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Objectives SARS-CoV-2 rapid antigen tests (RAT) provide fast identification of infectious patients when RT-PCR results are not immediately available. We aimed to develop a prediction model for identification of false negative (FN) RAT results. Methods In this multicenter trial, patients with documented paired results of RAT and RT-PCR between October 1st 2020 and January 31st 2021 were retrospectively analyzed regarding clinical findings. Variables included demographics, laboratory values and specific symptoms. Three different models were evaluated using Bayesian logistic regression. Results The initial dataset contained 4,076 patients. Overall sensitivity and specificity of RAT was 62.3% and 97.6%. 2,997 cases with negative RAT results (FN: 120; true negative: 2,877; reference: RT-PCR) underwent further evaluation after removal of cases with missing data. The best-performing model for predicting FN RAT results containing 10 variables yielded an area under the curve of 0.971. Sensitivity, specificity, PPV and NPV for 0.09 as cut-off value (probability for FN RAT) were 0.85, 0.99, 0.7 and 0.99. Conclusion FN RAT results can be accurately identified through ten routinely available variables. Implementation of a prediction model in addition to RAT testing in clinical care can provide a decision guidance for initiating appropriate hygiene measures and therefore helps avoiding nosocomial infections., Graphical abstract Image, graphical abstract

PURPOSE: Neutralizing antibodies can reduce SARS-CoV-2 cellular entry, viral titers, and pathologic damage. CT-P59 (regdanvimab), a SARS-CoV-2 neutralizing monoclonal antibody, was examined in 2 randomized, double-blind, placebo-controlled, single ascending dose, Phase I studies. METHODS: In study 1.1, healthy adults were sequentially enrolled to receive CT-P59 10, 20, 40, or 80 mg/kg or placebo. In study 1.2, adult patients with mild SARS-CoV-2 infection were enrolled to receive CT-P59 20, 40, or 80 mg/kg or placebo. Primary objectives of both studies were safety and tolerability up to day 14 after infusion. Secondary end points included pharmacokinetic properties. Study 1.2 also measured virology and clinical efficacy. FINDINGS: Thirty-two individuals were randomized to study 1.1 (6 per CT-P59 dose cohort and 8 in the placebo cohort). By day 14 after infusion, adverse events (AEs) were reported in 2 individuals receiving CT-P59 20 mg/kg (headache and elevated C-reactive protein levels) and 1 receiving CT-P59 40 mg/kg (pyrexia) (all Common Terminology Criteria for Adverse Events grade 1). In study 1.2, 18 patients were randomized (5 per dose cohort and 3 in the placebo cohort). Sixteen AEs were reported in 10 patients receiving CT-P59. No AEs in either study led to study discontinuation. Greater reductions in viral titers were reported with CT-P59 than placebo in those with maximum titers >105 copies/mL. Mean time to recovery was 3.39 versus 5.25 days. IMPLICATIONS: CT-P59 exhibited a promising safety profile in healthy individuals and patients with mild SARS-CoV-2 infection, with potential antiviral and clinical efficacy in patients with mild SARS-CoV-2 infection. ClinicalTrials.gov identifier: NCT04525079 (study 1.1) and NCT04593641 (study 1.2).

Background & Aims Microbiota dysbiosis and mucosa-associated bacteria are involved in colorectal cancer progression. We hypothesize that an interaction between virulent pathobionts and epithelial defense promotes tumorigenesis. Methods Chemical-induced CRC mouse model was treated with antibiotics at various phases. Colonic tissues and fecal samples were collected in a time-serial mode and analyzed by gene microarray and 16S rRNA sequencing. Intraepithelial bacteria were isolated using a gentamicin resistance assay, and challenged in epithelial cultures. Results Our study showed that antibiotic treatment at midphase but not early or late phase reduced mouse tumor burden, suggesting a time-specific host–microbe interplay. A unique antimicrobial transcriptome profile showing an inverse relationship between autophagy and oxidative stress genes was correlated with a transient surge in microbial diversity and virulence emergence in mouse stool during cancer initiation. Gavage with fimA/fimH/htrA-expressing invasive Escherichia coli isolated from colonocytes increased tumor burden in recipient mice, whereas inoculation of bacteria deleted of htrA or triple genes did not. The invasive E.coli suppressed epithelial autophagy activity through reduction of microtubule-associated protein 1 light-chain 3 transcripts and caused dual oxidase 2–dependent free radical overproduction and tumor cell hyperproliferation. A novel alternating spheroid culture model was developed for sequential bacterial challenge to address the long-term changes in host–microbe interaction for chronic tumor growth. Epithelial cells with single bacterial encounter showed a reduction in transcript levels of autophagy genes while those sequentially challenged with invasive E.coli showed heightened autophagy gene expression to eliminate intracellular microbes, implicating that bacteria-dependent cell hyperproliferation could be terminated at late phases. Finally, the presence of bacterial htrA and altered antimicrobial gene expression were observed in human colorectal cancer specimens. Conclusions Invasive pathobionts contribute to cancer initiation during a key time frame by counterbalancing autophagy and oxidative stress in the colonic epithelium. Monitoring gut microbiota and antimicrobial patterns may help identify the window of opportunity for intervention with bacterium-targeted precision medicine., Graphical abstract

Highlights • Non-severe COVID-19 patients have abnormal laboratory investigations. • Patients with prolonged pretreatment APTT have a longer isolation length. • Patients with elevated eosinophils after treatment have a shorter isolation length. • A nomogram could help to predict isolation probability at 11-, 16- and 21-day., Background Majority coronavirus disease 2019 (COVID-19) patients are classified as mild and moderate (non-severe) diseases. We aim to develop a model to predict isolation length for non-severe patients. Methods Among 188 non-severe patients, 96 patients were enrolled as training cohort to identify factors associated with isolation length via Cox regression model and develop a nomogram. Other 92 patients formed as validation cohort to validate nomogram. Concordance index (C-index), area under the curve (AUC) and calibration curves were used to evaluated nomogram. Results Increasing absolute eosinophil count (AEC) after admission was correlated with shorter isolation length (P = 0.02). Baseline activated partial thromboplastin time (APTT) > 30 s was correlated with longer isolation length (P = 0.03). A nomogram to predict isolation probability at 11-, 16- and 21-day was developed and validated. The C-indices of training and validation cohort were 0.604 and 0.682 respectively. Both cohorts showed a good discriminative ability (AUC, 11-day: 0.646 vs 0.730; 16-day: 0.663 vs 0.750; 21-day: 0.711 vs 0.783; respectively) and calibration power. Conclusions Baseline APTT and dynamic change of AEC were two significant factors associated with isolation length of non-severe patients. Nomogram could predict isolation probability for each patient to estimate appropriate quarantine length.

Background & Aims Yes-associated protein (YAP) and its paralog transcriptional co-activator with post synaptic density protein, drosophila disc large tumor suppressor and zonula occludens-1-binding motif (TAZ) are 2 co-activators downstream of Hippo tumor-suppressor cascade. Both have been implicated in the development of hepatocellular carcinoma (HCC). However, whether YAP and TAZ have distinct or overlapping functions during hepatocarcinogenesis remains unknown. Methods Expression patterns of YAP and TAZ were analyzed in human HCC samples. The requirement of Yap and/or Taz in protein kinase B (Akt)/ neuroblastoma RAS viral oncogene homolog (NRas) -driven liver tumorigenesis was analyzed using conditional Yap, Taz, and Yap;Taz knockout mice. Transcriptional programs regulated by YAP and/or TAZ were identified via RNA sequencing. Results We found that in human HCC samples, an almost ubiquitous activation of YAP or TAZ occurs, underlying their role in this tumor type. Intriguingly, 70% of HCC samples showed only nuclear YAP or TAZ immunoreactivity. In the Akt/NRas liver tumor model, where nuclear Yap and Taz can be detected readily, deletion of Yap or Taz alone only mildly delayed liver tumor development, whereas their concomitant ablation strongly inhibited tumor cell proliferation and significantly suppressed Akt/NRas-driven hepatocarcinogenesis. In HCC cell lines, silencing of either YAP or TAZ led to decreased expression of both overlapping and distinct sets of genes, with the most prominent gene signatures related to cell-cycle progression and DNA replication. Conclusions YAP and TAZ have overlapping and distinct roles in hepatocarcinogenesis. HCCs may display unique activation of YAP or TAZ, thus relying on either YAP or TAZ for their growth., Graphical abstract

Highlights • Cases of COVID-19 may be housed outside isolation wards prior to detection. • A bundle of infrastructural enhancements, improved PPE and social distancing was implemented. • Of 253 staff close-contacts and 45 patient close-contacts of 28 index cases, one patient contact tested positive. • Environmental sampling was positive for SARS-CoV-2 in 5 out of 28 cases. • An infection control bundle mitigated COVID-19 transmission in a general ward setting., Background During an ongoing outbreak of COVID-19, unsuspected cases may be housed outside of dedicated isolation wards. Aim At a Singaporean tertiary hospital, individuals with clinical syndromes compatible with COVID-19 but no epidemiologic risk were placed in cohorted general wards for COVID-19 testing. To mitigate risk, an infection control bundle was implemented comprising infrastructural enhancements, improved personal-protective-equipment (PPE), and social distancing. We assessed the impact on environmental contamination and transmission. Method Upon detection of a case of COVID-19 in the dedicated general ward, patients and healthcare workers (HCWs) contacts were identified. All patient and staff-close contacts were placed on 14-day phone surveillance and followed-up for 28 days; symptomatic contacts were tested. Environmental samples were also obtained. Findings Over a 3-month period, 28 unsuspected cases of COVID-19 were contained in the dedicated general ward. In 5 of the 28 cases, sampling of the patient's environment yielded SARS-CoV-2; index cases who required supplemental oxygen had higher odds of environmental contamination (p=0.01). A total of 253 staff close-contacts and 45 patient close-contacts were identified; only 3 HCWs (1.2%, 3/253) required quarantine. On 28-day follow-up, no patient-to-HCW transmission was documented; only one symptomatic patient close-contact tested positive. Conclusion Our institution successfully implemented an intervention bundle to mitigate COVID-19 transmission in a multi-bedded cohorted general ward setting.

Background & Aims Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. Methods We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. Results Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2–4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5–8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2–4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. Lay summary MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy., Graphical abstract, Highlights • Serum miRNA was sequenced in 183 NAFLD cases of varying severity and 10 population controls. • Plasma levels of miR-193a-5p were significantly increased in patients with advanced fibrosis, high NAS scores, or high SAF scores. • Other miRNAs including miR378d and miR378e were also significantly increased in certain comparisons. • The findings for miR-193a-5p were replicated in a cohort of 372 additional NAFLD cases.

The airborne transmission of SARS-CoV-2 remains surprisingly controversial; indeed, health and regulatory authorities still require direct proof of this mode of transmission. To close this gap, we measured the viral load of SARS-CoV-2 of an infected subject in a hospital room (through an oral and nasopharyngeal swab), as well as the airborne SARS-CoV-2 concentration in the room resulting from the person breathing and speaking. Moreover, we simulated the same scenarios to estimate the concentration of RNA copies in the air through a novel theoretical approach and conducted a comparative analysis between experimental and theoretical results. Results showed that for an infected subject's viral load ranging between 2.4 × 106 and 5.5 × 106 RNA copies mL-1, the corresponding airborne SARS-CoV-2 concentration was below the minimum detection threshold when the person was breathing, and 16.1 (expanded uncertainty of 32.8) RNA copies m-3 when speaking. The application of the predictive approach provided concentrations metrologically compatible with the available experimental data (i.e. for speaking activity). Thus, the study presented significant evidence to close the gap in understanding airborne transmission, given that the airborne SARS-CoV-2 concentration was shown to be directly related to the SARS-CoV-2 emitted. Moreover, the theoretical analysis was shown to be able to quantitatively link the airborne concentration to the emission., Graphical Abstract ga1

Introduction The Abbott ID Now COVID-19 assay is a point-of-care molecular diagnostic tool for the detection of SARS-CoV-2. We prospectively monitored implementation of the assay in a tertiary care hospital emergency department (ED) for the diagnosis of early symptomatic patients. Methods A total of 269 paired nasopharyngeal swabs were tested in parallel with the ID Now and laboratory-based molecular methodologies, 191 of which met selection criteria for testing based on symptoms description and duration. Results 46 and 48 samples were positive for SARS-CoV-2 with the ID Now and reference molecular assays respectively. Percent positive and negative agreement were high (93.8% and 99.6% respectively), as were the sensitivity and specificity (93.8% and 99.5%). ID Now results were available 17.47 hours earlier than qRT-PCR. Conclusion In symptomatic patients seen in ED within 7 to 10 days of symptoms onset, the ID Now COVID-19 assay allows for rapid and accurate detection of infection.

Background & Aims The inflammatory bowel diseases (IBDs), Crohn’s disease and ulcerative colitis, are caused in part by aberrant immune responses to resident intestinal bacteria. Certain dietary components, including carbohydrates, are associated with IBDs and alter intestinal bacterial composition. However, the effects of luminal carbohydrates on the composition and colitogenic potential of intestinal bacteria are incompletely understood. We hypothesize that carbohydrate metabolism by resident proinflammatory intestinal bacteria enhances their growth and worsens intestinal inflammation. Methods We colonized germ-free, wild-type, and colitis-susceptible interleukin-10 knockout mice (Il10-/-) with a consortium of resident intestinal bacterial strains and quantified colon inflammation using blinded histologic scoring and spontaneous secretion of IL12/23p40 by colon explants. We measured luminal bacterial composition using real-time 16S polymerase chain reaction, bacterial gene expression using RNA sequencing and real-time polymerase chain reaction, and luminal glucosamine levels using gas chromatography–mass spectrometry. Results We show that a consortium of 8 bacterial strains induces severe colitis in Il10-/- mice and up-regulates genes associated with carbohydrate metabolism during colitis. Specifically, Enterococcus faecalis strain OG1RF is proinflammatory and strongly up-regulates OG1RF_11616-11610, an operon that encodes genes of a previously undescribed phosphotransferase system that we show imports glucosamine. Experimental colitis is associated with increased levels of luminal glucosamine and OG1RF_11616 causes worse colitis, not by increasing E faecalis numbers, but rather by mechanisms that require the presence of complex microbiota. Conclusions Further studies of luminal carbohydrate levels and bacterial carbohydrate metabolism during intestinal inflammation will improve our understanding of the pathogenesis of IBDs and may lead to the development of novel therapies for these diseases.

In the wake of rising number of SARS-CoV-2 cases, the Government of India had placed mass-quarantine measures, termed as “lockdown” measures from end-March 2020. The subsequent phase-wise relaxation from July 2020 led to a surge in the number of cases. This necessitated an understanding of the true burden of SARS-CoV-2 in the community. Consequently, a sero-epidemiological survey was carried out in the central Indian city of Ujjain, Madhya Pradesh. This article details the processes of data acquisition, compilation, handling, and information derivation from the survey. Information on socio-demographic and serological variables were collected from 4,883 participants using a multi-stage stratified random sampling method. Appropriate weightage was calculated for each participant as sampling fraction derived from Primary Sampling Unit (PSU), Secondary Sampling Unit (SSU) and Tertiary Sampling Unit (TSU). The weightage was then applied to the data to adjust the findings at population level. The comprehensive and robust methodology employed here may act as a model for similar future endeavours. At the same time, the dataset can also be relevant for researchers in fields such as data science, epidemiology, virology and earth modelling.

Objectives A consensus guidance is provided for testing, utility and verification of SARS-CoV-2 point-of-care test (POCT) performance and implementation of a quality management program, focusing on nucleic acid and antigen targeted technologies. Design and Methods The recommendations are based on current literature and expert opinion from the members of Canadian Society of Clinical Chemists (CSCC), and are intended for use inside or outside of healthcare settings that have varied levels of expertise and experience with POCT. Results and Conclusions Here we discuss sampling requirements, biosafety, SARS-CoV-2 point-of-care testing methodologies (with focus on Health Canada approved tests), test performance and limitations, test selection, testing utility, development and implementation of quality management systems, quality improvement, and medical and scientific oversight.

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Background : South Africa was the African country with the most recorded cases of SARS-CoV-2 during 2020, experiencing 2 waves of infection. During the first wave, diagnostics were largely based on reverse transcription-linked PCR (RT-PCR). The Abbott PanBio antigen test was deployed during the 2nd wave which may have been driven by emergence of the B.1.351 variant. At the time of evaluation in mid-November 2020, B.1.351 was the dominant circulating virus in Nelson Mandela Bay, in the Eastern Cape Province. Methods : Used PanBio antigen swabs (collected from patients with genetically characterised virus) were first validated as suitable for PCR. A prospective study was then undertaken to evaluate assay performance in the field. Testing was conducted at mobile community testing centres on 677 ambulant patients. Used swabs were kept and tested by RT-PCR. Results : During initial validation, used swabs in proprietary lysis buffer were found to be suitable for PCR and secondly, the PB assay reliably detected patients infected with B.1.351. In the field study, of 146 RT-PCR positive individuals, 101 were RTD positive in the clinic. The RTD had a sensitivity of 69.2% (95%CI 61.4, 75.8) and specificity of 99.0% (95%CI 98.8, 99.3). Sensitivity was dependent on the amount of viral RNA in clinical samples, as reflected by the PCR cycle threshold (CT) value. Conclusions : The assay reliably detected B.1.351 infections in ambulatory ill patients during initial validation and in field testing. In the field, assay sensitivity was >90% in patients with high viral loads who are expected to be most infectious. Negative and positive predictive values were also >90%.

Highlights • SARS-CoV-2 virus was present in the air in the ICU. • Virus may be discharged in aerosol for days after patients test negative. • The finding may be one of the reasons for patients` re-detectable positive. • The clinical guidelines for recovered COVID-19 patients need to be improved., As coronavirus disease 2019 (COVID-19) is spreading worldwide, there have been arguments regarding the aerosol transmission of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Moreover, some re-detectable positive (RP) patients have been reported. However, little attention has been given to the follow-up of recovered patients, and there is no environmental evidence to determine whether these patients continue to shed the virus after they test negative. Therefore, with an objective to test the hypothesis of airborne transmission of SARS-CoV-2, it is necessary to 1) determine whether SARS-CoV-2 particles are present in the indoor air and 2) determine whether recovered patients are still shedding virus, thus providing much-needed environmental evidence for the management of COVID-19 patients during the recovery period. In this study, surface and air samples were collected from an intensive care unit (ICU) containing one ready-for-discharge patient. All surface samples tested negative, but the air samples tested positive for SARS-CoV-2. This implies that SARS-CoV-2 particles may be shed in aerosol form for days after patients test negative. This finding may be one of the reasons for the observation of RP patients; therefore, there is a need for improved clinical and disease management guidelines for recovered COVID-19 patients.

Background & Aims Treatment with non-selective beta-blockers (NSBBs) reduces the risk of ascites, which is the most common decompensating event in cirrhosis. This study aimed to assess the ability of a serum microRNA (miRNA) signature to predict ascites formation and the hemodynamic response to NSBBs in compensated cirrhosis. Methods Serum levels of miR-452-5p, miR-429, miR-885-5p, miR-181b-5p, and miR-122-5p were analyzed in patients with compensated cirrhosis (N = 105). Hepatic venous pressure gradient (HVPG) was measured at baseline, after intravenous propranolol, and 1 year after randomization to NSBBs (n = 52) or placebo (n = 53) (PREDESCI trial). miRNAs were analyzed at baseline and at 1 year. Results Nineteen patients (18%) developed ascites, of whom 17 developed ascites after 1 year. miR-181b-5p levels at 1 year, but not at baseline, were higher in patients that developed ascites. The AUC of miR-181b-5p at 1 year to predict ascites was 0.7 (95% CI 0.59–0.78). miR-429 levels were lower at baseline in acute HVPG responders to NSBBs (AUC 0.65; 95% CI, 0.53–0.76), but levels at baseline and at 1 year were not associated with the HVPG response to NSBBs at 1 year. Conclusions Serum miR-181b-5p is a promising non-invasive biomarker to identify patients with compensated cirrhosis at risk of ascites development. Lay summary Ascites marks the transition from the compensated to decompensated stage in cirrhosis and indicates a worsening in prognosis. There are currently no easily accessible tools to identify patients with compensated cirrhosis at risk of developing ascites. We evaluated the levels of novel molecules termed microRNAs in the blood of patients with compensated cirrhosis and observed that miR-181b-5p can predict which patients are going to develop ascites., Graphical abstract, Highlights • miR-181b-5p appears to be a useful serum biomarker to anticipate ascites onset. • Low serum miR-181b-5p indicates low risk of ascites in compensated cirrhosis. • Low serum miR-429 reflects acute hemodynamic response to non-selective beta-blockers.

The coronavirus disease 2019 drastically impacted solid organ transplantation. Lacking scientific evidence, a very stringent but safer policy was imposed on liver transplant (LT) early in the pandemic. Restrictive transplant guidelines must be reevaluated and adjusted as data become available. Prior to LT, the prevailing policy requires a negative SARS-Cov-2 real-time polymerase chain reaction (RT-PCR) of donors and recipients. Unfortunately, prolonged viral RNA shedding frequently hinders transplantation. Recent data reveal that positive test results for viral genome are frequently due to non-infectious and prolonged convalescent shedding of viral genome. Moreover, studies demonstrated that the cycle threshold of quantitative RT-PCR could be leveraged to inform clinical transplant decision-making. We present an evidence-adjusted significantly less restrictive policy for LT, where risk tolerance is tiered to recipient acuity. In addition, we delineate the pre-transplant clinical decision-making, intra- and postoperative management, and early outcome of two recipients of a liver graft performed while their RT-PCR of airway swabs remained positive. Convalescent positive RT-PCR results are common in the transplant arena, and the proposed policy permits reasonably safe LT in many circumstances., Highlights • Currently, a negative COVID-19 PCR is required prior to liver transplantation. • Prolonged convalescent non-infectious shedding is common in COVID-19. • Cycle Threshold could be leveraged to clinical transplant decision-making. • Less restrictive policy for LT, in PCR positive donors and recipients.

Background The viral shedding duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been fully defined. Consecutive detection of SARS-CoV-2 RNA from respiratory tract specimens is essential for determining duration of virus shedding and providing evidence to optimize the clinical management of coronavirus disease 2019 (COVID-19). Research Question What are the shedding durations of SARS-CoV-2 RNA in the upper and lower respiratory tract specimens? What are their associated risk factors? Study Design and Methods A total of 68 patients with COVID-19 admitted to Wuhan Taikang Tongji Hospital and Huoshenshan Hospital from February 10, 2020, to March 20, 2020, were recruited. Consecutive SARS-CoV-2 RNA detection from paired specimens of nasopharyngeal swab (NPS) and sputum were carried out. The clinical characteristics of patients were recorded for further analysis. Results SARS-CoV-2 RNA was detected from NPSs in 48 patients (70.6%), and from sputum specimens in 30 patients (44.1%). The median duration of viral shedding from sputum specimens (34 days; interquartile range [IQR], 24-40) was significantly longer than from NPSs (19 days; IQR, 14-25; P < .001). Elderly age was an independent factor associated with prolonged virus shedding time of SARS-CoV-2 (hazard ratio, 1.71; 95% CI, 1.01-2.93). It was noteworthy that in 9 patients, the viral RNA was detected in sputum after NPS turned negative. Chronic lung disease and steroids were associated with virus detection in sputum, and diabetes mellitus was associated with virus detection in both NPS and sputum. Interpretation These findings may impact a test based clearance discharge criteria given patients with COVID-19 may shed virus longer in their lower respiratory tracts, with potential implication for prolonged transmission risk. In addition, more attention should be given to elderly patients who might have prolonged viral shedding duration.

Background Little is known about the relationships between SARS-CoV-2, the respiratory virus responsible for the ongoing COVID-19 pandemic, and the upper respiratory tract (URT) microbiome. Objective Our objectives were 1) to compare the URT microbiome between SARS-CoV-2-infected and -uninfected adults, and 2) to examine the association of SARS-CoV-2 viral load with the URT microbiome during COVID-19. Methods We characterized the URT microbiome using 16S ribosomal RNA sequencing in 59 adults (38 with confirmed, symptomatic, mild-to-moderate COVID-19 and 21 asymptomatic, uninfected controls). In those with COVID-19, we measured SARS-CoV-2 viral load using quantitative reverse transcription PCR. We then examined the association of SARS-CoV-2 infection status and its viral load with the ⍺-diversity, β-diversity, and abundance of bacterial taxa of the URT microbiome. Our main models were all adjusted for age and sex. Results The observed species index was significantly higher in SARS-CoV-2-infected than in -uninfected adults (β linear regression coefficient=7.53, 95%CI=0.17-14.89, p=0.045). In differential abundance testing, 9 amplicon sequence variants (ASVs) were significantly different in both of our comparisons, with Peptoniphilus lacrimalis, Campylobacter hominis, Prevotella 9 copri, and an Anaerococcus unclassified ASV being more abundant in those with SARS-CoV-2 infection and in those with high viral load during COVID-19, whereas Corynebacterium unclassified, Staphylococcus haemolyticus, Prevotella disiens, and 2 Corynebacterium_1 unclassified ASVs were more abundant in those without SARS-CoV-2 infection and in those with low viral load during COVID-19. Conclusion Our findings suggest complex associations between SARS-CoV-2 and the URT microbiome in adults. Future studies are needed to examine how these viral-bacterial interactions can impact the clinical progression, severity, and recovery of COVID-19., Capsule Summary: In our study, we found substantial differences in the upper respiratory tract microbiome between SARS-CoV-2-infected and -uninfected adults. Furthermore, we show that the SARS-CoV-2 viral load is associated with the upper respiratory tract microbiome during COVID-19.

Highlights • The new antigen tests for COVID19 are an indispensable tool in the control of the pandemic due to their adequate sensitivity and specificity. • The implementation of the point of care technique in primary care is feasible and has good results. • Less than 5 days of evolution of the onset of symptoms and CT less than 27 in the PCR would define the results of the antigen techniques., Background RT-qPCR is the current recommended laboratory method to diagnose SARS-CoV-2 acute infection, several factors such as requirement of special equipment, time consuming, high cost and skilled staff limit the use of these techniques. A more rapid and high-throughput method is essential. Methods We analyzed clinical data and nasopharyngeal samples, collected during September 2020, from patients attended at the emergency department of a secondary hospital and in two primary healthcare centers in Madrid. The performance of the Panbio™ COVID-19 AG Rapid Test Device for the detection of SARS-CoV-2 antigen was compared to RT-qPCR. Results 255 nasopharyngeal swabs, including 150 from the emergency department and 105 from primary helthcare centers, were tested. 184 patients were symptomatic (72.1 %). Amongst the 60 positive RT-qPCR samples, 40 were detected by the rapid antigen test, given an overall sensitivity of 73.3 %. All the samples detected positive with the rapid antigen test were also positive with RT-qPCR. The median cycle threshold was 23.28 (IQR 18.5–30.16). Patients with less than seven days onset of symptoms showed a higher viral load, and sensitivity for rapid antigen test (86.5 %), compared to those with more days (sensitivity of 53.8 %)(p

Samples for diagnostic tests for SARS-CoV-2 can be obtained from the upper (nasopharyngeal/oropharyngeal swabs) or lower respiratory tract (sputum or tracheal aspirate or broncho-alveolar lavage - BAL). Data from different testing sites indicates different rates of positivity. Reverse-transcriptase polymerase chain reaction (RT-PCR) allows for semi-quantitative estimates of viral load as time to crossing threshold (Ct) is inversely related to viral load.ObjectivesThe objective of our study was to evaluate SARS-CoV2 RNA loads between paired nasopharyngeal (NP) and deep lung (endotracheal aspirate or BAL) samples from critically ill patients.MethodsSARS-CoV-2 RT-PCR results were retrospectively reviewed for 51 critically ill patients from 5 intensive care units in 3 hospitals ; Addenbrookes Hospital Cambridge (3 units), Royal Papworth Cambridge (1 unit), and Royal Sunderland Hospital (1 unit). At the times when paired NP and deep lung samples were obtained, one patient had been on oxygen only, 6 patients on non-invasive ventilation, 18 patients on ECMO, and 26 patients mechanically ventilated.ResultsResults collected showed significant gradient between NP and deep lung viral loads. Median Ct value was 29 for NP samples and 24 for deep lung samples. Of 51 paired samples, 16 were negative (below limit of detection) on NP swabs but positive (above limit of detection) on deep lung sample, whilst 2 were negative on deep sample but positive on NP (both patients were on ECMO).ConclusionsIt has been suggested that whilst SARS-CoV1 tends to replicate in the lower respiratory tract, SARS-CoV2 replicates more vigorously in the upper respiratory tract. These data challenge that assumption. These data suggest that viral migration to, and proliferation in, the lower respiratory tract may be a key factor in the progression to critical illness and the development of severe acute respiratory syndrome (SARS). Factors which promote this migration should be examined for association with severe COVID-19. From a practical point of view, patients with suspected severe COVID-19 should have virological samples obtained from the lower respiratory tract where-ever possible, as upper respiratory samples have a significant negative rate.

Introduction Responses of oral-microflora-exposed dental pulp to a triple antibiotic paste (TAP), a mixture of ciprofloxacin, metronidazole, and minocycline in ointment with macrogol and propylene glycol, remain to be fully clarified at the cellular level. This study aimed to elucidate responses of oral-microflora-exposed dental pulp to capping with TAP in mouse molars. Methods A cavity was prepared on the first molars of 6-week-old mice to expose the dental pulp for 24 h. The exposed pulp was capped with TAP (TAP group) or calcium hydroxide cement (CH group), in addition to the combination of macrogol (M) and propylene glycol (P) (MP, control group), followed by a glass ionomer cement filling. The samples were collected at intervals of 1, 2, and 3 weeks, and immunohistochemistry for nestin and Ki-67 and deoxyuride-5′-triphosphate biotin nick end labeling (TUNEL) assay were performed in addition to quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Results The highest occurrence rate of pulp necrosis was found in the control group followed by the CH group at Weeks 2 and 3, whereas the highest occurrence rate of healed areas in the dental pulp was observed in the TAP group at each time point. Tertiary dentin formation was first observed in the dental pulp of the TAP group at Week 2. In contrast, bone-like and/or fibrous tissues were frequently observed in the CH group. qRT-PCR analyses clarified that TAP activated the stem and dendritic cells at Weeks 1 and 2, respectively. Conclusions The use of TAP as a pulp-capping agent improved the healing process of oral-microflora-exposed dental pulp in mouse molars., Highlights • We established a mouse model to evaluate the pulpal responses to capping materials. • TAP induced odontoblast-like cell differentiation faster than calcium hydroxide. • Tertiary dentin was predominantly seen at the exposure site in the TAP group. • TAC-P tends to activate dental pulp stem cells earlier than calcium hydroxide. • TAP favored the repair process of the oral-microflora-exposed pulpal tissue.

Background Whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody tests are increasingly used to estimate the prevalence of SARS-CoV-2 infection, the determinants of these antibody responses remain unclear. Objectives To evaluate systemic and mucosal antibody responses toward SARS-CoV-2 in mild versus severe coronavirus disease 2019 (COVID-19) cases. Methods Using immunoassays specific for SARS-CoV-2 spike proteins, we determined SARS-CoV-2-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) in sera and mucosal fluids of two cohorts, including SARS-CoV-2 polymerase chain reaction (PCR)+ patients (n = 64) as well as PCR+ and PCR– healthcare workers (n = 109). Results SARS-CoV-2-specific serum IgA titers in mild COVID-19 cases were often transiently positive, whereas serum IgG titers remained negative or became positive 12–14 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers after symptom onset. Very high titers of SARS-CoV-2-specific serum IgA correlated with severe acute respiratory distress syndrome (ARDS). Interestingly, some healthcare workers with negative SARS-CoV-2-specific serum antibody titers showed SARS-CoV-2-specific IgA in mucosal fluids with virus-neutralizing capacity in some cases. SARS-CoV-2-specific IgA titers in nasal fluids inversely correlated with age. Conclusions Systemic antibody production against SARS-CoV-2 develops mainly in severe COVID-19, with very high IgA titers seen in patients with severe ARDS, whereas mild disease may be associated with transient production of SARS-CoV-2-specific antibodies but stimulate mucosal SARS-CoV-2-specific IgA secretion., Graphical abstract, Our study suggests a model where the extent and duration of SARS-CoV-2-related clinical symptoms correlates with the systemic and mucosal virus-specific humoral immune responses, with implications for seroprevalence studies.

Highlights • Detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing. • Direct metatranscritptome sequencing from nasopharyngeal swab of a COVID-19 patient. • Depletion of human rRNA and in-house bioinformatics strategy for Ion torrent sequencing., Herein, we describe the detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing directly from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil. Depletion of human ribosomal RNA and use of an optimized in-house developed bioinformatics strategy contributed to successful detection of the virus.

Almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (IBV) using live attenuated vaccines mass administered by spray at day of hatch. Although many different types of IBV vaccines are used successfully, the ArkDPI serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. In this study, we examined a different Ark vaccine strain (Ark99), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. Further attenuation of the Ark99 vaccine was achieved by passage in embryonated eggs but ArkGA P1, P20, and P40 (designated ArkGA after P1) were still too reactive to be suitable vaccine candidates. However, ArkGA P60 when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. In addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. The full-length genomes of viruses from egg passages 1, 20, 40, and 60 were sequenced using the Illumina platform and the data showed single nucleotide polymorphisms (SNPs) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. ArkGA P60 accumulated the most SNPs in key genes associated with pathogenicity (polyprotein gene 1ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. These results indicate that the ArkGA P60 vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic Ark-type virus.

Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.

Graphical abstract, More than a year after the SARS-CoV-2 pandemic, the Coronavirus disease 19 (COVID-19) is still a major global challenge for scientists to understand the different dimensions of infection and find ways to prevent, treat, and develop a vaccine. On January 30, 2020, the world health organization (WHO) officially announced this new virus as an international health emergency. While many biological and mechanisms of pathogenicity of this virus are still unclear, it seems that cytokine storm resulting from an immune response against the virus is considered the main culprit of the severity of the disease. Despite many global efforts to control the SARS-CoV-2, several problems and challenges have been posed in controlling the COVID-19 infection. These problems include the various mutations, the emergence of variants with high transmissibility, the short period of immunity against the virus, the possibility of reinfection in people improved, lack of specific drugs, and problems in the development of highly sensitive and specific vaccines. In this review, we summarized the results of the current trend and the latest research studies on the characteristics of the structure and genome of the SARS-CoV- 2, new mutations and variants of SARS-CoV-2, pathogenicity, immune response, virus diagnostic tests, potential treatment, and vaccine candidate.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Introduction : Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorisation for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in Sao Paulo, Brazil, for SARS-CoV-2 diagnosis Materials and Methods : A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays Results : The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets Conclusion : All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in Sao Paulo, Brazil.

The spike (S) protein mutations of SARS-CoV-2 are of major concern in terms of viral transmission and pathogenesis. Hence, we developed a PCR-based method to rapidly detect the 6 mutational hotspots (H49Y, G476S, V483A, H519Q, A520S, and D614G) in the S protein and applied this method to analyze the hotspots in the viral isolates from different geographical origins. Here, we identified that there was only the D614G mutation in the viral isolates. As of September 30, 2020, the analysis of 113,381 sequences available from the public repositories revealed that the SARS-CoV-2 variant carrying G614 has become the most prevalent form globally. Our results support recent epidemiological and genomic data demonstrating that the viral infectivity and transmission are enhanced by the S protein D614G mutation., Graphical abstract Unlabelled Image

The long-term cholesterol-lowering effect of replacing intake of SFA with PUFA is well established, but has not been fully explained mechanistically. We examined the postprandial response of meals with different fat quality on expression of lipid genes in peripheral blood mononuclear cells (PBMC) in subjects with and without familial hypercholesterolaemia (FH). Thirteen subjects with FH (who had discontinued lipid-lowering treatment ≥4 weeks prior to both test days) and fourteen normolipidaemic controls were included in a randomised controlled double-blind crossover study with two meals, each with 60 g of fat either mainly SFA (about 40% energy) or n-6 PUFA (about 40% energy). PBMC were isolated in fasting, and 4 and 6 h postprandial blood samples. Expression of thirty-three lipid genes was analysed by reverse transcription quantitative PCR. A linear mixed model was used to assess postprandial effects between meals and groups. There was a significant interaction between meal and group for MSR1 (P = 0·03), where intake of SFA compared with n-6 PUFA induced a larger reduction in gene expression in controls only (P = 0·01). Intake of SFA compared with n-6 PUFA induced larger reductions in gene expression levels of LDLR and FADS1/2, smaller increases of INSIG1 and FASN, and larger increases of ABCA1 and ABCG1 (P = 0·01 for all, no group interaction). Intake of SFA compared with n-6 PUFA induced changes in gene expression of cholesterol influx and efflux mediators in PBMC including lower LDLR and higher ABCA1/G1, potentially explaining the long-term cholesterol-raising effect of a high SFA intake.

Objectives To test our hypothesis that routine year-round testing of specimens from multiple body sites and genotyping of detected virus would describe seasonal changes, increase diagnostic yield, and provide a better definition of clinical manifestations of human parechovirus (PeV-A) infections in young febrile infants. Study design PeV-A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was incorporated in routine evaluation of infants aged ≤60 days hospitalized at Nationwide Children's Hospital for fever and/or suspected sepsis-like syndrome beginning in July 2013. We reviewed electronic medical records of infants who tested positive for PeV-A between July 2013 and September 2016. Genotyping was performed with specific type 3 RT-PCR and sequencing. Results Of 1475 infants evaluated, 130 (9%) tested positive for PeV-A in 1 or more sites: 100 (77%) in blood, 84 (65%) in a nonsterile site, and 53 (41%) in cerebrospinal fluid (CSF). Five infants (4%) were CSF-only positive, 31 (24%) were blood-only positive, and 20 (15%) were nonsterile site–only positive. PeV-A3 was the most common type (85%) and the only type detected in CSF. Although the majority (79%) of infections were diagnosed between July and December, PeV-A was detected year-round. The median age at detection was 29 days. Fever (96%), fussiness (75%), and lymphopenia (56%) were common. Among infants with PeV-A–positive CSF, 77% had no CSF pleocytosis. The median duration of hospitalization was 41 hours. Four infants had bacterial coinfections diagnosed within 24 hours of admission; 40 infants had viral coinfections. Conclusions Although most frequent in summer and fall, PeV-A infections were encountered in every calendar month within the 3-year period of study. More than one-half of patients had PeV-A detected at more than 1 body site. Coinfections were common. PeV-A3 was the most common type identified and the only type detected in the CSF.

Purpose To investigate the presence of SARS-CoV-2 RNA in tears of patients with moderate to severe coronavirus disease 2019 (COVID-19). Design Cross-sectional study. Participants Patients with laboratory-proven moderate to severe COVID-19. Methods Tears were collected within 48 hours of laboratory confirmation using 3 methods: conjunctival swab plus Schirmer’s test strips (group 1), conjunctival swab (group 2), and Schirmer’s test strips (group 3). Samples from both the eyes of each patient were transported in a single viral transport media for real-time RT-PCR. Detailed demographic profiles, systemic symptoms, comorbidities, and ocular manifestations were noted. Main Outcome Measures Viral load of a sample was determined using cycle threshold (Ct) value of E gene. A specimen was considered to show positive results if the amplification curve for the E gene crossed the threshold line within 35 cycles and if it showed positive results on an RNA-dependent RNA polymerase or open reading frame 1b gene assay. Results Of the 78 patients enrolled in the study, samples from 3 patients were found to be inadequate for analysis. Thirty-six patients (48%) had moderate disease, whereas 39 patients (52%) had severe disease, with no ocular involvement in any patient. In the 75 patients, RT-PCR analysis of tears showed positive results in 18 patients (24%), and 29 of 225 samples (12.9%) showed positive results. Positive results were found in 11 (14.7%), 11 (14.7%), and 7 (9.3%) patients in groups 1, 2, and 3, respectively (P = 0.3105). Mean Ct values in groups 1, 2, and 3 were 28.36 ± 6.15, 29.00 ± 5.58, and 27.86 ± 6.46 (P = 0.92), respectively. Five patients showed positive RT-PCR results by all 3 methods (mean Ct value, 25.24 ± 6.33), and 12 patients showed positive results by any of the 3 methods (mean Ct value, 32.16 ± 1.94), the difference in Ct values being statistically significant (P = 0.029). The median value of symptomatology in patients with positive RT-PCR results from tears was 5 days (range, 4–9 days). Conclusions SARS-CoV-2 RNA was detected in tears of 24% of patients with laboratory-proven moderate to severe COVID-19. Conjunctival swab remains the gold standard of tear collection for RT-PCR assay. A significantly higher possibility of viral transmission exists through tears in patients with moderate to severe COVID-19., SARS CoV-2 viral RNA was detected in tears of 24% of laboratory proven moderate to severe COVID 19 patients, in early course of the disease, posing risk of viral transmission through tears.

Background & Aims: The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic contexts in mice. However, the function of LGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail. Methods: We interrogated the function and expression of LGR family members using human pluripotent stem cellâderived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5-GFP-IRES-CreERT2 mice. Results: We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4 and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are humanâmouse species-specific differences at later time points of embryonic development. Conclusions: Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process. Keywords: Pluripotent Stem Cells, Endoderm, LGR5, WNT, Organoid, Intestine

The formyl peptide receptor 2 (FPR2) belongs to the family of seven-transmembrane G protein-coupled receptors (GPCR) and are expressed by many different cells but mainly studied in immune cells. FPR2 is involved in host defense against bacterial infections and clearance of damaged cells through the oxidative burst and chemotaxis of neutrophils. In addition, FPR2 has also been implicated as an immunomodulator in sterile inflammations, e.g. inflammatory joint diseases. Here we present data regarding FPR2 expression in human articular chondrocytes, isolated from healthy individuals and osteoarthritic patients, on both mRNA and protein level using qPCR and Imagestream flow cytometry. We also present data after receptor stimulation and monitoring of production of nitric oxide, reactive oxygen species, IL-6, IL-8 and MMP-3. The presented data show that human articular chondrocytes from patients with osteoarthritis as well as from healthy individuals express FPR2 both at mRNA and protein level. The biological relevance of FPR2 expression in chondrocytes needs to be further investigated.