Hypogeusia for sweet was statistically significant (p = 0.041), where it was more common among TCA (70%) than both SSRIs and the psychotherapy group (20%). Gustatory dysfunction was found to be mostly associated with TCA followed by SSRIs particularly for sweet taste thresholds. More attention has to be given to taste changes among these patients as oral health affects general health by causing considerable pain and by changing what people eat, their speech, and their quality of life and wellbeing. Proper awareness and evaluation of this problem will improve the quality of life for the depressed patients and avoid unnecessary treatment. This trial is registered with ClinicalTrials.gov ID: NCT03599011.It is quite clear that the ability to perceive taste sensations significantly affects food choice, which consequently affects health status in the long term. Gustatory dysfunction is a neglected symptom among the depressed patients and those under antidepressants therapy, although these patients are suspectable to oral problems, due to general self-negligence related to mental disease, fear of dental treatment, and side effects of varied medications utilized in psychiatry. This study is aimed at assessing gustatory thresholds (detection and recognition thresholds) among a sample of 30 depressed Egyptian adults under antidepressants therapy for at least 3 months or psychotherapy with age ranging from 20 to 50 years old, seeking the Psychiatric Clinic at the Faculty of Medicine, Cairo University, Egypt. These patients were distributed into three equal groups (tricyclic antidepressants (TCA), selective serotonin reuptake inhibitors (SSRIs), and psychotherapy) and were assessed for gustatory detection and recognition thresholds using the filter paper disc method through a scoring system. The participants were also divided into normal taste group in which both the detection and recognition scores were 1, while the scores from 2 till 5 were considered as hypogeusia group and the score of 6 was considered as dysgeusia group. The TCA group was statistically significant from the other 2 groups in sweet detection thresholds ( The depressed patients and those under antidepressants therapy are susceptible to oral problems, due to general self-negligence related to mental disease, fear of dental treatment, and side effects of varied medications utilized in psychiatry. Moreover, in recognition of a wide range of microbials, stress and damage signals, had been considered as the most important factors responsible for tissue damage linked with periodontitis .In this regard, it has been hypothesized that conditions such as periodontitis and Coronary heart diseases may determine the increased release at the serum and salivary levels of IL-1, IL-6, prostaglandins, metalloproteases, NO, and hs-CRP, which, in turn, negatively influences the tone of the endothelial wall and, finally, causes a high risk of endothelial dysfunction and CHD development . Oral anTaste changes can be classified into hypogeusia (diminished taste sensation), dysgeusia (taste distortion), phantogeusia (phantom taste), and ageusia (taste loss) . SystemiTaste changes are often drug related. For example, in one study, for approximately 25% of patients with taste disturbances at an ear, nose, and throat outpatient clinic, the problem was drug related . 12% of Data on medical variables suggested that mood disorders ranked first while anxiety disorders ranked second, 34.6 and 27.8%, respectively, followed by psychoactive substance use disorders (21.1%) and schizophrenia (6.8%). It was also suggested that 39.8% of patients had a duration of mental illness of 1–5 years, 88% of patients were independents for their daily life activities, and 65.4% of patients had no healthy practices Shah et al.  suggesteIn circumstances such as anxiety or depression, in which serotonin (5-HT) and noradrenaline (NA) are altered, they are associated with taste disturbances, indicating the necessity of these transmitters in the determination of taste thresholds in health and disease . Most ofIt has been reported clinically that both first-generation tricyclic antidepressants (TCA) and later-developed selective serotonin reuptake inhibitors (SSRIs) cause chemosensory complaints. Despite these helpful clinical observations in determining potential chemosensory side effects of drugs, it is essential to quantify the potential taste changes by carrying out experimental studies . Oral paThe rationale of conducting such study was to determine that gustatory thresholds (detection and recognition thresholds) among a sample of depressed Egyptian adults under antidepressants therapy (TCA and SSRIs) for at least 3 months were not affected as compared to those under nonpharmacological therapy (psychotherapy), seeking the Psychiatric Clinic at the Faculty of Medicine, Cairo University, Egypt.NCT03599011.STROBE statement checklist was followed in reporting cohort studies. This trial is registered with ClinicalTrials.gov ID: The study was approved by the Ethical Committee of the Faculty of Dentistry, Cairo University . The aim of the study and its benefits were explained to each participant with emphasis on confidentiality of the collected data. Each participant signed an informed consent before being enrolled in the study.This is a retrospective cohort study.The subjects had been recruited from the Psychiatric Clinic at the Faculty of Medicine, Cairo University, Egypt.A sample of 30 depressed patients under antidepressants therapy for at least 3 months or psychotherapy, with age ranging from 20 to 50 years old, seeking the Psychiatric Clinic at the Faculty of Medicine, Cairo University, was recruited during a period of almost 7 months starting from February 2020 to August 2020. Those who were of ages other than the mentioned, or under antipsychotics, hypnotics, or anticonvulsants therapies or suffered from olfactory dysfunction, were excluded.The thirty eligible patients were recalled using their phone numbers recorded in their follow-up records, were asked to come to the Diagnosis Center at the Faculty of Dentistry, Cairo University, in order to be assessed for gustatory functions and were given identification numbers and distributed into three equal groups , to which the operator was blinded to avoid selection bias. Nonrespondent bias was minimized by explaining to the participants the aim of the study and their importance and role in the study.The gustatory thresholds were assessed using filter paper disc method in which test discs were placed on the tongue . The guTest discs of 5 mm in diameter were wetted with a solution and placed on the left lateral part of the tongue at approximately 2 cm from the proglossis (the tip of the tongue), which is thought to be innervated by the chorda tympani nerve. During the tests, the participants were instructed to rinse their mouths with distilled water before testing the next concentration. A one-minute interval after water rinses is necessary before presenting further solutions.The five concentrations of the four tastes  used were starting from the lowest concentration then ascending to the highest possible concentration of each taste as follows: sweet—0.8 M, 1.2 M, 1.4 M, 1.6 M, and 2 M; salty—0.02 M, 0.1 M, 0.2 M, 0.3 M, and 1.2 M; sour—0.01 M, 0.02 M, 0.06 M, 0.1 M, and 1.2 M; and bitter—0.04 M, 0.15 M, 0.3 M, 0.6 M, and 0.9 M.The concentrations of each taste were serially scored from disc number 1 (lowest) to number 5 (highest). If the subject was not able to recognize the taste at the highest concentration, a score of 6 was given. The detection threshold was defined as the concentration at which the subjects clearly indicated it as different from deionized water, but not necessarily recognizing the quality of the taste, while the recognition threshold was defined as the solution at which the subjects clearly identified the quality of the taste. The participants were also divided into normal taste group in which both the detection and the recognition scores were 1, while scores from 2 till 5 were considered as hypogeusia group and a score of 6 was considered as dysgeusia group.  E1: Exposure 1—commonly prescribed tricyclic antidepressants   E2: Exposure 2—commonly prescribed selective serotonin reuptake inhibitors antidepressants This includes nonpharmacological treatment (psychotherapy).Age ranged from 20 to 50 years old. Gender was assessed as male or female. Social level was assessed by residence in urban or rural areas. Time since starting antidepressants therapy: at least 3 months ago. Presence of complications associated with antidepressants therapy: gustatory dysfunction (taste changes) was assessed by taste intensity measurements.The participant's mouth was rinsed with distilled water before testing the next concentration to avoid the affection of taste intensity measurements by the presence of residual stimuli from prior testing (Zverev ).It was quite clear that the ability to perceive taste sensations significantly affected food choice, which consequently affected health status in the long term (Snyder and Bartoshuk ).The eligible patients were recalled, given identification numbers and divided into three groups, to which the operator was blinded to avoid selection bias. Nonrespondent bias was minimized by explaining to the participants the aim of the study, their importance, and role in the study. Incomplete records were excluded from statistical analysis with reporting the cause of not completing the record.Based on the previous paper by Ramzy , taste dχ2) test and Fisher's exact test when appropriate. Adjustments of p value were done using the Bonferroni method for multiple testing. All p values are two-sided. p values ≤ 0.05 were considered statistically significant.All the results were subjected to statistical analysis. Data management and statistical analysis were performed using the Statistical Package for Social Sciences (SPSS) version 25. Numerical data were summarized using means and standard deviations and ranges. Categorical data were summarized as percentages. Data were explored for normality using Kolmogorov–Smirnov test and Shapiro–Wilk test. Comparisons between the 3 groups with respect to numeric variables were done using the one-way ANOVA. Pairwise differences were detected by Bonferroni's post hoc test. For categorical variables, differences were analyzed with chi square . Regarding the medical history of the patients, there was no significant difference between groups for systemic diseases (p = 0.531) and medications other than antidepressants (p = 1.000). Concerning the dental history of the patients, there was no significant difference between groups for dental history including oral hygiene status (p = 0.531) and dental care frequency (p = 0.406).As for the psychiatric disorders which the participants were diagnosed with , there wp = 0.043). The mean score for sweet detection threshold concentration was 0.9 ± 0.2, 0.8 ± 0, and 0.8 ± 0 for TCA, SSRIs, and psychotherapy, respectively. This was statistically significant (p = 0.040). The mean score for recognition threshold of sweet taste was 2.1 ± 1.0, 1.2 ± 0.4, and 1.2 ± 0.4 for TCA, SSRIs, and psychotherapy, respectively. This was statistically significant (p = 0.007). The mean score for recognition threshold of sweet taste concentration was 1.2 ± 0.3, 0.9 ± 0.2, and 0.9 ± 0.2 for TCA, SSRIs, and psychotherapy, respectively. This was statistically significant (p = 0.008); pairwise comparisons revealed that TCA group was statistically significant from the other 2 groups in sweet taste thresholds , salt detection threshold concentration (p = 0.247), recognition threshold of salt taste (p = 0.121), and recognition threshold of salt taste concentration (p = 0.129) for TCA, SSRIs, and psychotherapy groups were statistically not significant (p = 0.680), sour detection threshold concentration (p = 0.572), recognition threshold of sour taste (p = 0.880), and recognition threshold of sour taste concentration (p = 0.073) for TCA, SSRIs, and psychotherapy were statistically not significant (p = 0.284), bitter detection threshold concentration (p = 0.246), recognition threshold of bitter taste (p = 0.286), and recognition threshold of bitter taste concentration (p = 0.358) for TCA, SSRIs, and psychotherapy groups were statistically not significant ; 70% of TCA patients suffered from hypogeusia while 20% in SSRI and Psychotherapy group as shown in Hypogeusia for sweet was statistically significant (p = 0.043). The mean score for sweet detection threshold concentration was 0.9 ± 0.2, 0.8 ± 0, and 0.8 ± 0 for TCA, SSRIs, and psychotherapy, respectively. This was statistically significant (p = 0.040). The mean score for recognition threshold of sweet taste was 2.1 ± 1.0, 1.2 ± 0.4, and 1.2 ± 0.4 for TCA, SSRIs, and psychotherapy, respectively. This was statistically significant (p = 0.007). The mean score for recognition threshold of sweet taste concentration was 1.2 ± 0.3, 0.9 ± 0.2, and 0.9 ± 0.2 for TCA, SSRIs, and psychotherapy, respectively. This was statistically significant (p = 0.008); pairwise comparisons revealed that TCA group was statistically significant from the other 2 groups in sweet taste thresholds.The results of the present study revealed that the mean score for sweet detection threshold was 1.4 ± 0.7, 1 ± 0, 1 ± 0 for TCA, SSRI, and psychotherapy, respectively. This was statistically significant (p = 0.220), salt detection threshold concentration (p = 0.247), recognition threshold of salt taste (p = 0.121), and recognition threshold of salt taste concentration (p = 0.129) for TCA, SSRIs, and psychotherapy groups were statistically not significant. Also, the mean scores for sour detection threshold (p = 0.680), sour detection threshold concentration (p = 0.572), recognition threshold of sour taste (p = 0.880), and recognition threshold of sour taste concentration (p = 0.073) for TCA, SSRIs, and psychotherapy were statistically not significant (p = 0.284), bitter detection threshold concentration (p = 0.246), recognition threshold of bitter taste (p = 0.286), and recognition threshold of bitter taste concentration (p = 0.358) for TCA, SSRIs, and psychotherapy groups were statistically not significant. These findings indicated that regarding the four taste qualities thresholds, TCA group differed from the other two groups mainly in sweet taste thresholds.However, in the present study, the mean scores for salt detection threshold (nificant . Similarp = 0.041); 70% of TCA patients suffered from hypogeusia while 20% in SSRI and Psychotherapy group. Abnormalities for other tastes were comparable between groups.Regarding taste abnormalities between groups, the significant difference was detected in the detection of phantogeusia. 70% of psychotherapy group reported phantogeusia, in contrast to only 20% in TCA and SSRIs groups. Hypogeusia for sweet was statistically significant (p = 0.043) and concentrations (p = 0.040) and also sweet recognition thresholds scores (p = 0.007) and concentrations (p = 0.008). This significance could partially be in agreement with Kampov-Polevoy et al. [The results of the present study showed that TCA group was statistically significant from the other 2 groups in sweet detection thresholds scores . Berlin et al.  mentioneHowever, there was a contradiction to the above mentioned studies and the present study in a study by Dichter et al.  to examiConcerning SSRIs group in the present study, clinical reports mentioned that some patients associate SSRIs treatment with an experience of emotional blunting through which emotional responses to both aversive and pleasurable experiences are decreased , 27. ExpConsidering TCA group, it was found to be statistically significant from the other 2 groups in sweet taste thresholds in the present study. This finding was in agreement with Maddox et al.  who repoMoreover, in a randomized controlled study by Ramzy , the comp = 0.220), (p = 0.247), (p = 0.121), and (p = 0.129), respectively. Also, the mean scores for sour detection threshold, sour detection threshold concentration, recognition threshold of sour taste and recognition threshold of sour taste concentration for TCA, SSRIs, and Psychotherapy were statistically not significant (p = 0.680), (p = 0.572), (p = 0.880), and (p = 0.073), respectively. Similarly, the mean scores for bitter detection threshold, bitter detection threshold concentration, recognition threshold of bitter taste, and recognition threshold of bitter taste concentration for TCA, SSRIs, and psychotherapy groups were statistically not significant (p = 0.284), (p = 0.246), (p = 0.286), and (p = 0.358), respectively. These findings indicated that regarding the four taste qualities taste thresholds, that the most affected taste is sweet taste thresholds and TCA group differed from the other two groups particularly in sweet taste thresholds. These findings were in contrast to a study performed by Schiffman et al. [However, in the present study, the mean scores for salt detection threshold, salt detection threshold concentration, recognition threshold of salt taste, and recognition threshold of salt taste concentration for TCA, SSRIs, and psychotherapy groups were statistically not significant , where 70% of psychotherapy group reported phantogeusia, while only 20% in TCA and SSRIs groups which is considered in agreement with Dess and Edelheit [The present study revealed a significant difference between groups that was detected in phantogeusia (Edelheit  who founEdelheit  scored hp = 0.041), where 70% of TCA patients had hypogeusia while only 20% in both SSRI and psychotherapy groups. This is also in agreement with Schiffman et al. [Moreover, clinical reports mentioned that antidepressant drugs could cause taste disturbances including dysgeusia (distortion of taste), hypogeusia (diminished sensitivity of taste), and ageusia (absence of taste)  which isn et al.  who repon et al.  reportedThe oral health should not be separated from the remainder of the body as it affects general health by causing considerable pain and by changing what people eat, their speech, and their quality of life and well-being. Gustatory dysfunction should not be neglected in depressed patients and those under antidepressants therapy. It should be considered as a real pathophysiological symptom in depressed patients, and more work should be done on the mechanisms through which gustatory function is disturbed in depression. More experimental studies should be done to essentially quantify potential taste changes among patients under antidepressants therapy.Health care providers should know the causes of taste disorders in order to be capable of reaching correct diagnosis and planning treatment without unnecessary dental treatment. It should be noted that before taste changes assessment for any causes, dental causes should be first ruled out and the diagnosis of a gustatory dysfunction must be supported with a thorough medical history, the patient's subjective reporting, and psychophysical testing.The results of the present study can conclude that gustatory dysfunction is a neglected system among the depressed patients and those under antidepressants therapy till now, where gustatory dysfunction was found to be mostly associated with TCA followed by SSRIs and the depressed patients undergoing psychotherapy particularly for sweet taste thresholds. Moreover, phantogeusia was common among depressed patients undergoing psychotherapy while hypogeusia for sweet was more common among TCA than SSRIs. More attention has to be given to taste changes among these patients as oral health affects general health by causing considerable pain and by changing what people eat, their speech, and their quality of life and wellbeing. Proper awareness and evaluation of this problem will reduce its impact and improve the quality of life for the depressed patients.


A significant reduction in epitope variability was instead observed for some of the most immunogenic proteins . Analysis over longer evolutionary time frames indicated that this effect is not due to differential constraints. These data indicate that SARS‐CoV‐2 evolves to elude the host humoral immune response, whereas recognition by T cells is not actively avoided by the virus. However, we also found a trend of lower diversity of T cell epitopes for common cold coronaviruses, indicating that epitope conservation per se is not directly linked to disease severity. We suggest that conservation serves to maintain epitopes that elicit tolerizing T cell responses or induce T cells with regulatory activity.Analysis of the bat viruses most closely related to SARS‐CoV‐2 indicated that the virus probably required limited adaptation to spread in humans. Nonetheless, since its introduction in human populations, SARS‐CoV‐2 must have been subject to the selective pressure imposed by the human immune system. We exploited the availability of a large number of high‐quality SARS‐CoV‐2 genomes, as well as of validated epitope predictions, to show that B cell epitopes in the spike glycoprotein (S) and in the nucleocapsid protein (N) have higher diversity than nonepitope positions. Similar results were obtained for other human coronaviruses and for sarbecoviruses sampled in bats. Conversely, in the SARS‐CoV‐2 population, epitopes for CD4 The netmhcpan el 4.0 method  database . All protein sequences were retrieved and several filters were applied. Only complete genomes flagged as “high coverage only” and “human” were selected. Positions recommended to be masked by DeMaio and coworkers  were also removed.SARS‐CoV‐2 protein sequences were downloaded from the GISAID Initiative , generating a set of at least 23,625 sequences for each ORF. Proteins with <60 amino acids were excluded from the analyses.The list of GISAID IDs along with the list of laboratories which generated the data is provided in Table http://www.ncbi.nlm.nih.gov/). For all human coronaviruses, the only filter we applied was host identification as “human”. SARS‐CoV strains sampled during the second outbreak were excluded from the analyses. NCBI ID identifiers are listed in Tables For all the other human coronaviruses, as well as for a set of nonhuman infecting sarbecoviruses, sequences of either complete genomes or single ORFs  were retrieved from the National Center for Biotechnology Information database  index using the Shannon Entropy‐One tool from the HIV database (https://www.hiv.lanl.gov/content/index), with ambiguous character  excluded from the analysis. For SARS‐CoV‐2 strains, H was calculated on alignments of 10,000 randomly selected sequences for each protein. For each protein we evaluated the difference D between average H values at epitope and nonepitope positions.Variability at each amino acid position was estimated using the Shannon's entropy  . Specifically, only predicted proteins longer than 60 amino acids were analysed. Because most positions in SARS‐CoV‐2 genomes are invariable across the sampled genomes, the distribution of H is zero‐inflated, making the use of conventional statistical tests inappropriate  with positions not mapping to any of these epitopes. A significant reduction of variability was observed for S, N, ORF8, nsp15 and nsp16, whereas higher diversity was still evident for nsp8  antigen processing within host cells results in the preferential presentation of diverse viral epitopes to T lymphocytes depending on the MHC gene repertoire and on distinct preferences of the antigen processing pathway , HCoV‐229E (Duvinavirus subgenus), and HCoV‐NL63 (Setracovirus subgenus), have been transmitting within human populations for at least 70 years . Irrespective of the host, most variants are expected to be deleterious for viral fitness, or to have no consequences , suggesting the two latter viruses elicit mainly nonneutralizing responses  class I and class II molecules. Different mammals have diverse MHC gene repertoires and thus present distinct antigens. In particular, recent data from various bat species have indicated that many MHC class I molecules have a three‐ or five‐amino acid insertion in the peptide binding pocket, resulting in very different presented peptide repertoires compared to the MHC class I molecules of other mammals , both of which cause chronic infections in humans (Comas et al., + T cell trans‐infection (Sanjuán et al., + T cells targeting different virus proteins have distinct cytokine profiles, suggesting that the virus can modulate the host immune response to its benefit (Peng et al., Unexpected conservation of T cell epitopes was previously observed for HIV‐1 and Conceptualization, D.F. and M.S.; formal analysis, M.S., U.P. and D.F.; investigation, D.F., R.C., C.P., A.M. and M.S.; visualization, D.F., R.C; writing—original draft, M.S. and D.F.; writing—review & editing, M.S., M.C., R.C., U.P.; funding acquisition, M.S. and D.F.; supervision, M.S. and M.C.Table S1Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Appendix S1Click here for additional data file.Supplementary MaterialClick here for additional data file.


SARS‐CoV‐2 has emerged as a human pathogen, causing clinical signs, from fever to pneumonia—COVID‐19—but may remain mild or asymptomatic. To understand the continuing spread of the virus, to detect those who are and were infected, and to follow the immune response longitudinally, reliable and robust assays for SARS‐CoV‐2 detection and immunological monitoring are needed. We quantified IgM, IgG, and IgA antibodies recognizing the SARS‐CoV‐2 receptor‐binding domain (RBD) or the Spike (S) protein over a period of 6 months following COVID‐19 onset. We report the detailed setup to monitor the humoral immune response from over 300 COVID‐19 hospital patients and healthcare workers, 2500 University staff, and 198 post‐COVID‐19 volunteers. Anti‐SARS‐CoV‐2 antibody responses follow a classic pattern with a rapid increase within the first three weeks after symptoms. Although titres reduce subsequently, the ability to detect anti‐SARS‐CoV‐2 IgG antibodies remained robust with confirmed neutralization activity for up to 6 months in a large proportion of previously virus‐positive screened subjects. Our work provides detailed information for the assays used, facilitating further and longitudinal analysis of protective immunity to SARS‐CoV‐2. Importantly, it highlights a continued level of circulating neutralising antibodies in most people with confirmed SARS‐CoV‐2. The setup of a versatile COVID19 serology ELISA system is described, allowing Yes/No screening with a two‐step method that reduces false‐positives. Accurate antibody titer screening indicates a classic pattern of antibody production, peaking at 3 weeks post‐infection and remaining detectable for at least 6 months in 90% of the subjects tested. The antibody response does not correlate with age, but with severity of disease, and show an initial higher response in men. SARS‐CoV‐2 infection causes a wide variety of disease symptoms, from fever, asthenia, or myalgia, to pneumonia and in most severe cases acute respiratory distress syndrome referred to as COVID‐19. Yet, a large amount of SARS‐CoV‐2 infected patients remains asymptomatic. SARS‐CoV‐2 rapidly spread around the world and was declared a global pandemic in March 2020. It remains a continuing threat to health and socio‐economic well‐being. Despite the global number of infections reaching tens of millions, including almost one million fatalities, due to mitigation measures, the overall infection rate is relatively low with local infection hotspots. Although scientific progress is rapid, there remains a pressing need to understand the immune response that follows SARS‐CoV‐2 infection, including its role during disease and especially its potential long‐term protective effects.A prime immune target during coronavirus infections is the Spike (S) protein, closely associated with and targeted by neutralizing antibody responses and protective immunity, in contrast to most other viral proteins , 2, 3, 4Attempts to curtail and control the SARS‐CoV‐2 virus rely on increasing inter‐personal distance, including the closure of much social and economic activity, as well as testing for acute infection and personal hygiene measures. This was implemented early during the outbreak, with the University of Lisbon closing after March 13th, 10 days after the first recorded cases in Portugal. However, during the subsequent transition phase, restrictions have steadily been lifted. The gradual return to social and economic activity requires active surveillance to determine local outbreaks, contact tracing, and quarantine. In addition, those most vulnerable to COVID‐19 will need to remain under enhanced protection. Important information is how protective immunity develops in the population at large and in specific groups such as healthcare professionals. A thorough assessment of the duration of protective immunity is critical to determine the measures that need to be taken to prevent and handle future waves of SARS‐CoV‐2. Such information will need to be gathered widely, in different locations around the world, reflecting local conditions, such as containment measures and their timing. The data obtained will need to be accurate and the methods used transparent and reproducible to enable comparisons between locations and countries. The recent SARS‐CoV‐2 outbreak brings limitations with respect to exposure time but also gives us the opportunity to acquire real‐time data and develop reliable longitudinal follow‐up studies.To determine the cumulative rate of infection in communities and gaining insight into the potential protection against re‐infection, serological assays are critical. Depending on the aims of the study, the setup of such assays can be used for the detection of exposure to SARS‐CoV‐2 as well as gaining insights into neutralization activity, since antibody titers for both the S protein and RBD have been shown to correlate well with neutralizing activity , 9–11. WThe gold standard for antibody detection remains the ELISA, offering high flexibility and sensitivity, but limited scalability . SARS‐CoHuman sera pose a biological hazard to laboratory workers and can potentially contain not only SARS‐CoV‐2, but also other infectious viruses. Therefore, all ELISA steps were performed at biosafety level (BSL) 2, with BSL3 personal protective equipment. Inactivation procedures are recommended but can have uncertain effects on the accuracy of serological testing . We testThe S protein has a trimeric structure, while the in vitro expression of RBD results in the generation of monomeric and dimeric protein. However, when we tested the ability of RBD mono‐ and dimeric protein for antibody binding, both performed similar and comparable to the total protein fraction Figure . AdditioIn order to prepare for diagnostic use, the generation of quality control (QC) serum is critical to validate each assay run. Sera from exposed patients is most desirable as it will contain antibodies with a range of avidities and isotypes, providing more stable binding properties. However, sufficient volume needs to be obtained to ensure that there is enough material to complete the ELISA validation process and the study or series of studies to be undertaken. Sera from four SARS‐CoV‐2 exposed but healthy volunteers were assessed and pooled to serve as quality control for subsequent assays Fig. . UltimatWe used 100 pre‐COVID‐19 sera from healthy volunteers collected between October 2012 and November 2017 as negative controls Table . ReceiveIn order to increase pre‐COVID‐19 sample size and reflect a broader spectrum of the population, we obtained 61 samples of individuals with food allergies and 20 samples from individuals with bee and wasp allergies, because these contain increased levels of antibodies . Serum fWe subsequently analyzed 307 samples from hospitalized patients who tested positive for SARS‐CoV‐2 by PCR for the presence of anti‐SARS‐CoV‐2 IgG. Samples were acquired between April 6 and August 12, 2020 and at different times after the development of COVID‐19 symptoms. The patients demonstrated a variety of symptoms and underlying medical conditions Table . Taking Some patients who were SARS‐CoV‐2 PCR positive within an average of 8.7 days ‐1 ‐ 53) after blood was taken, did not show any classic COVID‐19 symptoms  Table . Within Within the hospital cohort, we obtained 29 sera from patients who received immunosuppressive drugs, including 13 of 29 (40%) receiving the glucocorticoids Prednisolone or Dexamethasone. Sera of these patients were collected between days 2 and 35 post‐COVID‐19 symptom onset. In this cohort, all patients tested SARS‐CoV‐2 positive by PCR, 20 of 29 (69%) did not seroconvert Fig. . Out of Twelve samples were from patients who previously contracted HIV1 or HIV2 and were treated with antiretroviral therapy  and one with HBV receiving Entecavir. All patients were male and SARS‐CoV‐2 seroconversion was analyzed within 11 days of symptom onset. Most patients  seroconverted Fig. . Of the hi and QClo) run in each plate. Although day‐to‐day plate variability is present, this is of very modest amplitude . To determine the seroprevalence in University staff, we screened 2571 employees, across all divisions, with serum obtained between May 13 and July 10, 2020. As mentioned, our assay is based on Stadlbauer et al.  that recude Fig. . The aveTo determine antibody responses accurately, we performed serum titrations using RBD protein to assay the anti‐SARS‐CoV‐2 IgM, IgG, and IgA responses. In agreement with earlier results, not all subjects assessed early (pre‐day 7) after the onset of COVID‐19 or those asymptomatic show seroconversion, but anti‐RBD antibodies rise swiftly during the first days of infection. This is the case for all three isotypes assessed. As with many reported antibody responses, including SARS , the antIn addition to early responses from healthcare workers and hospitalized patients, we analyzed 209 potential plasma donors for convalescent plasma therapy via the Portuguese Blood and Transplantation Institute (IPST). The volunteers were predominantly male (67%) and on average 38 years old. All were reported to be SARS‐CoV‐2 PCR positive, on average 107 days prior to serum sample collection Table . COVID‐1Analysis of the IgM, total IgG, and IgA responses confirmed a rapid and near‐simultaneous response of the three isotypes tested during the first weeks of SARS‐CoV‐2 infection Fig. . AntibodSince titers 2 months after COVID‐19 reduced, we next determined the antibodies' potential to neutralize SARS‐CoV‐2. We found neutralization activity in all tested sera in which anti‐SARS‐CoV‐2 IgG was determined, across 2–5 months after initial SARS‐CoV‐2 PCR‐positive testing Fig. . The levFor many pathogens and vaccines, antibody titers have been established over the past decades, accumulating detailed knowledge of average antibody responses. However, every microorganism is different, and level, neutralization activity, and longevity of antibody responses can be different between pathogens, vaccines as well as between individuals. Therefore, it remains important to acquire data for each novel infection, especially those posing a threat to human health such as SARS‐CoV‐2. In addition, initial antibody levels after vaccination, generally correlate well with a significantly reduced risk of (re‐)infection and pathology. Thus, the global collection of data from many different geographic locations, patient cohorts, and under different local conditions will contribute to a holistic understanding of the new pandemic.We explain the setup of an ELISA system in detail, as described previously , to faciWe show that neither frequently used methods of viral inactivation nor the complexity of RBD, monomeric, dimeric, or a mixture of both affects antibody determination. Although the reported presence of SARS‐CoV‐2 is limited in blood , the inaWithin the patient cohort, there were additional groups of interest. With SARS‐CoV‐2 being a positive‐stranded RNA virus, the use of anti‐retroviral medication had no major effect on antibody responses against the virus. The mode of action of these drugs is not known to interfere with RNA viruses or antibody production. Our data show the successful management of the eleven patients previously infected with HIV, with all of those patients showing a robust SARS‐CoV‐2 antibody response after the second week of COVID‐19 symptoms, in line with previous works , 19. TheWe and others  found hihttps://www.fda.gov/media/137029/download). We show that using only one protein for a large population screen picks up some false positives . Especially when the infection rate is low, the two‐step method is highly beneficial and strongly recommended [In many countries implementing mitigation strategies, infection prevalence remained modest at the time of sampling, May–June 2020, with a low frequency of infection , 28, 29.ommended . FurtherThe question of long‐lasting and protective immunity against SARS‐CoV‐2 is a focus of current research. We show that the initial antibody response against SARS‐CoV‐2 raises the three main isotypes in close concert as previously reported for SARS as well as SARS‐CoV‐2 , 30. TheRBD and Spike protein constructs were obtained from Dr. Florian Krammer, Icahn School of Medicine at Mount Sinai, New York, USA.Production and purification of recombinant proteins were performed at Instituto de Biologia Experimental e Tecnológica (iBET) Oeiras, Portugal as part of the Serology4COVID consortium as previously described by Stadlbauer et al. . BrieflyoC, and upper 6 × 0.25 ml of serum placed into six cryotubes. Samples are stored in a temperature‐controlled ‐80oC ultra‐low freezer at the iMM Biobank until analysis.Upon informed consent, blood was taken by vein puncture and two BD Vacutainer CPT tubes of blood and one serum tube were collected per patient. For serum collection, tubes were centrifuged at 2200 rpm, 10 min at 4Serum samples were obtained from the iMM biobank COVID‐19 collection and pre‐pandemic control sera from two allergy collections. Patients were recruited from the COVID‐19 unit and the Immunoallergology Department of Hospital de Santa Maria, Centro Hospitalar Lisboa Norte. The COVID‐19 collection and scientific use were approved by the Lisbon Academic Medical Center Ethics Committee (Ref. no. 155/20) as was the staff screening (Ref. no. 181/20). The allergy studies were approved with reference 116/13. Potential plasma donors registered voluntarily via the IPST website. Criteria for registration were a diagnosis of COVID‐19 documented by a positive PCR test for SARS‐CoV2 followed by two negative or one negative PCR tests 14 or 28 days prior to collection, respectively. Medical interviews were conducted to ensure that the criteria for apheresis plasma donation were fulfilled and that a full recovery from COVID‐19 had been achieved.Signed informed consent was obtained from all volunteers. All data were treated confidentially and anonymous, according to (EU) 2016/679 of 27 April 2016 . A professional obliged to confidentiality with guarantee appropriate information security measures carried out the data collection under the terms of the GDPR paragraph 2, article 29 Law no. 58 /2019, 8 August.To reduce risk from any potential residual virus in the serum, three different virus inactivation methods were tested: incubation for 1 h at 56°C (heat inactivation), the addition of 0.1% Triton X‐100 (a non‐ionic surfactant), or the combination of both (H+ Tx).Anti‐SARS‐CoV‐2 ELISAs were performed as described previously . BrieflyAnti‐SARS‐CoV‐2 ELISAs were performed as described in detail recently . SARS‐Cot‐test or two‐way ANOVA were used as stated in the figure legends, calculated using GraphPad Prism 6.0 software.A Kruskal–Wallis test (non‐parametric test) was done to compare the geometric ratios between groups with a significance level of 0.05 (Dunn's multiple comparisons test), Student's P.F.‐C. and B.B. setup the ELISA, tested it and run many samples. C.M. and P.G. provided blood samples from COVID19 patients, A.G. and M.S. run all antibody titrations, S.A., C.C. and J.P.S. setup and ran the neutralisation assays, H.N.‐C. and A.M. generated sera from COVID19 patients, M.C.P.‐S. provided allergy sera, F.R. and Â.A. catalogued all samples and generated sera, J.C., M.A.E., M.S. and E.V. coordinated potential plasma donor blood sample collections, M.R. and J.M.‐C. coordinated staff blood collection, M.V. coordinated the work, secured funding and wrote the manuscript.The authors have no commercial or financial conflict of interest.COVID‐19Coronavirus Disease 2019ELISAEnzyme‐linked immunosorbent assayIgImmunoglobulinODOptical densityPCRPolymerase chain reactionQCQuality controlRBDReceptor binding domainROCReceiver operating characteristicSARS‐CoV‐2Severe acute respiratory syndrome coronavirus 2S proteinSpike proteinSupporting InformationClick here for additional data file.


The complexity of left atrial appendage (LAA) in patients with nonvalvular atrial fibrillation (NVAF) is closely related to LAA thrombosis and stroke incidence. But the classification of LAA morphology is not uniform and controversial.This study divided the LAA into two categories according to the LAA morphology to investigate the risk of thrombosis related to the LAA structural complexity in NVAF patients.A total of 336 NVAF patients were enrolled continuously in this study. The patients were divided into thrombosis group and non‐thrombosis group according to whether the thrombus presence in LAA. Through computer LAA three‐dimensional reconstruction, LAA morphology was divided into the complex type and simple type according to with or without the clearly lobulated structure judged by imaging experts. The relationship between LAA thrombosis and various potential risk factors was analyzed.P < .001) was associated with the presence of LAA thrombus, independently of other enrolled risks.A total of 19 potential risk factors for LAA thrombosis in NVAF patients were enrolled into statistical analysis. The coincidence rate of LAA morphology classification was 96.4% (324/336) between two imaging experts. Multivariate logistic regression analysis showed that complex LAA morphology (OR 4.168, 95% CI 1.871‐9.288, It is a concise and reliable method to divide the LAA morphology into complex type and simple type according to whether with the clearly lobulated structure. The complex LAA is an independent risk factor for LAA thrombosis in NVAF patients. All patients were diagnosed with AF by conventional surface electrocardiogram or Holter examination, in line with “2014 AHA/ACC/HRS Guideline for the Management of Patients with Atrial Fibrillation.”Exclusion Criteria: patients with valvular AF,2.2A third‐generation force dual‐source CT  was used, as was an IE33 echocardiography .2.32.3.1Patients were recruited through our institution's Computerized Patient Record System. The following patient characteristics were documented upon participant recruitment: gender; age; type of AF; duration of AF (accurate to the month); oral anticoagulant use before admission; blood pressure during hospitalization; the presence of coronary heart disease, hypertension, diabetes, or vascular disease ; a history of stroke/transient ischemic attack (TIA)/thromboembolism (TE); and presenting either with or without symptoms and signs of congestive heart failure (CHF).2.3.2The following biochemical criteria were recorded: fasting serum glucose, brain natriuretic peptide (BNP), creatinine (Scr), plasma fibrinogen (Fbg) concentration, and prothrombin time international normalized ratio (INR) of every patient. All of which were measured for the first time after admission to the hospital.2.3.3First, patients with left atrial anterior‐posterior diameter (LAd), left ventricular end‐diastolic diameter (LVEDd), and left ventricular ejection fraction (LVEF) were measured by thoracic echocardiography. Moreover, chest X‐rays or computed tomography (CT) scans were used to determine whether aortic sclerosis was present. In addition, head CT or magnetic resonance imaging (MRI) was used to determine the presence or absence of cerebral infarction. Finally, coronary artery CT imaging or angiography was used to determine the degree of coronary artery stenosis, which aided in the diagnosis of coronary heart disease.2.4Based on the results of TEE and left atrial CTA, which were employed to assess whether a patient had a thrombus, the patients were subsequently divided into one of two groups: the thrombus group or non‐thrombus group. TEE is used as a “gold standard” for the diagnosis of LAA in patients with thrombus.2.52.5.1According to the analysis requirements, AF was divided into two categories: paroxysmal AF and non‐paroxysmal AF , which refer to the 2016 European society of cardiology (ESC) guidelines for the management of AF.2.5.2The definition of effective anticoagulation is that the INR value of warfarin taken orally before TEE is ≥2.0 for at least 3 weeks, or when patients had taken the recommended dose of a new oral anticoagulant for at least 3 weeks.2.5.32DS2‐VAScThe CHA2.5.4The three‐dimensional reconstruction of the left atrial CTA image was performed by CT image post‐processing system to obtain the LAA morphology of each patient; the image was classified based on the LAA's morphological characteristics. In this study, the LAA morphology was divided into two categories: simple‐LAA and complex‐LAA. Simple‐LAA was defined as only one backbone structure and no other distinct branch structures Figure ; a compl2.6t tests. If the measurement data did not conform to be the normal distribution, the median was used. The rank‐sum test was used for comparison between groups. Counting data use cases and percentage (%), the comparison between groups using χ2 tests. Multivariate logistic regression analysis was used to investigate independent risk factors for LAA thrombosis. The raw data of AF course and BNP does not conform to the normal distribution, the natural logarithm transformation for these parameters were performed before logistic regression analysis.Statistical analysis was performed using SPSS 19.0 software. Each group of variables was tested for normality and homogeneity of variance. The measurement data conformed to be the normal distribution as mean ± SD; the univariate analysis used two independent sample 33.1Among 336 patients, 162 (48.2%) cases had complex LAA morphology. The coincidence rate of LAA morphology classification was 96.4% (324/336) between two imaging experts, only 12 (3.6%) cases need the third expert to participate in the judgment.χ2 = 5.282，P = .022). In simple‐LAA, 89.1% of them were chicken wing‐like LAA, the remainder was wind bag‐like LAA. No cactus‐like and cauliflower‐like LAA belonged to simple‐LAA. The complex‐LAA included 14.8% of wind bag‐like, 56.2% of cauliflower‐like, and 29.0% of cactus‐like LAA, and without chicken wing‐like LAA. There were not statistically different to the proportion of LAA thrombus between simple‐LAA patients and chicken wing‐like LAA patients , as well as between complex‐LAA patients and non‐chicken wing‐like LAA patients .According to Di Biase's LAA morphological categories,3.2P = .013). Age (P < .002), AF course (P < .001), LAd (P < .001), and serum BNP concentration (P < .001) in the thrombus group were higher than those of patients in the non‐thrombus group, and the LVEF in the thrombus group was lower than that in the non‐thrombus group (P = .018). Compared with the non‐thrombus group, patients with thrombus had higher prevalence rates of male (P < .024), NPAF (P < .001), high‐risk CHA2DS2‐VASc score (P < .001), complex‐LAA (P < .001), hypertension (P = .025), diabetes mellitus (P < .001), coronary heart disease (P = .025), CHF (P < .001), stroke/TIA/TE, and vascular disease (P = .01). There were no significant differences in LVEDd, serum sCr concentration, plasma Fbg concentration, and effective anticoagulant rate between the two groups .As shown in Table 3.3P < .001), NPAF , and AF course  were the independent risk factors for the presence of LAA thrombosis.Univariate analysis showed that there were three factors related to LAA thrombosis, such as AF course, LAd, NPAF, complex‐LAA, and LVEF Table . With th4The LAA is a long, curved, blind cavity‐like structure that extends from the anterior lateral wall of the left atrium and has many uneven trabeculars, which have active systolic and diastolic functions that play an important role in relieving internal pressure in the left atrium. The anatomical and functional characteristics of LAA are prone to thrombosis in the AF state. At present, there is no unified LAA morphology‐based classification method in the academic community. In 2012, Di Biase et alP = .286). There were no cauliflower‐like and cactus‐like LAA in simple‐LAA group and no chicken wing‐like LAA in complex‐LAA group. The complexity of LAA in patients with AF is closely related to LAA thrombosis and stroke incidence.P = .959). In this study, wind‐like LAA in simple‐LAA patients accorded with the characteristics of no obvious branches. The above results indicated that our LAA form classification is reasonable and helpful to distinguish the simple structure LAA and the complex structure LAA in wind bag‐like LAA patients.This study divided the LAA into two categories (simple‐LAA and complex‐LAA), and compared with the four classifications of Di Biase et al.Di Biase et alP < .001).We performed a statistical analysis of 19 potential risk factors for LAA thrombosis in NVAF patients and found that a total of 14 risk factors were statistically different between the thrombosis group and non‐thrombosis group , while the rate of effective anticoagulation, LVEDd, LVEF, and serum Fbg concentration were not statistically different between the two groups. Multivariate logistic regression analysis showed that complex LAA was an independent risk factor for presence of LAA thrombosis . Moreover, complex‐LAA was an independent risk factor for LAA thrombosis , which is consistent with the findings from previous studies.In previous studies, cauliflower‐like LAA was regarded as the most complex LAA structure. This form of LAA has a short overall length, a large number of lobulated structures, and has been confirmed as an independent risk factor for stroke/TIA and LAA thrombosis by several studies.The reason why complex‐LAA is more likely to form a thrombus is related to LAA's lobulated structure, as well as its abundance of trabecular and lower LAA blood‐flow rate. Khurram et alP < .001), and the proportion of NPAF was significantly higher than that in the non‐thrombotic group . The two factors were independent risk factors for thrombosis in LAA, which was consistent with the findings of previous studies.The results of this study also showed that the course of AF among those patients in the thrombosis group was significantly longer than that in the non‐thrombosis group [30 (1‐180) months vs 22 (1‐120) months, 5In this study, of all 19 potential risk factors, only AF course, NPAF, and complex‐LAA were independent predictors of LAA thrombosis. However, several high‐risk independent risk factors of stroke proved by a large number of previous studies, such as heart failure, high‐risk CHA2DS2‐VASc score, and shock/TIA/TE history, were not risk factors of LAA thrombosis according to the results of this study. The main reason may be that the patients included in this study are limited to these with radiofrequency ablation indications (except for LAA thrombosis) in a single center, and only 336 patients enrolled, which cannot enough to represent the whole NVAF population.6In conclusion, it is a concise and reliable method to divide the LAA morphology into complex type and simple type according to with or without the clearly lobulated structure. The complex LAA is an independent risk factor for LAA thrombosis in NVAF patients.The authors declare no potential conflict of interests.Table S1 Results of univariate analysisClick here for additional data file.AppendixS1 Correction detailsClick here for additional data file.


The V‐shaped structure characteristic peaks occurred obviously in CS‐SI complexes detected by XRD patterns, which formed a new crystalline structure. The thermal stability was improved in CS‐SI complexes revealed by TGA and DTG curves that the thermal cracking temperature increased from 315°C to 320°C. The enthalpy (ΔH) of CS‐SI complexes decreased from 2.34 J/g to 1.75 J/g showed by DSC data, which indicated that the ordered structure of starch was destroyed. Furthermore, the content of resistant starch increased from 10.53% to 21.78% and predicted glycemic index (pGI) reduced in CS‐SI complexes. In conclusion, the digestibility and pGI of starch can be improved by complexed with soy isoflavone.Rapid starch digestion rate is negative for the normal level of human blood glucose. This study investigated the protective effects of corn starch (CS) complexed with soy isoflavone (SI) on the control of starch digestibility and glycemic index (GI). The structure of the corn starch–soy isoflavone (CS‐SI) complexes was characterized by Fourier transform infrared (FT‐IR), X‐ray diffraction (XRD), Thermogravimetric analysis (TGA), and Differential scanning calorimetry (DSC), and the complexes digestibility was evaluated using in vitro digestion model. The results of FT‐IR spectrum showed that, compared with corn starch, new characteristic peaks were not occurred in CS‐SI complexes, and the value of R Soy isoflavone entered the spiral cavity of amylose through hydrophobic force, and a V‐shaped crystalline structure was formed in CS‐SI complexes, which increased SDS and RS content, and the starch digestibility and predicted glycemic index were reduced. Starch has a positive effect on maintaining human life and movement, which play a key role obtaining energy from the diet for human. However, long‐term consumption of high glycemic index food was positively associated with obesity, hyperglycemia, diabetes typeⅡ, and other metabolic complications  was purchased from Shanghai Jinsui Biological Technology Co., LTD company. Soy isoflavone  was purchased from Xian Tianfeng Biotechnology Co., LTD company. α‐amylase from porcine pancreas (8 × USP) and glucosidase from aspergillus niger (≥260 U/ml) were purchased from Sigma‐Aldrich. D‐glucose assay kit was purchased from Megazyme International Ireland. Other reagents were of analytical grade.2.2g for 5 min, washed three times with ethanol solution , and centrifuged to remove soy isoflavone that were not complexed with corn starch, and the samples were freeze‐dried. The samples obtained are marked as SSI‐0, SSI‐1, SSI‐2, SSI‐3, and SSI‐4.The preparation of CS‐SI was slightly modified with reference to the published method  curves of samples were recorded.2.70T, the peak temperature of gelatinization pT, the final temperature of gelatinization CT, and the enthalpy value ΔH were known.Using DSC to determine the thermal properties of samples, refer to published methods for minor modifications , slowly digestible starch (SDS), and resistant starch (RS) of CS‐SI complexes was calculated by the following formula:Based on the Englyst method with slight modification ] was applied to investigate hydrolysis kinetics and pGI of the CS‐SI complexes .The hydrolysis index (HI) is the ratio of the area under hydrolysis curve (AUC) of samples to the AUC of fresh white bread. According to the equation, pGI = 8.198 + 0.862HI, get pGI , and the variances are calculated using SPSS 21 software (SPSS Inc). Analysis measurement, the difference is statistically significant. The significant differences were evaluated and displayed by different letters.33.12 present in starch molecule. The band at 1642/cm is attributed to the C = O stretching vibration in a carbohydrate group. Strong absorption peak appeared in all samples. Compared with SSI‐0, in the spectral range of 4,000 ~ 400/cm, the shape and position of infrared absorption peaks were similar and offset not obviously, new characteristic peaks not appeared or disappeared, which indicated that new group not generated in CS‐SI complexes and they were not combined by a covalent bond.FT‐IR spectroscopy was performed to investigate the structure of CS‐SI complexes, as Figure 1045/1022 and R995/1022 decreased, shown in Figure FT‐IR spectroscopy has been widely applied to investigate the short‐range molecular structure changes in starch double helices, and the differences in the crystalline and amorphous regions were observed between complexes. The ratio of FT‐IR spectrum (1045/1022)/cm and (995/1022)/cm absorption peak intensity can reflect the change of ordered structure in starch. The characteristic of ordered structure in aggregated structure of starch crystalline region is attributed to absorption peak near 1,054/cm, the characteristic of random coil structure of starch in the amorphous region is represented to absorption peak near 1,020/cm, and the absorption peak near 995/cm is an ordered structure of hydrogen bonds formed by the hydroxyl groups of anhydroglucose units between starch molecules Shingel, . Phenoli3.27 helical‐type diffraction ; pT and cT both showed an upward trend. The increase in gelatinization temperature could be attributed to interaction between amylose–amylose or amylose–amylopectin chains, and the formation of complexes between amylose and other substances. In our present research, ‐OH groups in SI might bind to starch granules, the redistribution of water between soy isoflavone and corn starch, which in turn affected the starch gelatinization transformation and hydration of amorphous region in starch granules, resulted in starch gelatinization temperature increased.Starch gelation and phase change can be quantitatively analyzed by DSC. Table ΔH) reflects the energy required for destruction of starch double helix during starch gelatinization, and before gelatinization of starch, measures the order of molecular chains inside starch granules. The destruction of ordered structure in starch granules will lead to a decrease in enthalpy value. The data in Table ΔH of CS‐SI complexes decreased significantly (p < .05). Similar to the result of Han , indicated that digestibility of corn starch after complexing with soy isoflavone was significantly reduced. The maximum hydrolysis degree C∞ of CS‐SI complexes ranged from 69.482 to 82.098, which was significantly lower than SSI‐0 (92.875). It is further shown that starch hydrolysis rate was reduced complexed with soy isoflavone, thereby digestibility of starch was reduced. In addition, the hydrolysis kinetic constant K value was decreased gradually. V‐shaped crystal structure of starch shows more resistance to α‐amylase hydrolysis, and the rate of starch hydrolysis can be reduced , as the concentration of soy isoflavone increased, pGI of CS‐SI complexes decreased gradually, and SSI‐4 had the lowest pGI value (68.09). Relatively higher SDS and RS content and lower pGI values in SSI‐1, SSI‐2, and SSI‐3, however they were still considered high GI (GI > 75) foods, while SSI‐4 was considered as medium GI food (Babatola et al., 4In this experiment, structural and in vitro digestion characteristics of corn starch incorporated with soy isoflavone were analyzed systematically, and glycemic index was predicted. The change mechanism of digestibility in CS‐SI complexes was revealed, and the non‐covalent interaction between corn starch and soy isoflavone was verified. CS‐SI complexes lost its original crystal, and a unique V‐shaped crystal structure was formed. By complexed with soy isoflavone, short‐range ordered structure and enthalpy value of starch were reduced, thermal stability of starch was improved, the content of SDS and RS in starch was increased, and pGI was decreased. From the structure characterization and digestibility results of CS‐SI complexes, it could be concluded that soy isoflavone entered amylose spiral cavity through hydrophobic force, and digestibility of starch was reduced by the change of CS‐SI complexes crystal structure. Therefore, complexed with soy isoflavone will open a pathway to modulate digestibility of starch.The authors declare no conflict of interest.


Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics SocietyThe coverage of the fifth‐generation network has increased steadily since the network was introduced in 2019. However, public protests around the globe against the construction of 5G network base stations have continued to occur for fear that electromagnetic (EM) waves emitted from the stations would cause adverse health effects. To identify factors that have contributed to such increased risk perception, we conducted a cross‐sectional study using data obtained from a survey that assessed Korean adults’ risk perception of EM wave‐related objects. We found that female gender, high level of perceived exposure to EM waves, evaluation of public policies as ineffective, and high level of objective knowledge on EM waves were associated with increased risk perception. Furthermore, we found that higher ratings on a few risk characteristics such as “personal knowledge,” “seriousness of the risk to future generations,” “dreadfulness,” and “severity of consequences” were also associated with increased risk perception as well. Bioelectromagnetics. © 2020 The Authors.  The recent development of the fifth‐generation (5G) network has led to widespread expectations that it would revolutionize mobile communications. As such, demand for the 5G network has been high; Korean mobile carriers began distributing the service in April 2019 [France‐Presse, In contrast to its apparent popularity, however, the new technology has not been welcomed by everyone. Many members of the public have perceived that electromagnetic (EM) waves from 5G network base stations are potential health risks, which has been shown by the protests in Korea that have delayed construction of base stations Jun, , and antWhat are the factors that have contributed to such public risk perception? Risk perception research has answered such a question by analyzing people's ability to make health‐related choices [Fischhoff et al., However, perhaps owing to the newness of the 5G network's development, no study has been published on risk perception of EM waves from 5G network base stations. Therefore, we conducted a cross‐sectional study using the data from a survey that assessed Korean adults’ risk perception of EM wave‐related objects. Our aims were to assess the degree of risk perception of EM waves from 5G network base stations and identify variables that significantly influenced the risk perception.From 1.1 million Koreans who were registered in the access panel of Truis , 207,809 adults over the age of 19 were selected by stratified random sampling  and contacted by e‐mail and KakaoTalk (a messaging application used widely in Korea) to participate in our survey. The response rate was 2.7%, as 5,677 responded to the survey. After taking quality control measures including exclusion of those who left the survey unfinished, data on 3,393 Korean adults were available for analysis. For the purpose of quality assurance, study subject selection, and coding of the data were conducted by Truis.The survey was designed to measure the degree of Korean adults’ risk perception of EM wave‐related objects, and it consisted of eight questionnaires, which were as follows: SQ (screening questions), DQ (demographic questions), section A (EM wave‐related sources), section B (objective knowledge), section C (risk perception and risk characteristics), section D (protective measures), section E (symptoms associated with use of EM wave‐related sources), and section F (media exposure and public policies). The study subjects completed the survey by accessing a web‐based program set up by Truis in 2019.Questions from the survey were screened to identify variables that would be relevant for our analysis. Demographic variables including gender, age group, marital status, level of education, monthly household income, smoking status, drinking status, whether study subjects lived with seniors with chronic disease, whether they lived with juniors younger than middle school age, and whether they lived with females (or were the ones) who were either pregnant or planning to become pregnant were selected.EM wave exposures and health‐related variables including whether study subjects used a mobile phone, whether they placed a mobile phone charger nearby while asleep, level of perceived exposures to EM waves, evaluation of public policies that provide protection from EM wave exposures, level of objective knowledge on EM waves, and self‐reported health were selected. Of note, the level of objective knowledge on EM waves was assessed by section B from our survey, and its details are provided in Supplementary Table 1.Risk characteristics Slovic,  assessedLastly, risk perception score was included; the score was rated on a 10‐point scale where 1 signified the least severe risk and 10 the most severe risk. In our survey, the study subjects rated risk perception score on a total of 19 objects. Thirteen objects were EM wave‐related; they were EM waves from 5G base stations, EM waves from mobile phones, EM waves from microwaves, EM waves from air fryers, EM waves from hair dryers, EM waves from massage chairs, EM waves from electronic foot baths, EM waves from low‐frequency therapy devices, EM waves from electric shavers, EM waves from radars, EM waves from transmission lines, EM waves from Bluetooth devices, and EM waves from electric heaters. On the contrary, six were not EM wave‐related; they were household chemical products, climate change, micro‐dust, drinking water pollution, electronic cigarettes, and cigarette smoking.T test was conducted to check for the presence of significant differences in risk perception scores of EM wave‐related objects between the two groups classified based on the level of objective knowledge.Descriptive statistical analysis on demographic variables, EM wave exposure and health‐related variables, risk characteristics, and risk perception score was carried out. Multiple linear regression was conducted to identify factors that significantly influenced risk perception of EM waves from 5G network base stations. Risk perception score of EM waves from 5G network base stations was chosen as the dependent variable; demographic variables, EM wave exposure and health‐related variables, and risk characteristics were used as covariates. Slopes and 95% confidence intervals (CIs) were calculated to quantify the magnitude of impact that each covariate exerted on the risk perception score of EM waves from 5G network base stations. All statistical analyses were performed using SPSS ver. 22 .This study was conducted after obtaining approval from the Institutional Review Board (IRB) of Korea University (KUIRB‐2019‐0240‐01).n = 1,760) were male, and 48.1%  female. For age group, 22.5% (n = 762) were between 20 and 29 years of age, 23.8% (n = 807) between 30 and 39, 27.9% (n = 945) between 40 and 49, and 25.9% (n = 879) between 50 and 59. For marital status, 43.5%  were single, 53.6%  married, and 2.8% (n = 94) either divorced or widowed. For level of education, 17.5% (n = 593) were high school graduates or less, 52.1%  either college students or graduates, and 30.4%  more than college graduates. The distribution of monthly household income was unimodal with 46.9%  earning between 3,000 and 6,000 dollars. For smoking status, 55.8%  were never‐smokers, 18.4% (n = 624) past smokers, and 25.8% (n = 875) current smokers. For drinking status, 17.2% (n = 584) were never‐drinkers, 25.8% (n = 874) past drinkers, and 57.0%  current drinkers. For living with vulnerable individuals, 17.5% (n = 593) lived with seniors diagnosed with chronic disorders, 23.5% (n = 799) with juniors younger than middle school age, and 7.2% (n = 243) with females (or were the ones) who were pregnant or planning to become pregnant  used a mobile phone, and 66.4%  placed a mobile phone charger nearby while asleep. About eighty percent  perceived that they were exposed to a high level of EM waves. In evaluating public policies that provide protection from EM wave exposures, 19.9% (n = 675) considered them effective, 54.3%  average, and 26.8% (n = 877) ineffective. The mean score on section B, which tested the level of objective knowledge on EM waves, was 4.6 problems correct out of 13 problems; 49.5%  scored better than the mean, and 50.5%  lower than the mean. For self‐reported health, 45.2%  considered themselves to be healthy, and 54.8%  unhealthy . The mean score on “controllability” was 4.26 (1: uncontrollable and 10: very controllable). The mean score on “seriousness of the risk to future generations” was 6.33 (1: not serious and 10: very serious). The mean score on “dreadfulness” was 5.77 (1: not dreadful and 10: very dreadful). The mean score on “severity of consequences” was 5.91 (1: not severe and 10: very severe). The mean score on “risk known to science” was 4.84 (1: not known to science and 10: well known to science). The mean score on “immediacy of effect of risk” was 4.60 (1: happens slowly and 10: happens immediately). The mean score on “familiarity” was 4.64 (1: unfamiliar and 10: very familiar) (Supplementary Table 2).The distribution of risk perception scores of EM waves from 5G network base stations was unimodal and approximately symmetric Fig. ; the meaThe mean risk perception score of EM waves from 5G network base stations was significantly higher for study subjects with a high level of objective knowledge than those with a low level of objective knowledge. And the same pattern was observed in regards to twelve other EM wave‐related objects (Supplementary Table 3).β: 0.188, P: 0.001; reference: male) study subjects who perceived that they were exposed to a high level of EM waves , those who regarded public policies as ineffective , and those with a high level of objective knowledge . Higher ratings on risk characteristics such as “personal knowledge” , “seriousness of the risk to future generations” , “dreadfulness” , and “severity of consequences”  were also associated with higher risk perception scores , never‐drinkers , and study subjects who were between 20 and 29 years of age . Higher ratings on risk characteristics such as “controllability”  and “risk known to science”  were also associated with lower risk perception scores   . Furthermore, when multiple linear regression (work not shown in Table) was conducted using the number of questions that the study subjects answered correctly, the risk perception score increased by 0.03 (P: 0.003) per one more question that they answered correctly.Lastly, we focus our discussion on the association between objective knowledge and risk perception. Objective knowledge can be enhanced effectively by education; hence, it is likely to become a policy target for risk communication efforts in the future. Past studies on the association between objective knowledge and risk perception have shown mixed results. Cousin and Siegrist [On the basis of the above findings, we interpreted that study subjects with greater risk perception have likely been more proactive in gathering relevant information, which led them to perform better on section B. However, the information that study subjects depended on was probably not of high quality as the low mean score on section B (4.6 questions correct out of 13) suggests. Our analysis (work not shown in Table) on sources of information that the study subjects looked up to learn more about EM waves seemed to support our contention; impartial and verified sources of information, such as government or authoritative agencies, were relied on less than exchanges of information with acquaintances. Furthermore, while the study subjects responded as resorting to the internet most frequently to learn more about EM waves, they were not aware of a credible webpage set up by the National Radio Research Agency to improve public knowledge of EMF sources.The limitation of our study was that its epidemiologic design as a cross‐sectional study limited causal inference. And the study subjects were invited to participate in our survey via e‐mail and KakaoTalk (a widely used messaging application in Korea), which might have resulted in selection bias of over‐representing people who might have been more tolerant to wireless technologies. Furthermore, although section B, which was used to assess the level of objective knowledge in our survey, was more extensive than some other measures used in the past, it might not have functioned as a perfect test of objective knowledge.However, despite such limitations, several strengths of our study exist. To our knowledge, our study was the first in the literature to investigate factors affecting risk perception of EM waves from 5G network base stations, and our analysis was conducted on a sufficiently large‐sized sample. It is hoped that the results of our study will be reflected in constructing an appropriate risk communication strategy so that reasonable dissemination of technologies can occur.Supporting information.Click here for additional data file.


The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid‐phase peptide synthesis strategy that is based on a water‐compatible 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real‐time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non‐natural Smoc‐protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions. Peptide synthesis goes green: A water‐compatible 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl (Smoc) protecting group has been developed that enables solid‐phase peptide synthesis under aqueous conditions as well as efficient postsynthetic purification of the peptides. This protecting group is fluorescent both when attached to an Nα atom and when cleaved and so also allows real‐time monitoring of building block coupling. Peptides, natural biopolymers that comprise a chain of up to 100 covalently linked amino acids, are elementary components in all living systems and regulate many biological processes. Currently, synthetic peptides are produced industrially for the treatment of cancer, diabetes, as well as cardiovascular and neurodegenerative diseases,The history of peptide synthesis dates back to the turn of the twentieth century, when CurtiusN‐methyl‐2‐pyrrolidone (NMP), which are currently the solvents of choice in SPPS, is undesirable as they are classified by the European Reach Regulation as substances of very high concern because of their carcinogenic, mutagenic, or toxicity properties towards reproduction (CMR). Although a sustainable, green alternative to the existing SPPS scheme is highly desirable,To date, peptide synthesis has gained almost optimal efficiency in terms of coupling yield and duration. However, solvent consumption, which has been recognized as an economic and environmental issue for over a decade, makes SPPS one of the most inefficient approaches known.A number of alternative approaches have been proposed to overcome this hurdle, among them peptide synthesis in less harmful organic solvents or those referred to as “green” ones ,tert‐butyloxycarbonyl, Fmoc=9‐fluorenylmethoxycarbonyl), the latter allows one to follow an N‐terminal deprotection, but only when the coupling step has already been accomplished and, therefore, cannot be repeated when there is insufficient coupling.Effective online monitoring at each synthetic step is another challenge in SPPS, as the final yield strongly depends on the coupling efficiency. Of the two main approaches for the solid‐phase assembly of peptides, which are named according to the respective transient protection groups as Boc‐ and Fmoc‐SPPS  was most likely due to steric hindrance. NMR studies performed with Nα‐Smoc‐amino acids 3–32 confirmed their structures (see Section 1.3 in the Supporting Information).The direct conversion of Nα‐Smoc group with the standard side‐chain protecting groups or, with the exception of Lys, Glu, Asp, Ser, Thr, and Cys, were used unprotected in aqueous SPPS , Smoc‐Ile‐OH (15), Smoc‐Phe‐OH (20), Smoc‐Pro‐OH (21), and Smoc‐Ser‐OH (22) with 3 equiv aqueous NaHCO3. HPLC analysis after 7, 14, and 21 days showed that the examined constructs were sufficiently stable (see Section 1.5 in the Supporting Information). The fact that all the Smoc‐protected amino acids were soluble and stable at concentrations typically used in peptide synthesizers, makes them suitable building blocks for automated SPPS.After the conditions for the Smoc group cleavage had been thoroughly examined, the stability of the Since the early years of peptide synthesis, it has been generally accepted that the formation of a peptide bond necessitates anhydrous coupling conditions as otherwise the required active species are hydrolyzed and the equilibrium is shifted towards the initial reagents. However, in water the acidity of carboxylic acids is increased compared to that in polar aprotic solvents commonly applied in peptide synthesis.Nα‐Smoc‐protected natural and some noncanonical amino acids possessing significant stability in basic aqueous solutions, but readily losing their terminal amino protection upon mild treatment with particular water‐soluble bases, we expanded the classic Merrifield procedure3, piperazine, or ethanolamine. Coupling of the next activated amino acid was then performed, followed, if required, by capping any free amines with special sulfo‐tags based on sulfoacetic acid or similar compounds , EDC‐HCl , and Oxyma  in aqueous NaHCO3. Smoc was cleaved with 1 m NaOH, 25 % aqueous ethanolamine, or 5–10 % aqueous piperazine. Coupling of the amino acids was performed in a similar manner as the loading of the first amino acid (for details see Section 1.7 in the Supporting Information). As expected, the lack of side‐chain protection significantly reduced the duration of the global cleavage. Indeed, even in the case of peptides with multiple arginine residues50, 51, 68), the cleavage time did not exceed 1 h, and the purity of the crude peptide was reasonable  to facilitate the formation of wrong, shortened sequences  and H‐ANKPG‐NH2 . Briefly, the racemization of the amino acids during the ASPPS corresponded to that observed in Fmoc‐based SPPS in DMF. The formation of aspartimide, a well‐documented side reaction occurring frequently at Asn‐R or Asp‐R sites, where R is an amino acid such as Gly, Ala, or Ser,2 (70), H‐VK(d‐D)GYI‐NH2 (71), H‐VKNGYI‐NH2 (72) and H‐VK(β‐D)GYI‐NH2 (73). Our data (see Section 1.10 in the Supporting Information) clearly indicate that aspartimide formation in water is temperature‐dependent, with the ratio of the β‐ to an α‐product being 1:3 at 4 °C  and 2.5:1 at 40 °C. Therefore, the deprotection of sequences prone to aspartimide formation in water should be performed at reduced temperature. Interestingly, β‐branching is the only aspartimide side reaction detected in ASPPS.In addition, two important side reactions commonly occurring in SPPS were studied. Racemization upon ASPPS was assessed by the assembly of the two model peptides H‐CYEIS‐NHα‐atom and when cleaved (see Sections 1.11 and 1.12 in the Supporting Information for details). These properties allow detection both in solution and when bound to the solid support, thus enabling for the first time a real‐time monitoring of both the coupling and deprotection steps. An assessment of the fluorescence at each coupling and deprotection step during the synthesis of the pentapeptide H‐LVAIG‐NH2 was performed by excitation at λ=280 nm and observing the emission at λ=340 nm , Oxyma (39), and 2‐hydroxypyridine‐N‐oxide  being the most efficient ones. Our experiments showed that although the coupling of amino acids in pure water gave peptides in reasonable yields and purity, the addition of organic co‐solvents enhanced the coupling performance significantly. Additional studies on the enantiomeric composition showed no increased racemization levels during the ASPPS process. The ionic properties of the Smoc protecting group gave rise to an elegant approach for the reliable purification of synthetic peptides. Merrifield had already shown that a monosulfonated Fmoc derivative could be applied for peptide isolation with ion‐exchange chromatography.To summarize, we have developed a working concept for efficient aqueous solid‐phase peptide synthesis and demonstrated its applicability to the synthesis of 22 biologically active peptides. The coupling efficiency was assessed in water‐based systems by applying the respective It is important to mention that the development of specific, water‐optimized resins and respective linker systems still remains a challenge that must be addressed. However, this issue definitely does not apply to the current work. The next goal in the frame of this study is the development of automated procedures for the small‐ and large‐scale sustainable synthesis of peptides.The authors declare competing financial interests. S.K., H.K, and C.U. are the founders of Sulfotools GmbH, a small chemical company interested in aqueous peptide synthesis. N.K. is an employee of Sulfotools GmbH. O.A., S.K., H.K, and C.U. are named inventors on a patent application (WO 2016 050764) filed by the Technische Universität Darmstadt and Sulfotools GmbH on the aqueous peptide synthesis methodology described in this work. R.M. declares no competing financial interest.S.K. initiated, designed, and coordinated the project; S.K., C.U., N.K. designed the experiments, performed all the experiments and analysis; O.A., S.K. wrote the manuscript; R.M. performed NMR studies and acquisition. O.A. and H.K. advised on all aspects. All authors discussed the results and commented on the manuscript.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and the associated coronavirus disease (COVID‐19) have spread throughout China. Previous studies predominantly focused on its place of origin, Wuhan, causing over estimation of the disease severity due to selection bias. We analyzed 465 confirmed cases in Zhejiang province to determine the epidemiological, clinical, and virological characteristics of COVID‐19.Epidemiological, demographic, clinical, laboratory, and management data from qRT‐PCR confirmed COVID‐19 patients from January 17, 2020, to January 31, 2020, were collected, followed by multivariate logistic regression analysis for independent predictors of severe/critical‐type COVID‐19 and bioinformatic analysis for features of SARS‐CoV‐2 from Zhejiang province.Among 465 COVID‐19 patients, median age was 45 years, while hypertension, diabetes, and chronic liver disease were the most common comorbidities. History of exposure to the epidemic area was present in 170 (36.56%) and 185 (39.78%) patients were clustered in 77 families. Severe/critical‐type of COVID‐19 developed in 49 (10.54%) patients. Fever and cough were the most common symptoms, while diarrhea/vomiting was reported in 58 (12.47%) patients. Multivariate analysis revealed eight risk factors for severe/critical COVID‐19. Glucocorticoids and antibiotics were administered to 60 (12.90%) and 218(46.88%) patients, respectively. Bioinformatics showed four single amino acid mutations and one amino acid position loss in SARS‐CoV‐2 from Zhejiang province, with more similarity to humans than to viruses.SARS‐CoV‐2 showed virological mutations and more human transmission in Zhejiang province, indicating considerable epidemiological and clinical changes. Caution in glucocorticoid and antibiotics use is advisable. People worldwide commonly get infected with the human coronaviruses 229E, NL63, OC43, and HKU1. Two other strains, severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) and Middle East respiratory syndrome coronavirus (MERS‐CoV), are zoonotic in origin and have been linked to lethal diseases.The first case of COVID‐19 in Zhejiang Province was diagnosed on January 17. Zhejiang province is 1000 kilometers from Wuhan. In contrast to the initial cases from Wuhan who had a history of close contact with Huanan seafood market, the majority of patients in Zhejiang province only had a history of working, studying, or travel to Wuhan. Nevertheless, knowledge about the specific epidemiology and clinical characteristics on a large scale still remains incomplete. In this study, we performed a comprehensive investigation of the epidemiology, clinical, and virological characteristics of 465 patients with laboratory‐confirmed COVID‐19 in Zhejiang province. To the best of our knowledge, this is by far the largest number of COVID‐19 patients reported in Zhejiang province with distinct clinical features compared to Wuhan.22.1We performed a retrospective study focusing on the epidemiological, clinical, and virological characteristics of confirmed cases of COVID‐19 from January 17, 2020, to January 31, 2020. The data were uniformly collected by the Health Commission of Zhejiang Province. All patients were assigned to specific hospitals for unified treatment according to Zhejiang Province's emergency rule. All patients who were diagnosed with COVID‐19 based on the WHO interim guidance were successfully enrolled in this study.For in‐depth data analysis, the subtype definition of COVID‐19 patients was according to the Chinese diagnosis and treatment scheme for SARS‐CoV‐2 (5th edition) with minor modification based on the WHO standards. Specifically, the severity of COVID‐19 was categorized as mild, common, severe, or critical. Mild type was defined as mild symptoms with no pneumonia on imaging. Common type was defined as having respiratory tract symptoms and pneumonia on imaging. Severe type was characterized by dyspnea, respiratory rate ≥30/minute, blood oxygen saturation ≤93%, PaO2/FiO2 ratio <300, and/or lung infiltrates >50% within 24‐48 hours. Critical cases were those that exhibited respiratory failure, septic shock, and/or multiple organ dysfunction/failure.2.2We obtained epidemiological, demographic, clinical, laboratory, management, and outcome data from the patients’ medical records. Clinical outcomes were followed up to January 31, 2020. If data were missing from the records or clarification was needed, we obtained data by direct communication with the attending doctors and other healthcare providers. All data were checked by at least two doctors. Laboratory confirmation of SARS‐CoV‐2 was done in the following institutions: the Zhejiang province CDC, the first affiliated hospital, School of Medicine, Zhejiang University, and the local CDC at the city level. Throat swab specimens from the upper respiratory tract and sputum samples that were obtained from all patients at admission were maintained in a viral‐transport medium. SARS‐CoV‐2 infection was confirmed by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) using the same protocol described previously.2.3We collected and described the epidemiological data ; anthropometrics; demographics; symptoms and signs on admission; laboratory and chest X‐ray/CT results; comorbidity; coinfection with other respiratory pathogens; treatment ; and clinical outcomes.2.4https://bigd.big.ac.cn/ncov/), except for ZJ01 which was the name of the SARS‐CoV‐2 isolated separately from Zhejiang patients. The coronavirus samples came from all over the world, including Wuhan (n = 4), Guangdong (n = 3), USA (n = 2), and Australia (n = 1). There were also SARS specimens (n = 3) and other animal coronaviruses (n = 10). Multalin  was used to compare the differences between these sequences. Further SWISS‐MODEL online server (https://swissmodel.expasy.org/) was used to reconstruct the three‐dimensional structure of protein according to gene or amino acid sequence. The reconstructed protein model was put in PyMol to explore molecular mechanic and protein function. Phylogenetic tree establishment, evolutionary analysis, and other bioinformatic analyses were performed by MEGA7.0. The heat map of relative synonymous codon usage (RSCU) was drawn with MeV 4.9.0, where Codon W1.4.2 was used to estimate the RSCU of these coronaviruses. Hierarchical clustering was used to analyze RSCU of these strains, with utilization of Euclidean distance selection and average linkage clustering.All published sample sequences (n = 23) were obtained from the NCBI viral genome database and NGDC  was used for all analyses.For continuous variables, mean (SD) and median (IQR) were used for normally and abnormally distributed data. Categorical variables were expressed as numbers (%). For laboratory results, it was determined whether the measurement was outside the normal range. Univariate logistic regression analysis was used to identify the risk factors of severe/critical‐type patients. All significant variables in univariate analysis were included in a multivariate logistic regression model with Forward:Wald method to identify independent predictors of severe/critical‐type patients. No adjustment for multiple testing was performed. A two‐sided 33.1This study analyzed 465 patients with confirmed SARS‐CoV‐2‐infected pneumonia from January 17, 2020, to January 31, 2020, in Zhejiang province. As shown in Table The situation in Zhejiang province presented the scenario of family clustering, where we found 185 infected patients from 77 families. For rare case analysis, two pregnant women were diagnosed with COVID‐19 in our study. Both of them were mild type, with one in the first trimester and the other in the last trimester. One 5‐year‐old child was diagnosed with mild‐type COVID‐19 due to family exposure. In contrast to SARS, MERS, and H7N9, most COVID‐19 cases were diagnosed as common type according to the SARS‐CoV‐2 Chinese (5th edition) guidelines. Only 8.82% patients developed the severe type, and 1.72% patients progressed to the critical type, showing lower tendency severity when compared to data from Wuhan.3.2The clinical characteristics of the patients are shown in Table Concerning infection‐related parameters, over 50% patients had increased erythrocyte sedimentation rate (ESR) and C‐reactive protein (CRP). Intriguingly, none of the patients had increased procalcitonin (PCT) levels. Since radiography is pivotal for disease identification and diagnosis, 462 patients underwent chest radiography on admission, among which 408 88.31%) were diagnosed with pneumonia. Bilateral ground‐glass opacities and consolidation were the most common radiological findings inEdits our cases  patients received methylprednisolone within 6.93 ± 3.34 days of onset of illness with daily dosage of 40 to 80 mg. Besides, only 218 (46.88%) patients received antibiotics therapy, which is much lower than the 71% from the Wuhan 99 cases study.2O. None of the patients underwent continuous blood purification due to renal failure or extracorporeal membrane oxygenation (ECMO). Liver injury was the most common complication (61 patients), followed by acute respiratory distress syndrome (ARDS) (11 patients) and shock (one patient). By the end of the study period on January 31, all patients had survived and were being treated in hospital, except for one patient who was discharged.Oxygen therapy plays an important role in the supportive care of patients. In this study, 445 (95.70%) patients received oxygen therapy, and eight patients underwent mechanical ventilation (four non‐invasive and four invasive). Only five patients were admitted to intensive care unit (ICU), where four patients received invasive mechanical ventilation till January 31 for 4‐9 days. The ventilator was set at P‐SIMV mode, with inhaled oxygen concentration of 35%‐100% and positive end‐expiratory pressure of 6‐12 cm H3.4There were total 49 patients with severe/critical COVID‐19 in this study. When compared with mild and common COVID‐19, initial univariate analysis of epidemiological, clinical, and laboratory variables identified 25 significant risk factors for severe/critical COVID‐19 Table . Based o3.5). The S protein of SARS‐CoV‐2 is responsible for recognizing angiotensin‐converting enzyme 2 (ACE2) receptor and mediating membrane‐enveloped fusion for invasion. By sequencing the S protein base of ZJ01, we found that ZJ01 was very similar to MN908947.3 from Wuhan . There were only four single amino acid mutations and one amino acid position loss in this sequence, as shown by 3D protein reconstruction . Except for one mutation point exposed on the protein surface (blue), the other mutations occurred within the S protein conformation.We confirmed that ZJ01 is a SARS‐CoV‐2 with 29,381 bases by utilizing biological methods Appendix . Howevern Figure . There n Figure . Except). Though there were variations among the members of the SARS‐CoV‐2 family, they were not enough to allow them to evolve into additional subspecies. The most surprising result was the RSCU heat map  and shock (0.22%), at a much lower rate than the previously reported rates of 17% of ARDS and 4% of shock.SARS‐CoV‐2 is an enveloped virion with approximately 50‐200 nm diameter and a single positive‐sense RNA genome.The incubation period and family clustering were found to be important for COVID‐19 control and prevention, which has been rarely reported in previous studies. According to latest available data based on 10 confirmed cases, the mean incubation period was estimated to be 5.2 days .The risk factors for severe/critical COVID‐19 were calculated by multivariate logistic regression analysis. The eight factors are presented in Table There are several limitations of this study that need to be acknowledged. Firstly, the retrospective nature of this study may decrease its credibility and future prospective cohort studies should be considered. Secondly, a complete analysis of COVID‐19 on a national level is urgently needed, which might provide more solid data. Thirdly, although we summarized the risk factors for severe/critical type of COVID‐19, there is still lack of a prediction model for disease fatality, since most of the enrolled cases are currently under treatment. Finally, cytokine change is common in coronavirus infectionsIn summary, Zhejiang province was the first province that exhibited the highest response level for SARS‐CoV‐2 infection and quarantined every suspected patient, followed by immediate virus detection. All these measures were effective and helped to control virus dissemination. On the basis of our experience with the virus, we revealed the novel epidemiological, clinical, and virological features of SARS‐CoV‐2 as summarized in this paper. The changes in these features may be due to virus mutation during dissemination, as we observed on comparison of the viral sequence between Zhejiang and Wuhan with focus on the invasion‐related S protein. We have also provided large‐scale preliminary data pertaining to our treatment experience, including low and appropriate glucocorticoid usage, early and timely antiviral therapy, avoiding unnecessary antibiotics, and ample oxygen supply. Further in‐depth analysis on the therapeutic interventions is under way.Jiangshan Lian: Conceptualization ; Data curation ; Methodology ; Project administration ; Writing‐original draft (lead). Xi Jin: Data curation ; Formal analysis ; Methodology ; Writing‐original draft . Shaorui Hao: Investigation ; Methodology ; Software ; Visualization . Hongyu Jia: Data curation ; Methodology ; Software ; Visualization . Huan Cai: Formal analysis ; Methodology ; Software ; Visualization . Xiaoli Zhang: Formal analysis ; Visualization . Jianhua Hu: Methodology . Lin Zheng: Investigation ; Methodology . Xiaoyan Wang: Data curation . Shanyan Zhang: Software . Chanyuan Ye: Data curation ; Validation . Ciliang Jin: Formal analysis (supporting); Methodology (supporting). Guodong Yu: Data curation (supporting); Formal analysis (supporting); Methodology (supporting). Jueqing Gu: Data curation (supporting); Investigation (supporting); Visualization (supporting). Yingfeng Lu: Formal analysis (supporting); Investigation (supporting). Xiaopeng Yu: Validation (supporting); Visualization (supporting). Dairong Xiang: Formal analysis (supporting); Validation (supporting). Lanjuan Li: Conceptualization ; Investigation ; Project administration ; Resources ; Writing‐review & editing . Tingbo Liang: Conceptualization ; Project administration ; Supervision ; Writing‐review & editing . Jifang Sheng: Conceptualization ; Project administration ; Resources ; Supervision ; Writing‐review & editing . Yida Yang: Conceptualization (lead); Funding acquisition (lead); Project administration ; Resources (lead); Supervision (lead); Writing‐review & editing (lead).Supplementary MaterialClick here for additional data file.