Severe hypoglycaemia may pose significant risk to individuals with type 2 diabetes (T2D), and evidence surrounding strategies to mitigate this risk is lacking.Data was re-analysed from a previous randomised controlled trial studying the impact of nurse-led intervention on mortality following severe hypoglycaemia in the community. A Cox-regression model was used to identify baseline characteristics associated with mortality and to adjust for differences between groups. Kaplan-Meier curves were created to demonstrate differences in outcome between groups across different variables.A total of 124 participants  were analysed. In univariate analysis, Diabetes Severity Score (DSS), age and insulin use were baseline factors found to correlate to mortality, while HbA1C and established cardiovascular disease showed no significant correlations. Hazard ratio favoured the intervention  and in multivariate analysis, only DSS demonstrated a relationship with mortality. Comparison of Kaplan-Meier curves across study groups suggested the intervention is beneficial irrespective of HbA1c, diabetes severity score or age.While DSS predicts mortality following severe community hypoglycaemia in individuals with T2D, a structured nurse-led intervention appears to reduce the risk of death across a range of baseline parameters. Treatment intensification which aims to reduce hyperglycaemia decreases risk of microvascular complications and long-term macrovascular risk. However, intensification of glycaemic treatment increases hypoglycaemic exposure, which is believed to be associated with an increased risk of cardiovascular events.6 As such, guidelines suggest individualising HbA1c targets while also focusing on hypoglycaemic avoidance and acknowledging that more research in this area is required.Type 2 diabetes (T2D) is characterised by chronic hyperglycaemia, resulting in increased risk of vascular complications and reduced life expectancy.Recently, we conducted a randomised controlled trial (RCT) investigating the impact of a structured nurse-led intervention on mortality in people with T2D following severe hypoglycaemia. The intervention, which focused on patient education, initiation of self-monitoring of blood glucose, and pragmatic medication changes resulted in a reduction in all-cause mortality. Mean baseline HbA1c tended to be higher in those who survived till study completion compared with patients who died at any point, suggesting that outcome can be influenced by pre-treatment patient characteristics. However, the use of individual metrics in isolation, such as HbA1c, fails to capture the complexity of T2D and have limited application for risk stratification and predicting clinical outcome. Conversely, tools that combine routine clinical metrics to better capture global comorbidity and overall diabetes severity have been shown to yield greater predictive value than individual proxy indicators, including HbA1c.Therefore, the aim of this exploratory post-hoc analysis was to understand risk factors for mortality following severe hypoglycaemia in T2D and investigate possible heterogeneity in response to the intervention, which would help to identify subgroups of patients who show enhanced, or reduced, benefits from nurse-led support. . The RCT received ethical approval from the UK National Health Service Health Research Authority (reference 100244), and all participants gave written informed consent. Detailed information about study procedures have been published previously. In brief, patients with diabetes who suffered an episode of severe community hypoglycaemia requiring the assistance of emergency services in the area surrounding Leeds, UK, were randomised to either standard care or a structured nurse-led intervention. The intervention centred on the initiation of self-monitoring of blood glucose, education surrounding the avoidance of hypoglycaemia and adjustment of glycaemic medication, as appropriate. The main intervention took place in the 3 months following recruitment with further limited involvement of the research nurse for a further 9 months. Data surrounding mortality were collected using electronic records, and death, including cause, was confirmed using death certificates, a statutory requirement in the UK. In the present analysis, we included patients with T2D randomised to standard care and intervention arms, given that the intervention failed to show an effect on those with type 1 diabetes (T1D).We performed an exploratory post-hoc analysis using data from a previously published RCT The severity score is a composite of hard clinical endpoints and biochemical variables and represents an assessment of existing, as well as risk of developing, diabetes-related complications. Scores range from 1 to 4 with 4 categorised as the greatest degree of diabetes severity. Scores were then compressed to provide dichotomous variables (groups 1 + 2 and 3 + 4). The matrix used to calculate diabetes severity score is available as Supplementary material, which is taken from the original journal article by Gibson et al.Participants were stratified into subgroups using an established Diabetes Severity Score which has previously been shown to be associated with mortality and healthcare costs.p < 0.2 from the univariate analysis were put into the multivariate analysis for further adjustment. Statistical significance was determined as p < 0.05 for all analyses. Graphpad (Prism 9) USA was used to construct Kaplan-Meier curves comparing survival between groups and a logrank test used to ascertain differences in trends among curves.Data analysis was performed using SPSS version 27  and assessed for normality. Continuous variables are reported as mean ± SD and categorical variables reported as frequency (%). Differences between dichotomised variables were assessed using independent t-tests or chi-square test. We employed univariate and multivariate analyses using a Cox-regression model to identify factors associated with an increased risk of mortality. Only factors with p value = 0.52.) Additional information on baseline characteristics is available in A total of 124 participants were included in this analysis. Of these, 60 were in the intensive group and 64, the standard of care group. Of those in the intensive group, 33 (55%) were male, mean age was 74.2 (± 10.7) years and mean HbA1c was 58.5 (± 13.4) mmol/mol. Of those in the standard of care group, 37 (57.8%) were male, mean age was 74.8 (± 10.2) years and mean HbA1c was 60.0 (± 16.2) mmol/mol. Mean follow-up for the intensive study arm was 40.9 (± 15.5) months and in the standard of care arm 39.0 (± 15.9) months, which was not significantly different  in favour of the intervention  and 20 (33%) deaths occurring, respectively. Using a Cox-regression model, a comparison between groups was made after adjustment for factors shown to correlate with mortality , with HR = 0.68  and Kaplan-Meier curves were constructed to depict differences in mortality between study groups. In order to depict differences, severity scores 1 + 2 and 3 + 4 were grouped together as were quartiles of age and baseline HbA1c. A logrank test was used to compare trends between curves, with significance shown for severity score, HbA1c quartile and age quartile. This data is displayed in  The main cause of death in the whole group was infections, followed by cardiovascular disease. There was a clear difference in cardiovascular death, comparing the two study arms with a single cardiovascular death occurring in the intervention arm (1.7%) vs 12 (18.8%) in the standard arm (p = 0.002). The intervention had no effect on other causes of death including infection, renal disease or dementia.We studied cause of death across the two groups using death certificates, and full details can be found elsewhere.Severe hypoglycaemia in those with type 2 diabetes necessitating the assistance of emergency services results in high mortality, which can be modulated following a simple and pragmatic intervention focussed on patient education, glucose monitoring and medication review. From this exploratory analysis, we show that some pre-treatment characteristics influence mortality following severe hypoglycaemia, yet the studied intervention was beneficial even following adjustment for such variables. Overall, single variables do not adequately predict which patients may benefit from intervention with classical markers of risk, such as HbA1c and the presence of established cardiovascular disease, failing to provide significant impact on mortality, albeit in a relatively small sample size. The strongest predictor of mortality was a composite Diabetes Severity Score which incorporates hard clinical endpoints and biochemical data from individuals. and suggest the potential utility of severe hypoglycaemia risk stratification for assessing treatment response to subsequent intervention.These results highlight the significant risk to those with type 2 diabetes following severe hypoglycaemia; the benefit of a relatively straightforward and non-invasive intervention; and raise the question as to whether diabetes severity score, calculated through a validated scoring system, should be incorporated into the management of such patients, prioritising higher risk individuals. Our findings extend recent work investigating the impact of multimorbidity on hypoglycaemia riskImportantly, this exploratory analysis suggests that the intervention is beneficial in all study participants, regardless of baseline characteristics, although statistical significance was not always reached due to small sample size. Given the small sample size and the fact that this was a single centre study, larger multicentre studies are urgently needed to provide more robust data that can be incorporated into national guidelines in order to improve outcome in this high-risk group of patients with severe hypoglycaemia.These data add to the existing literature in the field and also provide reassurance that the intervention is beneficial across a wide range of patients and is not limited to certain subgroups. In particular, the reduction in cardiovascular mortality in the intervention arm of the study carries an important clinical message and emphasises the need for adequate glycaemic support in the older population with diabetes. However, it should be acknowledged that this work was not a definitive outcome trial but supports the need for further research in this field through large multicentre studies with the ultimate aim of optimising care in this vulnerable group of individuals with diabetes.A strength of this study is the uniformity of patient care, the completeness of data regarding baseline characteristics and the simplicity of study intervention. There are a number of drawbacks that should be acknowledged, including small sample size and single geographical location, preventing generalisation to different healthcare systems and different ethnic groups, particularly as > 80% of participants were of Caucasian ethnicity. There are also numerical differences in study groups despite randomisation, namely, a higher percentage of participants in the standard care group who had established cardiovascular disease and who had the highest diabetes severity score of 4, although higher BMI and longer diabetes duration in the intervention group may have balanced out the risks.For patients with T2D sustaining an episode of severe hypoglycaemia in the community, a structured intervention appears to be helpful in all patients regardless of their baseline risk factors and Diabetes Severity Score. However, those with higher diabetes severity score appear to be at particular risk and may require prioritisation of the intervention. A large multicentre randomised controlled trial is urgently needed to provide conclusive data that a similar intervention should be part of routine clinical practice.Click here for additional data file.Supplemental Material, sj-pdf-1-dvr-10.1177_14791641211067415 for Risk factors associated with mortality in individuals with type 2 diabetes following an episode of severe hypoglycaemia. Results from a randomised controlled trial by Sam M Pearson, Noppadol Kietsiriroje, Beverley Whittam, Rebecca J. Birch, Matthew D Campbell and Ramzi A Ajjan in Diabetes and Vascular Disease Research


Bradyrhizobium strains to enhance soybean productivity in different growing conditions of Northeast Germany. Three indigenous Bradyrhizobium isolates  were tested in combination with different soybean cultivars of different maturity groups and quality characteristics in one field trial and two greenhouse studies. The results showed a highly significant strain × cultivar interactions on nodulation response. Independent of the Bradyrhizobium strain, inoculated plants in the greenhouse showed higher nodulation, which corresponded with an increased N uptake than that in field conditions. There were significantly higher nodule numbers and nodule dry weights following GMF14 and GMM36 inoculation in well-watered soil, but only minor differences under drought conditions. Inoculation of the soybean cultivar Merlin with the strain GEM96 enhanced nodulation but did not correspond to an increased grain yield under field conditions. USDA110 was consistent in improving the grain yield of soybean cultivars Sultana and Siroca. On the other hand, GMM36 inoculation to Sultana and GEM96 inoculation to Siroca resulted in similar yields. Our results demonstrate that inoculation of locally adapted soybean cultivars with the indigenous isolates improves nodulation and yield attributes. Thus, to attain optimal symbiotic performance, the strains need to be matched with specific cultivars.Commercial inoculants are often used to inoculate field-grown soybean in Europe. However, nodulation efficiencies in these areas are often low. To enhance biological nitrogen (N) fixation and increase domestic legume production, indigenous strains that are adapted to local conditions could be used to develop more effective inoculants. The objective of this study was to assess the ability of locally isolated  Glycine max (L.) Merr.] consumption as food and feed for livestock in Europe has increased significantly in recent decades, although domestic production has increased marginally. The European Union encourages domestic grain legume production to reduce reliance on imports, to minimize the negative environmental impacts associated with intensive cereal production  ensures the utilization of atmospheric nitrogen (N), which translates into improved soil fertility, increased crop productivity, and an economically sustainable production system  to enhance soybean productivity under varying growing conditions in Northeast Germany in greenhouse and field studies.Thus, for the selection of effective strains for future use as commercial inoculants to improve soybean production, there is a need to test the efficacy of the isolated strains of Two greenhouse experiments and one field trial were conducted at the experimental station at the Leibniz Center for Agricultural Landscape Research, Müncheberg, Germany . Soils for both greenhouse and field studies were from sites with no previous soybean cropping history. The soil in the area is classified as Podzoluvisol to Arenosol and is located 62 m above sea level. The area is characterized by droughts in spring and summer. The mean annual precipitation is 533 mm, and the mean long-term annual temperature is 8.5°C.Bradyrhizobium isolates on nodulation and biomass development. Five soybean cultivars of different maturity groups, growth and quality characteristics, namely, Comandor (000), Merlin (000), Shouna (000), Sultana (000), and Siroca (00)  and compared with a non-inoculated control (Bradyrhizobium isolates were previously isolated from arable soils in Northeast Germany (Fehrow and Müncheberg). A 4 × 5 factorial combination involving Bradyrhizobium isolates and soybean cultivars, respectively, in a complete randomized design was set for the study. Each treatment was replicated 3 times.This greenhouse experiment was an initial screening intended to study the interaction effects of representative soybean cultivars of Central Europe and the different oca (00) , were te control . These aBradyrhizobium isolate was cultured in yeast extract mannitol (YEM) liquid medium for 5 days at 28°C. The cells were then concentrated and washed twice with sterile distilled water. Each bacterial suspension was adjusted to a concentration of 108 cells ml–1.Each Seeds of each cultivar were first surface-sterilized with 70% alcohol for 1 min, followed by 3% sodium hypochlorite for 2 min. The seeds were rinsed 8 times with sterile distilled water to remove traces of alcohol and sodium hypochlorite. Seeds were then dried in sterile towel paper.Each surface sterilized seed was inoculated by thoroughly mixing 65 ml liquid culture with 1 kg seeds and allowing them to dry at room temperature for 2 h before sowing. Sowing was carried out on 10th September 2019. Three healthy seeds of uniform size were sown in each box (10 cm × 8 cm × 8 cm) containing 1 kg soil and were later thinned to one plant per box of comparable height and vigor after 2 weeks. Soybean plants were grown for 40 days (V5 growth stage) at an average temperature of 22°C under a 16-h light and 8-h dark photoperiod as employed by Bradyrhizobium strains is impacted by varying soil moisture levels. Sultana, one of the cultivars that showed a moderate response to inoculation in greenhouse Experiment 1 in terms of nodulation and shoot biomass growth was used. The three Bradyrhizobium strains used in greenhouse Experiment 1 plus a reference strain (Bradyrhizobium diazoefficiens USDA110) were inoculated to Sultana under two moisture levels. Non-inoculated soybean seeds were used as a control.This study was conducted to evaluate the extent to which the symbiotic performance of the Seed sterilization and inoculum preparation were performed as explained in greenhouse Experiment 1. In this study, inoculation was performed by mixing 10 g autoclaved peat (141°C for 24 min) with 65 ml liquid culture to form a slurry. Peat was added to reduce the excessive softening of the seed coat, which affected germination in greenhouse Experiment 1. Then, 1 kg soybean seeds were thoroughly mixed with the slurry until each seed was sufficiently coated. Sowing was carried out on 13th June 2020 as explained in greenhouse Experiment 1. The pots were arranged in a complete randomized design and each treatment was replicated four times.Three days after sowing, two moisture levels namely well-watered conditions (irrigation at 80% soil moisture) and drought conditions (irrigation at 40% soil moisture) were imposed on the respective treatment until termination of the study (50 days after sowing). Irrigation drippers were installed in each pot, and moisture was supplied when necessary. The well-watered treatment received 70 ml of water 4–5 times a week. The drought treatment received 35 ml of water 1–2 times a week. Soil moisture conditions were monitored with a UMP-1 BT soil moisture sensor  . SimilarBradyrhizobium strain inoculation under field conditions. Three soybean cultivars adapted to Northeast Germany from the list of cultivars used in greenhouse Experiment 1 were selected for this study. They were tested in combination with four Bradyrhizobium strains  and a non-inoculated control. The treatments were laid out in a randomized complete block design with 5 × 3 factorial combinations of Bradyrhizobium and soybean cultivars, respectively, and each treatment was replicated six times. The trial was conducted in the 2020 cropping season (May–October) at the site where soil for both greenhouse experiments was collected.This study was intended to evaluate different soybean cultivar responses to –2 at a spacing of 37.5 cm between rows on 5th May 2020. To avoid cross-contamination, the plot seeder was thoroughly cleaned after each treatment with a different inoculant by following a modified approach of –1 before sowing.Soybean seed sterilization, inoculum preparation, formulation, and inoculation were performed as explained in greenhouse Experiment 2. Each plot size measured 11.5 m × 1.8 m, and alleys of 3.5 and 0. 5 m were left between blocks and plots, respectively. Soybean seeds were sown at 80 seeds mIn both greenhouse studies, plant shoots were cut with scissors at 1 cm above the soil surface. Then, the soil in each pot was emptied on a white sheet covered with transparent plastic material. The plant was carefully removed to obtain unharmed roots and nodules. All adhering soil particles were rinsed off with a stream of water, and nodules were gently detached from the roots. The number of nodules on the roots was manually counted. All fresh nodules and shoot samples were dried in an oven at 60°C for 48 h. The dry weights of all samples were determined with a weighing balance. Dry shoot samples were later milled for the determination of shoot N uptake.–1) and thousand grain weight (TGW) were determined at 86% dry matter. Shoot N analysis was performed by the dry combustion method using a CNS elemental analyzer (Leco Instruments GmbH). The crude protein and oil contents in the grains were measured with near-infrared reflectance spectroscopy (NIRS) .In the field trial, aboveground biomass samples were collected from two inner rows of each plot 0.5 m in length at the R3 growth stage. Five plants from the inner rows of each plot were randomly selected, and their roots together with nodules were gently excavated with a spade. The shoot dry weight, nodule number, nodule dry weight, and shoot N uptake were determined as was conducted in both greenhouse studies. At physiological maturity, all soybean plants were harvested with a plot combine harvester. Grain yield  via the Mehlich-3 extraction method . Soil pH Na4P2O7 .Analysis of variance (ANOVA) was performed using the generalized linear model procedure (GLM) of the software package SAS 9.2 (SAS Institute 2008) after checking for normality and homogeneity of variances. In the field study, nodulation parameters, shoot dry weight, N uptake, TGW, pod number, grain weight, protein content, and oil content were analyzed using the two-way ANOVA model below:where,Yikl = responses μ = intercept ia = the soybean cultivar effect (fixed effect) of the thi level of αk = the Bradyrhizobium strain effect (fixed effect) of the thk level of γγik = interaction effect of soybean cultivar by Bradyrhizobium strains of the thi level of α with the thk level of γαγl = the block effect (random effect) of the thl level of ββl = the restriction error associated with the thl blockδeikl = the random errorBradyrhizobium strain were considered fixed factors, whereas, in greenhouse Experiment 2, Bradyrhizobium strain and moisture regimes were modeled as fixed factors. All pairwise comparisons were performed using the Tukey-test (p ≤ 0.05). The effects of all factors and their interactions were assessed using the standard error of difference (mean SED).The same model was used to analyze the responses in greenhouse Experiments 1 and 2. However, the blocking effect was not considered in analyzing the responses in either greenhouse experiment. In greenhouse Experiment 1, soybean cultivar and Bradyrhizobium, and soybean cultivar × Bradyrhizobium interactions  and Siroca (26.1 g kg–1) had the highest shoot N content, while the lowest was in Merlin (23.7 g kg–1) irrespective of Bradyrhizobium inoculation.Shoot N content was significantly influenced by cultivar, riment 1 . Inoculariment 1 . GMM36 aBradyrhizobium inoculation but not by the moisture regime  and nodule dry weight (0.08 g plant–1) (–1) and nodule dry weight (0.09 g plant –1) were observed when Merlin was inoculated with GEM96  . The higth GEM96 . In contBradyrhizobium strain  irrespective of Bradyrhizobium inoculation  for human consumption and were within the range reported for early maturity soybean groups in Austria (Strain GEM96, GMF14, and USDA110 inoculation yielded acceptable protein contents (403–412 g kg Austria . The higBradyrhizobium. Our present study, which agrees with Bradyrhizobium strains that are tolerant to cold temperatures and drought and could potentially be employed to increase soybean BNF and yield in Central Europe. Our results also suggest the need to explore other areas in Europe with longer field-grown inoculated soybean cultivation histories to identify more adapted Bradyrhizobium symbionts to expand the pool of inoculum resources in Europe.The observed yield in the present study was lower than the average reported by Inoculation of soybean with the present isolates induced higher nodulation, which increased grain yield and protein content compared with the uninoculated control. In particular, the symbiotic performance of the isolates varied based on their combination with specific soybean cultivars under drought and well-watered conditions. There was consistently high nodulation and improved biomass growth in Merlin-GEM96 inoculation in drought conditions but not in well-watered conditions. This, however, did not translate into increased grain yield at harvest. Strain GEM96 inoculation consistently enhanced yield attributes and its combination with Siroca or Sultana resulted in increased grain yield to a similar extent as the standard USDA110 strain. Thus, GEM96 in combination with Siroca or Sultana is effective for grain production under drought-growing conditions in Northeast Germany.The original contributions presented in the study are included in the article/RO, KY, KA, and MR conceived and performed the experiments. RO performed all the analyses and wrote the manuscript with assistance from MH, DE, NO-O, and SB-K coordinated the transportation of isolates, and analysis, and provided insightful input into the manuscript write-up. All authors contributed to writing the final version of the manuscript.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.


Arterio-ureteral fistula (AUF) is an uncommon diagnosis, but potentially lethal. Although the number of reports has increased over the past two decades, the true incidence and contemporary urologists’ experience and approach in clinical practice remains unknown. This research is conducted to provide insight in the incidence of AUF in The Netherlands, and the applied diagnostic tests and therapeutic approaches in modern practice.A nationwide cross-sectional questionnaire analysis was performed by sending a survey to all registered Dutch urologists. Data collection included information on experience with patients with AUF; and their medical history, diagnostics, treatment, and follow-up, and were captured in a standardized template by two independent reviewers. Descriptive statistics were used.Response rate was 62% and 56 AUFs in 53 patients were reported between 2003 and 2018. The estimated incidence of AUF in The Netherlands in this time period is 3.5 AUFs per year. Hematuria was observed in all patients; 9% intermittent microhematuria, and 91% presenting with, or building up to massive hematuria. For the final diagnosis, angiography was the most efficient modality, confirming diagnosis in 58%. Treatment comprised predominantly endovascular intervention.The diagnosis AUF should be considered in patients with persistent intermittent or massive hematuria.The online version contains supplementary material available at 10.1007/s00345-021-03910-3. Arterio-ureteral fistula (AUF) is a rare but potentially lethal complication, where a direct connection between artery or vascular graft and ureter exists. First clinical presentation is often intermittent hematuria, without any additional symptoms, which frequently ceases without any treatment , 2. AUF Different risk factors for AUF have previously been described : a combiNeither the European Association of Urology (EAU) nor the American Urological Association (AUA) include recommendations on AUF diagnosis or treatment.We observed an increase in the incidence of AUF in our personal practice, which was confirmed by the reports in medical literature.The aim of the current study is to estimate true incidence in The Netherlands, contemporarily applied diagnostic, and therapeutical approaches.A cross-sectional questionnaire approach was applied, in which a questionnaire was sent to all registered Dutch urologists. We focused on the incidence in The Netherlands, and the contemporary diagnostic modalities and treatment options. Based on the results of the questionnaire and in the absence of guidelines, recommendations and algorithm for clinical urologic practice will be presented.n = 398) registered Dutch urologists in December 2018 via the Dutch Urology Association (NVU). A reminder was sent out in February 2019. Questions included years in practice, and the encounter with patients suggestive of AUF in the period 2003–2018 .A digital questionnaire was sent to all  by two independent reviewers (TL and KK). Details included patient details, and data on diagnostics and treatment.AUF was defined as the confirmed presence of an abnormal passageway between a ureter and any artery or a previously inserted vascular graft. Only cases of confirmed cases of AUF were included. Excluded from analysis were cases describing fistulas between the ureter and any venous structure, fistulas between the bladder and an artery, cases in which ureteral damage was observed but no true fistula, and fistulas between the intestinal part of an ileal conduit urinary diversion and an artery.End points were death or loss to follow-up. Death was defined as AUF specific (due to massive hemorrhage or infection directly post-diagnosis) or death due to other causes.The Ethics Committee of the University Medical Center Utrecht approved this study (reference number WAG/mb/19/042283). The Dutch Urological Association approved questionnaire distribution among urologists. The need for individual consent was waived.Descriptive statistics were used (SPSS version 27.0).A total of 247 urologists of the 398 registered urologists in The Netherlands completed the questionnaire, amounting to a response rate of 62%. The years of practice in this group ranged from 1 to 30 years, with a mean of 17.1 years.In total 56 AUFs were reported in 53 patients. The 6 AUFs in 6 patients diagnosed between 2003 and 2007 we presented earlier . The most frequent oncological diagnosis was bladder cancer in men (21%) and cervical cancer in women (18%). A medical history of (endo)vascular treatment was present in 33%. Indwelling ureteral stents were present in 29 patients (88%), reasons for placement were ureteral strictures due to radiation, retroperitoneal fibrosis or ureteral–ileal stenosis.Hematuria was observed in all patients; 9% intermittent microhematuria (3/33 patients), and 91% (30/33 patients) building up to or initially presenting with massive hematuria. Accompanying symptoms were clot retention (21%), flank pain (9%), shock (6%), sepsis (3%), melena (3%), and urosepsis (3%).All patients underwent a cumulative number of 40 investigations (mean 1.2 per patient) not leading to AUF diagnosis. For confirmation of the diagnosis of AUF, angiography was most decisive (19 times), followed by CT angiography (8 times), during surgery (4 times), ureterography (2 times), MRI (1 time), and postmortem (1 time).Based on the details of clinical presentation, applied diagnostics and treatment, we presented a clinical algorithm, as presented in Supplementary Fig. 3.Supplementary Fig. 4 shows the AUF locations. The common iliac artery was predominantly involved (73%), right sided in 38% and left sided in 35%. At diagnosis, seven patients with a medical history of vascular surgery on one side, such as thrombendarteriectomy or stenting, showed the AUF on the ipsilateral side of the repair and in 4 out of 7 patients a pseudoaneurysm was observed. In all six patients after central vascular repair, the AUF was found on the right side a pseudoaneurysm seen in three cases.Open vascular repair was performed in 10/33 patients (30%), urological treatment in 7/33 patients (21%), and endovascular stent graft placement in 24/33 patients (73%) with embolization of the internal iliac artery in eight patients.A combined urological and (endo)vascular treatment was performed in 12/33 patients (36%).In two patients nephroureterectomy was performed, as hematuria was considered to be of renal origin. Afterwards, AUF appeared to be the cause of the hematuria.Follow-up ranged from 1.5 days to 92 months with a median of 35 months. Fifteen patients died during follow-up; two were AUF related, five were sepsis related, and eight were due to other causes.The first AUF-related death was a patient with Chronic Obstructive Pulmonary Disease (COPD) Gold IV, who died 6 days after placement of an endovascular stent, due to sepsis and weaning difficulties on the ICU. The second AUF-related death was due to severe hemorrhage which was inadequately controlled. This patient died within 2 days after presentation and the definitive diagnosis was found at autopsy.The five sepsis-related deceased patients died after 1–4 months (mean 3 months). Their deaths were possibly related to the initiation of infection after insertion of endovascular stent grafts (3×) or vascular prosthesis during AUF treatment.The causes of death not related to AUF were: metastatic disease in three patients after 11–34 months (mean 25 months), cardiovascular causes in two patients after, respectively, 35 and 50 months, a car accident in one patient after 84 months, probably COVID-19 in one patient after 41 months, and intestinal pneumatosis in one patient after 1 month.This results in an overall mortality rate of 45% after a mean of 23 months, with a AUF-specific mortality rate of 6%.In four patients, a second AUF developed after 1 day and 4, 8, 12 months, respectively. All four patients were treated with an endovascular stent graft and survived with a follow-up of 92, 32, 50, and 15 months, respectively. One patient developed an arterio-conduit fistula (ACF) after 6 months, which was treated with an endovascular stent, and had an uneventful follow-up of 30 months. Besides these five patients with second AUF or ACF development, none of the other patients showed persistent hematuria.This nationwide cross-sectional questionnaire analysis comprehensively presents the occurrence of AUF in The Netherlands. Based on the results, risk factors, location, and clinical presentation of AUF was described. We also presented an overview of performed diagnostic modalities, and treatment of AUF in a modern healthcare system as in The Netherlands.Our main finding is the incidence of AUF in The Netherlands per year. The calculated incidence in The Netherlands of AUF was 3.5 AUFs/year. The true incidence is likely to be higher, considering the non-response by 38% of urologists. In case the incidence of AUF in the practices of non-responders was comparable to those of the responders, incidence of AUF would actually be 5.6 per year (90/16). Calculations have to be interpreted with caution. The questionnaire was only sent to urologists. Some cases of AUF where urologists were not involved might have been missed. In addition patients may have died because of AUF in whom the diagnosis has not been made at all. Finally, recall bias may have played a roll.In the literature, no figures of incidence changes over the past few years can be found. However, larger series have been published in recent years.Krambeck et al.  only desThis increase may be due to more extensive pelvic treatments for varying oncological and vascular procedures in current surgical medicine, increased treatment with ureteral indwelling stents, more frequent radiation therapies, and overall longer life expectancy. Other possible reasons are improving imaging modalities, and improvement of medical registration and electronic patient files, leading to easier identification of AUF cases. It is difficult to estimate the true incidence based on the numbers of published cases, because publication bias cannot be ruled out.All our patients had a history of oncological treatment, irradiation therapy, vascular pelvic surgery, or chronic ureteral indwelling stents. Thus, all our patients had fistulas secondary to other conditions. This is in accordance with the findings described in Bergqvist and Madoff , 8, who Although the pathophysiology of AUF is still uncertain, different hypotheses exist on the development of AUF, and the role of risk factors Fig. . SurgeryThe male to female ratio was 55%:45%. This is in contrast with most cohort studies, such as Subiela et al. , who fouThe clinical presentation in the majority of our patients was long-lasting intermittent (micro)hematuria that promptly shifted to massive hemorrhage, especially during ureteral stent extraction or replacement; only three cases (9%) had intermittent microhematuria without clinical consequences as the only symptom. This clinical presentation mirrors that of previous studies .Several abdominal imaging modalities failed to confirm the diagnosis AUF. In our patient population cohort 38 investigations/imaging procedures were performed which not lead to the correct diagnosis. In two CT angiographies renal clots suggested a renal bleeding, leading to unnecessary nephroureterectomy. Angiography seems to be the most efficient modality, confirming diagnosis in 19/33 patients (58%). However, a negative angiography could not exclude AUF, because active bleeding is necessary for reliable angiography . In caseTherapeutic management in The Netherlands is predominantly endovascular (73%) with the placement of stent grafts during angiography. In this century there has been a shift in the therapeutic management of AUF from a surgical toward a less invasive approach . In the The AUF-specific mortality rate in our cohort was 6%, which is at the lower end of the spectrum. Five patients had sepsis-related death likely due to the endovascular stent grafts placed during AUF-treatment. However, this occurred after more than 30 days, so was not calculated in the AUF-specific mortality.In previous studies the reported AUF-related mortality was rather variable: Escobar et al. found 10–13% and 22% in the hemodynamic instable patients , van denThe overall mortality was 45% after a median of 4 months. A possible explanation for this high rate is limited overall prognosis due to oncological and/or vascular comorbidity .Noteworthy, in our cohort four new secondary AUFs were seen within 1 year and one ACF, all successfully treated by an endovascular stent. Simon et al.  and OmraTo our knowledge this is the first nationwide cross-sectional questionnaire analysis for AUF and the largest series of new AUF patients. One of the main strengths is that the response rate of 62% is quite high compared to overall response rate on similar questionnaires. This adequately reflects all urologists in The Netherlands.Our study is the first to address the occurrence of AUF and contemporary experience by urologists on a national level. However, our study is limited by recall bias, possible missed diagnosis, and the retrospective nature.The estimated incidence of AUF in the time period 2003–2018 in The Netherlands is close to 4 AUFs per year, but the true rates may be higher. In 56 cases described in this series, hematuria, sometimes massive, was the most predominant initial symptom. Angiography appears the best diagnostic modality. Treatment was predominantly endovascular intervention.We identified previous oncological and vascular treatment as well as indwelling ureteral catheters as major risk factors. Reported mortality rate was 6% and may be underestimated due to missed diagnoses.AUF should be considered in patients with persistent intermittent or massive hematuria, especially in combination with history of oncologic (radiation) therapy, as well as vascular surgery.Clinical awareness of AUF is important because of its possibly life threatening nature. A multidisciplinary approach is important in diagnosis and treatment.Supplementary file1 (DOCX 97 KB)Supplementary file2 (DOCX 47 KB)Supplementary file3 (DOCX 49 KB)Supplementary file4 (DOCX 81 KB)Below is the link to the electronic supplementary material.


Open burning is illegal in Ukraine, yet Ukraine has, on average, 300 times more fire activity per year (2001–2019) than most European countries. In 2016 and 2017, 47% of Ukraine was identified as cultivated area, with a total of 70% of land area dedicated to agricultural use. Over 57% of all active fires in Ukraine detected using space-borne Visible Infrared Imaging Radiometer Suite (VIIRS) during 2016 and 2017 were associated with pre-planting field clearing and post-harvest crop residue removal, meaning that the majority of these fires are preventable. Due to the small size and transient nature of cropland burns, satellite-based burned area (BA) estimates are often underestimated. Moreover, traditional spectral-based BA algorithms are not suitable for distinguishing burned from plowed fields, especially in the black soil regions of Ukraine. Therefore, we developed a method to estimate agricultural BA by calibrating VIIRS active fire data with exhaustively mapped cropland reference areas (42 958 fields). Our study found that cropland BA was significantly underestimated (by 30%–63%) in the widely used Moderate Resolution Imaging Spectroradiometer-based MCD64A1 BA product, and by 95%–99.9% in Ukraine’s National Greenhouse Gas Inventory. Although crop residue burns are smaller and emit far less emissions than larger wildfires, reliable monitoring of crop residue burning has a number of important benefits, including (a) improving regional air quality models and the subsequent understanding of human health impacts due to the proximity of crop residue burns to urban locations, (b) ensuring an accurate representation of predominantly smaller fires in regional emission inventories, and (c) increasing awareness of often illegal managed open burning to provide improved decision-making support for policy and resource managers. The vas~13 000 km2 (~5600 fire counts) of BA in 2009 (1.7% of the country area) compared to a maximum of ~45 000 km2 (~53 000 fire counts) in Ukraine in 2008 (7.5% of the country area). In addition, the higher number of average annual MODIS active fires per km2 in Ukraine compared to the whole of Russia (RU)—where there are a substantial number of wildfires spread over large areas—further highlights the scale of this problem  and European Russia (RU_EUR). For example, Turkey (TR) has seen an increasing trend in annual MODIS BA and active fire counts since 2002 with a maximum of  problem . Therefo~$200–$340 US) and up to 5 years in prison; the fines have since increased to ~$230–$5880 US. In addition, if a burn becomes uncontrollable and causes either fatalities or infrastructure damage, violators can face up to 10 years in prison. Despite these penalties, Ukraine has on average ~300 times more active fire pixel counts per year than most other European countries and has the highest active fire count per unit area even when compared to Russia (RU) . In 2016 and 2017, 47% of Ukraine was identified as cropped/sown area—i.e. land used for arable production . A large° N) can be transported and deposited in the Arctic during seasons when land and sea ice melting can be affected by deposition of absorbing particles on the ice surface [In 2001, a ban on buying and selling certain types of agricultural land was implemented in Ukraine, with the result that the majority of land users now rent their lands and therefore have no investment in the long-term quality of the soil. With the recent March 2020 decision to open the land market in Ukraine in 2021 , it is i surface –25. Ther2; stacks.iop.org/ERL/16/064019/mmedia)), multiple orbital overpasses, and comparatively intense burns due to high yield. However, the black Chernozem soil causes additional complications when using traditional spectral reflectance-based BA mapping methodologies [Satellite-based cropland BA mapping is notoriously difficult due to the small fire sizes and rapid changes within each field during the transition from harvesting to planting –31. In adologies . Unfortu2.Here we describe a new technique to estimate cropland BA in Ukraine by calibrating 375 m Visible Infrared Imaging Radiometer Suite (VIIRS) active fire observations (VNP14IMGML ) with ex2.1.www.planet.com), in conjunction with filtered VIIRS active fire point data  for two time periods (January–June and July–December). As in previous studies [α is in effect a conversion factor that can be used to extrapolate our reference areas to much larger regions. Because our high resolution reference areas include an indeterminate label (class 3) for fields that could not be unambiguously labeled as burned or unburned, we calculated lower (αL) and upper (αH) limits for α which treated (a) only the fields with definitive burns (class 1 and class 2) as actually having burned, and (b) the definite burned fields (class 1 and class 2) as well as the ambiguous fields (class 3) as actually having burned, respectively. Each burned field’s area  was weighted by its BA fraction, summed, and then divided by the total number of filtered VIIRS active fire points within the spatial and temporal constraints of each reference area  as an uncertainty) and applied them to the appropriate monthly filtered active fire counts to calculate the effective cropland BA within Ukraine  were used to calculate the effective BA per fire pixel  is the BA per oblast (o) during month t, Nf is the monthly filtered VIIRS active fire count within the Oblast, and αL and αH are the average seasonal minimum and maximum effective BA per fire pixel conversation factors.Conversion of the filtered cropland VIIRS active fires to estimated BA was undertaken at the oblast level. This spatial unit was chosen as oblasts represent an administrative boundary within Ukraine and are often used as the basis for resource management and policy decisions. Although the analysis was conducted at a monthly time step, the same method applies to any temporal timeframe. Monthly BA per oblast was calculated by multiplying the appropriate seasonal L and αH  to the fL and αH )(2)A as opposed to including all months within the analysis (January–June = spring and July–December = summer). With only a 2%–4% difference in total cropland BA, we opted to include all months as the burning patterns changed slightly between 2016 and 2017 , 24%–57% , and by 95%–99%   . This seA potential solution to reduce open burning in croplands is the incentivization of excess crop residue for the production of biofuel , 37, 42.4.Understanding both the magnitude and spatio-temporal patterns of burning in croplands is essential for resource management and policy initiatives. This study not only provides a more realistic understanding of the extent of BA within Ukraine croplands, but it also shows the importance of using the best available data and methods in policy documents. For example, the Ministry of Environment Protection and Natural Resources (MENR) of Ukraine, which is responsible for the creation of Ukraine’s Greenhouse Gas Inventory, is restricted from using remote sensing imagery in their analysis and therefore produces cropland BA estimates based on sporadic and incomplete reports from the State Emergencies Service of Ukraine. Despite significantly underreporting the extent of cropland burning, the satellite-based MCD64A1 BA maps are far more realistic than the BA estimates  used within the UNFCCC document. Furthermore, methodologies should also be updated as technology and knowledge improve. For instance, the MENR of Ukraine is constrained by the emissions methodology and definitions set out in the IPCC chapter 2: Generic methodologies applicable to multiple land-use categories section 2.4, equation 2.27 . This ge2 of former Soviet Union kolkhoz lands (state-owned cooperative farm land run by farm laborers) gave 6.92 million citizens a piece of property on average between 0.036 and 0.04 km2 (3.6–4.0 ha). As a result, 74.95% of all agricultural lands  became privately owned, 24.06% state-owned, and 0.99% collectively/communally owned. Since 2001, the land market has been banned in Ukraine, leading to the majority of land users  renting their lands and therefore having no investment in the long term quality of the soil [2 on average), whom often cannot afford environmentally clean technologies and thus typically burn their fields, will be able to sell their lands, thereby leading to an increase in the average area owned by land-users that can afford to invest in new machinery.Ukraine is in the midst of a transitional period encompassing numerous social, economic, environmental, and climatic changes that have heavily impacted both the past and present fire regimes. Since Ukraine’s independence in 1991, drastic changes have occurred in land use/land structure and ownership –51. Specrding to  is 414 8the soil . Howeverthe soil . It is e2 fire in Zhytomyr and the Chernobyl Exclusion Zone, and a 50 km2 fire in the Lugansk oblast (far eastern region of Ukraine) with five fatalities and up to 40 people injured. These large fires attracted the attention of authorities to this often overlooked problem. While the new law has increased fines for open burning to more than 20 times from the previous fines, it is unclear if these expanded penalties will be effective in the reduction of a culturally-ingrained practice.Although this is a step in the right direction, the lack of financial and technical support for farmers and land-users who do not want to sell their lands from the government and private industry will continue to further exacerbate open burning in Ukraine. Specifically, the absence of an effective, national-scale, financial incentivization program to help discourage farmers from burning crop residue and instead adopt more environmentally sound field-clearing technologies will likely continue to be a hindrance in the quest for reducing open burning. Fortunately, a new law aimed at reducing this practice was passed in Ukraine in April 2020  followin4.1.2). Other agricultural lands are also subject to both intentional and unintentional burning, including abandoned lands (2290 km2), hay (23 990 km2), pasture (54 210 km2), and almost all small, private fields (vegetable gardens) near villages [2 (2010) to 52 700 km2 (2015) for all land cover types in Ukraine [Open burning in Ukrainian agricultural lands spans beyond sown cropped fields (325 540 kmvillages . Due to  Ukraine . This st~2016). However, these data are only acquired in the visible and near-infrared wavelengths and therefore identifying active burns through short-wave infrared imagery is not possible. While Landsat-8 and Sentinel-2 are alternate options, the ~3–5 d overpass  leads to an increase in the misinterpretation of burned and plowed versus plowed-only fields, and many of the more subtle changes caused by burning are also indistinguishable at 20 and 30 m resolution. Finally, while coarse-resolution sensors such as MODIS and VIIRS are currently the best options for identifying active fires in Ukraine, many burned fields will have no proximate coarse-resolution active fire pixels  has helped provide evidence to support the development of a new national strategy for landscape fire management. Actionable steps are required to start moving Ukraine toward reducing cropland burning, for example, through the development of extension services for sustainable agricultural practices or the creation of viable markets for the commercialization of crop residue for bioenergy .Supplementary


Dirofilaria immitis) disease continues to increase and spread, remaining one of the most important and pathogenic parasitic diseases of dogs, despite the regular use of macrocyclic lactones (MLs) in preventive products. Dogs harboring strains of D. immitis resistant to MLs, the only drug class available for heartworm prevention in the United States, have been documented and proven. As no new products are available utilizing a novel drug class for the prevention of this disease, the only options for combating ML resistance include increasing the dose and/or changing the dosage regime of current MLs, or by optimizing the formulations of MLs currently available. Moxidectin provides a unique opportunity for optimization of the dose and formulation, which may provide improved efficacy against ML-resistant strains. Currently there are oral, topical, and injectable moxidectin products approved for heartworm prevention in the USA. Two new products (ProHeart® 12 and Simparica Trio®), available in many countries around the world including the USA, take advantage of the unique attributes of moxidectin for providing robust heartworm prevention against the strains of heartworm to which most dogs in the USA will likely be exposed. Both products have demonstrated 100% preventive efficacy in laboratory studies against recently collected field strains of heartworm, and also in large field studies, where the majority of dogs were living in the southern USA in areas where ML resistance has been confirmed to occur, therefore under elevated heartworm challenge. Based on the data summarized here, these products offer important advances in heartworm prevention and provide additional options for veterinarians and pet owners to protect their dogs from developing heartworm disease.Heartworm ( Dirofilaria immitis, infects many mammals including dogs, cats, and ferrets  and ProHeart® 12 [0.5 mg/kg (500 µg/kg) moxidectin] are the only injectable heartworm preventives, and these products offer extended-release moxidectin for the continuous prevention of heartworm disease in dogs for 6 and 12 months, respectively. ProHeart 6 and 12 offer a safe and reliable heartworm preventive option without owners having to comply with monthly administration. Data show that, on average, owners using a monthly product for heartworm prevention administer only half of the prescribed monthly doses necessary to provide complete heartworm prevention [ProHeartevention . Once Prevention . The perevention . AnaphylD. immitis L3 30 or 165 days after initiating treatment, respectively [n = 6) were either treated with a single dose of ProHeart 12 on day 0 or administered six consecutive monthly oral treatments of Heartgard Plus or Interceptor Plus on days 0, 30, 60, 90, 120, and 150. All dogs were necropsied on day 185, approximately 8 months post-inoculation. The preventive efficacy of ProHeart 12 was 100%, with no dogs having any adult worms at necropsy. In comparison, all six dogs in both the Heartgard Plus- and Interceptor Plus-treated groups, had adult worms present at necropsy, demonstrating an overall preventive efficacy of 10.5% and 14.6% respectively (Table n = 6 for each treatment group) were either treated with a single dose of ProHeart 12 on day 0 or administered six consecutive oral monthly doses before and after inoculation with Heartgard Plus or Interceptor Plus, for a total of 12 consecutive monthly treatments [The efficacy of ProHeart 12, Heartgard Plus, and Interceptor Plus against the ML-resistant strain JYD-34 was evaluated in two different laboratory studies, in which dogs were either inoculated with 50 ectively . In the eatments . All dogeatments . These sAs claims of lack of efficacy for MLs, and confirmed cases of ML resistance continue to increase, field efficacy trials become extremely important in evaluating the efficacy against current natural strains circulating that could be a mixture of susceptible and potentially ML-resistant strains. A field efficacy study evaluated the preventive efficacy of Heartgard Plus and ProHeart 12 with client-owned dogs from 19 veterinary clinics across the USA. Dogs received either 20 consecutive monthly doses of Heartgard Plus or two annual doses of ProHeart 12, with a total of 218 dogs receiving Heartgard Plus and 236 dogs receiving ProHeart 12 . The stu® Multi, Bayer Animal Health, Shawnee Mission, KS, USA) is the only ML FDA-approved for the elimination of circulating MF in canine heartworm disease, with a > 99% reduction in MF observed compared to untreated controls 7 days post-treatment [The transmission of heartworm disease is dependent on the ingestion of MF by female mosquitoes from a patent host. Therefore, the reduction and elimination of circulating MF in infected animals is important in reducing the transmission and prevalence of this disease. Topical moxidectin . The increased dose of moxidectin administered in Advantage Multi/Coraxis, 2.5 mg/kg (2500 µg/kg), provides efficacy for the treatment and control of gastrointestinal nematodes, including hookworm, roundworm, and whipworm species in dogs.Moxidectin can be administered topically for the prevention of heartworm disease and is available for dogs, cats, and ferrets  to a dog with the MDR-1 defect can result in severe adverse effects, resulting in a black box warning on the label.D. immitis to MLs has been confirmed in the USA. Despite continued research and investigation, products containing novel compounds for the successful prevention of heartworm disease have not been brought to market. Therefore, currently the management of ML resistance is likely best achieved through the use of optimized formulations of MLs that focus on increased dose rate, and selection of MLs with optimal physicochemical and pharmacokinetic properties. Moxidectin is one such ML with favorable attributes, such as high potency against heartworms, long half-life, and greater margin of safety, that make it ideal for optimization in various formulations. With the continued and increasing threat of ML resistance in the USA, veterinarians play a critical role in educating clients on the importance of proper heartworm preventive administration to protect their pets from all strains of heartworms to which their pets are potentially exposed, especially considering the potential for the further spread of ML-resistant heartworm strains in the USA.Resistance of some strains of


Bio-nanotechnology is considered as one of the low-cost approaches that have been utilized in production of nanomaterials. The current research aimed at investigating the influence of different types of Actinomycete strains on the final properties of silver nanoparticles (AgNPs) such as size, shape, polydispersity, and antibacterial properties. For this purpose, the following techniques were employed UV spectrophotometer, SDS-PAGE electrophoresis, TEM, FTIR, antibacterial agar diffusion test, and Zetasizer.Streptomyces spiralis and Streptomyces rochei were able to reduce silver nitrate into sliver nanoparticles. The diversity and molecular weights of extracellular proteins secreted by these stains were different as proved by SDS-PAGE technique. This consequently resulted in differences in polydispersity of AgNPs which indicate that the sizes of AgNPs were highly dependent on the amount, molecular sizes, and diversity of extracellular matrix proteins of the microorganism.It was found that among 34 Streptomyces isolates collected from the soil, This article might give an insight about the importance of molecular sizes of biomacromolecules such as proteins on the physical properties of biogenic synthesized nanoparticles. Aspergillus flavus [Penicillium italicum isolated from Iraqi lemon fruits [The expression of bio-nanotechnology has been originated for nanotechnology field recently . In bio-s flavus . The synn fruits . Promisin fruits . Zinc oxn fruits .Punica granatum peel extract was also reported. The formation of Fe oxide NPs was optimized using different concentrations of peel extract  to achieve small size and better morphology [Nerium oleander leaf extract in the one step green synthetic method. The gold nanoparticles synthesized by this method were pure and showed good antioxidant activity [2) nanocomposite was synthesized by a novel and ecofriendly green deposition method using phytochemicals from the aqueous extract of Ranunculus muricatus [Salvadora persica [streptomyces spiralis being capable of synthesizing nanoparticles [CoMnCrO4 nanoparticles were prepared via green sol–gel method by using the natural gel . A greenrphology . The redactivity . The goluricatus . A novel persica . Very fearticles . Actinobarticles , 17. In articles , 19.+ particle to Ag0 stage prompting the arrangement of silver nanoparticles (AgNPs) [Practically, it was confirmed that bioactive molecules such as exopolysaccharides are delivered by a variety of living beings either prokaryotes or eukaryotes including microorganisms, plants, and animals. Among these living beings, actinomycetes produce huge amounts of exopolysaccharides . In bio- (AgNPs) –24. Sinc (AgNPs) . Additio (AgNPs) . TherefoSoil samples were collected from Halayeb and Shalatene along Red Sea Coast. The samples were collected in sterile polythene bags from the depth of 10–15 cm. Then, the samples were dried at room temperature and stored in sterile container for further process . Thirty-A loop full of each isolate was cultured into the flask (100 mL) containing 50 mL sterile starch nitrate broth and incubated in the shaker incubator at 150 rpm at 28 °C. The cells were harvested by centrifugation (at 6000 rpm for 10 min) and the supernatant (5 mL) was challenged with 5 mL of 1 mM silver nitrate solution  . The tubSolutions of AgNPs were examined by UV–visible spectroscopy, were carried out in a UV–vis spectrophotometer , operating in a wavelength from 200 to 900 nm.A growth curve was built to assess growth time of both strains of Streptomyces isolates and relate it with the production period of the silver nanoparticles. The isolates were grown in submerged culture for 10 days. The growth medium in one flask was filtered through a Whatman no. 1 filter paper, every 24 h within 10 days of growth period. Dry weight of cell mass was set up on the contrast between the final weight of the filter paper, after filtration and drying and the initial weight .oC, followed by 30 cycles of 1 min denaturation at 95 oC, 1 min primer annealing at 55 oC, 1 min extension at 72 oC and a final 10 min extension at 72 oC. The amplified DNA fragment was separated on 1% (w/v) agarose gel electrophoresis, using TBE buffer containing ethidium bromide (1 μg/mL). A single ~ 800 bp DNA fragment was cut and extracted from the gel, using a Core Bio Gel Extraction Kit. The sequence was determined by the CinnaGen Company. Sequence data of partial 16S rDNA was aligned and analyzed for finding the closest homologous bacteria. The 16S rRNA nucleotide sequence was compared to nucleotide databases using BLASTN program that is available from the National Center for Biotechnology Information  and retrieved ligned using GeneDoc software version 2.6. 002. Phylogenetic trees were built utilizing the neighbor-joining.The genomic DNA was extracted from the cultures of two Streptomyces isolates by using kit  according to the manufacturer’s instructions. Amplification of 16S rDNA by PCR was done using universal bacterial primer forward F (5′-CGGGCGGTGTGTAC-3′) and reverse R (5′-CAGCCGCGGTAATAC-3′) which amplify a ~ 800 bp. Amplification was carried out in a final volume of 50 μL containing; PCR buffer (1×), Taq DNA polymerase (2.5 U), dNTPs (4 mM), primers (0.4 μM), and template DNA (4 ng) with 100 bp ladder DNA marker. The thermal cycle (PCR) steps were applied as follows: 5 min initial denaturation at 95 Screening for the extracellular proteins attached to AgNPs using SDS-PAGE.To examine the diversity of extracellular proteins involved in Ag NPs synthesis and secreted from both Streptomyces strains in their numbers and molecular sizes, electrophoretic analysis of concentrated proteins on 12% SDS-PAGE was used. Briefly, after 48 h of adding the extract of the two Streptomyces strains that has been grown for 8 days to silver nitrate, the media of both strains were compiled and concentrated utilizing Amicon Ultra centrifugal filter with cut-off of 10 KDa. The proteins that are larger than 10 KDa was retained and concentrated in the upper part of the filter and their concentration was assessed by using Bradford assay. The extracellular proteins that are associated with Ag NPs was diluted in phosphate buffer saline, pH 7.2 and then 20 μg of the proteins of each strain was added to the wells of 12% polyacrylamide gel containing 10% Sodium dodecyl sulphate (SDS). A low molecular weight marker  was used to show the distribution of molecular sizes of extracellular proteins associated with silver nanoparticles. Proteins on the gel were stained with 0.25% Coomassie Brilliant Blue R-250Transmission electron microscope (TEM) was utilized to examine the effect of the strain type on the morphology, diameter, and distributions of synthesized AgNps. Practically, copper grid was submersed in each solution of AgNPs samples and allowed to dry in room temperature. The dried AgNPs were then characterized by TEM instrument .−1.Infrared absorption spectroscopy (FTIR) estimations were completed independently for each treatment to discover the compound that interferes in the preparation of AgNPs. Spectra of AgNPs were recorded after mixing with potassium bromide (1:100), which were introduced in pellets form. They were obtained by Broker Tensor 27 spectrophotometer. The spectra measuring range was 400–4000 cmPseudomonas aeruginosa ATCC9027, Escherichia coli ATCC8739, Salmonella enterica ATCC14028, Staphylococcus aureus ATCC6538, Bacillus subtilis ATCC6633). Each microorganism was inoculated onto Muller-Hinton broth  and incubated at 37 °C in a shaker incubator at 200 rpm for 24 h. The fresh cultures were streaked onto the Muller-Hinton agar and wells (6 mm in diameter) were made in the medium using sterile sharp borer. Then 100 μL of each AgNPs were added into the wells. All plates were incubated at 37 °C for 24 h. Afterward, the clear zone of the growth inhibition for each AgNPs was measured and recorded [Antimicrobial activity of the AgNPs containing filtrate was done using the well diffusion method . AntibacColloidal solutions of AgNPs were investigated by Zetasizer nano-series (Nano ZS), Malvern, UK, to assess their particle distribution, sizes and charges. Practically, AgNPs was suspended in distilled water and underwent to Zetasizer measurement. Simultaneously, the particle charges of AgNPs were also determined.n = 3. The antibacterial results were analyzed analysed by Student’s t test, significance was set at p ˂ .0.05In this work, all the obtained experimental data are expressed as the mean ± standard deviation (SD) for Streptomyces spiralis, while isolate 7 exhibited 99–100% similarity with the genus Streptomyces rochei. The DNA sequences were published in the NCBI databases under the following specific accession numbers: 5 (MK559584) and 7 (MK559585). Phylogenetic analysis of the 16S rRNA sequence data showed that the streptomycete isolates 5 and 7 belonged to the genus Streptomyces spiralis and Streptomyces rochei, respectively, as shown in Figs. The amplified DNA products separated on agarose gel electrophoresis revealed a single band of about 800 bp are illustrated in Fig. Streptomyces spiralis and Streptomyces rochei, respectively, showed the ability to synthesis AgNPs. The Ag+ ions reduction was evidently noticeable when AgNO3 was added to the supernatant of actinobacteria, and the color changed from yellow to dark brown. In the control, there was no color development. The maximum production of silver nanoparticles was observed at day 8 as will be explained in the following sections. Further increase in incubation time resulted in gradual decrease in production of nanoparticles.The current study was focused on the extracellular synthesis of AgNPs from actinobacteria supernatant. A total of 34 actinomycetes isolates were isolated from collected soil sample from Halayeb and Shalatene area checked for their ability to produce AgNPs. The screening revealed that only two isolates numbers 5 and 7 identified as Streptomyces rochei starts reduction of silver nitrate in D3  earlier than Streptomyces spiralis, which stars the reduction process at D5  showed several bands at 600, 690, 820, 990, 1030, 1013, 1392,1575, 1640, 2900, 3300, and 3325 cm−1. In details, the bands observed at 1640 cm−1 is assigned to amide I and amide II linkages of proteins, while band 3300 cm−1 is attributed for binding vibration of N–H groups. The band at 1575 cm−1 assigned to N–H stretching band of the amide I protein group. Band recorded at 1013 cm−1 are attributed to C–N stretching vibrations of aromatic and aliphatic amines. The C–H blending of aldehyde was recognized at band 1392 cm−1. The peak at 3325 cm−1 is assigned to the O–H stretching group of alcohol or N–H stretching of secondary amine. The bands located at 600, 690, 820, 990, 1030 cm−1were only observed in sample 7 and its filtrate, and these bands are thought to be specific for the extracellular secretions of Streptomyces rochei.The absorption spectra were measured to introduce suggested interaction mechanism between the extracellular secretions and the AgNPs. The production of monodispersed AgNPs, capping, well-dispersed Ag-NPs in colloidal solution, stabilizing, and morphology are thought to be dependent on the utilized type of strain. In Fig. t test table and at degree of freedom 2, it was shown that the calculated t values were significant.The antibacterial activity of AgNPs synthesis by two potent Streptomyces strain was performed. The results in Table Streptomyces rochei. On the other side, sample 5   for each sample and they were found in the range from 200 to 350 nm. The higher diameter was recorded as 320 nm for sample 7 Fig. b with nais) Fig. a introduchei Fig d. While lis Fig. c exhibitColor changes during extracellular biosynthesis of AgNPs were previously reported in other studies , 35. It Streptomyces spiralis; SS) exhibited two peaks at 320 and 400 nm and sample B  showed one dominant peak with great intensity at 408 nm. After filtration, the spectrum of sample A is transformed into spectrum D and B is transformed into spectrum C. Spectrum C demonstrated no distinguished peaks for the AgNPs, while the spectrum D showed tow broad peaks 320 and 400 nm. These results suggest that the extracellular proteins SS and SR broths are different in their diversity, molecular sizes, and their ability to bind to silver nanoparticles. It has been reported that proteins can be used to stabilize the size distribution of nanoparticles by acting as a capping agent [Streptomyces spp. [Phomagardeniae [Streptomyces rochei tend to produce relative monodispersed AgNPs compared to those produced by Streptomyces spiralis, which reveals the influence of the strain type on the size homogeneity of the produced nanoparticles. It is thought that the rings of selected area diffraction (SAED) represent are attributed to the 111, 200, 220, and 311 planes starting from inner to outer ring and has bright intense spots clearly indicating the face-centered cubic (fcc) structure of elemental silver [Streptomyces rochei produced relatively monodispersed AgNPs due to the presence of single low molecular weight protein in its extracellular environment. While the diverse proteins in molecular sizes of Streptomyces spiralis contributed to the polydispersity in the sizes of Ag NPs. There is no significant impact on the antibacterial properties of Ag NPs against different gram positive and gram-negative bacteria.It can be noted that sample A  using the same incubation conditions. Two different AgNPs were obtained using these stains based on their investigated features such as time of production, particle diameter, charge, and dispersion of the particle sizes. Pronounced effect on these vital features of AgNPs is attributed to the different extracellular secretions of two different strains.


Green beans are a perishable crop, which deteriorate rapidly after harvest, particularly when minimally processed into ready-to-eat fresh-cut green beans. This study investigated the effectiveness of ethanol, ascorbic acid (AsA), tea tree essential oil (TTO), and peppermint essential oil (PMO) on the quality and storability of fresh-cut green bean pods samples stored at 5 °C for 15 days. Our results indicated that samples treated with ethanol, AsA, TTO, and PMO preserved appearance, firmness (except ethanol), chlorophyll content, and moisture compared with the samples without any treatment (control). Additionally, higher vitamin C, total soluble solids (TSS), total sugars, and total phenolic compounds (TPC) were observed in samples treated with ethanol, AsA, TTO, and PMO compared with the control. The most effective treatments for controlling microbial growth were ethanol followed by either TTO or PMO. All the treatments had positive effects on shelf life, maintained quality, and reducing microbial growth during 15 days of cold storage. A particular treatment can be selected based on the economic feasibility and critical control point in the value chain. Phaseolus vulgaris) belongs to the family of Fabaceae and is considered one of the most important legume crops worldwide. The pods of green bean can be harvested at an immature stage (as a fresh vegetable) or mature stage (for dried seeds). Green bean is a rich source of minerals, vitamins, and dietary fibre that play a significant role in the human diet and wellbeing [Green bean  and plant extracts as anti-microbial and shelf life enhancing agents in horticultural crops are well known ,5. The Urawberry  and lettrawberry . It has rawberry . New invrawberry  and dragrawberry .Ascorbic acid (AsA) plays an important role in plant antioxidant systems and human health ,14. PosiTo the best of the author’s knowledge, this is a novel investigation. There were no previous reports on the effect of ethanol, AsA, TTO, and PMO on postharvest behaviour and quality of green beans. The objective of the current study was to assess the effect of these natural compounds on the quality parameters and shelf life of fresh-cut green beans stored for 15 d at 5 °C.Phaseolus vulgaris L., variety Hama) were harvested from a local private farm and transferred under cooling condition (4 °C) within two hours to the postharvest laboratory. Green bean pods free from defects and damage, with uniform diameter and length, were prepared by cutting the two ends of the pod with a sterile sharp knife. The fresh-cut green bean pods were immersed in four different treatment solutions:A.Ethanol (0.5%),B.Ascorbic acid (AsA) (0.5%),C.Tea tree oil (TTO) (0.5%), andD.Peppermint oil (PMO) (0.5%).Green bean pods  polypropylene bags by using autoclaved forceps. The dimensions of the bags were as follows: length 22 cm, breadth 16.5 cm, and 1 mm thickness; the bags contained 250 g of samples each. Samples were stored at 5 °C and 90% RH for 15 d. Each treatment was carried out in triplicate and the whole experiment was repeated. For each treatment, samples were divided into two groups. One group was used to determine weight loss, decay, and general appearance throughout full storage time and the other was used to determine pod quality parameters (firmness and TSS), chemical compounds , mould, yeast, and total microbial count. All parameters were measured at time intervals of 0, 3, 6, 9, 12, 15 d after treatments.The control group left without any treatment. TTO and PMO concentrations were prepared by dissolving the required amounts of oils in 50 mL of 0.05 mL Tween-20 and then sterile distilled water was added to obtain 1000 mL of desire concentrations. The treated samples were then dried in a laminar airflow hood for 2 h, and then packed and sealed in micro-perforated (five perforations/cmv/v). The GC was equipped with an HP-5MS column . Analyses were carried out using helium as the carrier gas at a flow rate of 1.0 mL/min at a split ratio of 1:10, injection volume of 1 µL and the following temperature program: 40 °C for 1 min; rising at 4 °C min−1 to 150 °C and held for 6 min; rising at 4 °C min−1 to 210 °C and held for 5 min. The injector and detector were held at 280 °C and 220 °C, respectively. Mass spectra were obtained by electron ionization (EI) at 70 eV; using a spectral range of m/z 40 to 550 and solvent delay of 3 min. The identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.To analyse the chemical compositions of the TTO and PMO, the GC-MS system (Agilent Technologies) equipped with gas chromatography (7890B) and mass spectrometer detector (5977A)  was used. Samples were diluted with hexane ; then, the sample was mixed with 4 mL DPPH solution (0.24 mg L−1 in methanol). The control sample was prepared by mixing methanol with DPPH solution at the same volume. The mixture was mixed well and incubated in the dark at room temperature for 30 min; then, the absorbance was measured at 517 nm using a spectrophotometer . The free radical scavenging activity of each essential oil was calculated according to Equation (1).control = absorbance of control sampleA , and sample = absorbance of essential oil sampleA .The free radical scavenging activity of the TTO and PMO against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was measured according to the method described in . First, Green bean samples were weighed immediately after (drying in a laminar airflow hood for 2 h) and at every sampling time to measure weight loss by using a digital laboratory scale. Another set of the green bean samples of 250 g each in triplicates) were used for further chemical analysis. To determine general appearance, the following scale was used: 9 = excellent, 7 = very good, 5 = good, 3 = poor, and 1 = unacceptable. The appearance score was assessed by a group of three trained laboratory panelists.The firmness values of each pod were determined at three different points by using the digital penetrometer  with 6 mm diameter probe (range 0 to 1 kg*) . The fir2SO4) and mixed thoroughly. The sample was brought to boil using a boiling water bath. The solution was cooled to room temperature and absorbance was measured. The formation of the blue-green complex indicates the presence of total sugars. Glucose was used as a standard. Vitamin C content was determined using the titrimetric method with 2,6-dichlorophenolindophenol described by the Association of Official Analytical Chemistry [The total sugar content was determined by the anthrone method at 630 nm as described in . Brieflyhemistry .w/v) was added after incubation for 5 min. The final solution was mixed and incubated in the dark at room temperature for 1 h. The absorption was assessed at 765 nm using the spectrophotometer, and the results were expressed as milligrams of gallic acid equivalent (GAE) per 100 mg of fresh fruit weight.TPC was calculated by using the Folin–Ciocalteu reagent with some alteration by using gallic acid as a standard curve . Five grChlorophyll content was determined as described in . In brie−1).Ten grams of each treatment were homogenized with 90 mL sterile saline for 2 min by a stomacher . Total count and mould and yeast count (MY) were enumerated on plate count agar and potato dextrose agar after incubation at 37 °C for 48 h and 28 °C for 5 d, respectively . The resp ≤ 0.05 by using the Tukey test. Moreover, a second one-way analysis was carried out to investigate the storage time . The least significant differences among the treatment means were determined at e in the .The chemical composition of the EOs is shown in −1 for each PMO and TTO. PMO had lower radical scavenging activity (54%) compared to TTO (63.56%).In this study, the free-radical scavenging activity of PMO and TTO were assessed through the DPPH radical scavenging test. The radical scavenging activity was observed in EOs at the concentration of 50 µL mLThe appearance score was gradually lowered during storage time  and total microbial count increased with increasing storage time . In thisThe chemical composition of EOs agreed with Wu et al. , who sta−1, which is a very high concentration compared to the concentration used in this study (50 µL mL−1). Variations in the antioxidant potential of EOs have been reported, primarily due to the presence of conjugated double bond compounds, which serve as hydrogen/electron donors [Our results indicated that PMO had higher radical scavenging activity than the results reported by others  who founn donors . PMO andn donors .Appearance is the most important quality parameter for the consumer’s acceptance. Weight loss of fresh fruit and vegetables during cold storage is affected by factors such as the structure of the cuticle , transpiRegarding EOs treatments, our results shown in The results in AsA treatment retained the firmness of the green bean pods C; similaEOs maintained the firmness of treated samples with either TTO or PMO could be due to its properties to inhibit the pectin degradation on the surface of green beans . AdditioIt has been confirmed that ethanol retards chlorophyll degradation , repininOur results indicated that PMO and TTO treatments had a positive role in maintaining the chlorophyll content of fresh-cut green beans. This effect can be explained by a lower breakdown of chlorophyll pigments induced by PMO treatment, which acts as a coating agent . MoreoveVitamin C is classified as a natural antioxidant compound and has a potential role in reducing the risk of cancer by scavenging ROS in the human body . HoweverThe effect of postharvest treatment of TTO on vitamin C content of fruit and vegetable is not identified well due to limited research on this application. For example, a higher concentration of vitamin C was observed in lettuce plants treated before harvest with TTO and stored for 20 d at −2 °C ; howeverOur results shown in Treatment with either ethanol or PMO resulted in a higher TSS in green bean samples compared to control B. PrevioIt is well known that TPC are considered the most important antioxidant compounds, responsible for scavenging free radicals, resulting in higher antioxidant defense . Our resSamples treated with TTO showed higher TPC in this study; this agrees with previous work that observed higher TPC content during refrigerated storage of lettuce heads treated with TTO . They alIn this study, either ethanol or AsA slowed the reduction of total sugar during cool storage D. PrevioBotrytis cinerea was controlled in table grape [Controlling postharvest diseases by ethanol application has been tested by various previous reports; e.g., le grape  and anthle grape . The redle grape . In thisle grape , AsA trele grape .Previous works demonstrated the inhibited role of EOs and their several compounds for controlling human and plant pathogens . Our resOur study suggested that ethanol, AsA, PMO, and TTO can extend the life of fresh-cut green beans pods under refrigerated conditions by reducing weight loss and maintaining appearance, chlorophyll content, firmness, vitamin C, TSS, total sugar, and TPC. Moreover, a reduction in the mould and yeast count and total microbial count during storage was observed. Ethanol was the most effective application, followed by PMO and TTO, for preserving quality during refrigerated storage at 5 °C up to 15 d. Although all the treatments had positive effects, the best treatment shall be selected based on particular requirement and economic feasibility. For example, if there is a problem of higher microbial load along the value chain, we recommend the use of ethanol as it was the most effective, followed by PMP, TTO. Moreover, further research is required to enhance the storage ability of fresh-cut green beans and other legume crops including peas and board bean.