The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding ...preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal–Structural Analysis and Library Design (CSR-SALAD).
Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the ...synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of the intermediates shifts to favor covalently bound Trp adducts, which have increased thermodynamic stability. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High-resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a subdomain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.
The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein ...stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.
To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a ...case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.
The most recent version of the European Society for Medical Oncology (ESMO) Clinical Practice Guidelines for the diagnosis, treatment and follow-up of localised colon cancer was published in 2020. It ...was decided by both the ESMO and the Japanese Society of Medical Oncology (JSMO) to convene a special virtual guidelines meeting in March 2021 to adapt the ESMO 2020 guidelines to take into account the ethnic differences associated with the treatment of localised colon cancer in Asian patients. These guidelines represent the consensus opinions reached by experts in the treatment of patients with localised colon cancer representing the oncological societies of Japan (JSMO), China (CSCO), India (ISMPO), Korea (KSMO), Malaysia (MOS), Singapore (SSO) and Taiwan (TOS). The voting was based on scientific evidence and was independent of the current treatment practices and drug availability and reimbursement situations in the different Asian countries.
•This article provides ESMO expert recommendations adapted for the treatment of localised colon cancer in Asian patients.•The aim was to provide guidance for the optimisation of the management of such patients across Asia.•The availability and applicability of certain procedures as they relate to certain of the recommendations are discussed.
Women with atypical hyperplasia (AH) or lobular carcinoma in situ (LCIS) have a significantly increased risk of breast cancer, which can be substantially reduced with antiestrogen therapy for ...chemoprevention. However, antiestrogen therapy for breast cancer risk reduction remains underutilized. Improving knowledge about breast cancer risk and chemoprevention among high-risk patients and their healthcare providers may enhance informed decision-making about this critical breast cancer risk reduction strategy.
We are conducting a cluster randomized controlled trial to evaluate the effectiveness and implementation of patient and provider decision support tools to improve informed choice about chemoprevention among women with AH or LCIS. We have cluster randomized 26 sites across the U.S. through the SWOG Cancer Research Network. A total of 415 patients and 200 healthcare providers are being recruited. They are assigned to standard educational materials alone or combined with the web-based decision support tools. Patient-reported and clinical outcomes are assessed at baseline, after a follow-up visit at 6 months, and yearly for 5 years. The primary outcome is chemoprevention informed choice after the follow-up visit. Secondary endpoints include other patient-reported outcomes, such as chemoprevention knowledge, decision conflict and regret, and self-reported chemoprevention usage. Barriers and facilitators to implementing decision support into clinic workflow are assessed through patient and provider interviews at baseline and mid-implementation.
With this hybrid effectiveness/implementation study, we seek to evaluate if a multi-level intervention effectively promotes informed decision-making about chemoprevention and provide valuable insights on how the intervention is implemented in U.S. clinical settings.
NCT04496739
Helpers in cooperatively breeding groups can vary hugely in the variety and level of care they provide. Several studies suggest that kin selection alone cannot be invoked to explain variation in ...helping for many species, but there have been few explicit tests of this under controlled conditions. Here, we investigated whether relatedness to the breeding pair or consistent individual differences in behaviours explained variation in helping by the cooperatively breeding cichlid
Neolamprologus pulcher. We established standardized social groups consisting of a breeding pair and one related (
r = 0.5) and one unrelated (
r = 0) helper. Two forms of helping, territory maintenance and territory defence, were measured repeatedly under controlled conditions: helping was variable between, but consistent within, individuals. Furthermore, there was some evidence that helpers that carried out more maintenance also performed more defence. Contrary to the kin selection hypothesis, relatedness did not predict the amount or variety of helping executed. Risk responsiveness, activity levels and aggressiveness were repeatable within individuals, so constituted ‘behavioural types’ (or personality traits), but were uncorrelated with each other. More aggressive, risk-prone or more active helpers participated in more territory defence than submissive, risk-averse or inactive helpers. Risk-prone individuals contributed more to territory maintenance than risk-averse helpers. Overall, differences in behavioural type, rather than relatedness, explained most variation in helping behaviour in
N. pulcher. This study highlights the importance of considering consistent individual differences in behaviour for predicting participation and performance in complex social interactions.
By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a ...protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.
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•Chromophore substitution shifts Arch fluorescence to unprecedented NIR wavelengths•Directed evolution enhances NIR brightness and affinity for synthetic retinal•The NIR fluorescence of engineered Arch variants is pH sensitive•Membrane-localized Arch variants facilitate NIR imaging in bacterial cell culture
Using a combined approach of chromophore substitution and directed evolution, Herwig et al. engineered fluorescent Archaerhodopsin variants with unprecedented near-infrared excitation and emission. Evolved variants display pH sensitivity, enhanced fluorescent molecular brightness, and improved synthetic chromophore affinity.
Summary
Aims
Fibromyalgia (FM), a chronic disorder defined by widespread pain, often accompanied by fatigue and sleep disturbance, affects up to one in 20 patients in primary care. Although most ...patients with FM are managed in primary care, diagnosis and treatment continue to present a challenge, and patients are often referred to specialists. Furthermore, the lack of a clear patient pathway often results in patients being passed from specialist to specialist, exhaustive investigations, prescription of multiple drugs to treat different symptoms, delays in diagnosis, increased disability and increased healthcare resource utilisation. We will discuss the current and evolving understanding of FM, and recommend improvements in the management and treatment of FM, highlighting the role of the primary care physician, and the place of the medical home in FM management.
Methods
We reviewed the epidemiology, pathophysiology and management of FM by searching PubMed and references from relevant articles, and selected articles on the basis of quality, relevance to the illness and importance in illustrating current management pathways and the potential for future improvements.
Results
The implementation of a framework for chronic pain management in primary care would limit unnecessary, time‐consuming, and costly tests, reduce diagnostic delay and improve patient outcomes.
Discussion
The patient‐centred medical home (PCMH), a management framework that has been successfully implemented in other chronic diseases, might improve the care of patients with FM in primary care, by bringing together a team of professionals with a range of skills and training.
Conclusion
Although there remain several barriers to overcome, implementation of a PCMH would allow patients with FM, like those with other chronic conditions, to be successfully managed in the primary care setting.
Effect of various antimicrobial interventions on pork carcass cuts – skin-on and skinless, non-chilled and chilled – was studied. Carcass pieces were inoculated with Salmonella enterica, Shiga ...toxin-producing Escherichia coli (STEC), Escherichia coli pathogen surrogates or Campylobacter spp. Inoculated pieces were assigned to one of the following antimicrobial treatments: 2.5% and 5.0% room temperature lactic acid, 2.5% and 5.0% warm (55 °C) lactic acid, 400 ppm (0.4 mg/mL) room temperature peroxyacetic acid (PAA) or warm (55 °C) water. Treated pieces were sampled before antimicrobial treatment of non-chilled pork tissue, then at 30 m and 24 h post-treatment. For chilled pork, samples were collected after 24 h chilling and 30 m post-treatment. Lactic acid and PAA treatments reduced (P < 0.05) pathogenic and surrogate bacteria; warm water did not produce similar results. Objective and sensory color evaluations on treated pork indicated minimal negative impacts on pork color. Various antimicrobial interventions were effective in reducing surrogates on pork without diminishing quality.
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