The operative time and blood loss were used to evaluate the clinical outcomes. The delayed ligation group had significantly lower operative times than the regular surgery group (P < 0.05). Total, hidden, and intraoperative blood loss were all significantly lower in the delayed ligation group than in the regular surgery group (P < 0.05). However, there were no significant differences in postoperative blood loss. In conclusion, delayed femoral vein ligation could significantly reduce the operative time, hidden blood loss, and intraoperative blood loss in patients undergoing hip disarticulation.This study aimed to evaluate the effects of delayed femoral vein ligation on the clinical outcomes of hip disarticulation. We retrospectively reviewed 20 patients with extremity tumors (10 bone tumors and 10 soft tissue sarcomas [STS]) who underwent hip disarticulation. Patients treated for hip disarticulation with synchronous femoral vein ligation ( Most osseous and STS are now managed with limb salvage surgery, such as internal pelvic resection . HoweverAmputation has been extensively used in orthopedic clinics, mostly when the limbs cannot be preserved owing to various factors, such as trauma, infection, tumors, or vascular disease . AmputatMost amputations caused by distal limb diseases can be performed in the hemostatic band ,8. TournIn this study, we aimed to apply delayed ligation of the main limb vein in amputated patients and evaluate the curative effect from the aspects of operation time and blood loss. Our findings may provide an effective hemostasis method for hip disarticulation, which had positive clinical benefits.22.1n = 10, regular surgery group, n = 10, delayed ligation group, Twenty patients  with extremity tumors (10 with bone tumors and 10 with STS) were retrospectively reviewed from the Department of Orthopedics in the Tumor Hospital Affiliated to Harbin Medical University from December 2013 to February 2016. The study enrolled hip disarticulation patients treated with synchronous femoral vein ligation (Inclusion criteria: (1) The patient ages were in the range of 10–60 years and weighted 35–90 kg, (2) patients suffered from primary malignant limb tumors without limb preservation and had preoperative hemoglobin levels >10 g.Exclusion criteria: The patients diagnosed with primary or secondary coagulation dysfunction, patients who required massive preoperative blood transfusions, long-term oral anticoagulant therapy patients, or those who underwent interventional therapy for vascular emboli of the affected limbs, and excessive pressure fluctuations during operation, were excluded.The same experienced surgeons performed the two hip disarticulation procedures. The patients did not receive hemostatic or anticoagulant drugs intraoperatively, and the preoperative hemoglobin was greater than 10 g/dL. Ethics approval and consent to participate: This study was approved by the ethical committee of The Second Affiliated Hospital of Harbin Medical University, and consent was obtained from the patients.2.2The main difference between the regular surgery group and the delayed ligation group was the femoral vein ligation time. A 3 cm long transverse incision exposed the femoral artery and vein in the inguinal region when anesthesia was sufficient. First, we ligated the trunk artery of the limb, blocked the blood vessel with the ligation suture reserved in the main vein at any time, temporarily reserved the vascular sheath to avoid vascular tearing, successively cut off the muscle, fascia, and other soft tissues, and finally ligated and cut off the main vein until osteotomy.The femoral artery and vein were occluded using the double ligation technique in the regular group. Only the artery was occluded in the delayed ligation group , and bot2.3The main outcomes in the regular surgery group and the delayed ligation group were mainly evaluated according to operation time and blood loss. The operation time included the time spent on the skin incision, hip disarticulation, and skin suturing. All patients underwent routine blood examinations before and after surgery, respectively. Additionally, blood loss included predicted blood volume (PBV), total blood loss (TBL), hidden blood loss (HBL), intraoperative blood loss, and postoperative blood loss.H indicates the height (m) and W indicates the weight (kg).PBV was calculated with the following formulas, as described previously ,PBVmaleThe allowable blood loss was calculated by the following formula as described previously :VL=PBV×HO indicates the preoperative hematocrit or hemoglobin levels, HF indicates the hematocrit or hemoglobin levels postoperatively, and HAV indicates the mean value of the difference between pre- and postoperative hematocrit or hemoglobin levels.TBL and HBL were then calculated as follows:VL + blood transfusions (1 U packed red blood cells = 200 mL whole blood),TBL = HBL = TBL − (intraoperative blood loss + Postoperative blood loss).2.4t-test. A P-value <0.05 was considered statistically significant.All data were analyzed using SPSS software  and were expressed as mean ± standard deviation with minimum and maximum values. Comparison between the regular surgery group and the delayed ligation group was determined with an independent 33.1A total of 20 patients were included in this study in which 10 patients (28 ± 13 years) were treated using regular surgery, while the other 10 patients (29 ± 15 years) were treated by delayed ligation surgery. Furthermore, in the regular surgery group, the preoperative hemoglobin of patients was 132 ± 16 g/L, while in the delayed ligation group, the preoperative hemoglobin of patients was 121 ± 14 g/L. There are no significant differences in the age, weight, gender, height of patients between the regular surgery group, and the delayed ligation group.3.2Operative time is one of the main observation indices for hip disarticulation.P < 0.05).The average operative time of the delayed ligation group was 158 min and that of the regular surgery group was 183 min. As shown in 3.3P < 0.01).TBL was in the range of 521–1,141 mL in the delayed ligation group, and the average was 831 mL. In addition, TBL was in the range of 960–1,745 mL in the regular surgery group, and the average was 1,357 mL. TBL decreased significantly in the delayed ligation group compared with that in the regular surgery group .The HBL volume was 270–737.4 mL in the delayed ligation group, and the average HBL volume was 503.7 mL. The HBL volume was 457.6–1247.6 mL in the regular surgery group, and the average HBL volume was 852.6 mL. In the delayed ligation group, the volume of intraoperative blood loss was in the range of 220–420 mL, and the average volume of intraoperative blood loss was 320 mL. In the regular surgery group, the intraoperative blood loss volume was 361–629 mL, and the average volume of intraoperative blood loss was 495 mL. HBL and intraoperative blood loss also decreased significantly in the delayed ligation group than in the regular surgery group .In the delayed ligation group, the volume of postoperative blood loss ranged from 57 to 111 mL, and the average volume of postoperative blood loss was 84 mL. In the regular surgery group, the volume of postoperative blood loss ranged from 69 to 137 mL, and the average volume of postoperative blood loss was 103 mL. However, there were no significant differences in postoperative blood loss and PBV between these two groups (4Hip disarticulation is common for patients with limbs affected by serious trauma, vascular gangrene, infection, and malignant tumors . In hip Regular techniques are effective for the high-level amputation of lower limbs affected by trauma or gangrene ,24. DuriIn this study, a novel hip disarticulation with delayed femoral vein ligation was performed. The main artery was ligated first during this surgery, and the vascular sheath was reserved to avoid vascular tearing. The main vein was amputated and ligated until the backbone, and joint capsule of the truncated bone was exposed. Hip disarticulation with this novel ligation technique may reduce blood loss. In turn, it will reduce operation time based on a clear surgical field and fully visible anatomy. As expected, significantly reduced operative time, TBL, HBL, and intraoperative blood loss were observed in patients who underwent hip disarticulation with delayed vein ligation, indicating that delayed femoral vein ligation effectively reduced hemorrhage. During amputation, intraoperative blood loss can be influenced by various factors, such as the operator’s proficiency, the age and weight of the patients, and the size and location of the limb tumor . EffectiThis study has some limitations. First of all, the sample size we collected was relatively small, the observation indicators were relatively limited, and many bias factors affected the experiment. In our research, we did not consider possible complications or the use of other drugs that may have interfered with blood clotting. Future experiments need to expand the sample size and consider multiple factors for more in-depth research.5In conclusion, delayed femoral vein ligation during hip disarticulation could significantly reduce operation time and hidden and intraoperative blood loss. Although this novel ligation technique could reduce the perioperative bleeding risk for hip disarticulation, additional clinical studies on its application were needed.PBVpredicted blood volumeTBLtotal blood lossHBLhidden blood lossSDstandard deviation


Coughing caused by tracheal extubation is common following general anaesthesia. Heavy aerosol production by coughing during recovery from general anaesthesia in patients with respiratory infections  may be one of the highest risk factors for infection in healthcare workers. The application of local anaesthetics to the endotracheal tube is an effective method to reduce coughing. The most commonly used anaesthetics are compound lidocaine/prilocaine cream and tetracaine spray. However, coughing still occurs when the two anaesthetics are used alone. We speculated that the application of compound lidocaine/prilocaine combined with tetracaine spray would better prevent coughing caused by tracheal extubation.Patients scheduled for laparoscopic cholecystectomy or cholecystectomy combined with common bile duct exploration under general anaesthesia were randomly assigned to Group C , Group L (2 g compound lidocaine/prilocaine cream contains 5 mg of lidocaine and 5 mg prilocaine)), Group T (tetracaine) and Group F (compound lidocaine/prilocaine cream combined with tetracaine). The incidence of coughing, the endotracheal tube tolerance assessment, the incidence of agitation, the active extubation rate, the incidence of postoperative pharyngeal pain and the incidence of postoperative cough were recorded and analysed. Systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and the plasma concentrations of epinephrine and norepinephrine were measured immediately before extubation and 1 min after extubation.P < 0.001), Group T  and Group F . Group F had a significantly reduced incidence of cough compared to Group L and Group T . The secondary results were assessed. The endotracheal tube tolerance score in Group C  4, P < 0.001) was higher than Group L  2), Group T  3) and Group F  1). Group F had a significantly lower score than Group L and Group T . The incidence of agitation and the active extubation rate were also higher in Group C  than Group L , Group T  and Group F . Blood pressure, HR and plasma concentrations of epinephrine and norepinephrine were significantly higher in Group C than in all other groups at the time of extubation and 1 min after extubation (P < 0.001). Group F exhibited significantly reduced blood pressure, heart rate and plasma concentrations of epinephrine and norepinephrine compared to Group L and Group T . The incidence of postoperative pharyngeal pain and the incidence of postoperative cough were not significantly different among the groups.A total of 211 patients were randomly assigned to Group C (53 cases), Group L (52 cases), Group T (52 cases) and Group F (54 cases). The primary result is assessment of the incidence of cough. The patients emerged from general anaesthesia, 96% of Group C had cough, which was significantly reduced in Group L  “retrospectively registered”. Coughing caused by endotracheal tubes in general anaesthesia is one of the most common reflexes in clinical medicine, especially when the trachea is extubated and the incidence is between 38 and 96% . PatientThe Affiliated Hospital of Yan’an University, China organised this RCT. The trial was performed according to the CONSORT-2010 guidelines. The Ethics Committee of The Affiliated Hospital of Yan’an University approved the study protocol (NO. 2020042), and all subjects provided written informed consent before participating in the trial.Patients were included in the trial when they were 18 ~ 64 years old and scheduled for laparoscopic cholecystectomy or cholecystectomy combined with common bile duct exploration under general anaesthesia using endotracheal tube intubation. The following major exclusion criteria were used: American Society of Anaesthesiologists (ASA) grade greater than III; preoperative chronic pharyngitis, cough or other upper respiratory tract lesions; concurrent hypertension with or without drug therapy; difficult airway; allergies to lidocaine, tetracaine, or any other ingredients in the test product; intraoperative bronchospasm; operation time greater than 2.5 h; intraoperative bleeding (> 300 ml). We randomly assigned the patients to placebo , compound lidocaine/prilocaine cream, tetracaine spray, or compound lidocaine/prilocaine cream combined with tetracaine treatment at a 1:1:1:1 ratio. The primary end points included the overall incidence of cough, the endotracheal tube tolerance cough and cardiovascular reactions during extubation after anaesthesia during the recovery period, the sore throat within the first 24 h after extubation. Figure A total of 236 random numbers were generated by IBM SPSS Statistics 25 , and the software randomly divided the 236 numbers into four groups. Cases were enrolled according to the order of enrolment time corresponding to random numbers from small to large, and a random number corresponded to the admission ID number of the patient. A full-time anaesthesiologist (Investigator A) performed these assignments. The patient entered the operating room, and investigator A induced general anaesthesia according to the conventional method. Tracheal intubation was performed after smearing and/or spraying the tracheal tube according to the enrolment information. After completion of tracheal intubation, another anaesthesiologist (Investigator B) performed anaesthesia management according to conventional methods and collected data until the end of the study. All of the collected data were handed over to Investigator A for sorting into different groups, and Investigator C performed statistical analyses. The patients and Investigators B and C were all blinded to the grouping information.Anaesthesiologist A treated the surface of the tip of the endotracheal tube according to the random number table. In Group C, normal saline (2 ml) was evenly sprayed onto the front end of the tracheal tube up to the two black marked lines near the cuff. In Group L two grams of compound lidocaine/prilocaine cream  was evenly applied onto the front end of the tracheal tube up to the two black marked lines near the cuff. In Group T, 2 ml of 10% tetracaine  was evenly sprayed onto the front end of the tracheal tube up to the two black marked lines near the cuff. In Group F, two grams of compound lidocaine/prilocaine cream were first applied onto the front end of the tracheal tube up to the two black marked lines near the cuff then a small container with a spray function was used to evenly spray 2 ml of 10% tetracaine injection onto the front end of the tracheal tube where compound lidocaine adhered. The method of applying compound lidocaine/prilocaine cream onto the front end of the tracheal tube up to the two black marked lines near the cuff was performed according to a previous study  or mean [95% CI], as appropriate. Measurements were calculated as means (± standard deviation). ANOVA was used to compare different groups. Non-parametric one-way ANOVA  was used for rating data. Pearson's chi-squared test was used to compare the categorised data in the groups. We set A total of 236 patients at the Affiliated Hospital of Yan'an University were enrolled in the randomised trial. Twenty-five patients were excluded from this study, including 6 cases in Group C , 7 cases in Group L , 7 cases in Group T , and 5 cases in Group F . The study started on March 1, 2020 and ended on December 31, 2020. There were no significant differences in the baseline characteristics  of the patients among the groups . However, the incidence of coughing was significantly higher in Group L and Group T than in Group F (p < 0.001) . However, the incidence of agitation was significantly higher in Group L and Group T compared to Group F  . However, the active extubation rate was only significantly higher in Group T compared to Group F (p < 0.05)  . However, the scores of tracheal tube tolerance were significantly higher in Group L and Group T than in Group F        (Table p < 0.05)  Table . The incy) Table . The act5) Table . The incy) Table . We furtly) Fig. . We alsoly) Fig.  f. △DBP ly) Fig.  g. △HR wly) Fig.  h, and △1)  Fig.  i, j. Thy) Table , and onl5) Table .Table 2CThe current study compared the application of compound lidocaine/prilocaine cream, tetracaine spray or compound lidocaine/prilocaine cream combined with tetracaine to the front end of the surface of the tracheal tube and demonstrated a significant reduction in the incidence of induced coughing, agitation caused by sputum aspiration and extubation and the active extubation rate, which suggest significantly improved tolerance to the tracheal tube in patients during emergence from general anaesthesia. At least 65.1% and 75.0% of patients had induced coughing in the lidocaine/prilocaine cream and tetracaine group, respectively. However, the incidence of induced coughing was significantly reduced to 22.2% by the combination of compound lidocaine/prilocaine cream with tetracaine. The incidence of emergence agitation and active extubation rate were also significantly reduced to 16.7% and 5.6% in the compound lidocaine/prilocaine cream combined with tetracaine group, respectively. Compound lidocaine/prilocaine cream combined with tetracaine was also the most effective method to prevent extubation-induced increases in SBP, DBP, HR, E and NE plasma concentrations in patients emerging from general anaesthesia. These findings demonstrated that compound lidocaine/prilocaine cream combined with tetracaine had a better airway surface anaesthesia effect and significantly increased tolerance to the tracheal tube in patients under general anaesthesia.Endotracheal intubation-related mechanical stimulation is clinically common and associated with airway complications including, severe coughing, laryngeal injury and postoperative sore throat , 12. CouDuring emergence from general anaesthesia, sputum aspiration and tracheal tube extubation are the strongest stimulators of the tracheal mucosa, which is the most likely to induce cough. Our study showed that compound lidocaine/prilocaine cream combined with tetracaine significantly reduced the incidence of induced coughing caused by sputum suction and extubation by approximately 39% compared to the use of lidocaine/prilocaine cream alone. Although the spraying of tetracaine alone also significantly reduced the incidence of cough, the reaction was lowest in the lidocaine/prilocaine cream combined with tetracaine group. One systematic review and meta-analysis of dexmedetomidine, remifentanil, fentanyl, lidocaine i.v., intracuff lidocaine, and lidocaine via tracheal or topical route, demonstrated that in pair-wise comparisons all study medications were equivalent in reducing moderate and severe emergence coughing incidence, and were better than placebo or nothing . This meDuring emergence from general anaesthesia, the patient may exhibit emergence agitation. The main factor of emergence agitation is delayed extubation. Study showed that patients with delayed extubation developed a cough reaction was 16.7 times higher than patients without delayed extubation . We founTherefore, we though that compound lidocaine/prilocaine cream combined with tetracaine inhibited the airway reflexes caused by the tracheal tube and further enhanced the body's tolerance to the tube. Our results showed that compound lidocaine/prilocaine cream or tetracaine significantly improved tracheal tube tolerance. The tracheal tube tolerance scores were reduced to 2  and 3 , respectively. Compound lidocaine/prilocaine cream combined with tetracaine further significantly increased tracheal tube tolerance, and the tracheal tube tolerance score was reduced to 1 . Study showed that remifentanil 0.025–0.05 microg.kg(-1).min(-1) achieves satisfactory tracheal tube tolerance in awake and spontaneously breathing patients performed under general anaesthesia, the respiratory response subscore of comfort scale of patients is all 3 . Our finThese findings may induce beneficial effects on cardiovascular reactions during the course of extubation. The current study found that hemodynamic changes (blood pressure and/or HR) exceeded 20% of the baseline at 1 min after extubation. Compound lidocaine/prilocaine cream or tetracaine significantly reduced the SBP increase from baseline (△SBP), the DBP increase from baseline (△DBP) and the HR increase from baseline (△HR) about 8% ~ 12% compared to the use saline. However, compound lidocaine/prilocaine cream combined with tetracaine significantly reduced the average values of △SBP, △DBP and △HR about 30% ~ 50% compared to the use saline.When the endotracheal tube is removed, this stimulates the receptors of the airway mucosa, which results in the release of catecholamines , 24 and We also assessed the incidence of postoperative cough and postoperative pharyngeal pain. We found that the use of local anaesthetic significantly reduced these incidences compared to saline. The incidence of postoperative cough in the compound lidocaine/prilocaine cream (13.5%), tetracaine (21.2%) and compound lidocaine/prilocaine cream combined with tetracaine (13.0%) groups were not significantly different from each other. However, the incidence of postoperative pharyngeal pain was higher in the tetracaine group than in the compound lidocaine/prilocaine cream combined with tetracaine group. Soltani, H.A. and O. Aghadavoudi suggested that the use of lidocaine to inflate the endotracheal tube cuff at the end of surgery decreased the frequency of postoperative cough and sore throat by approximately 41.4% and 17.7% respectively, compared to the use of saline to inflate the endotracheal tube cuff . Their rThere are some potential risks in the application of local anaesthetics to the airway of a patient who is under general anaesthesia: 1) if the operation time is too long, the effect of the local anaesthetic will insufficient to suppress the cough response caused by extubation; 2) the local anaesthetic effects may extend to block part of the vocal cords and affect vocalisation; 3) compound lidocaine includes prilocaine, although we used safe doses, prilocaine may increase blood methemoglobin levels, which should be monitored in clinical practice.And there are some limitations to our study. 1) This study did not observe the duration of the continuous effect of compound lidocaine combined with tetracaine on airway topical anaesthesia. 2) This study did not consider the patient's anxiety state, which will affect the release of catecholamines during awakening, and may affect the evaluation of catecholamines released during tracheal tube extubation. 3) During recovery from general anaesthesia, the patient exhibits emergence agitation due to poor tolerance to the tracheal tube, which could not be well revealed in this study based on other factors, such as preoperative anxiety or bladder irritation caused by urine retention.In summary, we demonstrated that the application of compound lidocaine/prilocaine cream combined with tetracaine to the surface of the tracheal tube prevented the cough caused by extubation, increased the body's tolerance to the tracheal tube and reduced the release of catecholamine hormones into the blood and the increase in blood pressure during extubation in patients undergoing laparoscopic cholecystectomy and cholecystectomy combined with biliary exploration. Therefore, compound lidocaine cream combined with tetracaine may be a more effective approach for preventing coughing and stabilising circulation during extubation following emergence from general anaesthesia. This may play an important role in preventing medical staff from contracting respiratory infectious diseases, especially during the COVID-19 epidemic.


In addition, subgroup analyses were performed to reduce the heterogeneity of the initial meta-analyses. The results provided strong evidence that inhaling radon in the indoor environments is closely associated with the development of lung cancer and childhood leukemia in patients living in Europe and areas with high radon levels (≥100 Bq/m3).This study investigated the impact of residential radon exposure on human cancers  through a systematic review and meta-analysis of case–control studies. A total of 9724 articles obtained from electronic databases were assessed; however, only 55 case–control studies were eligible after manually screening and eliminating unnecessary studies. The causal associations were addressed by determining the meta-analysis’s estimated size effects  of the meta-analysis. Residential radon was revealed to significantly increase the incidence of lung cancer and childhood leukemia with pooled ORs of 1.38  (I However, the heterogeneity was primarily high owing to the significant variation in the individual study regions, sample size, and other effect factors .In the sensitivity analysis, removing one study at a time did not significantly affect the pooled OR; it ranged from 1.30  (I2 = 0% and p = 0.51). In contrast, the pooled RR suggested no association between radon exposure and cancer . The heterogeneities did not exist in the comparison based on OR values, while the comparison based on RR values exhibited a moderate degree of heterogeneity (I2 = 0% and 52%).The pooled estimate of 13 case–control studies for the increase in childhood leukemia induced by residential radon exposure was divided into subcomparisons based on the estimated size effects, namely, OR and RR . The res3, 100–150 Bq/m3, and ≥200 Bq/m3. Finally, the included studies were categorized regarding smoking status of patients, including nonsmokers and smokers.In this study, a subgroup analysis was designed to assess the impact of residential radon inhalation on human health based on estimated OR values corresponding to four subcriteria . First, 2 > 50%). In the analysis of the study population, a statistically significant increase in lung cancer due to residential radon exposure was observed in the case–control studies conducted in Europe , but not in those conducted in North America  and Asia  (3 and ≥200 Bq/m3)  . The res0 Bq/m3) . Regardi2 < 50%). Statistically significant associations were observed in European studies and studies conducted for <10 years, with pooled ORs of 1.36  and 1.49 , respectively. Moreover, the adverse effect of a residential radon level of ≥200 Bq/m3 on childhood leukemia was confirmed. Notably, all subgroup analyses achieved high statistical significance without heterogeneity (I2 = 0%–42.35% and p ≥ 0.1).The subgroup analyses of studies of childhood leukemia were performed using fixed-effect models because of insignificant heterogeneity . With a 100 Bq/m3 increase in the residential radon level, the risk of lung cancer was not significantly different between the case and control participants . Therefore, a dose–response relationship was concluded to exist between all lung cancers and residential radon exposure.In this study, only 15 included studies provided relevant data regarding lung cancer risk due to a 100 Bq/m studies ,46,48,59Funnel plots were constructed to detect publication bias. The results indicated that articles with positive OR/RR values have been published, while articles with negative OR/RR values have not been published, leading to an unsymmetrical publication bias A,B. In c3 increase in the residential radon level, the lung cancer rates were not significantly different between the case and control patients.In this study, the causal associations between residential radon exposure and the increasing incidence of human cancers were assessed through a systematic review and meta-analysis of case–control studies. According to the eligibility criteria, 55 case–control studies with a total of 150,175 participants were enrolled and evaluated, and the results showed that radon exposure was strongly associated with increased lung cancer and childhood leukemia. However, with a 100 Bq/m2 = 42.28% < 50%), but not in North America and Asia. One out of 10 lung cancer cases in Europe is reportedly attributable to indoor radon exposure, and naturally occurring indoor residential radon may have caused around 19,000 lung cancer deaths in Europe in 2019  and 1.21 , respectively. Finally, the analysis of smoking status indicated a strong association between radon and lung cancer. The results revealed that in the same areas with high radon concentrations, smokers had higher lung cancer incidence than nonsmokers . Thus, it can be said that radon and smoking can be considered to have a synergistic effect on the occurrence and development of lung cancer.For assessing the relationship between radon exposure and lung cancer, 42 case–control studies that provided estimated size effect values (39 ORs and 3 RRs) were selected to perform a meta-analysis. However, the strong relationship was only confirmed through the meta-analysis based on OR values with a pooled OR of 1.36 , even though the analysis had high heterogeneity. The significant heterogeneity in the statistical analysis can be explained by the fact that these included studies were conducted in various regions of the world and under different radon exposure concentrations with different periods of investigation. To reduce the heterogeneity, a subgroup analysis was then designed to reevaluate the relationship based on subcriteria such as the study population, study period, and residential radon exposure. The results revealed a significant difference in lung cancer rate between the cases and controls in Europe  without heterogeneity (I in 2019 . The hig in 2019 . Specifi in 2019 . Owing t2 = 0% and p = 0.51). In contrast, the pooled RR of 1.15  (I2 = 52% and p = 0.08) from five case–control studies suggested no significant difference between childhood leukemia in the case and control groups. The controversial results may be due to the small size of the analysis, with eight studies reporting OR values versus five studies reporting RR values. Thus, adverse effects must be analyzed when larger sample sizes are obtained in the future. Similar to the lung cancer issues, the impact of radon on childhood leukemia was analyzed based on three subcriteria: study population, period of investigation, and radon level. The results also indicated that European children were more affected by residential radon than North American children, with pooled ORs of 1.36  and 1.89 , respectively. Investigations with a shorter duration (≤10 years) seemed to yield highly accurate results for predicting adverse effects compared with a longer duration (≥20 years). Notably, the impact of radon exposure was confirmed in participants exposed to high radon levels (≥200 Bq/m3) with OR of 1.56  (I2 = 42.35% and p = 0.188). Therefore, a high residential radon level over a long period is concluded to be positively associated with childhood leukemia; it mutates cells in the immune system and bone marrow, leading to DNA damage and influencing other biologic mechanisms, and consequently inducing childhood leukemia   determined the risk of lung cancer associated with radon exposure through a collaborative analysis of 13 case–control studies in nine European countries [ determinn Europe . Li et an Europe . Moon etn Europe . Poolingleukemia . All of The meta-analysis performed had three primary limitations. First, this study included only case–control studies; these may provide an incomplete overview of the causal association between residential radon and human health. Therefore, this topic should be addressed by analyzing all types of studies, such as cohort studies, ecological studies, and randomized clinical trials in the future. Second, the results may be affected by the classification of radon exposure. Given the different radon concentrations in other regions, the radon exposure classifications designed for each study are also different. Third, because it extracted only the OR/RR values of all lung cancers and all childhood leukemias, the study only investigated the overview of radon-induced cancers. Therefore, detailed conclusions about the incidence of each histological type of lung cancer  and childhood leukemia  could not be drawn.3) had a higher incidence of cancers compared to those in other regions . Thus, appropriate technical and policy solutions should be recommended to reduce radon exposure to ensure the health of environmental conditions and residents. Further, although the meta-analysis revealed the high impact of residential radon on human health, the results were limited by their high heterogeneity and unsymmetrical publication bias. Thus, this investigation should be repeated when more relevant articles are available in the future to overcome the current limitations.This meta-analysis of 55 case–control studies, including a total of 49,805 case patients and 100,370 control patients, suggested that residential radon was a risk factor for lung cancer and childhood leukemia when using estimated OR values for the analysis versus RR values. Notably, participants who lived in Europe or an area with high radon levels (≥100 Bq/m


BRAF has been found to be altered in > 50% of cases. Most of these have been BRAF V600E mutations, whereas the incidence of BRAF V600K may vary from 10% to 30%. Little is known about the clinical prognostic correlations of BRAF V600K MMs. We evaluated the clinical and dermoscopic features, incidence, therapy response and outcomes in the medium to long term.A number of mutations related to malignant melanoma (MM) have been identified, and of the mutated genes, BRAF V600E and BRAF V600K mutations.To compare the clinical and dermoscopic characteristics, the response to systemic therapies and the prognosis among MMs with BRAF V600E and BRAF V600K mutations were included. Any MMs positive for BRAF V600K mutation were collected, and the number of V600K cases and their features were used to extract the same number of patients with BRAF V600E from our database using a matching method. The clinical and dermoscopic presentation, therapy response and disease progression of the two groups were then evaluated.We retrieved the data of patients tested in our centre for MM from 2012 to 2015, including clinical features, dermoscopic pictures, clinical history and tumour mutations. Only patients with BRAF V600E‐mutated MMs were identified, and then randomized with a propensity‐score method to match the 10 retrieved cases of BRAF V600K mutation. Both groups had a nodular appearance to the tumours and an advanced disease stage, and no significant differences in dermoscopic features were highlighted. During the follow‐up period, four patients with BRAF V600K died of disease‐specific causes. Moreover, we found a higher frequency of metastasis, a faster disease progression and more rapid mortality in patients with BRAF V600K.In total, 132 cases of BRAF V600E MMs, BRAF V600K MMs seem to be less responsive to therapy and have a worse prognosis.Despite the small size of this study, the results show similar clinical and dermoscopic characteristics between V600E and V600K mutations, but compared with  BRAF V600K variant have a similar clinical and dermoscopic presentation but a more aggressive behaviour than those with BRAF V600E.Our work shows that melanomas affected by the  Malignant melanoma (MM) is a neoplasm that results from uncontrolled proliferation of melanocyte cells.RAS, BRAF, MEK and ERK.BRAF ranges between 40% and 60% in patients with MM. The most prevalent mutations in MM are the BRAF V600E variant (about 80%) and BRAF V600K (5–30%), with other subtypes found at lower frequencies: V600M 4%, V600R 5% and V600D < 5%.Uncontrolled activation of the mitogen activated protein kinase (MAPK) signalling pathway is principally involved in MM pathogenesis.BRAF mutations within the same patient has been described, with differences between primary tumours and metastases, and between different metastases.BRAF mutation status is usually performed on the most recently resected or biopsied tumours. It is important to note that although BRAF inhibitors show comparative efficacy in both the V600E and V600K mutations, the two tumour types have sometimes been considered as distinct entities.BRAF V600K MMs, in contrast to BRAF V600E MMs, seem to increase with age and have a higher risk for brain and lung metastases and a shorter time from diagnosis to metastasis onset and patient death.BRAF V600K‐mutated MMs also described significant differences in sex, age, primary MM location, the interval from MM first diagnosis to Stage IV disease and the overall survival after the diagnosis of Stage IV disease. In particular, the V600K mutation was significantly associated with older age, male sex, head and neck as the primary MM site, short overall survival from the time of diagnosis of Stage IV disease and a higher degree of chronic sun damage, which might explain the variable geographical frequency of BRAF V600K.Heterogeneity in et al.BRAF and NRAS mutation status in primary cutaneous MM, very little is known about the dermoscopic and prognostic differences between V600E and V600K variants and the correlation of dermoscopic features and mutational burden.Finally, although Pozzobon The study was approved by the ethical committee at our institution (no. DERM‐MTC 2017). No informed consent was required.We conducted a retrospective analysis of patients registered at our Melanoma Unit at the Policlinico Sant'Orsola‐Malpighi, Bologna, Italy. The data are available on request from the authors.BRAF mutations. Only patients with at least 5 years of follow‐up during the period 2012–2015 and treated with immune or targeted therapies were included.We built a database using data collected by primary search from our internal register of anatomopathological reports, collecting all the cases of MMs to analyse for We used next‐generation sequencing technique for mutation analysis, using a multigenic panel .Patients considered as having been treated with targeted and immunotherapies only if a cycle of treatment, consisting of the standard dosage and with a duration of at least 12 months, had been performed with at least three control computed tomography scans for evaluation.Independent variables such as sex, age, tumour location on the skin, treatment type, TNM stage and Breslow thickness were registered in this purpose‐built database. Body sites for tumour location was included the limbs, trunk, face, scalp, back, hands, feet and anogenital area.BRAF V600K mutation were collected. The number of V600K cases and their features were used to extract the same number of patients with BRAF V600E from the database using a matched propensity score method. Matching criteria including patient sex and age, Breslow thickness, tumour site, TNM and therapies in use. Dermoscopic images of cases belonging to both groups were downloaded from our videodermoscopy database.All MMs positive for We compared the two groups for the characteristics of interest. The clinical presentation of the tumour was assessed as one of three patterns: nodular, ulcerated nodular and superficial.The evaluated dermoscopic characteristics included vascular pattern, blue–white veil, chrysalids, blue–grey blotches, reticular grey–blue areas, white regression structures, peppering, and presence of > 3 colours. Three trained dermoscopists have evaluated magnified images registered in our database. Each dermoscopic criteria have been singularly evaluated and each dermatologist was blinded to patient identity, clinical information and mutational status at the time of the assessment.Response to therapy was evaluated by analysing the trend of metastasis numbers and volume on three consecutive CT scans after therapy administration. Outcomes considered were no metastasis; tumour in remission, stable, with partial improvement or no improvement; mortality rate; time to metastasis onset (months); and time to death (months).BRAF V600E group was extracted from a larger group using a matched propensity score method. Approximation values for independent variables with a tolerance of 20% were used to reach 1 : 1 ratios in both groups. The two groups were then rechecked for homogeneity using the Mann–Whitney U‐test for quantitative variables and Fisher exact test for qualitative variables. Quantitative data were then expressed as mean ± SD and an independent samples Mann–Whitney U‐test was used to compare the two groups. Qualitative data were described using the Fisher exact probability method. P < 0.05 was considered statistically significant. Kaplan–Meier curves were used to represent the mortality and metastasis onset between the two groups of patients. Data processing and statistical analysis were performed using an Excel  database and SPSS software .The BRAF V600 mutations and with at least 5 years of follow‐up. Of these, 132 patients were positive for the BRAF V600E variant and 10 patients for the BRAF V600K mutation. Matching these groups, we identified 10 patients whose independent variables were not statistically different from that of patients with BRAF V600K.From the search in our hospital database, we found 223 patients positive for BRAF V600E group, and 65.90 years for the BRAF V600K groups  there was no difference in TNM stage evaluated between the two groups for the same Breslow thickness.The mean Breslow thickness was 2.35 mm for The tumours were classified in accordance with their appearance, with ulcerated nodular being the most common, followed by nodular and then superficial.BRAF V600K group and four images in the BRAF V600E group, making a total loss of seven cases (35%). However, analysis of the remaining samples did not demonstrate any noteworthy characteristics for either group.There was lower statistical power for the dermoscopic feature due to the lack of three images in the BRAF V600E group, 70% of the cases were treated with a combination of dabrafenib and trametinib, while 10% were treated with nivolumab and 10% with ipilimumab; for the remaining 10% of patients, it was not possible to identify the therapy because of incomplete medical records. The BRAF V600K groups also had 70% treated with a combination of dabrafenib and trametinib, while 10% were treated with nivolumab and 10% with a combination of vemurafenib and cobimetinib; again, it was not possible to identify the therapy for the remaining 10% of patients because of incomplete medical records.In the BRAF V600E group; eight patients in this group had stable response, compared with partial or no response in six patients in the BRAF V600K group.The study identified an interesting difference in the response to therapies, with a better outcome achieved in the BRAF V600E and nine patients with BRAF V600K died . Finally, a similar but not statistically significant outcome was observed in metastasis onset, which developed more rapidly in the BRAF V600K group than in the BRAF V600E group  nodular or ulcerated appearance.BRAF V600 mutations.Because of the missing dermoscopic images, our sample appears too small for analysis to confirm a significant pattern between the two groups; however, the analysis of the remaining cases did not highlight any specific differences between the groups. Furthermore, also white regression and peppering has been reported by other groups, we did not find such a high prevalence of this feature in our study, casting doubt on its correlation with BRAF V600E group, although it was challenging to achieve remission of metastases, 8 of the 10 patients had at least a stable response, whereas in the BRAF V600K group, 6 of the 10 patients achieved little or no response. These results strongly suggest stronger resistance to targeted therapies by BRAF V600K‐mutated tumours compared with the more widespread BRAF V600E variant, which agrees with previous studies.BRAF V600K, with 50% of the patients developing metastases in the first year after the first diagnosis, indicating a more aggressive course. The observed mortality rate was in accordance with the metastasis rate, with higher mortality from disease‐related causes occurring in the BRAF V600K compared with the BRAF V600E group, with a ratio of 4 : 1, respectively, and approximately half of the patients in the BRAF V600K group died within 3 years of diagnosis.By contrast, the analysis of the response to systemic treatments was clearer; in the Despite the small size of our study being a limitation the results allowed us to identify some interesting elements and distinctive traits between the two groups. There were no differences in clinical features or dermoscopic patterns between the two variants. We could not identify any clinical feature that, without molecular analysis, could lead to one mutation being suspected over the other.BRAF V600K‐mutated MMs showing lower response rate to therapies and a more rapid and frequent tendency to develop metastasis, implying greater resistance to the proposed treatments. Consequently, patients with BRAF V600K‐mutated MMs also had a more rapid and increased mortality rate during follow‐up.However, we did find differences in therapy response, metastasis development and mortality rate, with BRAF V600K mutation have a similar clinical presentation but more aggressive behaviour than those with the BRAF V600E mutation.•BRAF V600K MMs have different clinical and dermoscopic features, a higher risk for developing metastases and a worse prognosis compared with BRAF V600E MMs.•BRAF V600K is more resistant to systemic therapies in comparison to BRAF V600E.•We found nonsignificant differences in the clinical and dermoscopic presentations of the two principal mutations.•BRAF V600K tumours showed significantly greater resistance to systemic therapies and increased development and progression of metastases and mortality.In summary, this work shows that MMs with the


ABS) and P use efficiency (PUE), and consequently biomass accumulation and nutrient uptake. The significant correlations established between leaf stomatal density, PIABS, and PUE supported the above hypothesis. We concluded that the Poly-P fertilizers applied in well-watered conditions (I1) performed the best in terms of chickpea growth improvement, nutrient uptake and use efficiency. However, their effectiveness was greatly reduced under water stress conditions, unlike the Ortho-P form which kept stable positive effects on the studied parameters.Water shortage and soil nutrient depletion are considered the main factors limiting crops productivity in the Mediterranean region characterized by longer and frequent drought episodes. In this study, we investigated the interactive effects of P fertilizer form and soil moisture conditions on chickpea photosynthetic activity, water and nutrient uptake, and their consequent effects on biomass accumulation and nutrient use efficiency. Two P fertilizer formulas based on orthophosphates (Ortho-P) and polyphosphates (Poly-P) were evaluated under three irrigation regimes , simulating three probable scenarios of soil water content in the Mediterranean climate , and compared to an unfertilized treatment. The experiment was conducted in a spilt-plot design under a drip fertigation system. The results showed significant changes in chickpea phenotypic and physiological traits in response to different P and water supply regimes. Compared with the unfertilized treatment, the stomata density and conductance, chlorophyll content, photosynthesis efficiency, biomass accumulation, and plant nutrient uptake were significantly improved under P drip fertigation. The obtained results suggested that the P fertilizer form and irrigation regime providing chickpea plants with enough P and water, at the early growth stage, increased the stomatal density and conductance, which significantly improved the photosynthetic performance index (PI The impact of climate change on agriculture in the arid and semi-arid regions is increasingly felt, through the negative impacts of the decrease in precipitation, the high temperatures and radiation, and the longer and frequent drought on crop yield and quality3. Under these challenging pedoclimatic conditions, plants showed a series of morphological, physiological, and biochemical modifications to adapt to different types of abiotic stress and improve their capacity to absorb and use water and mineral resources efficiently6. It is widely documented that plants exposed to drought and nutrient deficiency reduce their photosynthetic activity and gas exchanges with the environment, by reducing the leaf area, closing their stomata, and increasing assimilates allocation to the root system9.Drought and soil nutrient depletion are considered the main factors influencing crop productivity in the Mediterranean region12. Although the application of P fertilizers has significantly improved crop yields through the enhancement of several biophysiological processes in plants, P is still one of the main nutrients showing low use efficiency14. In some cases, excessive amounts of P fertilizer are used to ensure high crops yield, which can consequently lead to abusive use of this non-renewable resource and damage the ecosystem15. P use efficiency is mainly dependent on P mobility and availability in the soil17. The capacity of plants to take up P is strongly related to many soil characteristics and parameters as well as to the fertilizer properties such as pH, fertilizer form and solubility and its interaction with the soil moisture and compounds20. For example, the application of orthophosphate (Ortho-P) fertilizers under high alkaline soil conditions resulted in high interaction between P ions and other bivalent cations, which can lead to the formation of some insoluble P forms, and consequently reduce P uptake and recovery by plants21.Phosphorus (P) is the second macronutrient limiting crops productivity in alkaline soils, mainly under arid and semiarid areas, due to its rapid complexation and precipitation with soil cations23. With this regard, increasing attention was paid to polyphosphates (Poly-P) fertilizers due to their high solubility and slow-release properties26. The Poly-P, which are defined as a condensed form of P, are usually less reactive in soil compared to the Ortho-P forms and present some interesting properties such as their capacity to chelate some micronutrients, especially under alkaline soil conditions27. Despite, these beneficial effects of Poly-P on nutrient availability and crops yield, their effectiveness is drastically influenced by soil properties and agricultural practices such as soil moisture and water irrigation amount and scheduling28.To deal with this major challenge, several strategies have emerged to improve P use efficiency, including the development of new fertilizer formulas and the implementation of high-frequent drip fertigation practices, as well as P foliar application and the use of plant growth-promoting rhizobacteria (PGPR) and biostimulant31. However, its response to P and water supply is greatly dependent on P fertilizer forms and water application strategies33. Even though drip fertigation is a highly efficient technic to maximize chickpea production, some issues related to the choice of fertilizers forms, rates, application timing and their interaction with soil moisture content are still of high interest for farmers, agronomists, and plant physiologists. It should be noted that most studies dealing with the interaction between water and P have mainly focused on P availability in soils and its direct effect on crops yields35. More attention can be drawn to the effect of P fertilizer forms, in different irrigation scenarios, on the photosynthetic apparatus, leaf stomatal activity, nutrient uptake and allocation, and their consequent effects on biomass accumulation and crops yield.Chickpea is a legume crop cultivated in the Mediterranean basin for its high nutritional value and its capacity to grow in harsh environmentsThe objective of this study was to investigate the interactive effect of P fertilizer form and soil moisture content on the main biophysiological process influencing chickpea growth. Three P fertigation regimes  were evaluated under three irrigation scenarios simulating three soil water content levels . The drought stress regimes were applied on 1-month-old plants and maintained for 32 days. Emphasis was placed on how Poly-P fertilizers can contribute to improving P use efficiency and crops yield under Mediterranean conditions characterized by frequent drought events.−1 of total nitrogen, 26 mg kg−1 of Olsen-P, 246 mg kg−1 of exchangeable K2O, 10.6 g kg−1 of CaO, 427 mg kg−1 of MgO, pH 8.4, 1.4% of organic matter, and a bulk density of 1.2 g cm−3. Further details regarding soil physicochemical properties are reported in Chtouki et al.25. The chickpea was planted at a density of 9 seeds per pot on February 11th, 2021, then thinned 3 days after plant emergence to ensure a homogeneous population density of 3 plants per pot, equivalent to 30 plants m−2. Chickpea plants were harvested on April 14th. Chickpea seeds (Cicer arietinum L.) were obtained from the Moroccan National Seed Marketing Company (SONACOS). This variety used for the experiment was “Kabuli Moubarak”, which is widely planted in Morocco.The experiment was conducted under greenhouse conditions at the experimental farm of the Mohammed VI Polytechnic University, in Benguerir central Morocco (32° 13′ 11.5′′ N 7° 53′ 29.9′′ W) which is a semi-arid region, with a mean monthly rainfall of 13.18 mm, of which approximately 7.75 mm fall in spring mm. The experiment was conducted in the 2021 spring chickpea growing season, with an experimental design composed of 54 pots  containing 10 kg of sandy–clay–loam soil per pot. The studied soil is characterized by 0.9 g kg2O5 per kg of dry soil  and compared to unfertilized treatments (control: zero P application). During the growing period, chickpea plants were fertigated with N and K2O at the rate of 8 and 14 mg kg–1 of dry soil respectively . Ammonium nitrate (33.5% N) and potassium nitrate  were used to adjust the nitrogen and potassium to the desired levels. Total quantities of the NPK fertilizers, including the Ortho-P and the Poly-P forms, were fractioned on 4 equal applications, and applied through a drip fertigation system at the rate of one application every two weeks. One dripper with a flow rate of 4 L h−1 was installed in each pot. The experiment was conducted in a split-plot design with six replicates per treatment.During the first 30 days from sowing, chickpea plants were grown under adequate soil moisture conditions (75% of soil FC). After that, three different irrigation regimes: irrigation at 75% of field capacity (FC) (I1), irrigation at 50% of FC (I2), and irrigation at 25% of FC (I3) were applied (until the harvest), based on regular measurements of soil volumetric water content (VWC). The soil VWC was measured by the soil moisture meter WET-2 sensor  that reads and stores measurements. The amount of required water for each irrigation regime was calculated every 2 days to maintain the soil at the desired field capacity level. In each irrigation regime, two inorganic P fertilizers, orthophosphate (Ortho-P) or polyphosphate (Poly-P) were applied at rate of 16 mg of PAfter 55 days from sowing, nine leaflets per treatment were sampled on fully expanded leaves, located at the middle of the canopy, to assess the interactive effect of P fertilizer form and soil water content on chickpea plant gas exchange. Nine fingernail polish imprints were taken from the abaxial surface of the leaflets for each treatment, then the imprints were observed under an optical microscope  equipped with a digital camera . Three images were randomly selected from each imprint (27 images per treatment) and the number of stomata was counted. Using the image analysis software , the stomatal density was calculated by dividing the number of stomata by the area of each image. The stomatal conductance was also measured on six young and fully developed leaves per treatment using SC-1 leaf porometer  after 56 days from sowing.a fluorescence (ChlF) was also evaluated to assess the impact of P fertilizer form and irrigation regime on the photosynthetic efficiency and the electron transfer into the photosystem II (PSII). Eighteen ChlF a measurements per treatment were performed on the youngest fully expanded leaves after 20 min of dark adaption (57 days after sowing). The measurements were taken by a portable Handy PEA fluorimeter , with 3000 µmol photons m−2 s−1 of light intensity provided by an array of six light-emitting diodes (peak 650 nm) and focused on the sample surface (4 mm2). For each studied treatment, the fluorescence transient (OJIP curve) was recorded during the first second of leaf illumination and two photosynthetic parameters were calculated: the photosynthetic performance index (PIABS) which give quantitative information about the energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors36, and the total driving force DFABS as suggested to estimate the partial driving force of photosynthetic processes evaluated by the corresponding PIABS37.To assess the impact of P fertilizer form on the photosynthesis apparatus of chickpea plants grown under different soil moisture conditions, the chlorophyll content index (CCI) was measured on mature and fully expanded leaves after 56 days from plant sowing. The CCI measurements were taken by portable chlorophyll meter , considering nine CCI leaf measurements per treatment. Further, the Chlorophyll The interactive effect of the irrigation regime and P fertilizer form on chickpea growth was assessed by the measurements of two variables: specific leaf area and total leaf number per plant. The specific leaf area was measured 54 days after plant sowing on nine mature and fully expanded leaves per treatment, using a portable leaf-area meter , while the total leaf number per plant was taken at the harvest stage. Chickpea plants were harvested after 62 days from sowing, then shoot and root parts were carefully separated and washed with deionized water. The dried biomass was taken after 72 h of oven drying at 60 °C and the root/shoot ratio was calculated to assess the impact of different fertigation regimes on assimilates partitioning in chickpea plants.3 for the determination of macro and micronutrients content  using the ICP-AES . The total N content in shoot and root samples was measured by Kjeldahl method, in K-375 auto-analyzer  according to O’Dell38. Total macro and micronutrient uptake by the plant was calculated for each treatment as the sum of the product of root and shoot dry biomass with the corresponding nutrient concentrations.The dried biomass of shoot and root were separately grinded and subjected to acid digestion with 4 M HNOTo evaluate how P fertilizer form and soil water content influence P acquisition and use by chickpea plants, P use efficiency (PUE) was calculated according to the following equation:39.The impact of P fertilizer supply, drought stress and their interaction on the irrigation water productivity was calculated by dividing the produced shoot dry weight of each treatment on the total irrigation water used for the corresponding irrigation regime40.The interactive effects of the irrigation regime and P fertilizer form on the studied variables were analyzed through factorial design (two-way ANOVA) in SPSS data processing software (SPSS 20.0), and mean differences between treatments were evaluated by Duncan’s new multiple range test at 0.05 probability levelAll the methods were carried out in accordance with relevant guidelines and regulations. Furthermore, regulations and all experimental protocols were approved by a named institutional and/or licensing committee.To regulate the gas exchange with its environment, the chickpea plant significantly changed its stomatal density and conductance depending on soil moisture conditions and P nutrition regime. The obtained results in Fig. Chlorophyll content, as assessed by SPAD measurements on fully expanded leaves, was significantly increased with P supply for all studied irrigation regimes Fig. b. Under O, which refers to minimal fluorescence when RCs are open, was slightly increased under drought stress regimes (I2 and I3) comparatively to that under well-watered conditions (I1), while the maximum fluorescence Fm, referring to maximal fluorescence when RCs are closed, was significantly decreased. A positive effect was also observed on the Fm in response to different P fertilizer applications, especially under adequate irrigation and medium water stress regimes. The changes in the O-J-I-P transients were more pronounced at the K-steep (300 µs) and the I-P phase, particularly under moderate drought conditions and P fertigated treatments.Data in Fig. ABS, in all studied irrigation regimes, with pronounced values under full irrigation (I1) and medium water stress conditions (I2). Regarding the mean values per irrigation regime, we observed a significant decrease in the PIABS of plants exposed to medium and severe water stress , in response to the different combinations of water and P fertilizer regimes  when applied under well-watered conditions (I1), although under moderate and severe water stress regimes the Poly-P fertilizer recorded values like those of the Ortho-P.Regarding the impact of the studied irrigation regimes on the irrigation water productivity (IWP), it can be seen in Fig. Soil water content is one of the main factors influencing plant nutrient uptake, and it is widely known for its primordial role in solutes transport to roots, nutrient solution equilibrium, and microbial activity improvement. The obtained results revealed that chickpea plants responded differently to P supply, P fertilizer form, and their interactions with the irrigation regimes. Data in Table 43. It is well documented that the individual or the combined effects of drought and P deficiency affect photosynthetic activity, nutrient uptake, and plant growth and yield45. The previous studies dealing with the interaction between water and P were mainly focused on the effect of P rate applied or P concentration in the soil solution on crop growth and development46. To the best of our knowledge, this is the first study that investigated the interactive effects between P fertilizer form (with a focus on Ortho-P and Poly-P) and soil water content on chickpea photosynthetic activity, plant growth, water and nutrient uptake, and their use efficiency. The response of the chickpea plants to Poly-P and Ortho-P fertilizers was studied by the simulation of three probable scenarios of soil water content  in the Mediterranean region, strongly characterized by longer and very frequent drought episodes.Under nutrient and water stress conditions, plants manifest multiple morphological and physiological changes to improve their capacity to absorb and use water and mineral resources46, hence, the combination of Poly-P and full irrigation regime (I1) resulted in the highest stomata density and conductance. Contrary to the Poly-P which greatly reduced the stomatal density under drought stress conditions (I2 and I3), the Ortho-P fertilizer kept its positive effect on stomatal density and conductance even under water stress regimes. These differences between P fertilizer forms can be explained by the impact of soil water content on the availability of P in soil. Under drought conditions, available P from the Ortho-P fertilizer is expected to be higher than that of the Poly-P since the hydrolysis of Poly-P is greatly reduced under water stress conditions48. Similar results were found by Sekiya and Yano46, who reported that an increase in superphosphate (which are Ortho-P fertilizer) application rate improved cowpea stomatal density under different water supply conditions. The authors revealed also that the improvement of the stomatal density with P supply was more pronounced under elevated atmospheric CO2 concentrations. According to Sharma et al.49, chickpea plants grown under drought and low P supply conditions reduced significantly their stomatal conductance to avoid leaf turgor loss via the transpiration process.The present study showed significant effects of P fertilizer form and irrigation regime as well as their interaction on the chickpea leaves. The application of P fertilizers significantly increased the stomata density under well-watered I1) and medium drought regimes (I2) compared to the unfertilized treatment , and photosynthetic performance index PIABS (r = 0.87)  to supply chickpea plants with sufficient amount of P and water and also by their indirect effects on the uptake of other nutrients, mainly those directly involved in the electron transport59. As shown in Table 24, who reported that Poly-P fertilizer had the capacity to chelate micronutrients , which significantly improved their uptake in maize plants under alkaline soil conditions. However, in our study, we demonstrate that strong chelation of micronutrients by the Poly-P may greatly reduce their availability for plants, especially when the hydrolysis process of the Poly-P fertilizer is impacted due to the low soil moisture content  Fig. a,b. Thes62, their effectiveness is greatly reduced under water stress conditions, unlike the Ortho-P fertilizer which exhibits a stable effect on plant growth even under water stress conditions. These changes in chickpea plants’ phenotype (specific leaf area and canopy) and growth  can be associated with the impact of different water and P supply regimes on photosynthesis efficiency65. Plants exposed to prolonged drought stress significantly reduced their stomatal density and conductance which greatly decreased the photosynthesis activity as assessed by the PIABS index and consequently reduced the CO2 fixation and biomass accumulation46. Moreover, the allocation of the photosynthetic assimilates may be changed. As revealed in Fig. 68.Our findings related to chickpea growth attributes revealed that an adequate P and water supply resulted in higher leaf specific area, higher leaf number per plant, and greater shoot dry matter. The combination of the Poly-P form with the I1 irrigation regime recorded the best results in terms of canopy growth and biomass accumulation. Significant interactive effects between P fertilizer form and soil water content were observed. Regarding results in Figs. ABS) and P use efficiency supported our hypothesis  significantly improved stomatal conductance, photosynthetic activity, biomass accumulation, and nutrient uptake, their effectiveness is strongly influenced by soil water content. The obtained results clearly showed the significant effect of polyphosphate fertilizer forms on chickpea response to P supply (greater plant growth and P use efficiency). Therefore, water availability remains an important point to be considered for any eventual integration of the Poly-P fertilizer forms in the crop fertilization programs under Mediterranean conditions, especially for annual crops, highly dependent on P nutrition at their early growth stages.


We compare the optical quality and design characteristic a new low cost solar powered binocular indirect ophthalmoscope (BIO), Holo, to Keeler BIO.Twenty-four participants each examined 10 simulation eyes using both the Holo and the Keeler BIO with a 30-diopter condensing lens. Number of Lea symbols printed on the retina of simulation eyes seen and time taken to identify them was recorded. Stereoacuity of 12 participants was tested while using the BIOs. Using 7-point Likert scale, participants gave feedback on design characteristic of both BIOs.p = 0.366, paired t test) or time taken to correctly identify each symbol  using each device. 12 out of 12 participants achieved stereoacuity of 60 arcsec using the Holo while with the Keeler BIO 11 achieved 60 arcsec and one 90 arcsec. There was no statistically significant difference in the scores for clarity of view, quality of illumination, field of view, binocularity, eye strain and robustness between the two devices. The Holo, scored higher for ease of use , comfort of wear  and portability .There was no statistical difference in number of Lea symbols correctly identified (15.63/20 for Holo vs. 15/20 for Keeler BIO, The Holo has the potential to be a clinically useful yet affordable diagnostic tool suitable for the first time of equipping eye care workers in low resource settings with a BIO at volume. Globally, 553 million people have some degree of visual impairment with an estimated 43.3 million being blind . Nine ouThe binocular indirect ophthalmoscope (BIO) is an important diagnostic tool for assessing posterior segment eye diseases. The BIO, in contrast to the view achievable with the more commonly available direct ophthalmoscope, offers a wide stereoscopic view of the retina, often despite hazy media . TraditiConsequently, a low cost solar powered BIO Fig.  called tIn this study we compare the optical quality and design characteristics of the Holo ($75) with a widely available traditional spectacle mounted device, the Keeler Spectra Iris Indirect Ophthalmoscope (Keeler BIO - $3203.90) amongst a range of eye care practitioners.University Teaching and Research Ethics Committees of the University of St Andrews granted approval to undertake the study which followed the tenets of the Declaration of Helsinki.Twenty-four participants who used a BIO in their daily work were recruited . They examined simulated eyes  instead of images of fundi.After using the two BIOs a 7-point Likert scale was used to rate the following characteristics: ease of use, quality of illumination, robustness, portability, comfort of wear, clarity of view, binocularity, field of view and eye strain.Participants were finally asked how frequently they used the additional features of a BIO including: safety filter, cobalt blue filter, red-free filter and variable aperture using a 5 option response; never and not aware of the feature, never but aware of the feature, less than 50% of the time, half of the time or more, and 100% of the time.t test was performed for the paired continuous variables. Wilcoxon signed-rank test was performed for the paired ordinal variables. All tests were two-tailed with type I error set at α = 0.05 and it was ensured that data met the particular test’s assumptions.Using IBM SPSS Statistics V.26, paired p = 0.366, paired t test) was statistically the same. There was also no difference in time taken to correctly identify each symbol  and mean time per symbol for Keeler = 14.83 s (SD = 5.86)).There was no statistical difference in the diagnostic and optical performance of the two devices. The number of Lea symbols correctly identified using either device (15.63/20 (Standard deviation (SD) = 3.74) for Holo vs. 15/20 (SD = 3.35) for Keeler BIO, There was no statistically significant difference between the different participants in either the number of symbols seen or time taken to identify.12 out of the 12 participants achieved stereoacuity of 60 arcsec using the Holo while with the Keeler BIO 11 achieved 60 arcsec and one 90 arcsec.p = 0.00488, Wilcoxon signed-rank test), comfort of wear  and portability .There was no statistically significant difference in the scores recorded for clarity of view, quality of illumination, field of view, binocularity, eye strain and robustness between the two devices. The Holo however scored statistically significantly higher for ease of use  and Conformitè Europëenne (CE) accreditation for this , 9. In aIn conclusion, this study demonstrates that the Holo has the potential to be a clinically useful yet affordable diagnostic tool suitable for the first time of equipping eye care workers in LMICs with a BIO at volume. The need to develop frugal technologies using innovative designs and manufacturing techniques to equip healthcare workers in low resource setting is more urgent than ever. The Holo represents such a device that can complement the many other strategies being established globally to reduce avoidable blindness .Binocular indirect ophthalmoscope is an important device in diagnosing retinal disease.Traditional binocular indirect ophthalmoscope is too expensive to be widely available in low and middle income countries.Holo, a frugal binocular indirect ophthalmoscope, offers users a comparable view of the retina to that achieved with a more expensive traditional binocular indirect ophthalmoscope.Holo can be used to equip eye care workers in low resource settings with a binocular indirect ophthalmoscope at volume.Holo – A frugal solar powered binocular indirect ophthalmoscope


GR), garlic (GC), oregano (O), and cinnamon (C) extracts. The experiment was divided into 2 stages, at preliminary in vitro stage antimicrobial susceptibility of plant extracts (PEs) was tested against reference strains from the American Type Culture Collection. O and GC extracts had the best antimicrobial properties (P < 0.05). Then in in vivo stage 2,400 Japanese quails hatching eggs were divided into 6 groups, 400 eggs each. Eggs from first group were not disinfected , eggs from second group were disinfected by formalin fumigation , in other groups 5% aqueous solutions of plant extracts of GR, GC, O, C were applied by spraying respectively. After standard incubation fertility, hatchability and periodical embryonic mortality were calculated as well as the body weight and livability of chickens during 14 d of rearing. Egg disinfection by aqueous solution of PEs led to maintain the hatchability, chick weight at hatch and post hatch body weight and early mortality of birds. Exclusion of any fungal isolates on eggshell surface was induced by GC followed by O and C groups (P < 0.05). In case of the bacteria colonies reduction only GR extract was effective. Chosen plant extracts may be treated as safe and alternative substances to traditional disinfectants of hatching eggs.The aim of study was to evaluate microbial and hatchability traits as well as chicks quality after hatching eggs disinfection with aqueous solutions of ginger ( E. coli, Proteus spp., Enterobacter spp., Pseudomonas spp., Klebsiella spp., Staphylococcus spp., Streptococcus spp., Clostridium spp., Bacillus cereus, Salmonella typhimurium, Micrococcus spp., Enterococcus spp., molds and yeasts are the most common microorganisms that have been isolated from hatching eggs. Therefore, the shell cleanness and the effectiveness of its disinfection is critical to achieve a high level of hatchability, reducing the burden of pathogens and ensuring the production of high quality chicks , prior to egg incubation  activity probably depends on pH, chemical composition, concentration or the specific bioactive compounds, also on population and types of affected pathogens. It has been found that plant extracts , garlic , oregano , and cinnamon  extracts.The aim of study was to evaluate microbial and hatchability traits as well as chicks quality after hatching eggs disinfection with aqueous solutions of ginger , garlic , oregano , and cinnamon  were used. The content of chosen phytochemical in PEs were analyzed as it was described before . The commercial, chemically pure powdered plant extracts of ginger  were used in this study.The PEs were used as powdery extracts, which was dissolved in distilled water by volumetric flask to produce 5% concentration. The experiment was divided into 2 stages. At preliminary stage the antimicrobial susceptibility of PEs was tested. The in vitro antimicrobial activity of the prepared plant extracts was tested against reference strains from the American Type Culture Collection (ATCC): MHB) in 96-well microtiter plates. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated. First, solutions of 200 mg/mL were prepared by dissolving individual plant extracts in dimethyl sulfoxide (DMSO). Then, a basic solution with a concentration of 40 mg/mL of the test compounds in MHB medium was prepared. Using the previously prepared solution, serial twofold dilutions were made in MHB obtaining extract concentrations ranging from 40 to 0.02 mg/mL.Tests were performed using the microdilution method in Mueller-Hinton Broth broth  was performed to check the viability of the strain and a negative control  was performed to check the sterility of the medium.Bacterial inoculum  was prepared in sterile saline from each strain. Then, 2 µL of bacterial suspensions were transferred to the wells each, obtaining a concentration of approximately 10MHA). The media were incubated at 36 to 37°C for 18 to 24 h. After incubation, MBC concentrations at which no bacterial growth occurred were determined. MBC/MIC ≤4 was taken as bactericidal effect and MBC/MIC >4 as bacteriostatic effect. The tests were performed in triplicate.The MIC value was determined by observing the lowest concentration at which no bacterial growth was observed in the well after incubation at 36 to 37°C for 24 h. Spectrophotometric method was used for this purpose by reading the absorbance at 570 nm using EL × 808 microplate reader . For the determination of MBC, 5 µL of the contents of each well were transferred onto Mueller-Hinton Agar medium  obtained from Japanese quailslayers at the age of 14 wk. The quails were housed in battery cages system with sex ratio (1♂:4♀) and fed with a breeder diet containing 2,900 kcal of metabolizable energy/kg and 20% of crude protein with free access to feed and water. Eggs were collected twice a day (in the morning and evening), taking into consideration that dirty eggs  and eggs with visible cracks of shell were discarded as hatching eggs. After collection, eggs were stored for no longer than 3 d at 15 to 18°C and 75% relative humidity before the start of research.4 and 21 mL of water . For disinfection in groups third, fourth, fifth, and sixth, aqueous extracts of GR, GC, O, and C were used respectively (5% distilled water solution).Eggs were divided randomly into 6 groups before incubation, 400 eggs per group (4 replication subgroups in each). Eggs from first group were not disinfected , eggs from second group were disinfected by fumigation with formaldehyde gas composed of 21 ml formalin (40%), 17 g KMnOPrior to eggs placing in the incubator, eggs were numbered according to sanitization treatments and disinfected by each aqueous extract of PEs using hand sprayer and covering the whole egg surface with solutions. After spraying, the egg trays were kept outside the incubator for 15 min at room temperature (24°C) to dry after disinfection procedures. With respect to egg fumigation by formaldehyde gas, it was implemented inside special chamber.-setting compartment–37.6 to 38.0°C temp. and 50 to 65% relative humidity.-hatching compartment–37.0 to 37.5°C temp. and 75 to 80% relative humidity.Eggs were hatched artificially using a Jarson hatching apparatus  under standard conditions of incubation:K) was determined as criterion of egg weight loss (moisture loss from egg) using the formula of PBS) with 3 drops of TWEEN 80. Containers with eggs were left on the stirrer for 1 h. Samples were serially diluted in PBS and plated on sterile medium in order to obtain the total number of aerobic mesophilic bacteria, the total number of bacteria, coliform bacteria, hemolytic bacteria, Salmonella spp., Staphylococcus spp., yeast, and mold fungi. After incubation colonies were counted and presented as cfu/1 mL of liquid from the egg. To identify the bacterial colonies, a microscopic examination was performed as well as Gram's staining method and API biochemical tests (bioMérieux Polska). Molds were identified using special keys . On 14th d of incubation the eggs were candled to determine the number of fertile and infertile eggs and dead embryos and moved from the setter to the hatching compartment. Fertility was calculated as the percentage of set eggs. Also, all eggs including fertile, infertile and unhatched eggs and hatched chicks were weighed and eggshell conductance constant  and survivability of chicks were registered on 1st, 7th, and 14th d of their age.After 17.5 d of incubation the number of hatched chicks, dead embryos, healthy and crippled chicks was recorded to determine the hatchability and mortality for both fertile and set eggs. Chicks were individually identified with numbered leg ring and raised (4 pens/group) according to their eggs disinfection treatments. They were reared under standard conditions of cage system for 14 d , a balanced diet  was provided P ≤ 0.05). The one-way ANOVA with Tukey's test was carried out. The mortality, hatchability and bacterial number of colony forming units were verified using nonparametrical χ2 test. All of the used tests were mentioned in tables.The data were analyzed with the use of statistical package SPSS 24.0PL . The norB. cereus ATCC 10876. The highest bactericidal activity of all tested extracts was observed against S. epidermidis ATCC 12228 (MBC/MIC = 1). Gram-negative bacteria were more sensitive to the tested extracts compared to Gram-positive bacteria. MIC values ranged from 10 to 20 mg/mL. All extracts showed bactericidal activity (MBC/MIC ≤4), except cinnamon extract, which showed bacteriostatic activity (MBC/MIC > 4) against S. typhimurium. The most sensitive Gram-negative bacterium was found to be P. aeruginosa ATCC 27853 with MIC = 10 mg/mL for all extracts tested.As indicated in the P = 0.001) and fertile eggs (P = 0.011) was recorded by NC and low total mortality on set eggs (P = 0.043) was in PC. All disinfectant groups and NC did not significantly differ in eggshell conductance const. (K) value and healthy chick proportion in egg. In weight loss of fertile eggs GR, GC and O did not differ from NC and PC in this trait. However, disinfectant solution of C led to positive reduction of weight loss of fertile eggs (%).Depending on group, E. coli in GC, Salmonella spp. in , Staphylococcus aciuri in , Staphylococcus epidermidis in , Staphylococcus spp. and nonidentified bacteria in (GC and O) have not been detected.No differences between GR, GC, O were stated as well as between NC and PC in body weight at hatch and at 14th d. Also, GR and GC did not differ from PC and NC with respect to BW at seventh day. However, C reduced BW at hatch and 14th d and O and C reduced BW at seventh day. Lack of differences in mortality and survivability in post hatch depending on group was recorded .Table 5BK values of fertile eggs compared to NC and PC. It can be reasoned that C increased the amount of metabolic water in embryonic tissues through oxidizing energy-containing substances found in C and also for its antioxidative properties to protect the growing embryo tissues  up to 6 wk of age were not influenced by spraying hen eggs with 0.55 or 0.75 μL of O/cm3 for 2 exposure times, 3 and 6 h compared to formalin and NC.All disinfectant solutions of PEs maintained stably the hatchability, total mortality on fertile egg, weight loss (%) of eggs, and embryos and healthy chick (%) as well as survivability of birds in post hatch. The C solution disinfectant exerted a potential effect to reduced water loss and  tissues . Egg wei tissues . The var tissues . The higy embryo  whereas Candida albicans, Aspergillus flavus, Aspergillus niger, Curvularia lunata, Cryptococcus neoformans, Trichophyton, Epidermophyton, Microsporum, and other fungi (DAS), diallyl disulfide (DADS), diallyltrisulfide (DAT), and diallyltetrasulfide (DATS) in GC oil had a powerful antifungal activity in vitro against 3 Candida spp.  and 3 Aspergillus spp.  and antimicrobial capability of these sulphides were determined by disulfide bonds. Allicin in GC was found to antimicrobial agent against wide range of strains of E. coli, C. albicans fungus, and intestinal protozoan parasites, such as Entamoeba histolytica and Giardia lamblia parasites  in eggshell as % of total isolates in most of PEs disinfectant solutions. It was also thought that the chemical structure, such as the existence of the functional hydroxyl (–OH) group attached to a phenyl ring in phenolic compounds of secondary metabolites in essential oils (EOs) of PEs have the greatest antimicrobial activity. Probably, its aromatic specificity is also responsible for the disruption of cell homeostasis, leading to growth suppression and cell death  led to maintain the hatchability, chick weight at hatch and post hatch body weight and early mortality of birds. Exclusion of any fungal isolates on eggshell surface was induced by garlic followed by oregano and cinnamon groups. In case of the bacteria colonies reduction only ginger extract was effective. Chosen plant extracts may be treated as safe and alternative substances to traditional disinfectants of hatching eggs.All authors declare that they have no conflicts of interest.