The development of pyoderma gangrenosum (PG) after surgical site trauma is a rare, poorly understood immunologic phenomenon. PG is an immunologic disorder characterized by lymphocytic infiltration of the dermis that can manifest with skin necrosis and ulceration. This rare phenomenon can mimic surgical site infection (SSI) when it occurs in the perioperative period and in the region of surgical wounds. Within the neurosurgical literature, only two cases of postoperative PG have been reported to our knowledge. We describe the clinical features and treatment of PG in the case of a 65-year-old man who underwent a three-stage surgical approach for intractable mechanical low back pain on hospital days (HDs) 1 and 2, and who subsequently developed PG around all three surgical sites in the immediate postoperative period (HD 8). The physical and laboratory findings and surgical and pharmacologic treatments are detailed. The patient was initially treated for presumed SSI, started on broad-spectrum antibiotics, and underwent surgical wound debridement twice, without resolution of symptoms. The diagnosis of PG was ultimately made by a consulting dermatologist on HD 17. The patient was started on systemic immunosuppression with steroids during his initial hospitalization; symptoms resolved within two weeks of the index surgery. Although PG is a rare entity, we suggest that it be considered in the differential diagnosis of nonhealing surgical wounds. Familiarity with PG may help mitigate unnecessary surgical morbidity and reduce the length of hospital stays and unnecessary use of antibiotics. Pyoderma gangrenosum (PG) is a rare immunologic phenomenon that mimics a surgical site infection (SSI). PG typically manifests as painful skin pustules that can rapidly progress to large necrotic ulcers with raised violaceous borders . BecauseThis study details the case of postoperative wound dehiscence after spine surgery that was initially treated as an SSI with two debridements and washouts; these treatments ultimately did not result in improvement of the wound. A diagnosis of PG was made nearly two weeks after index surgery, and the patient immediately improved after empiric treatment for PG. Explicit patient consent was obtained to disclose the health information in this study. Institutional review board review was not sought because a single case report does not meet the minimum criteria for institutional review board review.A 65-year-old man without significant medical history or history of prior surgery presented after conservative management failed to alleviate his chronic mechanical back pain, left leg radiculopathy, and paresthesia. Preoperative imaging obtained at presentation demonstrated levoscoliosis of the lumbar spine with spondylolisthesis, severe stenosis, and advanced degenerative findings at multiple lumbar segments Figures -1F. The A three-stage surgical approach was recommended with L4-5 and L5-S1 anterior lumbar interbody fusion, L2-3 and L3-4 extreme lateral interbody fusion, and posterior L2-S1 percutaneous screw placement and arthrodesis to correct the patient’s degenerative spondylolisthesis and to indirectly decompress the neural foramina. The patient underwent stage 1 and 2 procedures on hospital day (HD) 1 and stage 3 on HD 2 without any acute complications and with satisfactory postoperative images Figures , 2B. A t9 cells/L, although he was afebrile. On HD 8, the patient’s anterior wound began to express purulent fluid, with the wound edges showing dehiscence and ulceration  but not steroids. On HD 5, the patient’s white blood cell count was elevated at 12.0 x 10n Figure . Initialn Figure , 3C. Then Figure , 3E. DerAfter initiating intravenous prednisolone at a dosage of 50 mg, the patient’s abdominal wound improved within several hours with a noticeable decrease in erythema and edema. The patient was discharged home on HD 18 on oral amoxicillin/clavulanate (875/125 mg twice daily), oral doxycycline (100 mg twice daily), and a tapering dose of steroids starting at 50 mg for the first of seven days. At the six-week postoperative follow-up, the patient showed significant improvement in preoperative back pain, and his wounds were completely healed Figures -4C.This case illustrates the development of a nonhealing wound in the immediate postoperative period after spine surgery. Only three cases of postoperative PG after spine surgery have been reported, with only one of these reported in the neurosurgical literature ,15,16. TWhen patients present with possible SSIs in the immediate postoperative period, the possibility of PG should be considered, especially in patients with a medical or family history of systemic inflammatory disease, such as inflammatory bowel disease, although the majority of cases develop without such history ,7. PG leIt is not uncommon for patients ultimately diagnosed with PG to first undergo treatment for presumed SSI ,17. HoweAlthough postoperative PG is exceedingly rare, our case suggests that familiarity with this entity may mitigate costly and untimely surgical morbidity, the length of hospital stays, and the unnecessary use of antibiotics. Continuous wound ulceration, purulence, and erythema surrounding the surgical site in the immediate postoperative period despite antibiotic therapy and surgical reoperation, as well as a medical or family history of systemic inflammatory disease, should raise the suspicion of a noninfectious etiology. Prompt dermatologic consultation and treatment with immunosuppression should be pursued in such cases. Biopsy may prove useful at the time of wound washout to obtain a histologic diagnosis, though this was not pursued in our case. Ultimately, a short tapering course of steroids to treat PG should not have implications for bony fusion in patients who have undergone spine surgery and treatment for PG should be safe to pursue.


Moreover, male health educators aged 45–54 years had a positive attitude towards vitamin D (3.8 ± 0.7) (p = 0.007). In addition, female health educators who were divorced (3.8 ± 0.7) and widowed (3.6 ± 0.5) (p = 0.04) and those who were administrators (3.3 ± 0.7) (p = 0.01) had a positive attitude towards vitamin D. The Ministry of Education (MOE) in the Kingdom must educate health educators through educational programmes that aim to increase the knowledge of and develop a positive attitude towards vitamin D intake.Health educators in schools are a very important part of the education system. Considering the significant prevalence of vitamin D deficiency in the Kingdom of Saudi Arabia, it is important to investigate the knowledge of and attitude towards vitamin D among health educators. This study aims to examine the knowledge of and attitude towards vitamin D among health educators in public schools in Jeddah as well as to identify the associated sociodemographic factors. A cross-sectional study was conducted between May and December 2021 among 231 health educators. Data were collected via a self-administered online questionnaire. The results revealed that only 45% of health educators had good knowledge of vitamin D, and approximately 43% had a positive attitude towards vitamin D. Additionally, those who had good knowledge of vitamin D were males (58.7%) ( Vitamin D deficiency is a key public health concern worldwide. Recent studies have revealed the importance of vitamin D in maintaining good health and preventing numerous diseases . AlthougHealth educators in schools are a very important part of the education system due to their direct contact with children in schools at an early age; these educators play an important role in establishing a strong knowledge of health among future generations. The Ministry of Education (MOE) in Saudi Arabia has appointed health educators for all schools. The role of these health educators is to provide the necessary health education for young students and to teach them the concepts and impart them with the knowledge required to develop the value of health and well-being among future generations .Additionally, health educators are in contact with teachers and parents and, thus, extend their impact to the entire community. Considering the significant occurrence of vitamin D deficiency in the Kingdom, the scarcity of knowledge of and negative attitudes towards the health benefits of vitamin D and its sources might be the reason for the high prevalence of vitamin D deficiency. In addition, only a few studies in the extant literature have examined the knowledge of and attitudes towards vitamin D in Saudi Arabia ,23,24. PA cross-sectional study design was employed for this study, which was conducted in Jeddah between May and December 2021. Ethical approval was obtained from the Biomedical Ethics Research Committee of King Abdul-Aziz University (Reference No. 128-21). Ethical approval was also obtained from the Department of Education in Jeddah. All participants were health educators who worked in different stages of public schools  for boys and girls. According to the statistics from the Ministry of Education (MOE), public schools in the city of Jeddah belong to one of four educational administrative offices for girls  and six educational administrative offices for boys . The number of public schools for boys was 507, including 243 primary schools, 164 middle schools, and 100 secondary schools . The numThe inclusion criteria for study participants included adults (18 years old and above) from both genders who work as health educators in public schools in Jeddah city. The exclusion criteria included health educators who work in private schools. Two hundred and thirty-one  health educators were involved in this study. The majority of these health educators were from East Jeddah (32%) and North Jeddah (25.5%), followed by South Jeddah (18.2%) and Centre (15.6%). The lowest percentage of participants came from the administrative offices of Asafa (3.9%) and Naseem (4.8%).The data collection was conducted through a self-administered online questionnaire that was sent to the six above-mentioned educational administrative offices. Subsequently, the online questionnaire was distributed among the schools in each administrative area. The online questionnaire had a short introduction explaining the research goal and emphasized that participation was voluntary and anonymous. Additionally, it explained that completion of the questionnaire indicates accepting participation in the study. Questions regarding knowledge and attitude towards vitamin D were adapted from previous validated questionnaires ,28,29 whWith regard to knowledge of vitamin D, participants were asked whether they had heard of vitamin D and whether they believed vitamin D was important for their health. They were also asked about their source of information on vitamin D and where they believe the body acquires the vitamin from. The questions also included the food sources rich in vitamin D, benefits of vitamin D, people at risk of developing vitamin D deficiency, factors affecting the synthesis of vitamin D in the human body, vitamin D and calcium absorption, and vitamin D supplements . To consFor questions pertaining to the attitudes towards vitamin D questions, participants were asked whether they like to expose themselves to sunlight, believe vitamin D is important for health, and agree that exposure to sunlight is harmful to the skin. They were also asked whether they were concerned that their current vitamin D levels might be too low and whether they were willing to undergo tests for determining their vitamin D levels. Questions 1–4 had three possible responses: ‘Agree’, ‘Disagree’, and ‘Neither agree nor disagree’. The response ‘agree’ was given one point, and the other responses were awarded zero points. Additionally, question 5 had only two responses: ‘yes’ and ‘no’. The response ‘yes’ was awarded one point, and the response ‘no’ was awarded zero points; thus, attitude scores ranged from 0 to 5. These attitude scores were categorized as negative attitudes (0–3) and positive attitudes (3–5).p-value of 0.05 was considered statistically significant.All statistical analyses were performed using SPSS statistical analysis software (version 22). The sociodemographic characteristics and answers pertaining to the knowledge of and attitude towards vitamin D are presented as frequencies and percentages. Additionally, scores pertaining to the knowledge of and attitude towards vitamin D are presented as mean ± SD. The chi-square test was used to examine associations between sociodemographic characteristics and knowledge of and attitude towards vitamin D. Furthermore, a linear regression was conducted to examine the association between sociodemographic characteristics and knowledge of and attitude towards vitamin D scores. A p < 0.001) and had longer work experience (p < 0.001) than female health educators; moreover, male health educators more commonly worked in secondary schools (p < 0.001). The highest percentage of health educators from both genders were married, the proportion of single males was higher than that of females, and the proportion of divorced and widowed females was higher than that of males (p = 0.05).The sociodemographic characteristics of health educators classified by gender are presented in Health educators’ knowledge regarding vitamin D questions and responses are presented in Almost all of the participants were aware of vitamin D and believed it is important for their health. Moreover, 75% of health educators knew the sources of vitamin D, including diet, sun exposure, and supplements. Additionally, 70% of participants knew that fatty fish  are a good source of vitamin D, while one-third knew that milk and eggs are a good source of vitamin D. However, a few participants recognized vegetables and fruits (30%) and olive oil (10%) as good sources for the vitamin. Additionally, 94% of health educators were aware that vitamin D is important for bone health, and 27% were aware that it can prevent heart diseases. Additionally, the majority of participants correctly identified people at risk of developing a vitamin D deficiency; however, only a few (3%) knew that people with dark skin can be at greater risk of developing vitamin D deficiency. Similarly, almost one-third were aware that the use of sunscreen and time of day might affect vitamin D synthesis; however, only a few recognized other factors that affect vitamin D synthesis, including seasons (11.7%), cloud cover (16.5%), skin pigmentation (12%), latitude (3%), and pollution (10%). Furthermore, a small proportion of participants specified a high-fat diet (18.6%) and smoking (8.7%) as factors affecting vitamin D synthesis. The majority of health educators believed that vitamin D helps in the absorption of calcium in the body (76%) and that the intake of vitamin D supplements helps reduce the risk of vitamin D deficiency (90%). The most frequent sources of information regarding vitamin D among study participants were doctors (64%), family/friends (50%), the internet (48.5%), and television (32.5%). Overall, 45% of health educators had good knowledge of vitamin D.Health educators’ attitudes regarding vitamin D questions and responses are presented in p = 0.005) (= 0.005) . In addi= 0.005) . Overallp = 0.007). Male health educators aged 45–54 years had the highest scores for attitude (3.8 ± 0.7) compared to male health educators in other age groups. However, there was no significant association of age with knowledge and attitude scores among female health educators. Further, marital status also showed a positive association with attitude scores among female health educators (p = 0.04). Divorced and widowed females had higher scores for attitude  compared to single and married females . However, marital status did not show any significant association with knowledge and attitude scores among male health educators.Attitude scores indicated a positive association with age among male health educators (p = 0.001). Male health educators with postgraduate degrees had the highest knowledge scores (13.5 ± 4.6) compared to those holding diplomas and bachelor’s degrees . However, educational level did not have a significant association with knowledge and attitude scores among female health educators.Knowledge scores showed a positive association with educational levels among male health educators (p = 0.01). Furthermore, school stage, work experience, and specialization did not exhibit any significant association with knowledge and attitude scores among either male or female health educators.Additionally, female health educators who were administrators had the highest attitude scores (3.3 ± 0.7) compared to female health educators who were teachers (3 ± 0.6)  among health educators in public schools is considered insufficient in terms of how important it is to communicate such knowledge to students and teachers. However, other studies among different populations, including the general population and university students within Saudi Arabia have reported inconsistent findings ,30,31. FFurthermore, the results of the current study revealed that the most common sources of information regarding vitamin D in this study were doctors (who are the primary source of knowledge), then family and friends, and then the internet; this is consistent with a study in France  but contFurthermore, in this study, almost 95% of study participants were aware of the traditional role of vitamin D in strengthening bones and bone health, and 76% were aware that vitamin D helps in the absorption of calcium in the body; however, only 25% were aware of the role of vitamin D in protecting against heart disease. In fact, recent research has indicated that vitamin D deficiency is associated with a risk of cardiovascular disease, plays a protective role against cardiovascular disease and associated risk factors, and reduces the risk of heart failure . FurtherWith regard to health educators’ attitudes towards vitamin D, only 43% of health educators in this study had positive attitudes towards vitamin D. However, almost all health educators admitted the importance of vitamin D and agreed to test for the serum levels of vitamin D in their bodies if their health condition required it. Similarly, two-thirds were concerned about having vitamin D deficiency. In contrast, only 29% of health educators agreed to expose themselves to sunlight regularly. Similarly, almost 60% of health educators believed that exposure to sunlight is harmful to the skin or were nonaligned with this belief. This attitude towards exposure to sunlight might be the reason for the high prevalence of vitamin D deficiency in the Kingdom. Another recent study conducted among pre-menopausal women in Jeddah, Saudi Arabia, reported that approximately 50% of study participants did not like to expose themselves to sunlight . These fWith regards to the sociodemographic factors associated with knowledge of and attitude towards vitamin D, there were significant differences in the knowledge of vitamin D between males and females, as 59% of males had good knowledge of vitamin D compared to 41% of females. A previous study in Riyadh, Saudi Arabia, reported similar results, where 54% of males had good knowledge of vitamin D compared with 49% of females with such knowledge; however, the result was not statistically significant . This isTo the best of our knowledge, this is the first study to examine the knowledge of and attitude towards vitamin D among health educators in public schools in Saudi Arabia. Health educators in schools are neglected in terms of scientific research and health education, even though they are a very important and integral part of the education system, as health educators promote community health through their communication with teachers and parents. Consequently, they have a great influence in transferring knowledge, imparting scientific health concepts, and correcting misconceptions among students, teachers, and parents. Therefore, the findings of this study provide the MOE in the Kingdom with recommendations to enhance the roles of health educators and enhance the quality of health education provided in schools. Nevertheless, the current study has several limitations. First, this was a cross-sectional study, as it only demonstrated an association. Second, it is possible that selection bias was introduced when collecting data via a self-administered online questionnaire; however, validated questionnaires were used for data collection. Third, data regarding vitamin D practices were not collected from health educators, which will reflect the actual vitamin D-related behaviour such as exposure to sunlight, and the intake of vitamin D supplements and diet rich of vitamin D among this group; we recommend that future studies bridge this gap. Fourth, because serum vitamin D was not collected from study participants, correlations of knowledge of and attitudes towards vitamin D with serum vitamin D levels were not determined. Last, it may not be possible to generalise the findings of the current study to all health educators in the Kingdom; however, the findings are believed to be representative of health educators in Jeddah city, as health educators who participated in this study were recruited from the six educational administrative offices in Jeddah.The results of the current study revealed that over half of the health educators in Jeddah city had poor knowledge and negative attitudes towards vitamin D, which is insufficient when considered against the background their significant role in improving health awareness among future generations, including enhancing awareness of the importance of vitamin D and the effects of the deficiency of vitamin D on human health. The MOE must consider implementing educational programmes for health educators in public schools to improve their knowledge regarding vitamin D, particularly among female health educators and in aspects such as sources of vitamin D and individuals at risk of developing vitamin D deficiency. Additionally, educational programmes should emphasize the importance of exposure to sunlight as the main source of vitamin D and assure that sunlight exposure is a safe and healthy practice, particularly during certain specific times of the day, which will help to establish positive attitudes towards vitamin D and sun exposure among this significant group. Furthermore, future studies should examine vitamin D-related behaviour among health educators and investigate the correlations among knowledge of and attitude and behaviour towards vitamin D and serum vitamin D levels among health educators in schools. In addition, it is important to conduct qualitative research in the future to uncover the reasons for poor knowledge and a negative attitude towards vitamin D, which have possibly influenced the high prevalence of vitamin D deficiency in the population in Saudi Arabia.


It is unknown how ABCC6 genotype affects plasma PPi. Methods: We studied the association of ABCC6 genotype  and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity. Results: Our findings confirm low PPi in PXE compared with healthy controls . The PPi of patients correlated with increasing age  and was higher in females . However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found. Conclusions: Our data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.Background: Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PP In the e organs . Calcifie organs . Periphee organs .i levels, and disruption in one of these proteins results in increased ectopic calcification [i. The ABCC6 protein is responsible for increasing extracellular ATP, which is converted to PPi and AMP [i levels therefore seem to be the causal link between ABCC6 variants and the multi-organ calcification observed in PXE. However, currently there are no data available on how ABCC6 genotype affects plasma PPi levels. The present study therefore aimed to compare plasma PPi between a large cohort of PXE patients and controls, and to investigate arterial and ophthalmological phenotypes of PXE with special emphasis on any association between genotype and plasma PPi levels of the PXE patients who had biallelic pathogenic ABCC6 mutations.Several proteins are involved in maintaining local and systemic PPfication . In PXE,fication . ABCC6 p and AMP . PPi levi analysis, and clinical and molecular data from regular care were used . This cross-sectional study was conducted in adherence to the tenets of the Declaration of Helsinki and approved by the medical ethical review board of the University Medical Center Utrecht . All participants gave written informed consent for blood collection and all the PXE patients gave written informed consent for the use of their medical files for research purposes.PXE patients were recruited from the Dutch Expertise Center for PXE in the University Medical Center Utrecht, the Netherlands. Controls were recruited from the acquaintances of the PXE patients, excluding first and second-degree relatives. All patients had a clinical diagnosis of PXE based on the criteria of Plomp et al. . In shorhttps://www.mrcholland.com/), accessed 14 January 2021). Specific primers were used to avoid the amplification of ABCC6 pseudogenes ψ1 and ψ2 [Genomic DNA was isolated from whole blood. All ABCC6 exons and the flanking intron sequences were analyzed as part of the genetic screening for regular clinical care. Sanger sequencing was performed to identify rare sequence variants and small deletions and insertions, and multiplex ligation-dependent probe amplification (MLPA) was performed to screen for larger deletions in the ABCC6 gene . Blood plasma was transferred to a Centrisart ultrafiltration tube , centrifuged for 30 min  and the filtered plasma was stored at −80 °C until PPi analysis. Plasma PPi concentrations were determined as previously described [i was converted into ATP using ATP sulfurylase in the presence of excess adenosine 5′-phosphosulfate (APS). For each 10 μL of plasma, 70 μL of a mixture containing 32 mU ATP sulfurylase , 16 μmol/L APS , 80 μmol/L MgCl2, and 50 mmol/L HEPES (pH 7.4) was added. Following incubation at 37 °C for 30 min, ATP sulfurylase was inactivated at 90 °C for 10 min. Generated ATP was quantified using the ATP-monitoring reagent BacTiter-Glo . An equal volume of BacTiter-Glo reagent was added to the samples. Bioluminescence was determined in a microplate reader . Each sample was measured twice in triplicates.Blood was collected in 4.5 mL CTAD vacutainers . 50 µL of a 15% K3-ethylenediaminetetraacetic acid (EDTA) solution was added and the tubes were centrifuged for 15 min , whole body CT scanners  in the carotid siphon, common carotid artery, coronary arteries, thoracic and abdominal aorta, iliac arteries, and femoral and crural arteries. Arterial calcification was defined as hyperdense arterial wall lesions with a density above 130 Hounsfield Units (HU). Calcification mass scores were computed as the product of the volume of the lesion in ml and the mean attenuation in HU. A peripheral artery score was calculated as the sum of the femoral and crural arteries, and a total arterial calcification mass score was calculated as the sum of all different arterial beds.The ankle-brachial index (ABI) after a treadmill test was used to measure PAD. To calculate the ABI, systolic blood pressure was measured in the left and right brachial arteries, the tibial posterior arteries and the dorsal pedal arteries. During the treadmill test, patients were encouraged to walk on a treadmill with a 10% slope and a speed of 3.5 km/h for 6 min. Patients who stopped prematurely due to claudication were encouraged to continue walking as soon as possible. During recovery, the ABI was measured for 10 min in a supine position. The lowest value of these ABI measurements was used as the exercise ABI. The ABI measurements were performed by experienced technicians. PAD was defined as an ABI < 0.90. Patient-reported claudication was measured with the Fontaine classification and patients were classified as having no PAD (ABI > 0.90), asymptomatic PAD (ABI < 0.90 without claudication) and symptomatic PAD (ABI < 0.90 with claudication).All patients underwent routine ophthalmological examination, including best-corrected visual acuity (BCVA), macular spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF), near-infrared reflectance imaging (NIR)  and color fundus photography .The length of angioid streaks on 55° near infrared (NIR) imaging was used as a proxy for the extent of Bruch’s membrane calcification, as previously described [The presence of CNV and the severity of macular atrophy were scored in both eyes by three experienced graders . The presence of (in)active CNV was based on assessment of SD-OCT, fundus photography and fluorescein or indocyanine green angiography. The presence of macular atrophy was based on FAF, SD-OCT, color fundus photography and NIR. The largest atrophic area was graded as ‘no atrophy’, ‘mild atrophy’ (<twice the area of the optic disc) or ‘severe atrophy’ (> twice the area of the optic disc). An atrophic zone surrounding an (in)active CNV was considered independent of macular atrophy. BCVA was measured on Snellen charts or Early Treatment of Diabetic Retinopathy Study (ETDRS) charts and converted to the logarithm of the minimum angle of resolution (logMAR).Abcc6−/− female mice by hydrodynamic tail vein injection (HTVI) of ABCC6 in pLIVE plasmid  as described [Animal protocols were approved by the Food Chain Safety and Animal Health Directorate of the Government Office of Pest County, Hungary (XIV-I-001/707-4/2012). ABCC6-expressing liver samples were obtained from 12-week-old escribed . Brieflyescribed .i and continuous outcome measures was tested with the Pearson correlation.Characteristics are presented as mean ± standard deviation for normally distributed continuous variables, median (interquartile range (IQR)) for non-parametric distributed continuous variables and number (%) for categorical variables. For the ophthalmological phenotypes, data for the right eye were used for descriptive and regression analyses. If imaging of the right eye was not assessable, the left eye was used. The correlation between plasma PPi as the outcome, and adjusted for age and sex. Log10 transformation was performed on the calcification mass scores. The models including arterial calcification mass scores were additionally adjusted for differences in scanner and settings since this has been shown to affect calcification mass scores [p-value < 0.05 was regarded as statistically significant. All analyses were performed in RStudio version 1.1.456.Linear regression models were built with genotype or the individual phenotypes as the determinant and plasma PPs scores ,14. A p-p = 0.23), but more of the PXE patients were females (125 (60%) vs. 82 (40%) males, p = 0.02). In all 207 patients and 45 controls, PPi was measured. In addition, 189 patients underwent CT scanning, all patients underwent an exercise ABI measurement and 193 patients underwent an ophthalmological examination. Of the 207 patients, 192 were found to have biallelic pathogenic ABCC6 mutations.In total, 207 PXE patients from 175 families and 45 controls were included in this study. Age was similar between the groups . The female PXE patients had slightly but significantly higher PPi than the males . However, when adjusted for age and sex, no association between plasma PPi and peripheral  and total  arterial calcification mass was found. Patients with PAD  had higher plasma PPi than patients without PAD , but this difference attenuated after adjustment for age and sex , had higher plasma PPi than patients without CNV  and worse visual acuity was weakly correlated with higher PPi , those with two non-truncating mutations (n = 10), and a third group who had one truncating and one non-truncating ABCC6 mutation (n = 61).Of the 207 patients, 192 were included in the ABCC6 genotype and plasma PPIn our PXE cohort, a total of 81 non-truncating missense alleles were identified: 61 of these were in the ‘mixed’ group , and the remaining 20 were the alleles of 10 patients carrying missense mutations on both chromosomes. Altogether we found 34 different missense mutations, which represented a spectrum with high variety.i levels of the groups with two truncating ABCC6 alleles, or two non-truncating alleles, or those who had one truncating and one non-truncating allele  and that the ABCC6 genotype of these patients apparently did not account for these differences. Even the three patients from the same family (shown as squares) had different plasma PPi.We analyzed a few mutations in detail: for instance, the mutation R1141* was present in homozygous form in 21 (10.9%) of the 192 PXE patients, making R1141*/R1141* the most common ABCC6 genotype in the cohort examined. This high prevalence is not unexpected, since this mutation is quite common in the European PXE population ,16,17. I−/−Abcc6 mice [i level varied in these five patients between 0.40 μM to 0.96 μM, lacking any apparent correlation between this specific missense mutation and the plasma PPi measured.We studied the cellular localization of two missense mutants by overexpressing the human ABCC6 protein variants in the liver of −/− mice . A stren−/− mice ,19. Missi concentration, and one who, in contrast, had 0.7 ± 0.04 μM of plasma PPi  and 107 patients [In this large cohort of PXE patients, we show that PPi levels were not associated with disease severity in PXE. Only a small association between plasma PPi and CNV was found, and other ophthalmological outcomes were not associated with plasma PPi. In the recent publication of Leftheriotis et al. also no association between plasma PPi and lower limb arterial calcification was found [i and peripheral artery calcification score. Total body arterial calcification, as well as ophthalmological outcomes, were not studied by these authors. Contrary to PPi, age seems to be the most important determinant for disease severity in PXE. More ectopic calcification, as seen in older patients, leads to more need for inhibitors of it, of which PPi is one of the most important, but as we did not find an association between PPi and disease severity it remains difficult to explain the association between PPi and age that we and others have found.More importantly, we found that plasma PPas found , comparai [i [Our findings add to a growing body of evidence showing that both systemic and local factors contribute to the pathophysiology of PXE. The ABCC6 protein is thought to account for approximately 60% of circulating extracellular PPi . Althougi [i ,21,22, Pi [i . We recei [i .i of patients with inborn calcification disorders caused by inactivating mutations of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) encoding the nuclease acting upstream to ABCC6 protein [i concentrations were substantially decreased, similar to the two individuals who had biallelic mutations in their ENPP1 genes and the much more severe phenotype of GACI. This observation also suggests that the relationship between reduced plasma PPi and ectopic calcification symptom severity may not be as direct as previously thought.An interesting study has been published recently regarding the genotype, disease phenotype and plasma PP protein   the level of ABCC6 protein expression in the liver, controlled by e.g., oxidative stress [i, which may significantly influence plasma PPi. In addition, there is competition for ATP in the circulation: ENPP1 is the only nuclease producing PPi from ATP, while ectonucleoside triphosphate diphosphohydrolase (eNTPD) enzymes ‘consume’ ATP without generating PPi [The lack of association between genotype and plasma PP PPi see  also mitby ENPP1 . Factorse stress ,26; and e stress  and ENPPting PPi . This isi is not the sole possible inhibitor of ectopic calcification, and disease severity in PXE may be influenced by other calcification inhibitors, e.g., fetuin A [i as an additive), or smoking, which could enhance disease severity.Furthermore, PPfetuin A . We alsoi is a major cause of ectopic mineralization in PXE, it is not the only factor that prevents ectopic calcification [i are currently under development for the treatment of PXE and related disorders. Orally delivered PPi has been shown to increase plasma PPi in humans and mice, and to inhibit cardiac calcification and calcification of the vibrissae, kidneys and arteries of the legs in mice [i in PXE. The recombinant ENPP1 enzyme INZ-701 was shown to increase plasma PPi, inhibit cardiac and aortic calcification and increase survival in Enpp1−/− mice [i and partially prevented calcification in −/−Abcc6 mice [Abcc6−/− mice [i, inhibits systemic arterial calcification in PXE patients [Utilizing animal models, it was found that although reduced plasma PPfication ,31. In s in mice . These l−/− mice , and in −/− mice . Inhibit−/− mice . Finallypatients .i levels when compared with controls. Plasma PPi levels were correlated with increasing age and were higher in female PXE patients. Contrary to expectations, no clear association between plasma PPi levels and disease severity was found in PXE. Another unexpected finding of our study is that no correlation between ABCC6 genotype and plasma PPi level was found in the 192 pseudoxanthoma elasticum patients with known pathogenic phenotypes. Our findings should trigger future studies of the role of PPi in disease progression, and possible genetic or environmental modifiers, which might shed more light on the complex pathophysiology of PXE.Our results support that PXE patients have approximately 60% reduced plasma PP


Cyperus aromaticus (Ridley) Mattf. & Kukenth) is a significant agricultural and environmental weed found in tropical island countries including north Queensland, Australia. It is a prolific seed producer and consequently forms a high-density seedbank, and therefore understanding the longevity and persistence of the seeds can provide critical information required for the management of this species. A laboratory-controlled artificial ageing experiment was conducted where the seeds were exposed to a temperature of 45 °C and 60% relative humidity for 125 days. Seeds were removed at various times  and their viability determined through standard germination tests. It took 20 days in the artificial ageing environment for the seeds to decline to 50% viability which indicates that Navua sedge has relatively short-lived persistent seeds. These findings will assist in developing a better understanding of the seedbank dynamics of this invasive species, allowing managers to tactically implement control strategies and prepare budgets for ongoing treatments, and have implications for the duration and success of management programs.Navua sedge ( Cyperus aromaticus (Ridley) Mattf. & Kukenth), is an invasive perennial C4 sedge of tropical environments which forms dense stands, has prolific seed production and has a creeping rhizome. This aggressive weed is a major cause of concern for the agricultural industry, causing productivity losses in pastures, which affects milk and livestock industries, and also in crops such as rice, sugarcane and banana [Navua sedge  may produce viable seeds ranging from 44,400 to 56,700 seeds/m2 [Navua sedge is an extremely aggressive persistent weed, which competes with pasture species and crops for light, water, nutrients and space and has the ability to quickly smother competing plants . The pla2 in the soil seedbank in grazing pastures of tropical north Queensland [Management efforts are often focused to reduce the above-ground cover of weeds while efforts to manage their seedbank are not given comparable importance. However, recent study has shown the presence of more than 35,000 viable Navua sedge seeds/meensland  which iseensland . Seed deeensland . The inteensland ,11. Henceensland ,13. Two most used methods for testing seed longevity are seed burial in the field (in situ) and artificial ageing (ex situ). Studies on seed longevity in the field provide accurate information since they naturally involve both biotic and abiotic elements. These investigations, however, are not always feasible due to lengthy time requirements and considerable financial resource demands . By comp50) was 20 days (p < 0.05) was observed between these two groups and other ageing times. Mean germination time also increased with the number of artificial ageing days . In the field seeds were placed in labelled paper bags and transported to Federation University, Mount Helen campus, Victoria, Seed Ecology lab and stored in a dark cupboard until commencement of the experiment. The experiment was carried out between September 2021 and March 2022 at Mount Helen campus of Federation University Australia.Seed bags for artificial ageing were prepared by placing twenty mature Navua sedge seeds into 2 cm × 2 cm square bags made of fine (0.05 mm) nylon mesh, which were sealed using hot glue. A total of 72 seed bags were used in the experiment, out of which six bags were used to test for rehydration and 66 bags for the artificial ageing.Protocol for comparative seed longevity testing developed by the Millennium Seed Bank Project, Royal Botanical Gardens, Kew, United Kingdom was used in this study for artificial ageing of the seeds . For theFollowing the rehydration step, the 66 selected seed bags were moved to a second electrical enclosure box with the ageing solution (prepared by adding 300 g lithium chloride to 1 L water) at 60% RH. This box was then placed in the incubation chamber maintained at 45 °C in darkness for 125 days. Six replicate bags were randomly selected and removed at each of the following ageing times: 0, 1, 2, 5, 10, 20, 30, 50, 75, 100 and 125 days. ® No 10-filter paper and moistened with sterilised RO water to provide adequate moisture for germination and sealed with parafilm to ensure moisture retention. The Petri dishes were then placed in the incubation chamber equipped with cool-white fluorescent lamps to provide a photosynthetic photon-flux of 40 µmol m−2 s−1, maintained at 25/15 °C day/night temperature and 12 h light/12 h darkness [The seeds removed at each removal time were tested for viability using a standard germination test. Seeds from each bag were placed on separate 9 cm Petri dishes lined with sterilised Whatmandarkness . The obsdarkness ,29. 10 transformed and a Probit analysis performed in SPSS statistical software to find the P50 value, which is the time for viability to decline by 50% [The data was Loge by 50% . Seed vin represents number of seeds germinated on day D, and D is the number of days counted since the beginning of the germination trial.Mean germination time, which expresses the rate of seed germination, was calculated with the formula described by Kader : (2)MGT=G1, G2 and Gx are the germination percentages × 100 at 1, 2 and x days after sowing, respectively.A second formula given by Kader  was used


To investigate the role of tracheal wall injury in the development of benign airway stenosis in rabbits. Prospective study. We injured the tracheal walls of 28 New Zealand white rabbits using four different methods. Experimental group: Group A ; Group B ; Group C ; Group D . Bronchoscopy was performed on each experimental rabbit at 1, 2, 3 and 4 weeks after operation. High-resolution computed tomography (HRCT) and endobronchial optical coherence tomography (EB-OCT) were performed at 4 weeks, and the rabbits were sacrificed after the examination. Their gross and histological findings were comparatively determined whether the experimental rabbit stenosis was established. No airway stenosis was observed in group A. In group B, 28.57% of experimental rabbits developed tracheal stenosis . Fourteen rabbits in group C and group D had tracheal stenosis caused by granulation tissue proliferation at 1, 2 and 3 weeks after operation. At the fourth week after operation, 71.43% of experimental rabbits had tracheal stenosis due to granulation tissue hyperplasia, 7.14% of experimental rabbits had tracheal stenosis due to scar contracture and granulation hyperplasia, and 21.43% of experimental rabbits had tracheal stenosis due to scar contracture. EB-OCT scan showed that the cartilage layer with low signal reflection band was discontinuous. The injury of cartilage is the key factor of benign airway stenosis. Acute injury of airway mucosa alone is unlikely to cause airway stenosis, but combined with cartilage injury may aggravate airway stenosis. EB-OCT can clearly identify the airway layers of rabbits, which is helpful to evaluate the damage of tracheal cartilage and mucosa. The diagnostic potential of this technique makes EB-OCT a promising approach for the study and monitoring of airway diseases. In recent years, some retrospective studies in China have found that iatrogenic injuries such as tracheal intubation and tracheostomy are the primary causes of benign airway stenosis in China2. Airway stenosis is also the most common complication after tracheal intubation or tracheotomy, occurring in 10% to 22%3. The pathogenesis of airway stenosis is complex. Hillel et al.4 showed that epithelium plays an important role in the formation of idiopathic subglottic stenosis (iSGS) and iatrogenic laryngotracheal stenosis. Increasing evidence supports that iLTS is an active fibroinflammatory response mediated by dysregulated host immunity5. Specifically, M2 macrophages and Th2 cellshave been implicated in the pathophysiology of iLTS8, upregulation of immune checkpoints, programmed cell death protein 1 (PD-1) receptor (CD279) and its major protein ligands, PD-L1 , binding to CD4 T cells has also recently been observed in LTS patients, and TGFβ1 (transforming growth factor β1) has been identified as a potential mechanism of PD-L1 upregulation on fibroblasts in iLTS9. These immune alterations ultimately lead to fibroblast activation and increased extracellular matrix deposition, which results in airway stenosis11. A number of methods for inducing airway stenosis in animal models have been described, including brushing, thermal ablation damage, chemical damage, endotracheal intubation, and partial cartilage ring excision18. Trauma is an important cause of benign airway stenosis. However, the degree of airway wall injury in the formation of benign airway stenosis needs to be further explore. In this study, we used different methods to damage the tracheal walls of rabbits, and explored the role of airway wall injury in the formation of benign airway stenosis in rabbits.Benign airway stenosis is caused by various non-malignant tumor lesions, including tuberculosis, benign airway tumors, laryngomalacia or tracheomalacia, and iatrogenic laryngotracheal stenosis (iLTS). In the past, tuberculosis was considered the leading cause of benign airway stenosis in ChinaTwenty-eight 4-month-old New Zealand white rabbits, weighing 2.5–3.5 kg, from Suzhou Huqiao Biotechnology Co., LTD. , were randomly divided into four groups (7 rabbits/group): A, B, C, and D. Random numbers were generated using the standard = RAND function in Microsoft Excel. All protocols in this study were approved by the Committee on the Ethics of Animal Experiments of Dushu lake hospital to Soochow University Animal, Suzhou, China (IACUC permit number: 220048), in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . All methods were carried out in accordance with the ARRIVE guidelines and regulations. We employed four different tracheal injury methods, and rabbits were groups as follows: Group A (n = 7), tracheal mucosa was slightly damaged by an ordinary brush through tracheoscopy; Group B (n = 7), tracheal mucosa was severely damaged by a nylon brush after tracheotomy; Group C (n = 7), tracheal cartilage was injured with a vascular clamp after tracheotomy; and Group D (n = 7), tracheal mucosa was injured with a nylon brush and the tracheal cartilage was damaged by a vascular clamp. Severe mucosal injury was defined as obvious mucosal floating when the brush was immersed in normal saline after use.All rabbits were fasted for 6 h before surgery, intramuscularly anesthetized with 0.1 mL/kg of xylazine hydrochloride, and placed in the supine position on the operating table. After anesthesia, we intubated group A rabbits to the glottis using an endotracheal tube with an inner diameter of 6.5 mm in order to avoid destruction of the bronchoscope by the rabbit molars  and computed tomography (CT). At the end of the examination, the experimental rabbits were sacrificed by air embolization, and larynx and trachea specimens were obtained. The specimens were fixed in 10% buffered formalin, and some fresh tracheal specimens were placed in a freezer at −80 °C.19. The degree of tracheal stenosis was evaluated using the following two indexes: the transverse diameter, and the longitudinal diameter of the trachea. The former index (%) is defined as20:21.Thin-slice scans were performed on a Philips 64-row 128-slice CT . The scanning parameters were as follows: layer acquisition thickness: 1 mm, tube voltage: 120 kV, and tube current: 78 mA. The inner diameter of the trachea was measured in a fat window P-value < 0.05 was considered statistically significant.Results statistical analysis was performed using SPSS 23.0 software . Fisher's exact test was used to compare stricture formation rates between groups. The degree of stenosis was compared using the Mann–Whitney. A Obtained from the Medical Ethics Committee of Dushu lake hospital to Soochow University (220048).Comparison of the incidence of stenosis Fig. BP < 0.05), and there was no difference between Groups A and B (P > 0.05). The degree of stenosis in Groups C and D was 16.59–76.29% and 50.59–87.42%, respectively. There was no difference between Groups C and D (P > 0.05).The total tracheal stenosis rate in the four groups was 57.14% (16/28). The airway stenosis rates of group A, B, C and D were 0.00%, 28.57%, 100% and 100%, respectively. The formation rate of airway stenosis in Groups C and D was higher than that in Groups A and B. Groups C and D were significantly different from Groups A and B , some layers projecting inward, and discontinuity of the cartilage layer . In group B, the tracheal wall of the 5 rabbits without tracheal stenosis was cut and sutured along the smooth muscle and elastic fibers between the cricoid cartilage, but no stenosis was found by bronchoscopy, HRCT, EB-OCT and tracheal anatomy. It can be seen that simple tracheotomy and incision suture do not cause stenosis of the rabbit tracheal. In group A and group B, the tracheal mucosa was injured with a brush. The postoperative pathology showed that the mucosal surface of the rabbits without tracheal stenosis was pseudostratified ciliated columnar epithelium, and the cartilage structure was intact, indicating that the tracheal mucosa could regenerate after simple acute injury, and there was no stenosis formation with the repair of the mucosa. The histopathological results of rabbits with tracheal stenosis showed that when the tracheal wall was damaged to the cartilage layer, the ability of mucosal regeneration was poor, a large number of inflammatory cells infiltrated in the submucosa, capillaries increased, granulation tissue formed, and fibroblasts proliferated. EB-OCT and tracheal histopathology showed that cartilage injury was a key factor in the formation of benign tracheal stenosis. This is consistent with the results of previous studies31. Hillel et al.4 showed that compared with non-scar epithelium, the epithelium within iSGS and iLTS is morphologically abnormal. Although both iSGS and iLTS have reduced epithelial thickness, ciliary cells, and secretory cells, only iSGS had signifificant increases in pathological basal cell expression.This suggests that the epithelium plays an important role in the pathogenesis of both types of stenotic fibrosis. In this experiment, the rabbit tracheal mucosa can regenerate after acute injury alone, but the ability of mucosal regeneration is weakened when there is both cartilage and mucosal injury. Chronic, persistent damage to the mucosa involving the cartilage may result in airway stenosis.The degree of stenosis in group C and group D were 16.59–76.29% and 50.59–87.42%, respectively. Although there was no significant difference between the two groups, the proportion of grade 3 or above stenosis was 42.86% in group C and 100% in group D. This suggests that the formation of tracheal stenosis may be related to the function of tracheal mucosa. An intact tracheal mucociliary muco-system barrier removes exogenous stimuli; however, mucosal injury reduces clearance capacity. A mouse model of airway obstruction has shown that airway epithelial cells can regulate the growth of fibroblasts and prevent the overgrowth of fibroblasts from causing lumen obstructionThe tracheal stenosis was mainly caused by granulation tissue hyperplasia at 1 week after operation, and scar contracture was observed in some rabbits at 4 weeks after operation. This was also confirmed by gross anatomy. This suggests that we can choose different types of stenosis according to different time points for animal studies of benign airway stenosis, and provide different types of stenosis animal models for the study of interventional therapy of benign airway stenosis.In conclusion, cartilage injury is the key factor in the formation of benign airway stenosis in rabbits. Mucosa plays a certain role in the formation of airway stenosis, but acute injury of airway mucosa alone is unlikely to cause airway stenosis, and combined with cartilage injury may aggravate the degree of airway stenosis. Chronic, persistent damage to the mucosa with involvement of cartilage can cause airway stenosis.


Bunyavirales pose a continuous background threat to global health, but the fact remains that they are usually neglected and their distribution is still ambiguously known. We aim to map the geographical distribution of Bunyavirales viruses and assess the environmental suitability and transmission risk of major Bunyavirales viruses in China.Viral pathogens belonging to the order Bunyavirales viruses detected in humans, animals and vectors from multiple sources, to update distribution maps of them across China. In addition, we predicted environmental suitability at the 10 km × 10 km pixel level by applying boosted regression tree models for two important Bunyavirales viruses, including Crimean-Congo hemorrhagic fever virus (CCHFV) and Rift Valley fever virus (RVFV). Based on model-projected risks and air travel volume, the imported risk of RVFV was also estimated from its endemic areas to the cities in China.We assembled data on all Bunyavirales viruses in China from January 1951 to June 2021. Nineteen viruses were shown to infect humans, including ten species first reported as human infections. A total of 447,848 cases infected with Bunyavirales viruses were reported, and hantaviruses, Dabie bandavirus and Crimean-Congo hemorrhagic fever virus (CCHFV) had the severest disease burden. Model-predicted maps showed that Xinjiang and southwestern Yunnan had the highest environmental suitability for CCHFV occurrence, mainly related to Hyalomma asiaticum presence, while southern China had the highest environmental suitability for Rift Valley fever virus (RVFV) transmission all year round, mainly driven by livestock density, mean precipitation in the previous month. We further identified three cities including Guangzhou, Beijing and Shanghai, with the highest imported risk of RVFV potentially from Egypt, South Africa, Saudi Arabia and Kenya.Here we mapped all 89 species of Bunyavirales viruses are widely distributed in China, and the two major neglected Bunyavirales viruses including CCHFV and RVFV, both have the potential for outbreaks in local areas of China. Our study can help to promote the understanding of risk distribution and disease burden of Bunyavirales viruses in China, and the risk maps of CCHFV and RVFV occurrence are crucial to the targeted surveillance and control, especially in seasons and locations at high risk.A variety of The online version contains supplementary material available at 10.1186/s40249-022-00993-x. Bunyavirales is a large RNA virus order and comprises more than 560 viruses divided into twelve families using data collected up to June 30, 2021  lacked geographical information of viruses or clinical information of cases, (ii) had unspecified details , or (iii) other irrelevant studies including efficiency trails of virus infections, drug or vaccine trials, system reviews, or meta-analysis . Two authors (AYT and YYZ) conducted the initial searches independently and screened the retrieved studies following the inclusion and exclusion criteria as defined above. All conflicts of opinion and uncertainties were discussed and resolved by consensus with a third author (TLC). Some literature only identified the viruses in a broad scope, such as using “hantavirus” in lieu of reporting the specific genospecies, and thus we referred to these viruses as unclassified . The detailed procedures of viral data extraction and management were shown in the Additional file Studies were eligible if they described laboratory detections of sis Fig. . A recorIn addition, we supplemented another database of RVFV occurrence in RVF endemic areas in Africa and the Arabian Peninsula  from theHyalomma asiaticum presence in China was also included, because H. asiaticum was a major vector of the CCHFV in China .Forty-two explanatory covariates were chosen, mainly including meteorological data, volume of water in soil, surface runoff, minimum distance to the nearest bodies of water, elevation, land cover, Normalized Difference Vegetation Index (NDVI), mammalian richness, livestock density, and population density on global range. These covariates were used due to their potential effect on the environmental suitability for CCHFV and RVFV transmission based on the previous studies , 21–28. Bunyavirales viruses at the city level if data were available or at the province level otherwise. Data included the overall number of human confirmed cases, the overall positive rate of humans with serological evidence, and the distribution of vectors and both livestock and wild animals. We also summarized information regarding the first identification of Bunyavirales virus infections in China and drew the matrix diagram of these Bunyavirales viruses and their infected vectors and animal hosts. It should be mentioned that PCR detection technology was not popularized in China until the twentieth century and the detection results of viruses during this period were mostly obtained by serological detection. As such, there is the possibility of false positive results resulting from immune cross-reactions. We have flagged the records obtained by serological testing on the results of this study.We summarized and mapped the distribution of 2 were excluded in the modeling analysis due to the potential bias caused by uneven spatial scale. The locations of human cases and infections with CCHFV detected in animals and vectors by molecular or isolation assay records were included as presence data, but serological records in humans and animals were excluded due to the unguaranteed specificity of serological testing. The pseudo-absence data was generated by random sampling, the number was set to 15 times the presence data, and a weight of 1/5 was assigned.The BRT models were used to examine environmental suitability of CCHFV occurrence in China. The algorithm of logistic structure was used to fit the presence/absence of CCHFV at the 10 km × 10 km pixel level in the BRT models. We divided all CCHFV records in humans, animals or vectors into point data with exact coordinates or polygon data with non-exact coordinates. The former was located by a 10 km × 10 km pixel for points, and the latter by administrative districts, such as counties, cities or provinces. The point data was directly connected with the explanatory covariates at the 10 km × 10 km pixel level, and the polygon data was connected with mean value of covariates at all 10 km × 10 km pixels in this polygon. Polygon records larger than 4,000 kmTo reduce potential overfitting and improve predictive accuracy of the models, we first evaluated pairwise Pearson correlations to reduce multicollinearity among the 36 variables including 19 ecoclimatic, 10 environmental, and 7 biological covariates , and made projections using explanatory covariates from China to examine the environmental suitability in comparison. The occurrence of RVF was closely tied to meteorological conditions, such as the impact of El Niño on the RVF outbreak, and the impact of mosquitoes as important vectors of RVF. Thus, we established the BRT models on a month-year scale from 2000 to 2020, and included the monthly mean in temperature, precipitation, volume of water in soil, and surface runoff as covariates in the model  maps and calculated the area and population at risk for both viruses. The thresholds of CCHFV and RVFV models were determined by the cut-off values at which the Youden index of the test set was the maximum. We also calculated and mapped the number of months with environmental suitability for RVFV per year at the 10 km × 10 km pixel level. Considering that the suitable temperature for mosquitoes' survival from eclosion to adult emergence is at least 15 ℃, we adjusted the risk maps to prevent overestimation by converting all pixels with monthly temperatures of the previous month below 15 ℃ to no-risk . In addiAll analysis was performed in R 3.6.3  and ArcGIS 10.7 .Bunyavirales virus occurrence. An additional 1972 records of SFTS with 16,339 cases and 27,893 records of HFRS with 431,010 cases were collected from CISDCP, and 1389 unpublished records of viral sequences with geographic information were extracted from GenBank. In sum, 32,186 records were identified for this analysis  increased dramatically since 2010, and Hubei (36 species), Zhejiang (17 species), Yunnan (10 species) and Xinjiang (8 species) were the 4 provincial level administrative divisions (PLADs) with the largest number of newly-identified viruses reported , Songling virus (SGLV), Tacheng tick virus 1 (TcTV-1) and Tacheng tick virus 2 (TcTV-2)], 4 viruses of Hantaviridae , 4 viruses of Peribunyaviridae , 4 viruses of Phenuiviridae  and 2 viruses of Arenaviridae   were the most frequently determined illness with 431,010 cases reported in 31 PLADs of the mainland of China, followed by SFTSV with 16,339 cases in 26 PLADs, CCHFV with 331 cases in Xinjiang Autonomous Region of northwestern China  and fatigue (7.5–52.9%) , mosquitoes (11) and mites (10) acting as the main vectors, followed by spiders (3), sandflies (1) and other vectors (11)  , also scattered over the whole nation  of 0.963  in Xinjiang [Bunyavirales viruses. Shandong province is located in the North China Plain with more grassland vegetation and rich plant and animal resources. Shandong province was proved to be a high-suitable area for Haemaphysalis longicornis presence, which was the most important vector of SFTSV in China [H. longicornis were mainly concentrated in Liaoning province of northeastern China, Jiangsu and Zhejiang provinces in the eastern coastal areas, and some areas in central China, which was also consistent with the geographical distribution of SFTSV [Bunyavirales viruses. The distribution of mosquitoes carrying Bunyaviridae viruses was relatively scattered, except in northeastern and southern China. With global warming, mosquitoes have longer life cycle and larger range of activities. In addition, the risk of mosquito-borne infectious diseases is also increasing due to the increase of population density due to rapid urbanization and the frequent population flow under economic globalization, displaying the need for the monitoring of mosquitoes and mosquito-borne viruses.Ticks carried the highest variety of Xinjiang , and higXinjiang . These fin China . In addiof SFTSV . TherefoNairoviridae and Phenuiviridae were mainly carried by ticks, Peribunyaviridae mainly carried by mosquitoes, and Hantaviridae and Arenaviridae mainly carried by Rodentia. Hantavirus and SFTSV have been determined from over two vector types, which means that both have more potential for transmission, more difficulties to control, and higher risks to humans, showing a greater need for their consideration. In addition, we found that the Bunyavirales viruses carried by ticks were not detected in mosquitoes, and the converse was also true. One study examined the putative receptor-binding domains of tick-borne and mosquito-borne flaviviruses, the envelope (E) protein domain III (D3), and found that tick-borne recombinant D3s (rED3s) exhibited different spectral properties distinct from mosquito-borne viruses, that tick-borne viruses are more stable than mosquito-borne viruses [There were also significant differences in the types of viruses carried by different vectors, among which  viruses . Flavivi viruses . Thus, wBunyavirales have caused a variety of diseases and attracted much attention. Our results showed that except for HFRS and SFTS, the two widely distributed infectious diseases caused by Bunyavirales viruses in China, other Bunyavirales virus-related infectious diseases were mainly distributed in Xinjiang, northeastern and central China  in 2015 [It is worth emphasizing that of the human cases included in our study, there were 5 viruses reported only once in China, including BJNV 68 cases) , SGLV , we updated the knowledge regarding their ecological niche and distribution, and for the first time mapped the seasonally potential-risk areas of RVFV occurrence. The risk maps created by this study could guide future refinement of surveillance of CCHFV and RVFV in humans, vectors and animals, especially at targeted high-risk times and regions, and provide guidance for when and where the local government should remain vigilant for potential spread or imported events.Our study provides a full picture of the distribution of all Additional file 1. Additional information includes Methods, 14 Figures, 6 Tables and References. Table S1. The specific references for all 89 Bunyavirales viruses in China and Rift Valley fever virus in Africa and the Arabian Peninsula. Table S2. Search terminology used for the systematic review. Table S3. Information of explanatory covariates sources in this study. Table S4. Description of 42 potential explanatory covariates used in the modelling efforts. Table S5. Clinical characteristics of human cases infections with different viruses (species) belonging to the order Bunyavirales in China. Table S6. The model-predicted areas and population sizes at risk by monthly for RVFV occurrence in China. Fig. S1. The flow diagram of data collection for Rift Valley fever virus. Fig. S2. Recorded and predicted risk distribution of H. asiaticum presence in China. Fig. S3. Correlation matrix for 36 covariates in CCHFV model. Fig. S4. Correlation matrix for 18 covariates in RVFV model. Fig. S5. Distribution of the first identification site of 89 Bunyavirales viruses in China. Fig. S6. Geographical distributions of Bunyavirales viruses detected in animals. Fig. S7. The relationship matrix of Bunyavirales virus species and involved animals. Fig. S8. ROC curves and AUC values of CCHFV model. Fig. S9. The marginal effect of explanatory covariates for CCHFV occurrence risk in the model. Fig. S10. Recorded distribution of RVFV occurrence in Africa and the Arabian Peninsula. Fig. S11. ROC curves and AUC values of RVFV model. Fig. S12. The marginal effect of explanatory covariates for RVFV occurrence risk in the model. Fig. S13. Mean environmental suitability and monthly imported risk for RVFV from January to June in China. Fig. S14. Mean environmental suitability and monthly imported risk for RVFV from July to December in China.  Additional file 2. The dataset for CCHFV records in China used for BRT models.Additional file 3. The dataset for RVFV records from 2000 to 2020 in Africa and the Arabian Peninsula used for BRT models.


Background: Climate change results in warmer air temperatures and an uncertain amount and distribution of annual precipitations, which will directly impact rainfed crops, such as the grapevine. Traditionally, ancient autochthones grapevine varieties have been substituted by modern ones with higher productivity. However, this homogenization of genotypes reduces the genetic diversity of vineyards which could make their ability to adapt to challenges imposed by future climate conditions difficult. Therefore, this work aimed to assess the response of four ancient grapevine varieties to high temperatures under different water availabilities, focusing on plant water relations, grape technological and phenolic maturity, and the antioxidant capacity of the must. Methods: The study was conducted on fruit-bearing cuttings grown in pots in temperature-gradient greenhouses. A two-factorial design was established where two temperature regimes, ambient and elevated (ambient + 4 °C), were combined with two water regimes, full irrigation and post-veraison deficit irrigation, during fruit ripening. Results: There were significant differences among the ancient varieties regarding plant water relations and fruit quality. Conclusion: This research underlines the importance of evaluating the behavior of ancient grapevine varieties that could offer good options for the adaptation of viticulture to future climate conditions. Under this situation, associated variables determining the weather are also modified, resulting in both warmer air temperatures and an uncertain amount and distribution of annual precipitation. The IPPC data recorded in 2022 indicate that, in the Mediterranean area, air temperature and heat waves will increase throughout the 21st century above the global average, precipitation will decrease between 4 and 22%, episodes of torrential rains will increase in the northern part and droughts will be more prevalent in most areas. As a result, the increased risk of unexpected drought periods will impact directly most cultivated species, mainly in rainfed crops, such as the grapevine [According to the Intergovernmental Panel on Climate Change , climaterapevine . Air temrapevine ,6. Moreorapevine , low acirapevine ,9,10; alrapevine ,12. In southern Europe, the grapevine has been traditionally grown under rainfed conditions, thus leading to plants with low yield and low vigor but with high fruit quality. However, the growing episodes of water scarcity associated with climate change are modifying this procedure and the area of irrigated vineyards has exponentially increased in the last two decades to mitigate the negative impact of increasingly severe and unpredicted droughts on grapevine quality ,3. For iGiven the climate change predictions of increasing air temperatures and more frequent drought episodes, the selection of genotypes with better adaptability to environmental changes will be of great interest for perennial crops, such as grapevine . TraditiThe volumetric soil water content was monitored from veraison to berry maturity in the pots of the full irrigation (FI) and deficit irrigation (DI) treatments . Data ofpd measured in plants subjected to DI from veraison to maturity compared with full-irrigated plants (pd were recorded at the maturity (E-L 38 stage) for ambient (ATDI) and elevated (ETDI) temperature, respectively. The elevated temperature also modified plant water status this factor being especially relevant in Graciano (GRA) and Tinto Velasco (TV), in which Ψpd was always significantly lower in plants subjected to ETDI compared with ATDI.Four genotypes with oenological potential were chosen for the study . Irrigatd plants . Under DUnder our experimental conditions, the varieties exhibited remarkable diversity in the length of the ripening cycle and plant growth, as well as bunch and berry traits . TEM andRegarding the behavior of the grapevine varieties under the climate change scenarios assayed, phenology, plant growth, and most of the fruit characteristics were not affected by increased temperature and/or DI in TEM . Only elp ≤ 0.01), tonality , TPI , and total anthocyanins . In addition, the effects of high temperature had different intensities depending on the variety, as indicated by the significant interactions found for colour density , TPI , total anthocyanins , and antioxidant capacity . Similarly, varieties also responded in a different way to DI as highlighted by the significant interaction (V × I) for titratable acidity (p ≤ 0.01), tonality (p < 0.05), TPI (p < 0.05), and total anthocyanins (p ≤ 0.01). As expected, the variety was the main factor in modifying berry properties and the antioxidant capacity of must, the teinturier GNO being the cultivar with the highest concentration of total anthocyanins, TPI, and antioxidant capacity . Altogetp < 0.05) for titratable acidity. DI, as a single factor, hardly affected the phenolic maturity of the TEM. The temperature was the main factor that contributed to the decrease in total anthocyanins and cellular extractability of anthocyanins (EA), although such effects were more pronounced under water deficit (ETDI) .Regarding the responses of each variety, in TEM, the parameters related to technological maturity were more affected by temperature than by the irrigation level . High tep < 0.05) and for titratable acidity . In this variety, phenolic maturity was mainly modulated by irrigation, increasing the accumulation of total anthocyanins under water deficit (ATDI and ETDI treatments) regardless of the temperature regime. The concentration of extractable anthocyanins also improved in those plants grown under ETDI while the highest EA was obtained under ATDI. In TV, analyses of technological maturity showed that temperature was the main factor increasing must pH, whereas DI contributed to increased TSS and titratable acidity . The twoBerry attributes of GRA were clearly modified by both temperature and irrigation, acting alone or in combination . RegardiConcerning GNO, all the treatments with elevated temperature and DI resulted in high must pH, low titratable acidity, and low total anthocyanins , while EThe total antioxidant capacity was assessed by the mean of the DPPH assay and showed that the temperature rise was the main factor accounting for the enhanced total antioxidant capacity of berry extracts of TV . However2. To find out more about some of these varieties, the present study analyzes their response to deficit irrigation under warm temperatures, in terms of plant water relations, the technological and phenolic maturity of berries, and must antioxidant properties. The predicted increase in air temperatures and in the frequency and intensity of droughts are becoming challenges for most of the wine regions of southern Europe . Climatepd of plants subjected to DI ranged between −0.3 and−0.8 MPa  and at stage E–L 38 (in TEM and GNO), suggesting that the former varieties reacted earlier to soil water restriction than the later ones, possibly due to their higher leaf area  ,22, due Phenolic maturity includes not only the overall concentration of phenolic compounds but also their structure and capacity to be extracted from grapes during winemaking . AdequatPhenolic compounds and their antioxidant properties have generated significant interest related to human health benefits . In our It has been suggested that the application of DI can help to synchronize the technological and phenolic maturity of grapes . In our Vitis vinifera L.) varieties included in this study were selected from more than 65 genotypes recovered, from 2002 to 2020, in old vineyards (older than 65 years) that were identified using molecular markers [The grapevine  and placing them in a warm bed (27 °C) in a cold room (4 °C) for 30 days. After rooting was successful, the cuttings were planted in 0.8 L plastic pots containing perlite and peat (1:1 v:v) and then, they were transferred to a greenhouse where the environmental conditions were 25/15 °C and 50/90% relative humidity (day/night) regime and natural daylight  supplemented with high-pressure sodium lamps  to increase the photoperiod to 15 h with a minimum PPFD of 350 μmol m−2 s−1 at the inflorescence level. After 7–8 days bud-break took place and from this moment to flowering plant growth was controlled to leave one inflorescence and 4 leaves per plant. While flowering in Tempranillo (TEM), Tinto Velasco (TV), Graciano (GRA), and Grand Noir (GNO) started, respectively, on 4, 7 and 9 May 2021, the fruit set occurred on 11, 14 and 16 May 2021. The 400–500 mm long cuttings were prepared for fruit-bearing following the methodology developed by Mullins , which cv:v). Plants grew freely until reaching 16 leaves per plant, and then, the plants were regularly pruned to maintain an appropriate leaf area to fruit mass ratio for berry ripening [After the fruit set (Eichhorn and Lorenz (E-L) growth stage 27) , which tripening . Subsequripening . No planripening . Plants 3 H2O m−3 soil) × 100). In the DI treatment, plants were watered when the sensor value reached 50% of the value of FI  × 100) from veraison to maturity (E-L 38 stage). Previously, pot capacity was determined as water retained after free-draining water had been passed through the holes in the bottom of the pot. Plants were watered twice a day with irrigation doses ranging from 2.5 L to 3.5 L per pot according to the plant’s needs according to the daily measurements of the EC 5 water sensor. The irrigation (both at the pre-treatment greenhouse and the TGGs) was carried out using a nutritive solution in accordance with viticultural requirements [When berries began to color and enlarge , plants within each temperature regime were subdivided into two groups that were subjected to different irrigation schedules: (1) plants under full irrigation (FI), and (2) plants that received 50% of the water given to FI plants from veraison to maturity (E–L 38 stage) . Soil moisture sensors  were placed in each pot. FI plants were maintained at ca. 80% of pot capacity  ambient temperature (T) and full irrigation (ATFI); (2) ambient temperature and deficit irrigation (ATDI); (3) elevated temperature (T + 4 °C) and full irrigation (ETFI), and (4) elevated temperature and deficit irrigation (ETDI). There were 5 biological replicates (plants) for each combination of temperature, irrigation, and variety. Plants were kept in the TGGs until a commercial maturity of berries was attained (E-L 38 stage). The minimum, mean, and maximum daily temperatures were registered throughout the experiment. Weather data were obtained from the Pamplona Airport station  and the reference period from 2001 to 2021 . Data shParticularly, September was warmer with the maximum and minimum daily air temperatures being 2.5 and 2 °C higher than the average registered for the same month during the period 2001–2021. Moreover, treatments of ambient (AT) and elevated temperature (ET) experienced 11 and 24 days, respectively, at a maximum temperature above 35 °C . It is cpd) was measured with a SKYE SKPM 1400 pressure chamber  on fully expanded leaves just before irrigation. Determinations were made at three stages of berry ripening: (1) seven days after veraison (E–L 36 stage); (2) fourteen days after veraison (E–L 37 stage) and (3) fruit maturity (E-L 38 stage). Afterwards, every plant was harvested when the ratio of sugars to acidity ranged between 4 and 6. The main phenological phases of berry ripening were registered as the number of days from fruit set (E–L 27 stage) to veraison (E–L 35 stage), and from veraison (E–L 35 stage) to maturity (E–L38 stage). Predawn leaf water potential . Afterwards, the length and weight of bunches were recorded, and the bunches were destemmed to record the number of berries and total berry weight. Bunch compactness was calculated as the quotient between the weight and the squared length of the bunch. From each plant, 10 berries were separated into skin and flesh to calculate the relative skin mass as the quotient between skin fresh matter (FM) and whole berry FM. The rest of the berries were stored at −20 °C for subsequent analyses. −1 tartaric acid). For each plant, a sample of 20 berries was pressed and extracts were centrifuged at 4100× g at 4 °C for 10 min. The supernatant was used for the following determinations: TSS measured with a temperature-compensating refractometer ; must pH measured with a pH meter  standardized to pH 7.0 and 4.0; titratable acidity measured by titration with NaOH [Parameters included in the technological maturity were berry total soluble solid (TSS) concentration (expressed as °Brix), pH, and total acidity  by reading absorbance at 520 nm after reaction with bisulfite [Grape phenolic maturity parameters included total and extractable anthocyanins, cellular extractability (EA) of anthocyanins, total polyphenol index (TPI) and phenolic seed maturity (SM). A 20-berry sample per plant was utilized for the analysis of anthocyanins, total phenols, and chromatic properties. For berry anthocyanin extraction, two samples of the non-filtered, triturated berry homogenate were macerated for 4h at pH 1 (hydrogen chloride) and pH 3.2 (tartaric acid), respectively . Then, tisulfite . The pH isulfite . The celisulfite . The total polyphenol index (TPI) was estimated from the absorbance reading at 280 nm in the supernatant obtained after maceration at pH 3.2 . During −1). Total antioxidant capacity was analyzed on the must samples used for technological maturity determinations by using the free-radical scavenging activity  assay . The reap < 0.05), the differences among groups were tested with Duncan’s posthoc test. Results were considered statistically significant if p < 0.05. Statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) software  version 21.0 for Windows. After establishing that the data met the assumptions of normality (Shapiro–Wilk’s test) and homoscedasticity (Levene’s test) with a threshold of 0.05, two and three-way analyses of variance (ANOVA) were performed to assess the main effect of the factor variety, temperature, and irrigation level, and the interaction between them. When ANOVA was statistically significant , oenological potential, and antioxidant properties indicating that such two minor varieties stand out as alternatives to widespread varieties to be exploited. Our study underlines the importance of evaluating the behavior of the ancient grapevine varieties that could offer good options for the adaptation of viticulture to future climate conditions. However, studies carried out under controlled conditions in potted young plants have limitations to interpretation. Further studies under natural conditions are required before extrapolating the present results to field-grown ancient varieties tested. Moreover, the selection of resilient grapevine varieties to environmental constraints should be combined with adequate rootstocks whose root system architecture and root transcriptomic regulations make them tolerant to abiotic stresses.