Prospective blood donors are routinely screened for blood borne infections but medical illnesses and haemoglobin genotype are overlooked despite a high prevalence of haemoglobin AS among Nigerian donors.To determine the prevalence of haemoglobin AS and its association to renal function, if any.Apparently healthy donors were studied between February and December 2018. Their haemoglobin genotype and, estimated glomerular filtration rates were determined.2 and 99.9 ± 21.2ml/min/1.73m2 respectively . Eighty one (79.4%), 20 (19.6%) and 1 (1.0%) subjects had renal function at >90ml/min/1.73m2, 60–89ml/min/1.73m2 and 30–59ml/min/m2 respectively. There was no significant difference in the mean eGFR between subjects with haemoglobin AA and AS .There were 96 males (94.1%) and 6 (5.9%) females with mean age of 26.7±4.5 years (range 19–44 years) and mean eGFR of 103.97±19.00ml/min/1.73m2. Eighty one (79.4%) and 21 (20.6%) subjects had haemoglobin AA and AS genotypes respectively. The mean eGFR for subjects with haemoglobin AA and AS were 105.2±18.6ml/min/1.73mThe prevalence of sickle cell trait among Nigerian blood donors is high. There is no significant difference in the renal function status of blood donors with SCT and normal haemoglobin genotype. Blood transfusion, by all standards should be a safe, easy and fast life-saving procedure. It should be conducted in a manner that the health of recipients and donors alike is protected. The World Health Organization (WHO) guidelines on assessing donor suitability provides clinicians and blood bank personnel with protocols for acceptance and/or deferment of blood donors with sickle cell trait (SCT) and those with chronic kidney disease (CKD).3This approach often times gives an impression that the focus of the laboratory personnel is directed more at protecting would-be recipients without taking into consideration the adverse effects of blood donation on prospective donors, especially those who may have background medical illnesses. For instance, in one large study, a significant number of donors had measurable untoward systemic adverse effects such as fatigue, vasovagal symptoms, nausea and vomiting after donation for blood transfusion.4Current studies indicate that the prevalence of haemoglobin S among blood donors in Nigeria is high, ranging between 19.8% and 26.1%.2There are indications for physicians to pay closer attention to the haemoglobin genotype of would-be blood donors as there are new evidences suggesting that individuals with SCT are prone to developing chronic kidney disease (CKD).14Other established indications for excluding such people from blood donation include neonatal transfusion, exchange blood transfusion, leukofiltration and in cases where blood transfusion is required for a sickle cell anaemia patient.th February 2018 to 31st December 2018.This was a descriptive, cross-sectional study conducted at the blood bank unit of the kidney care centre, University of Medical Sciences Teaching Hospital, Ondo State, Southwest Nigeria from 5A representative sample of one hundred and two consecutive, apparently healthy individuals aged between 19 and 44 years who came to donate blood were selected by convenience sampling method.A minimum sample size of 74 for this study was calculated from the prevalence of 5.1% obtained for renal dysfunction among young adults with SCT.2 for sample size determination in study population less than 1,000 subjects was usedThe formula, z2pq/dp = proportion in the target population estimated to have a particular characteristicsz = the standard normal deviation (using 95% confidence level = 1.96)d = degree of accuracy desired, set at 0.05q= 1.0 – pall pregnant and lactating women, persons younger than 18 years or older than 60 years and persons with the following: haemoglobin concentration < 13.5g/dl for males and 12.5g/dl for females, positive serology testing for Human Immunodeficinecy Virus I and II (HIV I and II), hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus (Anti-HCV), persons who appear generally unwell, breathless, or having an active fever, cough or undergoing treatment for a medical illness, persons who donated within the last 12 weeks for males and 16 weeks for females were excluded from the study.1Blood samples were obtained for blood group, haemoglobin genotype and serum creatinine. Venous blood sample for analysis was collected at the media-cubital fossa from each participant. Two milliliters (ml) of blood was dispensed into the ethylene diamine tetra-acetate (EDTA) blood collection tubes for determination of haemoglobin genotype using the standard haemoglobin electrophoresis machine . Assessment of serum creatinine was performed using 3 ml of venous blood dispensed in lithium heparin collection tubes. Samples were centrifuged in the laboratory to separate the serum which was then analysed by the modified kinetic Jaffe method using the RANDOX, UK commercially prepared reagent.The estimated glomerular filtration rate (eGFR) was determined using the chronic kidney disease-epidemiology (CKD-EPI) calculator. The CKD-EPI was developed from a large database of people, including those with and without kidney disease, diabetes, and a history of organ transplantation and it takes into consideration gender in the estimation of eGFR.17Data was analyzed using SPSS version 20. Continuous variables were presented as mean ± standard deviation for un-skewed data. Discrete variables were presented as frequency and percentage. Comparison of means was carried out using the independent samples T test. P value < 0.05 was taken to be significant at 95% Confidence Interval.Ethical clearance for this research was sought from the Ethics and Research Committee of State Specialist Hospital, Akure, Ondo State. All the participants gave their informed consent before their samples were collected.2, 60–89ml/min/1.73m2 and 30–59ml/min/m2 respectively  and 6 (5.9%) females with mean age of 26.7±4.5 years (age range 19–44 years) and mean serum creatinine value of 100.5umol/L. Seventy three (71.6%), 15 (14.7%) and 9 (8.8%) had O Rhesus D positive, A Rhesus D positive and B Rhesus D positive blood groups respectively . Sixty sectively . There wSeventy seven (75.5%), 4 (3.9%) and 21 (20.6%) subjects had haemoglobin AA, AC and AS genotypes respectively and, the proportion of females with haemoglobin AS was higher than males .2 and 40.4±2.3%  respectively. The mean eGFR for subjects with haemoglobin AA, AC and AS were 105.2±18.6ml/min/1.73m2, 102.6 ± 16.1 ml/min/1.73m2 and 99.9±21.2ml/min/1.73m2 respectively (The mean packed cell volume and mean eGFR were 103.97±19.00ml/min/1.73mectively . There wMajority (94.1%) of the study participants were males which is similar to reports from within and outside Nigeria. 22The prevalence of blood donors with SCT 21.6%) was high in our study. This figure falls within the range already established from earlier studies in Nigeria.% was hig26In 2018, the Federal Government of Nigeria approved an Executive bill for the establishment of a National Blood Service Commission but this is still awaiting passage into law.It has been postulated that SCT may directly lead to loss of renal function through episodic sickling leading to chronic parenchymal ischaemia. Low oxygen content of the renal medulla may provide an enabling setting for intravascular sickling which may lead to chronic microvascular damage and, consequently to chronic hypoxia and tubule-interstitial fibrosis.322) while 1% had renal function below 60ml/min/1.73m2. The relatively lower mean eGFR among donors with SCT even though not to the significant level  suggests that there may be an association between SCT and CKD. It is possible that with a larger number of subjects, the mean difference may become significant. This finding suggests that there may be a larger number of individuals with the SCT who have concomitant CKD who are in the donor pool who require screening, detection and exclusion from blood donation. This process need not be limited to haemoglobin genotype but extended to cover other medical illnesses such as hypertension, diabetes, dyslipidaemia etc. For instance, there are reports indicating that co-existence of SCT with other chronic medical diseases  is associated with higher risk of developing CKD.34A little over one-fifth of the subjects in our study had renal function below optimum (90ml/min/1.73mThis was a cross-sectional study with the data skewed towards the male gender. Confounding variables such as hypertension, diabetes, obesity, smoking and other cardiovascular risk factors for chronic kidney disease were not excluded at the blood transfusion unit.The prevalence of SCT among Nigerian blood donors is high and there is no significant difference in the renal function status of blood donors with normal haemoglobin and those with the SCT. There is a need to conduct high powered, larger prospective studies to improve the reliability of outcome when determining relationship between SCT and renal dysfunction among blood donors.


Secondary structures provide a deep insight into the protein architecture. They can serve for comparison between individual protein family members. The most straightforward way how to deal with protein secondary structure is its visualization using 2D diagrams. Several software tools for the generation of 2D diagrams were developed. Unfortunately, they create 2D diagrams based on only a single protein. Therefore, 2D diagrams of two proteins from one family markedly differ. For this reason, we developed the 2DProts database, which contains secondary structure 2D diagrams for all domains from the CATH and all proteins from PDB databases. These 2D diagrams are generated based on a whole protein family, and they also consider information about the 3D arrangement of secondary structure elements. Moreover, 2DProts database contains multiple 2D diagrams, which provide an overview of a whole protein family's secondary structures. 2DProts is updated weekly and is integrated into CATH.https://2dprots.ncbr.muni.cz. The web interface was implemented in JavaScript. The database was implemented in Python.Freely accessible at Bioinformatics online. Nowadays, there are more than 170 000 structures in Protein Data Bank (PDB)  such as α-helices and ß-sheets. The most straightforward way to compare protein structures within a protein family might be its visualization using secondary structure 2D diagrams.Several tools for the 2D visualization of protein secondary structure have been developed , the list of its domains , and the PDB structures of these domains.Step 1: For each domain in the given family, find its SSEs (via SecStrAnnotator (nnotator ) and annStep 2: For each group of SSEs with the same name, compute average length and frequency of SSE occurrence.Step 3: For each domain in the family:Step 3.1: Try to select an appropriate starting layout among the previously computed domains.Step 3.2: Group all β-strands into sheets and compute a 2D model of each individual sheet.Step 3.3: Divide the helices and sheets into primary (common for most of the domains) and secondary (the remaining ones).Step 3.4: Place all primary helices and sheets into the 2D diagram.Step 3.5: Adjust the angles of the primary helices and sheets.Step 3.6: Add all secondary helices and sheets into the 2D diagram.Step 3.7: Adjust the angles of the secondary helices and sheets.Step 4: Draw an individual 2D diagram for each domain and a common multiple 2D diagram for the whole family.Please note, that steps 3.2 to 3.7 employ an optimization algorithm to minimize the error of projection from 3D to 2D and restrict the deviation from the starting layout. Details of the algorithm are described in the https://2dprots.ncbr.muni.cz.For each PDB structure, 2DProts provides 2D diagrams of all its domains . The last example also demonstrates the possibilities of a more detailed clustering.Each protein domain is represented in the 2DProts database via its identifier in the CATH format (e.g. 1r9nA01), and its 2D diagram is findable using this identifier. It is also possible to search for all domains of a specific protein using its PDB ID (e.g. 1r9n). Protein families can be found via a CATH identifier (e.g. 2.140.10.20) that can be used in the search field to obtain a multiple 2D diagram of the family.In comparison with other tools generating 2D diagrams of protein secondary structure, 2DProts has the following three marked advantages: Firstly, 2DProts reflects 3D arrangement of SSEs see , whereashttps://gitlab.com/jhutar/2dprot-web.Applicability of 2DProts is demonstrated by the fact that 2D diagrams and multiple 2D diagrams from 2DProts were recently integrated into the CATH database ) is planThis work was supported by Ministry of Education, Youth and Sports of the Czech Republic under the ELIXIR CZ research infrastructure project, including access to computing and storage facilities [grant number LM2018131]; and European Regional Development Fund—project ELIXIR-CZ [grant number CZ.02.1.01/0.0/0.0/16_013/0001777].Conflict of Interest: none declared.https://www.ebi.ac.uk/pdbe) and the CATH database (https://www.cathdb.info). The SecStrAnnotator used by 2DProts is available at https://sestra.ncbr.muni.cz. Database 2DProts with all computed 2D diagrams is freely accessible at https://2dprots.ncbr.muni.cz. Source code of the website of 2DProts is available at https://gitlab.com/jhutar/2dprot-web.The input data for the 2DProts database are sourced from the P DB database (btab505_Supplementary_DataClick here for additional data file.


M = 13.6, SD = 2.7, range 5–18 years; 50.9% girls), including 294 (12.2%) with ADHD and 2,124 (87.8%) without ADHD. The proportion with prior concussion among youth with ADHD (43.9%) was significantly greater than youth without ADHD . A significantly higher proportion of boys with ADHD had a prior concussion history (48.1%) than boys without ADHD , but this difference was not observed for girls . Youth with ADHD did not differ with regard to their estimated longest duration of symptoms from a prior concussion  and the proportion who reported taking longer than 28 days to recover from a prior concussion did not differ between those with ADHD (15.3%) and without ADHD (12.2%), χ2 = 2.20, p = 0.14. Among youth presenting to a specialty clinic, ADHD was associated with greater lifetime history of concussion but not a greater duration of symptoms from a prior injury.Child and adolescent student athletes with attention-deficit/hyperactivity disorder (ADHD) report a greater lifetime history of concussion than those without ADHD. This case-control study compared youth with and without ADHD presenting for care at a specialty concussion clinic on their lifetime history of concussion. We hypothesized that a greater proportion of youth with ADHD would report a history of prior concussion. Archival clinical data from patients presenting to a specialty concussion clinic in Montreal, Québec, Canada between September 2015 and August 2019 were analyzed. The sample included 2,418 children and adolescents (age:  N = 10,585) from the U.S. general population ages 9 and 10 years old. Namely, the prevalence of prior concussion among children with ADHD was 7.2% compared to 3.2% among children without ADHD, meaning ADHD is associated with twice the odds of experiencing a prior concussion before age 11 (Numerous studies have found that children and adolescents with attention-deficit/hyperactivity disorder (ADHD) report a greater lifetime history of concussion than those who do not have ADHD –5. For ee age 11 . MoreoveStudies have primarily examined samples of uninjured student–athletes who complete health history surveys at the beginning of sporting seasons. To our knowledge, no prior study has examined the association between ADHD and lifetime history of concussion among youth who present for clinical care after sustaining a concussion. Several studies of concussion specialty clinic patients have reported lifetime history of concussion in their samples. We extracted the proportions from 16 of these studies and determined, on average (weighted mean proportion), about 35% of youth presenting to concussion clinics report having a prior concussion –22, withUsing a large archival dataset, we examined the proportions of youth with and without ADHD who report a lifetime history of concussion when they present for treatment at a Canadian specialty concussion clinic. We hypothesized that (i) youth with ADHD would have a greater lifetime history of concussion than youth without ADHD, and (ii) boys with ADHD would have a greater lifetime history of concussion than girls with ADHD.SD = 2.7, range: 5–18) and equally split between girls (50.9%) and boys (49.1%). The primary  language spoken was French (57.8%), English (41.7%), and bilingual/equal fluency in both languages (0.5%). Data regarding racial and ethnic group affiliation were not available. A trauma coordinator, who is a licensed health care professional, prospectively gathered this information during a customary intake interview using standardized data collection forms, with the parent and child collaboratively providing answers. Specifically, youth and their caregivers were asked whether youth had a pre-injury diagnosis of ADHD (yes or no) and also reported the youth's concussion history (yes/at least one prior concussion or no prior concussion). Those with previous concussions estimated the longest duration of symptoms that they experienced from a prior concussion.Participants included concussed children and adolescents who presented for treatment at a specialty concussion clinic in Montreal, Quebec, Canada consecutively between September 2015 and August 2019. The sample included 2,418 children and adolescents see . The mea2-tests. The proportions of girls and boys with ADHD who experienced a prior concussion were also compared using χ2-tests. To characterize the magnitude of group differences, an odds ratio (OR) was calculated for each analysis as an effect size  (The proportions of youth with and without ADHD who had a history of concussion (one or more prior injuries) were computed and compared using χect size  and inte= large) . Analyse2 = 1.80, p = 0.09, OR = 1.12 . A total of 294 youth (12.2% of the sample) reported having been diagnosed with ADHD. Additionally, sex differences were not observed amongst children and adolescents with ADHD . No overall differences in mechanism of injury or referral source were observed, as 67.4% of youth without ADHD and 63.3% of youth with ADHD were concussed through a sport-related mechanism  and 56.2% of youth without ADHD and 52.7% of youth with ADHD were referred to the concussion clinic from the pediatric emergency room compared to other referral sources .In the total sample, roughly 2 out of 5 (38.3%) concussed youth presenting for care at a specialty concussion clinic had sustained a previous concussion. Boys (39.7%) and girls (37.0%) did not differ with regard to their personal history of concussion, χ2 = 4.41, p = 0.04, OR = 1.30 (1.02–1.67). The proportion with a prior concussion among boys with ADHD [48.1% (40.2–55.9%)] did not significantly differ from girls with ADHD [39.1% (31.0–47.2%)], χ2 = 2.38 p = 0.12, OR = 1.15 (0.91–2.29). However, the proportion with a prior concussion among boys with ADHD [48.1% (31.6–63.1%)] differed significantly from boys without ADHD [38.4% (35.4–41.3%)], χ2 = 5.33, p = 0.02, OR = 1.49 (1.06–2.09). This finding was not observed among girls .A significantly greater proportion of youth with ADHD [43.9% (95% CI 38.2–49.6%)] reported a prior concussion history compared to youth without ADHD [37.5% (35.5–39.6%)], χ2 = 2.56, p = 0.11, OR = 1.28 (0.94–1.75). Of those youth receiving care for a non-sporting injury, the proportion of lifetime history of prior concussion did not differ in youth with ADHD [39.8% (30.6–49.1%)] compared to youth without ADHD [32.2% (28.7–35.7%)], χ2 = 2.46, p = 0.12, OR = 1.39 (0.92–2.12). For individuals referred from the pediatric emergency department, the proportion of prior concussion significantly differed in youth with ADHD [47.7% (39.9–55.6%)] compared to youth without ADHD [38.0% (35.2–40.7%)], χ2 = 5.48, p = 0.02, OR = 1.49 (1.07–2.09). For individuals referred from sources other than a pediatric emergency department, the proportion of prior concussion did not differ in youth with ADHD [39.6% (31.4–47.7%)] compared to youth without ADHD [36.9% (33.8–40.0%)], χ2 = 0.36, p = 0.55, OR = 1.12 (0.78–1.61).We also examined lifetime history of concussion among youth with and without ADHD stratified by the mechanism of their index injury, i.e., their most recent injury, for which they were receiving care (sport-related index injury vs. non-sporting injury), as well as stratified by the referral source for their index injury (emergency room vs. another source). Of those youth presenting for care following a sport-related concussion, the proportion of lifetime history of prior concussion did not differ in youth with ADHD [46.2% (39.1–53.4%)] compared to youth without ADHD [40.1% (37.6–42.7%)], χn = 702), the median longest symptom duration from a previous injury was 21 days . Youth with ADHD  did not differ from youth without ADHD  with regard to longest duration of symptoms from a prior concussion, Z = 1.52, p = 0.13. Further, the proportion of youth who reported taking longer than 28 days to recover from a prior concussion did not differ between those with ADHD (15.3%) and without ADHD (12.2%), χ2 = 2.20, p = 0.14.Among all children and adolescents who reported a prior history of concussion  reporting a prior diagnosis of ADHD compared to about 6% in student athlete studies , 4. Simincussion , and theAs seen in This study is novel in that it is the first to compare the estimated longest symptom duration from previous concussions between youth with and without ADHD. Despite consistent findings that youth with ADHD experience greater lifetime concussion histories, youth with ADHD did not differ from youth without ADHD with regard to longest duration of symptoms from a prior concussion (median for both groups was 21 days). Similarly, the proportion of children and adolescents who reported taking longer than 28 days to recover (commonly considered the demarcation point for “persistent” or prolonged recover) from a prior concussion did not differ between those with ADHD (15.3%) and without ADHD (12.2%). This novel result aligns with conclusions from systematic reviews , 31 as wThis study has several methodological limitations. First, both ADHD status and concussion history were reported by the parents and youth; neither were verified using a clinical evaluation or by reviewing medical records. With that said, caregivers were present with the youth and this health history information was collected during a clinical interview with a trauma coordinator, and thus likely represents a more valid data collection process than many studies on this topic, which rely exclusively on unaided youth self-report of health history. Second, we do not know the subtype or severity of ADHD, age of ADHD onset, current ADHD symptom burden, or whether the youth were on medications for ADHD. Third, we did not have access to racial or ethnic group identification, and more refined examination and understanding of sociocultural factors in concussion research is an important future direction for the field. That said, this study examined Canadian youth whose predominant preferred or dominant language was French, a clinical population that, to this point, has not been characterized with respect to potential associations between pre-existing ADHD and lifetime concussion history. Finally, we do not know how many prior concussions the children and adolescents experienced (the data were coded as present/absent), how long ago the concussion(s) occurred, or the severity of those injuries.Among samples of student athletes and children from the U.S. general population, ADHD is consistently associated with greater lifetime history of concussion. This finding is consistent among the current sample of youth who presented for care at a specialty clinic in Canada, such that among children and adolescents who are seeking health care for an index concussion, those with ADHD report greater lifetime history of concussion than youth without ADHD. However, youth with ADHD did not differ from youth without ADHD in their estimated longest duration of symptoms from a prior concussion—consistent with past studies suggesting that ADHD, per se, does not appear to be a risk factor for prolonged symptoms following concussion.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author. The statistical code, syntax, output, analyses, and minimum dataset are available to qualified researchers upon request.The studies involving human participants were reviewed and approved by Ethics approval was provided by the McGill University Health Centre . 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.NC helped conceptualize the study, conceptualized the statistical analyses, drafted sections of the manuscript, assisted with the literature review, edited the manuscript, and approved the final manuscript. ET helped design and coordinate data collection, conducted the statistical analyses, drafted sections of the manuscript, critically reviewed, and edited/approved the final manuscript. GI helped conceptualize the study, assisted with the literature review, drafted sections of the manuscript, edited the manuscript, and approved the final manuscript. DF and LG critically reviewed and edited/approved the final manuscript. IG helped design and coordinate data collection, wrote the IRB, conceptualized the overall project, critically reviewed, and edited/approved the final manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.® Applications, Inc., National Rugby League, and the Spaulding Research Institute.NC acknowledges support from the Louis V. Gerstner III Research Scholar Award from Massachusetts General Hospital. GI acknowledges unrestricted philanthropic support from the Mooney-Reed Charitable Foundation, Heinz Family Foundation, Boston Bolts, ImPACTGI has a clinical practice in forensic neuropsychology, including expert testimony, involving individuals who have sustained mild TBIs. He has received research support from the Harvard Integrated Program to Protect and Improve the Health of NFLPA Members, and a grant from the National Football League. He serves as a scientific advisor for NanoDx™, Sway Medical, Inc., and Highmark, Inc. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This quality improvement study assesses the comorbidities associated with COVID-19 diagnostic codes in US health insurance claims. We analyzed the comorbidities associated with COVID-19 diagnosis to assess the specificity of the legacy code and the importance of using both codes.Accurate identification of COVID-19 diagnosis in patient medical records is essential for studies using administrative data to examine morbidity, mortality, and risk factors associated with COVID-19.5 Our analytic sample contained the longitudinal medical records of 28 853 694 patients across the US from January 1, 2018, to September 30, 2020. We examined the frequency of B97.29 and U07.1 in 2020 to understand their adoption by hospital and nonhospital health care professionals. We only considered the first encounter to avoid double counts. Because the legacy code may be used for other coronavirus incidents, we identified the most common co-occurring diagnoses before and after January 1, 2020, and calculated the correlation of their frequency using the SciPy package in Python, version 1.5.2. The same analysis was conducted for U07.1 for comparison. This study was approved by the Indiana University institutional review board and followed the Standards for Quality Improvement Reporting Excellence (SQUIRE) reporting guideline. Owing to the use of deidentified patient data, the need for informed consent was waived by the institutional review board.6In this quality improvement study, we used the deidentified Clinformatics Data Mart Database (Optum), which comprises commercial and Medicare Advantage health plans members who are similar to the US commercially insured population with respect to demographic characteristics.3 whereas in 2018 and 2019, only 1 out of 10 most frequent diagnoses that co-occurred with B97.29 was associated with COVID-19  than those with B97.29 in 2018 and 2019 . Using only U07.1 to identify patients with COVID-19 after April 1, 2020, missed 9714 patients diagnosed only with B97.29, consisting of 3.4% among 286 161 patients with either the legacy or new codes. However, the number of false positives due to screening B97.29 for patients with COVID-19 would be small, because the code’s drastic increase in 2020 can be attributed to COVID-19-related symptoms  in the data set between January 1 and September 30, 2020, 26 414 (0.14%) were diagnosed with B97.29, and 279 066 (1.47%) were diagnosed with U07.1. The number of patients with a B97.29 code increased in March 2020 but rapidly diminished after the introduction of U07.1 . AlthougCOVID-19 . The fresymptoms .4 showed that the legacy code B97.29 was quickly replaced by U07.1, and its use decreased to prepandemic levels. Our quality improvement study confirmed their findings about hospitals by using large-scale medical claims data, but our results suggest that some nonhospital health care professionals have continued to use B97.29 for COVID-19 diagnoses in 2020. It is possible that patients diagnosed with B97.29 were infected with coronavirus diseases other than COVID-19, and our findings may not generalize to other data sources. However, future research on COVID-19 using claims data should consider both B97.29 and U07.1 when identifying patients with COVID-19 to avoid introducing a systematic bias across hospital and nonhospital health care professionals given the strong socioeconomic disparity in rates of COVID-19 testing and infection.Using a hospital discharge data set, Kadri et al


The spleen was measured via ultrasonic imaging every minute for volume calculations, and venous blood samples were drawn before and after exposure for hemoglobin concentration (Hb). Mean (SD) spleen volume was 279 (115) mL before exposure, 219 (75) mL  at 3 min of exposure, and 201 (93) mL after 10 min exposure to hypoxia . Hb was 138.8 (7.6) g·L−1 before and 142.9 (8.1) g·L−1 after 10 min of exposure . SpO2 was 96.4 (1.7)% before exposure and 74.7 (8.4)% during the last minute of exposure . HR increased from 80 (14) to 90 (17) bpm during exposure . MAP remained unchanged. After 10 min recovery on oxygen, values had been restored for spleen volume and Hb, while SpO2 was higher and HR lower compared with before hypoxia exposure. We concluded that acute normobaric hypoxia of only 10 min caused significant spleen volume contraction with Hb increase. This rapid spleen response, evident already after 3 min of exposure, could have a protective effect during sudden exposure to severe hypoxia.The spleen contracts progressively during moderate normobaric hypoxia exposure of 20 min, which elevates hemoglobin concentration (Hb). However, acute hypoxia exposure could be shorter and more severe when oxygen systems fail during, e.g., high-altitude sky diving, aircraft cabin pressure drop, balloon flights, extreme altitude climbing, and in some maladies. We aimed to evaluate the speed and magnitude of spleen contraction during short exposure to extreme eupneic hypoxia and its subsequent recovery on oxygen. Eight female and seven male volunteers were exposed to normobaric hypoxia (10% oxygen) for 10 min during sitting rest, followed by 10 min on 100% oxygen. Heart rate (HR), arterial oxygen saturation (SpO Sudden decreases in oxygen partial pressure can occur in an abrupt loss of cabin pressure in an aircraft  age of 25 (3) years, height 177 cm , and weight 76 kg . The participants were all healthy non-smokers involved in physical training 6.6 (3.5) h per week. None of the participants had been at an altitude above 2500 m in the past 6 months. The participants received detailed information about the test protocol and signed an informed consent form. The protocol had been approved by the Regional Human Ethics Board of Umeå University, Sweden, and complied with the Helsinki declaration.The participants arrived at the lab after a period of 12 h without any alcohol consumption and 2 h without eating or drinking caffeine-containing beverages before the testing started. Height and weight were measured, and vital capacity was measured in the standing subject in triplicate with the largest volume used . The participant was seated and had a venous catheter  inserted in an antecubital vein of the left arm after which all other measurement equipment was attached. The protocol, which started after at least 20 min of sitting rest, consisted of three phases: (1) 5 min of normobaric normoxic respiration (PRE), (2) 10 min of normobaric hypoxic respiration of gas containing 10.0% oxygen in nitrogen  equivalent to 5,793 m altitude (EXP), and (3) 10 min of normobaric hyperoxic respiration (POST) at 100% oxygen, mimicking rescue oxygen breathing. The different gas mixtures were administered via face mask.Minute-to-minute measurements of spleen size were obtained via ultrasonic imaging  where three-axial spleen diameters were measured. Blood samples (3 mL) were drawn during the last minute of each phase and analyzed directly for Hb in triplicate . In two participants, capillary samples were taken in triplicate, as catheter insertion was not possible, and Hb was analyzed with HemoCue .2) were measured continuously across all phases with a pulse oximeter with a finger probe , and mean arterial blood pressure (MAP) was measured with a photoplethysmometer via finger cuff placed on the right hand . The end-tidal oxygen concentration (EtO2) and the end-tidal carbon dioxide concentration (EtCO2) in the face mask were measured via a gas analyzer . To avoid hypoxic syncope in the participants, the minimum SpO2 accepted was set at 65%; if the value was reached, phase 2 was terminated, and the participant was moved to phase 3. Continuous data were stored in a computer, together with an event-marker, via a BioPac MH100A CE multichannel data acquisition system  and were analyzed further using AcqKnowledge Software . Breath-by-breath data were analyzed for respiratory frequency and inhaled and exhaled oxygen fraction.Heart rate (HR) and peripheral oxygen saturation  and sex on the dependent variables  were conducted by a two-way mixed ANOVA with Bonferroni corrections for repeated measures. Assessments of associations between dependent variables were conducted with bivariate correlation tests using Pearson product-moment correlation coefficient (r). Meaningfulness of effects was estimated by the standardized mean difference  computed as the mean difference divided by the pooled SD. ES was presented along with 95% confidence intervals [CI]. Statistically significant difference was accepted at P < 0.05. Interpretation of ES was based on three categories: 0.0–0.5 was considered a small effect, 0.6–1.1 was considered a medium effect, and ≥1.2 was considered a large effect.Participants served as their own controls. The normal distribution of all data was tested using the Shapiro-Wilk test. Paired Student's n indicated for the respective variable.All participants completed the study protocol. In one participant, due to an unusual shape of the spleen, measurements were not possible, and in another participant, Hb samples could not be obtained. These two participants were withdrawn from the respective analysis and entirely from the correlation analysis. Results are presented as mean ± standard deviation (SD) with 2, and HR). There was no two-way interaction effect between males and females on the dependent variables during the experimental intervention, showing that these variables change equally between sexes in response to the hypoxia. Results are therefore presented as mean (SD) for the whole group.Males had a larger vital capacity and a larger pre-exposure spleen volume than females but Hb baseline levels were similar between sexes . The two2 was 98.6 (17.2) mmHg at PRE, and it decreased to 46.5 (6.3) mmHg during EXP . The EtCO2 was 32.7 (4.3) mmHg at PRE, and it decreased to 28.3 (4.7) mmHg during EXP .The EtO2 was 96.4 (1.7)% during PRE, and it decreased to 74.7 (8.4)% by the end of EXP % , which was also higher compared with the PRE value  beats·min−1 in EXP , which was also lower compared with PRE  beats·minP = 0.005; ES = 0.62 [−0.16–1.39]), and during the last 3 min of EXP, the volume decreased to 201 (93) mL , which was the same as the PRE value  mL. After 3 min of EXP, the spleen contracted to 219 (75) mL  g·L−1, which was 4.1 g·L−1 higher than PRE  g·L−1  g·Lr = 0.692, P = 0.006), but no correlation between initial spleen volume and relative volume change  and also between SpO2 Δ and Hb Δ .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Syphacia obvelata 13(4.83%), Aspiculuris tetraptera 13(4.83%), Heterakis spumosa 12 (4.46%), Hymenolepis spp. 9(3.34%), H.diminuta 8(2.97%), Hymenolepis fusa 4(1.48%), Lutziella microacetabularae 4(1.48%) and Lutziella spp. 1 (0.37%). No significant difference (P < 0.4289) was found in prevalence of parasites among areas, crops, crop stages and sex of the host while adult rats were found more infected than sub-adults. S. obvelata and A. tetraptera were the most common species of helminths while Lutziella sp., 1 (0.37%) was found only in one host. Rattus rattus (the black rat) was regarded as the host of helminth parasites of zoonotic importance, therefore the hidden health hazards of this rodent species needed to be considered to prevent infectivity of humans. Current study was concluded that Rattus rattus harbored a wide variety of helminth parasites which shows a hidden risk to inhabitants of the region. Monitoring rats’ population in settle areas and educating the local community about the risk of rat borne parasitic diseases transmission through rats appears to be absolutely essential.Present study was conducted to get information on helminth parasites of zoonotic importance among the black rats of district Swat, Pakistan. Two hundred and sixty nine rats were captured from agricultural ecosystem of the district using live captured traps from 2011 to 2013. Captured rats were anesthetized and surveyed for the presence of ectoparasites, then were carefully dissected for investigation of endoparsites. Helminth parasites of 8 species were identified. Presence of parasite was noticed in 23.7% of sampled rats. The infection rates of sampled rats was given in order of their infectivity as  Rats anR. rattus, R. norvegicus and R.r. rufescens have been trapped from different regions of Pakistan and screened for helminth parasites. Protospirura muris in nematodes and H. nana (fraterna) in cestodes were the most prevalent helminths isolated from R. rattus, however, trematodes and acanthocephalans were the least groups of parasites reported from R. rattus in Pakistan.The helminth parasites of rats have been studied in many parts of the world with special emphasis on zoonotic parasites. In Pakistan R. rattus in Pakistan such as: Lahore, Pakistan  were transported to vertebrate Pest Control Institute, Pakistan Agricultural Research Centre, University Campus, Karachi, Pakistan for confirmation of species identification.2.3Each of the rat specimens was anaesthetized and weighted through Precisa balance model No.18220 Switzerland. Each of the rats was identified up to species level and the information concerned was gathered relating the sex and age by using valid identification keys . Chlorof2.4Collection and processing of the rat specimens was performed in accordance with the procedures approved by the Ethical Committee of Pakistan Agricultural Council Islamabad and University of Karachi.2.5Collected helminth parasites were identified by Yamaguti’s method . Inciden3Two hundred and sixty nine rats were caught. Eight species of helminthes were recorded from the gastrointestinal tract contents of the rats. Sixty four specimens of the reported helminthes were observed, resulting in a prevalence of 23.7% with single or multiple infection.3.1R. rattus) were caught in this study. Of the total 23.7% (n = 64/269) were found infected. Slightly higher prevalence rate 12.6% (34/269) was found in the rats of irrigated areas than in the rain-fed 11.1 (n = 30/269) of sampled rats. No statistical difference  was noted between the areas and that of parasites recorded  was investigated in the rats of rice fields followed by maize rain-fed 631% (n = 17/269) while the least infection was observed in the rats caught in potatoes 4.83% (n = 13/269). No statistical difference  was noted between the crops and that of parasites recorded .Table 2P3.3The rats captured during mature/harvesting period were found more infected 9.29% (n = 25/269) followed by flowering/fruiting stage 8.55% (n = 23/269) while the least 5.94% (n = 16/269) rate of infection was found in the rats caught at vegetative stage of the crops studied. No statistical difference  was noted between the crop stages and that of parasites recorded .Table 3P3.4Of the sampled rats males were more infected 15.9% (n = 43/269) than females 7.80% (n = 21/269). No statistical difference  was noted between the sex of the rats and that of parasites recorded .Table 4P3.5Adult rats were more infected 20.0% (n = 54/269) than sub-adults 3.71% (n = 10/269). No statistical difference  was noted between the age of the rats and that of parasites recorded .Table 5P3.6H. nana 4/269 (1.48% and H. diminuta 8/269 (2.97%).A total of 23.7% (64/269) of the sampled population was infected with helminth parasites, of them 4.46% (12/269) have been identified as zoonotic parasites. Detected helminth parasites of zoonotic importance comprised 2 species such as: 3.7Hymenolepis sp. 9/269 (3.34%); S. obvelata and H. spumosa 13/269 (4.83% each); A. tetraptera 12/269 (4.46%); L. microacetabularae 4/269 (1.48%) and Lutziella spp. 1/269 (0.37%) species of helminths with no medical importance were observed during microscopic examination of the fecal wet and stained smears  were detected as non-zoonotic species. d smears . Eggs we4R. rattus are scarce in Pakistan. In general, 23.7% of the rats were infected with at least one species of helminth parasites. Seeing the variation of parasites, eight species of helminths were found in R. rattus presently with A. tetraptera, S. obvelata, H. spumosa, H. diminuta, H. nana, H. fusa, L. microacetabularae and Lutziella sp. Findings of the current study were similar to some extent with those of previous studies in Pakistan.Studies on intestinal helminth parasites from P > 0.05). This might be owing to more activity or higher number of the males. The highest parasite infestation rate was detected in sampled rats of adults (20%) than in sub-adults (3.71%). Higher parasite infestation rate in adults can be due to higher activity in the habitat. Rats trapped in the harvesting stage of the crops were most infected (9.29%) than others. Crop wise rats from potatoes fields were slightly more infected (7.06%) as compared to others and 12.6% highest infection rat was noted in the rats captured in the irrigated areas.The highest prevalence of infection with helminth parasites was seen in males, although no significant difference was seen between male and female rats ; Moniliformis karachinensis n.sp. Acanthocephalis murinis n.sp. and Strobiocercus of Taenia taeniformis (Euparyphium lobata sp.n (T. taeniformis and Trichuris sp. (Lutztrema (Lutziella) microacetabularae Rohde, 1966  and that of H. diminuta (2.97%) were reported zoonotically important. In a study conducted in Kashan, the zoonotic parasites H. nana fraterna (10.8%), H. diminuta (4.2%), and Trichuris muris (1.7%) were identified in 120 rodents including Meriones persicus, M. libycus, Gerbillus nanus, M. musculus, R. norvegicus, R. rattus and Jaculus blanfordi  with the highest infestation rate in R. rattus. This is consistent with the results of (H. nana was reported 1.48% in prevalence in the present study. This parasite was reported in different murid in Pakistan as Rawalpindi-Islamabad (H. diminuta. It has been observed that prevalence rate of infection with H. diminuta is not similar in rats of at different localities.The most commonly recognized zoonotic parasite in the present study was sults of  in Karacsults of . H. nanaslamabad ; Faisalaslamabad . Currentslamabad  with 11.5Considering of rodents’ parasitic fauna in diverse regions of the world can bridge the gap of information about the possible potentials for transmission of zoonotic helminths to humans in the region. North-western Pakistan is a part with unanswered questions upon this issue. The study of parasitic fauna in rodents in North-Western of the country is an attracting subject to parasitologists interested to recognize the role of rodents in zoonotic infection transmission. The results suggested that the range of various parasites recovered from the fecal droppings of rats collected from agricultural habitats can make human habitation more vulnerable to parasitic zoonosis and may thus be of significant public health importance. Further studies are required to understand the cross species transmission among the rats and human beings.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Endodontic infection is a biofilm disease that is difficult to irradicate with current treatment protocols, and as such, persistent micro-organisms may lead to ongoing or recurrent disease. The potential for the use of enhanced filling materials to modify biofilm regrowth is a promising strategy. This current study aimed to evaluate the anti-biofilm efficacy of calcium silicate cements modified with chitosan. The development of mono-species and multi-species biofilms on ProRoot MTA, Biodentine and bovine dentine discs were explored using quantitative microbiology analysis. The effect on regrowth of biofilms was assessed following the addition of chitosan to each cement. In comparison to a dentine substrate, both materials did not show the ability to inhibit biofilm regrowth. Biodentine incorporated with chitosan displayed a dose-dependent reduction in multi-species biofilm regrowth, unlike MTA. Notably, interkingdom biofilms were shown to enhance bacterial tolerance in the presence of chitosan. This study demonstrates the potential to enhance the antimicrobial properties of Biodentine. The findings highlight the need for appropriate model systems when exploring antimicrobial properties of materials in vitro so that interspecies and interkingdom interactions that modify tolerance are not overlooked while still supporting the development of innovative materials. Endodontic infection, in the form of biofilms, have been visualised colonising necrotic and treated root canals ,2,3. It In response to the challenges faced in sealing the root canal space, calcium-silicate-based materials have grown in prominence. The first member of the calcium-silicate-based materials to be introduced was mineral trioxide aggregate (MTA). MTA principally consists of tricalcium silicate, dicalcium silicate, tricalcium aluminate and tetracalcium aluminoferrite with bismuth oxide powder added as a radiopacifying agent . Subsequ2 pastes used as endodontic medicaments [Chitosan is a modified natural carbohydrate polymer produced by deacetylation of chitin . Chitosaicaments . Other sicaments ,34. We aicaments ,35,36. TCandida and bacteria formed on controls (dentine discs) were approximately 1 × log10 less when compared with colonies formed on the tested materials, although this did not reach statistical significance for viable cells.Biofilm regrowth and composition on dentine, MTA and Biodentine discs was assessed using live/dead qPCR. It was evident that both MTA and Biodentine did not show an ability to inhibit biofilm regrowth of any of the biofilm models after 24 h of incubation, compared to the control dentine discs A–C. NotaC. albicans was present on a dentine substrate (4.28 × 105 compared to 1.48 × 106 CFE), while on Biodentine, a much smaller 1.4-fold increase was apparent with respect to bacterial numbers in the absence of C. albicans. Meanwhile, no change was noted for bacterial loads on an MTA substrate  for bacteria and C. albicans in each mixed biofilm are shown in When determining the potential effect of interkingdom interactions, there was approximately a 3.5-fold increase in viable bacteria when ubstrate . The resbacteria . These rC. albicans was grown as a mono-species biofilm, the live CFE following 2.5 wt% and 5 wt% chitosan incorporations was reduced by 83% and 71%, respectively, compared to the unmodified Biodentine. However, this reduction was not statistically significant  to 7.12 × 105 and 1.42 × 105 CFE/mL. The microbial reduction was dose-dependent, and the greatest reduction observed at 5% chitosan was significant (** p < 0.01). A decrease in bacterial load (p > 0.05) by 67% was also observed at 5% compared to the control (bovine dentine)  B.6 CFE/mL in the unaltered material to 3.6 × 106 and 7.8 × 105 CFE/mL in the chitosan-treated material  was significant in the 5% added material (** p < 0.01). A decrease in the live CFE/mL of the mixed biofilm (p > 0.05) by ~55% was also observed at 5% compared to the control (bovine dentine). Of interest, the addition of chitosan preferentially targeted C. albicans in mixed-species biofilms and a concomitant significant (** p < 0.01) inhibition of regrowth of four-species biofilms (p > 0.05) when C. albicans was present  at 5% chitosan (4B).For the four-species biofilm model, adding 2.5% and 5% chitosan to Biodentine was able to effectively reduce live CFE by 56% and 90.5%, respectively, compared to the unaltered Biodentine C. The rebiofilms . In cont present . HoweverC. albicans grown on Biodentine with 2.5% CNPs was decreased by approximately 2-fold when the three bacterial species were added. On the other hand, an approximate 12-fold reduction was noted for C. albicans on Biodentine with 5% CNPs (8.39 × 105 compared to 6.79 × 104), compared to C. albicans mono-species biofilm, in the presence of bacteria  of chitosan, which subsequently favours electrostatic interactions between the formed positively charged chitosan molecules and negative residues at biological sites [It is clear from our findings that ProRoot MTA and Biodentine exhibited different antimicrobial behaviours when chitosan was added. One mechanism by which calcium silicate cements have been said to exert an antimicrobial effect is through modifying environmental pH. It has been postulated that increased alkalinity, resulting from the release of calcium hydroxide upon setting of MTA preparations and its subsequent dissociation into calcium and hydroxide ions, may be responsible for any observed antimicrobial action ,58. To uolutions . Other solutions ,61. It hal sites ,62. AccoCandida albicans SC5314 (ATCC MYA-2876), Streptococcus gordonii (ATCC 35105), Porphyromonas gingivalis (ATCC 33277) and Fusobacterium nucleatum (ATCC 10953) were constructed. C. albicans was grown on Sabouraud’s dextrose agar (SAB) at 30 °C aerobically for 24–48 h; S. gordonii was grown on Columbia agar supplemented with 5% horse blood (CBA) at 37 °C in 5% CO2 for 24 h. The other two anaerobic organisms were maintained on fastidious anaerobic agar (FAA) plates containing 5% defibrinated horse blood at 37 °C in an anaerobic chamber  with an atmosphere of 85% N2, 10% CO2 and 5% H2 for 24–48 h. All agar bases were supplied by Oxoid, UK. Standardised cultures of C. albicans and bacteria  standardised at 1 × 108 CFU/mL were first diluted to 1 × 106 CFU/mL and 1 × 107 CFU/mL in culture broth, respectively. The broth consisted of 1:1 mixture of Roswell Park Memorial Institute-1640 (RPMI) with Todd Hewitt Broth (THB) supplemented with 0.01 mg/mL hemin and 2 µg/mL menadione. Four mixed-species biofilms were grown in pre-sterilised polystyrene 24-well flat-bottom plates  for 24 h in 5% CO2 at 37 °C. Two derived models were also used in parallel, one of which contained bacterial species only  and one contained C. albicans only. This was to assess the importance of C. albicans in maintaining biofilm tolerance or otherwise.An established interkingdom endodontic biofilm model, previously described by our group, was used throughout this study . BrieflyBovine dentine was used, which is an appropriate substitute for human dentine due to its availability and its great similarity to the human dentine . Bovine To investigate the effect of chitosan on the antimicrobial properties of tested materials, chitosan was incorporated into MTA and Biodentine. Medium molecular weight Chitosan ) was used throughout this study. Briefly, chitosan powder was disinfected using UV for 15 min. Following this, the chitosan powder was incorporated into the Biodentine™ and MTA powders using two different concentrations (2.5 wt% and 5 wt%) and resultant powder was mixed with the manufacturer liquid component. Materials were then placed into aseptic moulds and allowed to set in a moist atmosphere at 37 °C for 3 h and 1 h, respectively.C. albicans only) biofilms were grown in RPMI/THB in 24-well plates for 24 h, as previously described. After incubation, the spent biofilm media was discarded, and biofilms were washed with PBS, mechanically disrupted in 1 mL of media and diluted to 1:10 in fresh RPMI/THB and then inoculated on MTA and Biodentine discs  into 24-well plates. Mechanical disruption of the biofilms serves the purpose of simulating mechanical debridement of the root canal. Plates were then incubated for an additional 24 h in 5% CO2 at 37 °C to allow biofilm growth.The antimicrobial ability of ProRoot MTA and Biodentine was assessed against biofilm regrowth on the materials placed in a 24-well plate. Bovine dentine discs were used as positive controls. Four-species, three-species (bacteria only) and mono-species  for DNA extraction. The composition of the regrown biofilms on dentine, MTA and Biodentine discs was assessed using live/dead qPCR, a technique that uses propidium monoazide (PMA), a DNA-intercalating dye, to differentiate biofilm viable and dead microorganisms. Samples were prepared as previously described by . Briefly2 incubator at 37 °C. Measurement of pH change of the storage solution was taken using a calibrated pH meter  at the following time points: 1, 3 and 24 h. The pH measurement of the manufacturer Biodentine liquid was also taken.Material discs 1 mm in thickness with 7 mm diameter were prepared of Biodentine and ProRoot MTA. Chitosan powder was incorporated into both materials at concentration of 2.5% and 5% by weight. The materials were allowed to set in a moist atmosphere at 37 °C for 3 h and 1 h, respectively. Each disc was then placed in a bijoux tube containing 3 mL sterile water. All samples were kept in a 5% COp values for non-parametric multiple comparisons . Differences were considered statistically significant when p < 0.05.Data distribution, graphs and statistical analysis were performed using GraphPad Prism version 8 . A D’Agostino-Pearson omnibus normality test was used before analysis to assess data distributions. Kruskal–Wallis with Dunn’s tests were used to determine the The present study demonstrates limited intrinsic antimicrobial abilities for the tested calcium silicate cements. This contrasts with previous studies but is likely a result of the use of multi-species biofilm models in the present study. This further highlights the need for the use of appropriate model systems in assessment of therapeutics to account for the often synergistic/protective relationships that exist in complex microbiological systems. The present study highlights the potential to enhance the biological properties of an existing calcium silicate cement, which may serve to reduce the likelihood of persistence or re-establishment of infections within the treated root canal space. Although some insight may have been gained into the mechanism through improvements in antimicrobial effects, further work is needed to fully elucidate this mechanism and understand the contribution of chitosan. Clearly, the modification of Biodentine to enhance antimicrobial properties has the potential to modify physical properties which may affect clinical handling and appropriateness. Further studies into material characteristics will be required to assess the use of chitosan as an active antimicrobial supplement in calcium silicate cements.


Staphylococcus aureus (MRSA) have reached epidemic proportions globally. Therefore, there is an urgent need for a continuous supply of antibiotics to combat the problem. In this study, bacteria initially identified as species belonging to the Bacillus amyloliquefaciens operational group were re-identified based on the housekeeping gene, gyrB. Cell-free supernatants (CFS) from the strains were used for antimicrobial tests using the agar well diffusion assay against MRSA and various types of pathogenic bacteria. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and physicochemical characteristics of the CFS were determined. Based on gyrB sequence analysis, five strains  were identified as Bacillus velezensis. The CFS of all B. velezensis strains showed broad inhibitory activities against Gram-negative and -positive as well as MRSA strains. Strain PD9 against MRSA ATCC 33742 was chosen for further analysis as it showed the biggest zone of inhibition (21.0 ± 0.4 mm). The MIC and MBC values obtained were 125 μl/ml. The crude antimicrobial extract showed bactericidal activity and was stable at various temperatures (40–80°C), pH (4–12), surfactants  and metal ions  when tested. However, the crude extract was not stable when treated with proteinase K. All these properties resembled the characteristics of peptides. The antimicrobial compound from the selected strain was purified by using solvent extraction method and silica gel column chromatography. The purified compound was subjected to High Performance Liquid Chromatography which resulted in a single peak of the anti-MRSA compound being detected. The molecular weight of the anti-MRSA compound was determined by using SDS-PAGE and zymogram. The size of the purified antimicrobial peptide was approximately ~ 5 kDa. The antimicrobial peptide produced from B. velezensis strain PD9 is a promising alternative to combat the spread of MRSA infections in the future.Infections caused by methicillin-resistant  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.  . The plates were incubated at 37°C for 16 to 24 h. The growth of the MRSA was observed; growth of MRSA was interpreted as bacteriostatic, while no growth meant bactericidal.in vitro growth inhibition assay was performed as described by Cao et al. [3 cfu/ml, 104 cfu/ml, 105 cfu/ml and 1 ml of MRSA ATCC 33742 suspension with final cell density 103 cfu/ml were independently inoculated into 98 ml of nutrient broth. The mixture was then incubated at 37°C with shaking at 200 rpm. Shake flask with MRSA ATCC 33742 only was used as control. Cell growth of MRSA ATCC 33742 was measured using mannitol salt agar at 24 h interval by using the serial dilution-agar plate method.The o et al.  with sli7 cfu/ml was grown in nutrient broth at 37°C with shaking at 200 rpm for 20 h. CFS obtained by centrifugation of bacterial culture at 8 000 x g for 10 min was used in each physiochemical analysis which was performed in triplicates.Strain PD9 at final cell density of 10Thermal stability assay was performed as described by Lee et al.  with sli2O at ratio 1:1 was used as a reference.Influence of pH on the activity and stability of strain PD9 CFS was determined as described by Lee et al.  with sli2O was used as a reference.The enzyme stability assay was performed as described by Shekh and Roy  with sli2, NaCI2, CuSO4, NiSO4, KCI, ZnNO3 at final concentration 1 mg/ml [The strain PD9 CFS were tested with surfactant: sodium dodecyl sulphate (SDS), Tween 20, Tween 80 and Triton X-100 at final concentration of 1% (v/v) and metal salts: MgCI 1 mg/ml . Treatedg. The organic layer formed was collected and evaporated using a rotary evaporator. Antimicrobial activity of the extract obtained was tested against MRSA ATCC 33742 by using disc diffusion assay. The extract obtained was subjected to silica gel column chromatography. The 1-butanol extract was prepared with minimum amount of dichloromethane (DCM). The sample was loaded to a silica gel (G60) column  and eluted by eluent with various ratios of DCM and methanol . About 25–26 fractions were collected. The disc diffusion method was adopted to evaluate the antimicrobial activities of all the fractions eluted against MRSA ATCC 33742. The active fraction was evaporated and diluted in glycine-NaOH buffer for SDS-PAGE analysis.Solvent extraction method was performed as described by Bordoloi et al  with sli7 cfu/ml of MRSA ATCC 33742. The plate was incubated at 37°C for 24 h and band with antibacterial activity was observed. The other part of the gel was stained using a staining solution made up of 45% (v/v) methanol, 10% (v/v) acetic acid and 1% (w/v) Coomassie Brilliant Blue R250 for 10–15 min. The protein bands which appeared on the gel was observed.Molecular weight of the anti-MRSA metabolite was determined by using tricine SDS-PAGE with stacking gel pH 6.8 (4%), overlaid 1-cm spacer gel pH 8.5 (10%) and separating gel pH 8.5 (16%) as described by Schägger  with sli2O and 5% acetonitrile  and B (100% acetonitrile). The flow rate of the mobile phase was 1 ml min-1. The elution gradient was as follows: 0% solvent B for 3 minutes, 0–40% solvent B for 5 min, 40–60% solvent B for 5 min, 60–80% solvent B for 5 min, 80–100% solvent B for 5 min and then back to 100% solution A. The purity of the peptide was determined by the number of peaks which appeared on the chromatogram. The peak was collected and tested against MRSA ATCC 33742.The active fraction obtained from the silica gel column chromatography was introduced into a reverse phase high performance liquid chromatography (RP-HPLC) system  using C18 reverse phase column . The separation was performed by the gradient method using two solvents: A  test was used  . The resgyrB was amplified from the bacterial genomic DNA for reassessment of bacterial strain identity . The amplified fragments were sequenced and compared to the sequences available in the GenBank database. The gyrB sequence of the bacterial strains showed ≥ 98% similarity to the closest known species in the database. The gyrB sequences were used for the phylogenetic analysis. The results demonstrated that strains PD9, B7, PU1, BP1 and L9 belonged to the B. velezensis clade and thus were closely related to B. velezensis. All the bacterial strains gyrB sequences had been deposited in the NCBI database under the accession numbers: MT338276 (B. velezensis PD9), MT338277 (B. velezensis B7), MT338278 (B. velezensis PU1), MT338279 (B. velezensis BP1) and MT338280 (B. velezensis L9). All the bacterial strains were subjected to antimicrobial activity screenings.An approximately 1.2-kbp partial fragment of B. velezensis strains against MRSA strains were determined (Table 1). All strains of B. velezensis showed inhibitory activities against all the MRSA strains tested including the MSSA strain ATCC 12228. Overall, strains ATCC 33741, ATCC 33742 and ATCC 700699 were highly inhibited by all B. velezensis strains. The inhibitory activities of all the B. velezensis strains observed against MSSA strain ATCC 12228 were quite similar to those with the tested MRSA strains. Strains PD9 and BP1 showed high inhibition activities against all tested MRSA strains and MSSA strain ATCC 12228. The biggest zone of inhibition recorded was from strain PD9 against MRSA 33742 (21.0 ± 0.4 mm) and the lowest zones of inhibition recorded were from strains PU1 and B7 against MRSA ATCC 25922 (11.0 ± 0.5 mm). It was noted that there was no significant difference in the inhibitory effects of the B. velezensis strains towards different MRSA strains. Therefore, a study on the activity of strain PD9 against MRSA ATCC 33742 was chosen for further analyses.The antagonistic activities of the B. velezensis strains were also used for antimicrobial screenings against several pathogenic bacteria including Gram-positive, and Gram-negative bacteria (Table 1). All B. velezensis strains showed broad-spectrum inhibitions across different groups of pathogenic bacteria. Strains PD9 and PU1 were able to inhibit the growth of Bacillus cereus, Vibrio parahaemolyticus, V. alginolyticus, Aeromonas hydrophila and Alcaligenes faecalis. Strains B7, BP1 and L9 showed similar patterns of inhibition to the strains PD9 and PU1, except against Alcaligenes faecalis.The cell-free supernatant of 1 to 23. Growths of MRSA ATCC 33742 were detected in wells with CFS of 24 to 210 dilution factors . Therefore, the MIC values for both treatments were at 23 dilution factor (125 μl/ml). After 24 h of incubation, no MRSA ATCC 33742 growth was observed , indicating the bactericidal activity of the antimicrobial compound present in the CFS of strain PD9. The MBC values for both treatments were found at 23 dilution factor (125 μl/ml).The MIC value of CFS from (i) 20 h-grown strain PD9 culture and (ii) 20 h-grown strain PD9 and MRSA ATCC 33472 (co-culture) were determined. In both samples, no growth of MRSA ATCC 33742 was observed in the wells with CFS dilution factor of 23 to 105 cfu/ml . The results indicated that the higher density of strain PD9 inoculated in the culture, the higher the reduction of MRSA ATCC 33742 growth. Also, the longer the incubation, the lower the growth of MRSA ATCC 33742. The logarithm of MRSA ATCC 33742 cell densities at 103,104 and 105 cfu/ml was reduced to 31.05, 49.87 and 53.60%, respectively as compared to the control (100%) after 96 h of incubation . Based on the results, strain PD9 could reduce the growth of MRSA ATCC 33742 in co-culture growth condition.The growth inhibition effect of strain PD9 on MRSA ATCC 33742 was determined. The growth of MRSA ATCC 33742 was reduced as compared to the control when strain PD9 was inoculated at final cell densities of 10B. velezensis strain PD9 CFS was not affected at temperatures of 40 to 80°C. There was no significant difference (P<0.05) in the inhibitory activity of the strain PD9 CFS to MRSA ATCC 33742 when incubated at different temperatures for 30 min . However, the inhibitory activity slightly declined at 100°C. There was a 15% reduction as compared to the control (untreated). The result showed that the antimicrobial compound present in the CFS was highly stable in a wide range of temperatures. The retained activity at 80°C indicated that the compound was thermally stable.The antimicrobial activity of the Fig 4). However, the relative activity was higher at pH 8–12. The highest activity was observed when the CFS was treated at pH 10 and 11. The results showed that the antimicrobial compound present in CFS was stable at various pH values and was not affected at acidic and neutral conditions. Interestingly, inhibitory activity was enhanced at alkaline condition.The strain PD9 CFS was treated with different pH values of buffer at 1:1 ratio. Based on the clearing zone measured, there was no significant difference in the inhibitory activities at pH 4–7. The relative zones of inhibition values were similar to the untreated sample . The inhibitory activity declined up to 8.92, 15.32, 33.33 and 42.34% after 0.5, 1.0, 1.5 and 2.0 h treatments, respectively.Treatment with proteinase K was done to test the stability of the strain PD9 CFS to the enzyme. Based on the zone of inhibition measured, the activity was not affected when the CFS was treated with proteinase K at 37°C. However, the inhibitory activity slightly declined when CFS was treated at 55°C for 0.5 to 2.0 h (Table 2). However, there were slight decline of the inhibitory activities of CFS treated with Triton X-100 and with MgCl2. Triton X-100 and MgCl2 reduced the inhibitory activities of CFS by 8.35 and 6.48%, respectively.The strain PD9 CFS was treated independently with different types of surfactants and metal ions. Based on the clearing zones observed, there was no significant difference (P<0.05) in the inhibitory activities of the CFS treated with a majority of the tested surfactants and metal ions . The 1-butanol extract obtained were subjected to silica gel column chromatography. The antimicrobial activities of the eluted fractions from different ratios of DCM:MeOH  were tested against MRSA ATCC 33742. The fractions eluted from the eluents with ratios of DCM:MeOH  exerted antimicrobial activities . Only the eluent with ratio of DCM:MeOH (1:1) did not show any inhibition activity. The eluent with ratio 5:1 showed the highest inhibition activity and three active fractions were collected. Among the three fractions, fraction number 24 showed the biggest zone of inhibition (19.8±0.5 mm). The active fractions obtained from the silica gel purified product were used for HPLC analysis. A single peak was observed on the chromatogram at retention time 13.932 min . The results indicated that the purified product only contained a single anti-MRSA compound. The eluted peak showed inhibitory activity against MRSA ATCC 33742 (result not shown).The extracellular metabolite of strain PD9 was extracted using 1-butanol as the organic solvent. The antimicrobial activities of the 1-butanol extract before and after evaporation were tested against MRSA ATCC 33742. The zones of inhibition observed before and after evaporation were 12.7±0.8 mm and 26.3±0.3 mm, respectively . However, the inhibitory effects were slightly different across the strains. The results indicated that the antimicrobial compound produced could be used as a universal therapeutic agent against different strains of MRSA. This inhibitory activity against MRSA was further explored as MRSA strains are considered as one of the most common antibiotic resistant pathogens in the world [B. velezensis strains were subjected to the antimicrobial activity assay against various Gram-positive and Gram-negative bacteria. All strains of B. velezensis especially strains PD9, B7 and PU1 showed a broad-spectrum of inhibition across the different types of bacteria tested. This could be due to the production of diverse types of antimicrobial compounds. As had been reported, B. velezensis encoded high genetic capacity for antimicrobial compound production such as of polyketides , cyclic lipopeptides  and others [All the strains of he world . In addid others .Fig 2). In another related study, bacterial supernatant from Lactobacillus strains isolated from commercial yogurt showed MIC values of 50 to 128 μl/ml against several pathogens including S. aureus, S. typhii and E. coli [L. casei against P. aeruginosa, where both values reported were 62.5 μl/ml (24 dilution factor) [B. velezensis against MRSA. Based on the result obtained, the growth of MRSA ATCC 33742 was lower  when MRSA ATCC 33742 was treated with CFS from strain PD9 + MRSA ATCC 33742 mix cultures. These results indicated that, in the presence of the pathogen (MRSA ATCC 33742), strain PD9 produced more antimicrobial metabolites. The induction of antimicrobial metabolite in the presence of competing species or pathogens was also observed in related studies of B. subtilis B38 against MRSA [E. coli and S. aureus [B. amyloliquefaciens MMS-50, antimicrobial peptide from B. subtilis URID 12.1 and anti-MRSA compound from marine Streptomyces exhibited bactericidal activities against MRSA [For further evaluation, the inhibitory activity of strain PD9 against MRSA ATCC 33742 was used as a reference. MIC was determined by using the CFS of strain PD9 culture and CFS was obtained from mixed strain PD9 and MRSA ATCC 33742 cultures. The MIC and MBC values obtained for both treatments were at 125 μl/ml ( E. coli . Similar factor) . Howevernst MRSA  and soil. aureus . From thnst MRSA , 49, 50.in vitro effect of strain PD9 on the growth inhibition of MRSA ATCC 33742 was determined. A significant reduction of MRSA ATCC 33742 was observed after 48 h of incubation after the inoculation of strain PD9 at high final cell density (104 and 105 cfu/ml). This could be due to the higher amounts of antimicrobial metabolite produced by the higher cell density [The  density .B. velezensis BS2, amysin from B. amyloliquefaciens SP-1-13LM and an antimicrobial peptide from B. firmus H2O-1 [B. subtilis strains URID 12.1 and RLID 12.1 [Streptococcus thermophilus [For further understanding of the characteristics and more information on the expected antimicrobial metabolite present in strain PD9 CFS, physicochemical analyses were performed. The effects of temperature, pH, proteinase K, metal ions and surfactant treatments were tested. The antimicrobial compounds were stable at high temperatures as well as at acidic and alkaline pH. The property of thermal stability would be useful during industrial process handling. Heat stable property was also reported in cyntibiotics (class II bacteriocins). These are the small peptides (<10 kDa) containing one or more disulfides bond that are necessary for their activities . In thisus H2O-1 , 51, 52.LID 12.1 , 49 as wmophilus . Based oB. subtilis 168 [B. cereus BC7 [Candida intermedia LAMAP1790 [B. velezensis which exhibits anti-MRSA activity. Another anti-MRSA metabolite produced by B. velezensis was identified as an aminoglycoside with a molecular weight around 418.32 Da [In this study, the anti-MRSA metabolite was successfully extracted using 1-butanol as the organic solvent. Higher inhibitory activity (bigger zone of inhibition) of the extract was observed after the evaporation process. The antimicrobial metabolite was purified using silica gel column chromatography. To further evaluate the purity of the silica gel purified product, HPLC analysis was performed. A single peak appeared on the chromatogram which indicated the success of using silica gel column chromatography for the purification process of the anti-MRSA peptide. The method successfully eliminated other unwanted metabolites or proteins in the extracts. The size of the purified anti-MRSA peptide obtained was approximately ~5 kDa. Although, there are reports on antimicrobial peptides with size of ~5 kDa isolated from ilis 168 , B. cerereus BC7  and CandAMAP1790 , to the 18.32 Da .B. velezensis strains from stingless bee products are important bacteria that can produce antimicrobial metabolites with inhibitory activities against MRSA strains and various types of pathogenic bacteria. B. velezensis strain PD9 produces an anti-MRSA metabolite with high thermal and pH stabilities. The present characterizations reveal the interesting properties of the antimicrobial peptide which show its promising potential for controlling pathogenic MRSA strains. Further efforts on identification, structural elucidation and characterization as well as mechanism studies are needed to allow for clinical applications of the anti-MRSA peptide from this bacterial strain.S1 FigBacillus velezensis PD9.AMP is the purified antimicrobial peptide from (DOCX)Click here for additional data file.S2 FigBacillus velezensis PD9. Halo zone was observed on the antimicrobial peptide band indicating MRSA growth inhibition.AMP is the purified antimicrobial peptide from (DOCX)Click here for additional data file.


In this study, we used daily air quality monitoring data for 28 air pollution transmission channel cities in the Beijing–Tianjin–Hebei region during 2014–2019 to quantify the relative contributions of meteorological factors on spatiotemporal variation in PM2.5 concentration by combining time series and spatial perspectives. The results show that annual mean PM2.5 concentration significantly decreased in 24 of the channel cities from 2014 to 2019, but they all still exceeded the Grade II Chinese Ambient Air Quality Standards (35 μg m−3) in 2019. PM2.5 concentrations exhibited clear spatial agglomeration in the most polluted season, and their spatial pattern changed slightly over time. Meteorological variables accounted for 31.96% of the temporal variation in PM2.5 concentration among the 28 cities during the study period, with minimum temperature and average relative humidity as the most critical factors. Spatially, atmospheric pressure and maximum temperature played a key role in the distribution of PM2.5 concentration in spring and summer, whereas the effect of sunshine hours increased greatly in autumn and winter. These findings highlight the importance of future clean air policy making, but also provide a theoretical support for precise forecasting and prevention of PM2.5 pollution.Understanding the spatiotemporal characteristics of PM It was not until the air quality standard was published that real-time hourly PM2.5 and PM10 concentration data for the major cities were collected and released online, which is essential for providing detailed information about particulate matter pollution and helpful for understanding how local air quality has changed over time.Meteorological conditions and synoptic circulation patterns exert important influences on PMariation ,32. ReceH region . Gonzalehe 2050s . Howeverhe 2050s . Statistribution . Howeverion [2.5 ,38. On t complex ,39. PM2. heating  and temp heating  and chemeneities ,41. Ther2.5 concentration during 2014–2019 in the 28 cities of the air pollution transmission channel in the BTH region of China at seasonal and annual scales. Then we explored the spatial heterogeneity of the relationship between PM2.5 concentration and meteorological variables based on geographical weight regression (GWR) analysis. Finally, we performed partial correlation and multiple linear regression (MLR) analyses to quantify PM2.5 concentration response to meteorological variables and their contributions to the PM2.5 concentration variation from a time-series perspective. The findings of this study will contribute to a more comprehensive understanding of the influence of meteorological variables on PM2.5 concentration and will provide strong theoretical support for precise forecasting and prevention of PM2.5 pollution in the BTH region.In this study, we examined reductions in PM2 [The study area includes 28 air pollution transmission channel cities of the BTH region, covering an area of 275,000 km2 ,39. The 10 and PM2.5 concentration data (μgm−3) between 1 March 2014 and 28 February 2019 for the 28 cities in the air pollution transmission channel of the BTH region (Jincheng data started from 1 March 2015) were obtained from an online database . This dataset was constructed by averaging hourly concentration data for all national air quality monitoring sites within each city listed on the official website of the China National Environmental Monitoring Center [2.5 and PM10 concentration obtained by the continuous automated monitoring system based on the micro-oscillating balance method and the β absorption method. The dataset contained very few missing records  and very few zero values for PM10 concentration. We interpolated missing values from the mean values of the two closest days to maintain time series data continuity and comparability. In addition, although PM2.5 has been paid special concern, it was not systematically observed and reported for most cities in China until 2014. Meanwhile, it was unreasonable to effectively consider the relative contributions of different particles to local air quality and the composition characteristics of particulate matter based only on fine particles (and without consideration of coarse particles). Therefore, we removed zero values of PM10 concentration and the corresponding PM2.5 concentration values from the time series in order to calculate the PM2.5/PM10 ratio, which provided crucial information related to particle origin and allowed retrospective prediction of PM2.5 concentration without direct PM2.5 measurements.Daily PMg Center . In accomaxT, °C) and minimum temperature , average atmospheric pressure , sunshine hours , average relative humidity , maximum wind speed  and wind direction (WD) for the 28 meteorological stations were collected from a Chinese meteorological data sharing service system . The 28 meteorological stations were generally located at the center of each city  means that the city and surrounding cities have high PM2.5 concentrations; High–Low (H–L) represents that the city has a high PM2.5 concentration, but those of surrounding cities are low; Low–High (L–H) shows that the city has a low PM2.5 concentration, but those surrounding cities are high; Low–Low (L–L) means that both the city and surrounding cities have low PM2.5 concentrations; Not significant (NS) indicates no significant difference in PM2.5 concentration among cities. Global Moran’s I and Local Moran’s I statistical tests used a z-test at 95% confidence level. Finally, the coefficient of variation (CV) was calculated to investigate fluctuation in the temporal variability of PM2.5 concentration. We also explored the spatiotemporal variation in the PM2.5/PM10 ratios using the same methods.The variation in PMsoftware ,42. The 2.5 concentration and meteorological factors for each city at seasonal and annual scales; this method has been successfully applied in previous environmental studies to remove covariate effects among multiple influential factors. Next, we performed multiple linear regression analysis of daily PM2.5 concentration and meteorological factors at seasonal and annual scales for each city, and the explanation ability of meteorological factors on PM2.5 variability was evaluated using the coefficient of determination (R2) [p < 0.05.Partial correlation analysis was performed to determine the relationships among temporal variations in daily PMion (R2) ,43. All 2.5 concentration based on site data for the 28 cities. First, we normalized mean seasonal and annual PM2.5 concentration and meteorological variable data during 2014–2019 for each city; we then conducted the geographically weighted regression analysis using the ArcGIS 10.4.1 software. The geographically weighted regression was run for an observation point by using a spatial kernel that centered on the point and weighted other observation points by a distance decay function. In this study, the Gaussian model was selected as a weighting function, and the corrected Akaike’s information criteria (AICc) was used to obtain the optimal bandwidth.The geographically weighted regression was developed to deal with this non-stationarity, which allows relationships between independent and dependent variables to vary spatially by producing a set of local parameters to reveal spatial relationships by assuming heterogeneous influence of the same factor on dependent variables in different spatial units ,45. We p2.5 concentrations were observed in Xingtai (83.69 μg m−3), Handan (80.98 μg m−3), Shijiazhuang (84.16 μg m−3) and Baoding (85.35 μg m−3), and the lowest PM2.5 concentration was observed in Shanxi Province, with a minimum of 56.77 μg m−3 in Yangquan (2.5 concentration exceeded the CAAQS (GB3095-2012) Grade II standard of 35 μg m−3. Mean PM2.5 concentration showed wide seasonal variation that was similar among all cities. The highest pollution levels were observed in winter and the lowest values in summer. However, the seasonal fluctuation range seriously differed among cities. For instance, Beijing, Tianjin, Tangshan, Langfang, Binzhou, Zibo and Yangquan showed lower CV values, whereas Baoding, Anyang, Puyang and Kaifeng had larger CV values (>40%). CV values were higher in winter and lower in spring. From 2014 to 2019, significant reductions in annual mean PM2.5 concentration occurred across 24 of the 28 channel cities , with a mean rate of −7.14 μg m−3 year−1  Grade II standard in all cities, and PM2.5 concentration variation showed significant seasonal and spatial distribution differences among all 28 cities. Compared with other seasons, PM2.5 concentration in spring and summer exhibited the clearest obviously decreasing trend, with significant reductions in 22 and 26 cities, respectively, which were mainly located in the Hebei and Shandong Provinces. Significant downward trends were also observed in 20 cities in autumn and only 4 cities in winter.During 2014–2019, the highest annual mean PMYangquan . In all 3 year−1 . About h2.5 concentrations during 2014–2019 had a Global Moran’s I of −0.05 (p = 0.95) in spring, 0.09 (p = 0.35) in summer, 0.20 (p = 0.08) in autumn, and 0.30 (p = 0.01) in winter, indicating spatial patterns in winter across all channel cities. Local spatial patterns of average PM2.5 concentrations in winter displayed clear spatial agglomeration characteristics, with H–H spatial clusters from Dezhou in 2015, to Handan Anyang, Hebi and Puyang in 2019. The L–L spatial clusters appeared in Beijing and Tianjin and persisted thereafter , Tianjin (0.62) and Binzhou (0.61) and lower ratios observed in Taiyuan (0.49), Yangquan (0.51) and Jincheng (0.50), all of which are located in Shanxi Province. By contrast, despite similar spatial distributions of mean PM2.5/PM10 ratios in each season, clear seasonal differences were observed. Among the 28 cities, 27 cities displayed their highest mean PM2.5/PM10 ratios in winter (excluding Hengshui), whereas almost all cities showed their lowest mean ratios in spring. From 2014 to 2019, the annual mean PM2.5/PM10 ratios in most cities of Hebei and Shandong Provinces exhibited a clearly decreasing trend, with significant decreases in Langfang, Shijiazhuang, Dezhou and Heze, whereas most of the other cities fluctuated over time , Zhengzhou (p = 0.03) and Kaifeng (p = 0.01).Annual mean PM of 0.56 . PM2.5/Pver time . Compare2.5/PM10 ratios demonstrated that the Global Moran’s I ranged from 0.005 to 0.15, with no significant differences at the seasonal or annual scale, indicating that the PM2.5/PM10 ratio of each city did not influence those of the other cities in aggregate. However, local spatial autocorrelation analysis exhibited L–L spatial clusters of mean PM2.5/PM10 ratios in Taiyuan and Yangquan during spring and winter and H–H spatial clusters in Tianjin in summer and in both Tianjin and Beijing in autumn  and WS (except in Yangquan); it was positively correlated with maxT in 23 of the 28 cities and with H throughout the study period. By comparison, the correlation between WD and PM2.5 concentration was positive over the northeastern part of the study area , but negative over the southwestern region . Generally, minT and H  were critical factors affecting variation in PM2.5 concentration throughout the year.Partial correlation analysis results for PM2.5 concentration and meteorological variables were generally similar to those at the annual scale, with some exceptions  were much stronger than those at the annual scale in most cities, whereas those correlations between PM2.5 concentration and minT became slightly weaker. In spring, minT and H were the two most important factors affecting the variation in PM2.5 concentration in most cities, whereas the relationship between AP and PM2.5 concentration was significant only in seven cities. Among the four seasons, PM2.5 concentration showed the weakest correlation with AP and H in summer and was more strongly correlated with maxT and SH for more cities in summer than in spring and autumn. In autumn, PM2.5 concentration was significantly negatively correlated with minT in 27 cities, with AP in 25 cities and with H in 24 cities; minT had the greatest influence on PM2.5 concentration variation. Interestingly, partial correlation analysis also showed that PM2.5 concentration was positively related to WD in winter in eastern cities such as Tianjin, Cangzhou, Xingtai, Binzhou, Dezhou, Zibo, Jinan and Jining, but positively correlated with WD in summer in some northern cities .Seasonal correlations between PMceptions . In wint2) of the variation in PM2.5 concentration among the 28 cities during the study period; Shijiazhuang had the highest R2 value (44.46%), and Jinan had the lowest value (21.30%). At the seasonal scale, the mean R2 was highest in winter (0.43), with the highest R2 values in Beijing, Tianjin and the cities of Hebei and Shandong Provinces, whereas R2 was lowest in summer, with a mean value of 0.13 . WS and WD were negatively correlated with PM2.5 concentration in all cities except Tangshan, but had a smaller impact on PM2.5 concentration in Beijing, Tianjin, Langfang, Hengshui, Xingtai, Handan, Liaocheng, Jining, Heze, Anyang, Hebi and Puyang . Correlations between meteorological factors and PM2.5 concentration were generally similar in winter and autumn  in the BTH region during 2014–2019. Similar trends have been reported in previous studies. For example, average annual PM2.5 concentration was reported to have decreased throughout the BTH region during 2013–2016 [2.5 concentration can be attributed mainly to the Action Plan on Prevention and Control of Air Pollution in 2013 and the Air Pollution Prevention and Control Program in Beijing, Tianjin, Hebei and surrounding areas in 2017. However, it was noteworthy that the rate of decline in PM2.5 concentration slowed dramatically after 2017, even fluctuating upward in some cities (2.5 concentration of all channel cities in the BTH region remained much higher than the Grade II standard of CAAQS in 2019, which indicates that further measures are still strongly needed despite the improvements attributed to control strategies implemented within the past decade.In this study, we found that annual mean PM013–2016  and 2013013–2016  and to h013–2016 ,36. These cities . We also2.5 concentration exhibits a distinct seasonal cycle, with less severe PM2.5 pollution in summer and greater pollution in winter, probably due to the combined impact of unfavorable weather conditions and coal and biomass combustion for residential heating [2.5/PM10 ratio also showed a seasonal variation, decreasing in spring and peaking in winter, which can be attributed to increasing fuel consumption [2.5 in winter [2.5 concentration in only four cities in winter, the most polluted season, whereas PM2.5/PM10 ratios of most cites showed a fluctuating upward trend. The increase in PM2.5/PM10 ratios also have been reported at several monitoring sites in the UK [2.5 emissions and their formation mechanisms in winter.The results of this study demonstrate that PM heating ,47. The sumption  and the n winter . In addin the UK  and Indin the UK . These r2.5 pollution in Shijiazhuang, Xingtai and Handan, and found that regional contributions were larger in January than in July [2.5 concentration occurred in autumn and winter, indicating that PM2.5 pollution in adjacent cities may strongly interact among the 28 cities of the BTH region during the latter half of the year. However, the local spatial autocorrelation results of our study showed that regions with high agglomeration of PM2.5 concentration were mainly concentrated in the southern Hebei Province and northern Henan Province, but with some spatial variation, suggesting that joint variable control strategies should be emphasized for PM2.5 pollution over time.Air pollution has been suggested to be strongly affected by pollutants from adjacent cities . The int in July . In acco2.5 concentration. In this study, minT and H were the leading factors affecting PM2.5 concentration at the annual scale. minT was negatively correlated with PM2.5 concentration, which is consistent with the fact that air quality is better in summer and worse in winter. This negative correlation may be closely related to less dispersion and dilution of air pollutants at lower minT. At low minimum temperature, air convection is weak and temperature inversion can easily occur. These conditions are not conducive to the diffusion and dilution of air pollutants [H had the strongest positive influence on PM2.5 concentration. Increases in H increase the production of sulfate aerosols via aqueous reactions and also significantly affect the partitioning of HNO3 between the gas and particulate phases, leading to enhanced nitrate formation [Meteorological conditions played an important role in influencing PMllutants . By contormation . In addiormation . For exaormation . Howeverormation . This diormation ,57.2.5 concentration showed some interesting characteristics in different seasons. Generally, meteorological factors had a greater influence on PM2.5 concentration in all cities in winter, and the effects of these factors were much stronger in winter than in other seasons, which is consistent with the findings of a previous study [2.5 concentration in summer, when the variation in PM2.5 concentration depended mainly on changes in maxT and SH. Previous studies have reported that increases in temperature increased radical production rates and promoted aerosol formation, but the atmospheric photolysis occurred on organic carbon, reducing PM2.5 concentration [maxT and SH on PM2.5 concentration in this region.The correlations between meteorological factors and PMus study . In wintus study . Fewer mntration ,40 (Chenntration ,59. TherWS was negatively correlated with PM2.5 concentration in all cities, while maxT and minT exhibited a positive correlation with PM2.5 concentration in 82.14% and 60.71% of all cities, respectively, at the annual scale. The influence of temperature seems complex and differs regionally. We also found that SH strongly affected the PM2.5 concentration in areas east of the Taihang mountains, whereas AP strongly affected the PM2.5 concentration in cities west of this mountain range. These differences can be attributed partly to the topographical characteristics. Unique atmospheric circulation subject to the particular topography has been suggested to develop spatial distribution characteristics of air pollutants [The spatial distribution of air pollution has been associated with meteorological factors in previous studies . Liu et llutants .10 and PM2.5 concentration data for Jincheng was collected and analyzed from 1 March 2015 due to the limited monitoring record. In addition, there were about 44 missing air pollution records among 28 cities and few zero values for PM10 concentration, which were removed to calculate the PM2.5/PM10 ratios. Although we applied interpolation to maintain the continuity and comparability of these time series data, some uncertainty remains. Moreover, although the Local Moran’s I method used in this study could indeed shed light on the potential interdependence and specific spatial agglomeration patterns of PM2.5 concentration, the limited observations for 28 cities were slightly less than the restriction on the numbers required by this method. Additionally, the temporal and spatial variations of PM2.5 concentration were strongly correlated with meteorological factors, and their relationships varied significantly across seasons and geographical locations, probably relating to PM2.5 components. To better address this problem, GWR was applied in this study to capture the spatial nonstationary characteristics, but it could not simultaneously deal with temporal nonstationarity [This study had some limitations. First, PMionarity ,63, whic2.5 concentration in 28 cities within the atmospheric pollution transmission channel in the BTH region with associated meteorological influences during 2014–2019. Average PM2.5 concentration appeared to decrease significantly in most cities at the annual scale, but exhibited clear seasonal and spatial variation. The mean PM2.5 concentration demonstrated a significant decreasing trend in more than 20 cities in spring and summer, but only 4 cities in winter, indicating that strict measures should be continued, especially in the most severely polluted season, despite successful air pollution control plans implemented within the past decade. Temporal variation in daily PM2.5 concentration was strongly correlated with minimum temperature and average relative humidity. The higher the PM2.5 temporal concentration, the stronger the influence of the meteorological factors, with a mean explanatory power of 31.96% for the 28 cities. Spatially, wind speed was negatively correlated with PM2.5 concentration for all cities, while maximum temperature and minimum temperature exhibited a positive correlation with PM2.5 concentration in 82.14% and 60.71% cities, respectively. These results will provide valuable references for understanding the spatiotemporal variation of PM2.5 pollution, as well as for future clean air policy making.In this study, we investigated the spatial and temporal variation of PM