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The Fe protein of nitrogenase plays multiple roles in substrate reduction and metallocluster assembly. Best known for its function to transfer electrons to its catalytic partner during nitrogenase catalysis, the Fe protein is also a key player in the biosynthesis of the complex metalloclusters of nitrogenase. In addition, it can function as a reductase on its own and affect the ambient reduction of CO2 or CO to hydrocarbons. This review will provide an overview of the properties and functions of the Fe protein, highlighting the relevance of this unique FeS enzyme to areas related to the catalysis, biosynthesis, and applications of the fascinating nitrogenase system.

The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii ( Av NifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active Av NifH protein upon co-expression of this protein with Av IscS/U and Av NifM in E. coli . Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed Av NifH protein has a high [Fe 4 S 4 ] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that Av NifM could serve as a chaperone and assist the maturation of a cluster-replete Av NifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the “clean” genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Heterologous expression of nitrogenase has been actively pursued because of the far-reaching impact of this enzyme on agriculture, energy and the environment. However, isolation of an active two-component, metallocentre-containing nitrogenase from a non-diazotrophic host has yet to be accomplished. Here we report the heterologous synthesis of an active molybdenum-nitrogenase by combining genes from Azotobacter vinelandii and Methanosarcina acetivorans in Escherichia coli . Metal, activity and electron paramagnetic resonance analyses demonstrate the integrity of the metallocentres in the purified nitrogenase enzyme; whereas growth, nanoscale secondary ion mass spectrometry and nuclear magnetic resonance experiments illustrate diazotrophic growth and 15 N enrichment by the E. coli expression strain, and accumulation of extracellular ammonia upon deletion of the ammonia transporter that permits incorporation of thus-generated nitrogen into the cellular mass of a non-diazotrophic E. coli strain. As such, this study provides a crucial prototype system that could be optimized/modified to enable future transgenic expression and biotechnological adaptations of nitrogenase. Heterologous expression of an active, metallocentre-containing nitrogenase in a non-diazotrophic host is challenging. Now, the heterologous biosynthetic pathway of Mo-nitrogenase is pieced together in Escherichia coli using genes from Azotobacter vinelandii and Methanosarcina acetivorans .

Biological carbon fixation is currently limited to seven naturally occurring pathways. Synthetic carbon fixation pathways have the potential to surpass aerobic natural pathways in efficiency, but none have been realized in living cells. Here, we present the reductive methylaspartate cycles (rMASP), a novel family of 4 energy-efficient aerobic CO2 fixation pathway variants. These cycles have the potential to outperform the yields and/or rates of the Calvin cycle and previously proposed synthetic CO2 fixation cycles. To realize these designs, we adopted a modular engineering approach in Escherichia coli. Several pathway modules up to a cascade of 11 enzymes were realized via engineering and evolution. We demonstrate crotonyl-CoA carboxylase dependent growth for both elevated and ambient CO2 conditions, and show in vitro activity of the other CO2-fixing 2-oxoglutarate carboxylase, which requires further activity optimization for mesophilic temperatures. This work demonstrates important steps toward realizing efficient synthetic carbon fixation pathways in living organisms.

ObjectiveTo investigate the humoral and cellular immune response to messenger RNA (mRNA) COVID-19 vaccines in patients with immune-mediated inflammatory diseases (IMIDs) on immunomodulatory treatment.MethodsEstablished patients at New York University Langone Health with IMID (n=51) receiving the BNT162b2 mRNA vaccination were assessed at baseline and after second immunisation. Healthy subjects served as controls (n=26). IgG antibody responses to the spike protein were analysed for humoral response. Cellular immune response to SARS-CoV-2 was further analysed using high-parameter spectral flow cytometry. A second independent, validation cohort of controls (n=182) and patients with IMID (n=31) from Erlangen, Germany, were also analysed for humoral immune response.ResultsAlthough healthy subjects (n=208) and patients with IMID on biologic treatments (mostly on tumour necrosis factor blockers, n=37) demonstrate robust antibody responses (over 90%), those patients with IMID on background methotrexate (n=45) achieve an adequate response in only 62.2% of cases. Similarly, patients with IMID on methotrexate do not demonstrate an increase in CD8+ T-cell activation after vaccination.ConclusionsIn two independent cohorts of patients with IMID, methotrexate, a widely used immunomodulator for the treatment of several IMIDs, adversely affected humoral and cellular immune response to COVID-19 mRNA vaccines. Although precise cut-offs for immunogenicity that correlate with vaccine efficacy are yet to be established, our findings suggest that different strategies may need to be explored in patients with IMID taking methotrexate to increase the chances of immunisation efficacy against SARS-CoV-2 as has been demonstrated for augmenting immunogenicity to other viral vaccines.

Placement of an interatrial shunt device reduces pulmonary capillary wedge pressure during exercise in patients with heart failure and preserved or mildly reduced ejection fraction. We aimed to investigate whether an interatrial shunt can reduce heart failure events or improve health status in these patients. In this randomised, international, blinded, sham-controlled trial performed at 89 health-care centres, we included patients (aged ≥40 years) with symptomatic heart failure, an ejection fraction of at least 40%, and pulmonary capillary wedge pressure during exercise of at least 25 mm Hg while exceeding right atrial pressure by at least 5 mm Hg. Patients were randomly assigned (1:1) to receive either a shunt device or sham procedure. Patients and outcome assessors were masked to randomisation. The primary endpoint was a hierarchical composite of cardiovascular death or non-fatal ischemic stroke at 12 months, rate of total heart failure events up to 24 months, and change in Kansas City Cardiomyopathy Questionnaire overall summary score at 12 months. Pre-specified subgroup analyses were conducted for the heart failure event endpoint. Analysis of the primary endpoint, all other efficacy endpoints, and safety endpoints was conducted in the modified intention-to-treat population, defined as all patients randomly allocated to receive treatment, excluding those found to be ineligible after randomisation and therefore not treated. This study is registered with ClinicalTrials.gov, NCT03088033. Between May 25, 2017, and July 24, 2020, 1072 participants were enrolled, of whom 626 were randomly assigned to either the atrial shunt device (n=314) or sham procedure (n=312). There were no differences between groups in the primary composite endpoint (win ratio 1·0 [95% CI 0·8–1·2]; p=0·85) or in the individual components of the primary endpoint. The prespecified subgroups demonstrating a differential effect of atrial shunt device treatment on heart failure events were pulmonary artery systolic pressure at 20W of exercise (pinteraction=0·002 [>70 mm Hg associated with worse outcomes]), right atrial volume index (pinteraction=0·012 [≥29·7 mL/m2, worse outcomes]), and sex (pinteraction=0·02 [men, worse outcomes]). There were no differences in the composite safety endpoint between the two groups (n=116 [38%] for shunt device vs n=97 [31%] for sham procedure; p=0·11). Placement of an atrial shunt device did not reduce the total rate of heart failure events or improve health status in the overall population of patients with heart failure and ejection fraction of greater than or equal to 40%. Corvia Medical.

Christian Gilissen, John Niederhuber and colleagues examine de novo mutations with parent-of-origin information from whole-genome sequencing datasets from 816 parent–offspring trios. They find maternal and paternal specific mutation signatures that are more prominent with increased age of the parent as well as clustered mutation signatures with no parental bias. De novo mutations (DNMs) originating in gametogenesis are an important source of genetic variation. We use a data set of 7,216 autosomal DNMs with resolved parent of origin from whole-genome sequencing of 816 parent–offspring trios to investigate differences between maternally and paternally derived DNMs and study the underlying mutational mechanisms. Our results show that the number of DNMs in offspring increases not only with paternal age, but also with maternal age, and that some genome regions show enrichment for maternally derived DNMs. We identify parent-of-origin-specific mutation signatures that become more pronounced with increased parental age, pointing to different mutational mechanisms in spermatogenesis and oogenesis. Moreover, we find DNMs that are spatially clustered to have a unique mutational signature with no significant differences between parental alleles, suggesting a different mutational mechanism. Our findings provide insights into the molecular mechanisms that underlie mutagenesis and are relevant to disease and evolution in humans 1 .

Most patients with anaplastic lymphoma kinase (ALK)-rearranged or ROS proto-oncogene 1 (ROS1)-rearranged non-small-cell lung cancer (NSCLC) are sensitive to tyrosine kinase inhibitor (TKI) therapy, but resistance invariably develops, commonly within the CNS. This study aimed to analyse the safety, efficacy, and pharmacokinetic properties of lorlatinib, a novel, highly potent, selective, and brain-penetrant ALK and ROS1 TKI with preclinical activity against most known resistance mutations, in patients with advanced ALK-positive or ROS1-positive NSCLC. In this international multicentre, open-label, single-arm, first-in-man phase 1 dose-escalation study, eligible patients had advanced ALK-positive or ROS1-positive NSCLC and were older than 18 years, with an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Lorlatinib was administered orally to patients at doses ranging from 10 mg to 200 mg once daily or 35 mg to 100 mg twice daily, with a minimum of three patients receiving each dose. For some patients, tumour biopsy was done before lorlatinib treatment to identify ALK resistance mutations. Safety was assessed in patients who received at least one dose of lorlatinib; efficacy was assessed in the intention-to-treat population (patients who received at least one dose of study treatment and had either ALK or ROS1 rearrangement). The primary endpoint was dose-limiting toxicities during cycle 1 according to investigator assessment; secondary endpoints included safety, pharmacokinetics, and overall response. This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865. Between Jan 22, 2014, and July 10, 2015, 54 patients received at least one dose of lorlatinib, including 41 (77%) with ALK-positive and 12 (23%) with ROS1-positive NSCLC; one patient had unconfirmed ALK and ROS1 status. 28 (52%) patients had received two or more TKIs, and 39 (72%) patients had CNS metastases. The most common treatment-related adverse events among the 54 patients were hypercholesterolaemia (39 [72%] of 54 patients), hypertriglyceridaemia (21 [39%] of 54 patients), peripheral neuropathy (21 [39%] of 54 patients), and peripheral oedema (21 [39%] of 54 patients). One dose-limiting toxicity occurred at 200 mg (the patient did not take at least 16 of 21 prescribed total daily doses in cycle 1 because of toxicities attributable to study drug, which were grade 2 neurocognitive adverse events comprising slowed speech and mentation and word-finding difficulty). No maximum tolerated dose was identified. The recommended phase 2 dose was selected as 100 mg once daily. For ALK-positive patients, the proportion of patients who achieved an objective response was 19 (46%) of 41 patients (95% CI 31–63); for those who had received two or more TKIs, the proportion of patients with an objective response was 11 (42%) of 26 patients (23–63). In ROS1-positive patients, including seven crizotinib-pretreated patients, an objective response was achieved by six (50%) of 12 patients (95% CI 21–79). In this phase 1, dose-escalation study, lorlatinib showed both systemic and intracranial activity in patients with advanced ALK-positive or ROS1-positive NSCLC, most of whom had CNS metastases and had previously had two or more TKI treatments fail. Therefore, lorlatinib might be an effective therapeutic strategy for patients with ALK-positive NSCLC who have become resistant to currently available TKIs, including second-generation ALK TKIs, and is being investigated in a phase 3 randomised controlled trial comparing lorlatinib to crizotinib (ClinicalTrials.gov, NCT03052608). Pfizer.

Lorlatinib is a potent, brain-penetrant, third-generation inhibitor of ALK and ROS1 tyrosine kinases with broad coverage of ALK mutations. In a phase 1 study, activity was seen in patients with ALK-positive non-small-cell lung cancer, most of whom had CNS metastases and progression after ALK-directed therapy. We aimed to analyse the overall and intracranial antitumour activity of lorlatinib in patients with ALK-positive, advanced non-small-cell lung cancer. In this phase 2 study, patients with histologically or cytologically ALK-positive or ROS1-positive, advanced, non-small-cell lung cancer, with or without CNS metastases, with an Eastern Cooperative Oncology Group performance status of 0, 1, or 2, and adequate end-organ function were eligible. Patients were enrolled into six different expansion cohorts (EXP1–6) on the basis of ALK and ROS1 status and previous therapy, and were given lorlatinib 100 mg orally once daily continuously in 21-day cycles. The primary endpoint was overall and intracranial tumour response by independent central review, assessed in pooled subgroups of ALK-positive patients. Analyses of activity and safety were based on the safety analysis set (ie, all patients who received at least one dose of lorlatinib) as assessed by independent central review. Patients with measurable CNS metastases at baseline by independent central review were included in the intracranial activity analyses. In this report, we present lorlatinib activity data for the ALK-positive patients (EXP1–5 only), and safety data for all treated patients (EXP1–6). This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865. Between Sept 15, 2015, and Oct 3, 2016, 276 patients were enrolled: 30 who were ALK positive and treatment naive (EXP1); 59 who were ALK positive and received previous crizotinib without (n=27; EXP2) or with (n=32; EXP3A) previous chemotherapy; 28 who were ALK positive and received one previous non-crizotinib ALK tyrosine kinase inhibitor, with or without chemotherapy (EXP3B); 112 who were ALK positive with two (n=66; EXP4) or three (n=46; EXP5) previous ALK tyrosine kinase inhibitors with or without chemotherapy; and 47 who were ROS1 positive with any previous treatment (EXP6). One patient in EXP4 died before receiving lorlatinib and was excluded from the safety analysis set. In treatment-naive patients (EXP1), an objective response was achieved in 27 (90·0%; 95% CI 73·5–97·9) of 30 patients. Three patients in EXP1 had measurable baseline CNS lesions per independent central review, and objective intracranial responses were observed in two (66·7%; 95% CI 9·4–99·2). In ALK-positive patients with at least one previous ALK tyrosine kinase inhibitor (EXP2–5), objective responses were achieved in 93 (47·0%; 39·9–54·2) of 198 patients and objective intracranial response in those with measurable baseline CNS lesions in 51 (63·0%; 51·5–73·4) of 81 patients. Objective response was achieved in 41 (69·5%; 95% CI 56·1–80·8) of 59 patients who had only received previous crizotinib (EXP2–3A), nine (32·1%; 15·9–52·4) of 28 patients with one previous non-crizotinib ALK tyrosine kinase inhibitor (EXP3B), and 43 (38·7%; 29·6–48·5) of 111 patients with two or more previous ALK tyrosine kinase inhibitors (EXP4–5). Objective intracranial response was achieved in 20 (87·0%; 95% CI 66·4–97·2) of 23 patients with measurable baseline CNS lesions in EXP2–3A, five (55·6%; 21·2–86·3) of nine patients in EXP3B, and 26 (53·1%; 38·3–67·5) of 49 patients in EXP4–5. The most common treatment-related adverse events across all patients were hypercholesterolaemia (224 [81%] of 275 patients overall and 43 [16%] grade 3–4) and hypertriglyceridaemia (166 [60%] overall and 43 [16%] grade 3–4). Serious treatment-related adverse events occurred in 19 (7%) of 275 patients and seven patients (3%) permanently discontinued treatment because of treatment-related adverse events. No treatment-related deaths were reported. Consistent with its broad ALK mutational coverage and CNS penetration, lorlatinib showed substantial overall and intracranial activity both in treatment-naive patients with ALK-positive non-small-cell lung cancer, and in those who had progressed on crizotinib, second-generation ALK tyrosine kinase inhibitors, or after up to three previous ALK tyrosine kinase inhibitors. Thus, lorlatinib could represent an effective treatment option for patients with ALK-positive non-small-cell lung cancer in first-line or subsequent therapy. Pfizer.

Femtosecond resolution X-ray fluorescence spectroscopy is shown to track the charge and spin dynamics triggered when an iron coordination complex is excited by light, and establishes the critical role of intermediate spin states in the de-excitation process. Tracking 3 d excitations in transition metal compounds The absorption and dissipation of light energy by 3 d transition metal complexes has a crucial role in photosynthesis and many artificial systems that harvest solar energy, but understanding this complex phenomenon in detail remains challenging. Wenkai Zhang and colleagues now show that femtosecond resolution X-ray fluorescence spectroscopy can overcome the limitations of existing methods for probing the excited-state dynamics involved. This is illustrated for a prototypical iron complex, where tracking of the charge and spin dynamics triggered upon light-excitation establishes the critical role of intermediate spin states in the de-excitation process. The authors anticipate that their method will finally enable the detailed mapping — and hence understanding — of the fundamental processes that underpin many useful light-triggered molecular phenomena involving 3 d transition metal complexes. Crucial to many light-driven processes in transition metal complexes is the absorption and dissipation of energy by 3 d electrons 1 , 2 , 3 , 4 . But a detailed understanding of such non-equilibrium excited-state dynamics and their interplay with structural changes is challenging: a multitude of excited states and possible transitions result in phenomena too complex to unravel when faced with the indirect sensitivity of optical spectroscopy to spin dynamics 5 and the flux limitations of ultrafast X-ray sources 6 , 7 . Such a situation exists for archetypal polypyridyl iron complexes, such as [Fe(2,2′-bipyridine) 3 ] 2+ , where the excited-state charge and spin dynamics involved in the transition from a low- to a high-spin state (spin crossover) have long been a source of interest and controversy 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 . Here we demonstrate that femtosecond resolution X-ray fluorescence spectroscopy, with its sensitivity to spin state, can elucidate the spin crossover dynamics of [Fe(2,2′-bipyridine) 3 ] 2+ on photoinduced metal-to-ligand charge transfer excitation. We are able to track the charge and spin dynamics, and establish the critical role of intermediate spin states in the crossover mechanism. We anticipate that these capabilities will make our method a valuable tool for mapping in unprecedented detail the fundamental electronic excited-state dynamics that underpin many useful light-triggered molecular phenomena involving 3 d transition metal complexes.