We report on the public health response generated by an outbreak of coronavirus disease (COVID-19) that occurred during March 2020 at Bach Mai Hospital (BMH) in Hanoi, northern Vietnam’s largest hospital complex. On March 18, a total of 3 distinct clusters of COVID-19 cases were identified at BMH. Diagnosis of the initial 3 COVID-19 cases led to contact tracing, symptom screening, and testing of 495 persons and limited quarantine of affected institutes or departments. When 27 staff members in the catering company tested positive for SARS-CoV-2, the entire BMH staff  was put under quarantine. Contact tracing in the community resulted in an additional 52,239 persons being quarantined. After 3 weeks, the hospital outbreak was contained; no further spread occurred in the hospital. Rapid screening of cases, extensive testing, prompt quarantine, contact tracing, and social distancing contributed to prevent community transmission in Hanoi and northern Vietnam. In Vietnam, as of September 19, 2020, there were 1,068 laboratory-defined cases of the coronavirus disease (COVID-19) and 35 deaths. The outbreak in Vietnam consisted of 2 waves: the first was during January 22–July 24 with imported cases from countries in the Asia–Pacific region and Europe . In January, the first 6 positive cases in Vietnam were diagnosed by the whole-genome sequencing method, with an average of 3–4 days for returning the results. Nasopharyngeal samples were collected at the quarantine sites, then transferred to reference laboratories. Four national institutes act as reference laboratories for different regions of the country. Three of the 4 reference laboratories diagnosed the first 6 cases, including National Institute of Hygiene and Epidemiology (NIHE) in Hanoi, Pasteur Institute in Ho Chi Minh City, and Pasteur Institute in Nha Trang. From January 31 onward, the real-time reverse transcription PCR (RT-PCR) method was widely applied, which helped reduce the time for laboratory confirmation to 6 hours. The test kits were first donated by the World Health Organization (WHO), then provided by Viet-A Corporation  led to infection of many healthcare workers and other inpatients  in Hanoi were reported, leading to a widespread investigation and response effort at the hospital. We describe how a nosocomial COVID-19 outbreak in one of the largest hospitals in Vietnam was contained through rigorous testing, active case finding, contact tracing, and whole-hospital quarantine.BMH is Hanoi’s largest national tertiary general hospital, with nearly 3,000 inpatient beds and an average of 5,000 outpatients per day. The hospital has 34 clinical centers, institutes, and departments and 6 paraclinical departments, with >6,000 healthcare workers and nonclinical staff. Three affiliated national institutes are under BMH management: National Heart Institute (NHI), National Institute of Mental Health, and National Institute of Medical Expertise. The first branch of the National Hospital for Tropical Diseases (NHTD) is also located inside the BMH area, but has been a freestanding hospital since 2006. The NHTD has a second branch that was the designated hospital for COVID-19 patients in northern Vietnam, located in a suburban area of Hanoi. BMH has its own infectious disease facility, the Center for Tropical Diseases (CTD), separate from NHTD .>1 suspicious symptom  and having a history of traveling through epidemic areas or having close contact with a patient with confirmed COVID-19 during the preceding 14 days. Before March 12, nasopharyngeal swab specimens from the suspected cases were transferred to the NIHE for SARS-CoV-2 confirmation. Beginning March 13, the specimens were processed and confirmed at BMH itself. Patients with suspected cases were transferred immediately, in dedicated vehicles, to the second branch of NHTD, even if test results had not yet been received. In early January 2020, BMH established 2 dedicated COVID-19 screening triage clinics for suspected cases. These clinics were located in separate areas from other departments of the hospital: the first was next to the main gate, and the second was set up near the CTD . Healthchttps://ncov.vncdc.gov.vn). Symptoms and treatment data were systematically collected from the official MoH COVID-19 database (https://ncov.moh.gov.vn) and the MoH official press release website (https://suckhoedoisong.vn).We conducted a desk review of available documents, patient records, and public data collected during March 17–April 15, 2020. We retrieved demographic data from the official COVID-19 database provided by General Department of Preventive Medicine  was carried out.We established different definitions of suspected cases, as well as a hierarchy of contacts, between the BMH outbreak and standards management in Vietnam in general. The MoH’s general guidelines defined a suspected case as illness in a person who had In the outbreak at BMH, all persons who visited the hospital during March 10–March 20 were considered as the F1 group, regardless of their exposure to laboratory-confirmed cases. For contact tracing, 4 levels of contacts were followed up, from F1 to F4 (F4 comprised close contacts of F3), which is one level higher than the general guideline. F1 and F2 persons were quarantined at a centralized area, and F3 and F4 persons isolated at home . Affecte>2 negative tests before a person was released from quarantine, Hanoi Center for Disease Control conducted an additional retest for all confirmed cases before the removal of lockdown. The test kits were either donated by the WHO or provided by Viet-A Corporation. In total, an estimated 15,000 tests were analyzed for quarantined persons at BMH. Each test cost »$30 USD.We tested all F1 persons quarantined at BMH for SARS-CoV-2 using RT-PCR in the microbiology department; persons at the CTD, NHI, and neurology department (ND) were tested 3 times to confirm the situations in these departments, and all others at BMH were tested once. Because of the requirement of >1 test by the local Center for Disease Control. The numbers of tests per person depended on the decision of the local steering committee, which considered the occurrence of symptoms and laboratory capacity.F1 and F2 persons who were traced and quarantined in the community were provided The study was approved by the BMH ethics committee. We applied all ethics considerations needed according to MoH or by its designees.During March 18–April 14, a total of 46 laboratory-confirmed COVID-19 cases were detected at BMH. The mean age of the patients was 44.9 years, and 80.4% were female. Ten (21.7%) patients were symptomatic; 91.3% had a history of admission to BMH or working or visiting an institute in the BMH complex, including healthcare workers (4.4%), nonclinical staff (58.7%), patients (13.0%), and family caregivers (15.2%) .Case 86 was in a female nurse working at the HIV outpatient clinic of the CTD. On March 11, she had chest tightness and pain and was admitted to the NHI; her diagnosis was a clinical manifestation of preexisting hypertension illness. She had multiple contacts with CTD staff during lunch and break periods, including the patient with case 87, a 33-year-old female nurse working at the COVID-19 screening clinic who developed fever (38.5°C) and dry cough on March 18 and had a positive test for SARS-CoV-2 on the same day . QuarantCase 133 was in a 66-year-old woman who was admitted to Lai Chau General Hospital for stroke on February 29 and was transferred to the BMH neurology department . On MarcOf the 46 confirmed cases, 27 were from the hospital catering company . These pOn March 28, the quarantine was extended to all of BMH. A total of 7,664 persons were quarantined: 6,258 healthcare workers and other staff members, 793 inpatients, and 613 of the patients’ related family caregivers .>2 m apart, ensuring that all healthcare workers used personal protective equipment, and having healthcare workers using N95 masks when performing aerosol-generating procedures associated with viral spread. In addition, only 1 healthcare worker at a time was allowed contact with a patient, except when a medical intervention required >1 person. Family caregivers were not permitted to have direct contact with patients. Body temperature measurement and mandatory medical reporting for all persons in and out of the hospital, enhanced room air flow, and retraining in infection prevention and control (IPC) were implemented for all the staff. Separate entryways for new emergency cases and routine dialysis patients were set. Other routine outpatients, such as patients with diabetes, hepatitis, or cardiovascular disease, were asked to delay their regular visits and go to local hospitals for care and treatment.BMH stopped new admissions on March 20, except for patients with severe and critical conditions. A total of 5,113 inpatients were transferred to local provincial hospitals or other specialized hospitals in Hanoi. These patients had non–COVID-19-related illnesses and were considered safe to transfer; they were managed as F1 persons and received preventive measures from the local government and Center for Disease Control. A total of 793 patients with non–COVID-19-related illnesses required treatment at BMH because of the severity of their illness. These patients were managed with a high level of infection control, including spacing beds F1 persons were categorized into 7 groups. Four groups had registered information at BMH: healthcare workers, visiting scholars and students, nonclinical staff, and patients (both inpatients and outpatients). The other 3 groups, family, hired caregivers, and other persons who visited patients, could be found only by epidemiologic investigation, self-reported or reported in the local community. In addition, information on all cases was widely available on social media and media outlets, alerting members of the general community about potential exposure if they were at the hospital.Healthcare workers, visiting scholars and students, severely or critically ill inpatients, and their family and hired caregivers were quarantined and managed at BMH. All other BMH cases from March 10–20  were traced and managed by the local Hanoi steering committee. A total of 52,239 persons were followed up in the community. Among those who were traced, 27,893 F1 and F2 persons were put in quarantine . Nearly >2 weeks; contact tracing in the community; and quarantine of all contacts. From the beginning of the COVID-19 pandemic, testing strategy has been an essential intervention in preventing community spread of COVID-19 in Vietnam. As of May 13, 2020, >275,000 SARS-CoV-2 RNA tests had been conducted in Vietnam; the proportion of tests per confirmed case was »950 , were considered as having suspected cases, placed in centralized quarantine, and tested. Modeling suggested that active case tracing and early testing had a major effect on reducing the community transmission of COVID-19, up to 80% . A large population screening in Iceland showed that the positive rate among 13,080 nontargeted citizens was 0.8%, and 43% of SARS-CoV-2 positive cases were asymptomatic (https://doi.org/10.1101/2020.04.25.20079103). Only 10 symptomatic cases were found among the 46 laboratory-confirmed COVID-19 cases in the outbreak (21.7%); 1 patient needed intensive care with mechanical ventilation (2.2%), and there were no deaths. Several large investigations with a similar approach to active case tracking efforts also showed a high rate of asymptomatic patients among persons who tested positive for SARS-CoV-2. For example, in a cohort of 829 employees who worked at Rutgers University and associated hospitals in New Jersey, USA, the prevalence of asymptomatic SARS-CoV-2 cases was 65.9% . Second, because we did not interview all the patients who tested positive, the source and index cases were not fully interpreted. Finally, we did not perform a complete outbreak investigation, which reduced the validity of the containment outcomes and made it difficult to compare this study with other studies.In conclusion, the COVID-19 outbreak containment at BMH is a noteworthy example in which a major university hospital was quarantined to prevent further community transmission. Containment of the outbreak in BMH could serve as an example for other settings that are experiencing new outbreaks of this highly transmissible disease. We suggest several recommendations to prevent hospital COVID-19 nosocomial outbreaks. Strict triage stations should be established at all entrances; healthcare workers, nonclinical staff, and contract workers should be monitored and those with symptoms recommended to stay home if ill; and other key IPC measures should be instituted at the hospital according to the hierarchy of IPC controls. Protective equipment should be provided to all staff, both clinical and nonclinical, as well as training in how to use it correctly. In addition, cases of severe viral pneumonia should be monitored closely, with SARS-CoV-2 testing recommended when all other possible causes have been excluded. High-risk groups, such as patients with severe acute respiratory infections, healthcare workers, and elderly patients, should also be strictly monitored.


To identify and quantify associations between baseline characteristics on hospital admission and mortality in patients with COVID-19 at a tertiary hospital in Spain.This retrospective case series included 238 patients hospitalized for COVID-19 at Hospital Universitario Clínico San Cecilio  who were discharged or who died. Electronic medical records were reviewed to obtain information on sex, age, personal antecedents, clinical features, findings on physical examination, and laboratory results for each patient. Associations between mortality and baseline characteristics were estimated as hazard ratios (HR) calculated with Cox regression models.2/FiO2 ratio , SOFA score  and CURB-65 score .Series mortality was 25.6%. Among patients with dependence for basic activities of daily living, 78.7% died, and among patients residing in retirement homes, 80.8% died. The variables most clearly associated with a greater hazard of death were age , diabetes mellitus , SatO2/FiO2 ratio, and high SOFA and CURB-65 scores.The patients residing in retirement homes showed great vulnerability. The main baseline factors that were independently associated with mortality in patients hospitalized for COVID-19 were older age, diabetes mellitus, low SatO Since 11 March 2020, when the World Health Organization (WHO) declared a worldwide SARS-CoV-2 pandemic, there have been more than 213,000 confirmed cases in Spain (as of 25 April 2020) and 22,157 deaths, representing a mortality rate of 47 per 100,000 inhabitants [Among confirmed cases of infection, 48.7% patients have required hospitalization mainly for pneumonia and adult respiratory distress syndrome (ARDS). Among patients with pneumonia, men, people older than 60 years, and people with CVD and/or DM are overrepresented . Among SRadiographic findings in patients with pneumonia are highly variable depending on–among other factors–the time elapsed since symptom onset ,10. HoweFew studies to date have shed light on the main factors associated with fatality rates in hospitalized patients. Most notably, a retrospective cohort study of 191 patients at two hospitals in China reported 41 deaths, and analysed the association of sex, age, chronic obstructive lung disease, coronary disease, diabetes, hypertension, chronic kidney disease, and other comorbidities on the risk of death due to COVID-19. According to the multivariate models in this study, associations were detected only for patient age and two parameters measured on admission: Sequential Organ Failure Assessment (SOFA) score and D-dimer concentration greater than 1 μg/ml . Other sThe aim of this study was to identify and quantify the associations between baseline characteristics on hospital admission in patients with COVID-19 infection  and mortality at a tertiary hospital in Spain.This retrospective case series comprised 238 patients admitted for COVID-19 to Hospital Universitario Clínico San Cecilio, and who died or were discharged, between 16 March and 10 April 2020. This 480-bed tertiary hospital is located in the city of Granada (southern Spain), and provides specialized health care services to a total population of 291,797 inhabitants. Only positive PCR test was considered as diagnosis criteria for the patients included in our series. Therefore, patients presenting compatible symptoms and negative test were not included in the present study. The information detailed below was collected from electronic medical records for each patient.Sex, age , presence of comorbidities , dependence for basic activities of daily living (BADL) (yes or no), clinical findings on admission , smoking status  and place of residence (own home or retirement facility).2), externally supplied oxygen (litres), fraction of inspired oxygen (FiO2), SatO2/FiO2 ratio, haemoglobin, lymphocyte count, neutrophil count, platelet count, D-dimer level, glycaemia, total bilirubin, LDH, ferritin, CRP, procalcitonin, troponins, creatinine, kidney function (elevated urea and creatinine), partial O2 and CO2 pressure in arterial blood (PO2 and PCO2), CO2, arterial blood pH, SOFA score, CURB-65 score, and X-ray findings on admission (categorized as dichotomous variables), e.g. diffuse opacities, interstitial opacities, hilar congestion or pleural effusion.Systolic and diastolic blood pressure, heart rate, temperature, basal oxygen saturation , it cannot be considered a true cohort of admitted patients, given that by definition we excluded patients who, at the end of the recruitment period (10 April 2020), remained hospitalized (censured data). For these patients the mean duration of hospital stay can be assumed to be longer than for patients included in the present case series. Because the length of hospital stay tends to be inversely associated with mortality, this association would lead to selection bias, which in turn would result in overestimating the proportion of deaths in our sample. In addition, it could potentially skew (in either direction) the magnitude of the associations between patient characteristics on admission and death. To compensate for this source of bias we opted to use time to death as a dependent variable and used Cox proportional hazards regression models to calculate hazard ratios (HR) in order to quantify the magnitude of associations between hazard or instantaneous death rate and patients’ baseline characteristics.Because of the low number of patients compared to the number of independent variables considered in this analysis, we used a three-step modelling process. First, we developed separate models for each baseline variable and for age. Next we defined subgroups of baseline variables , and used a stepwise process to build multivariate models for each variable . The variables selected in each model where then used in a stepwise procedure to build a final model that included baseline variables that were retained. In all cases the P value for inclusion in the model was set at 0.05, and the P value for exclusion was set at 0.10. The 95%CI was calculated for each HR. All data were analysed with STATA software (version 15.0) .This study met the requirements for appropriateness to achieve the intended aims and complied with all ethical principles pertinent to this type of study design. All data were fully anonymized before the analyses. No informed consent was required due to the characteristics of the design. The study was approved by the Research Ethics Committee of Granada province (CEI) on 13 April 2020.Slightly more than half of our patients were males . The age range was 24 to 97 years with a mean of 64.7, standard deviation 15.4, and median 67 years. Most patients (60.5%) were 60 years old or more, and the largest age stratum was 70 to 79 years. At least one comorbidity was present on admission in 56.7% of the sample. Among the most prevalent diseases were HT (48.7%), DM (21.9%), and CVD (22.7%). About one fifth of the patients (19.8%) had dependence for BADL, and 10.9% lived in retirement homes . On admiAlso shown in Tables 2/FiO2 ratio was inversely related with mortality . None of the other baseline variables were independently associated with mortality.The sex and age distribution in our series of patients differs from that reported by the WHO mission in China and from data published by the Spanish National Epidemiology Centre, both of which found very similar figures ,20. In tRegarding the distribution of comorbidities and symptoms on hospital admission, our results are consistent with earlier studies in China ,24,22,25The models we obtained with our adjusted analysis showed that in our series of patients hospitalized for COVID-19, characteristics present before SARS-CoV-2 infection that were independently associated mortality were age  and DM. A study from China based on a retrospective cohort of 191 patients identified CVD and DM as chronic diseases associated with greater mortality. However, in contrast to our finding for DM, Zhou and colleagues found that this association disappeared in their adjusted analysis . In thisAt least one other review of comorbidities associated with COVID-19 found that CVD and DM were among the factors most strongly associated with an increased risk of mortality, although this was based only on crude data .2/FiO2 ratio as a result of ARDS) and high SOFA and CURB-65 scores. These findings are consistent with factors identified in earlier reports from countries other than Spain [With regard to baseline variables relating to the characteristics of SARS-CoV-2 infection on admission, those that yielded independent associations with mortality were greater need for externally supplied oxygen to maintain adequate saturation  knowledge of COVID-19. There is another potential bias concerning the selection of patients which are mainly elderly patients coming from a localized region in Spain. Therefore, the results could not be widely generalized. A final consideration is that we designed a monocentric study with a limited number of patients, while a great number of different factors were analysed as independent variables. This limited sample size necessitated a sequential process to develop our models, with a stepwise approach and low number of predictive variables.2/FiO2 ratio, and high SOFA and CURB-65 scores. These characteristics define a profile of patients in Spain with an elevated mortality rate for whom a specific approach to clinical management is urgently needed.In addition to the close association between mortality and comorbidities, dependence for BADL and living in a retirement facility in our crude analysis, the main baseline factors that were independently associated in our adjusted analysis with mortality in patients hospitalized for COVID-19 were older age, DM, low SatO


Escherichia coli-infecting phages (coliphages) from poultry faeces to gain a better understanding of the coliphage diversity in the poultry intestine. All phages belonged to either the Siphoviridae or Myoviridae family and their genomes ranged between 44,324 and 173,384 bp, with a G+C content between 35.5 and 46.4%. Phylogenetic analysis was performed based on single “marker” genes; the terminase large subunit, portal protein, and exonucleases, as well as the full draft genomes. Single gene analysis resulted in six distinct clusters. Only minor differences were observed between the different phylogenetic analyses, including branch lengths and additional duplicate or triplicate subclustering. Cluster formation was according to genome size, G+C content and phage subfamily. Phylogenetic analysis based on the full genomes supported these clusters. Moreover, several of our Siphoviridae phages might represent a novel unclassified phage genus. This study allowed for identification of several novel coliphages and provides new insights to the coliphage diversity in the intestine of poultry. Great diversity was observed amongst the phages, while they were isolated from an otherwise similar ecosystem.Despite phages’ ubiquitous presence and great importance in shaping microbial communities, little is known about the diversity of specific phages in different ecological niches. Here, we isolated, sequenced, and characterized 38  Phages are thought to play essential roles in shaping the microbial ecology, including driving the diversity of the bacterial communities5. As no single gene is present in all phages, their taxonomic classification is based on host-range, physical characteristics, including size and morphology, genetic structure and composition, and overall genome similarity7. The phage classification scheme is regularly updated, refined and approved by the International Committee on the Taxonomy of Viruses (ICTV)8. Furthermore, in recent years several genome-based phage taxonomy schemes have been proposed9. According to the National Center for Biotechnology information (NCBI), as of February 2020, 9,238 complete phage genomes have been sequenced. However, despite a continuously rising number of sequenced phage genomes, most of them remain unclassified and poorly characterized. According to the ICTV, a phage genus can be defined as a group of viruses with > 50% nucleotide sequence similarity, which is distinct from viruses of other genera. Moreover, defining characteristics can be determined for each genus, including average genome length and number of coding sequences (CDSs), percentage of shared CDSs, and the presence of specific signature genes in genus members10.Bacteriophages (phages) are viruses that have the ability to specifically infect bacteria. They are estimated to be the most abundant form of life on Earth , these families comprise five, eleven, three, two, and five subfamilies, respectively, and 87, 210, 48, three, and 15 genera, respectively. The currently analysed Myoviridae coliphages belong to four subfamilies, including Ounavirinae, Peduovirinae, Tevenvirinae, and Vequintavirinae, and 17 genera. Siphoviridae coliphages are found in only two subfamilies; Guernseyvirinae and Tunavirinae, and in 13 genera. Podoviridae coliphages belong to two subfamilies, the Autographivirinae and the Sepvirinae, and to ten genera. Ackermannviridae coliphages belong only to the Cvivirinae subfamily and the Kuttervirus genus. To date, there have been no Herelleviridae coliphages isolated. To understand the diversity, relationships, and dynamics among any group of phages, nucleotide sequence information is needed1. For tailed phages, it has been reported that conserved genes such as the terminase large subunit, the portal protein and major capsid protein, can be used as phylogenetic phylomarkers for the diversity as well as their evolutionary relationship13.Most phages that infect 14. Besides, despite the phages’ significant role and ubiquitous presence in various areas, little is known on the nature and extent of phage diversity in different ecosystems3. Recently, there has been an interest in the diversity of coliphages16. Here, we performed a detailed genome-based characterization and phylogenetic analysis of 38 fully sequenced coliphages, all isolated from a single, relatively unexplored environmental source: poultry faecal material.Compared to their bacterial hosts, relatively few phages have been fully characterizedE. coli C600 or K514 as host strain.In this study, 38 coliphages were isolated from poultry faecal samples collected from 27 Belgian poultry farms located in five different regions, including West Flanders, East Flanders, Antwerp, and Limburg. Between one and seven phages were isolated from each farm using Caudovirales order and either the Siphoviridae family or the Myoviridae family. Analysis of the isolated Siphoviridae phages showed a long flexible non-contractile tail with a length varying between ~ 100 and ~ 200 nm and icosahedral heads with widths ranging from ~ 52 to ~ 77 nm  Myoviridae coliphages. The Siphoviridae phages were compared with 146 published phages from this family. The Myoviridae phages were compared with 171 published Myoviridae phages. According to ICTV guidelines, phage family, subfamily and genus were predicted based on genome similarity. Results are shown in Table Siphoviridae phages belonged to the Tunavirinae subfamily, except for Phage 61. This phage was predicted to belong to the Tequintavirus genus, which do not have any ICTV subfamily. The ten phages, Phage 8, 53, 54, 63, 65, 68, 69, 71, 72, and 75 all belonged to the Hanrivervirus genus. Three phages belonged to the Rtpvirus genus, including Phage 17, 70, and 73. No existing ICTV genus could be assigned to the remaining 13 coliphages. Phage 28, 56_1 and 76 could be assigned to the same unknown genus. Phage 58 and Phage 74 were found to be in the same genus. Phage 47, 48, 59, 64, and 77 were predicted to belong to the same genus. Phage 52, 56_2, and 80 were predicted to belong to the same genus. The 11 Myoviridae belonged either to the Tevenvirinae or the Ounavirinae subfamily. Tevenvirinae phages included the six phages: Phage 10, 11, 15, 18, 30 and 55. Phage 10, 11 and 55 belonged to the Tequatrovirus genus, and Phage 18 and 30 belonged to the Mosigvirus genus. Ounavirinae phages included the remaining five phages; Phage 60, 62, 66, 78 and 79. All phages belonged to the Felixounavirus genus.Taxonomic classification of the 38 isolated coliphages was performed through multiple WGS genome comparisons. These coliphages included 27 71%) % SiphoviSiphoviridae and 182 Myoviridae coliphage genomes were included. Characteristics of selected reference genomes are listed in Supplementary Table Siphoviridae phages isolated in this study were found in five different (sub)clusters, cluster A1–3, B and C, with a cut-off value of 0.82  and accessory genes (shell and cloud). As cluster A phages had only five core genes , analysis of subclusters, A1, A2 and A3, were performed additionally. Results are summarized in Table Pan genome analysis of The level of synteny and genomic rearrangement within each cluster or subcluster of related phages was assessed by genome comparison. Results are summarized in Table Genes encoding the terminase large and small subunit, the major capsid protein, DNA primase, single-stranded protein, portal protein, recombinase, specific tail protein and holin were the most conserved genes between all phages, whereas genes with the lowest level of conservation included, specific tail fiber proteins, tail tape measure proteins and HNH homing endonucleases. Hypothetical proteins were found with large variation in level of conservation. Each phage genome comprised between four and 17 LCBs. Genome comparison of phages belonging to subcluster A1, A2 and A3 identified 16, seven and four LCBs, respectively. All phages in each cluster comprised all LCBs. All cluster B phages comprised all six LCBs. For the cluster C phages, between 6 and 10 LCBs were identified for each phage. Phage 61 isolated in this study comprised all nine regions. Variation in number of LCBs was due to a variable repeat region comprising multiple LCBs, which was found only in some of the cluster C phages. For the cluster D comparison, 14–17 different LCBs were identified for each phage. Variation in number of LCBs was due to four different small variable regions of which some of all were missing in the majority of the phages. Phages isolated in this study, including Phage 79, Phage 78, Phage 60, Phage 66, and Phage 62, comprised 17, 17, 16, 15, and 14 LCBs, respectively. Comparison of phages belonging to cluster E identified 18 LCBs. All phages lacked one or both of the same two LCBs. Phages isolated in this study, including Phage 30, Phage 15, and Phage 18, comprised 16, 17, and 17 LCBs, respectively. All 13 cluster F phages included in the comparison comprised all five LCBs. The comparison confirmed the presence of homologue regions between the phages within the clusters but also highlighted that re-arrangement and/or gain/loss of LCBs must have occurred at some point during the evolution of the phages. The region encoding the terminase large subunit and portal protein were present in a conserved region all genomes in all eight comparisons.Siphoviridae and Myoviridae families. However, one should be aware of the possible biases as the coliphages were isolated using two E. coli K12-derived host strains only and, as such, cannot be seen as the complete coliphage diversity. Both host strains are mutated for the FhuA  gene, which is used as receptor for some phages, including Siphoviridae coliphages T1 and T517. However, phage adsorption is not always restricted to one receptor. Accordingly, we were able to isolate a T5-like phage using the selected host strains. Also, as none of the host strains has the F pili, phages utilising a F pili encoded receptor for absorption, such as Inoviridae phages, will most likely not be isolated17. The reasons why we could not isolate coliphages from the Podoviridae family remains obscure, however, this is in accordance with the finding of Korf et al.15, who also did not isolate any Podoviridae coliphages from poultry while they could isolate them from sewage and surface water. On the other hand, other studies have successfully isolated Podoviridae phages from poultry faeces21. Several factors might play a role in the type of phage being isolated, including culture and isolation method, host strain, and isolation source. In Podoviridae studies mentioned above, phages were isolated using a single or multiple E. coli host-strains and the DLA method was similar to this study. However, a notable difference is the type of host-strain used. In our study, laboratory strains were used whereas the Podoviridae study host-strains only included E. coli strains isolated from poultry. As our 11 Myoviridae phages were isolated from samples from 11 different farms, no correlation between phage isolated and geographical location (poultry farm) was found (data not shown). In accordance to the findings of Olsen16, the most prevalent genus of the Myoviridae and Siphoviridae phages isolated in our study was the Felixounavirus (45.5%) and the Hanrivervirus (33.3%), respectively. Both studies used E. coli K-12 derived laboratory strains as host strain for phage isolation. While in a collection of 50 coliphages isolated from surface water, manure, sewage, or animal faeces, 29 different E. coli host strains were used15. No Felixounavirus phages could be isolated and most Myoviridae phages belonged to the Tequatrovirus genus, followed by the Mosigvirus genus. Those two genera were also found in our study.In this study, 38 coliphages were isolated from poultry faecal material, sequenced and characterized. The high number of coliphage genomes included in the analysis allowed for a better understanding of both coliphage diversity in poultry and the global coliphage diversity within the 23, and a similar approach combining multiple methods is recommended when working with phages12. However, studying phage taxonomy has proven challenging since no universal conserved marker gene, as the 16S rRNA gene used for bacteria, exists throughout all phage families. Several semi-conserved family-specific marker genes have been proposed as candidates to support taxonomical classification of tailed phages, including DNA packaging and head assembly genes25. Accordingly, in this study we used the terminase large subunit, portal protein and exonucleases as markers, and were able to determine the family and subfamily to all coliphages isolated in this study in accordance with the TEM-based and whole genome-based classification, respectively. Furthermore, the analysis based on the terminase large subunit and portal protein resulted in a clear distinction between the two Tevenvirinae clusters, indicating different genera (Mosigvirus and Tequatrovirus). Thus, single gene-based analysis provides good initial indication in which taxonomic cluster the phages belong to. However, a single gene does not provide a global view of the structural organization of the phage nor accounts for genomic rearrangements, mutations, and mosaicism. Moreover, in accordance with finding from previous studies the selected gene was not always detected in the phage genomes24, thus excluding these phages form classification. When using single genes, one should be aware of the possibility of multiple distinct variants of the same gene within one genome. Several of our phages encoded up to three different exonuclease genes. Depending on which gene variant used for analysis, the risk of “false” cluster formation and distance, and hereby a faulty classification at subfamily or genus level was present. Thus, for more comprehensive phage taxonomy, including genus classification, the single gene analysis should be accompanied by whole genome-based analysis as well as functional gene studies. When investigating the evolutionary relationship between phages studies have shown the advantage of combining different proteomic and comparative genomic approaches, including WGS data and well-characterized reference dataset, which take into account the effect of horizontal gene transfer (HGT) and recombination events on the phage genome evolution26.Currently, the polyphasic approach is the most commonly used for bacterial classificationSiphoviridae genus. The three unclassified A3 subcluster from this study clustered together with the Jahat_MG145 reference phage, which was a singleton16. Thus, we propose that this group of phages represents a new unclassified genus with currently four phages, including Phage 52, Phage 56_2 Phage 69, and Jahat_MG145.In this study, genome-based phylogenetic and taxonomic analysis were performed in combination with traditional morphological examination of the phage using TEM. Through the genome-based analysis we identified a potential new 27, lower genome size seemed to be correlated with an increase in G+C content. The largest variation in genome size and number of CDSs were observed for the group of Myoviridae phages, whereas the largest variation in G+C content (7.2%) was overserved for the group of Siphoviridae phages. Notably, the Tequintavirus Phage 61 showed to be more similar to the group of Myoviridae compared to the other Siphoviridae phages based on the above-mentioned characteristics. When omitting Phage 61, G+C content variation for the Siphoviridae phages was only 2.9%. Phage G+C content has been shown to be correlated with the G+C content of the phage host28. Accordingly, differences in G+C content observed for the coliphages might reflect phage-host interactions and co-evolution with past and current host(s). Gene content, including number of core genes appeared to be associated with the cluster. Moreover, number of exonucleases encoded by the phage appeared to be cluster associated as well, as only phages from cluster C and cluster F encoded multiple exonucleases. Encoding multiple exonucleases could be a result of adaptive evolution conferring fitness advantage over other phages. However, it could just as well reflect some of the challenges to accurate phage genome annotation, including false negatives (undetected genes) and incorrect functional annotation30. Gene content variation has been shown to be related to recombination events resulting in acquisition or loss of gene(s)31. Through our comparative genomics analysis of related phages, LCBs with modules with varying level of gene conservation were identified and highlighted the different levels of heterogeneity between different phage clusters. Repeat regions were observed in several of the phage genomes and resulted in variation of LCBs. The presence of these regions should be considered when assessing the gene content variation, as these regions are shown to be prone to genome assembly mistakes, and as such, might represent false level of variation32. LCBs modules are also called for mosaic section and the two terms have been used interchangeably throughout history. One definition is that mosaic sections refer to patches of low nucleotide similarity when two similar phage genomes are compared, and that modules refer to exchangeable sections between two or more phages in the population13. The genome comparison showed the mosaic nature of the phage genomes, with modules with high level of conservation interspersed with mosaic sections. The mosaic sections could be acquired though HGT from other phages, which is thought to happen when phages are found in the same host. Most often this happens through phage co-infection or single-phage infection of a host that carries one or more prophages34. Moreover, phages have been shown to acquire genes from their host35. The comparative genomics approach hereby underlines the continuous evolution of phage genomes as well as the great phage diversity.We aimed to expand our knowledge on the coliphage diversity, and observed great diversity among these phages, while they were isolated from a similar ecosystem. The diversity was characterised by a great span in genome size (44.3–173.1 kb) and G+C content range (35.5–46.4%), as well as cluster-specific characteristics of the six phage clusters, A–F. Cluster B phages had the smallest genomes and lowest number of CDSs followed by phages from cluster A, D, C, and E/F. Similar to findings from other studiesIn conclusion, this study has identified a potential new coliphage genus and several new species and provides insight not only to the coliphage diversity of the intestine of poultry but the global coliphage diversity as well. Moreover, classification of phages isolated in this study brings us one step closer to a more refined taxonomic understanding of coliphages. Our comparative genomic analysis showed different levels of heterogeneity between different phage clusters and highlighted the mosaic nature of the phage genomes as well as the continuous evolution of phages in a single environment source. However, to fully understand the complexity and underlying mechanisms of the phage diversity further studies are needed.37. Briefly, the samples (5 g) were emulsified in Luria Bertani (LB) broth . The decanted supernatant obtained from each emulsion was enriched by the addition of two early-log phase host bacteria, E. coli K-12 derived laboratory strains C60038 or K51439, a non-restricting, modifying  derivative of strain C600. Suspensions were incubated overnight at 37 °C, with shaking (120 rpm) and were then centrifuged at 4,000 rpm for 30 min to pellet the cellular debris. The supernatant containing the phage(s) was re-centrifuged and filtered using a 0.45 µm membrane filter followed by a 0.2 µm Minisart Filter . The enriched phage suspensions were enumerated and tested for lytic activity on the host bacteria using the double-layer agar (DLA) technique41. Briefly, phage suspensions were serial diluted and spotted on an overlay of the respective host bacteria on solid LB medium supplemented with 0.8% agar and 0.5 mM CaCl2. A clear zone in the plate, a plaque, resulting from the lysis of host bacterial cells, indicated the presence of virulent coliphage(s). Samples with lytic activity against the indicator strain were further processed for single phage plaque isolation, including three rounds of plaque purification, and propagation. All phage lysates were stored at 4 °C until required.The phages were isolated from poultry faecal material, collected randomly from 27 poultry houses in Belgium in 2013. Phages were propagated according to Adams and Bonilla et al. with minor modificationsThe morphology of unique coliphages (≤ 95% nucleotide similarity) isolated in this study was investigated using transmission electron microscopy (TEM). Phage suspension was applied to the surface of Formvar carbon-coated grids, the phages were fixed using paraformaldehyde (PFA) (4% w/v), washed, and negatively stained with UrAC (1% w/v). After drying, grids were examined using a JEM-1400 Plus transmission electron microscope .42. The concentration and quality of the DNA was assessed using the NanoDrop  and Qubit fluorometer  according to the manufacturer’s instructions. Preparation of paired-end 2 × 250 bp sequencing libraries was done using the Nextera XT Kit  with adaptations for phage genomes as shown elsewhere43 and sequenced on the Illumina MiSeq platform using MiSeq Reagent Kit v2 (500-cycles) and manufacturer’s instructions, yielding a total of 16,270–237,128 paired end reads for each phage lysate. Read-pair contigs were generated for each MiSeq cluster prior to assembly.DNA extraction from phage lysates was performed using DNeasy Blood & Tissue Kit  as previously describedhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/), version 0.11.3, was used for quality control validation of the raw reads sequence data. Low-quality sequences were excluded from further analysis. The raw reads were trimmed for quality, adaptor sequences were removed using default parameters. The sequence reads were de novo assembled using QIAGEN Bioinformatics CLC Genomic Workbench, version 11.0.1, using default settings, with minimum contig length changed to 250 bp. An overview of assembly statistics is provided in Supplementary Table https://blast.ncbi.nlm.nih.gov/Blast.cgi) using the Basic Local Alignment Search Tool (BLAST) software44, and from the custom PHAge Search Tool Enhanced Release (PHASTER) phage database45. Newly assembled phage sequences were compared using both BLAST and PHASTER to identify unique and identical (> 95% nucleotide sequence similarity) phages. Assembled contigs were submitted to the ResFinder database, version 3.246 and the VirulenceFinder database, version 2.047 to identify any acquired antimicrobial resistance and virulence associated genes, respectively. By default, selected threshold for %ID was 90% and 60% for minimum length. All 15 databases for antimicrobial resistance genes were selected. The taxonomic group of E. coli bacteria was selected for VirulenceFinder. PHASTER and The Rapid Annotation using Subsystem Technology (RAST) server and the SEED viewer, version 2.0, were used for identification of CDSs and initial annotation of the phage genomes, including identification of the phage terminase large subunit48. Gene function of genes defined as “hypothetical protein” was predicted by comparison to homologue genes with defined functions in other related phage genomes. The G+C content of the phages was calculated using the SEED viewer.FastQC software (Siphoviridae and Myoviridae coliphages. Known reference coliphages included were limited to isolates with complete genomes found in the ICTV database and the NCBI database (data of November 2019).Multiple genome alignment of the WGS sequences was performed using Applied Maths BioNumerics software, version 7.6. According to the ICTV taxonomy guidelines, the 38 coliphages were classified into phage family, subfamily and genera based on nucleotide similarity to known 49, for comparison of the coliphages with published Siphoviridae or Myoviridae reference phage genomes (accessed from the NCBI database). The trees were constructed using unweighted pair group method with arithmetic mean (UPGMA) from a distance matrix of binary distances calculated from either, gene presence/absence within the full genomes of the phages determined using Roary version 3.12.050, Prokka, version 1.13.751 and prodigal, version 2.6.352, or Kmer presence/absence (using 10 and 21mer) based on de novo assembled contigs, as calculated using a python script  was constructed and supported by bootstrap analysis  with default substitution model (Tamura-Nei model) to assess the diversity of the coliphages using the phylogenetic and molecular evolutionary analyses (MEGA) software, version X54. The phylogenetic trees were based on the nucleotide sequences of the CDSs of the following genes: phage terminase large subunit, phage portal protein, or phage exonucleases. The reference genomes included, represented the best matching published sequences to the phages in this study (selected based on the BLAST max score) and core reference genomes for comparison.For phylogenetic analysis based on single marker gene, phage gene sequences were aligned using Clustal X, version 2.155 and phangorn56.The degree of topological and branch length agreement between the different phylogenetic methods and between the three marker genes was investigated using the R packages Analysis of Phylogenetics and Evolution (ape)A more detailed analysis of the most closely related coliphage genomes was carried out. Genomes were re-annotated using Prokka and pan genome analysis was carried out with Roary using script “roary-e-n-s-p 20-i90*.gff”, including identification of core genome, including core and softcore genes, and accessory genome, including shell and cloud genes.57 with default parameters. No more than 19 of the most related phages were included in each comparison for simplification. Relatedness of the phages were based on percentage of nucleotide similarity and number of shared core genes. Reference genomes were included for annotation references.In order to investigate the level of synteny and genomic rearrangement, whole genome alignment and comparison of coliphages and related reference phages from each cluster or subcluster were performed with the Mauve software using progressiveMauveSupplementary Information.


The extracts were characterized by high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS), and 57 compounds, mainly flavonoids but also organic and phenolic acids, fatty acids, and terpenes, were identified. After analytical characterization, a multistep screening approach, including antioxidant, enzymatic, and photoprotective cellular studies, was used to select the best extract according to its benefits of interest to the cosmetics industry. The extract obtained with supercritical CO2 presented the best characteristics, including a wide antioxidant capacity, especially against lipid peroxyl and •OH free radicals, as well as relevant photoprotective action and antiaging properties, making it a potential new ingredient for consideration in the development of new cosmetics.In the cosmetic industry, there is a continuous demand for new and innovative ingredients for product development. In the context of continual renovation, both cosmetic companies and customers are particularly interested in compounds derived from natural sources due to their multiple benefits. In this study, novel and green-extractive techniques (pressurized solvent, supercritical CO Currently, society has a higher interest in skin care products obtained from natural sources compared to pharmaceutical compounds obtained by chemical synthesis . NaturalNatural extracts have been used traditionally in herbal medicines. The nutritional, chemopreventive, and pharmacological properties of natural extracts highlight the beneficial health effects of plant-derived compounds. In fact, extracts of many plant species have demonstrated antioxidant and anti-inflammatory capacities, and they can also present antitumoral, antimicrobial, or antiaging actions ,6,7,8.The biological activities of plant extracts are due to their high contents of potential bioactive compounds that can interact with different targets involved in molecular mechanisms related to alterations or diseases. These biological activities are mainly due to the secondary metabolites of plants, which can be classified mainly into polyphenols, terpenoids, alkaloids and sulfur-containing phytochemicals . SecondaPrunus avium L., a tree commonly known as SC, belongs to the Rosaceae family. SC trees are widely distributed around the world, with a higher prevalence in temperate climates. In Europe, Spain, Italy, Greece, Poland, Hungary, and Germany produce the most SC -Leu-Gly-Pro-Ala (FALGPA), as described previously [N-succ-(Ala) 3-nitroanilide (SANA) as the substrate and phenylmethanesulfonyl fluoride (PMSF) as the positive control, as described in [N-acetylglucosamine after sodium hyaluronate incubation was measured, and p-dimethylaminobenzaldehyde was used as the positive control for inhibition. The antiglycation assay was performed with bovine serum albumin (BSA) as a substrate. Fluorescence was measured 7 days after BSA incubation with threose and diethylenetriaminepentaacetic acid, as described previously [The inhibition of collagenase was studied through the degradation of eviously , and epieviously . The effribed in . The hyaribed in ; the amoeviously , using av/v) FBS and 1% (v/v) of antibiotics (0.1 mg/mL penicillin and 100 U/mL streptomycin) in a humidified atmosphere with CO2 (5% v/v) at 37 °C. The cells were trypsinized following the manufacturer’s instructions every third day and seeded in 96-well plates . Extracts were prepared and dissolved in dimethyl sulfoxide (DMSO) at 30 mg/mL for every assay.Human immortalized keratinocytes from HaCaT cell line were obtained from CLS Cell Lines Service GmbH . The cells were cultured as previously described ,23 in DM2DCF-DA) fluorescence probe  was used to determine the effect of the extract on intracellular reactive oxygen species (ROS) generation induced by UVA and UVB radiation. For this purpose, cells were cultured in black 96-well plates and maintained in medium for 24 h. The cells were washed with phosphate-buffered saline (PBS) and treated with 50 μL of PBS containing the extract  during UVB light treatment (800 or 1200 J/m2) or UVA radiation (3 or 6 J/m2) emitted from Bio-Link Crosslinker BLX-E312 and BLX-365, respectively . After irradiation, the PBS was replaced with fresh medium, and the cells were incubated with H2DCF-DA (10 μg/mL) for 2 h at 37 °C and 5% CO2 for the oxidative stress assay. The fluorescence of H2DCF-DA was measured using a Cytation 3 Cell Imaging Multi-Mode Reader microplate reader  with 495 nm excitation and 520 nm emission filters.The 2′,7′-dichlorodihydrofluorescein diacetate -2,5-diphenyltetrazolium (MTT) assay was used to determine cell viability after 24 h of UVB irradiation to determine the photoprotective effect of the extracts ,22,23. Tp < 0.05, detailed in the figures using the following symbols: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 [Statistical comparisons were developed using GraphPad Prism software v6.0 . One-way ANOVA and Tukey’s post-test were employed for data analysis. Differences were considered statistically significant when < 0.0001 ,23.2O (50:50), and H2O, respectively, up to a concentration of 1000 mg/L. Their chromatographs are shown in The scPLE, scSFE, and scSWE extracts were fully characterized by HPLC-ESI-QTOF-MS. For this analysis, the dried extracts were reconstituted in EtOH, EtOH-HThe compounds were tentatively identified using the information provided by the software , together with the fragmentation patterns obtained from tandem mass spectrometry (MS/MS) experiments in comparison with standards when available or data previously reported in the literature. A total of 57 compounds were identified from 4 different families: (1) organic acids, phenolic acids, and derivatives (8 compounds); (2) flavonoids and derivatives (36 compounds); (3) fatty acid derivatives (9 compounds); and (4) terpenes (4 compounds). Thus, 18 of these compounds are herein identified for the first time in this matrix. The identities of the obtained compounds are summarized in Semiquantitative comparisons among the different extraction techniques regarding the presence of individual compounds in those extracts can be observed in After the analytical processing of the extracts, a multistep screening of the three extracts was conducted in three stages to select the best extract with the greatest potential as a novel cosmetic ingredient . This screening was designed to evaluate the most relevant biological activities for the cosmetic industry using a collection of in vitro and cellular assays, all of which are described in the Materials and Methods section.The first step evaluated the total phenolic content (TPC). This assay was the first step because polyphenolic compounds are one of the main groups of compounds known to show anti-aging effects and present other biological activities ,34. Furtp > 0.05). scSFE showed the highest antioxidant effect in the TEAC assay, followed by scPLE. However, scSWE showed a remarkably lower TPC and antioxidant capacity . Thus, both assays showed similar trends for the three extracts, with the lowest antioxidant activity for that extract with the lowest polyphenolic content, as expected. On the basis of these results, scSWE was not included in for further screening steps due to its poorer results.The results for this first stage are presented in Cistus sp. plants obtained by aqueous and hydroalcoholic conventional extraction methods previously characterized by our group [In a second stage, additional antioxidant assays, FRAP and ORAC, were carried out to clarify the antioxidant activities of the selected extracts (scSFE and scPLE). FRAP estimates the Fe(III) reducing activity, whereas the ORAC assay determines the activity related to chain-breaking antioxidants, which is directly related to peroxyl radicals. These analyses are more closely related to the biological function of antioxidants ,39. The ur group ,38.Although scSFE and scPLE presented some differences, probably due to differences in their composition, as shown in · radicals, and a Griess nitrite-based assay for nitric oxide radicals were performed. The results for all these assays are shown in The third stage further elucidated the antioxidant capacity of scSFE through additional antioxidant assays. In this sense, TBARS for the specific study of lipid peroxidation, a modified ORAC method based on OHscSFE showed a significant antioxidant capacity in all the assays, suggesting that this extract is a good candidate for use as a bioactive ingredient against oxidative stress, as the extract has shown antioxidant capacity through different methods and against different targets, such as lipid peroxidation and different kinds of free radicals.Antioxidant activities are highly desirable for cosmetic ingredients for several reasons. On the one hand, this activity protects the final formula itself from oxidation, especially from oxidation related to its oily ingredients. In addition to this advantage, which is mainly related to the final product formulation, the antioxidant activity of the ingredients is probably one of the most commonly used claims in cosmetic products. In this sense, natural extracts have been shown to reduce oxidative stress, mainly due to their polyphenols ,41. scSFIn this third stage, the putative modulative activities of scSFE on some of the most relevant enzymes related to skin health and appearance were also tested. In this sense, the activities of collagenase, elastase, hyaluronidase, and tyrosinase were challenged with scSFE, as detailed in the Materials and Methods section. These experiments were concluded with a study of the inhibition of advanced glycosylation end product (AGE) formation, conducted as described in the Materials and Methods section. The inhibition of collagenase, tyrosinase, elastase, hyaluronidase, and AGE formation are related to the prevention of the degradation of extracellular matrix (ECM), skin preservation, and antiaging. In fact, plant extracts have been shown to inhibit tyrosinase, collagenase, elastase, and hyaluronidase activity ,50,51. TCollagen plays a critical role in the appearance and function of the skin; it confers tensile strength and resiliency to the skin and is the main protein in the ECM of the dermis. Its degradation is related to skin wrinkling and aging. Collagenase inhibition is related to the maintenance of skin tensile strength and elasticity, even more so in collagenase induction by ROS or irradiation, which are important factors in aging. In this case, scSFE did not present any collagenase inhibition activity, presenting a negative value, which means that its effect was weaker than that of the negative control but was not statistically significant.Tyrosinase is an enzyme involved in melanin production, the main defense of organisms against UV irradiation. Melanin absorbs UV radiation and reduces the formation of photoproducts that could be harmful to the skin . Tyrosinp < 0.01).Elastin is an extracellular matrix protein responsible for elasticity in the dermis and other connective tissues by forming elastin fibers. Elastase is an enzyme able to degrade elastin, leading to skin aging and wrinkles. Therefore, the inhibition of elastase is related to skin aging and wrinkle protection. The results in p-dimethylaminobenzaldehyde (positive control), and the effect was highly significant (**** p < 0.0001).Hyaluronic acid is found in connective tissue and is part of the ECM. Hyaluronic acid presents water holding properties and maintains the viscosity and the correct permeability of connective tissues and maintains skin hydration. Hyaluronidase degrades hyaluronic acid, and its inhibition is related to the maintenance of high levels of hyaluronic acid, improving the general aspect of skin and specifically skin hydration. scSFE presented potent hyaluronidase inhibition activity, reaching almost 100% of the level obtained for p < 0.0001).Finally, oxidative stress increases protein glycation, which is responsible for advanced glycosylation end products (AGEs) in skin. AGEs are one of the causes of collagen degradation, leading to skin aging. The inhibition of protein glycation is related to the prevention of aging and wrinkling. As expected by its antioxidant capacity shown in the previous section, scSFE was able to reduce AGE formation by 50% with high statistical significance  are inhibitors of collagenase, elastase, and hyaluronidase ,54,55,56th scSFE . These rth scSFE ,62.The overproduction or accumulation of melanin could lead to pigmentary disorders such as vitiligo, and it is related to skin aging and photoprotection. Tyrosinase is the enzyme that regulates the hydroxylation of L-tyrosine to form 3,4-Dihydroxy-L-phenylalanine (L-DOPA), a precursor of melanin. Inhibiting tyrosinase is a method for avoiding disorders related to skin hyperpigmentation, and in vitro enzymatic assays, as employed in the present study, are significantly related to melanin synthesis in melanocytes . Some po2) was first determined through MTT assay in the presence of different concentrations of scSFE  increased cell viability compared to untreated irradiated cells, whereas 100 µg/mL extract did not protect against the 1200 J/m2 dose. This protective effect was greater in the highest treatment concentration  after 800 and 1200 J/m2 irradiation, with a statistically significant protective effect compared to the untreated control. The highest photoprotection activities were observed for 800 J/m2, 14.61% with 100 µg/mL extract, and 36.53% photoprotection with 200 µg/mL extract. However, weaker effects were obtained after 1200 J/m2 of UVB irradiation, with 3.51% and 13.99% photoprotection, respectively, compared to control as shown in scSFE extract increased the viability of cells 24 h after UVB irradiation compared to the irradiated control. At 800 J/mThis photoprotective activity of scSFE could be due to various factors. On the one hand, scSFE showed a significant absorption in the UV range in a dose-dependent manner, and thus a substantial portion of the observed keratinocyte photoprotection could be due to the ability of the compounds present in this extract to absorb and scavenge UVB radiation, as many plant extracts have been shown to do ,67,68,692DCF-DA fluorescence, as described in the methods section. 2) or UVB (800 or 1200 J/m2) irradiation in the absence or presence of the extract compared to the control (without irradiation).As mentioned in the previous section, one of the putative mechanisms involved in scSFE photoprotection may be its antioxidant properties. To check if the antioxidant properties shown by scSFE in the previous sections were also present at the cellular level, the antioxidant capacity of the extract was evaluated in vitro through the determination of intracellular ROS in the HaCaT cell line. Ultraviolet (UV) A and B were used to induce oxidative stress, as determined by measuring dichlorofluorescein-diacetate HOxidative stress was inhibited by all the concentrations at all the UV doses (both UVA and UVB) tested in this assay. The reduction of fluorescence observed in all conditions is displayed in 2DCF-DA is a fluorescent probe that is particularly sensitive to H2O2, •OH, and peroxynitrite radicals at the intracellular level [OH assay showed that scSFE has a significant capacity to scavenge •OH, a harmful radical that can be derived from the Fenton reaction of H2O2 or from lipid peroxides, and a significant capacity to eliminate NO• radicals, which may form peroxynitrite upon reaction with O2•−. Therefore, the photoprotective properties of scSFE shown in this study may be related to its capacity to decrease the generation of intracellular radical species such as H2O2, •OH, or peroxynitrite, which can damage a wide range of molecules in cells, including proteins and DNA [Har level . The ORA and DNA ,80. The  and DNA ,45,46,47 and DNA ,73,78, c and DNA ,82,83, rNew cosmetic ingredient development is a long and costly process, but there is continuous demand for new products in the cosmetic market. This manuscript tries to address this need through the revalorization of agricultural byproducts such as SC stems.According to the obtained results, scSFE is a strong candidate for use as a new cosmetic ingredient, especially due to its antioxidant properties, especially against lipid peroxidation, its activity against skin aging-related enzymes, and its photoprotective capability. As indicated in previous studies, these actions are related its main polyphenolic compounds—catechin, chrysin, and naringenin. However, further studies must be performed on three main topics: the molecular mechanisms involved in these biological activities, the putative pharmacological interactions between the scSFE main compounds, and the compatibility and stability of these compounds or the whole extract when incorporated in a final cosmetic formula.


Alseodaphne hainanensis is an endangered species with inhabiting dense forest along valleys, and it is one of the representative species of tropical rain forest in Hainan Island. In this paper, we reported and characterized the complete chloroplast genome sequence of the species assembled from short reads generated by Illumina sequencing. The size of chloroplast was 152,829 bp, a pair of inverted repeats (IRs) separating a large single copy and a small single copy, the size of IRs, LSC and SSC were 20,036 bp, 93,872 bp and 18,885 bp, respectively. A total of 129 genes were predicted, including 81 protein-coding genes, 38 tRNA, 8 rRNA, and 2 pseudogene. Phylogenetic analysis confirmed the position of A. hainanensis within the order Laurales. Alseodaphne hainanensis Merrill (Lauraceae) is an evergreen tree which grows to a height of 25 m. It is distributed in parts of Hainan Island and north of Vietnam, inhabiting dense forest along valleys at an altitude of 1400–1700 m. Alseodaphne hainanensis is an endangered species and its wood is finely grained, heavy, and durable. The population has been drastically reduced due to long-term utilization and unreasonable logging  in China, and the specimen was deposited at the botany laboratory of Hainan normal University, Haikou, China. Total genomic DNA was extracted from five mixed fresh leaf tissues using the CTAB method (Doyle The plant samples of od Doyle . The genAlseodaphne gracilis (NC037489) as reference, the chloroplast genome of A. hainanensis was assembled with the program NOVOPlasty  region  separated the large single copy (LSC) region  and small single-copy (SSC) region . The content of GC is 39.12% and chloroplast genome contains 129 predicted genes, including 81 protein-coding genes, 38 tRNA genes, 8 rRNA genes, and 2 pseudogenes. The accession number on GenBank is MK867376.The complete chloroplast genome of A. hainanensis in the order Laurales, 9 complete chloroplast genome sequence in the order Laurales were aligned by MUSCLE, the sequences were inferred by the maximum likelihood methods with MEGA7 (Kumar et al. Machilus thunbergii as outgroup. The result shows that Cinnamomum camphora in Lauraceae is most closed with A. hainanensis (A phylogenetic analysis was conducted to analyze the position of nanensis . Our res


Sirex noctilio F. (Hymenoptera: Siricidae: Siricinae), a new invasive species in China, is a significant international forestry pest which, transported via logs and related wood packing materials, has led to environmental damage and substantial economic loss in many countries around the world. It was first detected in China in 2013, and since then infestations have been found in 18 additional sites. Using a 322 bp fragment of the mitochondrial barcode gene COI, we studied the genetic diversity and structure of S. noctilio populations in both native and invaded ranges, with a specific focus in China. Twelve haplotypes were found across the native and invaded distribution of the pest, of which three were dominant; among these there were only one or two mutational steps between each pair of haplotypes. No obvious genetic structure was found other than in Chinese populations. China has a unique and dominant haplotype not found elsewhere, and compared with the rest of the world, the genetic structure of Chinese populations suggested a multiple invasion scenario. The current primary route for alien forest species invading China is through human activity. This is mainly unintentional introduction; transported seedlings and their propagation materials carry 46% of human activity-generated invaders, with packaging materials or wood accounting for 21%  [Dendroctonus valens, fall webworm Hyphantria cunea and loblolly pine mealybug Oracella acuta, have already caused the destruction of forestry resources in China, negatively influencing species diversity and ecological security, and resulting in major economic loss [Biological invasions are increasing exponentially as globalization progresses, without any sign of saturation, especially in insects ,2. At prndtjgb/) . Some mamic loss ,10,11. IPlutella xylostella, moves within China from south to north, with rare effective travel in the reverse direction [Grapholita molesta, which originates in southern China and has dispersed to the north [Frankliniella occidentalis, Cai (2017) recently determined the 13-year history of its invasion of China, revealing multiple introductions and further human-assisted spread, using mitochondrial genes and 18 microsatellite loci. In order to infer the invasion routes of the Asian long-horned beetle (Anoplophora glabripennis) in Europe, Javal (2019) used a 485 bp fragment of the mitochondrial barcode gene COI, which revealed multiple introductions from China and a bridgehead effect [COI gene (mtDNA cytochrome oxidase I) has been of widespread use in tracing invasions [Molecular markers, such as mitochondrial, ribosomal, chloroplast, and microsatellite DNA, and others, have become increasingly popular for investigating phylogenetic relationships between insect populations. By using mitochondrial genes and microsatellite loci, Wei 2013) showed that the invasive diamondback moth, irection . Similar showed td effect . The COInvasions ,16,17,18Sirex noctilio, a woodwasp native to Eurasia and North Africa, is a significant forestry pest, subject to quarantine regulations around the world [S. noctilio outside its native Eurasian range was in the southern hemisphere during the 1900s [Pinus radiata planted there, killing trees across approximately 120,000 ha of the north–central region and thereby causing huge economic and ecological loss. During 1987–1989, an outbreak in Australia and Tasmania killed more than 5 million P. radiata trees [he world . Its larhe 1900s . Followita trees ; it was ta trees . SeveralS. noctilio was first detected in 2013 in the northeast, at Daqing, Heilongjiang Province [S. noctilio is found in China at similar latitudes to its native sites; moreover, its native regions in Russia are near the Chinese border. Based on climatic conditions and host distribution, domestic and foreign experts analyzed the potential woodwasp distribution area in China. Their results indicated that a band stretching from Yunnan to Heilongjiang represents a highly suitable area for its establishment [In China, Province ,24. Six Province . MoreoveProvince . S. noctlishment . Its potS. noctilio in nine populations from New York and Pennsylvania (US), Ontario (Canada), and Queensland , showing that multiple introductions occurred in northeastern North America. However, a more comprehensive understanding requires the collection of more samples from its native Europe and Asia.In order to investigate the origins and pathways of invasion, Bernard (2012) used microsatellite and sequence data collected from five continents. Two major sources were identified, although the invasion history was quite complex, with most of the populations containing admixtures of independently introduced European insects that subsequently spread through the invaded regions. Similarly, Bittner (2017) investigated the genetic diversity of 924 S. noctilio populations were surveyed using the mtDNA cytochrome oxidase 1 (COI) gene. Our objective was to investigate the genetic structure and genetic diversity of S. noctilio populations around the world, especially in relation to Chinese infestations. We compared patterns in invaded continents and native regions, analyzing for the first time 778 individuals from 6 continents. In particular, samples from original European populations and recent Chinese infestations were included. Overall, this study aims to elucidate the invasive routes of this pest as well as provide data enabling the prevention of the harmful consequences of S. noctilio spread.In this study, the genetic structure and invasion routes of S. noctilio samples were collected from native European, non-Chinese invaders, and Chinese populations, comprising 44, 127, and 607 specimens, respectively. A summary of sampling sites and common parameters of the genetic diversity of S. noctilio in this study is provided in https://datadryad.org/stash/dataset/doi:10.5061/dryad.37mm8; from Genebank: https://www.ncbi.nlm.nih.gov/nuccore/?term=sirex+noctilio. Finally, we obtained a total of 607 specimens from 19 Chinese populations , we used only 20 of the sequenced individuals, which included all the haplotypes found in each population. We selected individuals from each haplotype based on the selection ratio and the number of samples in each haplotype. The final total of 364 samples for analysis consisted of 44, 127, and 193 individuals from Europe, non-Chinese invaded areas, and China, respectively.COI barcoding fragment was amplified via PCR using the forward primer LCO1490 (5ʹ-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) and the reverse primer HC02198, (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) . PCR reagents were used and reactions performed following Sun [COI fragment was amplified at the Canadian Centre for DNA Barcoding  using a slightly different sequencing set (C-LepFolF/C-LepFolR), following the standard high-throughput protocol [For the native European samples not supplied as sequences directly from GenBank, DNA extraction, PCR amplification and sequencing of the barcode fragment were performed for 15 specimens at the Laboratory of Forest Zoology URZF, INRAE . DNA extracts were prepared from one adult hind leg or thorax muscle using NucleoSpin tissue XS kits  according to the manufacturer’s protocol. The wing Sun . PCR proprotocol .COI amplification primers were synthesized by a commercial company . PCR primers LCO1490 and HC02198 were used as above. Reactions were performed in a total volume of 25 μL, containing 12.5 μL of 2 × GoTaq® Green Master Mix , 1 μL of each primer, 1.5 μL of DNA template, and 9 μL of ultrapure water. The reaction procedure was as follows: initial denaturation for 2 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 30 s, and extension at 68 °C for 2 min, with a final extension at 68 °C for 10 min. PCR products (3 μL) were analyzed by electrophoresis on a 1.5% (w/v) agarose gel (1 × TAE), alongside a DNA marker . After electrophoresis at 130 V for 20 min, the PCR products were visualized using ethidium bromide and ultraviolet light. Finally, products clearly visible after electrophoresis were sent to a commercial company  for sequencing in both directions.DNA was extracted from the leg or thorax muscle of each Chinese specimen using the Multisource Genomic DNA Miniprep Kit , following the manufacturer’s protocol to prevent cross-contamination, all tools were sterilized by flame or 75% pure anhydrous ethanol. DNA was eluted using 70 μL of elution buffer and stored at −20 °C until use. Species-specific COI sequences were trimmed to the same length (322 bp) for final alignment.All electropherograms were checked manually in CodonCode Aligner v3.7.1.  to assess their quality. The alignment was done independently using Clustal W implemented in MEGA v6.06 with default parameters, and no pseudogenes or stop codons were detected in the sequences ,29. All www.boldsystems.org).Details on the collecting data for each specimen, as well as photographs, sequence records, trace files, and primer sequences used for PCR amplification, together with GenBank accession numbers, are available through the dataset in BOLD  were performed on native European populations and Chinese populations to measure the partitioning of genetic variation between populations and groups of populations in Arlequin 3.5.2.2 ,35. The There were 11 variable sites among all insects sampled. Three dominant haplotypes, H1, H2, and H9, were present among the total of 12 haplotypes; the haplotype diversity was 0.4207. Only one or two mutation sites existed across the different haplotypes . There wp < 0.01) was between geographic clusters, with a relatively small part of the genetic variance  between populations within geographic clusters. When sites were grouped by latitude (p < 0.01) between clusters was slightly higher than that  between populations within clusters. Within this study, no clear genetic structure emerged within native European populations.AMOVA analysis of the genetic structure of Chinese populations  showed tlatitude , the varP. sylvestris var. mongolica seedlings are taken for distribution and planting elsewhere. H3 is present in 5% of DM individuals, and so does H4, H5, H6, H7, these haplotypes occurred in 10%, 5%, 7%, and 25% of sampled insects of HG, FJ, YS and HHEJ, respectively. The latitude 46.204°N was used to group samples into the categories north and south. Haplotype diversity in the northern area is obviously higher than that in the south. The northern cluster has seven haplotypes, H1, H2, H3, H4, H5, H7, and H8, from nine populations, compared to two haplotypes (H1 and H6) from ten populations in southern China.The Chinese sampling sites are divided into four groups based on geographical proximity: west for one group, middle for two groups and east for one group. The middle and eastern groups, where there are cities, contained 4 haplotypes, whereas the western group, which is mainly mountainous, had 3 haplotypes. The first site discovered in China, DM, contained 40% H1 and a similar proportion, 55%, of H2. There is no H1 in the western group, although the proportion of H1 in the two middle area groups is quite high as is also found in the eastern group. There is a decreasing trend from west to east in the proportion of H2. The representative populations on this axis were HDQ, DM, and HG with 100%, 55%, and 20% H2, respectively. Among the other six unique haplotypes in China, H8 is located in MZL, on the border between China and Russia. Four of the remaining five haplotypes are distributed in the populations from which large samples were taken and are located in city areas. The sixth, HHEJ, is located in the area from which S. noctilio is rather large, although it is known that the pest is harmful to various kinds of conifers around the world, especially Pinus, Picea, Abies, Larix, and Pseudotsuga [P. sylvestris var. mongolica is in the area of Honghuaerji in Inner Mongolia and north of the Huma River in Heilongjiang [S. noctilio in the wild distribution areas of P. sylvestris var. mongolica in either mountain or sandy ecological zones  and Jinbaotun (Inner Mongolia). The former is a mixed forest of Pinus koraiensis, L. olgensis, Picea koraiensis and P. sylvestris var. mongolica. The latter contains Pinus tabuliformis and P. sylvestris var. mongolica. However, fieldwork has indicated that the only infested conifer species in China to date is P. sylvestris var. mongolica, even when other potential hosts are present in the same forest [S. noctilio appears to be harmless in the natural distribution area of its favorite host tree species.Currently, the main infestation zones in China are those where e forest . As is cP. sylvestris var. mongolica, trees from this area will be planted elsewhere. We speculate that H1 is an invasion haplotype carried by humans that have entered the central and eastern urban areas of China. As it existed on all continents, its source is unclear. Based on our data, H2, H7 and H8 were found only in China, and given that they are unlikely to have originated there, we are unable to identify their origin. H2 is the main haplotype of the western component, with a distribution ratio that gradually decreases from west to east; this is in line with the characteristics of spread from a point of origin to other regions. This, as noted above, may represent an invasion originating with Russian wood imported for construction at Honghuaerji and spread eastward by planting. Severely endangered woodlands in Dumeng, Hegang, Fujin, and other eastern areas may have been invaded in this way. H7 and H8 are also unique to the western region; H8 occurs on the border with Russia.There are currently three more frequent haplotypes, H1, H2, and H9, which have a worldwide distribution. It is thus unsurprising that they are in the center of the haplotype network and closely connected with other well-established haplotypes . InteresS. noctilio populations than in those to the south. Damage to woodlands in the northeastern region occurs discretely, in multiple forest farms, parks, and other wooded land. This damage was concentrated, as is characteristic of the middle to late stages of an invasion. In contrast, in newly-invaded southern areas, such as Jinbaotun, damaged forests were localized and individual affected trees were distributed discretely in the affected area, indicating the initial stage of introduction. Although the 1130 km between Honghuaerji and Jinbaotun is shorter than the 1400 km between Honghuaerji and the affected area in the northeast, as the haplotype H1 found in the southern region is not present in Honghuaerji, the south regional populations may originate from northern China.Grouped by latitude, significantly more haplotypes exist in northern Chinese S. noctilio has a relatively strong natural flight ability. In the southern hemisphere, the maximum spread rate of an invading population of S. noctilio is 82 km per year [per year . In Chinper year . The strCOI) could prove effective, showing its utility as a method for determining invasion pathways and spread.When analyzing biological invasions, knowledge of the genetic structure of populations from the area of origin is vital in determining the source. Our target species is native to Eurasia and North Africa, but it does not cause serious harm there and has consequently received less attention, leading to difficulties in collecting samples. In particular, the lack of samples from the Russian region caused sampling breakpoints between the European and northeast Chinese populations. The observation that H2 occurred only in China may be due to the large sample size from some previously unsampled sites from this area. More reliable results may be obtained from sampling efforts focused on this region and on western China. Finally, microsatellite marker use involving cytochrome C oxidase subunit I (COI was used to analysis the genetic diversity and structure of S. noctilio populations in both native and invaded ranges, with a specific focus in China. Twelve haplotypes were found across the native and invaded distribution of the pest, of which three were dominant; among these there were only one or two mutational steps between each pair of haplotypes. No obvious genetic structure was found other than in Chinese populations. China has a unique and dominant haplotype not found elsewhere, and compared with the rest of the world, the genetic structure of Chinese populations suggested a multiple invasion scenario.In this study, a 322 bp fragment of the mitochondrial barcode gene


Background and Objectives: An effective flushing technique is essential to reduce intravenous (IV)-related complications and improve patient care. New technology should contribute to such improvements, while reducing costs and increasing care efficiency. This study evaluated the efficacy, safety, and convenience of a new flushing technique using a Baro Flush™ controller. Materials and Methods: We evaluated the efficacy and safety of Baro Flush™ by measuring the infusion flushing volume and pressure in vitro. Afterwards, we prospectively enrolled 3000 patients with flushing and assigned 1500 patients with a new technique for flushing and 1500 with a conventional flushing method, which was performed by 48 registered nurses (RNs) at the Gil Medical Center in June 2018. The efficacy, safety, and convenience of the new flushing method were evaluated though a questionnaire survey. Results: The average flushing pressure was 12.5 ± 0.6 psi (86.18 ± 4.14 kPa) with 1.2 ± 0.2 mL per flush, as recommended by the Centers for Disease Control and Prevention based on 85 experiments. No IV-catheter-related complications were reported by the RNs during the study. More than 80% of the RNs reported that the new flushing method was easier to learn, improved care efficacy, and was more convenient than conventional flushing. Conclusions: The new flushing method using a Baro Flush™ controller showed improved efficacy, safety, and convenience compared with the conventional flushing method, and no IV-catheter-related complications occurred, including occlusion and inflammation. The new flushing method promises to reduce IV-catheter-related complications and shows improved efficacy, safety, and convenience. Venous cannulation via a central or peripheral intravenous (IV) catheter is one of the most frequently performed health care procedures, with up to 80% of all hospitalized patients requiring medication, nutrition, or transfusion through IV catheters ,2,3. AltAccording to the Centers for Disease Control and Prevention (CDC), it is important to flush the catheter under positive pressure to reduce IV-catheter-related complications, maintain patency, and prevent the mixing of incompatible medications ,7,8,9. AConventional flushing methods usually involve several steps: preparing the flushing fluid (sterile 0.9% sodium chloride) in a 10 mL syringe; cleaning the access port with a single-use 70% alcohol-impregnated swab or 2% alcohol chlorhexidine thoroughly for at least 15 s, and allowing it to dry before accessing the system . AccordiFlushing with an aseptic non-touch technique using a prefilled syringe was introduced as an alternative to conventional methods; although it eliminated preparation of the flushing fluid, it still needed several complex steps . PositivTherefore, we evaluated the efficacy, convenience, and safety of the new Baro Flush™ flushing method in a prospective study population.The new flushing set is a modification of a conventional IV set, especially the fluid volume controller. To manipulate the flushing controller, the flushing controller is pulled along a conventional fluid line and down and up twice to generate sufficient flushing volume and pressure .To evaluate efficacy, convenience, and safety of the new Baro Flush™, we conducted a study in two steps. In the first step, we conducted an in vitro study to find the proper flushing volume and pressure before applying it directly into patients. In the second step, we prospectively enrolled 3000 patients who were admitted into the Gil Medical Center (GMC) and assigned into 1500 patients with the conventional flushing method and 1500 patients with the new flushing technique.We evaluated the efficacy and safety of the new Baro Flush™ flushing method by measuring the infusion flushing pressure and volume in vitro. CDC guidelines recommend an infusion pressure of less than 25 psi and a minimum flushing volume of 2 mL for one flush ,8,22. ToWe repeated this experiment 85 times by having 13 RNs and four physicians perform the experiment five times each.We prospectively enrolled 48 RNs at the GMC who performed 3000 cases of IV-catheter flushes in June 2018: 1500 cases (1500 patients) using the Baro Flush™ and 1500 cases (1500 patients) using the conventional method.We prospectively enrolled 3000 consecutive patients who were admitted into the GMC during the study period, who agreed to be enrolled in this study, and who signed into written consent.The inclusion criteria for registered nurses is as below: First, they had to agree to participate in this study and sign written informed consent. Second, they had to work as registered nurses in the ward in the GMC. Third, they had to have experience using flushing sets for patients. Fourth, they had to have experience flushing for patients. The inclusion criteria for enrolled patients was as follows: they were supposed to have an intravenous catheter more than 48 h after admission.TM was made for any patient who uses an intravenous catheter, we did not set any other exclusion criteria for this study except for the aforementioned one to avoid allocation bias.Exclusion criteria for enrolled patients and registered nurses were as follows: first, those who refused to participate in this study; second, those who were not supposed to have an intravenous catheter in less than 48 h. Since Baro FlushUsing a questionnaire, the 48 RNs evaluated the efficacy, convenience, and safety of the new, and conventional flushing methods. The questionnaire included four items: (1) learnability, (2) efficacy, (3) convenience, and (4) safety. Complications of the new flushing technique using the controller were closely monitored by qualified RNs.We monitored patients with flushing-related complications during the admission period and more than one week after discharge.p-value ≤ 0.05 was deemed to be significant. All statistical analysis was performed using SPSS software .The surveys consisted of close-ended multiple-choice questions and hybrid questions , which are summarized descriptively in proportion with their 95% confidence intervals. The nonparametric Mann–Whitney U test was used for the analysis. Categorical variables were reported as a percentage and analyzed using the chi-square or Fisher’s exact test. A The study design, survey protocol, and related documentation were reviewed and approved by the GMC Research Ethics Board (GMC IRB number: GBIRB2017-124).Using Baro Flush™, the average ± standard deviation of flushing infusion pressure was 12.5 ± 0.6 psi, (86.18 ± 4.14 kPa), and the mean flushing volume was 1.2 ± 0.2 mL per flush based on 85 experiments.No case of occlusion or phlebitis was reported in either group during the study period.Regarding the convenience of Baro Flush™, 70.0% (n = 33) of the participants indicated that the new method had good to excellent convenience compared with the conventional method . Regardip < 0.01). Therefore, this method is a promising alternative to the conventional flushing method.This study evaluated a new flushing method using the Baro Flush™ controller without the need for flushing syringes and evaluated its efficacy, convenience, and safety compared with the conventional flushing technique. The new flushing method reduced the risk of IV-catheter-related irritation and infection compared with the conventional method. Moreover, the learnability, efficacy, and convenience of the new method were higher than for the conventional method (There were several differences between our newly developed flushing device and the conventional IV infusion pump system. First, our newly developed flushing device is to be used for effective flushing not to be used for continuous drug infusion. While the conventional device, the IV infusion pump, is generally used for continuous drug infusion or transfusion, our newly developed device ,24, the There have been several attempts to overcome the pitfalls of conventional flushing methods. Flushing with prefilled syringes and positive- or negative-pressure mechanical valve needleless connectors has been introduced in clinical practice. When using prefilled syringes, RNs and clinicians do not need to prepare the flushing syringes . OtherwiAs an alternative, we evaluated an external flushing controller, the Baro Flush™, which is a modified version of the conventional fluid volume controller with the added function of flushing. The Baro Flush™ procedure consists of pulling the controller down and up along the IV line twice. Consequently, no aseptic procedures are needed for managing the controller, and no flushing syringes or accessing ports are required. This simplified technique is easy to learn and use and is effective. RNs reported that the new method was superior to the conventional method in terms of learnability, efficacy, convenience, and safety.The average flushing pressure using Baro Flush™ in 85 experiments was 12.5 psi with 1.2 mL per flush. According to CDC guidelines , the infOur study had several limitations. Although 3000 cases of flushing were examined, the study was conducted in a single center. Therefore, a larger study population in a multicenter randomized controlled trial is needed. Second, we monitored complications only for up to 7 days. However, IV-catheter-related complications usually occur within 7 days [In conclusion, the new flushing method using a flushing controller is a promising alternative to the conventional flushing method in terms of efficacy, safety, and convenience of use to prevent IV-catheter-related complications.