Search

Objectives Methotrexate (MTX) is one of the most commonly used medications to treat rheumatoid arthritis (RA). However, the effect of MTX treatment on cellular immune responses remains incompletely understood. This raises concerns about the vulnerability of these patients to emerging infections and following vaccination.Methods In the current study, we investigated the impact of MTX treatment in patients with immune-mediated inflammatory disease on B and CD4 T cell SARS-CoV-2 vaccination responses. Eighteen patients with RA and two patients with psoriatic arthritis on MTX monotherapy were included, as well as 10 patients with RA without immunosuppressive treatment, and 29 healthy controls. CD4 T and B cell responses were analysed 7 days and 3–6 months after two SARS-CoV-2 messenger RNA vaccinations. High-dimensional flow cytometry analysis was used to analyse fresh whole blood, an activation-induced marker assay to measure antigen-specific CD4 T cells, and spike probes to study antigen-specific B cells.Results Seven days following two SARS-CoV-2 vaccinations, total B and T cell counts were similar between MTX-treated patients and controls. In addition, spike-specific B cell frequencies were unaffected. Remarkably, the frequency of antigen-specific CD4 T cells was reduced in patients using MTX and correlated strongly with anti-RBD IgG antibodies. These results suggest that decreased CD4 T cell activity may result in slower vaccination antibody responses in MTX-treated patients.Conclusion Taken together, MTX treatment reduces vaccine-induced CD4 T cell activation, which correlates with lower antibody responses.Trial registration number NL8900.

Signal transducer and activator of transcription 3 (Stat3) plays a role in various cellular processes such as differentiation, inflammation, cell survival and microtubule dynamics, depending on the cell type and the activated signaling pathway. Stat3 is highly expressed in the hair cells and supporting cells of the cochlea and is essential for the differentiation of mouse hair cells in the early embryonic stage. However, it is unclear how Stat3 contributes to the correct function of cells in the organ of Corti postnatally. To investigate this, an inducible Cre/loxp system was used to knock out Stat3 in either the outer hair cells or the supporting cells. The results showed that the absence of Stat3 in either the outer hair cells or the supporting cells resulted in hearing loss without altering the morphology of the organ of Corti. Molecular analysis of the outer hair cells revealed an inflammatory process with increased cytokine production and upregulation of the NF-kB pathway. However, the absence of Stat3 in the supporting cells resulted in reduced microtubule stability. In conclusion, Stat3 is a critical protein for the sensory epithelium of the cochlea and hearing and functions in a cell and function-specific manner.

The electron–electron, or zero-field interaction (ZFI) in the electron paramagnetic resonance (EPR) of high-spin transition ions in metalloproteins and coordination complexes, is commonly described by a simple spin Hamiltonian that is second-order in the spin S: H=D[Sz2−SS+1/3+E(Sx2−Sy2). Symmetry considerations, however, allow for fourth-order terms when S ≥ 2. In metalloprotein EPR studies, these terms have rarely been explored. Metal ions can cluster via non-metal bridges, as, for example, in iron-sulfur clusters, in which exchange interaction can result in higher system spin, and this would allow for sixth- and higher-order ZFI terms. For metalloproteins, these have thus far been completely ignored. Single-molecule magnets (SMMs) are multi-metal ion high spin complexes, in which the ZFI usually has a negative sign, thus affording a ground state level pair with maximal spin quantum number mS = ±S, giving rise to unusual magnetic properties at low temperatures. The description of EPR from SMMs is commonly cast in terms of the ‘giant-spin model’, which assumes a magnetically isolated system spin, and in which fourth-order, and recently, even sixth-order ZFI terms have been found to be required. A special version of the giant-spin model, adopted for scaling-up to system spins of order S ≈ 103–104, has been applied to the ubiquitous iron-storage protein ferritin, which has an internal core containing Fe3+ ions whose individual high spins couple in a way to create a superparamagnet at ambient temperature with very high system spin reminiscent to that of ferromagnetic nanoparticles. This scaled giant-spin model is critically evaluated; limitations and future possibilities are explicitly formulated.

Objective There is a lack of consensus regarding the optimal approach for patients with full-thickness rectal prolapse. The aim of this international survey was to assess the patterns in treatment of rectal prolapse.Design A 23-question survey was distributed to the Pelvic Floor Consortium of the American Society of Colorectal Surgeons, the Colorectal Surgical Society of Australia and New Zealand, and the Pelvic Floor Society. Questions pertained to surgeon and practice demographics, preoperative evaluation, procedural preferences, and educational needs.Setting Electronic survey distributed to colorectal surgeons of diverse practice settingsParticipants 249 colorectal surgeons responded to the survey, 65% of which were male. There was wide variability in age, years in practice, and practice setting.Main outcome measures Responses to questions regarding preoperative workup preferences and clinical scenarios.Results In preoperative evaluation, 19% would perform anorectal physiology testing and 70% would evaluate for concomitant pelvic organ prolapse. In a healthy patient, 90% would perform a minimally invasive abdominal approach, including ventral rectopexy (56%), suture rectopexy (31%), mesh rectopexy (6%) and resection rectopexy (5%). In terms of ventral rectopexy, surgeons in the Americas preferred a synthetic mesh (61.9% vs 38.1%, p=0.59) whereas surgeons from Australasia preferred biologic grafts (75% vs 25%, p

ABSTRACT Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD). However, the role of hepatic involvement in EVD progression is understudied. Medical imaging in established animal models of EVD (e.g., nonhuman primates [NHPs]) can be a strong complement to traditional assays to better investigate this pathophysiological process in vivo and noninvasively. In this proof-of-concept study, we used longitudinal multiparametric magnetic resonance imaging (MRI) to characterize liver morphology and function in nine rhesus monkeys after exposure to Ebola virus (EBOV). Starting 5 days postexposure, MRI assessments of liver appearance, morphology, and size were consistently compatible with the presence of hepatic edema, inflammation, and congestion, leading to significant hepatomegaly at necropsy. MRI performed after injection of a hepatobiliary contrast agent demonstrated decreased liver signal on the day of euthanasia, suggesting progressive hepatocellular dysfunction and hepatic secretory impairment associated with EBOV infection. Importantly, MRI-assessed deterioration of biliary function was acute and progressed faster than changes in serum bilirubin concentrations. These findings suggest that longitudinal quantitative in vivo imaging may be a useful addition to standard biological assays to gain additional knowledge about organ pathophysiology in animal models of EVD. IMPORTANCE Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD), but the contribution of hepatic pathophysiology to EVD progression is not fully understood. Noninvasive medical imaging of liver structure and function in well-established animal models of disease may shed light on this important aspect of EVD. In this proof-of-concept study, we used longitudinal magnetic resonance imaging (MRI) to characterize liver abnormalities and dysfunction in rhesus monkeys exposed to Ebola virus. The results indicate that in vivo MRI may be used as a noninvasive readout of organ pathophysiology in EVD and may be used in future animal studies to further characterize organ-specific damage of this condition, in addition to standard biological assays.

Introduction: Niclosamide (Nc) is an FDA-approved anthelmintic drug that was recently identified in a drug repurposing screening to possess antiviral activity against SARS-CoV-2. However, due to the low solubility and permeability of Nc, its in vivo efficacy was limited by its poor oral absorption.Method: The current study evaluated a novel prodrug of Nc (PDN; NCATS-SM4705) in improving in vivo exposure of Nc and predicted pharmacokinetic profiles of PDN and Nc across different species. ADME properties of the prodrug were determined in humans, hamsters, and mice, while the pharmacokinetics (PK) of PDN were obtained in mice and hamsters. Concentrations of PDN and Nc in plasma and tissue homogenates were measured by UPLC-MS/MS. A physiologically based pharmacokinetic (PBPK) model was developed based on physicochemical properties, pharmacokinetic and tissue distribution data in mice, validated by the PK profiles in hamsters and applied to predict pharmacokinetic profiles in humans.Results: Following intravenous and oral administration of PDN in mice, the total plasma clearance (CLp) and volume of distribution at steady-state (Vdss) were 0.061–0.063 L/h and 0.28–0.31 L, respectively. PDN was converted to Nc in both liver and blood, improving the systemic exposure of Nc in mice and hamsters after oral administration. The PBPK model developed for PDN and in vivo formed Nc could adequately simulate plasma and tissue concentration-time profiles in mice and plasma profiles in hamsters. The predicted human CLp/F and Vdss/F after an oral dose were 2.1 L/h/kg and 15 L/kg for the prodrug respectively. The predicted Nc concentrations in human plasma and lung suggest that a TID dose of 300 mg PDN would provide Nc lung concentrations at 8- to 60-fold higher than in vitro IC50 against SARS-CoV-2 reported in cell assays.Conclusion: In conclusion, the novel prodrug PDN can be efficiently converted to Nc in vivo and improves the systemic exposure of Nc in mice after oral administration. The developed PBPK model adequately depicts the mouse and hamster pharmacokinetic and tissue distribution profiles and highlights its potential application in the prediction of human pharmacokinetic profiles.

The German Bight located within the central North Sea is a hydro- and morphodynamically highly complex system of estuaries, barrier islands, and part of the world's largest coherent tidal flats, the Wadden Sea. To identify and understand challenges faced by coastal stakeholders, such as harbor operators or governmental agencies, to maintain waterways and employ numerical models for further analyses, it is imperative to have a consistent database for both bathymetry and surface sedimentology. Current commercial and public data products are insufficient in spatial and temporal resolution and coverage for recent analysis methods. Thus, this first part of a two-part publication series of the German joint project EasyGSH-DB describes annual bathymetric digital terrain models at a 10 m gridded resolution for the German North Sea coast and German Bight from 1996 to 2016 (Sievers et al., 2020a, https://doi.org/10.48437/02.2020.K2.7000.0001), as well as surface sedimentological models of discretized cumulative grain size distribution functions for 1996, 2006, and 2016 on 100 m grids (Sievers et al., 2020b, https://doi.org/10.48437/02.2020.K2.7000.0005). Furthermore, basic morphodynamic and sedimentological processing analyses, such as the estimation of, for example, bathymetric stability or surface maps of sedimentological parameters, are provided (Sievers et al., 2020a, b, see respective download links).

Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62–78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

Marine spatial planning requires reliable data for, e.g., the design of coastal structures, research, or sea level rise adaptation. This task is particularly ambiguous in the German Bight (North Sea, Europe) because a compromise must be found between economic interests and biodiversity since the environmental status is monitored closely by the European Union. For this reason, we have set up an open-access, integrated marine data collection for the period from 1996 to 2015. It provides bathymetry, surface sediments, tidal dynamics, salinity, and waves for the German Bight and is of interest to stakeholders in science, government, and the economy. This part of a two-part publication presents data from numerical hindcast simulations for sea surface elevation, depth-averaged current velocity, bottom shear stress, depth-averaged salinity, wave parameters, and wave spectra. As an improvement to existing data collections, our data represent the variability in the bathymetry by using annually updated model topographies. Moreover, we provide data at a high temporal and spatial resolution (Hagen et al., 2020b); i.e., numerical model results are gridded to 1000 m at 20 min intervals (https://doi.org/10.48437/02.2020.K2.7000.0004). Tidal characteristic values (Hagen et al., 2020a), such as tidal range or ebb current velocity, are computed based on numerical modeling results (https://doi.org/10.48437/02.2020.K2.7000.0003). Therefore, this integrated marine data collection supports the work of coastal stakeholders and scientists, which ranges from developing detailed coastal models to handling complex natural-habitat problems or designing coastal structures.

The neurological effects of Ebola disease in the acute stage are not well-described. Here, the authors use longitudinal in vivo neuroimaging and immunohistochemistry to assess pathological changes in the central nervous system in a non-human primate model of Ebola virus infection.

A broadband EPR spectrometer is an instrument that can be tuned to many microwave frequencies over several octaves. Its purpose is the collection of multi-frequency data, whose global analysis affords interpretation of complex spectra by means of deconvolution of frequency-dependent and frequency-independent interaction terms. Such spectra are commonly encountered, for example, from transition-metal complexes and metalloproteins. In a series of previous papers, I have described the development of broadband EPR spectrometers around a vector network analyzer. The present study reports on my endeavor to start from an existing X-band spectrometer and to reversibly re-build it into a broadband machine, in a quest to drastically reduce design effort, building costs, and operational complexity, thus bringing broadband EPR within easy reach of a wide range of researchers.

The development of tungsten biochemistry is sketched from the viewpoint of personal participation. Following its identification as a bio-element, a catalogue of genes, enzymes, and reactions was built up. EPR spectroscopic monitoring of redox states was, and remains, a prominent tool in attempts to understand tungstopterin-based catalysis. A paucity of pre-steady-state data remains a hindrance to overcome to this day. Tungstate transport systems have been characterized and found to be very specific for W over Mo. Additional selectivity is presented by the biosynthetic machinery for tungstopterin enzymes. Metallomics analysis of hyperthermophilic archaeon Pyrococcus furiosus indicates a comprehensive inventory of tungsten proteins.

Background: Patients affected by different types of autoimmune diseases, including common conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and cellular immunity induced by infection and/or upon vaccination is affected by immunosuppressants. Methods: The dynamics of cellular immune reactivation upon vaccination of SARS-CoV-2 experienced MS patients treated with the humanized anti-CD20 monoclonal antibody ocrelizumab (OCR) and RA patients treated with methotrexate (MTX) monotherapy were analyzed at great depth via high-dimensional flow cytometry of whole blood samples upon vaccination with the SARS-CoV-2 mRNA-1273 (Moderna) vaccine. Longitudinal B and T cell immune responses were compared to SARS-CoV-2 experienced healthy controls (HCs) before and 7 days after the first and second vaccination. Results: OCR-treated MS patients exhibit a preserved recall response of CD8+ T central memory cells following first vaccination compared to HCs and a similar CD4+ circulating T follicular helper 1 and T helper 1 dynamics, whereas humoral and B cell responses were strongly impaired resulting in absence of SARS-CoV-2-specific humoral immunity. MTX treatment significantly delayed antibody levels and B reactivation following the first vaccination, including sustained inhibition of overall reactivation marker dynamics of the responding CD4+ and CD8+ T cells. Conclusions: Together, these findings indicate that SARS-CoV-2 experienced MS-OCR patients may still benefit from vaccination by inducing a broad CD8+ T cell response which has been associated with milder disease outcome. The delayed vaccine-induced IgG kinetics in RA-MTX patients indicate an increased risk after the first vaccination, which might require additional shielding or alternative strategies such as treatment interruptions in vulnerable patients. Funding: This research project was supported by ZonMw (The Netherlands Organization for Health Research and Development, #10430072010007), the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532 and #860003), the European Commission (SUPPORT-E, #101015756) and by PPOC (#20_21 L2506), the NHMRC Leadership Investigator Grant (#1173871).

The application of model-informed drug discovery and development (MID3) approaches in the early stages of drug discovery can help determine feasibility of drugging a target, prioritize between targets, or define optimal drug properties for a target product profile (TPP). However, applying MID3 in early discovery can be challenging due to the lack of pharmacokinetic (PK) and pharmacodynamic (PD) data at this stage. Early Feasibility Assessment (EFA) is the application of mechanistic PKPD models, built from first principles, and parameterized by data that is readily available early in drug discovery to make effective dose predictions. This manuscript demonstrates the ability of EFA to make accurate predictions of clinical effective doses for nine approved biotherapeutics and outlines the potential of extending this approach to novel therapeutics to impact early drug discovery decisions.

Abstract Survival is a key demographic component that often varies as a result of human activities such as recreational harvest. Detailed understanding of seasonal variation in mortality patterns and the role of various risk factors is thus crucial for understanding the link between environmental variation and wildlife population dynamics and to design sustainable harvest management systems. Here, we report from a detailed seasonal and cause‐specific decomposition of mortality risks in willow ptarmigan (Lagopus lagopus) in central Norway. The analyses are based on radio‐collared (n = 188) birds that were monitored across all seasons, and we used time‐to‐event models for competing risks to estimate mortality patterns. Overall, annual survival was estimated at 0.43 (SE: 0.04), with no distinct difference among years (2015/16 to 2018/19) or between sexes. Analysis of mortality risk factors revealed that on the annual basis, the risk of harvest mortality was lower than the risk of dying from natural causes. However, during the autumn harvest season (September–November), survival was low and the dominating cause of mortality was harvest. During winter (December–March) and spring seasons (April–May), survival was in general high and did not vary between males and females. However, during the spring season, juveniles (i.e., birds born last year) of both sexes had lower survival than adults, potentially because they are more prone to predation. During the summer season (June–August), females experienced a higher hazard than males, underlining the greater parental investment of females during egg production, incubation, and chick rearing compared to males. Our analyses provide unique insight into demographic and seasonal patterns in willow ptarmigan mortality risks in a harvested population and revealed a complex interplay across seasons, risk factors, and demographic classes. Such insight is valuable when designing sustainable management plans in a world undergoing massive environmental perturbations.

Abstract Objectives High‐magnitude CD8+ T cell responses are associated with mild COVID‐19 disease; however, the underlying characteristics that define CD8+ T cell‐mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope‐specific CD8+ T cells remain largely unexplored and are essential for the development of next‐generation broad‐protective vaccines. This study identified a broad spectrum of conserved SARS‐CoV‐2 CD8+ T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS‐CoV‐2 infection. Methods CD8+ T cells from 51 convalescent COVID‐19 donors were analysed for their ability to recognise 133 predicted and previously described SARS‐CoV‐2‐derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in‐depth ex vivo profiling of CD8+ T cell responses at quantitative and phenotypic levels. Results A comprehensive panel of 49 mostly conserved SARS‐CoV‐2‐specific CD8+ T cell epitopes, including five newly identified low‐magnitude epitopes, was established. We confirmed the immunodominance of HLA‐A*01:01/ORF1ab1637–1646 and B*07:02/N105–113 and identified B*35:01/N325–333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N361–369 and A*02:01/S269–277, depended on the donors' HLA‐I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID‐19 donors. Conclusion SARS‐CoV‐2 infection induces a predominant CD8+ T memory response directed against a broad spectrum of conserved SARS‐CoV‐2 epitopes, which likely contributes to long‐term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross‐reactive immune response, which could aid future vaccine strategies.

In a world undergoing massive declines in the distribution and abundance of many wildlife species, documenting basic ecological characteristics is often needed to be able to understand and potentially mitigate current and future pressures. Species living in alpine areas might be particularly vulnerable to climate change, in part because they are less likely to be able to migrate to new suitable areas. Here we report from a two year case study of rock ptarmigan (Lagopus muta) in central Scandinavia. Ptarmigan were captured in winter (n = 84), and fitted with radio collars. We estimated the natural survival from mid-winter to late summer to be 0.55 (SE: 0.07), with no distinct differences between juveniles and adults, sex, or between the two years. Natural survival through late winter (February–April) was estimated at 0.77 (SE: 0.05), survival trough breeding season May–July at 0.65 (SE: 0.08), and harvest mortality through the February winter harvest at 9% (SE: 3%). Moreover, we documented large scale movement from the wintering grounds before the breeding season in the spring. The longest recorded movement was 79.5 km, and the mean distance from the capture site for birds still in the sample in May–July was 20.3 (SD: 18) km. We discuss the implications of the results in terms of ongoing climate change.

There is currently no approved vaccine for Lassa fever virus (LASV). Here, Abreu-Mota et al. develop an inactivated, adjuvanted vaccine candidate expressing LASV glycoprotein (GPC) in a rabies virus vector, and show that non-neutralizing LASV GPC-specific antibodies are a major mechanism of protection.

Hemorrhagic smallpox, caused by variola virus (VARV), was a rare but nearly 100% lethal human disease manifestation. Hemorrhagic smallpox is frequently characterized by secondary bacterial infection, coagulopathy, and myocardial and subendocardial hemorrhages. Previous experiments have demonstrated that intravenous (IV) cowpox virus (CPXV) exposure of macaques mimics human hemorrhagic smallpox. The goal of this experiment was to further understand the onset, nature, and severity of cardiac pathology and how it may contribute to disease. The findings support an acute late-stage myocarditis with lymphohistiocytic infiltrates in the CPXV model of hemorrhagic smallpox.

Cobalamin-containing reductive dehalogenases from organohalide-respiring bacteria play a key role in the degradation of halogenated organic compounds. Here the authors proposed a catalytic mechanism for dehalogenation that relies on a long-range electron transfer from the cobalt in the centre of the enzyme’s cofactor to the substrate.

ABSTRACT Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease. IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus.

The branchiomeric skeletal muscles co-evolved with new chambers of the heart to enable predatory feeding in chordates. These co-evolved tissues develop from a common population in anterior splanchnic mesoderm, referred to as cardiopharyngeal mesoderm (CPM). The regulation and development of CPM are poorly understood. We describe an embryonic stem cell-based system in which MESP1 drives a PDGFRA+ population with dual cardiac and skeletal muscle differentiation potential, and gene expression resembling CPM. Using this system, we investigate the regulation of these bipotent progenitors, and find that cardiac specification is governed by an antagonistic TGFβ-BMP axis, while skeletal muscle specification is enhanced by Rho kinase inhibition. We define transcriptional signatures of the first committed CPM-derived cardiac and skeletal myogenic progenitors, and discover surface markers to distinguish cardiac (PODXL+) from the skeletal muscle (CDH4+) CPM derivatives. These tools open an accessible window on this developmentally and evolutionarily important population.

The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 μs. The instrument employs a micro-mixer with a mixing time of 2.7 μs integrated with a 30 mm long flow-cell of 109 μm optical path length constructed from two parallel sheets of silver foil; it produces ultraviolet-visible spectra that are linear in absorbance up to 3.5 with a spectral resolution of 0.4 nm. Each spectrum corresponds to a different reaction time determined by the distance from the mixer outlet, and by the fluid flow rate. The reaction progress is monitored in steps of 0.35 μs for a total duration of ~600 μs. As a proof of principle the instrument was used to study spontaneous protein refolding of pH-denatured cytochrome c. Three folding intermediates were determined: after a novel, extremely rapid initial phase with τ = 4.7 μs, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 μs and 345 μs to a coordinatively saturated low-spin iron form in quasi steady state. The time-resolution specifications of our spectrometer for the first time open up the general possibility for comparison of real data and molecular dynamics calculations of biomacromolecules on overlapping time scales.