Halogenases for the synthesis of small molecules

Enzymatic halogenation is a rapidly developing tool in the synthetic chemist's toolbox. Utilizing oxygen, halide salts and operating at ambient temperatures in aqueous media, halogenating enzymes, particularly flavin- or Fe(II)/α-ketoglutarate dependent halogenases, allow the regio- and stereoselective installation of halogen atoms to yield valuable building blocks and uniquely derivatized complex molecules, including natural products. Apart from modulating the physico-chemical properties of molecules and, in consequence, their biological activity, halogen atoms can also serve as chemical linchpins for further derivatization of the molecular scaffold, for example in chemo-enzymatic cascades. Thanks to rapid advances in bioinformatic enzyme sourcing, biosynthetic pathway elucidation and enzyme engineering strategies, the palette of enzymatically produced halogenated structures is constantly growing. In addition, successful studies to improve catalytic performance of the halogenation biocatalysts are boding well for industrial applications

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants

Vaccinia Virus Protein C6 Is a Virulence Factor that Binds TBK-1 Adaptor Proteins and Inhibits Activation of IRF3 and IRF7

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7

"Eat and you will be eaten": a qualitative study exploring costs and benefits of age-disparate sexual relationships in Tanzania and Uganda: implications for girls' sexual and reproductive health interventions

BACKGROUND: Age-disparate sex is associated with increased HIV risk among adolescent girls and young women (AGYW) in sub-Saharan Africa. However, little has been done to understand the dynamics of such relationships from the perspectives of either AGYW or older men, and the communities in which these relationships are embedded. This article explores the motivations and perceived benefits of such relationships for AGYW and older men, plus the social and sexual and reproductive health (SRH) consequences. METHODS: This study held 37 participatory focus group discussions and 87 in-depth interviews with young people aged 14-24 and adult community members aged 25-49 in rural and urban Tanzania and Uganda. Participants were sampled using purposive and snowball techniques. Thematic analysis was conducted with the aid of NVIVO 10 software. RESULTS: Motivations, perceived benefits and costs for AGYW centred around four main themes: financial motivations, emotional support, meeting social expectations and reflections on sexual health. Specifically, AGYW noted that older partners gave gifts/money of higher value compared with younger men. Men's perceived benefits and costs revolved around the need to satisfy their sexual desire, the perception that AGYW were capable of engaging in new and creative sexual styles and their desire for prestige among male peers. Both AGYW and men recognised the social and SRH consequences as: risk of violence, social stigma, risk of unplanned pregnancy and risk of sexually transmitted infections including HIV. CONCLUSION: Interventions need to acknowledge the perceived benefits of age-disparate sexual relationships for AGYW and older men and engage them in critical reflection on the medium- to longer-term consequences versus the shorter-term satisfaction of needs, desires and aspirations, as a way to navigate the constrained opportunities they face given existing structural limitations. Interventions should also tackle the structural constraints AGYW face by helping them access resources, become empowered and challenge the expectation of having to depend financially on men. Interventions with men should unpack the assumption that men are naturally hypersexual. The role of peers for both girls and men should be acknowledged, and a shift from individual targeted interventions to changing norms at the community level should be considered

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection ar