances while controlling for possible confounders. RESULTS: Parental social capital was negatively associated with children's sleep disturbances (ß=-0.02, SE=0.01, P<.05), when controlling for demographic factors (sex of parent, household income, foreign-born status, parent's age, sex and age of child, suggesting that children of parents with higher social capital had fewer sleep disturbances. CONCLUSIONS: Parents with higher social capital tended to have children with few total sleep disturbances than did parents with lower social capital. Parental social capital may be a potentially modifiable aspect of the home environment that has implications for children's health.

Sequential Pd(0)-, Rh(I)-, and Ru(II)-catalyzed reactions in a nine-step synthesis of clinprost.

A step-economical synthesis of clinprost is reported that concludes with 3 different transition metal-catalyzed reactions: Pd-catalyzed decarboxylation with allylic rearrangement, Rh-catalyzed diene-ene [2+2+1] reaction, and Ru-catalyzed cross-metathesis reaction. The complexity bestowed to the molecule from these reactions converts a readily accessible ester to clinprost without using protecting groups in only 9 total steps.