A Dynamic Water Channel Affects O2 Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.

Profiling the dysregulated immune response in sepsis: overcoming challenges to achieve the goal of precision medicine.

Sepsis is characterised by a dysregulated host immune response to infection. Despite recognition of its significance, immune status monitoring is not implemented in clinical practice due in part to the current absence of direct therapeutic implications. Technological advances in immunological profiling could enhance our understanding of immune dysregulation and facilitate integration into clinical practice. In this Review, we provide an overview of the current state of immune profiling in sepsis, including its use, current challenges, and opportunities for progress. We highlight the important role of immunological biomarkers in facilitating predictive enrichment in current and future treatment scenarios. We propose that multiple immune and non-immune-related parameters, including clinical and microbiological data, be integrated into diagnostic and predictive combitypes, with the aid of machine learning and artificial intelligence techniques. These combitypes could form the basis of workable algorithms to guide clinical decisions that make precision medicine in sepsis a reality and improve patient outcomes.

Association of Asymmetric and Symmetric Dimethylarginine with Inflammation in the Population-Based Study of Health in Pomerania.

The amino acids arginine (Arg), asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are related to nitric oxide (NO) metabolism and potential markers of two different disease entities: cardiovascular disease such as atherosclerosis and systemic inflammation in critically ill patients with sepsis. Although very different in their pathophysiological genesis, both entities involve the functional integrity of blood vessels. In this context, large population-based data associating NO metabolites with proinflammatory markers, e.g., white blood cell count (WBC), high-sensitivity C-reactive protein (hsCRP), and fibrinogen, or cytokines are sparse. We investigated the association of Arg, ADMA and SDMA with WBC, hsCRP, and fibrinogen in 3556 participants of the Study of Health in Pomerania (SHIP)-TREND study. Furthermore, in a subcohort of 456 subjects, 31 inflammatory markers and cytokines were analyzed. We identified Arg and SDMA to be positively associated with hsCRP (ß coefficient 0.010, standard error (SE) 0.002 and 0.298, 0.137, respectively) as well as fibrinogen (ß 5.23 × 10-3, SE 4.75 × 10-4 and 0.083, 0.031, respectively). ADMA was not associated with WBC, hsCRP, or fibrinogen. Furthermore, in the subcohort, Arg was inversely related to a proliferation-inducing ligand (APRIL). SDMA was positively associated with osteocalcin, tumor necrosis factor receptor 1 and 2, and soluble cluster of differentiation 30. Our findings provide new insights into the involvement of Arg, ADMA, and SDMA in subclinical inflammation in the general population.

Agreement between cardiac output measurements by pulse wave analysis using the Pressure Recording Analytical Method and transthoracic echocardiography in patients with veno-venous extracorporeal membrane oxygenation therapy: An observational method comparison.

BACKGROUND: Measuring cardiac output (CO) is important in patients treated with veno-venous extracorporeal membrane oxygenation (vvECMO) because vvECMO flow and CO need to be balanced. Uncalibrated pulse wave analysis with the Pressure Recording Analytical Method (PRAM) may be suitable to measure CO in patients with vvECMO therapy. OBJECTIVE: To assess the agreement between CO measured by PRAM (PRAM-CO; test method) and CO measured by transthoracic echocardiography (TTE-CO; reference method). DESIGN: A prospective observational method comparison study. SETTING: The ICU of a German university hospital between March and December 2021. PATIENTS: Thirty one adult patients with respiratory failure requiring vvECMO therapy: 29 of the 31 patients (94%) were treated for COVID-19 related respiratory failure. MAIN OUTCOME MEASURES: PRAM-CO and TTE-CO were measured simultaneously at two time points in each patient with at least 20 min between measurements. A radial or femoral arterial catheter-derived blood pressure waveform was used for PRAM-CO measurements. TTE-CO measurements were conducted using the pulsed wave Doppler-derived velocity time integral of the left ventricular outflow tract (LVOT) and the corresponding LVOT diameter. PRAM-CO and TTE-CO were compared using Bland-Altman analysis and the percentage error (PE). We defined a PE of <30% as clinically acceptable. RESULTS: Mean ±â€ŠSD PRAM-CO was 6.86 ±â€Š1.49 l min -1 and mean TTE-CO was 6.94 ±â€Š1.58 l min -1 . The mean of the differences between PRAM-CO and TTE-CO was 0.09 ±â€Š0.73 l min -1 with a lower 95% limit of agreement of -1.34 l min -1 and an upper 95% limit of agreement of 1.51 l min -1 . The PE was 21%. CONCLUSIONS: The agreement between PRAM-CO and TTE-CO is clinically acceptable in adult patients with vvECMO therapy.

The SARS-CoV-2 Delta-Omicron Recombinant Lineage (XD) Exhibits Immune-Escape Properties Similar to the Omicron (BA.1) Variant.

Recently, a recombinant SARS-CoV-2 lineage, XD, emerged that harbors a spike gene that is largely derived from the Omicron variant BA.1 in the genetic background of the Delta variant. This finding raised concerns that the recombinant virus might exhibit altered biological properties as compared to the parental viruses and might pose an elevated threat to human health. Here, using pseudotyped particles, we show that ACE2 binding and cell tropism of XD mimics that of BA.1. Further, XD and BA.1 displayed comparable sensitivity to neutralization by antibodies induced upon vaccination with BNT162b2/Comirnaty (BNT) or BNT vaccination followed by breakthrough infection. Our findings reveal important biological commonalities between XD and Omicron BA.1 host cell entry and its inhibition by antibodies.

Renaissance of glucocorticoids in critical care in the era of COVID-19: ten urging questions.

The 40-year-old experience with glucocorticosteroids (GCs) in the context of severe infections is complex and troublesome. Recently, however, a clear indication for GCs in severe COVID-19 has been established. This may constitute a harbinger of a wider use of GCs in critical illnesses. A fundamental prerequisite of such an action is a better understanding of the heterogeneity of critical illness and GCs operationalization within the precision medicine approach. In this perspective, we formulate ten major questions regarding the use of GCs in critical illness. Answering them will likely facilitate a new era of effective and personalized GCs use in modern critical care.

Correction: The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal.

[This corrects the article DOI: 10.1039/D2SC00385F.].

The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal.

[FeFe]-hydrogenases catalyze the reversible conversion of molecular hydrogen into protons and electrons with remarkable efficiency. However, their industrial applications are limited by their oxygen sensitivity. Recently, it was shown that the [FeFe]-hydrogenase from Clostridium beijerinckii (CbA5H) is oxygen-resistant and can be reactivated after oxygen exposure. In this work, we used multifrequency continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy to characterize the active center of CbA5H, the H-cluster. Under oxidizing conditions, the spectra were dominated by an additional and unprecedented radical species. The generation of this radical signal depends on the presence of an intact H-cluster and a complete proton transfer pathway including the bridging azadithiolate ligand. Selective 57Fe enrichment combined with isotope-sensitive electron-nuclear double resonance (ENDOR) spectroscopy revealed a spin density distribution that resembles an H-cluster state. Overall, we uncovered a radical species in CbA5H that is potentially involved in the redox sensing of CbA5H.

Evidence for an ACE2-Independent Entry Pathway That Can Protect from Neutralization by an Antibody Used for COVID-19 Therapy.

SARS-CoV-2 variants of concern (VOC) acquired mutations in the spike (S) protein, including E484K, that confer resistance to neutralizing antibodies. However, it is incompletely understood how these mutations impact viral entry into host cells. Here, we analyzed how mutations at position 484 that have been detected in COVID-19 patients impact cell entry and antibody-mediated neutralization. We report that mutation E484D markedly increased SARS-CoV-2 S-driven entry into the hepatoma cell line Huh-7 and the lung cell NCI-H1299 without augmenting ACE2 binding. Notably, mutation E484D largely rescued Huh-7 but not Vero cell entry from blockade by the neutralizing antibody Imdevimab and rendered Huh-7 cell entry ACE2-independent. These results suggest that the naturally occurring mutation E484D allows SARS-CoV-2 to employ an ACE2-independent mechanism for entry that is largely insensitive against Imdevimab, an antibody employed for COVID-19 therapy. IMPORTANCE The interaction of the SARS-CoV-2 spike protein (S) with the cellular receptor ACE2 is considered essential for infection and constitutes the key target for antibodies induced upon infection and vaccination. Here, using a surrogate system for viral entry, we provide evidence that a naturally occurring mutation can liberate SARS-CoV-2 from ACE2-dependence and that ACE2-independent entry may protect the virus from neutralization by an antibody used for COVID-19 therapy.

SARS-CoV-2 variants C.1.2 and B.1.621 (Mu) partially evade neutralization by antibodies elicited upon infection or vaccination.

Rapid spread of SARS-CoV-2 variants C.1.2 and B.1.621 (Mu variant) in Africa and the Americas, respectively, as well as a high number of mutations in the viral spike proteins raised concerns that these variants might pose an elevated threat to human health. Here, we show that C.1.2 and B.1.621 spike proteins mediate increased entry into certain cell lines but do not exhibit increased ACE2 binding. Further, we demonstrate that C.1.2 and B.1.621 are resistant to neutralization by bamlanivimab but remain sensitive to inhibition by antibody cocktails used for COVID-19 therapy. Finally, we show that C.1.2 and B.1.621 partially escape neutralization by antibodies induced upon infection and vaccination, with escape of vaccine-induced antibodies being as potent as that measured for B.1.351 (Beta variant), which is known to be highly neutralization resistant. Collectively, C.1.2 and B.1.621 partially evade control by vaccine-induced antibodies, suggesting that close monitoring of these variants is warranted.

Kinetics of human leukocyte antigen receptor HLA-DR+ monocytes and T lymphocytes during remission induction therapy in ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is characterized by small vessel inflammation and the presence of autoantibodies against cytoplasmic proteases, most often proteinase-3 and myeloperoxidase. Peripheral blood monocytes are an important source of local macrophage accumulation within parenchymal organs, as evidenced by their presence in early lesions in ANCA-associated glomerulonephritis. Major histocompatibility complex (MHC) II cell surface receptor human leukocyte antigen receptor (HLA-DR) allows antigen presentation to T cells and is crucial for the initiation of an immune response. We herein report HLA-DR abundance in AAV and the kinetics of HLA-DR+ monocytes and T lymphocytes during remission induction therapy in AAV. Life-threatening AAV with pulmonary hemorrhage and renal involvement was associated with the presence of HLA-DR in a considerable population of peripheral blood monocytes and T lymphocytes, and relapsing disease manifested despite persistent B cell depletion after remission induction with rituximab. Moreover, remission induction in AAV with steroids, plasma exchange and intravenous cyclophosphamide, and improvement of clinical symptoms were associated with a decrease in HLA-DR+ differing between monocytes and T lymphocytes. Particularly, persistent suppression of HLA-DR+ monocytes was observed during remission induction, while an initial decrease in HLA-DR+ T lymphocytes was followed by recovery of this population during the further course. Detailed insights into HLA-DR kinetics could pave the way towards an increased understanding of immunopathology and identify patients that could mostly benefit from distinct remission induction regimens.

The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control of the COVID-19 pandemic.

The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike protein raised concerns that the virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent patients or individuals vaccinated with the BioNTech-Pfizer vaccine (BNT162b2) with 12- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1 (Astra Zeneca-Oxford)/BNT162b2 vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.

No evidence for increased cell entry or antibody evasion by Delta sublineage AY.4.2.

Since the beginning of the COVID-19 pandemic, multiple SARS-CoV-2 variants have emerged. While some variants spread only locally, others, referred to as variants of concern, disseminated globally and became drivers of the pandemic. All SARS-CoV-2 variants harbor mutations relative to the virus circulating early in the pandemic, and mutations in the viral spike (S) protein are considered of particular relevance since the S protein mediates host cell entry and constitutes the key target of the neutralizing antibody response. As a consequence, mutations in the S protein may increase SARS-CoV-2 infectivity and enable its evasion of neutralizing antibodies. Furthermore, mutations in the S protein can modulate viral transmissibility and pathogenicity.