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Abstract Background Although COVID-19 initially caused great concern about respiratory symptoms, mounting evidence shows that also the pancreas is productively infected by SARS-CoV-2. However, the severity of pancreatic SARS-CoV-2 infection and its pathophysiology is still under debate. Here, we investigate the consequences of SARS-CoV-2 pancreatic infection and the role of the host factor Placenta-associated protein (PLAC8). Methods We analyze plasma levels of pancreatic enzymes and inflammatory markers in a retrospective cohort study of 120 COVID-19 patients distributed in 3 severity-stratified groups. We study the expression of SARS-CoV-2 and PLAC8 in the pancreas of deceased COVID-19 patients as well as in non-infected donors. We perform pseudovirus infection experiments in PLAC8 knock-out PDAC and human beta cell-derived cell lines and validate results with SARS-CoV-2 virus. Results We find that analysis of circulating pancreatic enzymes aid the stratification of patients according to COVID-19 severity and predicts outcomes. Interestingly, we find an association between PLAC8 expression and SARS-CoV-2 infection in postmortem analysis of COVID-19 patients both in the pancreas and in other bonafide SARS-CoV-2 target tissues. Functional experiments demonstrate the requirement of PLAC8 in SARS-CoV-2 pancreatic productive infection by pseudovirus and full SARS-CoV-2 infectious virus inoculum from Wuhan-1 and BA.1 strains. Finally, we observe an overlap between PLAC8 and SARS-CoV-2 immunoreactivities in the pancreas of deceased patients. Conclusions Our data indicate the human pancreas as a SARS-CoV-2 target with plausible signs of injury and demonstrate that the host factor PLAC8 is required for SARS-CoV-2 pancreatic infection, thus defining new target opportunities for COVID-19-associated pancreatic pathogenesis.

Abstract Urothelial carcinoma (UC) of the urinary bladder has significant challenges in treatment due to its diverse genetic landscape and variable response to systemic therapy. In recent years, patient-derived organoids (PDOs) emerged as a novel tool to model primary tumors with higher resemblance than conventional 2D cell culture approaches. However, the potential of organoids to predict therapy response in a clinical setting remains to be evaluated. This study explores the clinical feasibility of PDOs for pharmacotyping in UC. Initially, we subjected tumor tissue specimens from 50 patients undergoing transurethral resection or radical cystectomy to organoid propagation, of whom 19 (38%) yielded PDOs suitable for drug sensitivity assessment. Notably, whole transcriptome-based analysis indicated that PDOs may show phenotypes distinct from their parental tumor tissue. Pharmacotyping within a clinically relevant timeframe [mean of 35.44 and 55 days for non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC), respectively] was achieved. Drug sensitivity analyses revealed marked differences between NMIBC and MIBC, with MIBC-derived organoids demonstrating higher chemosensitivity toward clinically relevant drugs. A case study correlating organoid response with patient treatment outcome illustrated the complexity of predicting chemotherapy efficacy, especially considering the rapid acquisition of drug resistance. We propose a workflow of prospective organoid-based pharmacotyping in UC, enabling further translational research and integration of this approach into clinical practice.

Background Pancreatic ductal adenocarcinoma (PDAC) is mostly refractory to immunotherapy due to immunosuppression in the tumor microenvironment and cancer cell-intrinsic T cell tolerance mechanisms. PDAC is described as a “cold” tumor type with poor infiltration by T cells and factors leading to intratumoral T cell suppression have thus received less attention. Here, we identify a cancer cell-intrinsic mechanism that contributes to a T cell-resistant phenotype and describes potential combinatorial therapy.Methods We used an unbiased screening approach of T cell resistant and sensitive murine KPC (KrasLSL-G12D/+; Trp53fl/fl; Ptf1aCre/+) PDAC cells in a three-dimensional co-culture platform with syngeneic antigen-educated T cells to identify potential cell-intrinsic drivers of T cell suppression in PDAC. Comparative transcriptomic analysis was performed to reveal promising candidates that mediate resistance to T cells. We investigated their contribution by shRNA-mediated knockdown and pharmacological inhibition in murine in vitro and in vivo studies, as well as in patient-derived organoids (PDOs). A combination of transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to validate the underlying T cell response phenotypes of PDAC cells. The action of TGM2 via interaction with tubulin and the impact of microtubule dynamics and vesicle trafficking were evaluated by protein analyses and live-cell imaging. Correlation analyses via TCGA data complemented the functional studies.Results We identified transglutaminase 2 (TGM2) as a mediator of T cell suppression in PDAC. We report that high levels of TGM2 expression in patients’ tumors correlate with immunosuppressive signatures and poor overall survival. We found that TGM2 regulates vesicle trafficking by modulating microtubule network density and dynamics in pancreatic cancer cells, thus facilitating the secretion of immunosuppressive cytokines, which impair effector T cell functionality. In TGM2-expressing PDOs, pharmacological TGM2 inhibition or treatment with nocodazole increased T cell-mediated apoptosis. Also, pretreatment of TGM2high PDOs with sublethal doses of the spindle poisons paclitaxel or vincristine increased CD8+T cell activation and sensitized PDOs toward T cell-mediated cytotoxicity.Conclusions These findings indicate that targeting microtubular function therapeutically may enhance antitumor T cell responses by impacting activity of immunosuppressive cytokines in the PDAC microenvironment.

Patients who survive critical COVID-19 frequently report post-acute sequelae of COVID-19 (PASC) such as psychosomatic and neurocognitive health problems. The goal of this study was to identify clinical risk factors and other predictors for such long-term consequences in severely ill COVID-19 patients. Adult COVID-19 intensive care unit (ICU) survivors from August 2020 to May 2021 were enrolled. A broad range of clinical, laboratory and chest computed tomography (CT) data was collected during their ICU stays. The association between ICU predictors and psychosomatic, respiratory, and neurocognitive assessments 12 months after ICU discharge was analyzed using univariate regression analysis. In 17 patients (mean age 58.9 ± 11.4 years), laboratory markers (CRP, lymphocytes, hemoglobin), ICU severity (SOFA, SAPS II, need for mechanical ventilation), complications (ARDS), and lung CT data (ground-glass opacity) were promising predictors of depressive and anxiety symptoms, fatigue, and sleep problems. Recovery of psychosomatic health such as fatigue, depression, and anxiety correlated with lower levels of inflammation and high hemoglobin levels. ARDS, mechanical ventilation, and worse SOFA and SAPS II scores were further risk factors for depressive and anxiety symptoms. Our study identified novel associations such as pulmonary ground-glass opacity being positively associated with depression, anxiety, fatigue, and insomnia levels.

The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

AbstractThe "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Abstract Background The coronavirus disease 2019 (COVID-19) pandemic led to a global increase in healthcare-associated infections (HAI) among intensive care unit (ICU) patients. Whether this increase is directly attributable to COVID-19 or whether the pandemic indirectly (via staff shortages or breaches in infection prevention measures) led to this increase, remains unclear. The objectives of this study were to assess HAI incidence and to identify independent risk factors for HAI in COVID-19 and non-COVID-19 ICU patients. Methods We established a monocentric prospective HAI surveillance in the medical ICU of our tertiary care center from September 1st 2021 until August 31st 2022, during circulation of the SARS-CoV-2 delta and omicron variants. We consecutively included patients ≥ 18 years of age with an ICU length of stay of > 2 calendar days. HAI were defined according to the European Centre for Disease Prevention and Control definitions. HAI rate was calculated per 1,000 patient-days or device-days; risk ratios (RR) and corresponding 95% confidence intervals (CI) for COVID-19 versus non-COVID-19 patients were calculated. We used multivariable Cox regression to identify independent risk factors for HAI. As a proxy for institutional COVID-19 burden, weekly COVID-19 density (i.e. percentage of COVID-19 patients among all ICU patients) was included in the model as time-dependent co-variable. Results We included 254 patients, 64 (25.1%) COVID-19 and 190 (74.9%) non-COVID-19 patients; 83 HAI in 72 patients were recorded, thereof 45 ventilator-associated lower respiratory tract infections (VA-LRTI) (54.2%) and 18 blood stream infections (BSI) (21.6%). HAI incidence rate was 49.1/1,000 patient-days in COVID-19 and 22.5/1,000 patient-days in non-COVID-19 patients (RR 2.2, 95%-CI 1.4–3.4). This result was mainly due to different VA-LRTI rates (40.3 vs. 11.7/1,000 ventilator days, p < 0.001), whereas BSI rates were not statistically different (9.4 vs. 5.6/1,000 patient days, p = 0.27). Multivariable analysis identified COVID-19 as main risk factor for HAI development, whereas age, mechanical ventilation and COVID-19 density were not significant. Conclusions These data from the fourth and fifth wave of the pandemic show a higher HAI incidence in COVID-19 than in non-COVID-19 ICU patients, mainly due to an increase in pulmonary infections. A diagnosis of COVID-19 was independently associated with HAI development, whereas institutional COVID-19 burden was not.

Abstract Background Autoresuscitation is the phenomenon of spontaneous return of circulation after cessation of CPR, also known as the Lazarus phenomenon. Most of the evidence is based on case reports and a few systematic reviews. The occurrence of autoresuscitation may lead to self-reproach and dismay in affected emergency personnel and may rise questions about the correct procedure after terminating resuscitative efforts. In contrast to existing cardiac arrest guidelines there is no standardized approach to terminating resuscitative attempts. Case We report a case of out of hospital autoresuscitation in a 67-year-old female after 60 min of advanced cardiac life support. After shock refractory shockable rhythm, we recorded pulseless electrical activity and fixed pupils, consequently resuscitation was terminated. About 50 min later the patient surprisingly showed signs of life. Due to the suggestive history a coronary angiography was performed, showing severe coronary heart disease which necessitated surgical intervention. After ACBP surgery and intensive care followed by treatment on the cardiological ward, she was finally discharged to neurological rehabilitation. Conclusion As already proposed by existing literature, there should be at least a 10-min interval of close monitoring after abandoning CPR. Transport of a deceased patient should only take place after secure signs of death can be detected. Further investigation is needed to determine which patients are most likely to benefit from an extended observation period. Our case reports highlights the difficulties in death declaration and the importance of close monitoring after abandoning CPR.

Genes involved in distinct diabetes types suggest shared disease mechanisms. Here we show that One Cut Homeobox 1 (ONECUT1) mutations cause monogenic recessive syndromic diabetes in two unrelated patients, characterized by intrauterine growth retardation, pancreas hypoplasia and gallbladder agenesis/hypoplasia, and early-onset diabetes in heterozygous relatives. Heterozygous carriers of rare coding variants of ONECUT1 define a distinctive subgroup of diabetic patients with early-onset, nonautoimmune diabetes, who respond well to diabetes treatment. In addition, common regulatory ONECUT1 variants are associated with multifactorial type 2 diabetes. Directed differentiation of human pluripotent stem cells revealed that loss of ONECUT1 impairs pancreatic progenitor formation and a subsequent endocrine program. Loss of ONECUT1 altered transcription factor binding and enhancer activity and NKX2.2/NKX6.1 expression in pancreatic progenitor cells. Collectively, we demonstrate that ONECUT1 controls a transcriptional and epigenetic machinery regulating endocrine development, involved in a spectrum of diabetes, encompassing monogenic (recessive and dominant) as well as multifactorial inheritance. Our findings highlight the broad contribution of ONECUT1 in diabetes pathogenesis, marking an important step toward precision diabetes medicine.

Genetic risk variants that have been identified in genome-wide association studies of complex diseases are primarily non-coding1. Translating these risk variants into mechanistic insights requires detailed maps of gene regulation in disease-relevant cell types2. Here we combined two approaches: a genome-wide association study of type 1 diabetes (T1D) using 520,580 samples, and the identification of candidate cis-regulatory elements (cCREs) in pancreas and peripheral blood mononuclear cells using single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) of 131,554 nuclei. Risk variants for T1D were enriched in cCREs that were active in T cells and other cell types, including acinar and ductal cells of the exocrine pancreas. Risk variants at multiple T1D signals overlapped with exocrine-specific cCREs that were linked to genes with exocrine-specific expression. At the CFTR locus, the T1D risk variant rs7795896 mapped to a ductal-specific cCRE that regulated CFTR; the risk allele reduced transcription factor binding, enhancer activity and CFTR expression in ductal cells. These findings support a role for the exocrine pancreas in the pathogenesis of T1D and highlight the power of large-scale genome-wide association studies and single-cell epigenomics for understanding the cellular origins of complex disease.

Transcutaneous auricular vagus nerve stimulation (taVNS) is becoming increasingly established in the treatment of various neurological and psychiatric diseases. However, only a few studies have focused on the overall influence of taVNS on cortical excitability in general. The planned study will investigate the effect of taVNS on the excitability of the motor cortex in young healthy subjects. The aim of the study is to gain better understand of the physiological mechanism of taVNS to contribute to new fields of application of taVNS in new areas such as the treatment of stroke or multiple sclerosis. This protocol describes a single-center, prospective, double blind, sham-controlled trial that evaluates the effect of taVNS on motor cortex excitability with a planned sample size of 30 participants. The effect of taVNS is investigated by neuronavigation and electromyography (EMG) coupled transcranial magnetic stimulation (TMS) applied before and after taVNS stimulation. The following parameters are assessed: resting motor threshold (RMT), active motor threshold (AMT), recruitment curve (RC), short intracortical inhibition (SICI), intracortical facilitation (ICF). All parameters will be assessed for taVNS on the basis of perception threshold and tolerance threshold. All investigations performed in the study were reviewed and approved by the local ethics committee of the University Medical Center Greifswald (study reference number: BB048/22).Clinical trial registrationwww.drks.de, number: DRKS00029937.

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

Abstract Homologous recombination deficiency (HRD) leads to DNA double‐strand breaks and can be exploited by the use of poly (ADP‐ribose) polymerase (PARP) inhibitors to induce synthetic lethality. Extending the original therapeutic concept, the role of HRD is currently being investigated in clinical trials testing immune checkpoint blockers alone or in combination with PARP inhibitors, but the relationship between HRD and immune cell context in cancer is incompletely understood. We analyzed the association between immune cell composition, gene expression, and HRD in 9,041 tumors of 32 solid cancer types from The Cancer Genome Atlas (TCGA). The numbers of genomic scars were quantified by the HRD sum score (HRDsum) including loss of heterozygosity, large‐scale state transitions, and telomeric allelic imbalance. The T‐cell inflamed gene expression profile correlated weakly, but significantly positively, with HRDsum across cancer types (ρ = 0.17). Within individual cancer types, a significantly positive correlation was observed only in breast cancer, ovarian cancer, and four other cancer types, but not in the remaining 26 cancer types. HRDsum and tumor mutational burden (TMB) correlated significantly positively across cancer types (ρ = 0.42) and within 18 cancer types. HRDsum and a proliferation metagene correlated significantly positively across cancer types (ρ = 0.52) and within 20 cancer types. Mismatch repair deficiency and HRD as well as proofreading deficiency showed a high level of exclusivity. High HRD scores were associated with an immunologically activated tumor microenvironment only in a minority of cancer types. Our data favor the combination of genetic markers, complex genomic markers (including HRDsum and TMB), and other molecular markers (including proliferation scores) for a precise and comprehensive read‐out of the tumor biology and an individually tailored treatment.

There is a growing interest in mimicking structural color in natural systems aiming for sustainable and long-lasting color. Here the authors report a platform for three-dimensional printing assembly of colloidal particles of silica and carbon that have programmable structural color.

Abstract Background It remains elusive how the characteristics, the course of disease, the clinical management and the outcomes of critically ill COVID-19 patients admitted to intensive care units (ICU) worldwide have changed over the course of the pandemic. Methods Prospective, observational registry constituted by 90 ICUs across 22 countries worldwide including patients with a laboratory-confirmed, critical presentation of COVID-19 requiring advanced organ support. Hierarchical, generalized linear mixed-effect models accounting for hospital and country variability were employed to analyse the continuous evolution of the studied variables over the pandemic. Results Four thousand forty-one patients were included from March 2020 to September 2021. Over this period, the age of the admitted patients (62 [95% CI 60–63] years vs 64 [62–66] years, p

A model of autoimmune pancreatitis is developed by blocking the activation of NF-κB in pancreatic stellate cells, via conditional deletion of NEMO (IKKγ), which presents strong pancreatic inflammation with eosinophilia after the induction of chronic pancreatitis by repeated caerulein challenges.

Abstract Background Despite the adoption of strict infection prevention and control measures, many hospitals have reported outbreaks of multidrug-resistant organisms (MDRO) during the Coronavirus 2019 (COVID-19) pandemic. Following an outbreak of carbapenem-resistant Acinetobacter baumannii (CRAB) in our institution, we sought to systematically analyse characteristics of MDRO outbreaks in times of COVID-19, focussing on contributing factors and specific challenges in controlling these outbreaks. Methods We describe results of our own CRAB outbreak investigation and performed a systematic literature review for MDRO (including Candida auris) outbreaks which occurred during the COVID-19 pandemic (between December 2019 and March 2021). Search terms were related to pathogens/resistance mechanisms AND COVID-19. We summarized outbreak characteristics in a narrative synthesis and contrasted contributing factors with implemented control measures. Results The CRAB outbreak occurred in our intensive care units between September and December 2020 and comprised 10 patients (thereof seven with COVID-19) within two distinct genetic clusters (both ST2 carrying OXA-23). Both clusters presumably originated from COVID-19 patients transferred from the Balkans. Including our outbreak, we identified 17 reports, mostly caused by Candida auris (n = 6) or CRAB (n = 5), with an overall patient mortality of 35% (68/193). All outbreaks involved intensive care settings. Non-adherence to personal protective equipment (PPE) or hand hygiene (n = 11), PPE shortage (n = 8) and high antibiotic use (n = 8) were most commonly reported as contributing factors, followed by environmental contamination (n = 7), prolonged critical illness (n = 7) and lack of trained HCW (n = 7). Implemented measures mainly focussed on PPE/hand hygiene audits (n = 9), environmental cleaning/disinfection (n = 9) and enhanced patient screening (n = 8). Comparing potentially modifiable risk factors and control measures, we found the largest discrepancies in the areas of PPE shortage (risk factor in 8 studies, addressed in 2 studies) and patient overcrowding (risk factor in 5 studies, addressed in 0 studies). Conclusions Reported MDRO outbreaks during the COVID-19 pandemic were most often caused by CRAB (including our outbreak) and C. auris. Inadequate PPE/hand hygiene adherence, PPE shortage, and high antibiotic use were the most commonly reported potentially modifiable factors contributing to the outbreaks. These findings should be considered for the prevention of MDRO outbreaks during future COVID-19 waves.

Introduction: While invasive and associated with risks, metabolic and bariatric surgery (MBS) can promote sustained weight loss and substantial health benefits in youths with extreme obesity. The path toward informed decision making for or against MBS is poorly characterized and postoperative follow-up to assess risks and benefits is inconsistent. In youths with extreme obesity, we aimed to evaluate decision making toward MBS, as well as MBS outcomes and adherence with follow-up and recommendations in the setting of a structured pre- and post-MBS program. Methods: Participants were recruited in the setting of the multicenter “Youth with Extreme Obesity Study” (YES). YES is a cohort study in adolescents and young adults aged 14–24 years with obesity (BMI ≥30.0 kg/m2) who were recruited at four medical centers and one job center in Germany between 2012 and 2018. Participants at two medical centers with BMI ≥35 kg/m2, aged 14–24 years, and interested in pursuing MBS were included in the subproject 3 “Safety and effectiveness of weight loss surgery in adolescents with severe obesity within a structured pre- and post-surgery treatment program – an observational study” that comprised a 2-months pre- and 12-months post-MBS program. Results: Twenty-eight of 169 youths (17%) with BMI ≥35 kg/m2 were interested in MBS. Twenty-six fulfilled published eligibility criteria for MBS and participated in the structured pre-MBS preparation program. Of these, 9 participants (2 females) decided against, and 17 (n = 11 females) decided for MBS (sleeve gastrectomy). The 12-month follow-up rate was high (16/17 [94%]) and all participants achieved significant weight reduction (ΔBMI: −16.1 ± 5.6 kg/m2). Eleven of 16 participants (69%) reported taking the prescribed dietary supplements in the first year after MBS, but only five of them (31%) did so daily. In contrast to the high 12-month retention rate, follow-up after completion of the structured program was low at 24-months (9/16 [56%]) and at 36-months (5/15 [36%]), respectively. Conclusion: Participants demonstrated active decision making for or against MBS and high adherence with the structured pre- and 12 months post-MBS program, but participation was low thereafter. These findings endorse the need for longer term structured post-MBS programs to capture long-term outcomes and provide adequate care in this vulnerable group at the transition to adulthood.

Abstract Porous materials are relevant for a broad range of technologies from catalysis and filtration, to tissue engineering and lightweight structures. Controlling the porosity of these materials over multiple length scales often leads to enticing new functionalities and higher efficiency but has been limited by manufacturing challenges and the poor understanding of the properties of hierarchical structures. Here, we report an experimental platform for the design and manufacturing of hierarchical porous materials via the stereolithographic printing of stable photo-curable Pickering emulsions. In the printing process, the micron-sized droplets of the emulsified resins work as soft templates for the incorporation of microscale porosity within sequentially photo-polymerized layers. The light patterns used to polymerize each layer on the building stage further generate controlled pores with bespoke three-dimensional geometries at the millimetre scale. Using this combined fabrication approach, we create architectured lattices with mechanical properties tuneable over several orders of magnitude and large complex-shaped inorganic objects with unprecedented porous designs.

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, but its spike protein doesn’t efficiently bind human ACE2. Here, the authors show that exchange of spike residue 403 between RaTG13 and SARS-CoV-2 spike proteins affects binding to human ACE2 and entry of pseudotyped viruses.