Conservative treatment of canine dystocia with oxytocin and occasionally denaverine hydrochloride is usually unsuccessful. In this study, we used an in vitro organ bath assay to investigate the potential ecbolic effects of oxytocin and denaverine on pregnant canine myometrium. The circular and longitudinal myometrial layer of canine uterine tissue obtained during emergency Caesarean section were dissected, cut into strips, and mounted in the organ bath. These strips were stimulated with different concentrations of oxytocin and denaverine hydrochloride and the resulting changes in contractility and contraction pattern were evaluated statistically. Our experiments showed different effects on contractility depending on the oxytocin concentrations used. We further identified differences in responses between layers, especially when high doses of oxytocin were applied. Repetitive stimulation with high doses of oxytocin caused the longitudinal layer to stop contracting completely. In contrast, low doses of oxytocin had a better effect on the contractility of both layers and, thus, are recommended for use in clinics. Experiments with denaverine hydrochloride showed no effect on myometrial contractility.In pregnant bitches, the response to oxytocin and denaverine hydrochloride in dystocia management is usually poor. To better understand the effect of both drugs on myometrial contractility, the circular and longitudinal muscle layers were examined in an organ bath. For each layer, three myometrial strips were stimulated twice, each with one of three oxytocin concentrations. The effect of denaverine hydrochloride was studied once in direct combination with oxytocin and alone with subsequent oxytocin administration. Contractions were recorded and evaluated for average amplitude, mean force, area under the curve (AUC), and frequency. Effects of different treatments were analyzed and compared within and between layers. In the circular layer, oxytocin significantly increased amplitude and mean force compared to untreated controls regardless of stimulation cycles or concentrations. In both layers, high oxytocin concentrations caused tonic contractions, while the lowest concentration created regular rhythmic contractions. Longitudinal layer tissue responded to oxytocin with a significantly decreased contractility when stimulated twice, presumably a sign of desensitization. Denaverine hydrochloride neither affected oxytocin induced contractions nor showed a priming effect to subsequent oxytocin. Thus, no benefit of denaverine hydrochloride on myometrial contractility was found in the organ bath. Our results suggest a better efficiency of low-dose oxytocin in canine dystocia management. Canine pregnancy is maintained by progesterone P4) solely of luteal origin. Around day 60, P4 drops below a critical threshold level ,2,3, res solely oROCK1 and ROCK2, the two effector kinases of the GTPase RhoA that regulates smooth muscle contractions through calcium sensitization [Knowledge about the underlying physiological endocrine and molecular changes during normal canine parturition has significantly increased. Nonetheless, understanding of the changes during canine dystocia, especially uterine inertia, remains limited. Primary uterine inertia, the lack of functional myometrial contractions at term, is responsible for more than 70% of canine dystocia cases and, thus, the most common maternal cause for labor complications ,15,16,17tization ,26, weretization .Medical approaches to attempt treatment of uterine inertia include administering oxytocin as an ecbolic drug. Various oxytocin concentrations have been described with especially recently lower dosages being recommended to effectively increase the quality of myometrial contractions  instead OXTR gene expression in primary uterine inertia compared to obstructive dystocia [OXTR expression in complete primary uterine inertia compared to obstructive dystocia. Interestingly, we further identified significant differences in OXTR protein expression between the inner circular myometrial layer and the outer longitudinal layer in primary uterine inertia as well as obstructive dystocia, allowing us to hypothesize that there might be a different responsiveness to oxytocin between the two muscle layers [The nonapeptide hormone oxytocin is one of the most potent uterotonic factors. Its release from the pituitary gland is triggered by intracervical pressure when a puppy enters the birth canal . The effdystocia , supportdystocia , who alse layers . These oDenaverine hydrochloride was formerly registered for use in dogs but is currently only available for cows. Data on denaverine hydrochloride are limited, not allowing for reliable statements to be made about its mode of action and effectiveness. Based on the mode of action of papaverine, the benzylic acid derivative denaverine is described as a “neurotropic-musculotropic spasmolytic agent”, decreasing the tone of the muscle of the soft birth canal  without being tocolytic ,37,38,39Despite the high failure rates of medical treatment attempts, no studies on canine uterine contraction patterns and contractile responses to ecbolic drugs have been conducted. However, such studies would be crucial for the understanding of canine uterine contractions during parturition. Furthermore, the oxytocin dose–response relationships of the canine myometrium are completely missing, although the clinical dosing recommendations have recently been adjusted ,43. TherAnimal experimentation was approved by the respective authorities (Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), permit AZ 20/3360).n = 6). In two cases, the incision was performed in the uterine body due to medical reasons. The fresh tissue was immediately transferred into physiological saline solution and stored at 4 °C until further preparation. Time between harvesting of the sample and start of the actual experiment varied between 3 and 25 h depending on the time points of the surgery.To assess the uterokinetic properties of oxytocin, interplacental uterine tissue was collected from pregnant bitches around term during medically indicated C-sections with/without ovariohysterectomy . Bitches were either presented for emergency C-section during parturition or for an elective C-section. Elective C-sections were performed between day 61–63 after ovulation with obvious fetal intestinal motility visualized by ultrasound confirming fetal maturity . DetaileTo investigate motility of the myometrial tissue, two organ bath set ups  were used, with eight tissue chambers each. The performed method of dissection has previously been successfully described for guinea pig gastrointestinal tissue .2, 2.5 CaCl2, 1.2 NaH2PO4, 117 NaCl, 25 NaHCO3, 11 glucose, and 4.7 KCl, which was exchanged every 10 min. Four strips of 1 cm length and 0.25 cm width aligned with the direction of the muscle fibers were prepared from each myometrial layer. The strips were then mounted one at a time in the eight chambers of the organ bath, filled with 12 mL of preheated 37 °C warm modified Krebs solution. The Krebs solution consisting of (in mmol/L) 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 117 NaCl, 20 NaHCO3, 11 glucose, and 4.7 KCl, was constantly oxygenated with 95% O2 and 5% CO2. The buffer temperature was maintained throughout the experiment by warm distilled water, which was passed through a heat exchanger  circulating around the chambers in a double Plexiglas wall.Following removal of the endometrium, the two myometrial layers  were carefully dissected using a stereomicroscope . Throughout the dissection process, the tissue was covered with ice cold, preoxygenated modified Krebs solution composed of (in mmol/L) 1.2 MgCl® Easy  as previously described [Myometrial strips were connected to an isometric force transducer  in order to measure myometrial contractions. A resting tension of 2 g (~20 mN) was applied to all strips, simulating the in vivo pre-tension to cause physiological phasic and tonic contractions. The contractility variations measured by the force transducer were converted from mechanical to electrical signals by a measuring transformer  and recorded with the software Catmanescribed ,46.Denaverine hydrochloride  was used in quantities of 1.97 nM and 2.97 nM, extrapolated from the manufacturer’s instructions  for in vDuring the experiments, raw data from all timeframes (TFs) were saved in an Excel file . In every TF (1–5), 10 contractions, if any, were evaluated. The peak of each contraction and the baseline tension before and after the contractions were determined manually, and the difference was used to calculate the amplitude, meaning the maximum force of each contraction. In some cases, the voltage did not drop back down to baseline tension before rising again to a peak. In this case, the contraction was defined as finished when the tension reached below 70% of the tension difference between the baseline tension and the peak. If a contraction was directly followed by a minor one, with less than 0.1 g above the baseline tension, it was not counted as an additional contraction but included in the previous one. Generally, increases in baseline tension below 0.5 g were not considered contractions. The time from the initial increase in tension to the return to baseline tension was determined as the duration of the contraction, and, thus, the mean force per contraction was calculated. Furthermore, the area under the curve (AUC) was used to measure the contractile effects of each agonist concentration and calculated as the integral of the time interval of one contraction. The frequency of contractions in 10 min was counted. Results were transformed from g into mN (amplitude and mean force), or from g·s into mN·s (AUC) by multiplying by 9.8. Finally, individual values for amplitude, mean force, and AUC were averaged for all the contractions examined per TF and used for the following statistical analysis. As 1 nM oxytocin, eliciting relatively equal contractions, was used in all denaverine experiments, the same method of analyzing mean values was retained here.In some strips exposed to 10–100 nM oxytocin, we observed an initial long-lasting contraction followed by many other very short, weaker contractions instead of steady and equal ones. Consequently, in a second approach, we analyzed only the initial response of the strips to the respective concentrations of oxytocin for the parameters described above.For all statistical tests and graphical presentations, Graph Pad Prism9 software  was used.Statistical analysis was performed to analyze the direct effect of oxytocin and denaverine on the parameters of contraction to be measured, including amplitude, contraction mean force, and frequency, as well as the integration of these values to generate mean integral force or AUC.n = 3 stripes), the mixed effects model was applied. The ANOVA/mixed effects model investigated the simple main effects of each independent factor included and checked for interaction between them. When the ANOVA/mixed effects model revealed significant differences, post hoc comparison of the means was performed. Tukey’s multiple comparison test was utilized to investigate the effects of the different concentrations. Additionally, Šídák’s multiple comparisons test was used to compare the first and second stimulation within the respective layer. In the following step, the responses of the two layers (factor “layer”) were compared at both stimulation TFs (factor “stimulation cycle”) for each oxytocin concentration separately. For this, we used a two-way RM ANOVA or a mixed effects model, followed by Šídák’s multiple comparisons test in case of significant differences. If not stated otherwise, all data are presented as mean ± standard deviation. Additionally, a possible influence of the covariant storage time of samples until experiment, age of bitches at C-section, and P4 serum concentration was investigated using SAS Studio .For the analysis of the oxytocin stimulation, a two-way repeated measurement (RM) ANOVA was used to investigate the effects of the three different oxytocin concentrations (factor “concentration”) and the two stimulation cycles (factor “stimulation cycle”) on the average amplitude/mean force/AUC/frequency of contractions in the two different myometrial layers, respectively. In the case of a missing value due to a loosened knot during the experiment  with the equilibration time frame before (TF1). To investigate the effect of prior denaverine-priming on oxytocin-induced contractility, strips stimulated with oxytocin at only the first stimulation (TF2) were compared to those stimulated with oxytocin (TF 4) after receiving only 2.97 nM denaverine (TF2). A paired p ≤ 0.05.All differences were considered to be significant if The typical responses of the two layers during oxytocin exposure are shown in Whereas both layers showed regular, rhythmic contractions in response to 1 nM oxytocin (24/34 stripes in the first and 28/34 stripes in the second stimulation), the response to 10 nM and 100 nM was quite different. In the circular layer, the high and medium oxytocin concentration caused a permanent tonic contraction in seven strips from five bitches. In three strips, these contractions lasted up to 20 min, maintaining a relatively constant mean force. Subsequent contractions were regular and shorter. After the wash out, all circular strips maintained uniform regular contractions. Once stimulated, no strip in the circular layer stopped contracting afterwards.In the longitudinal layer, both high oxytocin concentrations (10 nM and 100 nM) caused only a single strong spastic contraction in 18 of 31 strips. In contrast to the contraction observed in the circular layer, tension peaked following oxytocin application and then slowly declined within 5–10 min until settling at a level above the baseline tension. When applied for the second time, no response to 10 nM and 100 nM oxytocin was identified in 27 of 31 strips from the longitudinal layer.Covariance analysis showed no significant effect of sample storage time, age of bitches, or serum P4-concentrations on the parameters of amplitude, mean force, AUC, and frequency of contractions.p ≤ 0.001) but not in the circular layer.Two-way RM ANOVA revealed a significant interaction of the independent variables (concentration and stimulation cycle) in the longitudinal (p ≤ 0.001). All concentrations caused a significant increase in the amplitude of contractions compared to the non-stimulated control regardless of the stimulation time point  as well as the stimulation cycle (p ≤ 0.001) had a statistically significant effect on the amplitude. At the first stimulation, the longitudinal layer responded to all oxytocin concentrations, showing an increase in amplitude compared to the control. However, this increase was only significant for 1 nM  and was even lower than in the untreated control group. Consequently, comparison of the three concentrations showed no differen ces at first stimulation, but there was a significantly stronger response to 1 nM compared to 10 nM and 100 nM (p ≤ 0.01 for each) at the second stimulation in the longitudinal layer . A significant effect of the stimulation cycle on the amplitude was found for 10 nM (p ≤ 0.01) and 100 nM (p ≤ 0.001), with a significantly reduced amplitude in the longitudinal layer at the second oxytocin application. This explains the observed significant main effect of the factor “layer” , as the mean force of contractions increased significantly after each oxytocin stimulation compared to the control in both stimulations  of the independent variables was found. Furthermore, simple main effects analysis showed a significant effect of the oxytocin concentration (p ≤ 0.05) and the stimulation cycle (p ≤ 0.001) on the mean force of contractions. No significant differences between untreated control and stimulated strips were found, except for the first-time use of 1 nM oxytocin (p = 0.014). The second application of 10/100 nM oxytocin caused a significantly reduced mean force compared to the effect at the first stimulation (p ≤ 0.001) and the second use of 1 nM oxytocin  of variables was found for all oxytocin concentrations. Furthermore, for 10 nM and 100 nM, but not for 1 nM oxytocin a statistically significant main effect of “layer” (p ≤ 0.01 for each) and “stimulation cycle” (p ≤ 0.001 for each) was identified . Additionally, in the two layers, simple main effect analysis showed a statistically significant effect of each factor on the AUC of contractions .ANOVA/mixed-effects model analysis revealed interactions of “concentration” and “stimulation cycle” in both layers (p ≤ 0.001) and only at the first stimulation (p = 0.019)  c. Moreov= 0.019) c.p ≤ 0.05 for each), but a significant decrease occurred at the second stimulation , as the response was always stronger at the first application. Although AUC did not differ significantly between layers, it is noteworthy that using 1 nM and 10 nM oxytocin at the first stimulation the AUC was greater in the longitudinal than the circular layer, with even a trend to significance at 1 nM . At the first stimulation, the frequency of contractions increased significantly following stimulation with 1 nM and 10 nM oxytocin (p ≤ 0.01 for each). This response was not only reproducible at the second stimulation, but all three concentrations also accelerated contractions significantly compared to the untreated control . As already described for the amplitude and the mean force of contractions, when stimulated for the second time, the response to 1 nM oxytocin, accelerating the frequency of contractions, was significantly stronger than the responses following the two higher concentrations in the longitudinal layer  was found and, accordingly, when comparing the response of layers, a significantly lower frequency in the longitudinal layer was observed in both stimulation cycles using 10 nM and 100 nM oxytocin . Furthermore, in the longitudinal layer, the amplitude following stimulation with 1 nM oxytocin was now significantly higher compared to the control (p = 0.05). Additionally, the mean force in the longitudinal layer was not only significantly higher than the untreated control after 1 nM oxytocin (p ≤ 0.001), but also after 10 nM (p < 0.05). A significant difference in the mean force between both stimulations with 1 nM oxytocin was found for the longitudinal layer (p ≤ 0.05).When evaluating the first contraction only, the initial response to oxytocin, results differed only slightly. Where previously only a trend was apparent, the variable “stimulation cycle” now influenced the amplitude in the circular layer (p ≤ 0.05). When looking at the effects of the different oxytocin concentrations separately, a trend to significance when comparing the AUC between layers after the second dose of 10 nM emerged  , and alsper se (non-pregnant vs. pregnant) requires further investigation. In addition to these postulated differences between the cyclic and pregnant myometrium, Crankshaw et al. [Differences between the circular and longitudinal myometrial layers with regard to different innervation and responsiveness to various stimuli have been reported in several species, such as cattle ,76, pig w et al.  further Nevertheless, the markedly reduced responsiveness of the longitudinal myometrial layer after stimulation with high doses of oxytocin is a new finding in canines. In human obstetrics, exposure to high doses of oxytocin during labor augmentation is associated with uterine atony and consecutive risk of severe post-partum bleeding ,85. In aIt remains to be clarified why only the longitudinal layer ceased to respond to oxytocin stimulation. Interestingly, we recently identified a significantly reduced OXTR protein expression in the longitudinal compared to the circular myometrial layer of bitches at term , supportRegarding the clinical application of oxytocin, our results suggest that only low concentrations of oxytocin are suitable for (re)activating myometrial contractions in the (peri)parturient dystocia bitch. High concentrations might cause spasm-like tonic contractions of both myometrial layers, obviously not promoting labor, but reducing fetal oxygen supply and thereby threatening fetal vitality and outcome . FurtherA limitation of the study is the inhomogeneity of specimens included in terms of breed, age, body weight, litter size, serum P4 concentration, and medical indication for the C-section. However, on the other hand, samples represent the entirety of the animals presented in clinics. Additionally, the time between sample collection and start of the experiment differed, mainly for logistical reasons. Nevertheless, our statistical analysis disproved an influence of age of bitches, serum P4 concentrations, and sample storage time on the investigated parameters, supporting the suitability of the results and relevance of data.In summary, the organ bath technique is suitable for studying the contractile response of uterotonic substances on the isolated canine periparturient myometrium, namely the longitudinal and circular layer separately. Our results confirm different responses depending on the oxytocin concentrations used. Based on our findings, high doses of oxytocin, as often applied by some practitioners, contrary to the current recommendations, are not beneficial in dystocia management and should be avoided. Additionally, for the first time, we identified differences in the contractility of the longitudinal and circular myometrial layers, indicating different oxytocin sensitivity. All oxytocin concentrations induced or amplified existing contractility in the circular myometrial layer. The longitudinal layer showed an initial tonic contraction but did not respond subsequently when re-treated with higher doses of oxytocin . This effect might be attributable to a short-term desensitization of the OXTR, uncoupling it from the respective G-protein. Further investigations into the distinct differences between the myometrial layers are crucial for a better understanding of canine parturition, not only during normal parturition, but also during dystocia due to uterine inertia.


Prior to surgery, medication noncompliance and dietary measures failed to achieve adequate glycemic control or weight loss. Postoperatively, the patient lost 20 kg and achieved improved T2DM control (HbA1C reduction), allowing complete cessation of diabetic medications. The patient's case demonstrates bariatric surgery's potential to significantly alter the clinical course of obesity and T2DM versus standard care. National guidelines outline eligibility criteria for bariatric referral; however, utilization rates remain low (<1%) despite over two million eligible individuals in the United Kingdom. Improved access could reduce disease burden and healthcare costs from diabetes complications over the long term. This case report provides a real-world example supporting bariatric surgery as an effective intervention for appropriately selected patients with obesity and uncontrolled T2DM, with the potential to improve clinical outcomes and lower costs associated with diabetes management.Type 2 diabetes (T2DM) and obesity represent major global health burdens and economic costs to healthcare systems. T2DM management is challenging due to multiple comorbidities and limited drug efficacy. Bariatric surgery has emerged as an effective treatment approach. A 65-year-old man with refractory obesity (BMI > 35 kg/m Diabetes is a chronic condition, which has become a major public health concern approaching epidemic proportions globally. It is estimated that approximately 18 million people die every year from cardiovascular disease (CVD), for which diabetes and hypertension are major predisposing factors .Type 2 diabetes (T2DM) also known as adult-onset diabetes is characterized by hyperglycemia due to progressive impairment of pancreatic β-cell insulin secretion and/or insulin resistance. T2DM is estimated to affect more than 300 million individuals globally . T2DM haEffective diabetes management often presents many challenges. Clinicians and patients are often overwhelmed by the need to address comorbid chronic conditions in addition to patients’ diabetes-specific treatment goals .The economic burden posed by diabetes on national healthcare services such as the NHS is quite substantial. The burden of diabetes can be divided into direct and indirect costs. The direct cost burden of diabetes includes diagnostic and monitoring tests, GP consultations, medications, insulin pumps, continuous monitoring devices, hospital admission, and secondary professional referrals. The indirect costs include social care needs, lost productivity through sickness, absences from work, and hindered quality of life. Combined, the direct and indirect costs of T2DM have been estimated to cost the NHS 21.8 billion pounds annually and are predicted only to increase over the next 20 years. As of 2011, the burden of diabetes on the NHS expenditure was estimated to be 10%. Of this 10%, the indirect costs accounted for more than 60%. Specifically, the indirect cost is due to complications arising from long-term disease burdens such as foot ulcers and amputations, cardiovascular disease, end-stage renal disease, and acute admission due to diabetic emergencies .Studies looking into similar statistics in France and Germany have shown similar trends. The cost of 3000 to 15000 euro per patient increases as insulin therapy is initiated, and most of the costs are attributed to complications and comorbidities due to T2DM .Therefore, it has become of paramount importance to find alternative means of treating diabetes with a more robust outcome that not only minimizes the development of associated comorbidities but provides a more cost-effective alternative to current pharmaceutical drugs.Currently, the management of diabetes is via a multifaceted approach with lifestyle modifications being the first line. Obesity has been closely linked to poor diabetic control, and weight loss has been shown to lead to significant improvements in glycemic control and reduced medication use in patients with T2DM . This weBariatric surgery is a group of surgical procedures done with the intent of substantial weight loss in individuals with severe obesity. Bariatric surgery can be done via several techniques. The most common variants are gastric banding, Roux-en-Y gastric bypass, sleeve gastrectomy, and biliopancreatic diversion. They exert their effect by a combination of mechanisms including caloric restriction, reduced absorption, and hormonal changes, which alter the mechanics of appetite and satiety .It is widely accepted that bariatric surgery can help improve health outcomes in a range of metabolic diseases, including T2DM and obesity . BariatrHerein, we present a case of a patient with poorly controlled T2DM and obesity who was effectively managed with elective bariatric surgery. Despite the significant drop in the number of bariatric procedures performed in this country, this case highlights that bariatric procedures should be considered as not only a viable treatment option for patients but also a cost-effective means of treating these patients.This case was presented at the European Endocrine Society at the 22nd European Congress of Endocrinology, which took place from September 5th to 9th, 2020, as an abstract and poster.A 65-year-old man was referred to the endocrine team with poor diabetic control, ongoing chronic kidney disease, and previous history of non-alcoholic fatty liver and prostate hyperplasia, for which he was taking Metformin (1g BD), Forxiga (10 mg OD) (dapagliflozin), Finasteride (5 mg OD), and Alfuzosin (10 mg OD). On physical examination, the patient's weight was 131 kg, his heart rate was 99 bpm, and his blood pressure was 154/88 mmHg. The foot examination was unremarkable. There was no evidence of peripheral neuropathy or ulceration. Laboratory findings showed urea of 8.8, creatinine of 114 umol/L, eGFR > 60 ml/min, and hemoglobin A1C (HbA1C) of 77 mmol/mol.He indicated that he gained weight over the past few months. He also indicated that he felt more fatigued and tired. The patient was referred to a dietician to help with his weight loss and diabetic control. The patient was again seen by the endocrine team six months later, and his weight remained alarmingly high (126 kg) despite consulting a dietician. In addition, his diabetes remained uncontrolled. At this point, he was given an additional antidiabetic drug Victoza (1.2 mg OD) and was referred to Homerton Hospital NHS Trust for consideration of bariatric surgery in the form of a gastric band.Following the bariatric surgery, he lost a significant amount of weight and showed better glycemic control. His medication following bypass surgery included multivitamins, especially Vitamin B12 and Vitamin D with all previous diabetes medication stopped.The patient's glucose levels after undergoing gastric band bariatric surgery are shown in Figure The patient's glucose readings after undergoing gastric band surgery are shown in Table T2DM, obesity, hypertension, and dyslipidemia collectively carry a high risk of cardiovascular disease and mortality. Diabetes is also a leading cause of non-traumatic lower extremity amputation and blindness in the United Kingdom. The majority of diabetes-related amputations in England are preventable if patients get the right early preventative care upon recognition of uncontrolled diabetes.Medical management of T2DM and obesity is challenging as drug therapies have limited efficacy due to side effects and noncompliance. However, bariatric surgery has shown promise as an effective treatment. This case report details one patient who underwent gastric bypass surgery in December 2018. He lost 20 kg post-surgery and demonstrated significantly improved diabetic control, with glucose levels ranging from 4.5 to 6.0 mmol/L. His HbA1C gradually declined, and he was ultimately able to stop all diabetic medications , with lower BMI thresholds (reduced by 2.5 kg/m2) for Asian, Middle Eastern, Black African, or African-Caribbean family background [In 2014, the National Institute for Health and Care Excellence (NICE) established guidelines for the use of bariatric surgery. To be eligible, patients required a BMI ≥ 40 kg/mckground .Based on these criteria, over two million people in the United Kingdom were estimated to be eligible for bariatric surgery. However, a study found that less than 1% of eligible patients were referred for and received the intervention, highlighting a gap between clinical need and access . More coBariatric surgery is well-established as an effective treatment modality for patients with obesity and T2DM. Several studies and case reports have highlighted the key role bariatric surgery can have in achieving both weight loss and T2DM remission . A recenThis case suggests that bariatric surgery could significantly alter the course of patients with obesity and uncontrolled diabetes, reducing associated morbidity and mortality. If these findings were extrapolated to a larger population, offering the option of such an intervention to a greater number of individuals could effectively improve patient outcomes and significantly reduce the cost burden on the NHS.Chronic conditions such as T2DM are causing strain on national healthcare. Since 2010, there has been a one percent increase in healthcare spending in England. The cost of treating diabetes by the NHS has increased annually in connection with increased incidence of T2DM and obesity. Diabetic therapy is currently costing the NHS over one billion . Along wStudies estimate the cost of providing bariatric surgery for patients with obesity and T2DM can be recouped within three years via reduced prescriptions per patient and less hospitalization .LimitationsThis is a case study and thus cannot predict the confounding factors that may occur in a larger population. Additionally, there is only a limited time span that the patient was monitored. Long-term advantages may not be as significant as predicted. The case may lack generalizability to a wider population.Bariatric surgery is a robust means of treating diabetes and obesity. The use of this would limit both the overprescription of antidiabetic therapy and its cost, which at the moment is estimated to cost the NHS over a billion pounds. Moreover, it would improve the psychological and physical well-being of patients hindered by T2DM.


Strawberry cultivation is an important source of income for Brazilianfarmers. Cultivation can be done using the traditional model, in whichproducers must flex their trunk to handle seedlings, or using the hydroponicmodel, which involves an upright posture.To verify the influence of cultivation model on posture and back painprevalence among strawberry producers.t-test forindependent samples and the chi-square test were used to compare groupresults.A total of 26 strawberry producers who used traditional or hydroponic methodsparticipated in the study. The angular values of the curvatures of thethoracic and lumbar spine in the sagittal plane were obtained using the“flexicurve” method, while pain prevalence was determined with Souza &Krieger’s back pain questionnaire. The Growers using the traditional model had greater thoracic spine curvature than those who used the hydroponic model . There was an association between thoracic spine classification andcervical pain, with a higher prevalence of thoracic kyphosis and cervicalpain in the traditional model and a higher prevalence of normal curvature inthe hydroponic model. Both groups reported a higher prevalence of pain inthe lower back than in other locations.The cultivation model influenced posture and back pain prevalence instrawberry producers. Producers who use the traditional model have greaterangulations of the thoracic spine, hyperkyphosis, lumbar straightening, andcervical pain than those who use the hydroponic model. Fragaria x ananassa) is cultivated in the most variedregions of the planet,1 withincreasing production every year.2The fruit is an important source of income for many Brazilian farmers,3 especially smallproducers,4 having greatvalue commercially and for the rural economy.2Strawberry ,5 hydroponic, and semi-hydroponic.3 The traditional cultivation model . The study was also authorized by the Bom PrincipeAssociation of Strawberry Producers. All participants provided written informedconsent prior to inclusion.This quantitative study, which involved a descriptive, comparative andcross-sectional design,A total of 26 strawberry producers from Bom Princípio, state of Rio Grandedo Sul, Brazil, participated in the study. The participants used eithertraditional  or hydroponic  cultivation systems . ProspecData collection consisted of an initial interview regarding eligibility criteriaand sociodemographic data, followed by pain and postural assessments. Theevaluation was performed by an individual researcher according to theparticipant’s convenience.10 Instrumento deAvaliação da Dor nas Costas (back pain assessment instrument),whose objective is to identify the intensity and frequency of pain in sevenregions: the cervical spine, the shoulders, the shoulders and arm, the dorsalspine, the lower back, the gluteus muscles, and the gluteus muscles andlegs.Pain assessment was performed using Souza & Krieger’sThe questionnaire consists of 2 parts. The first part presents the instrument’sobjectives, identifies individual data and maps the human body, indicating the 7above-mentioned regions. The second part investigates pain intensity in theseregions according to the following scale: none, mild, average, strong, andunbearable. Pain frequency is assessed with the following alternatives: 1-4times per year, 1-4 times per month, 1-4 times per week, 4-6 times per week, and7 times per week. All those who answered “none” were considered pain-free; forany other option, the producer was classified as having pain.12 To do this, participants stoodin their usual posture with their back unclothed, their bare feet parallel, andtheir shoulders and elbows at 90° of flexion. To determine the thoraciccurvature, the spinous processes of six anatomical points  were located through palpation and marked with a skin pencil. Aftermarking the anatomical points of interest, the flexible ruler was molded to theparticipant’s back from the C7 spinous process to the S1 spinous process .14 For thelumbar curvature, angles > 54° were considered hyperlordosis, angles < 22°were considered straightened curvature, and angles between 22° and 54° wereconsidered intact physiological curvature.15Angular values > 40° were considered hyperkyphotic posture, values < 20°were classified straightened curvature, and angles between 20° and 40° wereconsidered intact physiological curvature.t-test for independent samples to compare the angle(mean and standard deviation) of lumbar and thoracic curvature betweencultivation models. The chi-square test was used to assess the associationsbetween frequencies (n and %) of pain , posturaldeviations  andcultivation model. The significance level was set at 5%.Statistical analysis was performed in IBM SPSS Statistics 20.0, includingStudent’s The thoracic curvature of traditional producers was significantly greater than thatof hydroponic producers . There was no difference betweencultivation models regarding lumbar curvature  .There was an association between thoracic  and lumbarspine  classifications and cultivation model, ie, ahigher prevalence of hyperkyphosis and lumbar straightening in traditionalcultivation, and a higher prevalence of normal curvature in hydroponic cultivation. There wThis study aimed to verify the influence of cultivation model on posture and backpain among strawberry producers. Those using the traditional model had greaterthoracic curvature and a higher prevalence of thoracic hyperkyphosis, lumbarstraightening, and cervical pain than hydroponic producers. However, growers fromboth models had a higher prevalence of pain in the lower back than the otherregions.16 of whichare reduced lumbar lordosis (straightening) and thoracic hyperkyphosis, preciselywhat we found in the present study , is considered the strawberry capital of thecountry.Our results are particularly relevant for professionals who deal directly with thespine and human movement , sincethey provide evidence of body posture and pain differences in different cultivationmodels. Based on these results, professionals can instruct producers to adapt theirseedling management processes to prevent or control pain and the overloadingassociated with the traditional model. To our knowledge, our study is the first toevaluate spinal curvature among strawberry producers using the flexicurve method,which can be easily applied in clinical practice.It can be concluded that the cultivation model influences the posture and back painprevalence of strawberry producers. Workers who use the traditional model havehigher thoracic spinal angles and a higher prevalence of hyperkyphosis,straightening of the lumbar spine, and cervical pain than those who use thehydroponic model. However, in both models the highest prevalence of pain was in thelower back.


The heat transfer remains a huge problem for industrialists and engineers because many production processes required considerable amount of heat to finish the process successfully. Although, conventional fluids have large scale industrial applications but unable to provide huge amount of heat transfer. Therefore, the study is organized to propose a new ternary heat transfer model using different physical constraints. The key applications area of nanofluid heat transfer are chemical, applied thermal and food processing engineering.and Methodology: The key purpose of this research is introduce a new ternary nanofluid model using the impressive effects of thermal radiations, surface convection and saddle/nodal points. The results simulated via RKF-45 and discussed in detail.2O3 nanoparticles form 1%–7% (keeping fixed CuO and Cu as 4% and 6%) and s1 = −0.2,-0.4,-0.6,-0.8 controlled the fluid movement while s1 = 0.2,0.4,0.6,0.8 boosted the velocity. Increasing the convection process Bi = 0.1,0.2,0.3,0.4 increased the temperature significantly. Further, shear drag is maximum for ternary nanofluid and thermal radiations Rd = 0.1,0.2,0.3,0.4 enhances the heat transfer rate.The strength of Al However, working fluids are the key ingredients for this purpose. The conventional fluids are inherently poor thermal performance due to weak thermal conductivity which fail to produce considerable heat for industrial purposes. Therefore, the new fluids introduced with outstanding thermal conductivity and termed as Nanofluids. These fluids are extension of simple fluids with addition of nanoparticles which dispersed in the host functional fluid uniformly and provide significant contribution in thermal transport. Based on the dispersion of nanoparticles, these further characterize as mono nano ,2, hybriMany industrial apparatuses can be configured by different geometries in which cylinder with plan or wavy surface is significant. In recent decades, researchers and engineers inspired by the applications of heat transfer for sinusoidal and plan cylinders and performed different studies. Recently, Waqas et al.  offered 2O3 nanoparticles while functional base solvent taken as water.In 2022, Khan et al.  extendedThe heat and mass transportation under magnetic dipole influence for hybrid Casson nanoliquid over an elastic cylinder is accomplished by Ahmad et al. . The Ag Inspired by the enhanced heat transfer rate and cooling performance of nanoliquids, the researchers put their efforts on the study of multiple type of nanoliquids. Recently, Gangadhar et al. , Marulas2O3, CuO and Cu used for the analysis. The heat transfer in ternary nanofluid will be discussed in this specific research under the variable ranges of the parameters involved. The model results would be advantageous for practical applications particularly in applied thermal, chemical and food engineering where enhanced heat transfer is essential. The core focus of this research will.•To introduce an advance and efficient ternary nanofluid model for sinusoidal cylinder with saddle and nodal stagnation regions.•To examine the impact of thermal radiation and convective thermal effects on the heat transport efficacy of the functional fluid.•To examine the influence of particles concentration factor •To analyze the comparative Skin friction and Nusselt number computation with increasing thermal radiation, Biot number and According to afore cited literature, it is examined that the previous attempted has been made only up to Newtonian, mono nano and hybrid nanofluids. Thus, there is a significant gap of heat transfer study using novel ternary nanofluid over a sinusoidal cylinder by considering novel ternary nanoliquid as functional fluid over saddle and nodal stagnation region subject to novel effects of convective thermal condition. Three distinct metals nanoparticles namely Al2The non-transient 3D laminar stream of ternary nanofluid is considered over a particular cylinder type with sinusoidal radius. Three distinct sorts of nanoparticles and host solvent are the components of functional nanofluid. Due to sinusoidal radius, there exist nodal and saddle points on the cylinder's surface subject to the utmost and minimum radius locations. Further, the coordinate directions  associated with the velocity components . Further, 2.1•The flow is laminar, non-transient and 3D over a cylinder with non-uniform radius.•The surface is considered with no thermal and velocity slip.•The flow is SPF and the streamlines separated through saddle and nodal points.•2O3–CuO–Cu) uniformly saturated in the host solvent.No chemical reaction takes place and the nanoparticles u_Further, the specified boundary and ambient location of the cylinder are as follows :(5)u_=0,Further, in order to transform the model in more simplified form, the following appropriate similarities for sinusoidal cylinder will be adopted:2.4The traditional model of newtonian fluid over a sinusoidal cylinder will be modified using the below advance ternary nanofluid  Table 1Table 1 a2.5This subsection deals with the final version of the enhanced heat transfer over a sinusoidal cylinder. For this, we used the appropriate model similarity variables and supporting ternary nanofluid expressions in the basic governing laws mentioned in Eqs. Further, physical quantities integrated in the model are Prandtl number  adoptedThe supporting transformations for the model conversion are as under:Now, proceed to the proper arrangement of the model and using the above transformations, below system achieved.The updated model is then analyzed using RK scheme with tolerance up to 4The effects of the physical constraints like particles strength 4.1The effects of concentration factors  on the heat performance of ternary nanofluid are presented. The formulated problem dealt using numerical scheme and simulated the results. It is examined that.Kamel SMIDA: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Abbasi Adnan: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data. Muhammad Umer Sohail: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data. Iskander Tlili: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Asma Javed: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.Data will be made available on request.There is no financial/competing interest regarding the publication of this work.


FOLR1. Onset typically occurs in late infancy and is characterized by psychomotor regression, epilepsy, and a hypomyelinating leukodystrophy on magnetic resonance imaging. If left untreated, progressive neurodegeneration occurs. However, early treatment with folinic acid has been shown to stabilize or reverse neurological features. Approximately thirty patients have been described worldwide. Here, we report the first two cases with genetically proven cerebral folate transport deficiency from South-Eastern Europe, describe the effect of oral folinic acid therapy on clinical and neuroradiological features and review the literature.Neurodegeneration due to cerebral folate transport deficiency is a rare autosomal recessive disorder caused by biallelic pathogenic variants in FOLR1 , confirming the diagnosis of neurodegeneration due to cerebral folate transport deficiency. Folinic acid treatment was promptly initiated in both patients. The younger sibling was treated early in disease course at 2 years of age, and demonstrated complete recovery in clinical and MRI features. The older sibling, who was 8 years of age at the time of diagnosis and treatment, demonstrated partial but substantial improvements.Two siblings presented in childhood with clinical and radiological findings consistent with a hypomyelinating leukodystrophy. Exome sequencing revealed a novel homozygous pathogenic variant in We present the first account in the literature that early treatment initiation with oral folinic acid alone can result in complete neurological recovery of both clinical and radiological abnormalities in neurodegeneration due to cerebral folate deficiency. Moreover, through the report of these patients along with review of the literature, we provide information about the natural history of the disease with comparison of treatment effects at different stages of disease progression. This report also reinforces the importance of universal access to genetic testing to ensure prompt diagnoses for treatable disorders. FOLR1 [FOLR1 (OMIM *136430) encodes for the folate receptor-alpha (FOLRα), which is abundantly expressed in the choroid plexus and considered the main folate transporter of 5-methyltetrahydrofolate (MTHF) across the blood–brain barrier. FOLRα is the only transporter responsible for cerebral folate supply via exosome-mediated delivery of MTHF from the CSF to the brain parenchyma [Neurodegeneration due to cerebral folate transport deficiency OMIM #613068), first described in 2009, is caused by biallelic pathogenic variants in 3068, firFOLR1 cause FOLRα deficiency, impairing cerebral folate transport and supply, leading to isolated cerebral folate deficiency and progressive neurodegeneration [Biallelic hypomorphic pathogenic variants in neration . This dineration .The late-infantile onset and absence of embryonic malformations in this disorder suggest preserved expression of folate receptor-beta (FOLRβ) in fetal choroid cells, which compensates for the lack of FOLRα function , 2, 4. HThe pathophysiological mechanisms by which MTHF deficiency causes neurological disease are still under investigation. The prevailing hypothesis links the lack of MTHF to impaired myelin formation through cerebral methylation processes, which results in a deficiency of phosphatidylcholine, sphingomyelin, and other methylated membrane phospholipids crucial for myelin formation and stability . AnotherFOLR1. We also present an in-depth report before and during folinic acid treatment, with clinical and MRI evolution, as well as a review of the previously published cases.Here, we report siblings with hypomyelination and neurodegeneration for whom exome sequencing revealed a homozygous novel pathogenic variant in This research was approved by the Institutional Review Boards of Clinic for Child Neurology and Psychiatry University of Belgrade (IRB number 1-48/3-2016) and the McGill University Health Center and Montreal Children’s Hospital Research Ethic Boards (11-105-PED and 2019-4972), and conducted following the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from the patients’ parents/legal guardians.Exome sequencing was performed using genomic DNA extracted from whole blood following standard protocols. DNA was prepared using the TruSeq library prep and samples were enriched using the IDT xGenv2 exome research panel supplemented with custom mitochondrial probes and sequenced to a minimum of 7 Gb for a mean of 80 × average coverage or greater on an Illumina NovaSeq 6000 (2 × 150 paired end reads). Bidirectional sequences were assembled, aligned to reference gene sequences based on human genome build GRCh37/UCSC hg19, and analyzed using the custom-developed software RUNES and VIKING [We retrospectively reviewed medical records and evaluated MRI studies conducted serially over 10 years for Patient 1 and over 4 years for Patient 2.FOLR1, considering their clinical features, neuroimaging results, genetic findings, treatment regimen, and response to treatment. This was completed by reviewing all biomedical literature available in the PubMed Medline database between September 2009-December 2022, using the following MeSH terms: FOLR1 gene, cerebral folate deficiency, hypomyelination, leukodystrophy, folinic acid.Further, we assessed the data from all published patients with biallelic pathogenic variants in Serum folate levels were measured using Abbott Architect i4000Sr test equipment and Abbott Architect Folate reagent using the Chemiluminescent Microparticle Immunoassay (CMIA) method.The patients described in this study are siblings, including a boy currently aged 12 years (Patient 1), and his younger sister currently aged 6 years (Patient 2). They were born to non-consanguineous unaffected parents of Serbian origin, with a family history negative for neurological disorders. Both patients were referred to our department for additional investigations at the ages of 8 years  and 2 years .The older male sibling (Patient 1) had uneventful early psychomotor development until 18 months of age, when he gradually developed an ataxic gait and speech regression. At 4 years of age, he started having epileptic seizures, which were treatment-resistant, occurred daily, and of multiple different types . Various combinations of ten standard antiepileptic drugs (AEDs) were tried without success. At the time, the patient was being treated in a department without resources for access to detailed metabolic investigations or genetic sequencing, resulting in his cause of illness remaining unknown. Cerebellar ataxia and hypotonia progressed, and at 6.5 years of age, he lost the ability to walk and sit without support, with poor head control. His expressive language consisted of up to five meaningful words and he showed autistic behavioral changes with outbursts of anger and poor social contact. No other abnormalities were found on physical examination. The severity of his seizures increased, frequently leading to status epilepticus. EEG showed diffuse disturbance in cerebral activity with slow background activity and multifocal epileptiform discharges. The patient’s neurological motor, cognitive, and language function progressively worsened, leading to dependency for all activities of daily living and severe neurological impairment at 7.5 years of age. His examination at the time was characterized by a complete loss of speech and social interactions, as well as significant cerebellar signs , axial hypotonia, and mild pyramidal and bulbar signs.The younger female sibling (Patient 2) had normal psychomotor development. At the age of 22 months, she started manifesting mild intention tremor in the upper limbs and mild truncal ataxia. She did not exhibit seizures or any other neurological abnormalities.In Patient 1, brain MRI at the age of 7 years Fig. A1–E1. BrFor both patients, routine blood analyses and urinalyses, including blood/urine metabolic screening  and vitamin B12 and homocysteine concentrations in serum, were normal. Of note, serum folate concentration values were also within normal range in both patients, measured at 17.8 ngr/ml in Patient 1, and 15.3 ngr/ml in Patient 2 .Lumbar punctures to measure CSF neurotransmitters were not performed due to lack of parental approval and resources in Serbia. Therefore, the most efficient and the least invasive method to investigate the genetic diagnosis was to promptly perform exome sequencing using patient DNA extracted from whole blood.FOLR1: c.465_466delinsTG; p.W156G (NM_016725.3), which we assessed for pathogenicity using the ACMG guidelines and classifications. Using Sanger sequencing, we validated the presence of the variant in both patients and confirmed the parents to be heterozygous carriers (PP1). This specific variant has not been reported in large population databases  (PM2), and a missense variant causing the same protein change is reported in heterozygous form in only 3 individuals, with no homozygous individuals reported . This variant is reported by in silico softwares to be pathogenic and is present in a conserved amino acid region (PP3). Additionally, this specific indel variant has not been reported in the literature, however a missense variant at the same position leading to the same protein change  has been reported in two affected siblings in a compound heteozygous form [Using exome sequencing, in both siblings we identified a homozygous novel pathogenic variant in ous form , and oneTreatment with folinic acid was initiated immediately after obtaining the genetic results,  and the response to therapy was monitored over 4 years. Specifically, the patients were treated with levofolinic acid, the L-isomer of folinic acid. Notably, folinic acid can also be prescribed as a mixture of both the biologically active L-isomer and the inactive D-isomer, however, reports show that a better outcome may be associated with the use of only the L-folinic acid compound .In Patient 1, the initial dose of oral folinic acid of 2 mg/kg/day did not lead to notable improvements, and therefore within a month, the dose was increased to 5 mg/kg/day, which resulted in a dramatic improvement of neurological features. The patient’s bulbar symptoms disappeared, weakness and ataxia began to subside, and over the next 8 months, he gradually began to walk independently, while his speech comprised 4–5 meaningful words. His dose of oral folinic acid was then slowly increased to 8 mg/kg/day. The severity and frequency of seizures decreased from dozens per day to 0–3 brief atonic and focal seizures with impaired awareness, and the antiepileptic therapy was reduced to two AEDs which are presumed to have a minimal anti-folate effect (levetiracetam and lamotrigine). Any attempt to further modify/reduce antiepileptic therapy would result in the aggravation of seizures. During folinic acid treatment, his serum folate concentration remained within the normal range, with values measured at 17.1 ngr/ml  at 12 years of age.On the latest neurological examination at age 12 years, the patient presented with cerebellar signs, while bulbar and pyramidal signs were completely resolved. Cerebellar ataxia and hypotonia appeared milder, and he could walk and perform simple motor tasks independently. His behavioral abnormalities subsided, however, no significant improvement in expressive language was observed. Follow-up brain MRI at 10 years of age showed progression of both supra- and infratentorial myelination  is the first reported patient with neurodegeneration due to cerebral folate deficiency who demonstrated complete recovery of both clinical features and brain MRI abnormalities following oral folinic acid treatment started just after symptom onset.The siblings we describe here provide strong support for the effectiveness and importance of folinic acid treatment initiation at a very early age in patients with pathogenic variants in Contrarily, the 6-year delay in diagnosis of Patient 1 can explain his incomplete clinical recovery. It is important to note that the main cause for the delay in diagnosis was the inability to provide timely access to metabolic and genetic testing, as the patients were treated in a department without the necessary resources to perform this testing on a clinical basis. Therefore, genetic analyses were only performed later on a research basis, and by the time of genetic diagnosis, his neurological impairments had more substantially progressed. In Patient 2, genetic analysis was performed near the beginning of symptom onset at 2 years of age, and the immediate initiation of folinic acid therapy led to the complete resolution of both clinical and MRI abnormalities.Additionally, the prolonged exposure of Patient 1 to a plethora of antiepileptic drugs for intractable epilepsy prior to establishing the correct diagnosis may have resulted in negative effects on his disease course. Indeed, some AEDs may have a harmful anti-folate effect, including valproate, phenobarbital, primidone, phenytoin, carbamazepine, oxcarbazepine, topiramate, gabapentin, and pregabalin , 26. HowBased on the published literature, psychomotor regression with cerebellar ataxia starting in the second to third year of life, along with refractory epilepsy with mostly myoclonic seizures and radiological findings of cerebral hypomyelination with or without cerebellar atrophy should raise suspicion of this disease. Other clinical signs such as autistic behavioral features, deceleration of head growth, frequent occurrence of status epilepticus, and radiological findings of brain calcifications can be seen as well and do not exclude the possibility of this disorder/diagnosis. Furthermore, due to the phenotypic variability associated with this disease and other inherited neurological diseases, all individuals with drug-resistant epilepsy should undergo genetic testing.Prior to our report, a complete resolution of neurologic symptoms was only accomplished in one patient, however, their MRI features did not fully resolve, with the preexisting cerebellar atrophy remaining despite the therapy , 2. ThisBrain MRI features improved in both of our patients with oral folinic acid therapy. Specifically, white matter T2-signal hyperintensity started to decrease and the volume of both supra- and infratentorial structures began to increase after treatment for two years in Patient 1, and one year in Patient 2. After four years of treatment, the brain MRI findings in Patient 1 did not yet normalize, but continued to improve. Moreover, brain MRS revealed normalization of the white matter choline peaks for age, in line with the reactivation of the myelination process . The 5-yThe route of folinic acid administration may be of importance when treating neurodegeneration due to cerebral folate deficiency, given that FOLRα is necessary for the transfer of MTHF from the CSF to the brain parenchyma , 3. In tFOLR1, they exhibited phenotypic variability as the older sibling demonstrated an earlier disease onset with a more severe disease course. It has been proposed that even in the presence of the same FOLR1 pathogenic variants, the variable phenotypic severity may reflect the individual variability in the timing of fetal FOLRβ inactivation, different potency of FOLRβ functioning, variable residual FOLRα functions, or variable capacity of alternative folate transport mechanisms [Although both siblings harbored the same homozygous pathogenic variant in chanisms . It may chanisms , 5. Perichanisms , 10. RegFOLR1 in two Serbian siblings with clinical and brain MRI presentations consistent with neurodegeneration due to cerebral folate transport deficiency, along with the response to treatment and long-term follow-up, therefore contributing to the literature delineating the natural history of the disease. The youngest sibling is the first patient reported for whom complete recovery of both clinical and brain radiological abnormalities was achieved with oral folinic acid treatment, suggesting that early oral therapy may be sufficient to treat this condition compared to other more invasive routes of administration.We report a novel pathogenic variant in FOLR1 should be investigated promptly and included in all leukodystrophy panels to ensure early treatment with folinic acid and optimize clinical outcomes. Finally, these cases highlight the importance of universal access to genetic testing, to ensure that treatable conditions are promptly diagnosed and treatment initiated early to optimize clinical outcomes.Furthermore, in patients with genetically undiagnosed hypomyelination,


Salmonella (9.6%) and Escherichia coli (6.5%) were detected, while all Enterococcus were resistant to at least one tested antibiotic. Fewer than 20% of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (17.3%) and carbapenem-resistant Enterobacteriaceae (CRE) (7.7%) were recovered. Six ARGs were detected using qPCR, primarily the erythromycin-resistance gene, ermB. Of the 26 antibiotics measured, almost all water samples (98.7%) had detectable levels of antibiotics. Analysis of wastewater samples from three WWTPs showed that WWTPs did not completely remove AR contaminants. ARGs and antibiotics were detected in all the WWTP effluent discharges, indicating that WWTPs are the source of AR contaminants in receiving water. However, no significant difference in ARGs and antibiotics between the upstream and downstream water suggests that there are other sources of AR contamination. The widespread occurrence and abundance of medically important antibiotics, bacteria resistant to antibiotics used for human and veterinary purposes, and the genes associated with resistance to these antibiotics, may potentially pose risks to the local populations exposed to these water sources.The aquatic environment has been recognized as a source of antibiotic resistance (AR) that factors into the One Health approach to combat AR. To provide much needed data on AR in the environment, a comprehensive survey of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues was conducted in a mixed-use watershed and wastewater treatment plants (WWTPs) within the watershed to evaluate these contaminants in surface water. A culture-based approach was used to determine prevalence and diversity of ARB in surface water. Low levels of AR  There is a growing recognition of aquatic environments as a reservoir and transmission route through which antibiotic resistance (AR) can be spread ,2,3,4. SEnterobacteriaceae are a clinical concern as they confer resistance to most β-lactam antibiotics , carbapenem [meropenem], cephalosporin , dihydrofolate reductase (DHFR) inhibitors [trimethoprim], glycopeptide [vancomycin], glycylcycline [tigecycline], lincosamide [lincomycin], lipopeptide [daptomycin], macrolide , oxazolidinone [linezolid], penicillins , quinolone , sulfonamide , and tetracycline [tetracycline].® hydrophilic–lipophilic balance  solid-phase extraction (SPE) cartridges. Afterwards, the cartridges were dried and stored in a −20 °C freezer until elution. Antibiotics were eluted sequentially with 6 mL methanol and 6 mL dichloromethane, then gently evaporated under nitrogen gas, and reconstituted in 10% acetonitrile for LC-MS/MS analysis.Each water sample was filtered through a 0.2 µm filter to remove any biologicals and stored at 4 °C. Within 24–48 h after filtering, each sample was spiked with 5 μL of internal standard  before extraction on a preconditioned (6 mL methanol then 6 mL of water) Oasis® 2.6 μM C18  high-performance liquid chromatography (HPLC) column . Injection volume for all samples was 20 μL. All samples were run with blanks and calibration curves at the beginning and end of the sequence with quality assurance/quality control (QA/QC) analyzed every 10 samples. The mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. Initial conditions were 0% B for 4 min, ramped to 60% B over 16 min, increased to 98% B, and held for 4 min before returning to starting conditions  with a flow rate of 200 μL/min. Quantification of all 26 antibiotics was performed in positive mode with multiple reaction monitoring (MRM). Instrument parameters were optimized for each compound prior to analysis (A TSQ Quantum Ultra™ accurate mass triple quadrupole mass spectrometer (Thermo Fisher Scientific) with an electrospray interface was used for the detection of antibiotics. Separation of compounds was achieved using a Kinetexanalysis .Bayesian generalized linear mixed models (GLMM) were fitted to the data from both surface water and wastewater samples. For presence–absence data of AR contaminants in surface water, a logistic GLMM was fitted with season, contaminant, and their interactions as fixed predictors. A separate logistic GLMM was fit for β-lactamase-containing bacteria pooled together. For copy numbers of ARGs in surface water, a GLMM was fitted with a hurdle gamma response distribution. For wastewater samples, we did not fit a formal statistical model to the presence–absence data due to the sparsity of presences for individual AR contaminants. Another hurdle gamma GLMM was fitted with sample type (influent and effluent), gene, season, and their interactions as fixed predictors to assess the ability of WWTPs to reduce ARG concentrations in wastewater. A similar model was fit for antibiotic concentration with sample type, drug, season, and their interactions as predictors. Additional models were fitted with the interaction of WWTP and sample type as predictors to compare the effectiveness of the treatment plants. Finally, models were fit comparing ARG copy numbers and antibiotic concentrations upstream and downstream of the NORO WWTP.In all cases, marginal means and trends were estimated using the medians of the posterior predictive distributions and uncertainty was characterized with the 66%, 90%, 95%, and 99% quantile credible intervals (QCI). These marginal means were contrasted by taking the ratio between different groups, again finding the median and QCI of the posterior distributions of the ratios. A complete description of the statistical methods, including model fitting details and information about software used, is found in the Enterobacteriaceae, which are increasingly reported in healthcare and community settings, were commonly found in surface water and wastewater effluents that were released into this water, indicating their prevalence in the community as well as the natural environment. These findings suggest that continuous surveillance of AR contaminants in the aquatic environment, identification of AR hotspots, and preventive interventions are needed to ensure public health. The comparison of wastewater influent and effluent samples has shown that wastewater treatment is not effective in removing all the AR contaminants present in the wastewater and, in fact, some antibiotics and resistance genes were enriched during the process. Treated effluents carried these AR contaminants, indicating that WWTPs are a source of AR in the receiving water. However, surface water samples that had not been impacted by WWTPs also had high levels of AR contaminants, suggesting that WWTPs are not the sole contributors of AR in the environment, and other sources of AR contaminants should also be considered. In our subsequent study, we will investigate the factors that could have led to high prevalence and widespread distribution of AR contaminants in the water.This study endeavored to provide an overall picture of AR, including ARB, ARGs, and antibiotics, in a mixed-use watershed. The current study has shown that antibiotic-related contaminants are prevalent in the freshwater environment, including commensal and pathogenic bacteria that are resistant to antibiotics used for human and veterinary purposes, medically important antibiotics, as well as the genes associated with resistance to these antibiotics. The wide dissemination and abundance of such AR contaminants may potentially pose health concerns to the populations exposed to contaminants in these water sources. ESBL-, AmpC-, and carbapenemase-producing


We analyzed a PPI network of potential angelicin targets in the treatment of osteosarcoma and identified hub targets. We systematically performed GO and KEGG enrichment analyses of the potential targets of angelicin, and we predicted it function in osteosarcoma treatment and the underlying molecular mechanism. Through molecular docking, the interactions between hub targets and angelicin were simulated, and then, the hub targets of angelicin were identified. Based on these results, we validated the effects of angelicin on osteosarcoma cells by conducting in vitro experiments. The PPI network analysis of potential therapeutic targets identified four apoptosis-related hub targets, namely, BCL-2, Casp9, BAX and BIRC 2. GO and KEGG enrichment analyses demonstrated that angelicin regulates osteosarcoma cell apoptosis. Molecular docking results indicated that angelicin can freely bind to the hub targets listed above. In vitro experiments showed that angelicin promoted osteosarcoma cell apoptosis in a dose-dependent manner and inhibited osteosarcoma cell migration and proliferation in a time- and dose-dependent manner. The RT-PCR results showed that angelicin simultaneously promoted the mRNA expression of Bcl-2 and Casp9 and inhibited the mRNA expression of BAX and BIRC 2. Angelicin promotes osteosarcoma cell apoptosis and inhibits osteosarcoma cell proliferation and migration by activating a signaling network that is composed of hub targets that link multiple signaling pathways. Angelicin could become an alternative drug for the treatment of osteosarcoma.To explore the antitumor effects of angelicin on osteosarcoma and the underlying mechanism. We aimed to elucidate the mechanism by network pharmacology, molecular docking, and  Osteosarcoma (OS) is the most common bone malignancy in children and adolescents , 2 and iAngelica and Angelica dahurica [Angelicin is one of the main active ingredients of dahurica . Existindahurica –12. Howein vitro experiments to explore whether angelicin affects the biological behavior of OS and its specific mechanism.Recently, network pharmacology has offered a new strategy for exploring the relationships between drugs and diseases by integrating systems biology, multidirectional pharmaceutical biology, bioinformatics, and computer science , 14. CurThe flow chart of the study design is shown in https://pubchem.ncbi.nlm.nih.gov/) and SymMap (http://www.symmap.org) databases with the keyword “Angelicin”. PubChem: that includes a substantial amount of data related to small organic molecule bioactivity and is funded by the National Institutes of Health (NIH). The SymMap database is a traditional Chinese medicine (TCM) evidence association database that includes an immeasurable amount of information about herbal medicines, their ingredients, and their drug targets; the TCM symptoms, Western medicine symptoms, and diseases associated with these symptoms that these medicines are used to treat; and correlations among these data. In this way, the SymMap database correlates TCM and Western medicine at levels ranging from the phenotypic level to the molecular level.All the active ingredients of Angelicin were obtained from the Pubchem (https://www.disgenet.org/) and the GeneCards database (https://www.genecards.org/) using the keyword “Osteosarcoma” and using “Homo sapiens” as the filtering criterion. In addition, the targets obtained from the DisGeNET database with a score >0.8 were screened. The GeneCards database is a search platform that retrieves genes that are associated with human diseases from 150+ web sources. Genes were searched on the two platforms using the keyword “Osteosarcoma”. The retrieved data include information about Osteosarcoma, such as gene names and gene IDs. The targets that were obtained from these two databases were then integrated, and duplicates were removed, ultimately yielding genes related to “Homo sapiens OS”.A search was performed in the DisGeNET database (Homo sapiens OS-related targets were imported into an online Venn diagram mapping platform (https://bioinfogp.cnb.csic.es/tools/venny/). The region where the two circles of the Venn diagram intersect indicates the potential targets of angelicin in the treatment of OS. The targets that were related to the mechanism by which angelicin treats OS were imported into the STRING database. A PPI network diagram was generated with a confidence level of 0.7, and it was imported into the STRING app in Cytoscape software for further processing and analysis.The identified angelicin-related targets and To further analyze the PPI network and identify hub targets, we imported the PPI network into Cytoscape 3.8.0 software and analyzed the network graph with Network Analyzer. Information on the degree, betweenness centrality (BC), and closeness centrality (CC) of the corresponding targets was obtained. We used degree≥degree median, CC≤CC median, and BC≥BC median as the screening criteria to identify the hub targets.To further analyze the potential mechanism underlying the function of angelicin in OS treatment, we enriched the targets mentioned above with the Bioconductor Cluster Profiler package in R software. According to the GO enrichment analysis, the potential therapeutic targets of angelicin in the treatment of OS are described in terms of biological processes, cellular components, and molecular functions. Additionally, the potential signaling pathways by which angelicin functions in the treatment of OS were explored by KEGG enrichment analysis.To investigate the potential interaction between angelicin and its potential targets for the treatment of OS, we performed molecular docking between angelicin and the hub genes based on the principle of semiflexible molecular docking. The leading software for molecular docking is AutoDock, which includes AutoDock tools and AutoDock Vina. Angelicin and hub targets were processed with AutoDock tools, and the docking center of the hub targets was determined. The 3D structures of the target proteins were obtained from the PDB online platform, and the 3D structures of angelicin were obtained from the PubChem online platform.The human OS cell line MG63 (Procell CL-0157) was kindly provided by Procell Life Science and Technology Co., Ltd. MG63 Cells were grown in 90% modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μmol/L streptomycin, and incubated at 37°C in a humidified atmosphere of 5% CO2.MG63 cells were treated with trypsin, grown in 96-well plates (3000 cells/well), and cultured with media supplemented with various concentrations of angelicin. The cells were incubated in a 37°C cell culture incubator for 24 h, 48 h or 72 h. At each time point, the plates were removed, the medium in the corresponding wells was gently aspirated, and the cells were gently washed with sterile PBS. CCK8 and serum-free medium were mixed at a ratio of 1:9, and 100 μl of the mixture was slowly added to each well. The cells were incubated in a cell culture chamber at 37°C for 1 h. The OD value of each well was measured at 450 nm by a multifunctional enzyme marker. Cell activity (%) = / × 100%.5 cells per well; when the cells had completely adhered to the well, the original medium was discarded, and the cells were washed 2 times with PBS. Angelicin was added at different concentrations and incubated for 24 h. The cells were stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). ACEA NovoCyteTM  equipment was used to perform the flow cytometry analysis. FlowJo  software was used to analyze the results.According to the instructions, we analyzed the effect of angelicin on the apoptosis of MG63 cells by flow cytometry. MG63 cells were seeded in 6-well plates at a density of 5 × 105 cells per well. Then, the cell culture plates were gently shaken to uniformly distribute the cells to form a monolayer, and the cells were prepared for scoring when they reached 95% confluence. A 200 μL pipette tip was used to form a vertical scratch along a sterile straightedge. Then, the treated wells were gently washed 3 times with PBS to remove the nonadherent cells, and the plates were imaged and photographed (0 h). Media supplemented with different concentrations of angelicin (50 and 200 μmol/L) were added, and the control group was incubated in the same volume of MEM supplemented with DMSO; the cells were cultured in an incubator at a constant temperature of 37°C with 5% CO2 for 24 h or 48 h, and then, the cells were photographed. The scratch area S was obtained using the scratch area analysis module of ImageJ software, and the scratch healing rate was calculated according to the following formula: wound healing rate = (1-(S24h/S0h)) × 100%.MG63 OS cells in the logarithmic phase of growth were seeded in 6-well plates at a density of 3 × 10Total RNA was extracted from cells using an RNA extraction kit, first-strand complementary DNA (cDNA) was synthesized from the total RNA using an RNA reverse transcriptase kit, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed using SYBR Green reagent  according to the instructions. The primer sequences are shown in in vitro experimental data was conducted using GraphPad Prism 9. The difference between two groups was assessed using one-way ANOVA. The statistical significance of the CCK8 results was assessed using two-way ANOVA. The data are presented as the means ± standard errors of the means (SEM), and a p value < 0.05 indicates statistical significance.Statistical analysis of the bioinformatics data and mapping was performed by R ×64 4.0.3, Cytoscape 3.8.0, and PyMOL software. Statistical analysis of the The original contributions presented in the study are included in the article Material, further inquiries can be directed to the corresponding authors.Using “angelicin” as the keyword, we screened its targets in the PubChem and SymMap databases. We identified 36 targets of angelicin by integrating and deweighting the screened targets, as shown in The keyword “osteosarcoma” was used to screen the DisGeNET and GeneCards databases. A total of 4389 relevant targets were identified after integration and removal of duplicates.Angelicin targets and OS therapeutic targets are shown in a Venn diagram . In totaThe obtained potential targets of angelicin in the treatment of OS were subjected to GO and KEGG enrichment analyses. The results of biological processes, cellular components, and molecular functions are shown in Molecular docking of the identified hub genes was performed using AutoDock software. The molecular docking results were visualized using PyMOL software, and the docking results of the four hub targets are shown in P < 0.0001). The differences in cell viability between the 50 μmol/L and 200 μmol/L groups were statistically significant (P < 0.0001)  . The viaP < 0.05). The apoptosis rate of OS MG63 cells gradually increased with increasing angelicin concentrations . The wound healing rate of OS cells gradually decreased with increasing angelicin concentration , and the results are shown in To measure the mRNA expression of pivotal targets in OS MG63 cells after treatment with different concentrations of angelicin, we performed RT-PCR experiments, and the results demonstrated that the expression of BAX and Casp9 gradually increased, while the expression of the antiapoptotic proteins Bcl-2 and BIRC2 gradually decreased with increasing angelicin concentration. The differences in mRNA expression among the different concentration groups were statistically significant (Angelica archangelica and Paeoniae Radix Alba [Osteosarcoma is the most common malignant tumor, accounting for approximately 55% of all bone tumors . OS is mdix Alba , 21. Exidix Alba , 10. InsAngelica affects the biological behaviors of osteosarcoma. When the NF-κB signaling pathway is inhibited, the biological behaviors of osteosarcoma, such as proliferation, migration, and apoptosis, are suppressed [in vitro results further confirmed the network pharmacological results. It was also demonstrated that angelicin inhibits osteosarcoma cell proliferation and migration in a dose-dependent manner and promotes osteosarcoma cell apoptosis in a dose-dependent manner.In this study, we intersected angelicin therapeutic targets and OS-related targets that were identified in several databases and found potential therapeutic targets through which angelicin functions in the treatment of OS. Furthermore, we found the molecular hub targets of angelicin. Then, we screened the hub targets, including Casp9, BIRC2, BAX, and BCL2, of which BCL2 and Casp9 are proapoptotic proteins, and BAX and BIRC2 are antiapoptotic proteins . All fouppressed , 28, 29.ppressed , 30, 31.in vitro experiments described above show that angelicin inhibits OS cell proliferation and migration and promotes OS cell apoptosis upregulating Casp9 and Bcl-2 gene expression and downregulating BAX and BIRC2 gene expression, thereby regulating apoptosis in multiple species, apoptosis, the P53 signaling pathway, the NF-κB signaling pathway, and the TNF signaling pathway to form a signaling pathway network.The network pharmacology, molecular docking, and in vitro experiments that angelicin inhibits osteosarcoma cell proliferation and migration and promotes osteosarcoma cell apoptosis. We are the first to observe these results. Network pharmacology is an efficient tool that is used for the investigation of the mechanisms of action of drugs. However, it is clear that the present study still has some limitations. This study did not examine the specific signaling pathways that are regulated by angelicin in osteosarcoma cells, but we have predicted which pathways are targeted by angelicin via network pharmacology; molecular biological validation is our next step. In addition, in vivo experiments were not conducted in this study, and the inhibitory effect of angelicin on osteosarcoma in vivo was not explored; we will pursue these experiments in future work. Third, the exploration of the loci of apoptotic proteins on which angelicin acts is another focus of our future work.Through pharmacological and molecular docking assays, we discovered the regulatory effect of angelicin on osteosarcoma cells. Based on these results, we verified with We found that via the regulation of multiple signaling pathways, angelicin inhibited osteosarcoma cell proliferation in a time- and dose-dependent manner, inhibited osteosarcoma cell migration in a dose-dependent manner, and promoted osteosarcoma cell apoptosis in a dose-dependent manner. Angelicin could be an alternative medicine for the treatment of osteosarcoma, and it still needs to be explored in depth.Supplementary Tables 1-2 and 4Supplementary Table 3