There are many injection methods for the treatment of knee osteoarthritis, but there is no comprehensive comparison, based on the fixed effect model.According to the retrieval strategy, we searched randomized controlled trials (RCTs) randomly from PubMed, the Cochrane Library, Embase, the China National Knowledge Infrastructure (CNKI), Wanfang Database from their inceptions to August 2020, and 2 members of us selected literatures and extracted data independently. Methodological quality was assessed by using the Cochrane bias risk tool, and meta-analysis was performed by using the Stat.14.0.This study will evaluate the effectiveness and safety of different injectable drugs for the treatment of knee osteoarthritis and rank the efficacies of drugs, then to determine the optimal treatment.This study will provide evidence for the choice of injection therapy for knee osteoarthritis.INPLASY202080099. Its main clinical symptoms include knee pain, impaired activity, late stage, and even disability, which seriously affects daily life and work.,6 IA injection has been proved to be effective in relieving pain and improving function in many studies.–9 However, injection therapy includes many drugs, such as hyaluronic acid, ozone, platelet-rich plasma (PRP), botulinum toxin (BT), and corticosteroids. The questions are, will different drugs have different effects? Which drug is the best choice for treating knee osteoarthritis?Currently, the treatment methods for knee osteoarthritis can be divided into surgical treatment and conservative treatment. Due to economic problems, surgical risks, postoperative complications, and other reasons, most patients choose conservative treatment. Conservative treatment mainly includes oral or external anti-inflammatory drugs , intra-articular (IA) injection, exercise therapy, physical therapy, etc.–11 only compared one treatment approach with blank control or another. On the contrary, in the previous network meta-analysis, scholars only compared the therapeutic effects of 4 IA injection therapies on knee osteoarthritis, without mentioning ozone, BT, and other treatments.In recent years, many scholars have carried out randomized controlled trials (RCTs) studies on these drugs, and many relevant systematic evaluations and meta-analyses had been published based on clinical research results, but most meta-analyses we believe that it is necessary to conduct a meta-analysis of the Network to explore the efficacy of various treatment methods in the treatment of knee osteoarthritis, so that to comprehensively evaluate the safety and efficacy of injection therapy and provide evidence for the clinical treatment of knee osteoarthritis.Considering the above-mentioned issues and the recent publication of a number of new RCTs,22.12.1.1All interventions, including at least 2 RCT injections for knee osteoarthritis, should be selected, with injections limited to BT, corticosteroid (CS), hyaluronate (HYA), peppering technique (PEP), PRP, placebo (PLA).2.1.2 “Practical Osteology,” “American College of Rheumatology (1987),” etc, the main contents are as follows: Recurrent knee pain in the past 1 month; Accompanied by any of the following at least 2 cases, X-rays in standing or weight-bearing positions showed narrowing of joint space, subchondral osteosclerosis or cystic changes, and joint marginal osteophyte formation; More than 50 years old; Joint stiffness time is less than 30 minutes after get up in the morning; Sound of bone friction or bone friction when the joints move.According to the diagnostic criteria of knee osteoarthritis stipulated in guidelines for the “Diagnosis and Treatment of Osteoarthritis,”2.1.3The interventions under study should include at least 2 different injection therapeutics, limited to botulinum toxin (BT), CS, HYA, peppering technique (PEP), placebo and ’wait and see’ (PLA), and bright-rich plasma (PRP). They were excluded from ozone therapy, Autologous Blood (AB), glycosaminoglycan polysulfate (GSGPS), Prolotherapy (PRO), stem cell therapy, etc.2.1.42.1.4.1Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) is used to evaluate the structure and function of the knee from the 3 aspects of symptoms and signs of pain, stiffness, and joint function, and evaluate the treatment effect through the score changes before and after treatment.2.1.4.2(1)Visual analog scale (VAS) for pain, A ruler marked with a number from 1 to 10 indicates pain, 0 is painless, and 10 is the ultimate tolerable pain. The patient marks his or her own pain on the ruler.(2)Scoring for Lysholm knee makes a preliminary assessment from the levels of different levels of exercise function, the rating is more inclined to the daily life activities.(3)Aims2-s is used to evaluate patients from 5 aspects, including body, symptoms, and emotions. There are total of 20 items, which are often used to evaluate patients’ quality of life.(4)The incidence rate of adverse events.2.1.5(1)Exclusion of reviews, animal experiments, case reports, and non-RCTs;(2)The comparison of efficacy between different doses or injection sites of only one injection therapy is excluded.(3)If there is any information loss or obvious error in the article, it will be screened out for further study.2.2Comprehensive searches of RCTS on injection therapy for knee osteoarthritis were conducted in 3 English databases of PubMed, Cochrane Library, Embase, and 2 Chinese databases of CNKI and Wanfangs, and the time of index was from their inceptions to August 2020 for each database. The retrieval strategy of PubMed is summarized in Table 2.32.3.1For the convenience of management, we imported the titles retrieved from the 5 databases into EndNote Software AQ8 (V.X9); we deleted the duplicate articles, 2 team members (YT and YSF) of us independently read the titles and abstractions, and deleted the nonconforming literatures, then read the full text of articles that cannot be identified by the title and identify the ones that will eventually be included. At the end of this step, the results of both parties are cross-checked, if any objections arise, a decision is made through group discussion and will be decided by a third reviewer (JL). The overall process and results are shown in Figure 2.3.2Microsoft Excel 2016 was used to establish information data extraction table, and pre-extraction was carried out to determine the feasibility of the table. Then, 2 team members (YT and YSF) will independently extract the following information after training:(1)Basic information: Title, author, country, year, language, etc.(2)Baseline information: Gender, age, number of persons, country, diagnostic criteria, etc.(3)Methodological information: Grouping method, allocation concealment, blind method, result bias, etc.(4)Intervention measures: Treatment measures, dosage, treatment time, frequency, etc.(5)Results: Data of primary and secondary results.After the work is completed, the results are cross-checked, if there are differences, a group discussion is conducted to determine the final result.2.4,20The 2 authors (YT and YSF) evaluated the article methodology of inclusive trials independently, by the Cochrane collaboration “bias risk” tool sequences generated from six aspects of allocation concealment, blind (or mask), incomplete data evaluation, evaluation reports, and other sources of bias selective results. Finally, for each items, we will made ranking of “Low-risk bias,” “High-risk bias,” and “Unclear” based on the Cochrane collaboration “bias risk” tool.2.52.5.1If data are insufficient from the selected study, we will contact the author via email for the required data. If baseline and outcome data or other data are included, the mean and standard deviation of the change will be manually calculated according to the Cochrane.2.5.2In the network diagram, each dot represents an intervention; The larger dot area means the bigger population of the studied intervention; The line between the 2 dots represents that there is direct comparison to RCT studies among 2 interventions; The line thickness represents the numbers of direct comparison to RCT studies among 2 interventions.2.5.3Transitivity and consistency are the prerequisites for reticular meta-analysis. The transitivity was evaluated qualitatively from the perspective of methodology and was evaluated according to the PICO principle. Consistency was mainly to check local and overall consistency. Local consistency can be checked by loop consistency test (Higgins model). The global consistency test was verified by the corresponding inconsistency model according to different data.2.5.4,23Heterogeneity tests for all included studies were performed by using Network prediction interval graph, then to study the relationship of the weighted mean difference (WMD) at a 95% confidence interval (95% CI) and estimation zone (95%Prl) to invalid line, only when invalid line crosses perpendicularly to estimation zone but does not to CI, then it means that heterogeneity exists.2.5.5If there is a direct comparison between the experimental interventions included in the data , the Stata14.0 will be used for pairwise meta-analysis based on a random-effects model.2.5.6Two team members (YT and YSF) used statistical software Stata  for analysis. A random effects model was used for network meta-analysis to compare the variables between different interventions. By comparing Surface Under the Cumulative Ranking Curve (SUCRA), the optimum intervention measures were determined. The range of SUCRA is 0% to 100%; the higher of the cumulative ranking curve means the better of the efficacy.2.5.7Funnel plots are used to detect publication bias. If the images are asymmetric, it indicates that there is publication bias.2.5.8If the analysis shows significant heterogeneity, then the root cause will be analyzed according to the PICOS principle, and the STATA 14.0 will be used for subgroup analysis.2.5.9 2 team members evaluate the quality of the research and divide it into 4 levels of “high,” “medium,” “low,” and “very low”; then the results will be exchanged. If there is any disagreement, the final option will be selected via group discussion.According to the standards in the Grading of Recommendations Assessment Development and Evaluation (GRADE) system,2.6The secondary literature study has no relationship to the personal data of the study, so the ethical approval is not required. Evaluation of the Comparative Efficacy and Safety of Injection therapies for Knee osteoarthritis may provide evidence for clinical treatment of this disease. The results of the study will be published in a peer-reviewed journal.3,26 At present, the pathogenesis of this disease is unknown, but now, it is mostly considered that joint structure and function failure are caused by senile degeneration, osteoporosis, inflammation, metabolic factors, and so on. Compared with the surgical treatment, conservative method is more acceptable, and the clinical efficacy is also worthy of attention. However, IA therapy has become an optimized method for conservative treatment, which can reduce local pain and improve joint's function and activity by injecting drugs directly into the lumen of the knee. In face of multiple injections, it is our common concern to select a high effective drug without side effects. Therefore, the purpose of this study is to evaluate the efficacy and safety of multiple injection therapies in the treatment of knee osteoarthritis, so that to provide ideas, methods, and evidence for clinical intervention KOA.Knee osteoarthritis is the main cause of lower extremity disability among senior people, and the main pathological changes are degenerative changes, destruction, and hyperosteogeny of articular cartilage.In this study, first, the limitations were discussed according to the different injection therapies. Ignoring to the dose, injection position and time would also affect the efficacy.Second, in terms of efficacy evaluation, we selected several criteria, among which VAS score was obtained according to the subjective feelings of patients, which was not objective.,15Finally, many high-quality RCTs were excluded because of the stringent inclusion criteria, and the feasibility of the articles depends on the methodological quality of the included articles.Data curation: ting yu, Shifan Yan.Formal analysis: ting yu, Shifan Yan.Investigation: Genping Zhong.Resources: Daocheng Zhu.Software: Daocheng Zhu, Siyu Qin.Supervision: Zhenhai Chi, Xilin Ouyang.Validation: Pan Cheng, Haiyan Li.Writing – original draft: Rixin Chen.Writing – review & editing: Lin Jiao.


At the end of 2019, peoples normal lives were disrupted by a sudden plague (COVID-19), the huge impact of COVID-19 on society has never been appeared. How to effectively prevent and treat COVID-19 is a concern for all health care workers. Exercise as a green and cheap complementary therapy, which has been proven to improve the immune capacity of the body and prevent infection. The main purpose of this study is to provide a reliable methodological guidance and credible evidence for exercise on COVID-19 therapeutic.This protocol is guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols. We will search the following database sources for the Randomized controlled trials: the Cochrane Library, PubMed, EMBASE, Web of Science, Chinese Biomedical Literature Database (CBM), Chinese National Knowledge Infrastructure Database (CNKI), Chinese Science and the Wanfang Database. All randomized controlled trials of exercise therapy for COVID-19 in the above database will be considered for inclusion, and high-quality articles will be screened for data extraction and analysis, to summarize the therapeutic effect of exercise on COVID-19 patients.In this study, we hope to find strong evidence for the treatment of COVID-19 by exercise.The conclusion of our study will provide credible evidence to judge whether exercise is an effective intervention on the COVID-19 patients therapeutic, and guide future researches.PROSPERO registration number: CRD42020200883. After study by Chinese health experts, the pathogen of unexplained pneumonia was identified as a novel coronavirus on January 9, 2020, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Subsequently, SARS-CoV-2 began an epidemic in China, and more than 80,000 Chinese residents were infected with SARS-CoV-2. On February 11, 2020, World Health Organization (WHO) officially named the pneumonia caused by SARS-CoV-2 as Corona Virus Disease 2019 (COVID-19). Subsequently, on March 11, WHO identified the COVID-19 outbreak characteristically as a pandemic. In the next 5 months, COVID-19 began to spread around the world, more than 20 million people infected with COVID-19 and more than 700,000 deaths worldwide by August 12, 2020, and over 210 countries and territories are involved. SARS-CoV-2 has 4 main ways of transmission.,3 First is droplet transmission, general people caused by inhalation droplets that is emitted by infected person when are coughing or talking. Second is transmission by close contact, that is contact with the mucous membrane or damaged skin of the COVID-19 patient or the virus carrier person, or contact with the droplets of the infected person who left on the surface of the object; the third is aerosol transmission, that is inhaling aerosol formed of droplets which emitted by infected people; the fourth is other possible means of transmission, including fecal-oral transmission etc. The main initial symptoms of COVID-19 patients include fever, dry cough, fatigue, and few people accompanied with pharyngeal pain, muscle soreness, nasal congestion, and runny nose, etc. The early clinical symptoms of COVID-19 are similar to common influenza and without specificity. Most people with COVID-19 experience are mild to moderate illness, but around 15% progress to severe pneumonia, and about 5% of patients progress to acute respiratory distress syndrome. Studies have shown that among the many critical patients with COVID-19, the proportion of the elderly is higher, which is closely related to the relatively low immunity of the elderly.,6SARS-CoV-2 is one of the coronaviruses. Coronavirus is a positive single-stranded RNA virus with a diameter of 80 to 120 nm, which can be divided into 4 genera, namely α, β, δ, and γ. The SARS-CoV-2 is β-coronavirus, and it is the seventh coronavirus have found that can infect human. Current studies have found that people susceptible to SARS-COV-2 include of all ages, and the main source of infection are confirmed COVID-19 patients, and those in the incubation period and asymptomatic infected persons. Regular exercise can significantly improve the immune ability of the body and reduce the incidence of infectious diseases, while long-term high-intensity exercise or lack of exercise is an important cause of immune decline.,10 Bernardi and Simpson found that regular moderate intensity training can significantly improve the immune ability of the body and effectively reduce the incidence of respiratory diseases.,12 Nieman and Grande also found that regular moderate-intensity physical activity can reduced incidence, duration and severity of upper respiratory tract  infections.,14 Shimojo et al study found that 1-hour swimming per day can attenuate serum levels of inflammatory cytokines and increased anti-inflammatory cytokines of mice with endotoxemia. Prospective studies have consistently shown that regular physical activity reduces the risk of infection and the burden of latent viral infections. In addition to improving immunity, regular exercise will also help to weight control, mood improvement, better sleep, and relieve anxiety, which are common concomitant symptoms in COVID-19 patients. Therefore, in this study, we aim to systematically review the positive role of exercise in promoting recovery of patients with COVID-19 and improving the quality of life of the COVID-19 cured population.As a kind of green and cheap complementary therapy, exercise has a positive effect on many diseases. In 2007, American College of Sports Medicine and the American Medical Association officially proposed “Exercise is Medicine”. Studies have shown that exercise is closely related to the immune ability of the body.22.1 statement guidelines.In accordance with the guidelines, this systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO) on July 27, 2020 (registration number CRD42020200883). The consent of this protocol report is based on the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 20152.22.2.1We will include researches related to exercise therapy of patients suffering from COVID-19. Due to language restrictions, we will search for articles in English and Chinese. In order to get a more objective and true evaluation, all articles must meet the following 4 conditions at the same time:1.Published documents with complete documents data;2.Participants were confirmed to have COVID-19;3.The type of trial is randomized controlled trial (RCT);4.The intervention group received exercise intervention for at least 2 months.2.2.2We will include all patients who suffering from COVID-19 regardless of sex, age, racial group, education, and economic status. Pregnant women, postoperative infections, psychopaths, patients with severe pneumonia or other reasons who cannot exercise, patients with severe cardiovascular and/or liver and/or kidney diseases will not be included.2.2.3The studies at least one of the groups received exercise intervention will be included. Exercise intervention include aerobic exercise, daily exercise, endurance training, high-intensity interval training, resistance, exercise. Other stimulation methods such as postoperative rehabilitation training, sports rehabilitation after fracture and athlete training will be excluded.2.2.4This systematic review will include studies comparing exercise to non-exercise intervention controls , studies comparing different exercise or physical activity protocols , and studies comparing an exercise-based intervention to another treatment approach .2.2.5Primary outcomes: Time of disappearance of main symptoms  recovery time, and serum cytokine levels. Secondary outcomes: Accompanying symptoms  disappear rate, CT image improvement, the results of COVID-19 nucleic acid test are negative on 2 consecutive occasions (not on the same day), average hospitalization time, occurrence rate of common type to severe form, clinical cure rate, and mortality.2.3The following electronic databases will be searched from inception to July 2020: the Cochrane Library, PubMed, EMBASE, Web of Science, Chinese Biomedical Literature Database (CBM), Chinese National Knowledge Infrastructure Database (CNKI), Chinese Science and the Wanfang Database. To ensure literature saturation, we will scan the reference lists of included studies or relevant reviews identified through the search. We will also search the authors personal files to make sure that all relevant material has been captured.2.4The search terms on PubMed are as follows: exercise ; COVID-19 ; convalescence ; randomized controlled trial . Combinations of Medical Subject Headings (MeSH) and text words will be used. The same search term is used in other electronic databases. These search terms are shown in Table 2.52.5.1We chose the PRISMA flow chart to show the process of selecting literature for the entire study Fig. . Before 2.5.2The reviewers will assess the eligibility of the studies independently using the inclusion and exclusion criteria. The following data then will be extracted from the studies selected for inclusion using a data collection form, and recorded onto an excel file: first author and year of publication, study design, participants , interventions , outcomes , adverse reactions, risk of bias assessment and funding sources. Three teams of reviewers will extract data independently and in duplicate from each eligible study, any disagreements will be resolved by discussion, with any ongoing differences in opinion being arbitrated by a third reviewer (YMY). We may also contact the authors of primary studies by telephone or email to ask for additional relevant information, if necessary. All data will be transferred into Review Manager Software (RevMan V.5.3) for analysis and synthesis.2.5.3Two authors  independently assessed the methodological quality of each trial according to the standards advised by the Cochrane Handbook For Systematic Reviews of Interventions. Any disagreements were resolved by discussion and reached consensus through a third reviewer (YMY). The risk of bias was evaluated for each study by assessing the randomization process, the treatment allocation concealment, blinding of participants and personnel, blinding of outcome assessment, the completeness of the data and selective outcome reporting or not. A judgment as to the possible risk of bias on each of the 6 domains will be made from the extracted information, the risk of bias is evaluated at 3 levels, namely, low risk, high risk, and ambiguity. If the information is vague, we will try to contact the corresponding author of the article.2.5.4In this protocol, we will use 95% confidence interval (CI) risk ratio (RR) to rigorously analyze the dichotomous data. And for the continuous data, weight mean difference or standard mean difference is used to measure the efficacy of 95% CI. Skewed data and non-quantitative data will be presented descriptively.2.5.5Preliminary analysis will be conducted by random grouping of individual participant. However, special problems often arise in studies that are not standard designed, such as cluster randomized trials or studies with multiple treatment groups. For clustered randomized trials, we will refine the results by extracting an interclass correlation coefficient based on the Cochrane Handbook For Systematic Reviews of Interventions. And for multiple treatment groups, we will present the additional treatment arms. Where the additional treatment arms are not relevant, they will not be taken into account.2.5.6We will try our best to ensure the integrity of the data. When there are missing data, we will try our best to contact the corresponding author of the article, including but not limited to sending emails or making a phone call. If the corresponding author cannot be contacted, we will use sensitivity analysis to assess the impact of the missing data on the final outcome, if the impact on the outcome is significant, we will remove the experiment with incomplete data. After data integrity is assured, intention analysis therapy and sensitivity analysis will be performed.2.5.72 test (significance level: 0.1) and I2 statistic will be used to test the heterogeneity among trials. When the I2 value is <40% means might not be important; 30%< I2 < 60% means represent moderate heterogeneity; 50%< I2 < 90% means substantial heterogeneity; 75%< I2 < 100% means considerable heterogeneity. If high levels of heterogeneity among the trials exist (I2 ≥ 50% or P < .1), the study design and characteristics in the included studies will be analyzed and subgroup analysis or sensitivity analysis will be try to use to explain the heterogeneity.For the detection of statistical heterogeneity, the Chi2.5.8I2 ≥ 50% or P < .1), the random effects model will be used. If heterogeneity is substantial, we will perform a narrative, qualitative summary.Each outcome will be calculated and combined using the RevMan 5.3. Specific implementation was based on the current version of the Cochrane Handbook for Systematic Reviews of Interventions. If tests of heterogeneity are not significant, the Mantel-Haenszel method will be chosen for fixed effect model, and if statistical heterogeneity is observed .2.Types of interventions .3.Duration and frequency of exercise.2.5.11Sensitivity analysis will be performed according to sample size, study design, heterogeneous quality, methodological quality and statistical model, the trials with quality defects will be excluded to ensure the stability of the analysis results.2.5.12This paper will use the evidence quality rating method to evaluate the results obtained from this analysis. GRADE will be assessed across the domains of risk of bias, consistency, directness, precision and publication bias. In the context of the system review, quality reflects our confidence in the effectiveness of assessment. It has 4 evaluation levels, namely, high (further research is very unlikely to change our confidence in the estimate of effect), moderate (further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate), low (further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate), or very low (very uncertain about the estimate of effect).2.6Ethics and dissemination: The content of this article does not involve moral approval or ethical review and will be presented in print or at relevant conferences.3 and reduce the probability of infection with infectious diseases, which also provides a new way for the treatment of COVID-19.The outbreak of COVID-19 is a disaster for humanity without a doubt. Although Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) have caused local epidemics before, the impact of COVID-19 is clearly greater this time. It has been more than 8 months since the began of COVID-19, during which the number of daily infections and deaths people has been on the rise. In order to reduce the spread between people, many countries and regions have adopted quarantine policies, which has sharply reduced the space of former participants of exercise. Existing evidence has shown that regular exercise can significantly improve the immune capacity of the bodyThis paper mainly describes how to systematically review the therapeutic effect of exercise on COVID-19, which mainly includes 4 aspects: literature collection, literature screening, data extraction and data analysis. This study can provide some methods guidance for other scholars to study the prevention and treatment effect of exercise on COVID-19 in the future. However, due to the limitation of language, we only searched Chinese and English literature, which is also the deficiency of this study.Conceptualization: Zhangmeng Xu.Data curation: Duoduo Yu, Dongdong Mao, Donghong Feng, Tao Li.Formal analysis: Zhangmeng Xu, Yong Chen.Funding resources: Yaming Yu.Investigation: Shengsong Yan, Ting Wang, Duoduo Yu, Donghong Feng.Methodology: Zhangmeng Xu, Yong Chen, Duoduo Yu, Dongdong Mao.Software: Zhangmeng Xu, Yong Chen, Yaming Yu.Writing – original draft: Zhangmeng Xu.Writing – review & editing: Yaming Yu, Yong Chen.


Marian Cove is experiencing some of the most rapid environmental changes in the Antarctic region; however, little is known about the response of bacterial communities to these changes. The main purpose of this study was to investigate the spatial variation of physical–biogeochemical–bacterial community features in the Marian Cove surface waters and the environmental parameters governing the spatial variation in the bacterial community composition during the summer of 2018. The Marian Cove surface waters are largely composed of two different characteristics of water masses: relatively low-temperature, -salinity, and -nutrient surface glacier water (named SGW) and relatively high-temperature, -salinity, and -nutrient surface Maxwell Bay water (named SMBW). The SGW bacterial communities were dominated by unclassified Cryomorphaceae, Sedimenticola, and Salibacter genera, while the SMBW bacterial communities were dominated by Sulfitobacter, Arcobacter, and Odoribacter genera. Spatial variations in bacterial community composition were mainly attributed to physical and biogeochemical characteristics, suggesting that the bacterial community composition of the Marian Cove surface waters is mainly determined by environmental characteristics. These findings provide a foundation to improve the understanding of bacterial community variations in response to a rapidly changing Marian Cove in the Antarctic. Antarctic regions have played an important role in regulating the earth’s climate system, not only on a glacial–interglacial timescale but also during the Anthropocene era ,2. In spMarian Cove, which is located between Weaver and Barton Peninsulas , is one Given that studies about the variations of microbial communities in various environments have profoundly increased our understanding of the responses of bacterial communities to environmental changes , Marian Therefore, the main purpose of this study was, for the first time, to (1) present the spatial distributions of physical–biogeochemical–bacterial community features in Marian Cove surface waters during summer, (2) investigate the environmental parameters governing the spatial variation in the bacterial community composition, and (3) provide significant insight into the alteration of the Marian Cove bacterial community in response to rapid Antarctic environmental changes.Marian Cove is a small cove, 4.5 km in length, 1.5 km in width, and up to 120 m deep  7]. Mar. Mar7]. 4), nitrate and nitrite (NO3 + NO2), silicate (SiO2), and ammonium (NH4) were measured using an autoanalyzer . Dissolved inorganic nitrogen (DIN) is represented as the sum of NH4 + NO3 + NO2.Physical and biogeochemical parameters of the water samples were measured at each station. Water temperature (T), salinity (S), and dissolved oxygen were measured using a conductivity–temperature–depth instrument . For nutrient analysis, the sample at each station was filtered through a 0.2-µm syringe filter , placed in a 50-mL conical tube, and stored at −20 °C until measurement. After thawing the frozen seawater, phosphate , according to the manufacturer’s protocol. Quantitative and qualitative analyses of DNA were carried out using PicoGreen and Nanodrop, respectively. Polymerase chain reaction (PCR) was carried out to amplify the V3–V4 regions from the extracted environmental DNA, a variable sequence of the 16S rRNA gene, using primers 341F and 805R [To assess the bacterial community composition, DNA from the filter paper was extracted using the PowerSoiland 805R . The PCRand 805R . The seqand 805R . QIIME (and 805R . Raw seqhttp://www.r-project.org/) [To analyze the relationship between stations and environmental parameters, principal component analysis (PCA) was performed using the R stats package  . Permutact.org/)  and thenct.org/) . Heatmapct.org/) ,31. The ct.org/) ,33.4 concentrations were also apparent between the SGW and SMBW regions  compared with those in the SMBW region   e,f. In sctively) .To characterize the difference in bacterial community compositions with space, the relative abundance (%) of bacterial communities at the class level was analyzed . In the 2, while SMBW stations were associated with T, S, DIN, and PO4. In line with PCA, PCoA also showed that the bacterial community composition along the stations was likely to be distinctly separated into SGW and SMBW groups , which commonly inhabits the guts of Antarctic penguins and birds ,38, was Polaribacter (of the class Bacteroidia) was one of the dominant bacterial groups in the surface of the Marian Cove waters c. AccordIt is known that alteration of bacterial community composition is attributed to environmental changes ,41,42. A4, and SiO2)  . Unclassnditions . These rBased on the results of this study, at present the marine bacterial community composition  was more dominant than the freshwater composition  in the Marian Cove surface waters. Rapid environmental changes in the Antarctic will directly and indirectly affect Antarctic bacterial community compositions. Marian Cove is one of the Antarctic regions undergoing rapid environmental changes due to global warming . In partWe investigated physical–biogeochemical properties and bacterial community composition observed during the summer of 2018 in the Marian Cove surface waters. The SGW region is characterized as having relatively low T, S, and nutrients, while the SMBW region has relatively high T, S, and nutrients. Thus, the Marian Cove surface water samples showed a physical and biogeochemical gradient between the SGW and SMBW regions. In the SGW region, unclassified Cryomorphaceae, Sedimenticola, and Salibacter at the genus level were the dominant groups, and Sulfitobacter, Arcobacter, and Odoribacter genera were dominant in the SMBW region. Overall, the SGW bacterial community composition showed negative correlations with environmental parameters, whereas the SMBW bacterial community composition was positively correlated with environmental parameters. This contrast supports that the bacterial community composition of the Marian Cove surface waters is significantly determined by environmental characteristics. Therefore, attention should be paid to how the Marian Cove bacterial community will be altered in the future in response to rapidly changing Antarctic environments.


Objective: To investigate the association between antibiotics administration timing with morbidity and mortality in children with severe sepsis and septic shock, presenting to a tertiary care center in a developing country.Methods: This is a retrospective study of children aged 14 years or younger diagnosed with severe sepsis or septic shock at a free-standing tertiary children's hospital in Saudi Arabia between April 2015 and February 2018. We investigated the association between antibiotic administration timing and pediatric intensive care unit (PICU) mortality, PICU length of stay (LOS), hospital LOS, and ventilation-free days after adjusting for confounders.Results: Among the 189 admissions, 77 patients were admitted with septic shock and 112 with severe sepsis. Overall, the mortality rate was 16.9%. The overall median time from sepsis recognition to antibiotic administration was 105 min (IQR: 65–185.5 min); for septic shock patients, it was 85 min (IQR: 55–148 min), and for severe sepsis, 130 min (IQR: 75.5–199 min). Delayed antibiotic administration (> 3 h) was associated with 3.85 times higher PICU mortality  in children with septic shock than in children who receive antibiotics within 3 h, after controlling for severity of illness, age, comorbidities, and volume resuscitation. However, delayed antibiotics administration was not significantly associated with higher PICU mortality in children diagnosed with severe sepsis.Conclusions: Delayed antibiotics administration in children with septic shock admitted to a free-standing children's hospital in a developing country was associated with PICU mortality. Sepsis is the most common cause of death in children . In deveThe ACCM-PALS guidelines recommend antibiotic administration within 1 h of sepsis recognition . The 202To the best of our knowledge, there are no epidemiological reports on sepsis in the pediatric population in Saudi Arabia. However, there are two reports addressing sepsis management. Hasan et al.  reportedThe goal of the present study was to investigate the association between antibiotic administration timing with morbidity and mortality in children with severe sepsis and septic shock.This retrospective cohort study was conducted at the King Abdullah Specialist Children's Hospital, a government tertiary and academic free-standing hospital in Riyadh, Saudi Arabia. The hospital's inpatient bed capacity is ~220, with 25 beds assigned for closed medical-surgical PICU. The unit's patient case-mix include general surgery, neurosurgery, solid organ transplant, bone marrow transplant, and trauma. The hospital was accredited by the Joint Commission International.Patients aged 0–14 years admitted between April 2015 and January 2018 were included in the study if they met the criteria for severe sepsis or septic shock as defined by the International Pediatric Sepsis Consensus Conference (IPSCC) . PatientChildren were excluded if they were diagnosed with an illness that might compromise the “volume resuscitation” component of the guidelines, which includes end stage renal disease, unrepaired congenital heart disease or dilated cardiomyopathy, or palliative cardiac procedures. In addition, children with do-not-resuscitate status, inter-hospital transferees after identification of sepsis and initiation of management, and children who were on antibiotics prior to the onset of sepsis were excluded. A total of 480 admissions were reviewed, of which 189 were included in the analysis . The stuAll the data were individually obtained from electronic medical records and were manually reviewed. Variables collected included demographic data, Pediatric Index of Mortality 3 (PIM 3) score , comorbiSepsis recognition time varied depending on the area of presentation. For emergency room patients (ER), it was triage time ; for inpIBM SPSS version 26 was used to analyze the data. The median and percentile (Q1–Q3) values were used to describe the quantitative variables of age, PIM 3 score, and organ dysfunction. Frequencies and percentages were used to describe the categorical variables, such as gender, comorbidities, source of infection, and antibiotics administration timing within 1, 2, and 3 h.U-test for non-parametric data were used for categorical and continuous variables, respectively. The associations identified in the analyses were expressed as odds ratios (OR) and 95% confidence intervals (CI).The primary analysis was the association between the timing of antibiotic administration with PICU mortality of children with septic shock and severe sepsis. Other outcomes included PICU LOS, hospital LOS, and ventilation-free days (VFD). VFD was defined as the number of days between successful extubation and day 28; thus, it is considered zero if the patient dies before 28 days. Based on previous findings, the predefined variables of interest were the time from meeting the sepsis criteria to the antibiotic administration at 1, 2, and 3 h and the volume of boluses in the first 2 h , 13, 14.P < 0.05 was considered statistically significant for all analyses.Multivariable logistic regression was used for assessing the timing of antibiotic administration with PICU mortality in the septic shock and severe sepsis subgroups. Finally, we built multivariate linear regression models for evaluating the impact of antibiotic administration within 1 h on continuous outcomes . Since PICU LOS, hospital LOS, and VFD were not normally distributed, data were log-transformed prior to the statistical modeling. Age, comorbidities, severity of illness (PIM 3 score), and volume of boluses in the first 2 h were included in the regression analysis based on previous findings , 13. A Pn = 155) were known to have several comorbidities. The overall PICU mortality rate was 16.9% (n = 32).During the study period, there were a total of 172 patients with 189 admissions meeting the inclusion and exclusion criteria. There was almost equal gender distribution, with 86 males (45.5%) and the median age was 19 months (IQR: 3–74.5 months). The median PICU LOS was 8 days (IQR: 2–23.5 days), and the median hospital LOS was 22 days (IQR: 9–30 days). Among the participants, 79.9%  had septic shock. The sources of infection were primarily respiratory , followed by bloodstream . Cultures were positive in 72 children. Almost half of the children  had the onset of sepsis (time zero) in the ER, while the remainder  developed sepsis while admitted in the hospital wards. Forty four children (23.3%) were diagnosed with hospital-acquired infections. Children with septic shock had more positive cultures, more multiorgan dysfunction syndrome on sepsis recognition, and lower VFD than children with severe sepsis n = 27 out of 189), while 51.3% (n = 97 out of 189) received antibiotics within 2 h (The percentage of children who received antibiotics within 1 h of the onset of sepsis was 14.3% (thin 2 h . The ovep = 0.131). In patients with septic shock, there was an increased risk of mortality for each hour of delay, which became more predominant at the 3-h mark. There was a 14.3% mortality rate in children receiving antibiotics within 3 h and a 33.3% mortality rate in children receiving antibiotics after more than 3 h. However, this was not statistically significant (p = 0.063). In the severe sepsis cohort, there was no difference in the mortality with antibiotics administration timing at 1, 2, or 3 h. After adjusting for the age, comorbidities, severity of illness (PIM 3 score), and volume of boluses ≥ 20 mL/kg in the first 2 h from sepsis recognition (time zero), the AOR for PICU mortality was 3.85 times higher in children with septic shock and with antibiotics administration beyond 3 h in comparison to within 3 h  (–14.374)  , while the reported median times from Boston Children's Hospital, Stollery Children's Hospital, Children's Hospital of San Antonio, and Children's Hospital of Philadelphia were 96, 115, 135, and 140 min, respectively .Administering antibiotics beyond 3 h from sepsis recognition in children with septic shock was found to be an independent risk factor for mortality. The available literature addressing the association between rapid antibiotic administration and mortality is scarce. Our findings are similar to those reported by Weiss et al.  which inp = 0.009); however, the patients who received early antibiotics might have been sicker, and this might not have been captured by their risk adjustment tool. van Paridon et al. (Although the 2020 SSC guidelines recommend starting antibiotics within 1 h from the recognition of septic shock and within 3 h of severe sepsis patients, we have only been able to show a statistically significant difference in mortality at 3 h from sepsis recognition . Severaln et al.  found noOur study demonstrated statistically significant shorter PICU LOS in children with severe sepsis who received antibiotics within 1 h. Evans et al.  showed sThis study has several limitations. First, this is an observational study, which makes it prone to bias and is not always able to establish causation. Second, this study is based on the data from a single center, which resulted in a relatively small sample size, and also, the difference in national health care systems, population demographics, resources, and staffing characteristics is a potential limitation to its generalizability. Lastly, we were unable to determine appropriate antibiotics because of the practical constraints and retrospective nature of this study. Nonetheless, our study strengthens the observation from the limited number of studies that delayed antibiotic administration is associated with higher mortality.In summary, our findings are in keeping with some available literature in pediatrics, which shows that adherence to antibiotics administration timing is low. In our hospital, a delay in administering antibiotics to children with septic shock is an independent risk factor for mortality and is associated with longer PICU LOS in children with severe sepsis. Further prospective research in the pediatric population with sepsis is essential to confirm the beneficial impact of antibiotic timing on patient outcomes.The datasets used analyzed during the current study are available from the corresponding author upon reasonable request.The studies involving human participants were reviewed and approved by King Abdullah International Medical Research Center. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.YK: conceptualization, methodology, formal analysis, investigation, data curation, writing original draft, visualization, and supervision. AA: conceptualization, investigation, and writing original draft. NA: conceptualization, methodology, writing, review, and editing. MA and BA: investigation and writing original draft. SA: writing original draft. AFA and RV: formal analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A translocation t leading to a fusion of IGH and BCL3 which is a rare cytogenetic abnormality in CLL patients, has a more aggressive clinical course with a shorter time to first treatment (TTT) and worse overall survival (OS). To date, there is no literature reporting the identification of the t in Chinese CLL patients and the reviewing the characteristic of all patients with this abnormality reported previously in the literature.We first demonstrate three cases of t translocation among the 200 CLL patients from 2017 to 2019 in our hospital. We investigated several aspects such as clinicopathologic features, cytogenetic analysis, IGHV mutations, next-generation sequencing technology (NGS), and histopathological characteristics in order to clearly define the features of this entity in Chinese patients and compare them with patients reported previously in western countries.The clinical and pathological features of our three cases resemble those of earlier reports. All patients had atypical morphologic features and atypical immunophenotypes with low CLL scores detected by flow cytometry. All cases were unmutated in the IGHV mutations. Two cases showed complex karyotype and one case demonstrate missense mutations of TP53 and FBXW7.In conclusion, this is the first report on IGH/BCL3-positive B-CLLs in Chinese people, which provided a comprehensive analysis of clinical and pathological characteristics. In addition to some similar clinical and laboratory features reported in the previous literature, we first found that CLL with t has a higher possibility of being accompanied with high complex karyotype (high-CK), which is now regarded as a novel negative prognostic marker. Early identification of this abnormality in CLL patients is so important that patients can benefit from the more aggressive treatments at the onset of the disease. BCL3 locus at chromosome 19q13 and the immunoglobulin heavy chain gene at 14q32, is a rare recurrent cytogenetic abnormality identified in chronic lymphocytic leukemia (CLL) cases, most of which are distinctive, classic CLL and are associated with poor prognosis.t, involving the Our three Chinese CLL patients with t also demonstrate atypical morphologic features, immunophenotypes, and progressive disease processes, similar to cases in previous reports from Western countries.It is noteworthy that CLL with t is often accompanied with high complex karyotype (high-CK), which is now regarded as a novel negative prognostic marker.BCL3 for fluorescence in situ hybridization (FISH) should be used to increase the detection of t in CLL patients, especially when the conventional chromosome banding analysis demonstrates add(14q32) or FISH indicates an abnormal IGH signal.Cytogenetic analysis should be performed in all newly diagnosed CLL patients. Furthermore, a probe for Active treatments, such as Bruton’s tyrosine kinase (BTK) inhibitors, should be considered to block this progressive process once this abnormality is identified in CLL patients.in situ hybridization (FISH) and chromosome banding analysis (CBA), play an important role in the prognostic evaluation of CLL. More than 80% of CLL patients harbor at cytogenetic aberrations  is a common clonal neoplasm of small, mature B lymphocytes among adults in Western countries; however, it is relatively rare in Asians . Recentlrrations . Among t in 1983 , has beesurvival –7.IGH/BCL3-positive cases in our department to clearly define the features of this entity in Chinese patients and compare them with those of patients from Western countries.To date, studies reporting the identification of t14;19) in Chinese CLL patients, use of next-generation sequencing (NGS) for this condition, and application of the novel drug, BTK inhibitor, in patients with this abnormality are lacking. Moreover, previous studies have revealed that Chinese CLL patients display different clinical and molecular features from CLL patients from Western countries , 9. We f in ChineTP53, t/IGH/CCND1. Peripheral blood or bone marrow aspirate samples were analyzed by flow cytometry , and the CLL score was calculated according to the system by Matutes and colleagues (http://imgt.org). The IGHV mutation statuses were designated as follows: unmutated if there were <2% mutations (≥98% homology) or mutated if there were >2% mutations (≤98% homology) compared to germline sequences. NGS was performed using Ion Torren PGM/Illumina NextSeq 550Dx platform with the targeted panel from 40 genes . Finally, we retrospectively screened and identified three cases with t in the cytogenetic database. IGH and BCL3 gene rearrangement probes  were used to prove that all the cases had IGH/BCL3 translocation. Clinical information and related laboratory testing data were collected together and are described in From June 2017 to December 2019, we received 200 blood and bone marrow samples from patients diagnosed with CLL according to the iwCLL criteria in our laboratory. Using the standard Wright-Giemsa staining protocol, peripheral blood smears were prepared for manual 100-cell differential white blood cell counting; manual 200-cell differential white blood cell counting was performed using bone marrow aspirate smears. Particular attention was given to the features of lymphocytes. Bone marrow core biopsy and excisional lymph node biopsy specimens, when available, were routinely processed, and H&E-stained sections were examined. Samples from all patients underwent conventional G-banding chromosomal analysis by culturing for 72 h with the stimulation of CPG mitogens (the combination of CpG-oligonucleotide DSP30 and interleukin-2). FISH was performed in all patients using a commercially available probe panel , including del(13q14)/D13S19, +12, del(11q22)/ATM, and del(17p13)/9 cells/L. The other parameters were absolute lymphocyte count, 25.95×109 cells/L; hemoglobin level, 101 g/L; and platelet count, 241×109 cells/L. The patient had no fever, cough, chest tightness, or other discomforts. No enlargement of lymph nodes or the spleen was found in the CT scan. Bone marrow aspirate showed extensive infiltration by a heterogeneous mixture of small and medium cells with indented nuclei. Leukemic cells expressed CD19, CD22, CD23, and kappa, whereas the expression of CD5 and surface Ig was dim-positive and that of FMC7 was negative; the CLL score was 3. By karyotyping, trisomy 12 was the only additional cytogenetic abnormality, and this was confirmed by FISH using a probe panel. Interphase FISH analysis using the dual-color BCL3 probe demonstrated one intact red/green fusion signal and 1 red and 1 green split signal, which indicated a BCL3 gene break-apart. The IGHV status was unmutated. The patient was placed in the Rai 0 category. The patient was observed for 18 months without any treatment until the lymphocyte count reached 55.95×109 cells/L. To date, the patient has received five cycles of the standard rituximab, fludarabine, and cyclophosphamide (FCR) treatment and the follow-up is being continued.Patient 1 was a 50-year-old man admitted to the hospital for hemorrhoidectomy in December 2017. The blood examination showed high white blood count, 30.75×109 cells/L. The other parameters were absolute lymphocyte count, 16.85×109 cells/L; hemoglobin level, 105 g/L; and platelet count, 258×109 cells/L. PET/CT showed multiple lymph node enlargements throughout the body. Lymph node biopsy demonstrated that the lymph node specimen was predominantly composed of small lymphocytes with a mixture of larger lymphocytes with scanty cytoplasm, clumped chromatin, and round or slightly irregular nuclei. These slightly large cells were diffused with an increased proliferation index. Immunohistochemical staining showed that the lymphoid cells were positive for CD5, CD20, CD23, and BCL2 and negative for CD3, CD10, BCL6, and Cyclin D1. The index of Ki-67 staining was about 30%. Bone marrow morphology displayed abnormal lymphocyte proliferation, diffuse or interstitial distribution, small cells mixed with increased large cells, round or irregular nuclei, dispersed chromatin, and insignificant nucleoli. Immunohistochemical staining results were as follows: CD20+, CD5-, CD23 , CD10-, CD3-, MPO-, BCL2-, and CyclinD1-. G-banding analysis showed a complex karyotype of near-tetraploid: 85~90, XXYY, del(6)(q21), -7, -8, -10, +12, -13, t×2, -15, -18[cp10] , t, add(17)(p13), -18, -20[2]/46,XY[18]. FISH further confirmed the existence of the deletion of the P53 gene. Flow cytometry showed 66.87% monoclonal B lymphocytes with the expression of CD5 and CD20, and dim Sig expression as well as negative expression of FMC7, CD79b/CD22, and CD103. The CLL score was 3–4. NGS demonstrated the missense mutations of the seventh exon of TP53 and the ninth exon of FBXW7. The IGHV status was also unmutated. The patient was placed in the Rai IV category. The standard FCR treatment was performed for 2 cycles. Due to the unsatisfactory efficacy of chemotherapy, the patient entered the clinical trial of BTK inhibitor. However, the patient died of serious pulmonary infection after two months of treatment with BTK inhibitor.Patient 3 was a 55-year-old man diagnosed with CLL at a local hospital and began treatment with chlorambucil without the related prognostic evaluation including CBA, FISH, IgHV status, and NGS in June 2016. One and a half years later, the patient presented to our hospital because of disease progression. After admission, routine blood examination showed a leukocyte count of 80.55×10IGH/BCL3 gene rearrangements is considered to be a rare and recurrent chromosomal abnormality in B-cell malignancies, especially in CLL patients   involving patients . Until npatients , 7.To our knowledge, our report is the ﬁrst report of Chinese CLL patients with t . The clinical and pathological features of our three cases resemble those of earlier reports. All three patients were very young, with an average age of 50 years, and had atypical cellular morphology with cleaved nuclei or lymphoplasmacytic features. The growth pattern in the bone marrow biopsy specimens was interstitial to diffuse. Flow cytometric immunophenotyping showed an atypical immunophenotype with low CLL scores. All our cases were unmutated based on the molecular genetic analysis of the IGHV somatic mutation status.Similar to previous studies, all of our cases harbored additional cytogenetic abnormalities in addition to t14;19), and two of the three cases coexisted with +12 (case 1 and case 2). Moreover, both cases 2 and 3 demonstrated high complex karyotype . Combined with the 86 previously reported CLL cases with t  in the literature, we found that 79 cases had complete karyotype information, and high-CK was observed in 20/79 (25.3%) of the cases Table 3,;19, and With the introduction of effective mitogens, sufficient metaphases of the CLL clone can be obtained, and thus the detection rate of chromosomal abnormalities has also increased. Cytogenetic abnormalities, particularly high-CK, have recently emerged as one of the independent novel biomarkers associated with an inferior outcome , 17. TheTP53 mutation and a FBXW7 mutation. The TP53 mutation had high concordance with 17p deletion and was an independent negative prognosis predictor of CLL  has not been addressed in previous studies. Interestingly, NGS was performed for case 3, and it identified a r of CLL . The fre has not BCL3 gene, which encodes an IKB-like protein and modulates the activity of the NF-KB transcription factors. BTK inhibitors play a role in blocking the NF-KB pathway, so this could also be an alternative treatment for this progressive group of patients. Our patient 3 was already in poor condition when he was enrolled in the BTK inhibitor clinical trial. The duration of drug treatment was too short to evaluate its efficacy. Although we are not yet able to draw a conclusion on the efficacy of BTK inhibitors in patients with t , we still suggest that this novel drug or an association of BTKi and anti-BCL2. is a good attempt for this kind of patients especially in the early time of the disease. Notably, an overall assessment of the patient is necessary at the onset of the disease. Meanwhile, we could consider the more aggressive treatment once the high-risk factor was identified.It is well-known that CLL has a highly variable clinical course, with some patients progressing rapidly within several months of diagnosis and others living without treatment for years. Therapy for CLL is currently undergoing a revolution with the introduction of B-cell receptor signaling inhibitors (BTK inhibitors), which have been recommended as the first-line treatment for CLL patients and relapsed refractory patients by NCCN guidelines. The molecular identification of t was illuminated by McKeithan et al. , and thiInevitably, as a retrospective analysis, there are still some limitations in our research, including unavailability of some examination information and short follow-up time, both of which could be overcome with the expansion of our sample size and targeted management of these patients.IGH/BCL3-positive B-CLLs in Chinese patients, which provided a comprehensive analysis of clinical and pathological characteristics. In addition to having some similar clinical and laboratory features to cases reported in the literature, we first found that CLL with t has a higher possibility of being accompanied with high-CK, which is now regarded as a novel predictive marker for refractoriness to not only chemotherapy-based treatment regimens but also novel agents such as the BCL-2 inhibitor venetoclax, independent of the presence of TP53 aberration. Further studies on the pathogenesis of t in CLL patients and subdivision of this group of patients combined with different karyotypes or NGS results are required to make more accurate assessments of these patients who could benefit from tailored treatment.In conclusion, this is the first report of https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA669583.The original contributions presented in the study are publicly available. This data can be found here: The studies involving human participants were reviewed and approved by Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.QL designed the study and wrote the manuscript. YW reviewed all related literature, performed bioinformatic analysis, and revised the manuscript in this study. SX performed FISH experiment and gathered related data. HZ made chromosome banding analysis. XM performed the flow cytometry test. MX performed high throughput sequencing experiment. JW collected clinical sample and related data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The clinical cases were periodically observed along an 18-month follow-up period via radiography. Data were expressed as focal size of the lesions (mean ± standard error (SEM) of all the radiographic outcomes) observed in each group at each interval point. Statistical analysis was performed using the Student’s t-test that allowed us to compare the control and Cupral groups; the statistical significance was set at p < 0.05 and p < 0.01, where the latter was highly significant. Before treatments, the focal sizes were 4.8 mm and 4.95 mm for control and Cupral-treated groups, respectively. After 6 months, the mean focal sizes were 3.9 mm and 2.14 mm for the control and Cupral groups, respectively. After 12 months, in the control group, the mean focal size was measured at 2.8 mm, while, in Cupral group, the lesion size decreased down to 0.31 mm and a highly dynamic regeneration of the destructive focal-bone occurred. After 18 months, the lesions were further significantly reduced in the control group , while they were barely detectable in the Cupral group (0.2 mm). In conclusion, we provide initial evidence that the Cupral-electrophoresis methodology is effective in treating destructive periodontitis of teeth with problematic canals up to 18 months, thus allowing teeth preservation.Endodontic failure has been and continues to be a problem for endodontics-specialists. Complicated anatomy, numerous foramens, and accessory canals are an environment for microorganisms to infect the teeth. The purpose of the present work was to evaluate the regeneration of copper–calcium hydroxide -endodontically treated teeth diagnosed with apical periodontitis using an electrophoresis technique. In total, 132 patients, aging from 19 to 65 years old, underwent endodontic treatment mono- and multi-radicular teeth, with complicated canals from January 2019 to June 2020. The patients were divided into two groups: (i) the control group—which included 54 patients ( H2O) was used. In the Cupral-treated group, we followed the minimally invasive protocol, recommended by Knappwost [A commercially available copper–calcium hydroxide—based  compound, containing calcium hydroxide (Ca(OH)nappwost . After t2O after each file, thus avoiding NaOCl, EDTA or CHX usage in order to not compromise the effects of Cupral paste.Here, we performed the crown-down technique up to 1/3 and 2/3  of the canal length according to the presented endodontic cases. The coronal part of the root was enlarged through using Gates-Glidden drills sizes 1-2-3  and was then mechanically processed with K-file sizes 40–15 . The canals were irrigated with dist. H2O to a creamy consistency. The paste was prepared thinner for mandibular canals, while for those maxillary was slightly thicker. The consistency was introduced into the canal by the help of lentulo spiral fillers size 1 . For both maxillary and mandibular distal teeth, Cupral paste was used; in the frontal teeth, a portion of Cupral and 9 portions of the Ca(OH)2-highly dispersed paste  were applied at a ratio 1:9. This combination-paste was due to the color effect of Cupral and also this paste was inserted into the root canal by the lentulo spiral fillers size 1 . After the paste was placed within the canal, the needle of cathode (K−) was inserted inside the endodontic cavity and anode (A+) clamp ; the current was applied by switching the apparatus COMFORT II . Before electrophoresis session, each canal was dried using sterile paper points , then a portion (size of 2–3 mm) of Cupral paste was taken and placed on a sterile glass plate and mixed it with dist. H−1, depending on the anatomic construction-canal  until the second session application. The latter was performed after 8 days, removing the provisional filling  and refreshing the canals by rinsing with dist. H2O, drying with sterile paper points (Meta Biomed) and a new portion of Cupral paste was restored with lentulo spiral filler size 1 (Dentsply Maillefer), thus creating a suspension of Cupral and followed the same technique as the first session for distal and frontal teeth (in combination with Ca(OH)2-highly dispersed paste (Humanchemie GmbH), performing in each canal 7.5 mA × min/canal.After the electrophoresis procedure, the cavities were closed provisionally with a sterilized cotton pellet and Cavit2O, dried with sterile paper points , and finally filled by means of Atacamit -alkaline cement (owning permanent sterilization effects) and gutta-percha points , using the lateral condensation technique. The final teeth crowns filling were completed with the help of a Competence Universal Composite® .After the second electrophoretic session, the treated canals were rinsed with dist. HThe follow-up for both groups was performed obtaining intraoral radiographic images before and after the final canals filling, after 6, 12 and 18 months. The parallelism technique associated with the Rinn centerers was performed for both periapical and bitewing radiographs. In addition, appropriate devices were used, such as receptor instruments with ring guides, cotton rolls, standard bite blocks, and bitewing tabs. The digital receptor was positioned vertically or horizontally parallel to the radiographed teeth. The X-ray beam was directed at right angles to the teeth and the receptor.Measurements of the focal sizes were obtained analyzing the radiographic data of both groups, considering the apical bone destruction. This was done with the help of a ruler  relying v/v buffered 4% w/v for histology, Titolchimica S.p.a, Pontecchio Polesine, Italy) for fixation and then delivered to the Histology Laboratory for histological examination. Then, they were resected and placed in 37% formaldehyde . After removal of all soft and hard tissues from the samples, they were next placed in 14% EDTA  for the decalcification process for 6 days, changing every day the solution. Additionally, to remove the decalcification solution, the samples were rinsed under a running tap water wash for 20 min following the dehydration process by immersion in 96 °C ethyl alcohol. The samples were then embedded in Paraplast paraffin  blocks at 58 °C. After this, the blocks were cut using the Leica microtome  to a thickness of 5 μm. The cuts were processed longitudinally and horizontally. The slides were stained with the Hematoxylin and Eosin (H&E) and then evaluated by the light microscope Nikon Eclipse E200  with 400× of magnification.A few Cupral-treated cases  during the 18 month-follow up showed a fracture, which was likely due to the functional overload and we were obligated to extract them. After the extraction, these roots were inserted into a labeled container with 10% neutral buffered formalin  of all the focal sizes outcomes obtained. Statistical analysis was performed using the Student’s TM cement.In the radiographs presented in An interesting fact is that X-rays observed a total disappearance of the apical infection, which could not be observed in all the cases. However, clinically the patients claimed to have no symptoms. In the periapex, there were radiolucency areas, which indicate that the regeneration is not over yet after 12 and 18 months but it continues at a slow rate (note: some of the cases are still under our observation).In all cases treated with Cupral and electrophoresis, a massive reduction of the clinical lesion was observed over time. After 12 and 18 months, a dense area around the periapex was seen, which indicates the complete regeneration of the destructed apical structure, as shown in In The focal areas, visible in the radiographs, were measured using a ruler and expressed in mm, before treatment (time 0) and after 6, 12, and 18 months .At time 0, the focal sizes were 4.8 mm and 4.95 mm for control- and Cupral-treated groups, respectively. After 6 months of observation, the mean focal sizes were 3.9 mm and 2.14 mm for control- and Cupral-treated groups, respectively. These data indicated that Cupral treatment was significantly more effective than the traditional treatment in reducing focal sizes down to about 45% after 6 months. After 12 months, in the control group, the mean focal sizes measured of 2.8 mm; in Cupral-treated group, a highly dynamic regeneration of destructive focal-bone occurred, since we observed a decrease of the lesion to 0.31 mm. Once again, Cupral treatment provided the best results, with a drastic reduction of 89% of focal sizes. After 18 months of treatment, the damaged bone structures were further significantly reduced in the control group, with lesions showing mean values around 2.62 mm; meanwhile, in the Cupral-treated group, the regeneration beyond apex reached 0.2 mm, with a focal area reduction of 92.4% respective to the control group.In this study, we described a novel method as a successful and safe alternative for endo-canalar treatment. The preparation and treatment techniques in the present work were different, because we wanted to emphasize which one improved the outcome in the treatment of apical periodontitis. The technique used in control group, the traditional method, was related to the mechanical and chemical preparation up to the physiological narrowing in cases with passable root canals; while in the Cupral group, the technique of mechanical processing of the canals was different, because the canal was prepared up to 2/3 of its length, or less as far as the complicated anatomy of the tooth root allowed. Here, we performed a mini-invasive treatment where the canals were expanded minimally and part of them up to 1/3 of the length remained untreated, which was sterilized by the electric current of copper hydroxide ions. A long-term sterility of the canals was obtained through impregnation with copper sulfite and as demonstrated by the abrogation of the destructive apical lesions, stimulation of the osteogenesis was obtained through hydroxyl ions.In the follow-up of the control group, a positive result was found, where we detected 4.8 mm prior to treatment, then 3.9 mm after 6 months, and 2.8 mm and 2.62 mm after 12 and 18 months, respectively. Interestingly, in the Cupral-treated group, a gradual and consistent improvement of the regeneration has been observed, where from 4.95 mm prior to treatment to 2.14 mm after 6 months, to 0.31 mm after 12 months and 0.2 mm after 18 months. The results obtained showed that the Cupral-electrophoresis technique was more effective in the treatment of cases with apical periodontitis compared to traditional ones. The healing process of inflammatory focal in various age groups progressed at different speeds in both groups. As can be predicted, younger ages (19–35 years old) had the maximum regeneration rate, with elder ages in both groups having a lesser regeneration rate. The minimal regeneration occurred in old patients over 51 years of age 2, CHX, etc. [2, regardless of the method of condensation of the latter in the root canal. Accordingly, Peters et al. [2 paste, the bacterial chance to infect root canals is high in the period between treatment sessions.The clinical and radiological follow-up of the patients showed in control group that the traditional method solved the pain but the apical regeneration was slow and unsure. In line with our results, the literature claims that the bactericidal activity of Ca(OH)HX, etc. . AccordiHX, etc. , bacteris et al.  stated t2 paste limits endodontic bacteria growth, but does not enable their complete elimination [The traditional Ca(OH)mination . With timination ,7. In momination . Despitemination . Endodonmination .Copper hydroxide ions penetrate the root area and enter into the dentin tubules, helping in disintegration and extracellular destruction of the biofilm matrix and destroying the membrane of microbes and spores, fusing the cellular residues and pulp fibrous elements . Cupral’Our data indicated that the Cupral-electrophoresis technique helped stimulate the regeneration of apical destructive focal sites, in infected, obliterated, and curved root canals over 60°. This was likely related to the emersion of Cupral during electrophoresis session into the periapical tissues through the permanent electrical current, thus achieving proteolysis, detaching the apical-cement associated microbial biofilm, which is impossible to achieve using those traditional methods. Accordingly, in vitro, Cupral is demonstrated being efficient against the formation and persistence of bacterial and fungal biofilms .It is relevant to underline, that during clinical Cupral-electrophoresis treatment, the patient does not feel any discomfort or pain. This is because of the very weak current, caused by a low voltage, and only focally used in the endodontic cavity. Upon treatment, high proteolytic activities of hydroxy-cuprate and hydroxyl ions occur in the endodontic cavities. This allows decontamination of the radicular canal and periapical tissue, and in turn will fasten debris absorption from radicular canal, by the organism. Upon such treatment, exudate production (sterile proteolysis) is facilitated in the endodontic environment, decreasing in this manner the pain during chewing process. The Cupral’s high alkalinity (pH = 13) and Atacamit too (pH = 12) does not jeopardize the reinfection of radicular canal, destroying any kind of microbial agent that can enter the endodontic cavity.2 has been known for many decades and has not sparked interest in our group.To observe specifically the penetration of copper hydroxide, it was decided with the patient’s approval to histologically examine the fractured extracted teeth after 18 months of follow-up. We also agreed on this act, because the body’s electrolyte environment is different from those treated in vitro/ex vivo. This phenomenon was seen quite clearly in the histological sections see , where a2-highly dispersed paste in the ratio of 1:9 [Often, intra- and post-endodontic procedures result in tooth staining and fracture, which is a major aesthetic concern for patients, especially when anterior teeth are involved ,37,38. Ao of 1:9 .According to the manufacturer, this method has some limitations; it cannot be applied to teeth with vital pulps, such as after vital extirpation, where devitalization must be performed first, nor in patients allergic to copper or other metals. Pregnant women and patients with cardiovascular disease and pacemaker cannot be treated, because of the current.Taking into account all the above-mentioned points, we can say that (1) the use of Cupral’s solution during the apical periodontitis treatment helped raise the medical success, in both near and distant times. Further, (2) according to ,42,43,44Our study provides the first evidence on the efficacy of Cupral in the treatment of apical periodontitis. By using both a clinical and radiological 18-month follow-up, we demonstrated the effectiveness of the treatment in the near and distant time. We favor the idea that the action of Cupral and its penetration are indeed optimized through electrophoresis.The intense periapical tissue regeneration observed paves the way for further in-depth studies to better comprehend Cupral’s clinical impact.


Three-month-old female mice were used, and they were randomly divided into four groups: control, model, CoQ10, and titanium dioxide  groups. Our results showed that body weight, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein expression were significantly decreased, while malondialdehyde (MDA) activity and metalloproteinase-1 (MMP-1) level were increased in UVB-treated mice. Besides, the stratum corneum was shed from the skin surface in the model group compared with the control group. In contrast, CoQ10 sunscreen prevented from UVB-induced skin damage, as well as reversing SOD, GSH-Px, and MDA activities, and MMP-1 and DNMT1 levels. Taken together, the current study provided further evidence on the prevention of UVB-induced skin damage by CoQ10 and its underlying mechanisms.The level of sun ultraviolet ray reaching the surface of the earth is increasing severely due to the rapid development of the society and environmental destruction. Excessive exposure to ultraviolet radiation causes skin damage and photoaging. Therefore, it is emerged to develop effective sunscreen to prevent ultraviolet-induced skin damage. This study was aimed at investigating the effects of Coenzyme Q The skin is the largest barrier that protects against the damage from the environmental risk factors and eventually results in skin aging. Skin aging can be categorized into intrinsic and extrinsic responses. The intrinsic skin aging occurs naturally as time passes , while e10  was found by Crane et al. in 1957 to be in the mitochondria of the beef heart, and broadly distributed in mammalian tissues [10, a necessary factor for healthy body, plays an important role in cardiovascular disorders and aging, including heart failure, hypertension, and endothelial dysfunction [10 also has a potential role for the prevention and treatment of heart ailments by improving cellular bioenergetics via scavenging free radicals [10 might be a useful preventive medication against skin photoaging [10-loaded conventional carriers, ultrasmall lipid nanoparticles containing CoQ10 exhibited reduced capacity in free radical formation compared with non-nanocarrier-treated cells. Therefore, ultrasmall lipid nanoparticles containing CoQ10 were shown to be suitable to increase the antioxidant capacity of the skin [10 significantly reduced the levels of myeloperoxidase (MPO), phospholipase A2 (PLA2), and MDA, while it increased SOD levels in vivo and in vitro [10 might rejuvenate a wrinkled skin through inhibiting the degradation of dermal fiber components and stimulating the paracrine of dermal fiber via upregulation of interleukin- (IL-) 6 and MMP secretion [10 sunscreen against skin damage induced by UVB as topical application on a mouse skin.Coenzyme Q tissues , 13. CoQfunction . Growingradicals . With reotoaging . Among Cthe skin . Moreovein vitro . A studyecretion , 21. The10 was provided by Runhe Biology Co. , while titanium dioxide (TiO2) was purchased from Kemao Chemical Co. . Ointment base was made by our laboratory, which contained purified water, petrolatum, polysorbate 80, and cetostearyl alcohol and did not contain any drug.CoQThis study was carried out according to the Guide for the Care and Use of Laboratory Animals of Guangdong Laboratory Animal Monitoring Institute, the National Laboratory Animal Monitoring Institute of China. All the procedures performed were in accordance with the ethical standards of the Academic Committee on the Ethics of Animal Experiments of Guangdong Medical University. 36 specific pathogen-free female Kunming mice were acclimatized to local vivarium conditions  and allowed to free access of water and diets containing 1.11% calcium and 0.74% phosphorus. The average weight of mice was about 27.66 g ± 0.56.10 sunscreen was composed of CoQ10 and ointment base at the concentration of 10 mg/g. TiO2 sunscreen was used as a positive control and composed of TiO2 and ointment base at the concentration of 50 mg/g [n = 9), aging model group , CoQ10 group , and TiO2 group . The hair on the back of each mouse was shaved, and 0.5 g of ointment was topically applied to 3 × 3 cm2 of the skin once daily for 8 weeks. Except the control group, the other groups were exposed to ultraviolet B radiation  under diffused UV light  for 30 mins every day.CoQ 50 mg/g , 19. 36 All the mice were weighed weekly. At the end of the treatment, the mice were sacrificed, and blood was collected from the eyeballs. Firstly, the serum was collected from the blood by centrifugation, and it was used for biochemical assays. And then, the dorsal skin, heart, liver, kidney, and brain were dissected, weighed, and normalized by body weight. Finally, dorsal skin tissues were also collected for histological, biochemical, and quantitative real-time PCR analyses.The degree of skin damage exposed to UVB could be determined by evaluating the activities of MDA, SOD, and GSH-Px . They ar2) was fixed with 4% paraformaldehyde for Van Gieson (VG) and hematoxylin and eosin (H&E) staining. H&E staining was used to assess the skin structure alteration, while VG staining was applied to detect the presence of collagen fibers. All the stained skin specimens were observed and photographed using an optical microscope .Each part of the skin samples  as recommended by the manufacturer. Total RNA was reverse transcribed to cDNA using a commercial kit  according to the protocol of the manufacturer. Target genes were amplified with SYBR Premix Ex Taq™  using a PikoReal 96 Real-Time PCR System . The sequences of the forward and reverse primers  are shown in T method .α-Tubulin antibody  was used as a housekeeping control.The total protein was extracted from the dorsal skin tissues with ice-cold lysis buffer. The protein concentrations of the lysates were measured by the bicinchoninic acid kit . An equal amount of proteins was used and separated by SDS-PAGE gels and then transferred onto the nitrocellulose membranes. Then, the membranes were incubated with primary DNMT1 antibody , and anti-rabbit-HRP antibody . The blots were developed by enhanced chemiluminescence  with a ChemiDoc™ MP System . p < 0.05 was considered statistically significant.The results were expressed as the mean ± standard deviation (SD). The data were analyzed using SPSS 17.0 software . The significance of differences between groups was evaluated by one-way or two-way ANOVA, and 10 and TiO2 mice were significantly increased compared to those of the model mice. The results also showed that liver weight was significantly decreased when the mice were exposed to UVB compared to control mice  radiation. Sunlight is the main source of UV radiation, which can induce skin senescence, inflammation, aging, and cancer . TherefoA damage . In thisIn this study, we found that the mouse skin was damaged by UVB. This was shown by the decrease in the growth rate of body and liver weights in UVB-treated mice. Besides, UVB decreased the activities of SOD and GSH-Px and increased MDA activity in the mouse skin. This suggested that the balance between oxidant and antioxidant systems was impaired when the mouse skin was exposed to UVB only. During the aging process, the skin dermis becomes thin and damaged , due to 10 sunscreen could alleviate the alterations of collagen in the mouse skin induced by UVB, and the stratum corneum of the mouse skin was not shed from the skin surface. MMP-1 expression was shown to be increased with age, which is a major factor that causes collagen breakdown and wrinkling problems [10 sunscreen treatment could inhibit MMP-1 upregulation and collagen degradation induced by UVB in the mouse skin. Similarly, CoQ10 sunscreen was also shown to reduce MMP-1 levels in dermal fibroblasts [10 sunscreen in the mouse skin. Furthermore, MDA is a biomarker of cell membrane damage caused by free radicals [10 sunscreen could reduce MDA activity induced by UVB. Taken together, we suggest that CoQ10 sunscreen has antioxidant activities against UVB damage in the mouse skin and prevents from collagen degradation by suppressing MDA activity and MMP-1 levels and enhancing SOD and GSH-Px activities.In the current study, we demonstrated that topical application of CoQproblems , 31. Indproblems . Therefoproblems . In factproblems . Interesroblasts . Moreoveroblasts . Our resradicals . We foun10 sunscreen prevents UVB-induced skin damage by enhancing the antioxidant capacity of the skin and delays the breakdown of collagen through suppressing MMP-1 level and MDA activity in the mouse skin. Therefore, we suggest that CoQ10 sunscreen might have beneficial effects in antiaging, and topical application of CoQ10 sunscreen could be potential to protect against UVB-induced photoaging.In conclusion, our findings indicated that topical application of CoQ


Coronavirus disease 2019 (COVID-19) has migrated to regions that were initially spared, and it is likely that different populations are currently at risk for illness. Herein, we present our observations of the change in characteristics and resource use of COVID-19 patients over time in a national system of community hospitals to help inform those managing surge planning, operational management, and future policy decisions. Demographic data were collected from the electronic health records of inpatients with discharged from facilities affiliated with a large healthcare system. The HCA Healthcare system consists of 186 hospitals and >2,000 sites of care located in 21 states and the United Kingdom. Acute-care facilities consist primarily of urban and suburban community hospitals as well as specialty and tertiary referral facilities. Collectively, these facilities provide ~5% of all inpatient hospital services in the United States. Affiliated facilities are distributed geographically across the country with a concentration in the southern United States.For this analysis, patients were included if they had laboratory-confirmed COVID-19 (using reverse-transcriptase polymerase chain reaction (RT-PCR) for severe acute respiratory coronavirus virus 2 (SARS-CoV-2)] and were discharged between March 1 and June 30, 2020 . Aggregated data for age, race, ethnicity, length of stay, level of care, discharge disposition, and select treatments were analyzed by month.P < .0001). The distribution of patient age also changed from a unimodal, nearly normal distribution in March with a peak between 44 and 64 years of age, to a bimodal and right-skewed distribution in June with peaks around 24–34 years and 44–54 years.Monthly volumes of hospitalized SARS-CoV-2–positive patients increased steadily between March and June, 2020 Table  while thThe distribution of patient race and ethnicity also varied over time, which may reflect the change in distribution of active COVID-19 surges and the service areas of affiliated facilities. Most notably, there was a substantial increase in the number of patients who identified as Hispanic ethnicity  generally increases with age. This trend held true over the 4-month time period, even as the mean LOS changed by month . By June, patients meeting this criteria accounted for 4,671 of 5,919 patients (78.9%).When segregated by month, patient age bracket (>65 years or <65 years), and severity at admission, the mortality rate declined month after month for all groups. For patients <65 years of age who were initially admitted to a noncritical care unit, the mortality rate decreased from 5.0%  in April to 2.1%  in June. For patients >65 years of age initially admitted to a noncritical care unit, the mortality rate decreased from 29.4%  in April to 13.8%  in June.1 The use of tocilizumab remained relatively constant and rare, consistent with the use of this treatment in patients with severe disease. The use of remdesivir increased, but this may be attenuated by access issues . Since March, ~40% of all COVID-19 patients received treatment with tocilizumab, remdesivir, or steroids .Between March and June, the use of hydroxychloroquine decreased precipitously, consistent with the revised recommendations against this therapy Table .1 The usAs the COVID-19 pandemic continues, there are observable changes in the patient population affected, hospitalization and resource use, and mortality rate. Using data from COVID-19 patients admitted to community hospitals across the United States during nonsurge conditions, we showed that although patient age and other characteristics changed over time, outcomes are improving for all patients. This finding suggests that knowledge gained from early experiences with this disease is allowing providers to better care for patients with COVID-19.