To examine the association between practice percentage coding of chronic kidney disease (CKD) in primary care with risk of subsequent hospitalisations and death.Retrospective cohort study using linked electronic healthcare records.637 general practitioner (GP) practices in England.2, separated by at least 90 days, excluding those with coded initiation of renal replacement therapy.167 208 patients with CKD stages 3–5 identified by 2 measures of estimated glomerular filtration rate <60 mL/min/1.73 mHospitalisations with cardiovascular (CV) events, heart failure (HF), acute kidney injury (AKI) and all-cause mortalityParticipants were followed for (median) 3.8 years for hospital outcomes and 4.3 years for deaths. Rates of hospitalisations with CV events and HF were lower in practices with higher percentage CKD coding. Trends of a small reduction in AKI but no substantial change in rate of deaths were also observed as CKD coding increased. Compared with patients in the median performing practice (74% coded), patients in practices coding 55% of CKD cases had a higher rate of CV hospitalisations (HR 1.061 (95% CI 1.015 to 1.109)) and HF hospitalisations (HR 1.097 (95% CI 1.013 to 1.187)) and patients in practices coding 88% of CKD cases had a reduced rate of CV hospitalisations (HR 0.957 (95% CI 0.920 to 0.996)) and HF hospitalisations (HR 0.918 (95% CI 0.855 to 0.985)). We estimate that 9.0% of CV hospitalisations and 16.0% of HF hospitalisations could be prevented by improving practice CKD coding from 55% to 88%. Prescription of antihypertensives was the most dominant predictor of a reduction in hospitalisation rates for patients with CKD, followed by albuminuria testing and use of statins.Higher levels of CKD coding by GP practices were associated with lower rates of CV and HF events, which may be driven by increased use of antihypertensives and regular albuminuria testing, although residual confounding cannot be ruled out. A large database of 167 208 patients with biochemical evidence of chronic kidney disease (CKD) was used for analysis, covering 637 general practitioner practices in England who volunteered to participate in an audit of care and which were representative of the general population in terms of age and sex.Risk of confounding due to patient characteristics is reduced by studying the association between practice level (rather than patient level) CKD coding and patient-level outcomes, where practice casemix is not expected to differ with practice coding rates.Practice behaviours associated with CKD coding performance that are not believed to occur as a consequence of CKD coding may confound associations but were adjusted for as far as possible.Average duration of follow-up between assessment of practice coding performance and end of data collection for outcomes was limited to approximately 4 years; longer term effects of CKD coding may, therefore, not be captured in this study.Chronic kidney disease (CKD) is a growing public health problem.In the UK, computer systems used by general practitioner (GP) practices allow electronic coding of patient clinical information, enabling consistent and specific recording and ease of access to coded data.11The National Chronic Kidney Disease Audit (NCKDA) conducted in England and Wales in 2015–2016 found that approximately 30% of biochemically confirmed CKD stages 3–5 cases were not given an appropriate CKD code in primary care electronic healthcare records (EHRs).In attempt to overcome the issue of unmeasured confounding experienced in original analyses of the NCKDA,We carried out a retrospective cohort study using routinely collected EHRs.The NCKDA database holds selected data from the EHRs of 695 GP practices in England and was used to identify a cohort of patients with CKD for analysis and to define exposure variables. Data extraction ranged from March 2015 to July 2016 as practices were gradually recruited into the audit, with the majority of practices recruited by July 2015. In brief, the audit is a snapshot of care at the time point of audit data extraction. Details of the audit and data collection strategy are specified elsewhere.2 separated by at least 90 days, with the most recent measure recorded within the last 2 years prior to data extraction and excluding any patients with coded initiation of renal replacement therapy.The analysis cohort included all adult patients in the NCKDA database extracted from eligible GP practices in England and with biochemical evidence of CKD (hereafter referred to simply as ‘CKD’ and/or ‘confirmed CKD’), defined as at least two records of estimated glomerular filtration rate (eGFR) <60 mL/min/mAt the time of data extractions (2015–2016), Read codes were used to electronically record patient findings in GP computer systems.Practice CKD coding performance was characterised as the percentage of patients with CKD in a practice with a CKD stages 3–5 Read code, hereafter referred to as practice CKD coding (performance) or percent coded CKD. Practice CKD coding performance was defined at practice data extraction, which marked the index date for commencement of follow-up for outcomes.Practices with fewer than 50 total CKD cases were excluded from analysis due to anticipated excess noise in measurement of the primary exposure.Four outcomes of interest were studied: (1) hospitalisation for CV events, (2) hospitalisation for heart failure (HF), (3) hospitalisation with AKI and (4) all-cause mortality, defined in 10.1136/bmjopen-2022-064513.supp1Supplementary dataFeatures of a practice that may confound the association between practice CKD coding and patient adverse outcomes were defined and categorised as: those reflecting overall practice risk profile; practice testing behaviours for CKD; and characteristics of the identified practice CKD population. Practice features that may improve CKD outcomes, some of which may lie on the causal pathway between practice CKD coding and patient adverse outcomes, were also defined. Practice percentage variables were defined by summing the number of patients meeting relevant risk factor criteria in each practice and dividing by relevant practice denominators. Practice list size data were used to determine size of the adult population in each practice.Patient-level risk factors identified in NCKDA data  were defined based on presence of any relevant Read code in the EHR prior to data extraction and included:Diabetes—any diabetes code not superseded by a diabetes resolved codeHypertension—any hypertension codeCVD—any CVD codeCKD stages 3b-5—confirmed CKD and latest eGFR <45Statin use—any statin prescription or contraindication codeAntihypertensive use—any prescription or contraindication code for angiotensin-converting enzyme inhibitors (ACEi) or angiotensin-II receptor blockers (ARBs)Blood pressure (BP) targets met—last BP within target range within last year before data extraction: systolic blood pressure (SBP) <130 mm Hg and diastolic blood pressure (DBP) <80 mm Hg in those with diabetes or last urine albumin to creatinine ratio (ACR) ≥70 mg/mmol or last protein to creatinine ratio (PCR) ≥100 mg/mmol; SBP <140 mm Hg and DBP <90 mm Hg in all other patientsInfluenza vaccination—any influenza vaccination code in the last yearPneumococcal vaccination—any pneumococcal vaccination code in the last 5 yearsAdditional patient-level risk factors not available in NCKDA data were defined using HES data and included:Recent chronic obstructive pulmonary disease (COPD) admission—admission in last 3 years prior to (NCKDA) practice data extraction with a COPD ICD-10 code (J44) as primary diagnosisRecent cancer admission—admission in last 3 years prior to (NCKDA) practice data extraction with a cancer ICD-10 code  as primary diagnosisGPs’ awareness on how to identify CKD may depend on the overall burden of conditions associated with CKD in their practice. Practice prevalence of diabetes, hypertension and CVD were determined by summing the number of adult patients meeting patient-level comorbidity definitions, with adult population size as denominator. Practice list size data stratified by age and sex were used to determine mean practice age and percent of adults that were male. Practice deprivation was summarised using the median rank of the Index of Multiple Deprivation (IMD) score among all patients extracted from the GP practice .Testing behaviours which may impact the patient vintage  and the types of patients with CKD selected for analysis were also defined within practices, including percentage of diabetes patients with a GFR test in the last year, percentage of patients with CKD with a GFR test in the last year and percentage of the practice adult population with confirmed CKD.Underlying practice morbidity and testing behaviours may impact the types of patients with CKD detected and therefore included in analysis. Percent CKD stages 3b-5, percent recent COPD admission and percent recent cancer admission were defined using patient-level risk factor definitions, with number of total CKD cases as denominator.Practice behaviours expected to be related to CKD coding, some of which may be on the causal pathway from CKD to improved outcomes were defined as: percent usage of ACEi/ARBs in hypertension, percent usage of statins in diabetes, percent usage of statins in CVD, percent meeting BP target in last year in CKD, percent ACR/PCR test in last year in CKD, percent influenza vaccination in last year in CKD and percent pneumococcus vaccination in past 5 years in CKD stages 4–5. Practice behaviour variables were dichotomised at the median value.Baseline characteristics of the study population were summarised by sextile of practice percent coded CKD to determine balance in patient characteristics according to primary exposure. Practice characteristics were also summarised by sextile of practice CKD coding to identify any associations with other practice characteristics.Cox proportional hazards regression (time to first event) was used to evaluate the association between practice CKD coding and each of the four patient outcomes. Hospitalisation outcomes were censored for death. A 5 knot spline was used for the primary exposure, providing flexibility to demonstrate the nature of association between practice coding and outcomes across the spectrum of practice CKD coding performance, without overfitting. The following adjustments for practice characteristics (included as continuous covariates) were carried out sequentially:Model 1: adjusted for practice characteristics reflecting overall practice risk profile Model 2: adjusted for practice characteristics reflecting overall practice risk profile, practice testing behaviours and practice characteristics of the detected CKD population .Adjusted HR curves for outcomes with 95% CIs were plotted across the spectrum of practice CKD coding, compared with average (median) practice CKD coding. Attributable fractions for the number of (first) events preventable by the median follow-up time among patients with CKD in practices at lower coding levels  if such practices instead coded at higher coding levels  were estimated under assumption of causality following adjusted Cox regression (model 2), detailed in Additional analyses with a single linear continuous covariate for percent coded CKD were carried out after visual inspection of an approximately linear relationship for some model 2HR curves, allowing a more convenient clinical interpretation. These models were restricted to sextiles 2–5 of practice coding only (representing the 67% of most averagely performing practices) where linear trends were most apparent. Descriptive likelihood ratio tests were used to assess improvement in model fit using spline terms vs a single linear covariate.Subgroup analyses were carried out by diabetes status and by CKD severity . Practice percent coded CKD was recalculated within each subgroup for analysis, since coding behaviour differed substantially between subgroups.Further Cox regression analyses aimed to identify practice behaviours associated with improvements in all four patient outcomes, with adjustment for all (model 2) confounders as well as practice behaviours that may improve CKD outcomes. Practice CKD coding covariates were excluded from analysis to identify practice factors most predictive of outcomes, regardless of CKD coding performance and not conditional on CKD coding.Kidney Care UK supported the research questions, grant applications and related record linkage applications of the NCKDA. After NCKDA discontinuation, Kidney Care UK helped with ethics and section 251 permissions to maintain database access for research purposes. A patient representative (Fiona Loud) was involved in the NCKDA from inception, is a co-author and critically reviewed content of this paper.Of 695 practices in England captured in the NCKDA database, 637 practices (92%) met criteria for analysis (at least 50 CKD cases), covering 99% of all patients with CKD from the original database (n=167 208) . CKD codStudy population characteristics were generally well balanced between sextiles of practice CKD coding . There wMedian practice percent coded CKD was 73.9% in the overall CKD population. It was higher in CKD stages 3b-5 (87.9%) than CKD stage 3a (64.8%), and higher in those with diabetes (78.6%) than those without diabetes (71.4%). Coding performance was more variable in early stage CKD and non-diabetic CKD .Practice characteristics were generally well balanced in coding sextiles 2–5 . At the Of 167 208 patients with CKD identified from the NCKDA database, after national linkage we found that 563 deaths had occurred but had not been reported on primary care systems at date of data extraction, leaving 166 645 eligible for outcomes analysis. Median follow-up duration was 3.8 years (range 1 day to 3.9 years) for HES outcomes and 4.3 years (range 1 day to 4.4 years) for mortality, with no meaningful differences in follow-up between sextiles. Crude event rates by sextile are shown in There are strong trends, particularly among sextiles 2–5 (55%–88% coded), of reduced rates of outcomes with improved practice CKD coding in fully adjusted analyses (model 2). Compared with patients in the averagely performing practice (74% coded), patients in practices coding only 55% of CKD cases had a significantly higher rate of CV hospitalisations (HR 1.061 (95% CI 1.015 to 1.109)) and HF hospitalisations (HR 1.097 (95% CI 1.013 to 1.187)). Patients in practices coding 88% of CKD cases had a significantly reduced rate of CV hospitalisations (HR 0.957 (95% CI 0.920 to 0.996)) and HF hospitalisations (HR 0.918 (95% CI 0.855 to 0.985)), compared with the averagely performing practice. The percentage of preventable events over a period of 3.8 years (median follow-up time) for an improvement in practice coding from 55% to 88% of CKD cases (attributable fraction) was 9.0% for first CV hospitalisations and 16.0% for first HF hospitalisations, under assumption of causality after Cox modelling (model 2). Trends of a small reduction in AKI but no substantial change in rate of deaths were also observed as CKD coding increased.  are shown in Subgroup analyses showed a steeper reduction in CV hospitalisations and HF hospitalisations with increasing practice coding among CKD stages 3b–5 than in CKD stage 3 both in spline regression analyses and those assuming linear effects of practice CKD coding .Analysis of the association between practice behaviours and CV hospitalisations adjusted for all confounders showed a significant reduction in rate of CV hospitalisations for patients with CKD belonging to practices with greater than average usage (median 76.6%) of ACEi/ARBs in hypertension compared with practices with lower than average usage (HR 0.956 (95% CI 0.929 to 0.983)) . PracticHigher levels of practice CKD coding were associated with lower rates of hospitalisation for CV and HF events among patients with confirmed CKD, after adjusting for practice characteristics. Reductions in hospitalisation rates were strongest in CKD stages 3b-5, although greatest opportunities for improvement are in CKD stage 3a where practice variation in coding was much wider. There was no difference in death rates according to practice CKD coding . Findings were limited by duration of follow-up with longer-term benefits of CKD coding not yet apparent. Practice behaviours associated with CKD coding including usage of ACEi/ARB therapy and ACR/PCR testing were independent predictors of reduction in hospitalisation rates.2 more than 3 months apart are not recognised by GPs as having CKD because of concerns of overdiagnosis, for example, in elderly patients without hypertension or in patients recovering from AKI, despite these patients meeting the accepted definition of CKD. Furthermore, numerous studies have demonstrated disparities in CKD coding efforts with younger patients, those from deprived backgrounds, and ethnic minorities being less commonly coded than their counterparts,There are very limited studies looking at the impact of recognition and diagnostic coding of CKD in primary care as many health systems use coded disease as opposed to laboratory records to identify patients. It is possible that some patients with 2 eGFR measures <60 mL/min/1.73 mA key strength of this study is the large sample size, including data from 167 208 patients with CKD. Data were extracted from GP practices in England with a similar age–sex distribution to the whole population, so findings are likely to be generalisable to the wider population. By studying the association between practice-level CKD coding and patient-level outcomes, we were able to eliminate a lot of confounding due to individual patient characteristics that would be present in a conventional study design using patient-level exposure with unmeasured confounders. This was demonstrated by the balanced risk profile in patient characteristics observed across sextiles . This is a major benefit over original analyses of the NCKDAA potential weakness is that included practices had volunteered to participate in an audit of care. CKD coding may have been higher in recruited practices than the general population which may have impacted on estimated strengths of associations; benefits of coding in the wider practice population may be larger than estimated. Risk factor evaluation mostly relied on comorbidity coding and it is not clear whether small differences in risk factor prevalence reflected true morbidity or GP behaviour. Assessment of eligibility of patients for analysis also relied on availability of repeat creatinine tests over time, which may depend on patient risk, and earlier stage CKD cases or more severe cases managed solely in secondary care may be disproportionately missing. Nevertheless, this identified CKD population may stand to benefit most imminently from improvements in primary care, assuming GPs target further coding efforts to patients already identified as at risk and with creatinine test results compatible with CKD. While there was a small signal of more frequent creatinine testing with increasing practice CKD coding, this was only at the extremes, and distribution of CKD severity appeared generally very well balanced across practice coding sextiles. Practice characteristics were analysed differently depending on whether they were likely to confound analyses or lie on the causal pathway, however, we could not verify if our assumptions were reasonable, and misspecification could affect reliability of conclusions. For example, practice management of hypertension with ACEi/ARB therapy may plausibly confound analyses  or lie on the causal pathway (if management of hypertension occurs as a consequence of CKD coding). Our findings for AKI are likely affected by outcome misclassification as hospital codes were used to detect AKI events, which may have led to underestimation of the number of events and lack of power to detect an association. We did not have enough dialysis events to allow evaluation of the impact of practice coding on outcomes. These data precede the use of SLGT2-inhibitor drug treatment in HF and albuminuric kidney disease in UK primary care.Rates of CV and HF events were lower for patients belonging to practices coding more CKD, supporting the argument that CKD coding in primary care may contribute to improvement in patient outcomes. While the presence of unmeasured confounding cannot be ruled out, this is in agreement with other studies conducted in this setting.


Do patients with tuberculosis (TB) have differential preferences for strategies to improve TB diagnostic services?In this cross-sectional study of 326 patients newly diagnosed with TB in Lusaka, Zambia, 3 groups with distinct preferences for different TB diagnostic service enhancements were identified. Groups 1 and 2 were far more likely to report waiting at least 4 weeks after symptom onset to seek care, and they demonstrated strong preferences for facilities providing same-day TB test results, financial incentives, and greater privacy.Tailored strategies that include features most preferred by persons with undiagnosed TB, who are likely to delay care seeking, have the potential to accelerate TB diagnosis and care engagement. This cross-sectional study of patients with tuberculosis in Zambia examines preferences for improving tuberculosis diagnostic services. Delayed engagement in tuberculosis (TB) services is associated with ongoing transmission and poor clinical outcomes.To assess whether patients with TB have differential preferences for strategies to improve the public health reach of TB diagnostic services.A cross-sectional study was undertaken in which a discrete choice experiment (DCE) was administered between September 18, 2019, and January 17, 2020, to 401 adults (>18 years of age) with microbiologically confirmed TB in Lusaka, Zambia. The DCE had 7 attributes with 2 to 3 levels per attribute related to TB service enhancements. Latent class analysis was used to identify segments of participants with unique preferences. Multiscenario simulations were used to estimate shares of preferences for different TB service improvement strategies.The main outcomes were patient preference archetypes and estimated shares of preferences for different strategies to improve TB diagnostic services. Collected data were analyzed between January 3, 2022, to July 2, 2022.P < .001). Strategies to improve TB diagnostic services most preferred by all participants were same-day TB test results alone  and combined with a small financial testing incentive , shortened wait times , or greater privacy . However, the most preferred service improvement strategies differed substantially by group.Among 326 adults with TB , 3 groups with distinct preferences for TB service improvements were identified. Group 1 (192 participants [58.9%]) preferred a facility that offered same-day TB test results, shorter wait times, and financial incentives for testing. Group 2 (83 participants [25.4%]) preferred a facility that provided same-day TB results, had greater privacy, and was closer to home. Group 3 (51 participants [15.6%]) had no strong preferences for service improvements and had negative preferences for receiving telephone-based TB test results. Groups 1 and 2 were more likely to report at least a 4-week delay in seeking health care for their current TB episode compared with group 3 (29 [51.3%] in group 1, 95 [35.8%] in group 2, and 10 [19.6%] in group 3; In this study, patients with TB had heterogenous preferences for TB diagnostic service improvements associated with differential health care–seeking behavior. Tailored strategies that incorporate features most valued by persons with undiagnosed TB, including same-day results, financial incentives, and greater privacy, may optimize reach by overcoming key barriers to timely TB care engagement. Collectively, delayed and missed TB diagnoses contribute to morbidity, catastrophic economic costs, and mortality among socioeconomically vulnerable individuals and propagate continued transmission, undermining progress toward global control TB targets.4Missed tuberculosis (TB) diagnoses represent the largest gap in the global TB care cascade.6 In most settings, patient-related delays in care seeking after symptom onset is the primary driver of delays in the overall TB care pathway; such delays are therefore an important area of focus for future TB interventions and program initiatives.8 To close existing gaps in TB detection and expedite care engagement, it is crucial that interventions and implementation strategies to reach persons with undiagnosed TB directly account for their differential barriers to engaging in TB services and their unique preferences by including them as stakeholders in the design process.Persons with TB living in high-burden, limited-resource settings may face several barriers at each step of their TB care journey, resulting in prolonged and complex care pathways.8 Overall, same-day TB test results, facilities with shorter wait times, and services closer to home were single-component strategies strongly preferred by most patients; no differences by HIV status or sex were found. However, exploring heterogeneity of preferences is often undertaken to optimize market shares  in commercial applications, and similar approaches could be applied to also enhance public health reach. It is unknown whether TB diagnosis and care engagement strategies may need to be tailored to appeal to distinct groups of patients with unique behaviors and preferences, including which strategies may be most preferred among persons with undiagnosed TB who are likely to substantially delay initial care engagement. Therefore, we undertook a latent class analysis of a DCE and simulated the predicted preferences of distinct TB patient preference groups for the hypothetical implementation of different single-component and multicomponent strategies to improve TB diagnostic services.We previously reported the results of a discrete choice experiment (DCE) among patients with TB in Lusaka, Zambia, evaluating their preferences for implementing possible TB service improvement strategies that could accelerate TB diagnosis and care engagement by overcoming barriers to seeking and accessing health care.8 Individuals eligible for study inclusion were those 18 years or older, who had microbiologically confirmed TB, who had their TB episode classified as a new or relapse case, and who began anti-TB therapy within the prior 2 weeks.8 Ethical approval was obtained from the University of Zambia Biomedical Research Ethics Committee and the institutional review board of the University of California, San Francisco. All participants provided written informed consent in their preferred language. This study is reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.9A prospective cross-sectional study of 401 patients with TB was undertaken between September 18, 2019, and January 17, 2020, at 2 public health facilities in Lusaka, Zambia.8 The DCE was developed to determine patient preferences for improving characteristics of TB diagnostic services to inform the design of future strategies that could facilitate improved TB care engagement. The development and design process has previously been described in detail.8 The DCE attributes and levels were based on a review of the literature and refined through discussions with key stakeholders.10 Seven attributes with up to 3 attribute levels were included in the final design  of attribute levels, overall and within each preference group  and Stata software, version 17.0 (StataCorp LLC). First, data cleaning was undertaken to improve the quality of individual-level data  and HIV positive (107 [56.3%] vs 31 [27.4%] in group 2 and 20 [39.2%] in group 3) . Group 3P < .001). Furthermore, 26 (14.0%) of those in group 1 and 11 (13.6%) of those in group 2 had prolonged health care–seeking delays of 8 weeks or more compared with 2 (3.9%) of those in group 3, although this finding did not reach statistical significance (P = .13).Large differences were found in health care–seeking behaviors according to preference groups . Groups Participants in group 1 were more likely to report that no one influenced their health decisions (81 [42.2%]) compared with groups 2 (21 [25.6%]) and 3 (4 [7.8%]). Among those reporting that certain persons influenced their health decisions, family members were the most influential (205 [93.6%]) and did not differ across groups , a strategy of providing diagnostic services closer to where participants lived substantially improved shares of preferences for all 3 preference groups  there are unique archetypes of patients with TB that cannot be predicted by demographic characteristics alone; (2) not all patients with TB require the availability of enhanced features or strategies to seek care for their symptoms in a timely manner ; and (3) to overcome their unique barriers and reduce the time to initial health care seeking, implementation of tailored care engagement strategies may be required to reach and appeal to different archetypes of persons with undiagnosed TB who are more likely to have prolonged delays in seeking health care.21 The availability of convenient, same-day test results was highly preferred among participants in groups 1 and 2. However, the feasibility of delivering same-day TB test results at health care facilities in many high-burden settings is currently challenged by the limitations of available diagnostic tools  and their resource requirements.23 Investments should be made to develop accurate, point-of-care TB triage and diagnostic tools that can be scaled up in community and lower-level health care settings where persons with undiagnosed TB may first present.26 We also found that provision of convenient, decentralized TB diagnostic services would be highly valued to individuals who voiced strong positive preferences for services closer to their home  and services that reduce their time waiting and being evaluated.28 Such participants also showed strong positive preferences for small financial incentives to undertake TB testing. Monetary rewards conditional on undertaking a positive health behavior are feasible, acceptable, and effective at improving care engagement for HIV in resource-limited settings31; they could similarly increase motivation to seek care for TB symptoms sooner by positively shifting the opportunity costs associated with seeking health care. Furthermore, group 1 and 2 archetypes also indicated strong preferences for a diagnostic strategy that incorporated greater privacy, where no one would recognize them. This finding suggests that TB-related stigma remains a key barrier that should be addressed as part of multicomponent strategies to accelerate TB care engagement and improve TB diagnosis.32Participants clustering into preference groups 1 (time is money) and 2  were much more likely to delay seeking initial health care for their symptoms compared with those in group 3 (status quo). Strategies that could reach, engage, and provide TB diagnosis among group 1 and 2 archetypes sooner may not only improve individual clinical outcomes and reduce the economic costs associated with prolonged care pathways but also substantially drive down community transmission by reducing periods of infectiousness.34 Religious leaders were also more likely to be cited as trusted persons who influenced the health-related decisions of groups 1 and 2. This finding suggests that trusted community members outside the health care system are important stakeholders to engage in the design and implementation of future TB control efforts to optimize reach.36In addition to differential preferences for TB diagnostic service improvements, group 1 and 2 archetypes had different trusted health influences and sources of information. Although family members were the most trusted source of health information across the 3 groups (>90%), friends were more likely to directly influence the health-related decision-making of groups 1 and 2. Thus, social network–based approaches in which patients with newly diagnosed TB directly reach out to their friends and family who are close contacts may represent a promising TB case–finding strategy.Group 3 comprised the smallest patient archetype, but most (>80%) of these participants reported seeking care within 4 weeks of TB symptom onset. This finding was somewhat surprising given that such persons were likely to have classically described TB risk factors, such as a history of smoking and alcohol use disorder, and may be expected to have poor health care–seeking behavior. However, participants in group 3 were much more likely to state that they personally knew at least 1 person who had previously been diagnosed with TB. Group 3 participants may therefore have had greater TB-related knowledge and TB risk perception, prompting earlier health care seeking after symptom onset. Although group 3 participants had weaker preferences for TB diagnostic service enhancements because they were the group most likely to seek care in a timely manner, it may be less important to account for their unique preferences, because usual care services appeared to largely meet their needs. Notably, we found that scaling up the delivery of TB test results by telephone would be completely unacceptable to this patient archetype and could unintentionally discourage already largely positive health care–seeking behaviors. It was not clear whether privacy concerns, lack of reliable access to a mobile telephone and/or data, or other factors drove strong negative preferences for this service feature among group 3 as well as group 2 participants. These findings require further exploration through qualitative research and provide a cautionary tale for intervention and program designers.37; however, health-related behaviors may be different among residents in rural settings, where geographic access and costs of seeking health care may serve as a stronger barrier to engaging in TB services. Furthermore, because surveys were administered in person with the help of a trained research assistant, some responses may have been subject to social desirability bias; however, DCEs are less prone to such bias given the indirect manner in which preferences are elicited.13 In addition, DCEs capture participants’ stated preferences under hypothetical conditions38; therefore, it cannot be known whether implementation of the most preferred strategies would translate into improved health care–seeking behaviors without additional studies conducted in community settings.40 Nonetheless, DCEs have been shown to have strong value for predicting actualized choices and represent an important tool for ensuring that health interventions and implementation strategies are aligned with the preferences of patients, community members, and other key stakeholders.41This study has some limitations. Patients with TB were enrolled from large facilities in Lusaka, a large urban population center that accounts for a large proportion of Zambia’s TB notificationsThis study revealed multiple archetypes of patients with TB in Lusaka, Zambia, defined by distinct preferences for implementing different strategies to improve TB diagnostic services. These archetypes also identified groups of patients who were substantially more likely to delay seeking health care as well as differential trusted sources of health information. The delivery of tailored multicomponent interventions and implementation strategies accounting for known health care preferences and preferred communication channels may optimize reach and acceptability and ultimately facilitate improved and expedited TB diagnosis and care engagement in settings with high TB burdens.


At present, less than 30% of researchers worldwide are women. Long-standing biases and gender stereotypes are discouraging girls and women away from science-related fields. Science and gender equality are, however, essential to ensure sustainable development as highlighted by UNESCO. In order to change traditional mindsets, gender equality must be promoted, stereotypes defeated, and girls and women should be encouraged to pursue careers as researcher. Therefore, Frontiers in Immunology is proud to offer this platform to promote the work of women scientists, across all fields of NK and Innate Lymphoid Cell Biology. The work presented here highlights the diversity of research performed across the entire breadth of NK and Innate Lymphoid Cell Biology research and presents advances in theory, experiment, and methodology with applications to compelling problems. Together, these 8 articles having women as first or last authors deal with distinct aspects of NK cell biology in health and disease.Three articles address different aspects of NK cell development.Wu et al., autologous transplantation of rhesus macaque hematopoietic stem cells (HSCs) was performed to track the clonal distribution of T, B, myeloid and NK cell populations in blood and across tissues, including liver, spleen, lung, and gastrointestinal (GI) tract. The experimental setting relies on clonal tracking via introduction of high diversity genetic barcodes together with GFP into HSCs to follow immune cell development. Interestingly, authors observed tissue resident (TR) NK cell clones specific for some tissues such as lung or GI tract as well as TR NK cell clones shared across liver and spleen, and distinct from other tissues. This experimental model could be further utilized to study clonal changes in circulating NK cells and in NK cells infiltrating tissues in response to infections, vaccines, or immunotherapy.In the manuscript by the research group directed from Persyn et al. studied the role of the transcription factor interferon regulatory factor (IRF)-2 in human NK cell differentiation. As such, in vitro NK cell differentiation cultures were settled by starting from cord blood-derived hematopoietic stem cells (HSC) transduced with IRF2 knockdown or IRF2 overexpression vectors. Of interest, authors found that IRF2 knockdown strongly reduced NK cell proliferation during development. Also, they demonstrated that IRF2 is necessary for functional maturation of NK cells, as NK IRF2-/-cells had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.Bourayou and Golub aims at explaining how inflammation could impact on NK cell development, maturation and effector functions. In particular, the authors firstly summarized known and emerging factors implicated in the regulation of NK cell maturation and then discussed how distinct inflammatory conditions could influence this process.Another cutting edge topic that was addressed by Xie et al. have elaborated an interesting overview about past and recent advances in human uterine NK (uNK) cell research highlighting the role of uNK cells in both normal physiological conditions and in pregnancy. The mechanism of action of uNK cells in various reproductive diseases including recurrent miscarriage, preeclampsia, and endometriosis is discussed from multiple perspectives. In addition, the potential clinical usage of uNK cells as a novel immunotherapeutic strategy for reproductive disorders is largely described.NK cells have emerged as crucial innate immune cells involved in the physiology and pathology of both non-pregnant and pregnant uteri. Fionda et al., the potential impact of age-related changes on NK cell phenotype and function during SARS-CoV-2 infection was investigated. Of interest, adult patients (below 65 years of age) had a reduced number of total NK cells, while elderly (over 65 years of age) showed a peculiar skewing of NK cell subsets towards the CD56lowCD16high and CD56neg phenotypes, expressing activation markers and check-point inhibitory receptors. IFN-γ production was severely compromised only in adult patients in a TGF-β−dependent manner. Authors suggest that TGF-β might limit an excessive NK cell activation that could amplify the systemic inflammatory response leading to a less severe infection course.In the manuscript by Mele et al.). The results obtained showed a NK cell phenotype predominantly characterized by increased expression of the inhibitory receptor NKG2A and by a concomitant reduction of a number of activating receptors . This inhibitory phenotype was associated with a reduced antibody-dependent cellular cytotoxicity (ADCC) activity evaluated either as degranulation or as IFN-γ production. An interesting observation derived from this work concerns an increment of glucocorticoid-induced tumor necrosis factor-related (GITR) costimulatory receptor not only on NK cells but also on tumor infiltrating CD4 and CD8 T lymphocytes. Thus, GITR could represent a potential therapeutic target for immunotherapeutic approaches combining GITR agonists and immune checkpoint inhibitors.In a model of head and neck squamous cell carcinoma (HNSCC), tumor infiltrating NK cells were defined based on the analysis of cell surface markers and on functional assays  and insulin-like growth factor binding protein 5 (IGFBP5) (i.e: SFRP4+IGFBP5hiNKT cells) was discovered by single cell RNAseq analysis in the foci of patients with adenomyosis, an estrogen-dependent gynecological disease, in which chronic pain is a clinically unresolved issue.In the manuscript by +IGFBP5hiNKT cells were capable of converting part of the stem cells found in affected tissues into neurogenic cells and were therefore, likely responsible of inducing pain.Of interest, it was observed that these SFRP4All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Spontaneous cervical internal carotid artery dissection (cICAD) is a common cause of stroke in young adults. Endovascular therapy is an indispensable treatment for cICAD in some cases, but it faces great challenges.A bilateral spontaneous cICADs with hypoperfusion-related AIS after 72 h from the onset was presented herein. The patient responded well to primary Solitaire stent detachment at the critical flow-limiting site.Primary stent implantation at the critical flow-limiting site rather than covering the entire dissection may be a therapeutic option in spontaneous cICAD complicated with cerebral hypoperfusion. The Solitaire stent may be a good choice at the acute and subacute stages of cICAD. Spontaneous cervical internal carotid artery dissection (cICAD) is a relatively common cause of stroke in young adults  and slurring speech on August 30, 2020. He was diagnosed to have a right internal carotid artery (ICA) territory infarct based on non-contrast computed tomography (NCCT) at local hospital. Despite antiplatelet therapy with aspirin 100 mg/d and clopidogrel 75 mg/d, over the next 3 days, left limb weakness progressively worsened (2/5), accompanied by somnolence (GCS 3 + 5 + 6). Therefore, he was transferred to our hospital on September 2, 2020. The Glasgow Coma Scale (GCS) score was 13/15. The head NCCT revealed new infarcts in the right internal watershed area and CT perfusion (CTP) showed a 100.3 mL mismatch of bilateral cerebral hemispheres, indicating that a considerable penumbral region was present in the supply area of each internal carotid artery (ICA)  was 1 at the 90-day follow-up. Follow-up neck CTA at three months showed no residual lesion in both ICAs. No relapse of cerebral ischemic events during the 15-month follow-up occurred. A recent neck CTA showed that both ICAs remained patent without relapse of dissection (Fig. This was a typical case of bilateral spontaneous cICAD complicated with bi-hemispheric hypoperfusion that responded well to primary stent implantation at the critical flow-limiting site rather than covering the entire dissection. The lesions of cICAD are usually long segmented and the usual procedure of EVT uses a partial overlap technique to implant multiple stents in tandem to reconstruct the entire dissected segment (Cohen et al. A very challenging task of EVT in cICAD is choosing the type of stent for deployment. Based on previous reports, specific types of intracranial/carotid self-expanding stents might be good choice (Moon et al. Furthermore, the present case taught us a lesson that we should be more active in intervening with the cICAD with cerebral hypoperfusion. If the left ICA lesion was treated earlier, the patient may have avoided the second ischemic event. There were reports on successful treatment of bilateral cICADs with one procedure (Sedat et al. The present case demonstrated that primary stent placement at the critical flow-limiting site rather than covering the entire dissection may be a therapeutic option in spontaneous cICAD complicated with cerebral hypoperfusion, and the Solitaire stent may be a good choice at the acute and subacute stages of cICAD. Moreover, we should be more active in intervening with EVT in spontaneous cICAD with hypoperfusion.


Service member exposure to explosive blast overpressure waves is common with considerable attention to traumatic brain injury (TBI) and neuropsychological sequalae. Less is known about the impacts on the respiratory system, particularly long-term effects, despite vulnerability to overpressure. Using a national registry, we previously observed an independent relationship between self-reported blast exposure and respiratory symptoms; however, the impact on objective measures of pulmonary function is poorly understood.1%; %∆FEV1), volume (TLC%), diffusion (DLCO%) and respiratory mechanics (forced oscillometry) were selected for analysis.307 Veterans referred to our national specialty center for post-deployment health concerns underwent a comprehensive multi-day evaluation that included complete pulmonary function testing (PFT), occupational and environmental medicine history, neuropsychological or psychological evaluation. We developed an a priori chart abstraction process and template to classify Veterans into blast exposure groups: (1) none, (2) single-mild, or (3) multiple-mild. This template focused primarily on clinician documented notes of blast related TBI that were used as proxy for blast overpressure injury to thorax. PFT variables characterizing flow .Veterans  were referred 8.6 ± 3.6 years after their last deployment and presented with considerable comorbid conditions and health problems . After chart abstraction, Veterans were assigned to none (n = 208), single mild (n = 52) and multiple mild (n = 47) blast exposure groups. Among the blast exposed, clinicians documented 73.7% were < 50 m from the blast and 40.4% were physically moved by blast. PFT outcome measures were similar across all groups (In this referred sample of deployed Veterans, PFT measures of flow, volume, diffusion, and respiratory mechanics were not associated with clinician documented blast exposure per the retrospective chart abstraction methodology applied. Yet, these clinical findings suggest future research should determine and assess distinction between Veteran recollections of perceived blast experiences versus overpressure wave exposure to the respiratory system.The online version contains supplementary material available at 10.1186/s12931-022-02071-0. Approximately 8 in 10 combat injuries in recent conflicts have an explosive blast etiology  and well−1) of IED blasts and controlled detonations. Investigators observed an unadjusted association between their blast exposure severity score and lung clearance index (marker of ventilation heterogeneity) in 71 deployed individuals that was interpreted as evidence for a link between blast exposure and small airways injury. Whereas scoring instruments and standardized interviews exist to assess blast-related TBI and associated neuro-psychological sequelae beyond self-report ),A chart reviewer template was developed to guide the chart abstraction process. This template consisted of preselected key variables: (1) proximity to blast ; (2) number of sub-concussive and concussive blasts; (3) severity of acute symptoms for TBI caused by blast  ; (4) PTSInitially, reliability, completeness, and consistency of the clinical notes for the key variables of interest of the chart abstraction process were unclear; upon further examination of the abstracted data, the variables for number of concussive blasts and TBI severity were selected to define blast exposure groupings. These variables were selected because they were the most complete and they provided the most consistent blast exposure assessment according to the interrater agreement of the chart abstraction process  codes present in the WRIISC clinician’s notes. ICD9 codes were converted to ICD10 codes to organize into comorbid condition and health problem groupings . ComorbiAssessments for differences among blast exposure groupings for patient characteristics were conducted with the Kruskal–Wallis test for continuous variables followed by Dunn test for post-hoc multiple comparisons. Fisher’s exact test was used to assess for association between categorical patient characteristics and blast grouping. The interrater reliability analysis for the chart abstraction was conducted using Gwet’s AC2 with linear weighting , 21. A mAfter the chart abstraction process, all subjects were categorized according to blast group as follows: none (n = 208), single mild blast exposure (n = 52), and multiple mild blast exposures (n = 47) Table . Due to 2. Median total deployment duration was found to be statistically significantly different across blast groups  with the multiple mild blast exposure group having a significantly longer total deployment length as compared to the group with no blast exposure . Blast exposure group was also associated with military branch (p = 0.001) and sex (p = 0.03). None of the remaining characteristics were statistically significantly different across groups. Also presented in Table Patient characteristics are presented in Table The overall trends and distribution of data, as indicated by the shape and location of the violin plots and inner boxplots, were similar across the three blast groups for each of the nine selected pulmonary function test outcomes Fig. . To suppWe hypothesized lung injury from mild blast exposure during deployment will result in impaired pulmonary function. In the absence of standardized instruments to classify blast exposure to the thorax, we first developed a standardized approach to characterize blast exposure derived from clinical interviews with emphasis on TBI as a proxy for physiological effect from blast exposure. After assigning Veterans to exposure groups , we evaluated whether group assignment was associated with select pulmonary function outcomes by comparing the overall trends and distribution of data Fig.  as well As highlighted by the National Academies of Sciences, Engineering, and Medicine, “…there is a striking absence of data on the long-term pulmonary outcomes of exposure to blast .” In a There are two major limitations with this study: (1) potential contributors to errors in blast exposure assessment, and (2) confounding due to underlying comorbid conditions and health problems. In assessing blast exposure, it is challenging to disentangle recollections of prior blast exposures versus experiences. Martindale et al.  highlighThe observed lack of an association between blast exposure and objective measures of pulmonary function in the present analysis may also be attributable to various confounding factors intrinsic to the examination of a clinical sample. Foremost, Veterans referred to the NJ WRIISC have chronic, unexplained symptoms, including dyspnea and many other symptoms and deployment-related concerns. The presence of multiple and diverse comorbid conditions and health problems may have compromised the ability to detect the association between blast exposure and measure of long-term pulmonary function. Similarly, almost 20% of the sample were current smokers and about one third were former smokers albeit with a minimal pack-year history Table . The useThis study exhibited strengths in its design and offers important insight for future research. This is a large well-described single site cohort evaluated by a multidisciplinary team of subspecialty clinicians obtained approximately 9 years after deployment. The high inter-rater reliability (> 92%) for the variables we abstracted from the medical record to define blast exposure are reflected in Table exposures versus experiences. Moreover, the clinical findings and experience presented herein may also aid the design and development of prospective controlled studies to better characterize potential blast exposure persistent and latent effects on cardiopulmonary health.In this retrospective analysis of medical records, Veterans deployed to Iraq and Afghanistan with or without clinician-documented mild blast exposure demonstrate similar pulmonary function. Reliance on clinical interviews tailored to evaluate blast-related TBI as a proxy for blast-related thoracic injury may have impacted our ability to differentiate between groups. However, our approach could be modified for future investigations designed to distinguish between blast Additional file 1. Contains the following supplemental tables as described in the manuscript text: (1) Table S1. Key variables used for retrospective chart abstraction process and interrater reliability. (2) Table S2. Number of subjects in regression models and additional number (%) of subjects excluded in adjusted models due to incomplete predictors data. (3) Table S3. Model fit results by outcome measure and model type. (4) Table S4. Model fit results ) by outcome measure and model adjustment for blast exposure group (any mild blast exposure) vs. reference group (no blast exposure). (5) Table S5. Additional pulmonary function test findings: lung volumes and diffusion. (6) Table S6. Additional pulmonary function test findings: spirometry. (7) Table S7. Additional pulmonary function test findings: forced oscillation technique.


In this Perspective,we highlight select systems in the hope of demonstrating the breadthof ArM design strategies and applications and emphasize how thesesystems address problems that are otherwise difficult to do so withstrictly biochemical or synthetic approaches.Artificial metalloproteins(ArMs) have recently gained significantinterest due to their potential to address issues in a broad scopeof applications, including biocatalysis, biotechnology, protein assembly,and model chemistry. ArMs are assembled by the incorporation of anon-native metallocofactor into a protein scaffold. This can be achievedby a number of methods that apply tools of chemical biology, computational  Fundamentalinsights from structural biology, biophysics, and the developmentof truncated small-molecule synthetic mimics of metalloprotein activesites have informed our understanding that both the primary coordinationsphere of the metal ion and the surrounding protein microenvironment,or secondary coordination sphere, are essential for proper function.Proteins with artificially engineered metal active sites is one approachthat has produced systems that has informed us on the subtle connectionsbetween structure and function. The advancement of synthetic methodologiesand biochemical tools such as protein engineering has recently reinvigoratedthe study of artificial metalloproteins (ArMs). This hybrid approachat the interface of chemical biology and synthetic chemistry aimsto prepare new systems that address problems in a wide range of applications,including biocatalysis,3 biotechnology,5 protein structure and assembly,7 and model chemistry.8Metalloproteins constitute an estimatedone-third to half of theproteome.de novo protein matrices,as summarized in 9 Synthetic metal complexes canbe incorporated by a variety of means, including (i) formation ofa direct dative, covalent bond to an amino acid ligand, (ii) covalentattachment to the protein by a bioconjugate moiety, or (iii) noncovalentanchoring in the protein environment. Thus, artificial metalloproteinsoffer the opportunity to incorporate both nonbiologically relevantmetals and designed ligands in a platform that alsobenefits from the aforementioned mutagenesis of proteins. Here, wepresent selected examples from the literature that survey the differentapproaches to prepare ArMs. In this Perspective, we have focused onthe choice of protein host in a manner that highlights how both nativemetalloproteins and proteins that do not contain native metal sitescan be applied to the design of artificial metalloproteins.Artificial metalloproteins are constructedby the introductionof non-native metallocofactors into a naturally occurring proteinor through the design of 20 Some of the best examples of this approach are found in heme metalloenzymes,which are a versatile class of proteins with functions ranging fromreversible dioxygen binding to C–H bond functionalization.Many re-engineering studies have left the heme cofactor intact andhave focused on determining the contributions of residues within theactive site toward functions. Using site-directed mutagenesis methods,single point mutations are made to change either the axial ligandsat the Fe centers or those around the Fe housed within the secondarycoordination sphere. In addition, the seminal work of Arnold on reengineeredcytochrome P450s (P450s) using directed evolution methods has stronglyaffected a variety of different fields, including approaches relevantto ArMs.26 Hartwig has also shown that the substitution of Fe porphyrins withunnatural metalloporphyrins within heme proteins produces an arrayof ArMs with new functions.28 There are a number of reviewsthat have already discussed the importance of these studies, and theywill not be reiterated here;30 instead, we will describethe utility of using heme metalloproteins to engineer additional metalion binding sites to produce new metallocofactors.One of the most obvious approaches for constructingArMs is tore-engineer a metalloprotein to produce new active sites with structuraland functional properties that differ from those of the native protein.2 to water, using the energy from that reactionto generate a transmembrane proton gradient which ultimately leadsto ATP synthesis. The site of binding and subsequent reduction ofO2 is the heme-CuB bimetallic center —these metalloproteinsare a family of terminal oxidases that are part of the respiratorypathways of eukaryotic mitochondria and bacteria. Their function isto reduce Oc center 2.31−33 Sc peroxidasewith a Cubinding site near the heme center (CuBCcP) was the first protein model of the heme-CuB site foundin cytochrome c oxidases that exhibited spin couplingbetween the Cu(II) and the heme-Fe(III) center without any additionof exogenous ligands.36 Later Lu redesignedsperm whale myoglobin with a Cu binding site near the heme center. CuB-Mb hasa low affinity for O2 in the absence of any metal ionsin the Cu binding site, but increased affinity for O2 wasfound in the presence of Ag(I) ions; however, this ArM was unableto further reduce dioxygen.37 In contrast,when CuB-Mb is treated with Cu(II) ions and a reductant,binding and reduction of dioxygen was observed. These findings suggestthat the Cu center is structurally important for efficient O2 binding to the heme center in HCOs and is essential for reduction.A redesigned cytochrome B-Mb reduced O2 in the presenceof Cu(II), and reductants formed a peroxy-heme intermediate that subsequentlyunderwent heme degradation to verdoheme—a reaction observedin heme oxygenases (HOs).38 This contrastswith the formation of high-valent Fe(IV)–-oxido intermediatesthat are observed in HCOs and P450 enzymes. Biochemical studies conductedon P450s suggest that a hydrogen-bonding network of water moleculesand hydrophilic residues in the distal pocket of the heme proteinsupplies protons to the peroxy-heme and facilitates conversion tothe reactive Fe(IV)–oxido species. Lu and co-workers thereforeproposed that CuB-Mb may lack a similar H-bonding network(and proton delivery mechanism), which may explain why CuB-Mb does not exhibit HCO activity. To test this hypothesis, H2O2 was added to the Fe(III)/Cu(II) CuB-Mb form (met-CuB-Mb), which resulted in the formationof an Fe(IV)–oxido species. They proposed that the additionalprotons liberated upon coordination of H2O2 facilitatedformation of the Fe(IV)–oxido species, similar to the roleof the hydrogen-bonding network in P450s, and suggested that similarhydrogen-bonding networks may be important to HCO function. With thiswork, Lu and co-workers have demonstrated the power of their approachby transforming an O2 carrier protein such as myoglobinand engineering it to display both HO and HCO activity in a mannerthat was informative of the probable mechanisms of native HCO enzymes.The ArM Cu39 In this system,the advantages of both synthetic chemistry and protein chemistry wereleveraged in order to visualize, through XRD methods, reactive intermediatesover the course of the catalytic reaction. The high solvent contentand molecular flexibility endogenous to protein crystals enables thepossibility to trap and structurally characterize reactive intermediatesduring catalysis. However, due to the highly evolved nature of enzymeactive sites, catalytic reactions occur very rapidly, which oftenmakes it difficult to trap reactive intermediates. Catalytic transformationsby small molecules typically proceed at much slower rates, but similar in crystallo reactions are challenging to perform becauseof their more rigid crystal lattices. To address these obstacles,Ménage et al. have successfully engineered a system that incorporatesa small-molecule catalyst into a protein host to structurally characterizevarious catalytic intermediates.Artificial metalloproteinshave also been prepared using native metalloprotein hosts and syntheticmetallocofactors to produce new active sites; this approach is incontrast to reproducing the native active site that was discussedabove. Marchi-Delapierre, Cavazza, and Ménage utilized thisapproach to develop an ArM capable of dioxygen binding, activation,and arene hydroxylation.− (L = N-benzyl-N′-(2-hydroxybenzyl)-N,N′-ethyldiamine diacetate) was incorporated into the nickel-bindingprotein NikA through a series of noncovalent intramolecularH-bonding interactions with the protein host OH]ein host 3A. The e2 adduct 3Band th2 adduct 3C. To su40 Theactive site was modified by redesigning the Fe complex used in theprevious study via substituting functional groups at the reactiveC–H bonds of the aromatic rings on L to prevent intramolecularhydroxylation  and the conversion of H2 to protons and electrons.44 Thereare three phylogenetically distinct classes of hydrogenase enzymes,which include the dinuclear [FeFe] and [NiFe] hydrogenases and a mononuclear[Fe] hydrogenase. The potential application of [FeFe] hydrogenasesas a renewable source of H2 generation has inspired significantstructural and mechanistic investigations by both biochemists andsynthetic chemists. The active site of [FeFe] hydrogenase containsan asymmetric dinuclear [2Fe] core with each low-valent Fe(I) centercoordinated by carbonyl (CO) and cyanide (CN–) ligandsand a bridging dithiolate ligand  without the need for the biological maturaseproteins .55 The synthetic complexesemployed in the study depicted in 2),58 N (2-NH),59 or O (3-O)60 atomat the bridgehead. In vitro addition of the syntheticcomplexes to the apoprotein produced artificial hydrogenases thatfeature a distinct atom  at the bridgehead positionof the thiolate ligand that is explicitly known because it was firstsynthesized and characterized outside of the protein. The hydrogenevolution activity revealed that only HydA-2-NH,featuring a N atom bridgehead, was capable of reproducing the activityof the native enzyme; HydA-1-CH2 and HydA-3-O did not demonstrate any H2 evolution.This finding provided key supporting evidence that [FeFe] hydrogenaseslikely contain an azadithiolate bridge that facilitates proton transferduring catalysis. The ability to leverage the atomistic control ofsynthetic chemistry and vary the identity of the bridgehead centralatom would otherwise be difficult, if not impossible, to biosyntheticallyengineer, especially considering that the biosynthetic origin of thisbridging moiety was previously unknown.45 Molecular structures of the artificial hydrogenases prepared withthe apoprotein from Clostridium pasteurianum (CpI) were obtained by protein X-ray diffraction experiments61 technology. Streptavidin is a homotetrameric protein .71 Notably, Savdoes not naturally bind a metallocofactor but artificial metalloproteinshave been constructed using biotin–Sav technology by employingbiotinylated ligands with metal-chelating moieties. Artificial metalloproteinsof this type were first realized by Whitesides, who treated streptavidin(Sav) with a biotinylated organometallic Rh complex to produce anasymmetric hydrogenation catalyst.72The above discussions all dealt with protein hostswhose naturalfunctions required the binding of metal ions. A complementary approachis to confine artificial metallocofactors within proteins that haveno prior capacity to bind metal ions. protein 7A consis protein 7B.69 The75 He has creatively modified the Sav host and the biotin binding pocketsurrounding the metal center in a manner that tunes the catalyticproperties of the ArMs. His work has demonstrated how protein-inducedlocal environments around metallocofactors can greatly influence thefunction, reminiscent of directed evolution previously developed byArnold.24 In one example, Ward and Rovis developedan ArM that catalyzed the asymmetric C–H bond functionalizationreactions between pivaloyl-activated benzhydroxamic acid and acrylateswith enantiometric ratios of 90:10 and overall yields of >90% (76 The ArM contains a biotinylated Rh(III)–Cp* complex(Cp* = pentamethylcyclopentadiene) as the artificial metallocofactorthat was known to catalyze this reaction in the presence of acetateion.80 The key findings were to re-engineer Sav to include two mutations—onechanged a lysine to a glutamate that provided a local base in theform of a carboxylate ion near the Rh(III) center that acceleratedthe reaction, and another changed a serine to lysine that establishedthe high enantioselectivity. Although no structural data were reported,the high degree of selectivity and yield supports the premise thatthe Rh complex is localized proximal to these two residues withina Sav subunit.Ward hassince greatly advanced the development of ArMs using Savas the protein host, and he has reported on a variety of novel systems. of >90% 8.76 The 2O2.81 However,like the Rh systems discussed above, selectivity is harder to achievewith these types of Fe complexes. A series of ArMs with biotnC–Fe(TAML) complexes  complexes (TAML = tetraamidomacrocyclic ligand)—these complexes were first introduced byCollins and are known to cleave C–H bonds when they are treatedwith Homplexes 9 were prK121′ = 2.3 Å), thelysine in the other subunit of the pair  moiety was lockedinto a specific position (100% occupancy) through interactions inboth the primary and secondary coordination spheres of the Fe center.The complex was positioned such that it could form a secondary coordinationsphere hydrogen bond between the arginine at 112 and a carbonyl groupon the TAML ligand. This localization places the Fe center near thesubunit interface of the Sav dimer and within bonding distance toK121′ that are associated with coordination of an endogenous ligand to aconfined metal complex. In this way, we can rapidly determine whichcombination of Sav variant and artificial metallocofactor gives thedesired positional match.We havebeen engineering ArMs with Sav hosts to emulate the activesites within metalloproteins. Our designs have used both hydrogenbonds and bonding interactions to endogenous ligands to localize theartificial metallocofactors within Sav. These bonding requirementsrequired the embedded metal complex to be reproducibly positionednear specific amino acid side chains of Sav, which was done througha process we denoted as 83 Oneproposed function of the constrained geometries of entatic states,known as the rack mechanism, is to preorient metalloenzymes towardcatalytic transition states. These constrained geometries exhibitedby metalloproteins are often difficult to reproduce synthetically,requiring thoughtful ligand design to mimic the steric constraintsof a protein environment.To illustrate this concept, we describeour work on engineering entatic states within ArMs. One manner thatnature has evolved to modulate the properties of metalloenzymes isthrough entatic states. Entatic states are uncommon geometries thatresult from structural restrictions placed upon the metal ion(s) andits ligands by the largely prearranged coordination environments dictatedby the protein secondary structure.2+ and Cu+ redox states.85 The activesites of cupredoxins exhibit a distorted-trigonal-monopyramidal coordinationgeometry for both Cu(I) and Cu(II) states. While it is known for Cu(I)complexes, this type of structure is rare for Cu(II) complexes becausethere is a strong electronic preference for the square-planar coordinationgeometry. The active sites within cupredoxins overcome this preferenceby constraining the endogenous ligands in such a manner so to forcethe Cu(II) state to adopt this unusual geometry  and the small hyperfinecoupling constant observed by EPR spectroscopy (A ≈ 180 MHz).86The canonical representation of entaticstates in bioinorganicchemistry is the active sites of cupredoxins, commonly referred toas blue copper proteins. Cupredoxins are electron transfer proteinscontaining mononuclear copper sites that cycle between Cugeometry 11A. Thes87 A biotinylated bis[2-(2-pyridyl)ethyl]amine(dpea) was used to prepare the CuII complexes [CuII(biot-n-dpea)(Cl)(H2O)]Cl containing a variable linkerlength  in theEPR spectra that were not observed in the complex alone or on introductioninto WT Sav. These absorbance features are typical of blue copperelectron transfer proteins such as stellacyanin and azurin and initiallysuggested the coordination of the cysteine thiolate residue to thesynthetic Cu cofactor. The molecular structures of the artificialmetalloproteins determined by high-resolution protein X-ray diffractionconfirmed the thiolate coordination and revealed important structuraldifferences in the Cu coordination sphere as a result of the ligandlinker length state.r length 11B,C. For length 11B,C. ThIII–(μ-OH)–FeIII] core in Sav from a mononuclear Fe complex. Dinuclear Fe cores areprevalent in biology and facilitate a range of processes includingO2 binding, O2 activation, and chemical functionalization,among others. Synthetic efforts have successfully produced many examplesof di-FeIII cores through self-assembly of mononuclearspecies or dinucleating ligands containing bridging moieties. Despitethe many different ligand systems that have been applied to constructdinuclear FeIII cores, these complexes demonstrate onlya small variation in the structural metric parameters of the di-Fecores as a result of the highly thermodynamic nature of the bridging[Fe–(μ-O)–Fe] moiety. Counter to the norm, wehave engineered ArMs that exhibit unusually long Fe···Fedistances as a result of being embedded within a protein host.We have also appliedthe concept of positional matching of aminoacid residues with a synthetic biotinylated iron complex to engineera dinuclear [FeIII(biot-n-dpa)(OH2)3]Cl3 , and a series of Sav variants  containing tyrosine mutations were selected with theintention of providing a readily visible optical assay for amino acidbinding due to FeIII–OY interactions indicative of formation of an FeIII–OY bond for only [FeIII(biot-bu-dpa)]⊂K121A/L124Y-Sav.Binding of the tyrosinate moiety to the FeIII center wasfurther supported by the observation of characteristic phenol ringvibrations and ν(Fe–OY) and ν(C–OY) modes for FeIII-bound tyrosinate species by resonanceRaman spectroscopy.Biotinylated dipyridylmethylamine(dpa) ligands containing variableethyl, propyl, or butyl linkers were used to synthesize the Fe complexes[Feractions13.88 IncIII(biot-bu-dpa)]⊂K121A/L124Y-Savwas silent, suggesting magnetic coupling of the immobilized Fe centers.The Mössbauer spectrum of enriched [57FeIII(biot-bu-dpa)]⊂K121A/L124Y-Sav exhibited a single quadrupoledoublet, confirming the antiferromagnetic coupling of the Fe centers.Solution X-ray absorption near edge spectroscopy (XANES) in concertwith variable-temperature Mössbauer spectroscopy of enriched[57FeIII(biot-bu-dpa)]⊂K121A/L124Y-Savcrystals indicated the presence of a bridging hydroxide moiety betweenthe two Fe centers to form a [FeIII–(μ-OH)–FeIII] core.The close proximity of the tyrosineresidues from neighboring subunitsin the assembled Sav dimer initially suggested the possibility toform bridged di-Fe cores in the vestibule. Indeed, the ⊥-modeEPR spectrum of [FeIII(biot-bu-dpa)]⊂K121A/L124Y-Savwas determined by X-ray diffraction at 1.30 Å resolution (3), and cyanide (CN) ligands.Surprisingly, the addition of exogenous ligands yields very smalldifferences in the observed Fe···Fe′ distanceswith slightly contracted distances of 3.91 Å (OAc), 3.82 Å(N3), and 3.73 Å (CN), respectively. In comparisonto both synthetic and biological precedents of [FeIII–(μ-OH)–FeIII] cores with Fe···Fe′ distances of3.3–3.6 Å, the ArMs discussed in this series feature uncharacteristicallylong distances between the two Fe centers. We assert that this unusualobservation is a direct result of immobilization of the complexeswithin the rigid Sav host. Due to both the strong affinity of Savfor biotin in concert with coordination of the Fe centers by the tyrosineamino acid residue, the Fe centers represent entatic states that areconstrained in space and incapable of forming classical, more contracteddi-Fe cores.The molecular structure of n (Mn(dpp)) cofactor. The computations included two additionalaxial ligands at the Mn center: a histidine residue to anchor theMn(dpp) to the protein through a Mn–N bond and a coordinateddioxygen fragment. Notably, the inclusion of dioxygen in the computationsensured that the structure provided adequate space for oxidant andsubstrate access at an axial position of Mn(dpp). The amino acid compositionwas further tuned to allow flexibility to accommodate the metallocofactorand to minimize the number of oxidizable bonds near the cofactor,thus preventing undesirable oxidation of the protein backbone. Thesequences of loops connecting the helices were optimized, resultingin multiple variants with stable folded cores, one of which was amenableto crystallization. Absorption and circular dichroism spectroscopyon Mn(III) MPP1 in solution supported MnIII(dpp) bindingan axial histidine residue as designed. In further studies, it wasfound that Mn(III) MPP1 could be oxidized to Mn(V) MPP1 with sodiumperiodate. The formation was monitored by changes in the Soret bandand supported by reactivity studies in which the Mn(V) MPP1 specieswas able to transfer an O atom to thioanisole to form a sulfoxide,as shown in Solutions to these challenges are found in the workof DeGrado, who has been a leader in the assembly of helix bundlesthat bind synthetic metallocofactors. In one recent report, he wassuccessful in installing a synthetic Mn–porphyrin in a III(dpp) MPP1 verifiedseveral of the design elements of this variant that included axialligation of the engineered histidine residue to MnIII(dpp)16. Moreo96 In one example, he designed a de novo di-Fe proteinto mimic the structural and functional properties of the metalloenzyme p-aminobenzoate N-oxygenase AurF, whichcatalyzes the hydroxylation of amines.97 A key structural feature was to increase the accessibility to thecatalytic di-Fe center, which was accomplished by the mutation ofseveral alanine residues within the interior of the protein to glycines.A second design change involved altering the primary coordinationsphere around one Fe center by incorporating an additional histidineresidue that coordinated to only one of the Fe centers—thisnew, unsymmetrical di-Fe site accurately modeled the active site inAurF and proved to be catalytic for the N-hydroxylationof arylamines.DeGrado has alsospearheaded thedevelopment of biomimetic active sites, and his work on di-Fe proteinshas provided accurate models for several natural proteins. He againutilized four-helix bundle proteins to produce endogenous Fe bindingsites housed within the interior cavity formed by the helices.de novo designsto prepare series of novel proteins with redox-active sites, manyof which are excellent probes for electron transfer proteins. Hisdesigns utilize three-helix bundles where the metal binding site isagain within the interior of the protein.98 He has demonstrated that these helical scaffolds may be used tomodel aspects of the secondary coordination spheres that surroundthe metallocofactors in native proteins. In order to regulate electrontransfer, many proteins have evolved networks of redox active residuesto enable hole hopping mechanisms to transfer electrons over largedistances. Pecoraro and Aukauloo implemented this concept in a de novo designed protein in which a tyrosine amino acidrelay in the protein scaffold mediates intramolecular electron transferbetween a [RuIII] unit and a FeIIS4 site embedded within the protein.100Pecoraro has used de novo designedcopper proteins. We have already described the properties of the activesites in cupredoxins, the electron transfer proteins that containCu sites and are found in numerous organisms. The active sites withincupredoxins have evolved to control both the primary and secondarycoordination spheres around the Cu center, and both are needed toensure proper function. Modifications of either sphere can lead todiffering electron transfer properties, which have been used to establishstructure–function relationships. Such changes also resultin pronounced changes in color and spectroscopic properties that canbe used as a rapid indicator for structure–function changes.Pecoraro has used this sensitive spectroscopic feature as a probeto rationally design artificial cupredoxins that traverse a spectrumof cupredoxin properties.101 He designedthe cupredoxin site within de novo three-helix bundleproteins, which takes advantage of the pseudo-C3 symmetry that is associated with this type of variant. Afterthe first report of a red copper protein that mimics the His2CysGlu Cu binding site in nitrosocyanin, this de novo construct was redesigned to reproduce the spectroscopic propertiesof green and blue copper proteins as well. In one redesign, the metalbinding site was rotated, which produced the green copper protein.This variant was in turn converted to a blue copper protein by removing an axial methionine. Additional spectroscopic studiesusing EPR and XAS methods provided additional evidence for the structuresof these sites and the structural heterogeneity present in some variants.Nevertheless, this work exemplifies the utility of de novo designed proteins and allowed for a comparison of these classesof copper proteins outside of the native cupredoxin fold.Thesehelical structures have also proven to be sensitive scaffoldsfor tuning electronic properties in 102 monomericproteins are mutated to create new binding sites for metal ions locatedat the protein surface that can self-assemble into ordered oligomersupon metal binding. The monomeric heme-binding protein cyt cb562 was mutated to include a dihistidine motif  at its surface that enabled theformation of new protein oligomers upon metal coordination. The resultingoligomers were demonstrated to self-assemble into unique supramoleculararrangements depending on the preferred coordination geometry of thetransition-metal ion coordinated by the engineered surface histidines,stidines17.103 Fo104 approach, artificialproteins could be evolvedto self-assemble, even in the absence of metal ions at the proteininterfaces. Notably, the designed interfaces of the artificial proteinoligomers exhibited selective binding of Zn(II)  in comparison to other transition-metal ions,including Cu(II), which should bind more strongly according to theIrving–Williams series.105 In oneexample, the engineered supramolecular association proved to be sofavorable that the MeTIR approach could be applied to an invivo assembly of the ArM. Tezcan and co-workers have sincedemonstrated that MeTIR can be used to design several ArMs that self-assembleinto complex hierarchical structures that demonstrate properties ofnative metalloproteins such as preorganized metal-binding interiorcavities and allostery that are difficult to achieve with the otherstrategies described herein. Furthermore, ArMs assembled by this approachhave been evolved to acquire novel hydrolytic reactivity toward ampicillin,a β-lactam antibiotic, that functions both in vitro and in vivo  in vivo 18.106 Th107 This approach is closely related to the aforementionedMDPSA, wherein protein surface mutations designed to coordinate ametal ion and an exogenous, metal-binding organic linker promote theassembly of 3D lattices with protein nodes. Due to the presence ofthe organic linker, the resulting materials do not feature the sameclose protein–protein interfaces as those previously describedand are highly porous materials that maintain the intrinsic structuraland functional properties of the incorporated protein node. Like metal–organicframeworks, the design of protein–metal–organic frameworksis highly modular, as the protein node, metal ion, and organic linkermay all be varied to yield materials with new topologies.108 Indeed, Tezcan has shown that the thermal propertiesof protein–metal–organic frameworks derived from ferritinnodes are tunable and variable depending on the identity of the interstitialmetal ion and organic linker.109Tezcan and co-workers have alsodeveloped protein–metal–organiccrystalline frameworks that can also be viewed as artificial metalloproteins.112 Within a larger context, ArMsoffer an ideal platform to test existing ideas on how these effectsregulate the properties of metal complexes that should lead to improvedcatalytic systems.This Perspective highlighted advances in engineering biologicallyrelevant active sites within protein hosts. These types of ArMs haveprovided a wealth of information on the properties of metallocofactorsand thus have proven to be useful models for natural proteins. Itis striking that, within this area of research, there are severalcomplementary methods to engineer new ArMs, and we focused on thedifferent types of protein hosts that have been used. One common featureof these ArMs is the role that the host has in regulating the microenvironmentsaround the metallocofactors. Protein-induced interactions, particularlythose involved in noncovalent interactions, are designed into manyof the active sites that have been discussed. We have previously arguedthat microenvironmental effects are crucial for function and are keyfactors in what distinguishes the functions of metalloproteins fromthose of synthetic systems.114 In one system, he introducedan ArM with two catalytic sites that acted synergistically to achieveenantioselectivities of up to 99% ee for a Michael addition reaction.113 The key design feature was the engineeringof two different catalytic sites, in which one was comprised of anunnatural p-aminophenylalanine residue to activatean enal and the second contained a Lewis acidic CuII complex.The first site activated an enal, and the second activated the Michaeldonor by enolization and delivered it to one preferred prochiral faceof the activated enal. These results show the promise of engineeringmultiple catalytic sites within one protein host and how increasedstructural complexity can be harnessed to enhance function.One idea for further development is to engineeradditional sitesfor the specific binding of external molecules. This concept is particularlyimportant for catalysis where selectivities and efficiencies can beamplified by designing substrate-specific binding site proximal toan artificial metallocofactor. For catalysis, the introduction ofan additional catalytic site would also be advantageous for many transformations.We point to the work of Roelfes, who recently reported ArMs with dualcatalytic centers.


Dear Sirs,Reversible cerebral vasoconstriction syndrome (RCVS) is an important differential diagnosis in patients with acute onset thunderclap headache , 2. ClinA 38-year-old Caucasian female nurse presented to our emergency department with persistent holocephalic headache after having had a first abrupt episode of thunderclap headache two weeks prior to admission. That first episode of thunderclap headache started 20 min after she was substituted with intravenous iron  for the first time in order to treat chronic iron deficiency anemia , the neurologic examination was unremarkable. Electrocardiogram detected sinus rhythm at a heart rate of 96/min without any further abnormalities. Blood pressure was slightly elevated with 149/82 mmHg. Laboratory test results showed hypochromic, microcytic anemia  and an elevated red cell distribution width (RDW) of 19.5%. By CSF analysis (performed on admission two weeks after the first episode) subarachnoid haemorrhage was definitely excluded . CSF opening pressure was slightly elevated with 31 cmHMultimodal CT scan with CT-angiography revealed multifocal segmental cerebral artery vasoconstriction, predominantly affecting both middle cerebral arteries (MCA) and the P1-segment of the posterior cerebral artery (PCA) Fig.  A–C. TraCombined, RCVS was diagnosed and treatment with oral nimodipine (3 × 60 mg daily) and additional analgesics (ibuprofen 1200 mg daily and metamizole 3 g daily) was started. Symptoms improved, no further episodes of thunderclap headache occurred at that time and control TCCD showed normalized PSV values after five days (149 cm/s in the left MCA). After discharge, sequential TCCD was performed every four weeks (for 3 months) recording normal PSV values. While tapering oral nimodipine gradually over 10 weeks, the patient experienced one more episode of holocephalic headache with an intensity of 8/10 (NRS), which resolved spontaneously. The symptoms completely resolved within three months and there was no second episode of thunderclap headache  thunderclap headache, (ii) vasoconstriction on cerebrovascular imaging, and (iii) rule out of subarachnoid haemorrhage by CT and lumbar puncture. A connection to the intravenous iron substitution seems very likely by its timely association in absence of other trigger factors of RCVS.Iron is a key-element and involved in the regulation of oxygen transport and mitochondrial function acting as a transition-metal ion and as a cofactor in various enzymatic processes  . However2+) versus ferric (Fe3+) ions] and their bioavailability, as overall plasma-free and non-transferrin-bound iron is higher after direct intravenous iron administration [Oral and intravenous iron preparations differ regarding the valency of iron ions [ferrous  and ferric (Fe3+) ions in vasoconstrictive syndromes: iron ions have been shown to have vasoconstrictive effects itself on the aortal system and on cerebral arteries in experimental studies [Up to now, there is limited data on the role of ferrous (Fe studies , 12 and  studies . Further studies . Thus, iNew insights in the effect of iron in the pathophysiology of intracranial vasospasm could also be interesting for iron-binding treatment options of patients with SAH and vasospasms: in a previous study of rabbits with SAH and intracranial vasospasm the use of iron chelators reduced vasospasm by decreasing oxidative stress and endothelial cell death . In primFurthermore, cigarette smoke exposure promotes free radical-mediated oxidative stress and is linked to endothelial dysfunction, inflammation and atherosclerosis and thus is an important cause of vascular disease . TherefoStill, it remains unclear why the CSF opening pressure was slightly increased in our patient. The initially seen elevation of CSF opening pressure could be caused by overweight, and there have been cases reporting increased CSF pressure due to iron deficiency anemia , 19. FurOur case might give a new perspective into the underlying pathophysiology of RCVS that is probably more frequent than previously thought. An increased understanding of the role of iron homeostasis in the regulation of cerebral vascular tone and spasms is necessary and may lead to novel insights into etiology and treatment options for RCVS. Further investigations are warranted to disentangle the complex interplay of iron homeostasis, redox balance and endothelial function in the pathophysiology of RCVS.