Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions.

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

De Novo Design of Integrin α5ß1 Modulating Proteins for Regenerative Medicine.

Integrin α5ß1 is crucial for cell attachment and migration in development and tissue regeneration, and α5ß1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5ß1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5ß1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine. One-Sentence Summary: A de novo-designed fibronectin substitute, NeoNectin, is specific for integrin α5ß1 and can be incorporated into biomaterials for regenerative medicine.

Evolution and Impact of a Diagnostic Point-of-Care Ultrasound Program in a PICU.

OBJECTIVES: To evaluate the impact of point-of-care ultrasound (POCUS) use on clinicians within a PICU and to assess infrastructural elements of our POCUS program development. DESIGN: Retrospective observational study. SETTING: Large academic, noncardiac PICU in the United States. SUBJECTS: Patients in a PICU who had diagnostic POCUS performed. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Between January 1, 2017, and December 31, 2022, 7201 diagnostic POCUS studies were ordered; 1930 (26.8%) had a quality assurance (QA) record generated in an independent POCUS QA database. The cardiac domain was most frequently imaged (81.0% of ordered studies, 81.2% of reviewed studies). POCUS images changed clinician understanding of pathophysiology in 563 of 1930 cases (29.2%); when this occurred, management was changed in 318 of 563 cases (56.5%). Cardiac POCUS studies altered clinician suspected pathophysiology in 30.1% of cases (472/1568), compared with 21.5% (91/362) in noncardiac studies (p = 0.06). Among cases where POCUS changed clinician understanding, management changed more often following cardiac than noncardiac POCUS (p = 0.02). Clinicians identified a need for cardiology consultation or complete echocardiograms in 294 of 1568 cardiac POCUS studies (18.8%). Orders for POCUS imaging increased by 94.9%, and revenue increased by 159.4%, from initial to final study year. QA database use by both clinicians and reviewers decreased annually as QA processes evolved in the setting of technologic growth and unit expansion. CONCLUSIONS: Diagnostic POCUS imaging in the PICU frequently yields information that alters diagnosis and changes management. As PICU POCUS use increased, QA processes evolved resulting in decreased use of our initial QA database. Modifications to QA processes are likely necessary as clinical contexts change over time.

A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS-CoV-2-Implications for Future Drug Discovery Efforts.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

Delocalized quinolinium-macrocyclic peptides, an atypical chemotype for CNS penetration.

Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.

Retrospective Outcomes Comparison by Treatment Location for Pediatric Mild and Moderate Diabetic Ketoacidosis.

OBJECTIVES: Pediatric diabetic ketoacidosis (DKA) is often treated in a PICU, but nonsevere DKA may not necessitate PICU admission. At our institution, nonsevere DKA was treated on the floor until policy change shifted care to the PICU. We describe outcomes in pediatric mild to moderate DKA by treatment location. METHODS: Patients aged 2 to 21 with mild to moderate DKA (pH <7.3 but >7.1), treated on the floor from January 1, 2018 to July 31, 2020 and PICU from August 1, 2020 to October 1, 2022 were included. We performed a single-center, retrospective cohort study; primary outcome was DKA duration (from emergency department diagnosis to resolution), secondary outcomes included hospital length of stay, and complication rates, based on treatment location. RESULTS: Seventy nine floor and 65 PICU encounters for mild to moderate pediatric DKA were analyzed. There were no differences in demographics, initial pH, or bicarbonate; PICU patients had more acute kidney injury on admission. Floor patients had a shorter DKA duration (10 hours [interquartile range 7-13] vs 11 hours [9-15]; P = .04), and a shorter median length of stay (median 43.5 hours [interquartile range 31-62] vs 49 hours [32-100]; P < .01). No patients had clinical signs of cerebral edema; other complications occurred at similar rates. PICU patients received significantly more intravenous electrolyte boluses, but there were no differences in dysrhythmia or electrolyte abnormalities on final serum chemistry. CONCLUSIONS: Our study did not find a clear benefit to admitting patients with mild to moderate DKA to the PICU instead of the hospital floor. Our findings suggest that some children with nonsevere DKA may be treated safely in a non-PICU setting.

Binding and sensing diverse small molecules using shape-complementary pseudocycles.

We describe an approach for designing high-affinity small molecule-binding proteins poised for downstream sensing. We use deep learning-generated pseudocycles with repeating structural units surrounding central binding pockets with widely varying shapes that depend on the geometry and number of the repeat units. We dock small molecules of interest into the most shape complementary of these pseudocycles, design the interaction surfaces for high binding affinity, and experimentally screen to identify designs with the highest affinity. We obtain binders to four diverse molecules, including the polar and flexible methotrexate and thyroxine. Taking advantage of the modular repeat structure and central binding pockets, we construct chemically induced dimerization systems and low-noise nanopore sensors by splitting designs into domains that reassemble upon ligand addition.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane ß-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.

De novo design of alpha-beta repeat proteins.

Proteins composed of a single structural unit tandemly repeated multiple times carry out a wide range of functions in biology. There has hence been considerable interest in designing such repeat proteins; previous approaches have employed strict constraints on secondary structure types and relative geometries, and most characterized designs either mimic a known natural topology, adhere closely to a parametric helical bundle architecture, or exploit very short repetitive sequences. Here, we describe Rosetta-based and deep learning hallucination methods for generating novel repeat protein architectures featuring mixed alpha-helix and beta-strand topologies, and 25 new highly stable alpha-beta proteins designed using these methods. We find that incorporation of terminal caps which prevent beta strand mediated intermolecular interactions increases the solubility and monomericity of individual designs as well as overall design success rate.

Screening patients in general practice for advanced chronic liver disease using an innovative IT solution: The Liver Toolkit.

BACKGROUND: Identifying patients with undiagnosed advanced chronic liver disease (ACLD) is a public health challenge. Patients with advanced fibrosis or compensated cirrhosis have much better outcomes than those with decompensated disease and may be eligible for interventions to prevent disease progression. METHODS: A cloud-based software solution ("the Liver Toolkit") was developed to access primary care practice software to identify patients at risk of ACLD. Clinical history and laboratory results were extracted to calculate aspartate aminotransferase-to-platelet ratio index and fibrosis 4 scores. Patients identified were recalled for assessment, including Liver Stiffness Measurement (LSM) via transient elastography. Those with an existing diagnosis of cirrhosis were excluded. RESULTS: Existing laboratory results of more than 32,000 adults across nine general practices were assessed to identify 703 patients at increased risk of ACLD (2.2% of the cohort). One hundred seventy-nine patients (26%) were successfully recalled, and 23/179 (13%) were identified to have ACLD (LSM ≥10.0 kPa) (10% found at indeterminate risk [LSM 8.0-9.9 kPa] and 77% low risk of fibrosis [LSM <8.0 kPa]). In most cases, the diagnosis of liver disease was new, with the most common etiology being metabolic dysfunction-associated steatotic liver disease (n=20, 83%). Aspartate aminotransferase-to-platelet ratio index ≥1.0 and fibrosis 4 ≥3.25 had a positive predictive value for detecting ACLD of 19% and 24%, respectively. Patients who did not attend recall had markers of more severe disease with a higher median aspartate aminotransferase-to-platelet ratio index score (0.57 vs. 0.46, p=0.041). CONCLUSIONS: This novel information technology system successfully screened a large primary care cohort using existing laboratory results to identify patients at increased risk ACLD. More than 1 in 5 patients recalled were found to have liver disease requiring specialist follow-up.

The Development and Evaluation of Novel Patient Educational Material for a Variant of Uncertain Significance (VUS) Result in Hereditary Cancer Genes.

A Variant of Uncertain Significance (VUS) is a difference in the DNA sequence with uncertain consequences for gene function. A VUS in a hereditary cancer gene should not change medical care, yet some patients undergo medical procedures based on their VUS result, highlighting the unmet educational needs among patients and healthcare providers. To address this need, we developed, evaluated, and refined novel educational materials to explain that while VUS results do not change medical care, it remains important to share any personal or family history of cancer with family members given that their personal and family medical history can guide their cancer risk management. We began by reviewing the prior literature and transcripts from interviews with six individuals with a VUS result to identify content and design considerations to incorporate into educational materials. We then gathered feedback to improve materials via a focus group of multidisciplinary experts and multiple rounds of semi-structured interviews with individuals with a VUS result. Themes for how to improve content, visuals, and usefulness were used to refine the materials. In the final round of interviews with an additional 10 individuals with a VUS result, materials were described as relatable, useful, factual, and easy to navigate, and also increased their understanding of cancer gene VUS results.

De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Computational design of soluble and functional membrane protein analogues.

De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.

Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.

Inhibition of Interleukin-33 to Reduce Glomerular Endothelial Inflammation in Diabetic Kidney Disease.

Introduction: Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Methods: Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling in vitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Results: Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. Conclusion: We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.

Engaging a community to focus on upper limb function in people with multiple sclerosis: the ThinkHand campaign case study.

BACKGROUND: Solving complex research challenges requires innovative thinking and alternative approaches to traditional methods. One such example is the problem of arm and hand, or upper limb function in multiple sclerosis (MS), a neurological condition affecting approximately 2.9 million people worldwide and more than 150,000 in the United Kingdom. Historically, clinical trials and research have focused on mobility and walking ability. This excludes a large number of patients who are wheelchair users, limiting their quality of life and restricting access to possibly helpful medications. To address this issue, the ThinkHand campaign was launched in 2016, aiming to raise awareness about the importance of upper limb function in MS and develop alternative ways to measure, record, and account for hand and arm function changes. MAIN BODY: The campaign utilised innovative strategies at scientific conferences and online surveys to engage people affected by MS, healthcare professionals, charities, and researchers in discussing the importance of preserving upper limb function. Through co-design and interdisciplinary collaboration, the campaign developed new tools like the low-cost cardboard version of the Nine-Hole Peg Test, facilitating remote monitoring of hand function. Additionally, the campaign co-created the "Under & Over" rehabilitation tool, allowing individuals with advanced MS to participate in a remote rehabilitation program.The impact of the ThinkHand campaign has been significant, helping to shift the focus of both academic and industry-supported trials, including the O'HAND and ChariotMS trials, both using upper limb function as their primary end point. The campaign's patient-centred approach highlighted the importance of recognising patients' perspectives in research and challenged established assumptions and practices. It demonstrated the effectiveness of interdisciplinary collaboration, systems thinking, and co-creation with stakeholders in tackling complex problems. CONCLUSION: The ThinkHand campaign provides valuable insights for health research practices. By involving patients at all stages, researchers can gain a deeper understanding of the impact of disease on their lives, identify gaps and focus research on their needs. Experimentation and iteration can lead to innovative solutions, and openness to unconventional methods can drive widespread change. The ThinkHand campaign exemplifies the potential of patient-centred approaches to address complex research challenges and revolutionise the field of MS research and management. Embracing such approaches will contribute to more inclusive and impactful research in the future.

Solving complex research challenges requires creative thinking and new ways of doing things. One such challenge is understanding the problems with arm and hand function in multiple sclerosis (MS), a neurological condition that affects more than 150,000 in the United Kingdom. In the past, research focused mainly on walking ability, leaving out many people who use wheelchairs.To tackle this issue, we created the ThinkHand campaign in 2016. Its goal was to raise awareness about the importance of hand and arm function for people with MS (pwMS) and find better ways to measure changes in these functions such that they can become outcomes in clinical trials. This could provide a pathway to better treatments for pwMS who cannot walk.The campaign used various methods, including surveys, social media posts, exhibitions and music to involve pwMS, healthcare professionals, charities, and researchers in discussions about the issues. Working together, they created tools to support pwMS, particularly those at an advanced stage of the disease (pwAMS), to take part in research and measure their hand and arm function. Through our collaborative approach focusing on patients' perspectives, the campaign challenged old ideas and deeply embedded practices. It showed that collaboration between different areas of expertise involving pwMS at all stages of research can help solve complex problems. This campaign teaches us valuable lessons for health research. When researchers listen to patients and try new things, they can better understand how a disease affects people's lives and develop better solutions.In conclusion, we show how embracing a patient-centred approach can address complex research challenges and improve how we study and manage MS and other conditions in the future.

Reconciling lesions, relapses and smouldering associated worsening: A unifying model for multiple sclerosis pathogenesis.

The failure of relapses and white matter lesions to properly explain long-term disability and progression in multiple sclerosis is compounded by its artificial separation into relapsing remitting, secondary progressive, and primary progressive pigeonholes. The well-known epidemiological disconnection between relapses and long-term disability progression has been rediscovered as "progression independent of relapse activity", i.e. smouldering multiple sclerosis. This smouldering associated worsening proceeds despite early and prolonged use of disease modification therapies, even those that are highly effective at preventing relapses and new/enhancing white matter lesions on MRI. We recognise that smouldering associated worsening and relapse/lesion associated worsening coexist, to varying extents. The extent of cortical demyelination has been shown to correlate significantly with the severity of diffuse injury in normal appearing white matter (post mortem histopathologically (r = 0.55; P = 0.001), and in vivo with MRI (r = -0.6874; P = 0.0006)) and does so independently of white matter lesion burden. Axon loss in the normal appearing white matter explains disability in multiple sclerosis better than focal white matter lesions do. Smouldering associated worsening typically manifests as a length-dependent central axonopathy. We propose a unifying model for multiple sclerosis pathogenesis, wherein accumulation of cortical lesion burden predisposes associated normal appearing white matter to diffuse injury, whilst also intensifying damage within white matter lesions. Our novel two-hit hypothesis implicates cortical disease as a culprit for smouldering multiple sclerosis, abetted by active focal inflammation in the white matter (and vice versa). Substantiation of the two-hit hypothesis would advance the importance of specific therapeutic intervention for (and monitoring of) cortical/meningeal inflammation in people with multiple sclerosis.

Immunogenicity of biologics used in the treatment of asthma.

PURPOSE OF THE REVIEW: Asthma is a major global disease affecting adults and children, which can lead to hospitalization and death due to breathing difficulties. Although targeted monoclonal antibody therapies have revolutionized treatment of severe asthma, some patients still fail to respond. Here we critically evaluate the literature on biologic therapy failure in asthma patients with particular reference to anti-drug antibody production, and subsequent loss of response, as the potential primary cause of drug failure in asthma patients. RECENT FINDINGS: Encouragingly, asthma in most cases responds to treatment, including the use of an increasing number of biologic drugs in moderate to severe disease. This includes monoclonal antibody inhibitors of immunoglobulin E and cytokines, including interleukin 4, 5, or 13 and thymic stromal lymphopoietin. These limit mast cell and eosinophil activity that cause the symptomatic small airways obstruction and exacerbations. SUMMARY: Despite humanization of the antibodies, it is evident that benralizumab; dupilumab; mepolizumab; omalizumab; reslizumab and tezepelumab all induce anti-drug antibodies to some extent. These can contribute to adverse events including infusion reactions, serum sickness, anaphylaxis and potentially disease activity due to loss of therapeutic function. Monitoring anti-drug antibodies (ADA) may allow prediction of future treatment-failure in some individuals allowing treatment cessation and switching therefore potentially limiting disease breakthrough.

The reduced net carbon uptake over Northern Hemisphere land causes the close-to-normal CO2 growth rate in 2021 La Niña.

La Niña climate anomalies have historically been associated with substantial reductions in the atmospheric CO2 growth rate. However, the 2021 La Niña exhibited a unique near-neutral impact on the CO2 growth rate. In this study, we investigate the underlying mechanisms by using an ensemble of net CO2 fluxes constrained by CO2 observations from the Orbiting Carbon Observatory-2 in conjunction with estimates of gross primary production and fire carbon emissions. Our analysis reveals that the close-to-normal atmospheric CO2 growth rate in 2021 was the result of the compensation between increased net carbon uptake over the tropics and reduced net carbon uptake over the Northern Hemisphere mid-latitudes. Specifically, we identify that the extreme drought and warm anomalies in Europe and Asia reduced the net carbon uptake and offset 72% of the increased net carbon uptake over the tropics in 2021. This study contributes to our broader understanding of how regional processes can shape the trajectory of atmospheric CO2 concentration under climate change.