Attenuated total reflectance infrared spectroelectrochemistry at a carbon particle electrode; unmediated redox control of a [NiFe]-hydrogenase solution

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.We report a versatile infrared spectroscopic method for studying redox chemistry of metalloproteins, and demonstrate for the first time electrochemically-induced changes to the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. A carbon particle network working electrode allows control over a wide potential window without the need for solution mediators.EC/FP7/258600/EU/Understanding and Exploiting Biological Catalysts for Energy Cycling: Development of Infrared Spectroelectrochemistry for Studying Intermediates in Metalloenzyme Catalysis/ENERGYBIOCATALYSISDFG, EXC 314, Unifying Concepts in Catalysi

Comparison of carbon materials as electrodes for enzyme electrocatalysis:hydrogenase as a case study

We present a study of electrocatalysis by an enzyme adsorbed on a range of carbon materials, with different size, surface area, morphology and graphitic structure, which are either commercially available or prepared via simple, established protocols. We choose as our model enzyme the hydrogenase I from E. coli (Hyd-1), which is an active catalyst for H2 oxidation, is relatively robust and has been demonstrated in H2 fuel cells and H2-driven chemical synthesis. The carbon materials were characterised according to their surface area, surface morphology and graphitic character, and we use the electrocatalytic H2 oxidation current for Hyd-1 adsorbed on these materials to evaluate their effectiveness as enzyme electrodes. Here, we show that a variety of carbon materials are suitable for adsorbing hydrogenases in an electroactive configuration. This unified study provides insight into selection and design of carbon materials for study of redox enzymes and different applications of enzyme electrocatalysis

Electrochemical control of [FeFe]-hydrogenase single crystals reveals complex redox populations at the catalytic site

Elucidating the distribution of intermediates at the active site of redox metalloenzymes is vital to understanding their highly efficient catalysis. Here we demonstrate that it is possible to generate, and detect, the key catalytic redox states of an [FeFe]-hydrogenase in a protein crystal. Individual crystals of the prototypical [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) are maintained under electrochemical control, allowing for precise tuning of the redox potential, while the crystal is simultanaously probed via Fourier Transform Infrared (FTIR) microspectroscopy. The high signal/noise spectra reveal potential-dependent variation in the distribution of redox states at the active site (H-cluster) according to state-specific vibrational bands from the endogeneous CO and CN- ligands. CpI crystals are shown to populate the same H-cluster states as those detected in solution, including the oxidised species Hox, the reduced species Hred/HredH+, the super-reduced HsredH+ and the hydride species Hhyd. The high sensitivity and precise redox control offered by this approach also facilitates the detection and characterisation of low abundance species that only accumulate within a narrow window of conditions, revealing new redox intermediates

Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site‐specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other “non‐canonical” amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non‐canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an u nbiased p hosphopeptide enrichment strategy based on strong a nion ex change (SAX ) chromatography (UPAX ), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry‐based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non‐canonical phosphosites is approximately one‐third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high‐throughput exploration of non‐canonical phosphorylation in all organisms

STAT-HI: A Socio-Technical Assessment Tool for Health Informatics Implementations

This paper proposes a socio-technical assessment tool (STAT-HI) for health informatics implementations. We explore why even projects allegedly using sound methodologies repeatedly fail to give adequate attention to socio-technical issues, and we present an initial draft of a structured assessment tool for health informatics implementation that encapsulates socio-technical good practice. Further work is proposed to enrich and validate the proposed instrument. This proposal was presented for discussion at a meeting of the UK Faculty of Health Informatics in December 2009

Geographical variation in rates of surgical treatment for female stress urinary incontinence in England: a national cohort study.

OBJECTIVE: To examine geographic variation in use of surgery for female stress urinary incontinence (SUI), mainly midurethral mesh tape insertions, in the English National Health Service (NHS). DESIGN: National cohort study. SETTING: NHS hospitals. PARTICIPANTS: 27 997 women aged 20 years or older who had a first SUI surgery in an English NHS Hospital between April 2013 and March 2016 and a diagnosis of SUI at the same time as the procedure. METHODS: Multilevel Poisson regression was used to adjust for geographic differences in age, ethnicity, prevalence of long-term illness and socioeconomic deprivation. PRIMARY OUTCOME MEASURE: Rate of surgery for SUI per 100 000 women/year at two geographic levels: Clinical Commissioning Group (CCG; n=209) and Sustainability and Transformation Partnership (STP; n=44). RESULTS: The rate of surgery for SUI was 40 procedures per 100 000 women/year. Risk-adjusted rates ranged from 20 to 106 procedures per 100 000 women/year across CCGs and 24 to 69 procedures per 100 000 women/year across the STP areas. These regional differences were only partially explained by demographic characteristics as adjustment reduced variance of surgery rates by 16% among the CCGs and 35% among the STPs. CONCLUSIONS: Substantial geographic variation exists in the use of surgery for female SUI in the English NHS, suggesting that women in some areas are more likely to be treated compared with women with the same condition in other areas. The variation reflects differences in how national guidelines are being interpreted in the context of the ongoing debate about the safety of SUI surgery

Follow-up care for men with prostate cancer and the role of primary care: a systematic review of international guidelines

The optimal role for primary care in providing follow-up for men with prostate cancer is uncertain. A systematic review of international guidelines was undertaken to help identify key elements of existing models of follow-up care to establish a theoretical basis for evaluating future complex interventions. Many guidelines provide insufficient information to judge the reliability of the recommendations. Although the PSA test remains the cornerstone of follow-up, the diversity of recommendations on the provision of follow-up care reflects the current lack of research evidence on which to base firm conclusions. The review highlights the importance of transparent guideline development procedures and the need for robust primary research to inform future evidence-based models of follow-up care for men with prostate cancer

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

The IPBES Conceptual Framework - connecting nature and people

The first public product of the Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES) is its Conceptual Framework. This conceptual and analytical tool, presented here in detail, will underpin all IPBES functions and provide structure and comparability to the syntheses that IPBES will produce at different spatial scales, on different themes, and in different regions. Salient innovative aspects of the IPBES Conceptual Framework are its transparent and participatory construction process and its explicit consideration of diverse scientific disciplines, stakeholders, and knowledge systems, including indigenous and local knowledge. Because the focus on co-construction of integrative knowledge is shared by an increasing number of initiatives worldwide, this framework should be useful beyond IPBES, for the wider research and knowledge-policy communities working on the links between nature and people, such as natural, social and engineering scientists, policy-makers at different levels, and decision-makers in different sectors of society