Non-tubal ectopic pregnancies (NT-EPs) are rare but potentially life-threatening conditions. The incidence ranges are between 5–8.3% of all ectopic pregnancies. For this retrospective observational study, 16 patients with NT-EP and treated from January 2014 to May 2020 were recruited. Demographic details, symptoms, Beta human chorionic gonadotrophin (β-hCG) levels, ultrasound findings, management and treatment outcomes were presented. In hemodynamically stable patients, diagnosis was made using ultrasounds and β-hCG levels. Laparoscopy was essential to identify and remove the ectopic pregnancy in clinical unstable patients. A radical laparoscopic approach was chosen in one case of cervical pregnancy diagnosed late in the first trimester. Medical treatment and minimally invasive procedure, alone or combined, resulted in effective strategies in asymptomatic women with an early diagnosis of NT-EP. We report cases of cervical pregnancies successfully treated by hysteroscopy alone or combined with medical treatment, the first case of scar pregnancy treated by mini-reseptoscope and curettage and the fifth case of interstitial pregnancy treated with Methotrexate and Mifepristone. In this manuscript we report a single center experience in the management of NT-EPs with the aim of outlining the importance of the early diagnosis for a minimally invasive treatment in order to reduce maternal morbidity and mortality and preserve future fertility. Ectopic pregnancies (EP) account for 2% of all pregnancies and in most of the cases gestational sac (GS) is implanted within the fallopian tube . PregnanThe institutional review board (RC 08/2020) approved this retrospective observational descriptive study in February 2020.n = 16 NT-EPs divided into four groups on the basis of the implantation site: cervical pregnancies (CP) (n = 6), interstitial pregnancies (IP) (n = 3), cesarean scar pregnancies (ScP) (n = 3), abdominal pregnancies (AbP) (n = 2), ovarian pregnancies (OvP) (n = 2)  . Diagnos (n = 2) .Diagnosis of IP was made by TVUS according to criteria outlined by Timor-Tritsch that consist of an empty uterine cavity, a myometrial layer of less than 5 mm surrounding the GS and a chorionic sac separated and laterally located 1 cm or more from the sideward portion of the uterine cavity . MoreoveCriteria used for the diagnosis of ScP included no fetal parts in the uterus or cervix, the presence of a GS in the anterior isthmic portion covering the scar site or entirely embedded within the myometrium and absence of contact between the bladder and GS  Figure . Abdominal and Ovarian pregnancy  were det2 of the body surface in addition to Mifepristone 600 mg and Misoprostol 400 mcg orally (n = 1), a combined treatment using a previous single dose of MTX IM injection at dosage of 50 mg/m2 of the body surface followed by hysteroscopy (n = 2) and a totally hysteroscopic approach (n = 2). One case of CP was diagnosed late in the first trimester, and it was treated by laparoscopy. A fertility sparing treatment was administrated in five cases of CP: A total medical management using a single dose of MTX IM 50 mg/mn = 1), the combination of systemic MTX with Mifepristone 600 mg (n = 2).Conservative medical management for IPs involved the use of MTX  or the combination of Mifepristone and Misoprostol (n = 1). Hysteroscopy alone was chosen in one case (n = 1)  . Additio (n = 1) .n = 16 NT-EPs were listed in our institution’s records. Background and clinical characteristics of the study patients are summarized in n = 4 cases, emergency laparoscopy was essential in order to identify and remove the EP in clinically not stable women with hemoperitoneum/acute abdomen symptoms. In particular, we identified n = 2 AbPs, n = 2 OvPs, n = 3 IPs, n = 3 ScPs, and n = 6 CPs  , n = 2 women had undergone a previous salpingectomy because of a history of tubal EP. Symptoms at diagnosis included abdominal pain and/or vaginal bleeding in n = 8 patients; hemoperitoneum and abdominal acute pain was recorded in n = 4 cases of OvPs and AbPs; no symptoms were recorded in n = 4 cases  and AbPs (n = 2). In n = 1 case of CP, the diagnosis had been made late in the first trimester because of the delayed discovery of the pregnancy status: the patient was totally asymptomatic with a cervical GS with a diameter of 78 × 60 mm. The radical laparoscopic approach with a total hysterectomy combined with uterine arterial embolization had been chosen in order to reduce the hemorrhagic risk. Among primary treatments, n = 7) with a diagnosis of CP and ScP at an early GA and low serum β-hCG level in order to preserve future fertility. Additionally, after the hysteroscopic procedure, curettage had been made in the group of ScPs disease. A detailed description of primary and secondary treatments performed is shown in Surgeons had decided for a conservative treatment using hysteroscopy, alone or in combination to medical treatment, in hemodynamically stables patients  has been estimated as high as 2–5% , with thWith the widespread availability and application of ultrasound, identifying earlier the location of the gestational sac has become possible, guiding the gynecologist in choosing how to manage ectopic pregnancies appropriately .In order to reduce morbidity and mortality related to a delayed diagnosis and the appearance of life-threatening complications, it might be useful to refer patients to centers of excellence. The availability of dedicated early pregnancy units has improved diagnosis and the follow-up for these patients .The management of each patient must be individualized based on clinical symptoms, viability of pregnancy, GA, hCG levels and women’s wishes.The acquisition of expertise in the care of NT-EPs ensures a faster diagnosis, which gives a better chance of successful medical therapy or minimally invasive surgery, such as the local injection of one of several agents MTX, potassium chloride (KCl), hyperosmolar glucose, etoposide (directly into the GS ), curettWe have presented data from a six-year review of all diagnosed NT-EPs and their subsequent management. This series adds to the growing body of evidence that the hysteroscopic approach, combined with systemic MTX or alone, is a safe and efficacious first-line treatment for women with high-risk NT-EPs desiring to preserve future fertility.2 body surface area) or local MTX (1 mg/kg body weight)) is preferred for ScP and CP. Expectant management is possible in asymptomatic patients with nonviable pregnancy and decreasing hCG levels. Women with heavy bleeding or failed medical treatment need surgical procedures [In general, the primary treatment option for most IP and OvP is surgery; medical treatment with systemic MTX  and the interstitial line are diagnostic of IP .Early diagnosis with TVUS allows conservative treatment with methotrexate; if it is made later in gestation, surgical treatment can be required.From our analysis it emerged that medical management with MTX IM regimen isolated or combined with mifepristone  can be considered a good option in women with IP and a strong motivation for future conceptions .This complication is likely to become more common with the increased rates of abdominal delivery increasing worldwide and in 72% of cases occurs in women who have had more than 2 CDs ,26.TVUS is likely to emerge as a future gold standard for the diagnosis of scar implantation. Diagnosis is relatively simple in early pregnancy, but as the pregnancy progresses, the distinction between ScP, CP and low intrauterine pregnancy becomes more difficult. In women with non-viable pregnancies, Doppler ultrasound and “sliding organs sign” should be used to confirm the diagnosis of a scar pregnancy .A range of treatment options are available to treat ScP; however, it is not clear which is the best option. Surgical procedures, alone or in combination with medical treatment, have high success rates, but greater surgical skill is required. Medical management is not considered treatment of choice for ScP probably because the absorption and efficacy of MTX is reduced by the fibrous tissue surrounding the GS, which is located in an unusual site inside the uterine cavity. When combined with curettage or hysteroscopy, MTX appears more efficient . From a We have recorded 3 ScPs successfully treated by hysteroscopy, followed by curettage in one of the cases, in order to remove trophoblastic residues and reduce risk of subsequent reintervention. This minimally invasive approach allows the direct visualization of the implantation site and the possibility to separate the gestational sac from the myometrium under operator view. In literature, a success rate for this procedure of 8/8 is reported . HysteroOvP is a rare event, with estimates of frequency ranging from 1 in 2100 to 1 in 7000 pregnancies or 3% of all EPs.A more echogenic wide ring on the ovary, compared with the ovarian tissue, a yolk sac or fetal parts are ultrasonographic findings for OvP. Surgical criteria where described by Spiegelberg: the fallopian tubes intact and separate from the ovary, the GS on the ovary must be attached to the uterus through the utero-ovarian ligament and the placental tissue appearing mixed with ovarian cortex. Surgical treatment is the most frequent approach, and an oophorectomy or a wedge resection of the ovary is usually required .Abdominal pregnancy is defined as pregnancy anywhere within the peritoneal cavity and represents around 1–1.5% of all EPs with an estimated incidence of 1:8000–10,000 pregnancies. Maternal mortality is estimated to be 2–30%, while perinatal mortality in those undiagnosed is 40–95%.Studdiford′s criteria are normal bilateral fallopian tubes and ovaries, the absence of utero-peritoneal fistula and pregnancy related exclusively to the peritoneal surface and early enough to eliminate the possibility of secondary implantation following a primary location in the tube.TVUS is the first-line tool for diagnosis, but also, MRI could be considered in cases of ambiguity, especially to define the extent of the placental tissue invasion to the abdominal and pelvic organs. When images are inconclusive or patients become hemodynamical instable, laparoscopy is essential. Laparoscopic surgery should be considered for early abdominal pregnancy to allow better access to deal with placental attachment and control the bleeding .According to the present literature, we report four cases enhanced with clinical symptoms of hemodynamical instability and acute abdomen and TVUS findings suggestive for OvPs and AbPs treated successfully with laparoscopy.The main limitations of this manuscript are small sample sizes due to the rarity of the disease and heterogeneity in treatments on the basis of clinical specific characteristics and with the aim of individualizing the optimum mode of management.It is imperative for a reference center for NT-EP management having well trained surgeons in minimally invasive surgery with specific skills, reducing risks of life-threating hemorrhage and hysterectomy and preserving future fertility.From our sample emerges the necessity of a fast diagnosis of NT-EP. This can help a conservative management with a medical or minimally invasive approach. The important progress in imaging technologies allows a faster diagnosis, permitting the transfer of the patient to a reference center where the choice of the best procedures may reduce the impact of surgery on the patient’s fertility.


Acute respiratory infections appear to precipitate vascular events. Acute myocardial infarction (AMI) and stroke are the leading cause of death and disability globally. This study was based on a cohort of patients admitted to Townsville University Hospital between January 2006 and December 2016. Using a self-controlled case series design, we investigated the risk of AMI or ischaemic stroke after an episode of pneumonia. We defined the ‘risk interval’ as the first 14 days after hospitalisation for pneumonia and the ‘control interval’ as one year before and one year after the risk interval. Among a population (N = 4557) with a median age of over 70, a total of 128 AMI and 27 stroke cases were identified within 1 year of an episode of pneumonia in this study. Ten and two admissions occurred during the risk interval, while 118 and 25 admissions occurred during the control period. The relative incidence ratios (RIR) of AMI increased after an episode of pneumonia  2.44–9.67). The risk for stroke after the exposure period of 14 days was 4.94 (95% CI 1.12–21.78) considering only the first stroke incidence. The RIR results for AMI and stroke were not altered by adjusting for age, sex or Indigenous status. The risk of AMI and stroke were significantly higher two weeks after an episode of pneumonia. When combined, the acute vascular events of acute myocardial infarction (AMI) and stroke are the leading cause of death  and disability globally5. The main risk factors for vascular disease are chronic and predominantly associated with lifestyle (smoking and obesity), diabetes, hypertension and familial factors8. These severe vascular events also appear to be precipitated acutely by lower respiratory tract infections10. As lower respiratory tract infections (LRTIs) are common, an awareness of the potential for subsequent AMI and stroke is required to direct additional preventive strategies such as vaccination  and the use of anti-platelet therapy. The traditional focus on primary cardiovascular risk factor prevention could be complemented by secondary prevention targeted at the post-infective period.Cardiovascular disease (CVD) is the number one cause of death globally with an estimated 17.9 million deaths  in 2016In this study, we sought to quantify the risk of AMI and stroke in patients living in the tropical regions of Northern Australia after an episode of hospitalisation due to lower respiratory tract infections (LRTI), caused by both bacterial pneumonia and influenza. The database used includes a large proportion of Australian Indigenous peoples, so we’re able to explore whether additional factors, including Indigenous status influence this association.12. This methodology allows for minimisation of fixed confounders.We have employed a self–controlled case series methodology to optimally investigate associations between transient exposures and linked eventsOut of a total of 4557 admissions with pneumonia as the primary diagnosis, we identified 128 unique AMI admissions from 117 patients and 27 unique stroke admissions from 26 patients, which occurred within an individual 2-year period determined by the discharge date of pneumonia-related hospitalisation  and LRTI-diabetes all events  were not statistically significant. Similarly, we found no evidence of an increased risk of cancer (ICD-10-AM codes C00-C75 excluding only C34 that encodes for lung cancer) after LRTI.In addition to sensitivity analysis, we performed robustness checks by examining associations between LRTI exposure and other health outcomes such as admissions to hospital for complications of diabetes and cancer diagnosis Table . SimilarWe explored the heterogeneity in the incidence ratios due to LRTI exposure with respect to gender, age, and indigenous status by including an interaction term Table . We also15. These studies recorded significant increases in incidence risks of stroke following infections with Streptococcus pneumonia and influenza.In this study, we provide evidence that, in a population of elderly patients living in the Australian tropics, there is a transient increase in the risk of AMI or stroke after hospitalisation for severe LRTI. The “at-risk” interval, in which the increased risk of AMI/stroke persisted, was 14 days. The incidence of admissions for AMI and stroke were more than four and seven times, respectively, during the 14–day interval after the LRTI stimulus. Our results also showed that an elevated incidence ratio is still present up to 3-months. However, the magnitude of relative risks was smaller than RIRs for 14-day period, which was in line with our expectations. Our findings are consistent with the previous studies suggesting respiratory infections, including pneumonia and influenza, might act as triggers for AMI and stroke events15.We also tested whether incidence ratios are heterogeneous with respect to sociodemographic characteristics, such as gender, age, and indigenous status. Although we reported the differences in the incidence ratio point estimates, the analyses had low power to identify statistically significant differences between the subsamples. This is similar to the previous studies where confidence intervals for the estimated incidence ratios stratified by age overlapped and no evidence of significant heterogeneity were reported18. Additionally, atherosclerotic disease has an inflammatory component that is enhanced by severe infection due to LRTI and other infections including urinary tract infections contribute to increased risk of these vascular events through a number of complex mechanisms19. Sepsis induces a procoagulant state through the action of cytokines, disturbance in coagulation pathway factors, activated platelets, and innate immune system activation20. Prevention of AMI and stroke that occur hyper-acutely in sepsis due to LRTI relies on restoring the normal physiological state with fluid resuscitation, supplemental oxygen, and antibiotics. Other interventions may be useful in the prevention of vascular events during the 2–4 weeks after LRTI. Long term use of aspirin has been associated with reduced cardiovascular events in cohort studies of patients with pneumonia21.From the clinical perspective, both AMI and stroke occur due to atherosclerotic disease and, particularly, unstable vascular plaques. The altered physiology of sepsis with an increased sympathetic tone, vasoconstriction, increased myocardial oxygen demands and hypovolaemia play major roles in precipitation of acute vascular events, and places increased demand on the heart22. This Turkish trial of 185 patients showed a large reduction in the incidence of ACS in patients randomised to 300 mg aspirin daily in the month after a pneumonic illness. Death due to acute coronary syndrome was also significantly reduced. This single trial requires validation with a larger study. Further analysis of the patients involved in the ANTISEPSIS primary prevention trial may be able to provide more evidence of benefit from low-dose aspirin for the prevention of coronary vascular diseases following pneumonia and other sepsis leading to hospitalisation23.A randomised controlled trial of aspirin for the prevention of acute coronary syndrome (ACS) following pneumonia was reported in 201324. A meta-analysis of observational studies shows that the use of statins reduces all-cause mortality after pneumonia25. Influenza vaccination appears to reduce mortality due to ACS26. Pneumococcal vaccination has also been associated with reduced risk of acute coronary syndromes and associated mortality27.Statin drugs have also been studied and may reduce ACS due to lipid-lowering and their immunomodulating effectsWe are cognisant of a number of limitations of our study. Firstly, we did not include the AMI or strokes that occurred during the admission for pneumonia because we were unable to calculate the time interval between these two events if they occurred in the same hospitalisation episode. This potentially underestimated the magnitude of the increased risk of AMI and stroke. The absence of these data also made it difficult to study the periods of risk less than 1 week. Overall, a total of 112 cases of AMI and seven cases of stroke were recorded in discharge ICD-10-AM coding as other diagnoses during a hospital episode where pneumonia was the primary diagnosis.Secondly, information on subsequent hospitalisations would not be available for patients who may have moved to the catchment of another health services after having an LRTI event. This may lead to the underestimation of our relative risk ratios. However, with the large catchment and no other tertiary hospital within 1300 km, we believe the underestimate will be relatively small compared with other hospitals.Lastly, the numbers of stroke events included in the study were small and, although significant, the association with preceding LRTI should be interpreted with care as the confidence intervals around the point estimates are wide. While the data used were from a single hospital, potentially limiting generalisation to the Australian population, other similar results from international studies support this result being a true association.This self-controlled cohort study of patients admitted to an Australian tertiary hospital demonstrates an association between hospitalisation with severe lower respiratory tract infection and increased risk of cardiovascular disease in the 2 weeks post-discharge. There are data from observational cohort studies and randomised controlled trials of benefits of aspirin, statins, influenza and pneumococcal vaccination in the prevention of cardiovascular disease associated with pneumonia. On this basis, it is imperative that optimal secondary prevention strategies are deployed in patients with severe pneumonia and influenza.28. The database includes detailed information on the cohort of patients admitted to Townsville University Hospital (TUH) with LRTI between January 1, 2006, and December 31, 2016. TUH is a regional tertiary hospital in Australia providing care for 670,000 people in Northern Australia. The complete database includes diagnostic codes (ICD-10-AM) for 41,367 patients with 378,487 admissions.The data used in this study is derived from a large relational database described in Eisen et al.We included in our study all inpatients with a primary diagnosis of viral and bacterial pneumonia defined by J09–J18 ICD-10-AM discharge codes. We limit our exposure dates to the period between January 1, 2007, and December 31, 2015, to allow each patient to have a 2-year interval (from 1 year before and 1 year after the exposure date) in which we observe AMI and stroke events.The outcome of interest in this study was all incidence of AMI (based on ICD-10-AM codes) codes: I21-I22 and ischaemic stroke  between January 1, 2006 and December 31, 2016 occurring in hospitalised patients. The risk event consisted of bacterial pneumonia or influenza occurring as the primary diagnosis for patient hospital admissions. For patients with more than one incident of AMI/stroke within the exposure window, we created two scenarios. The first scenario includes only single incidents of AMI or stroke (the first event for each patient). The second scenario includes the count of all events a patient experienced during the observation period.We define the observation period for each patient as the interval 1 year before and 1 year after the discharge date of the first LRTI exposure. To be included in the study, patients required at least one recorded inpatient AMI/stroke episode within this 2-year period. In the case where an AMI/stroke admission happened within 30 days from the discharge date of the previous episode, we consider the subsequent episode as a related admission and do not count it as a separate event. The schematic of the study design is presented in Fig. 29. We test the robustness of our results by running a likelihood ratio test for the models with and without interaction between exposure and censoring indicators. We conclude that this censoring approach does not produce any statistically significant differences and do not cause bias to our results.We note that few patients died within the 50-week period after the end of the exposure period. For these patients, the baseline period became smaller, and the observation period ended at the corresponding time of death. Because our study outcomes (AMI and stroke) carry a high mortality risk, the impact of death on the observation periods can create bias in the results, although it is often reported to be smallWe used separate conditional fixed-effects Poisson regressions to compute parameter estimates and interpret the results in terms of incidence rate ratios, i.e. relative incidence of AMI/stroke in “at-risk” or exposure periods to the incidence in baseline or unexposed periods. Because this methodology relies on within-person comparisons, it eliminates the problem of time-invariant (i.e. fixed) confounders. This is advantageous as some confounding factors may not be adequately recorded or captured in the dataset. Furthermore, we account for the time-varying confounders by including controls for calendar months.Although patient to patient variability  is avoided by including all individuals in both at risk and control groups, yet some individual-based factors could still modify the risk of the outcome given the exposure.We perform additional analyses by including interaction terms to examine whether heterogeneous effects due to age at AMI/stroke event (< 65 years vs ≥ 65 years), gender , and indigenous status (Indigenous vs non-Indigenous) are significant. The likelihood ratio test was used to test the importance of interaction terms.Experiencing an AMI/stroke may change the probability of subsequent AMI/stroke episodes that might violate the independence of events assumption for the self-controlled case series methodology. To account for this potential problem, we perform a robustness check by running separate analyses for first records of AMI/stroke and all AMI/stroke episodes. We also sought to study the specificity of the demonstrated relationship between the at-risk LRTI disease and AMI/stroke outcome. In this regard, we analysed whether a similar association was present between LRTI and diabetes/cancer, both common non-infective causes for hospitalisation, using the same self-controlled cohort study methodology.This study was part of the project, HREC/16/QTHS/221, approved by the Townsville Hospital and Health Service (THHS) Human Research Ethics Committee. A waiver of consent for access to anonymised data was approved under the Queensland Public Health Act (RD007802). The anonymised secondary data extraction does not require patient recruitment. All methods regarding human use in the manuscript were carried out in accordance with relevant guidelines and regulations.


The artificial pancreas is a closed-loop insulin delivery system that automatically regulates glucose levels in individuals with type 1 diabetes. In-silico testing using simulation environments accelerates the development of better artificial pancreas systems. Simulation environments need an accurate model that captures glucose dynamics during exercise to simulate real-life scenarios. We proposed six variations of the Bergman Minimal Model to capture the physiological effects of moderate exercise on glucose dynamics in individuals with type 1 diabetes. We estimated the parameters of each model with clinical data using a Bayesian approach and Markov chain Monte Carlo methods. The data consisted of measurements of plasma glucose, plasma insulin, and oxygen consumption collected from a study of 17 adults with type 1 diabetes undergoing aerobic exercise sessions. We compared the models based on the physiological plausibility of their parameters estimates and the deviance information criterion. The best model features (i) an increase in glucose effectiveness proportional to exercise intensity, and (ii) an increase in insulin action proportional to exercise intensity and duration. We validated the selected model by reproducing results from two previous clinical studies. The selected model accurately simulates the physiological effects of moderate exercise on glucose dynamics in individuals with type 1 diabetes. This work offers an important tool to develop strategies for exercise management with the artificial pancreas. Type 1 diabetes (T1D) is a chronic disease in which the immune system attacks and destroys the pancreatic beta cells resulting in the loss of insulin secretion . HoweverThe artificial pancreas system is a promising solution for individuals with T1D. It is a closed-loop delivery system composed of a glucose sensor, an infusion pump, and a control algorithm. The artificial pancreas can be a single-hormone system that infuses insulin or a dual-hormone system that infuses insulin and glucagon . The conRegular exercise is recommended by the American Diabetes Association  for its Enhancing the control algorithms can be accelerated by performing in-silico testing in simulation environments  before mA few models are reported that describe the exercise effect on glucose dynamics. In particular, Roy and Parker  extendedIn this paper, we proposed six physiologically-motivated variations of the Bergman Minimal Model  to captu2max and lasted for 60 minutes. Interval exercise consisted of 2-minute alternating periods of 50% and 85% VO2max for 40 minutes, with two 10-minute periods at 45% VO2max at the start and end of the sessions. VO2max values were obtained at the admission visit using a graded exercise test  is the expected value of D(θ) given y  and D(Eθ|y[θ]) is the deviance evaluated at the posterior mean of the parameters. The DIC is then formally defined as:Complexity is measured via the estimation of the ‘effective number of parameters’:The best model was chosen based on: (i) the DIC score, (ii) parameter’s identifiability and physiological plausibility, and (iii) a CV below 100%. Parameter values were used to generate virtual patients and conduct in-silico experiments.For the purpose of model validation, we performed in-silico simulations to reproduce results of two previously completed clinical studies , 54. We The protocol of the first simulated study reflected a clinical study conducted by Rabasa-Lhoret et al.  in 8 indmax (the exercise was divided into four 15-min bouts with 5-min rest periods in between). The insulin reductions included: 1) pump stop at exercise onset, 2) an 80% basal reduction set 90 min pre-exercise, and 3) a 50% basal reduction set 90 min pre-exercise.The second simulated study replicated a clinical study conducted by Zaharieva et al. , where 1We also performed in-silico simulation using the Hovorka model  to reproFor each subject, 100,000 iterations of WinBUGS were run. The last 20,000 samples were used to generate the posterior distributions. Model 1, having a fixed increase in both glucose effectiveness and insulin sensitivity, showed the DIC of 471. By neglecting the increase in glucose effectiveness, Model 2 produced a higher DIC of 731. Model 3 produced a better DIC compared with Model 2 by neglecting the increase in insulin sensitivity (731 vs 705). Model 4, incorporating the effect of exercise intensity on rate of glucose uptake, reduced DIC to 450. Model 5, incorporating the effect of exercise duration on rate of glucose uptake, did not reduce DIC compared with Model 4; its DIC was the highest with value 750 . Combining the effect of both intensity and duration in Model 6 produced the lowest DIC of 425 . As a result, Model 6’s performance suggests that during exercise there is an increase in glucose effectiveness based on exercise intensity, and an increase in insulin sensitivity based on exercise intensity and duration. Additionally, all of the parameters of Model 6 were identified with good sensitivity (CV < %30). Visual inspection of Model 6’s fits confirms good performance of the model to fit the data. The weighted residuals of Model 6 are shown in 2max. Similar to the clinical study’s conclusion, a 75% reduction in premeal bolus resulted in a safer glycemic profile with a decreased risk of hypoglycemia compared with a 50% reduction in premeal bolus. The remaining graphs can be found in the S2–S5 Figs in The comparison between our simulation results and the results of the published clinical study of Rabasa-Lhoret et al.  demonstrSimilarly, simulation results demonstrate that Model 6 was able to reproduce the results obtained during the clinical study of Zaharieva et al. . Fig 6 sThe quantitative comparison between our simulation results using the Hovorka model  and the Mathematical models that incorporate the effects of exercise on glucose dynamics play an important role in accelerating the development of safe and effective artificial pancreas systems usable in normal life conditions. In this paper, we proposed six variations of the Bergman Minimal Model , compare1 = 0), and Models 3 assumed a zero increase in glucose effectiveness (e2 = 0). Model 4 related the increase in glucose effectiveness and insulin sensitivity to exercise intensity; meanwhile, Model 5 related the increase in glucose effectiveness and insulin sensitivity to exercise duration. Finally, Model 6 related the increased glucose effectiveness to exercise intensity and related the increased insulin sensitivity to exercise intensity and duration. In our system identification problem, the input to our system is exercise, which is not modified by glucose levels, and thus, this system identification problem is considered in open-loop settings. The insulin infusion rate is indeed altered by glucose levels, but this is considered an inner loop to the system. In addition, insulin infusion rate has been mostly suspended during exercise.In people with T1D, exercise increases the rate of their glucose uptake . HoweverThe outcomes of our work are consistent with findings from literature. Models 1 produced a higher DIC compared with Models 4 and 6 which suggests that adding a fixed increase on both glucose effectiveness and insulin sensitivity for the duration of the exercise is inaccurate. This agrees with two previous studies , 47 thatA particularly interesting finding in our work is related to the comparison between the models that included the effect of exercise intensity vs exercise duration. Adding the effect of exercise intensity on the increases in glucose effectiveness and insulin sensitivity produced a low DIC value and good fits as shown in Model 4, confirming findings in literature , 48 that2max), durations (30–60 min), and pre-meal insulin bolus reductions (25–75% reductions). However, there was some quantitative differences with both studies that are likely due to the mismatch between the meals used in the clinical study and those modelled in the simulations, as well as the mismatch in insulin absorption kinetics and insulin sensitivities between the virtual patients and those specific to the real patients recruited in the clinical study. In-silico validation using the Model 6 integrated with Hovorka model showed good quantitative results. The comparison with the clinical study of Haidar et al. [In-silico validation showed that Model 6 successfully predicted changes in glucose levels observed in clinical studies of moderate exercise. The simulations used virtual patients with the Model 6’s parameters estimated from the single-hormone closed-loop moderate-exercise data. The comparison between our simulated results with Model 6 and the clinical study of Rabasa-Lhoret et al.  demonstrr et al.  demonstr1 and p2. Thus, a priori values, taken from literature, were used to produce plausible values. Another limitation is lacking a direct measure of the rate of glucose uptake in our data which require complex experimental procedures with the use of tracers and varying insulin doses in order to give more information about the effects of insulin-dependent and insulin-independent rates of glucose uptake, and thus help estimate e1 and e2 more accurately. Additionally, the data did not include multiple exercise durations which could help develop models with more complex relationship instead of the linear relationship of glucose effectiveness and insulin sensitivity on exercise duration (as is the case in Model-5 and Model-6). Finally, due to lack of post-exercise data, we were not able to model early and late post-exercise period.We used rich data to estimate model parameters including plasma glucose, plasma insulin, and oxygen uptake . HoweverIn this work, only the effects of moderate exercise were modeled. Interval exercise is associated with higher glucose levels than moderate exercise , 62. ThiThe current model does not take into consideration the effects of other factors, such as age and fitness levels as seen in other models . LiteratThe new exercise model proposed in this work was developed from real T1D clinical data and was compared with other models that simulate different physiological phenomena experienced by individuals with T1D during moderate exercise.Developing a model that is able to capture the changes in glucose dynamics during exercise is one of the challenges in improving closed-loop systems . Such moS1 File(DOCX)Click here for additional data file.


Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment. These vesicles are passaged through the endo-lysosomal system and their contents digested and absorbed.16 A major conceptual challenge is to understand how cups are shaped and close. This requires F-actin to be organized in space over a scale of microns, first to make a cup, and then to bring its lips together for closure, all in the absence of guidance from a solid particle or coat. However the signaling lipid phosphatidylinositoltrisphosphate (PIP3) plays a key, but poorly understood, role in organizing macropinocytosis across species.19Macropinocytosis is driven by actin polymerization beneath the plasma membrane, which in different cells produces cups or sometimes flaps of membrane as the primary macropinocytic structures.Dictyostelium amoebae are prolific macropinocytes, as well as phagocytes, and can imbibe their own volume of medium in a few hours.21 Macropinosomes form around discrete domains of PIP3 (an ether-linked lipid in Dictyostelium22) in the plasma membrane, which can be microns across24 and are genetically essential for macropinocytosis.19 Domains also include active-Ras, which activates PI3-kinase, and active-Rac, which activates actin polymerization25 and are regulated by RasGAPs such as (NeuroFibromatosis 1 (NF1) and RGBARG .2825 which they shape by attracting a ring of dendritic actin polymerization to their periphery. This ring is visualized by reporters of the Scar/WAVE complex—an activator of the Arp2/3 complex—which form an irregular necklace around PIP3 domains in all cases investigated. Ringed actin polymerization helps explain how cups are shaped but not necessarily how they close.We have proposed that PIP3 domains are templates for macropinocytic cups,Dictyostelium or any other cell type, and although we previously obtained static images of Scar/WAVE rings, we could not track them over the lifetime of cups to discover how they participate in expansion and closure.Limitations in microscopy have hindered investigation of PIP3 domains and macropinocytic cups. Although cups are readily visible, they are difficult to follow over time and in 3D due to their size, dynamism, and the light sensitivity of cells. It is not known how cups close in 29 To match this advance in microscopy, image-analysis methods need extending to 3D time series, to segment cells and PIP3 domains and to measure fluorescent intensities along the plasma membrane.30Light-sheet microscopy gives a new approach to these problems, allowing macropinocytic structures to be followed in 3D from birth to closure.Dictyostelium model with unprecedented detail. We describe how macropinocytic cups are structured and show that they have two distinct ways of closing: at lip or base. We find that PIP3 domains maintain a ring of Scar/ WAVE throughout expansion and closure and that cups close when their PIP3 domain stops expanding. We propose a conceptual model for the evolution of macropinocytic cup shape over time, whereby stalled expansion of domains causes cups to close. This “stalled wave” hypothesis is tested through a simple physical model.Here, using lattice light-sheet microscopy and custom computational analysis, we follow macropinocytosis and PIP3 domains in the Dictyostelium cells for periods of 5–10 min in 3D, at around 0.3 Hz, with two fluorescent channels. Cells were observed at low density in a simple medium, where they maintain macropinocytosis for at least a day.31 Using strain Ax2, with paired reporters for PIP3 (PkgE-PH-GFP), marking the membrane of macropinocytic cups,25 and F-actin (lifeAct-mCherry) revealing the actin cytoskeleton,32 we gained an overview of how macropinosomes form.Using lattice light-sheet microscopy, we followed macropinocytosis in 33 These cups expand and close, with maximum-intensity projections showing macropinosomes sealing and being released from the plasma membrane , which do not produce macropinosomes unless they escape up the side of the cell. We regard these as frustrated macropinosomes.Cups form on any exposed part of cells and frequently move over the surface as they develop. On average, cells have 2.71 ± 1.71 PIP3 domains at any moment and produce 1.8 ± 1.2 macropinosomes per min , rangingDictyostelium cells resemble macrophages in forming macropinosomes from cups,14 but differ in that we do not observe them forming from linear ruffles or flaps, nor is there a major involvement of F-actin “tentpoles.”16Overall, Unexpectedly, we found that cups can close in two different ways—lip and basal closures—of which lip closure is roughly twice as common  and S3. In basal closure, cups deepen and narrow, then constrict locally before pinching off a macropinosome near the base. A large proportion of the PIP3 domain remains in the plasma membrane, where it can yield further macropinosomes. This sequence generally terminates by closure at the lip and extinction of the PIP3 domain.Macropinosomes formed by either route frequently remain attached to the cell surface by a thin tether, which can persist for 10 s or more, before rupturing  and S1G.Cups can also fail. Most commonly, the PIP3 domain simply fades away, followed by its associated F-actin. Or there can be an abortive closure in which the cup constricts but does not close .D. discoideum isolate NC4, and parent to Ax2.36 Ax2 was selected from DdB for growth in standard liquid medium, which is largely due to deletion of the RasGAP NF1, resulting in Ras activation and massively upregulated macropinocytosis.26To test the generality of our observations, we examined macropinocytosis in strain DdB, which is an early derivative of the original Dictyostelium macropinocytosis (DdB cells (with intact NF1) were grown in enriched liquid medium to stimulate macropinocytosis and examined using the PIP3/F-actin reporter combination. They produce generally smaller PIP3 domains and macropinosomes than Ax2 cells. Both lip and basal closures, as well as tethers are observed, showing that these are common features of ocytosis .To understand how macropinocytic cups are structured and how they change during closure, we devised computational tools to map their individual components over space and time and to combine these into composite maps. For microscopy, a reporter of interest was combined with a constant PIP3 reporter and domains defined by first segmenting the cell surface using both reporters and then the domain itself, using only the PIP3 reporter . Intensi37) falls just inside the lip, with PIP3 levels elevated over the entire inner surface of the cup but falling off beyond the lip .The flexible plasma membrane requires mechanical support to shape it into a cup. To understand how this is achieved, we mapped F-actin and some of its binding proteins in cups. F-actin forms a continuous scaffold, extending from top to bottom of cups and some distance beyond the rim , and S4.40 It is strongly recruited to cups, concentrating toward the lip. Single-headed myosins link F-actin to membranes,41 with several strongly recruited to macropinocytic cups. These localize differentially, with myosin-1B recruited toward the periphery of cups, where its staining is often fragmented, whereas the PIP3-binding myosin-1E is evenly concentrated toward the base, closely matching the distribution of PIP3, as previously described.43 The abundance of myosin-1 proteins in cups suggests that the plasma membrane is more firmly attached to the actin cytoskeleton in cups than elsewhere. Carmil44 is only modestly enriched in cups.F-actin-binding proteins are recruited in distinct spatial patterns, broadly confirming previous work  and S3. Although microtubules  moved actively within the cytoplasm and make fleeting contacts with macropinocytic structures, we did not detect any prolonged association between them suggestive of a structural role in cups (not shown). We did not notice any appreciable differences between cups that close at lip or rim, and both were included in the analysis. Nor were any acute changes detected in the patterns of any of the reporters found before cups closed, except possibly in F-actin, whose signal intensified about 20 s before closure as shown by space-time plots –2G.25 we again found that Scar/WAVE forms striking rings around PIP3 domains, which could now be followed through the entire lifetime of cups  subunit instead of HSPC300 as used previously, of cups . This sh of cups  and 3G.  of cups , as alsoThese results show that macropinocytic cups are organized around PIP3 domains, which continuously activate a ring of actin polymerization at their periphery. This creates a cup-shaped F-actin scaffold, including specialist F-actin-binding proteins. In the concave parts of the cup, the membrane must be linked to the scaffold strongly enough to prevent it from detaching (delaminating). Myosin-1, and other cross-linking proteins, may perform this role. The general structure of the cup does not appear to change as it switches from expansion to closure, implying a biophysical rather than molecular mechanism for closure.15We examined cups closing at the lip in en face views, using the Scar/WAVE reporter. This clearly shows that these cups close by concerted inward movement of the lips, until the orifice is sealed . We rare46 However, we could not detect myosin-II at the lip, or elsewhere in cups , we made detailed measurements of domain area to test the correlation.First, we analyzed a set of simple domains that did not split and could be followed from their origin to their closure at the lip . All ori2 s−1 (n = 11) and 0.30 ± 0.34 μm2 s−1 (n = 27), respectively  Dendritic actin polymerization occurs in a ring around the perimeter of PIP3 domains. It is protrusive and pushes against the membrane. (2) Macropinocytic cups are supported by a continuous F-actin scaffold. (3)The plasma membrane adheres to this scaffold via cross-linking molecules, such as myosin-1, and therefore resists delamination. (4) Cups close when expansion of the PIP3 domain stalls.T). Dendritic actin polymerization is assumed to occur only at the domain boundary with its force given by the parameter (kP). Polymerization creates a reaction force that drives a retrograde flow of F-actin inward, potentially applying a stretching force to the membrane of the cup. We explored its importance by varying how strongly this force is coupled to the membrane of the cup. Actin polymerization within the cup due to formins49 and the signaling dynamics sustaining PIP3 domains are not considered.The main components of the model are summarized in 34 The zone of actin polymerization moves outward and does not remain under the same area of membrane for long, so that the cup remains shallow. As the domain stalls, actin continues to polymerize under the same ring of membrane but does not capture more membrane.In the first phase of its life , the PIPP; T) constricts the cup, elongating the PIP3 domain and pushing its boundary toward the lip, keeping pace with the protruding lip and maintaining the position of the polymerization zone. Tubes form when parameter values lie just outside the range giving lip closure, whereas values just within this range provides the largest vesicles for a given membrane tension, although these cups take longer to close  throughout the life of macropinocytic cups, we find that it is recruited around PIP3 domains at all stages and whatever the size of the domain. The Arp2/3 complex, which is activated by Scar/WAVE is similarly recruited in rings  as is vasodilator stimulated phosphoprotein (VASP), which interacts with Scar/WAVE and may accelerate actin polymerization.53This study strongly supports the hypothesis that macropinocytic cups are shaped by a ring of dendritic actin polymerization formed around PIP3 domains.55 Our work provides an alternative mechanism. We find that macropinocytic cups can close at the base, as well as at the lip. Vesicles forming from the base of ruffles have previously been reported in macrophages,16 although whether they also undergo delamination remains to be seen.When a macropinocytic cup closes, the opposing membranes must be brought together, although they are initially microns apart. A purse-string mechanism, driven by myosin-II contraction at the lip of the cup, has been proposed for phagocytocytic cups.In our unified model, continued actin polymerization at the lip drives both types of closure: lip closure results when the lip turns inward, and basal closure results when it continues to elongate the cup, stretching the membrane until it delaminates from the scaffold and pinches off a macropinosome.Although we observe no clear evidence for a role of actomyosin contraction or microtubule motors, we cannot completely dismiss the possibility that they contribute to the inward stretching of the cup into the cell body. Importantly in our model, the force is simply imposed along the membrane and can apply equally to any mechanism. The key point is that any force that pulls the cup base away from the lip helps promote closure under a broader range of biophysical parameter space.50 although enacted differently, also incorporates the propositions of actin polymerization around PIP3 domains and stalled expansion. It, likewise, reproduces cup formation and closure at the lip and base, indicating that this is a robust consequence of the initial assumptions.Our modeling treats macropinocytic cups in 2D and only considers mechanical forces. Yet, even within these limitations, it readily reproduces an expanding cup that closes at the lip, and one that does not close at the lip, but elongates, as in base closure. The theoretical model of Saito and Sawai,35 They are likely based on a reaction-diffusion system, with auto-catalysis and cross-inhibition of diffusing molecules, including Ras, PIP3, and Rac. PIP3 recruits and activates proteins genetically required for fluid uptake by macropinocytosis, including the protein kinases Akt and serum glucocorticoid regulated kinase 1 (SGK), Leep1 (a regulator of Scar/WAVE), C2GAP2 (a RasGAP), and myosin-1,57 but these are not essential to form PIP3 domains. Active-Ras, which activates PI3-kinase, may be at the core of the domain-forming system, because Ras domains form in the absence of PIP3 domains,25 whereas, conversely, PIP3 domains are slaves to Ras activity because they are greatly expanded when Ras activity is increased by deletion of the RasGAPs NF1 or RGBARG.27PIP3 domains are complex structures that form spontaneously in the plasma membrane, including at the base of cells.58 This is consistent with PI3-kinases producing PIP3 within domains from PI4,5P2, and the PIP3 diffusing out for reversion by PTEN, whereas PI4,5P2 is replenished in the domain by diffusion from the surrounding membrane. This spatial separation of PIP3 production and destruction may be one aspect of the reaction-diffusion system that helps limit domain size. Finally, the ability of PIP3 domains to attract actin polymerization to their periphery may be due to the creation of an annulus, where Rac activation outweighs its inhibition.27We find that domains produce opposing gradients of PIP3 and PI4,5P2, as also do basal waves.59 Other cupped structures organized by PIP3 domains could include large phagosomes in Dictyostelium60 and circular dorsal ruffles in mammalian cells.61Extending our work, we propose that cupped structures of the plasma membrane are more generally created by ringed actin polymerization around PIP3 domains. Mammalian macropinocytic cups also feature a central domain of PIP3, which may similarly organize the cup and guide its closure.Dictyostelium and more widely.In summary, we have described macropinocytosis in greater detail than hitherto and gained mechanistic insights, leading to a unified model for how cups close at lip or base. The stalled wave model provides a framework for understanding macropinocytosis both in KEY RESOURCES TABLE⚬Lead contact⚬Materials availability⚬Data and code availabilityRESOURCE AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS⚬Dictyostelium transformation⚬Lattice light-sheet microscopy⚬Deconvolution and registration⚬Cell segmentation⚬Fluorescence projection⚬Annotation⚬PIP3 domain segmentation⚬PIP3 domain grading⚬Fluorescence measurements⚬PIP3 domain geometry⚬3D maximum intensity projections⚬Surfaces⚬Modelling⚬Base closure⚬Quantification and statistical analysisMETHOD DETAILS Detailed methods are provided in the online version of this paper and include the following: jason.king@sheffield.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jason King  under the persistent URL: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.till.bretschneider@warwick.ac.uk).Requests for information on image processing and computation should be directed to Till Bretschneider (Dictyostelium discoideum strains used in this study derive from the axenic laboratory strain Ax2(Kay),26 with the exception of the non-axenic isolate Ddb. Reporter strains are listed in key resources table and derive from Ax2(Kay)26 unless otherwise indicated. They were grown at 22°C either in HL5 axenic medium  in shaken suspension at 180 rpm, or on tissue-culture plates; or in association with Klebsiella aerogenes on SM agar plates. Non-axenic or poorly growing axenic strains, such as DdB, were grown in HL5 reinforced with 10% dialysed FCS (Gibco).All 5 cells/cm2 in 6-well tissue culture dishes in SUM low fluorescent medium .31Cells for microscopy were grown for several days in HL5 medium to maximally up-regulate macropinocytosis. They were prepared for microscopy by incubation for 2-24 hr at approximately 1065 generally to express two reporters, one of which was for PIP3 (PH-PkgE25). The PI45P2 reporter used was Nodulin,62 PI34P2 was PH-TAPP124 and PI4P was P4M.66 Microtubules were imaged using GFP-a-tubulin67 and F-actin by LifeAct.32 Cells were transformed by electroporation and selected on pre-grown bacteria with the appropriate drug.64 Plasmids are available from Addgene, or from the authors by request.Cells were transformed with plasmids from the Paschke/Veltman series,LLSM was carried out using a 3i Lattice Light Sheet instrument with a 0.71 NA long-working-distance water-immersion excitation objective and a 1.1 NA water immersion emission objective with a total magnification of 62.5x. Images were captured on two Hamamatsu ORCA-Flash 4.0 v3 sCMOS cameras (single channel each). The sheet pattern was a Bessel lattice of 50 beams, with an inner and outer numerical aperture of 0.493 and 0.550 respectively. The thermoelectric cooler system was set to 17°C to achieve a temperature of 22°C in the sample chamber.Before imaging, 10 ml of TetraSpeck beads (Thermofisher) were dried to a 5 mm coverslip  and mounted on the sample holder using vacuum grease . 10 ml of medium (SUM) was added to the bath and the sample left to equilibrate in the microscope for 30 min before being used for alignment and PSF measurement. Cameras were aligned by exciting the beads at both 488 nm and 560 nm and a PSF measurement taken for localization.Dictyostelium cells in 6-well plates were detached by pipetting up and down and allowed to settle for at least 15 min onto 5 mm coverslips added to the wells. A coverslip was mounted on the sample holder and transferred to the microscope containing 10 ml SUM in the bath. Samples were allowed to equilibrate before recording. Images were recorded in two channels using 1-2% and 5-10% laser powers for 488 nm and 560 nm respectively, depending on the reporter. 3D volumes were recorded at 0.3 μm step size (0.163 μm deskewed) for 120-150 planes with a 5-10 ms exposure. This resulted in an overall speed of 2.24-3.12 seconds per volume, depending on the configuration, and an -resolution of 0.104 μm/pixel. Volumes were recorded at this rate for 3–10 min. All Videos and images are maximum intensity projections.68 (50 iterations).Because the same microscope was used for all experiments, deconvolution was made more efficient by condensing one PSF for each channel and applying it to all videos for deconvolution. Comparisons between these deconvolved images and a sample of videos deconvolved using PSFs generated immediately prior to the videos showed no substantial change in intensity but did yield a small translation of the image. This translation was accounted for by measuring cross-correlations of the channels within a range of 5 voxels (twice the largest observed translation) and selecting the translation that yielded the maximum average cross-correlation for the whole video. Deconvolution was performed using the Richardson-Lucy method30 The segmentation method itself requires only two parameters. However, some pre- and postprocessing is often required to enable accurate segmentation, which increases the number of parameters.All videos were segmented using the curvature-enhanced random walker.To identify parameter configurations for the segmentation, videos assigned one of three groups: (1) those with PIP3 in the red channel, (2) those with PIP3 in the green channel without LifeAct, and (3) those with PIP3 and LifeAct. A parameter configuration that yielded accurate segmentation for most videos in each group was identified. Segmentations were manually verified, and any errors to segmentation of PIP3 patches were excluded from subsequent analysis. Parameter configurations for videos with a low segmentation accuracy of PIP3 patches were adjusted to improve the accuracy.Videos in group (1), which contains the majority of the videos, were segmented using similar parameter configurations, with 70% using a single configuration. Similarly, group (2) mostly used the same parameter configuration (80%), with small variation from this configuration otherwise. Group (3) had the highest variation in parameter configurations, due to the fact that neither PIP3 nor actin have a strong presence in the cytoplasm leading to segmentation errors. However, there was a large overlap in parameter configurations, with at most four distinct values for any given parameter.The segmentation error rate for each PIP3 domain was calculated by manually grading the segmented surfaces (further details below). An average of 6.97% +/- 0.83% (standard error) of frames were identified as having a segmentation error and were excluded from subsequent analysis.v on the surface, the image voxel coordinates along a line l normal to the surface through v were calculated. Line scans were truncated to avoid including voxels containing membrane at another part of the cell surface; if a point p in l is closer to another point on the surface than to v,then l is truncated at p and any points beyond p are discarded from l. A minimum distance of 2 voxels (approx. 0.2 μm) was imposed on the truncation to prevent zero-length line scan in highly curved parts of the surface. Lines had a maximum length of 1 μm (1.5 μm for actin fluorescence). Each vertex was assigned the maximum fluorescence value along its line scan. Background subtraction of the fluorescence was applied to the image prior to projection using a top hat filter of radius 10 voxels for all images.Triangulated surface meshes were obtained from the segmented videos using Matlab’s isosurface algorithm. These meshes take the form of a set of vertices and a set of triangular faces that connect the vertices. In order to measure the fluorescence on the membrane, the fluorescence values in the image were projected onto the surface. This was performed using line scans along the direction normal to the surface. For a given vertex For the purpose of computing line scans, all surfaces were smoothed using Laplacian smoothing. Projecting actin fluorescence onto the surface additionally required shrinking the surface areas of high mean curvature. This is because the actin cortex lies slightly below the membrane, and therefore the peak fluorescence is farther from the surface than other markers. Given that highly curved areas introduce extreme truncation of line scans, this would lead to the actin at the lip of the macropinocytic cups being reduced. We solved this problem by shrinking the surface under mean curvature flow, which forces the surface to shrink in highly curved regions. In order to avoid simultaneously shrinking the cups during this process, mean curvature flow was only applied in regions of positive mean curvature. Positive mean curvature flow was applied until any point on the surface had moved up to a maximum of 5 μm from its original position.63 Markers were placed on the surface using the marker placement tool in MiCellAnnGELo at locations within PIP3 domains in each video. For de novo domain formation events, markers were placed in frames prior to this event at the approximate location of the forming domain. Similarly, after domain elimination events markers were placed in subsequent frames at the approximate location of the extinguished domain. When a single domain contained more than one cup, separate markers were placed in each cup. Markers were grouped in sequences by first measuring the distance from markers in each frame to markers in the following frame and pairing each marker to the closest marker in the following frame. This pairing process was repeated in reverse, and two markers were grouped together if they paired in both directions. Occasionally, markers moved large distances between frames, for example during a closure event, leading to them no longer pairing with the corresponding marker in the previous frame. These markers we regrouped later in the pipeline by comparing manual annotations of sequences and joining sequences with no overlapping frames, but with the same manual annotations in each frame.Surfaces were annotated using MiCellAnnGELo, a VR software for rapid annotation of surfaces.37 of the PIP3 fluorescence values on the surface. Threshold values were computed for each marker using the histogram from vertices nearest to the given marker in the geodesic sense. In cases where structures such as cup lips or ruffles present as thin protrusions with PIP3 on only one side of the structure, a low level of PIP3 will be projected onto the other side in the surface representation due to spreading of the fluorescence during image capture. In order to avoid including the opposite side of such structures in the domain, mean curvature is used to separate the two sides, while also providing geometric definitions for cups and lips.Domains were identified around each marker using Otsu thresholdingcup is defined as a connected area on the surface about the marker, where all vertices have PIP3 intensity above the Otsu threshold and mean curvature less than 0 (concave). The lip is defined as the band of vertices, grouped by geodesic distance from the cup boundary, with the highest average mean curvature (highly convex). The domain about the given marker is the set of all vertices within the lip contour with PIP3 value above the Otsu threshold.For a given marker, a cups, domains,or lips for two markers intersect, it is necessary to further segment these structures to maintain correspondence with the markers. This segmentation was performed by dividing the structures along the line of equal geodesic distance between the two markers. Domains that required segmentation in this manner were automatically graded as being split domains.In cases where domain for ease of processing. The frame when this false domain changes to a true domain were automatically marked as domain formation events, and the reverse as domain elimination events.For cases where the PIP3 intensity was below the threshold value at the marker, the marker vertex and all vertices sharing a face with that vertex were marked as the cups, domains, and lips were additionally smoothed by dilating , filling (adding connected components surrounded by the vertices of the given feature in the mesh graph), and eroding (the reverse of the dilation operation).Each of the De novo domain formation, domain elimination, lip closure, base closure, failed closure, and domain splitting. Domains were also assessed for cell segmentation and domain segmentation errors, which were noted and excluded from subsequent analysis. The automated grading above provided an initial set of gradings, which were adjusted where necessary.Each domain was subsequently graded manually for each frame to mark the events of 69 This notebook allows the user to change surface colours between fluorescence and domains, as well as providing controls for opacity and colour scaling. Buttons for grading allow the marking of base, lip, and failed closures, as well as domain splitting, segmentation and domain detection errors, and noting the start and end of the domain.Gradings were made by visual comparison of the surface and the 3D maximum intensity projection of the video. A custom Jupyter notebook was designed to allow rapid grading of domains, using ITKWidgets with PyVista to render the surfaces.For a given marker, frames between domain start and end gradings are taken to be the times when the domain is present. For domains with no start frame (either formed prior to the start of the video or formed from splitting), the domain is assumed to start from the first frame that the corresponding marker is present. Similarly, domains with no end frame are assumed to end when the marker is no longer present. Frames where the domain was not automatically detected for a given marker (see previous section) but manually marked as having a domain (and vice versa) were automatically graded as having a domain segmentation error.In the subsequent analysis, measurements of a domain graded as split or to have an error in a given frame were excluded for that frame. Where time-dependent measurements were taken, domains which were graded as split at any time in the interval being measured were excluded.Fluorescence measurements were made relative to the domain boundary. Additionally, the mean curvature of the surface was computed and processed in this manner, without the background subtraction step. The surface outside and inside this boundary was partitioned by geodesic distance to the boundary, where bands outside the domain were equally spaced at 0.2 μm, and bands inside the domains were equally spaced to 0.1*domain depth (defined below). Using a normalized distance inside the domain allows comparison between domains of different sizes. The average fluorescence values were taken in each band, giving a 1-dimensional profile of the fluorescence values for each time point. For cells with multiple domains, bands around a domain were restricted to points on the surface closest to the given domain’s boundary in the geodesic sense.For each domain, the background fluorescence measurement in each frame was taken to be the average fluorescence 5 μm outside the domain. The median background fluorescence over the lifetime of the domain was taken to be the overall background. Fluorescence values were normalized by dividing by this background value .To give an overall profile of fluorescence and curvature, the average normalized 1-dimensional profile was taken for each domain, and these averages aggregated to give the plots shown in For time-dependent analysis, closure events were taken to be the temporal reference points. The number of domain sequences was reduced to include only those that contained the closure event being investigated (either lip or base closure), and the required time interval preceding the event. Due to variable time steps of the videos and some frames being rejected in the quality control step above, fluorescence values were linearly interpolated at every 2.5 seconds prior to the closure time. The average interpolated 1-dimensional fluorescence profiles for each time point were thus computed and plotted as colour values on a 2D image. The image was distorted to account for distance normalization of the profiles by scaling the part of the image representing points inside the domain by the average domain depth along the distance axis.Geometric measurements for each domain were made using the surface geometry: the perimeter was taken to be the total length of edges along the boundary of the domain; the area was given by the total area of all faces in the domain, and the depth was taken to be the maximum distance of any point in the domain to the domain boundary.de novo formation, lip closure, and elimination were aggregated by normalizing in both time and the geometric measurement, allowing the observation of general trends in the geometry -plane) in the top left, the projection in y -plane) in the bottom left, and the projection in x -plane) in the top right. This yielded a view where the projections with lower resolution (x and y) could be easily compared to the high-resolution projection (z) due to the alignment of the common axes. These projections allowed easier identification of closure events than the projection in z alone because they provide some of the 3D information lacking in the single projection.Surface rendering for purely visualization purposes was performed in Matlab (see above for annotation using VR and grading using ITKWidgets in Jupyter). The surfaces were preprocessed by applying Laplacian smoothing (3 iterations), to provide a smoother surface to view. A customized Matlab live script was used to allow fast rendering of the surfaces. Surface colour and opacity can be linked independently to fluorescence, curvature, or PIP3 patches. These values can be scaled to allow better visualization of the surface for the length of the video.A mathematical model is used to simulate the behaviour of cells during macropinocytosis. Our aim with this model is to show the feasibility of cup formation and closure resulting from a polymerization force acting at the boundary of a PIP3 domain, given that the domain area expansion stalls at some time point.70 The total force, FT, at a given vertex is tension is the force due to membrane tension, modelled as elastic stress induced by stretching the contour, Fbending is the force due to bending of the membrane, Farea is a force preserving the area inside the contour, and Fpoly is the normal force due to actin polymerization. The first two of these forces are calculated in the same manner as previously,70 Farea=kA(A/A0-1) for area inside the contour A with initial value A0 and force coefficient kA, and Fpoly is applied along the normal direction at the domain boundary as described below. Bending and area force coefficients are kept constant in all simulations.The model is based on a previous contour model used to study blebbing.While a contour model is 2D in nature, we may interpret it as approximating a slice through a cell with rotational symmetry, allowing us to calculate values for the perimeter  from 2D P/d for a constant value vP. This gives a slowing rate of expansion in length, while the domain area, calculated using a surface of rotation, stops expanding when approaching lip closure due to the shape changes of the domain.We define a PIP3 domain on the contour as a portion of the contour with length d, expanding at a rate vpoly is applied as a constant multiple, kP, of a Gaussian function centered at the domain boundary on both sides with standard deviation 0.25 μm. A domain expansion speed of 0.1 / d μm s-1 for domain width d is used for all experiments.The contour is initiated as a circle with radius 5 μm, discretized using 1000 line segments, and PIP3 patch of initial width 0.02 μm. Throughout the simulation, the force FThis 2D model can produce range of behaviors of cups . If we a71 when setting the length of the delamination zone, allowing the membrane to continue to move inwards until closure. It is conceivable that smaller initial delamination lengths could also lead to base closure, since tension at the edges of the delaminated region would be increased, leading to further detachment from the actin cytoskeleton. A similar effect can also occur during lip closure, but is not essential for closure in this case.If a portion of the membrane in the cup, approximating a cylinder of length l and width w, delaminates from the actin cytoskeleton, then it will start to move under membrane tension. Here, we model the tension force as proportional to mean curvature, while assuming all other forces are negligible. If the aspect ratio l/w < 0.66 (approx.), then the membrane in the cylinder can attain a minimum surface area without closure. We therefore enforce an aspect ratio greater than 0.66Statistical results are presented in Figs S1-S5Vid S1Vid S2Vid S3Vid S4Vid S5Vid S6Vid S7Vid S8Vid S9


The COVID-19 pandemic has posed a huge challenge to healthcare systems and their personnel worldwide. The study of the impact of SARS-CoV-2 infection among healthcare workers (HCW), through prevalence studies, will let us know viral expansion, individuals at most risk and the most exposed areas in healthcare organizations. The aim of this study is to gauge the impact of SARS-CoV-2 pandemic in our hospital workforce and identify groups and areas at increased risk.This is a cross-sectional and incidence study carried out on healthcare workers based on molecular and serological diagnosis of SARS-CoV-2 infection. Of the 3013 HCW invited to participate, 2439 (80.9%) were recruited, including 674 (22.4%) who had previously consulted at the Occupational Health Service (OHS) for confirmed exposure and/or presenting symptoms suggestive of COVID-19. A total of 411 (16.9%) and 264 (10.8%) healthcare workers were SARS-CoV-2 IgG and rRT-PCR positive, respectively. The cumulative prevalence considering all studies (IgG positive HCW and/or rRT-PCR positive detection) was 485 (19.9%). SARS-CoV-2 IgG-positive patients in whom the virus was not detected were 221 (9.1%); up to 151 of them (68.3%) did not report any compatible symptoms nor consult at the OHS for this reason. Men became more infected than women , including when data were also classified by age. COVID-19 cumulative prevalence among the HCW assigned to medical departments was higher (25.2%) than others, as well as among medical staff (25.4%) compared with other professional categories (p<0.01).The global impact of the COVID-19 pandemic on HCW of our centre has been 19.9%. Doctors and medical services personnel have had the highest prevalence of SARS-CoV-2 infection, but many of them have not presented compatible symptoms. This emphasizes the performance of continuous surveillance methods of the most exposed health personnel and not only based on the appearance of symptoms. At the end of 2019 a cluster of patients with a severe respiratory syndrome, whose cause was finally identified as a new coronavirus (SARS-CoV-2) Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Study Design: This appears to be a cross-sectional study only, which looks at 1) the prevalence of COVID + results of all HCW within the institution from April to May, and 2) the prevalence of COVID + employees who have a known exposure or have presented with symptoms from March to May who have reported to OHS. Cross-sectional studies look for association in a defined time period, as described in this study, where as longitudinal studies have repeated measures on the same population over time to look at cause and effect, which this study does not do.Additional Discussion Paragraphs:1) A more in depth discussion on the specificity and sensitivity of both NP PCR and serology testing is needed. PCR has a high false negative rate while serology testing has both a high false positive and false negative rate. Furthermore, positive predictive value is impacted by the prevalence in the populations. All of these points should be discussed as they impact the interpretation of the results.2) Other than in figure 1, there is no mention of the drop in the number of cases after universal mask use for HCW was implemented. This is an important observation and emphasizes the importance of the use of PPE and source control.3) In limitations, state the limitations of cross sectional studiesThis study has shown an association between being a HCW and becoming COVID +, but has not demonstrated a cause and effect. This should be better described in the discussion and conclusion. Furthermore, the are rates and trajectories between the HCW and general population are very similar as shown in Fig 1, with HCW rates falling below that of the general population after mask usage was implemented, so it is difficult to describe a cause and effect of being a HCW working in a hospital when the community spread and rates follow similar curves.Grammatical corrections:The study of the impact of33 areas. *Exposed areas where? In the hospital?37 *deleter the work 'finally'39 the "Occupational Health Services (OHS)" for confirmed exposure and/or presenting symptoms suggestive of43 detection) "was" 485 (19.9%). SARS-CoV-2 IgG-positive patients in whom the52 Conclusions. "The" global impact of the COVID-19 pandemic on HCW of our centre67 means "such" as fomites and contaminated surfaces[3]. Asymptomatic carriers may play76 cases were reported to the national epidemiological surveillance network[9]";" they83 Health personnel are critical and have become a weak point of the health *explain what you mean by 'become a weak point"159 *delete the word 'finally'300 There are 163 of 411 in total (38.7%), of which there are even 12 (3%) in which the virus was detected by rRT-PCR. *Explain this sentence better. It does not do a good job interpreting the result.Reviewer #2: The paper is nicely written and gives an good overview of the spread of SARS-CoV-2 in a Spanish hospital.In the result section, the use of two data sources is a bit confusing. This should be clarified.The differences in prevalence in departments is really interesting. A further differentiation into medical departments (ICU vs. ER) would be interesting.In my opinion, the major flaw in this research is the inclusion of antibody-positivity into the overall SARS-CoV-2 rate in the study population. In the discussion the possibility of false-positive test results in IgG and IgM should be mentioned. According to this possibility using only one antibody-assay IgG-positive participants should not be counted as SARS-CoV-2-cases as done here.Small typos need to be corrected .**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  7 Dec 2020Response to reviewers1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.PLOS ONE’s style requirements have been revised.2. Please provide additional details regarding participant consent.We have added additional details regarding participant consent.Reviewer #1. Study Design: This appears to be a cross-sectional study only, which looks at 1) the prevalence of COVID + results of all HCW within the institution from April to May, and 2) the prevalence of COVID + employees who have a known exposure or have presented with symptoms from March to May who have reported to OHS. Cross-sectional studies look for association in a defined time period, as described in this study, where as longitudinal studies have repeated measures on the same population over time to look at cause and effect, which this study does not do.We agree with the reviewer that our study is not designed to establish a causal relationship, so no repeated measurements are performed over a period, nor two study cohorts exist, and therefore it cannot be defined as a longitudinal study itself. However, we believe that it is not just a cross-sectional study only because in order to know the real impact of the pandemic in our population, a continuous follow-up of the study population was carried out (through rt-PCR test) to know the incidence in HCWs who presented symptoms compatible with the COVID-19 infection or had close contact with a confirmed case. We think that perhaps it is more appropriate to define as an incidence study what we had previously defined as a longitudinal study. The incidence study is an observational longitudinal study whose main objective is to estimate the incidence of a disease in a given population. In this way, we have made the definition changes on the manuscript.1) A more in depth discussion on the specificity and sensitivity of both NP PCR and serology testing is needed. PCR has a high false negative rate while serology testing has both a high false positive and false negative rate. Furthermore, positive predictive value is impacted by the prevalence in the populations. All of these points should be discussed as they impact the interpretation of the results.We have included in the text the following paragraph in response to the reviewer's requirements:Although we have not evaluated the sensitivity and specificity of the diagnostic tools used in our study, there is evidence on the performance of the different platforms and targets. In the case of rt-PCR, sensitivity ranges of 43.6-100% and specificity of 98-100% depending on protocols and targets(35). These data are also influenced by the sample type and quality and the time since exposition(36). To resolve the risk of high false negative rate, we have established measures such as the repetition of molecular tests in the case of symptomatic HCW or confirmed case contacts that had been negative. We have also standardize sampling and reduced the sample extraction team reinforcing its training. On the other hand, serum IgG antibody detection have been validated by other researchers with excellent results, achieving 100% sensitivity at day 12 post infection. Test performance could also be affected by the prevalence of infection in the study population. However, as Lorentzen et al(39) have verified, with a prevalence> 10%, as seen in our center, the positive and negative predictive values remain high even with sensitivity values <80%.2) Other than in figure 1, there is no mention of the drop in the number of cases after universal mask use for HCW was implemented. This is an important observation and emphasizes the importance of the use of PPE and source control.The following text has been included in the discussion section:In the temporal evolution of confirmed cases registry among health personnel , the decrease in cases that began 7-14 days after the imposition of measures to contain the spread of the virus is striking. Actions included maintaining social distance, hand washing and hygiene, and especially mandatory use of a facial mask. The main health authorities, such as WHO (Advice), recommended these arrangements. Although the control of the viral expansion cannot be attributed in an absolute way to these measures, since the Madrid lockdown coincided in time, it is undoubted that this basic measure is one of the actions that have allowed the control of infection in a highly exposed population as HCW.3) In limitations, state the limitations of cross sectional studies.The following limitations of study design has been included in the text:Finally, our study design has introduced some limitations. Cross-sectional studies do not allow causal inferences and only association and no causation can be inferred. There may also be an overestimation of results as well as temporal bias, such as the appearance of new cases in a continuously evolving epidemic. With this design we are unable to investigate temporary relation between exposures and outcomes. Also incidence studies do not allow causal inferences since they lack a control group. In addition, there is the possibility of loss of individuals during follow-up.This study has shown an association between being a HCW and becoming COVID +, but has not demonstrated a cause and effect. This should be better described in the discussion and conclusion. Furthermore, the are rates and trajectories between the HCW and general population are very similar as shown in Fig 1, with HCW rates falling below that of the general population after mask usage was implemented, so it is difficult to describe a cause and effect of being a HCW working in a hospital when the community spread and rates follow similar curves.Indeed, we agree with the reviewer that the association shown between being hcw and becoming covid + has not been proven as cause and effect. This objective was beyond the scope of the study. In the introductory section, it is already stated that the objective of the study was to describe the impact of the pandemic among health workers. The appearance of positive cases between the general population and HCWs has in fact been almost parallel in terms of evolution but very different in terms of prevalence. HCWs are part of the general population and participate in social and family life in a similar way to the rest, so their evolution is highly influenced. But they have also been exposed to positive cases due to their work, which could explain their higher prevalence than in the general population. As the positive patients increased, so did the exposure of the workers and vice versa. Following the reviewer's indication, this will be better described in the manuscript.Grammatical corrections:The study of the impact of33 areas. *Exposed areas where? In the hospital?the most exposed areas in healthcare organizations37 *deleter the work 'finally'done39 the "Occupational Health Services (OHS)" for confirmed exposure and/or presenting symptoms suggestive ofdone43 detection) "was" 485 (19.9%). SARS-CoV-2 IgG-positive patients in whom thedone52 Conclusions. "The" global impact of the COVID-19 pandemic on HCW of our centredone67 means "such" as fomites and contaminated surfaces[3]. Asymptomatic carriers may playdone76 cases were reported to the national epidemiological surveillance network[9]";" theydone83 Health personnel are critical and have become a weak point of the health *explain what you mean by 'become a weak point"This means that from the point of view of healthcare system, HCW had been the weak link in chain: health personnel have had a great demand for work during this pandemic due to the avalanche of infected patients; as these workers are highly exposed and have more chances of becoming infected and falling ill, they endanger, even more if possible, the sustainability of the health system. We have changed that expression for “Healthcare personnel are a key element in managing this Covid-19 pandemic”.159 *delete the word 'finally'done300 There are 163 of 411 in total (38.7%), of which there are even 12 (3%) in which the virus was detected by rRT-PCR. *Explain this sentence better. It does not do a good job interpreting the result.This sentence has been changed: “One hundred and sixty-three of the 411 (38.7%) workers who presented IgG were asymptomatic. Even in 12 of them (3%) SARS-CoV-2 was detected by rt-PCR.”Reviewer #2: The paper is nicely written and gives an good overview of the spread of SARS-CoV-2 in a Spanish hospital.In the result section, the use of two data sources is a bit confusing. This should be clarified.As indicated by the reviewer, and to clarify the origin of data, several subsections have been created in the results section: cross-sectional study, incidence study and global impact.The differences in prevalence in departments is really interesting. A further differentiation into medical departments (ICU vs. ER) would be interesting.We agree with the reviewer that these differences are very interesting. According to his indications, we have also included the differences between the Emergency-Internal Medicine and ICU groups.In my opinion, the major flaw in this research is the inclusion of antibody-positivity into the overall SARS-CoV-2 rate in the study population. In the discussion the possibility of false-positive test results in IgG and IgM should be mentioned. According to this possibility using only one antibody-assay IgG-positive participants should not be counted as SARS-CoV-2-cases as done here.We agree with the reviewer that the performance of diagnostic tools may be one of the main limitations of the study. Also following the instructions of reviewer #1, the limitations and consequences of the lack of sensitivity and specificity in the diagnostic techniques used to classify the study participants, including serological techniques, have been yet introduced into the manuscript. Validation of serological techniques is beyond the scope of this study. However, the excellent results obtained with the kit used by our team by other researchers supports its use as a screening and classification method. Even taking into account the limitations already included in the manuscript, we believe that the positivity of IgG antibodies against SARS-CoV-2 is an excellent marker of previous infection. This parameter has been widely used in epidemiology and infection control as a unique marker because it is a powerful indicator of present or past contact with a microorganism.Small typos need to be corrected .Manuscript has been revised and corrected.AttachmentResponse to reviewers.docSubmitted filename: Click here for additional data file. 21 Dec 2020Impact of SARS-CoV-2 pandemic among health care workers in a secondary teaching hospital in Spain.PONE-D-20-22318R1Dear Dr. Garcia-Martinez,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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