The significance of IgM deposits in glomerular mesangium has been controversial since they were first described due to the variations in the both the definitions used and described impact on clinical outcome. The aim of our study was to evaluate the significance of the IgM deposits in the glomerular mesangium for outcomes of nephrotic syndrome (NS) in children.n = 18) and IgM-negative (n = 27).Forty-five children with NS who underwent renal biopsy at tertiary pediatric hospital from January 1st, 2000 to December 31st, 2015 and the pathology diagnosis of minimal change disease, focal segmental glomerulosclerosis and mesangial hypercellularity (MH) were retrospectively analyzed. IgM positivity was defined as ≥1+ imunofluorescence with predominantly mesangial distribution. The patients were stratified into IgM-positive (p = 0.186).At the end of the median follow-up 4.5 years (range 0.17–13.14), the IgM-positive group was represented by 11 patients (61.1%) in remission, 3 patients (16.7%) with active disease and normal kidney function, 2 (11.1%) patients with active disease and impaired kidney function, 2 (11.1%) patients on renal replacement therapy. Accordingly, the IgM-negative group included 13 patients (48.1%) in remission, 12 (44.4%) with active disease and normal kidney function, 1 (3.7%) with active disease and impaired kidney function, 1 (3.7%) on renal replacement therapy, with no statistical significance between groups (This study did not reveal significant differences of the disease outcomes between IgM-positive and IgM-negative groups. The incidence of nephrotic syndrome (NS) is 2–7 cases per 100,000 children per year , who repThe aim of our study was to evaluate the significance of IgM deposits in the glomerular mesangium for the clinical course, treatment strategy and outcomes of NS in children and to evaluate the histological disease progression based on follow-up biopsies.A retrospective chart analysis was done on children age 0–18 years) with NS who underwent renal biopsy at Children‘s Hospital, Affiliate of Vilnius University Hospital Santariskiu Clinics from January, 2000 to December, 2015 with the histological diagnosis of MCD, FSGS and MH. Indications for renal biopsy were: patient’s age < 1 year or >10 years at the time of first manifestation of NS or additional clinical features , and a frequently relapsing, steroid-dependent or steroid-resistant form of NS. Patients with systemic disease, causing IgM deposition in the kidney, were excluded from this study. Detail of the patient selection process is presented in Fig. 8 years wPatients were divided into two groups, IgM positive and IgM negative to compare the patient’s age, gender, clinical manifestation, treatment strategy, response to steroids and outcome. The median follow up time was 4.5 years (range 0.17–13.14).All biopsies were obtained by the same investigator and evaluated by light and immunofluorescence (IF) microscopy by the same pathologist, with 23 biopsies also evaluated by electron microscopy.Minimal change NS was defined as edema or nephrotic range proteinuria (≥ 3+ urine dipstick test) and hypoalbuminemia ≤25 g/l, and pathology findings of minimal change disease on light microscopy with or without mesangial proliferation, C1q and/or IgM mesangial deposition on IF microscopy, and with or without foot process effacement on electron microscopy.FSGS was characterized by at least one segmental lesion, with obliteration of capillaries with or without adhesion to the Bowman’s capsule.MH was defined as a uniform increase in mesangial cells  in more than 80% of the glomeruli .Glomerular IF findings were graded on a scale 0–4+. IgM positivity was defined as a grade of ≥1+ IF intensity with predominantly mesangial distribution Fig. .Fig. 2WeArterial hypertension was defined as systolic and/or diastolic blood pressure elevation above the 95th percentile based on the child’s age, gender and height or a patient currently on antihypertensive medication .Hematuria was defined as ≥1+ on dipstick test or >5 red blood cells present per high power field of spun urinary sediment.2 was defined as decreased kidney function. Serum creatinine was measured by Jaffe method during the study period.Glomerular filtration rate (GFR) was calculated using the Schwartz formula . The valRemission – < 1+ of protein on urine dipstick test for 3 consecutive daysRelapse – occurrence of >3+ protein on urine dipstick for 3 consecutive days for a patient who was previously in remissionSteroid-dependent nephrotic syndrome – patients who relapsed during the steroid treatment or within 2 weeks after the discontinuation of steroidsSteroid-resistant nephrotic syndrome – persistent proteinuria for ≥4 weeks while treated with steroids.Frequently relapsing nephrotic syndrome – patients who had 2 or more relapses in 6 months or 4 or more relapses in 12 months during the first year of treatment.Definitions used to evaluate response to therapy based on KDIGO 2012 guidelines :RemissioActive disease – persistent proteinuria and/or decreased kidney function during the last visit. This is divided to active disease with normal kidney function or active disease with decreased kidney function.Persistent proteinuria – any amount of protein found in urine with dipstick test at the time of last follow-up.Kidney replacement therapy – transplantation or dialysis.Cured – a patient who was relapse-free for at least 5 years.Definitions used to evaluate patient outcomes:p value of <0.05 was considered to be statistically significant.Statistical analysis was performed using the SPSS 23.0 software version for Windows . All data are reported as median (range) unless otherwise specified. Categorical variables are described as frequency and percentage. Means of quantitative variables were compared using the t-test or Mann-Whitney test if data is not normally distributed. Categorical variables were compared using the Person’s chi square test, Fisher exact test or likelihood ratio, as appropriate. A p = 0.95). Detailed biopsy results are displayed in Table Forty-five patients were included into the final analysis: 18 patients (40%) were IgM positive and 27 (60%) were IgM negative. Initial kidney biopsies were performed at a median of 0.71 years (0.1–10.03) in the IgM positive group and a median of 1.00 years (0.1–7.6) in the IgM negative group after the disease presentation with no statistically significant difference . In the IgM positive group, patients required more immunosuppressive medications to achieve remission: fifteen patients were treated with Cyclosporine, four with Mycophenolate mofetil, three patients received Rituximab, but the differences were not statistically significant.Three (16.7%) and eight (29.6%) patients were treated with steroids only in the IgM positive and negative group, respectively (p = 1.0). 3 patients (16.7%) in IgM positive group and 2 patients (7.4%) in IgM negative group had a third kidney biopsy performed during their follow-up period. The third biopsy was performed at a median of 5.45 years (2.18–7.06) in IgM positive group and a median of 4.41 years (4.31–4.51) in IgM negative group with no statistically significant difference (p = 1.0). Histological disease progression from MCD to FSGS was accounted for in 2 patients. One patient was IgM positive and initially steroid sensitive, and after three adjuvant immunosuppressive medications, he is currently in remission. The other patient was IgM negative, initially steroid-dependent, and after treatment with one adjuvant immunosuppressive medication, and she currently has active disease but with normal kidney function. All four patients who progressed to end stage renal disease revealed FSGS on their first biopsy.Eighteen follow-up biopsies were performed for thirteen patients. Second biopsy was performed for 8 patients (44.4%) in IgM positive group and for 5 patients (18.5%) in IgM negative group. Follow-up biopsies were performed at a median of 2.08 years (1.52–5.58) in IgM positive group and a median of 2.32 years (2.02–2.32) in IgM negative group with no statistically significant difference  in the IgM positive group (median 4.52 years) compared to the IgM negative group (median 3.55 years). Although more patients in IgM positive group had impaired kidney function at last follow-up (22.2% vs 7.4%), the difference did not reach statistical significance.No statistically significant differences were found in renal outcome between the groups Table . There wIgM deposits have been implied to have diverse significance of the clinical course and outcomes for NS. Some researchers have questioned the significance of IgM deposits alone. It has been proposed, that these deposits could occur due to passive entrapment in the glomeruli due to glomerular sclerosis . This coIn our study, IgM deposits were found in 40% patients. This result is is higher than in previous studies, but this could be explained by different inclusion criteria. The reported range of IgM deposits in idiopathic NS varies from 24.3%  to 62.5%p = 0.033). However, this could be due to a larger patient group (IgM+ 30 patients) and the authors do not demonstrate the rate of dense deposits present on electron microscopy.Numerous studies have suggested that IgM deposits in the glomeruli found in patients with NS are associated with poor steroid response. Our data showed higher frequency of steroid resistance in the IgM-positive group compared to IgM-negative (33.3% vs. 18.5%), but the difference was not significant. These results correspond to several other studies , 22. Kanp < 0.05), while none of the 7 IgM-positive patients progressed to ESRD as reported by Spreitzer et al. [IgM deposits have been associated with the development of impaired kidney function. Although our data did not produce statistically significant findings, there is a tendency towards impaired kidney function at the last visit in IgM-positive patients. Furthermore, three patients out of 45 (6.6%) progressed to chronic kidney disease (CKD), however the progression of renal disease did not correlate with the presence of the electron dense deposits: none of the 3 patients with electron dense deposits developed CKD. Similar results were found in the Swartz et al.  study: or et al. . Neverthp = 0.049), therefore a tendency towards poor steroid response and disease progression could be caused by FSGS alone. It has been shown by a few studies that IgM deposits could be also found in patients in FSGS, with a frequency ranging from 19.4% [We have also taken into account that biopsy results in our IgM-positive patient group were heterogeneous, consisting of MCD, MH and FSGS. In the IgM positive group FSGS was a more common finding than MCD  since some patients received angiotenzin-converting-enzyme (ACE) inhibitors for the renal protective effect. Therefore, based on the definition of arterial hypertension, all of these patients were included into arterial hypertension group.This study has demonstrated that clinical course and disease outcomes did not differ significantly between IgM-positive and IgM-negative groups.


Do adversarial environmental conditions create social cohesion? We provide new answers to this question by exploiting spatial and temporal variation in exposure to earthquakes across Chile. Using a variety of methods and controlling for a number of socio-economic variables, we find that exposure to earthquakes has a positive effect on several indicators of social cohesion. Social cohesion increases after a big earthquake and slowly erodes in periods where environmental conditions are less adverse. Our results contribute to the current debate on whether and how environmental conditions shape formal and informal institutions. There is widespread agreement that social cohesion can be crucial to a society’s cultural and economic development. Social cohesion lowers the transaction costs of working together and hence facilitates cooperation. People have the confidence to invest in collective activities, knowing that others will also do so –3. They Proponents of “environmentalism” believe that environmental conditions are key determinants in shaping human formal and informal institutions (e.g. ) and thucomunas  in Chile. The regions and comunas we are comparing have very similar historical, cultural and traditional roots, share the same formal institutions and, unlike e.g. in [In this paper, we report evidence on a case where harsher environmental conditions have led to more social cohesion, as measured by a variety of indicators. Our study exploits regional and temporal variation in exposure to earthquakes across 15 regions and 346  e.g. in , there aChile is a long narrow country extending approximately 4300 km from north to south. It is bordered by the South Pacific Ocean to the west and by the Andes Mountains to the east. The country lies on the boundary between the Nazca plate and the South American continent, one of the most seismically active areas in the World .Measuring Earthquake Impact. The severity of an earthquake is traditionally measured by either its magnitude or its intensity. Magnitudes and intensities measure different characteristics of an earthquake. Earthquake magnitude quantifies the maximum energy released at the quake epicenter. Data provided by the National Seismological Center in Chile and the U.S. Geological Survey mostly report two magnitude scales: surface-wave magnitude  and moment magnitude (Mw). We focus on these two throughout this study and report one or both, depending on their availability. Local magnitude, also known as Richter scale, is another measure of earthquake magnitude. All magnitude measures should yield approximately the same value for any given earthquake [Mw tends to be higher than Ms for Chilean earthquakes (see below).rthquake , but Mw The problem of magnitude scales is that they do not necessarily reflect the impact of an earthquake on the surface nor how far-reaching a quake is. Earthquake intensity, by contrast, measures the impact of an earthquake on earth’s surface, on humans, and their structures. Intensities thus allow us to quantify the effect on an event in areas located far from the epicenter and the effect of the same event can differ across regions. The most commonly used intensity measure is the Modified Mercalli scale, which we also employ.Ms at the epicenter is denoted by Ms7.5 and all earthquakes of magnitude 7 or higher as Ms7.0+. The notation is analogous for Mw. In case of the Modified Mercalli scale, we write, say, intensity VI in the Mercalli scale throughout.In what follows, we follow the notation in the literature on earthquakes. Magnitudes are written in Arabic letters, while intensities are denoted by Roman numerals. For example, an earthquake of magnitude 7.5 according to Ms/Mw ≥ 7.0 or of intensity higher than or equal to VII in the Modified Mercalli Scale. Earthquakes with Ms8.5+ are referred to as megaearthquakes, but not treated differently in the analysis. A threshold of Ms7.0 is used because the National Seismological Center provides a series of historic important and/or destructive earthquakes of magnitude 7.0 or greater. Additionally, magnitudes higher than or equal to 7.0 are typically associated with intensities VII or greater. An intensity greater or equal to VII entails damages that go from “Damage negligible in buildings of good design and slight to moderate in well built ordinary structures” to “Damage Total” [Ms7.0+ threshold, earthquakes are considered severe enough to entail important human and economic losses over large areas. Combining magnitudes and intensities to evaluate the impact of an earthquake is crucial because even areas at large distance from the epicenter can be severely affected. For example, even though the epicenter of the 2010 Maule megaearthquake was located 335 km south of Santiago de Chile, its aftershocks covered an area that extends from the coast to the trench for a length of over 600 km. As a result, Regions VIII and IX located to the south of Maule as well as regions VI and XIII (the Metropolitan Region) located to the north of the epicenter in Maule suffered intensities higher than or equal to VII in the Modified Mercalli Scale.Our analysis focuses on major earthquakes which we define to be of either e Total” . For theHistory of Earthquakes in Chile. Over the past six decades, several Ms7.0+ earthquakes have been registered in the area  [the area , includirthquake . In May  (Mw9.5) . This me (Mw9.5) .Ms7.8 (Mw8.0) earthquake and its associated tsunami struck the south coast of Valparaíso (Region V). The epicenter was located about 125 km west of Santiago de Chile but the aftershock zone extended about 200 km in length north-south and was at least 100 km wide from east to west along the dip of the subducted Nazca plate [Ms7.3 (Mw8.0) earthquake shook Antofagasta in the North of Chile (Region II), approximately 1400 km north of Santiago de Chile. In 2005, a Ms7.8 earthquake struck Tarapacá (Region I). The epicenter was located 49 km north of Pica and about 1,800 km north of Santiago de Chile. In November 2007, the northern region of Antofagasta suffered another earthquake of Ms7.5 (Mw7.7), the epicenter being located 43 km west of the comuna of María Elena about 1500 km north of Santiago de Chile. The 2007 shock caused two deaths, 4451 homes were damaged and 3012 destroyed.In between the two megaearthquakes in 1960 and 2010, Chile has experienced several other important earth tremors. We briefly describe the most relevant in terms of impact. In March 1985, a comuna-level analysis. It represents the sixth largest earthquake ever recorded worldwide, with a magnitude of Mw8.8 [In February 2010, Chile experienced its second megaearthquake in the last six decades, the 2010 Maule earthquake, which plays a crucial role in our of Mw8.8 . This evof Mw8.8 . This ilof Mw8.8 ).Mw8.8 Maule earthquake in our statistical analysis, as this more recent event overlaps with available data on social cohesion. comuna level for three specific regions that were heavily affected by the 2010 Maule event .As we can observe in comuna level. Chile is divided in 15 regions. These regions are further divided into provinces, and the provinces into a total of 346 comunas. The reason to analyze both regions and comunas is the following. More information on social cohesion is available at the regional level. However, there are only 15 regions in Chile. Comunas, by contrast, provide more cross-sectional units but fewer variables are available at the comuna level.Our aim is to assess whether and how adverse environmental conditions affect indicators of social cohesion. Since earthquakes are exogenous to social cohesion and since their occurrence and strength are precisely recorded, they are a good measure of adverse environmental conditions. Our analysis is performed from two geographical perspectives, at the regional level and at the comunas. At the regional level we consider two different measures. We construct a dummy variable  the epicenter of a Ms7.0+ earthquake was located there or (ii) the earthquake was felt in region j with an intensity equal to or higher than VII in the Modified Mercalli Scale.Using data from the Chilean National Seismological Center and the US Geological Survey we construct several measures of exposure to earthquakes for both regions and The particular threshold of three years was selected because it entails a time span that matches the availability of our measures of social cohesion. There also exists a technical reason. If we select a threshold much larger than three years , j was last affected by a major earthquake. For regions unaffected by any major earthquake in the last 30 years we set this variable equal to 30. Compared to shorter-term measures, both We also analyze a recency measure  it is identified by the seismological service as a comuna hit by the 2010 Maule earthquake ; (ii) it suffered at least one fatal victim; (iii) it asked for economic aid. The variable Since social cohesion data at the i) Life satisfaction, (ii) Trust, (iii) Social behavior, (iv) Suicide, and (v) Voting. We perform our analysis using eight indicators of social cohesion, one for each class (i), (ii), (iv) and (v), and four different indicators of social or anti-social behavior , which measures the extent of agreement to the question “Are you satisfied with your life?” The second indicator is Trust, which measures the extent of agreement with the statement “Generally speaking most people can be trusted”. Both Life Sat and Trust are taken from the Latinobarómetro, a survey conducted every year in 18 different Latin American countries that gathers information on individuals’ attitudes and beliefs (iii)). The first one is charitable giving (Charity), which is viewed as part of social behavior [Teletón, that aims to raise funds to help children with disabilities. Our second indicator of social behavior is volunteering (Volunteering). This variable measures the percentage of people in an area who self-report having done any voluntary work in the Chilean household survey CASEN. The third indicator is criminality (Crime) which we expect to be inversely related to social behavior. This index encompasses crimes both reported to the police by the citizens and discovered by any police officer per each 100000 inhabitants. The index considers various forms of crimes, such as aggravated assault, murder, rape, robbery, burglary, motor vehicle theft, etc. Our fourth indicator is corruption (Corruption), also considered inversely related to social behavior [iv) we use an index of suicides (Suicides) that measures the number of suicides per 100000 inhabitants according to national statistics. Finally, electoral participation (Voting) is our eighth indicator, which measures the percentage of people who showed up at the polls.There is no consensus on how to measure social cohesion . The OEC beliefs . The twobehavior  and defibehavior . To consCharity, Volunteering, Voting, Life Sat, Trust) and negative  according to their relation with social cohesion. For instance, more volunteering is associated with more cohesive societies while higher crime is considered a sign of lower social cohesion. Finally, we would like to mention that due to the lack of available data only Volunteering, Crime, and Suicides are available at both regional and comuna level. Charity, Voting, and Corruption are not disaggregated at the level of comunas, whereas Life Sat and Trust can solely be constructed at the comuna level due to the sampling strategy of Latinobarómetro, which focuses intensively on specific regions.The units and time spans of all these variables are summarized in Since regional differences in social cohesion could come about by a number of factors other than exposure to earthquakes, it is imperative to account for this possibility. To do so we use two different approaches: (i) Fixed Effects regressions which control for some key indicators  and (ii) Principal Component Analysis  and Xjt is a vector of controls. αj is a region fixed effect, which can be arbitrarily correlated with the controls Xjt. This term captures unobserved variables that do not vary over time, such as e.g. cultural or geographical factors. A key implication of this approach is that, since the unobserved variables do not change over time, any change in the outcome variable cannot be attributed to these fixed characteristics. The coefficient of interest is β, which shows how earthquake exposure affects the social cohesion indicator in question on average across affected regions. Coefficients γ are not reported explicitly in Volunteering goes in the expected direction but is not statistically significant at the regional level.Odd regressions in fake earthquake exposure. Our test shows that the probability of obtaining the result in Negative indicators, by contrast, decrease with exposure to earthquakes. Crime rates are 45% of a SD lower in affected compared to unaffected regions (column (7)). This amounts to approximately 271 crimes per 100000 inhabitants. Although the estimated coefficients for corruption and suicides show the expected direction, they are not statistically significant (columns (9) and (11)). Overall, the results suggest that adversarial conditions create social cohesion. People in affected regions display more positive and less negative indicators. As one way to check that our results are not caused by factors unrelated to earthquakes we perform the following Placebo test. We first randomly distribute the value of DISTEQ, whether social cohesion erodes if there is a prolonged period without earthquakes. The results, reported in even columns in Charity approx. 13 years after the last earthquake (≈1 SD of DISTEQ), whereas the corresponding reduction in Volunteering is DISTEQ (slightly more than 13 years) leads to an increase of 464 crimes per 100000 inhabitants on average and to an increase of three suicides per 100000 inhabitants. The effect on corruption is not statistically significant, but goes in the expected direction. All regional-level results are robust to performing the principle component analysis that allows for controlling for a larger set of regional characteristics  approach, in which we compare the affected and unaffected comunas before and after the 2010 Maule earthquake. The estimated model isPOSTt is a dummy variable that equals 1 if t ≥ 2011 (the year after the earthquake) and 0 for years before the earthquake, comunac was hit by the Maule earthquake in 2010, and Life Sat and Trust were collected six months after the February 2010 Maule earthquake, estimations with these indicators set POSTt = 1 if t ≥ 2010 and 0 otherwise. For Volunteering, Crime and Suicides there are no available data for 26 comunas (out of 346) and we only have reliable data on Life Sat and Trust for 98 comunas.β1’s reflect the average increase or decrease in the indicator between the period before and after the 2010 Maule event for the unaffected comunas and the coefficients β2 measure the difference between affected and unaffected comunas before the 2010 Maule earthquake. Our main interest lies in the parameter β3 that estimates the average difference in the evolution of the indicator between affected and unaffected comunas from before the event to post-earthquake.POST = 0) affected communities display 40% of a SD lower rates of volunteering compared to unaffected comunas. This changes after the 2010 Maule earthquake, where affected comunas display 27% higher rates of volunteering compared to others. Life satisfaction rates are no different between affected and unaffected comunas pre-earthquake, but after the Maule earthquake affected comunas exhibit 74% higher rates of life satisfaction compared to unaffected comunas. Likewise, crime rates are no different before the earthquake, but 16% lower in affected comunas post-earthquake. The diff-in-diff analysis does not yield statistically significant results for Trust and Suicides. As for the former, even though this variable is standard across a number of household surveys worldwide, it has been criticized as a measure of trust. In particular, the authors in [Prior to the 2010 Maule earthquake  shows null effects as well. Both tests suggest strongly that the 2010 Maule earthquake is the reason behind the detected differing evolution of social-cohesion indicators between the affected and unaffected comunas.We performed several robustness tests for these findings see . First, Volunteering and two particular aspects of Crime. The estimates are consistent with our previous comuna-level results and demonstrate that they are robust to migratory movements, which suggests that our results are not driven by systematic movements of the population from and to (un)affected regions.Finally, comunas. People in more affected regions give more to charity, are more likely to engage in volunteering and vote, are less likely to engage in crime, and seem to be more satisfied with their lives. Since these differences erode over time, it further points to an interaction between earthquake exposure and social cohesion.Increased exposure to earthquakes seems to be consistently associated with higher levels of positive and lower levels of negative social cohesion indicators both across 15 Chilean regions and 320 Chilean This shows that, apart from the significant and well studied economic consequences of natural disasters –35, therOur results complement the emerging literature documenting positive effects of natural disasters. Toya and Skidmore  provide Furthermore, our findings add to the resilience research that has explored how communities develop strategies to deal with (frequent) natural disasters. The need for a better understanding of the linkages between ecosystems and human societies has been pointed out by ; see 4646 for a Our results support the “environmentalist hypothesis” that environmental conditions shape social organization , 11, 12.Even though our data have unusually rich variation in earthquake exposure, our study still presents several limitations. The data on social cohesion indicators are still limited at smaller geographical scales. Understanding the mechanism underlying the interaction between environmental conditions and social cohesion would greatly benefit from complementing our analysis with extensive experimental analysis of social cohesion indicators. Future research is needed to uncover the precise social mechanisms behind our findings. In this direction, one recent study has shown that, after being exposed to a hurricane, people seem to create networks associated with higher social cohesion . One couS1 AppendixOnline Supplementary Material contains additional information concerning our data and analysis.(PDF)Click here for additional data file.


Drosophila melanogaster provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, we have defined the pattern of cell death in regenerating discs. Our data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. We also present results which suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, our results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously.Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of  Drosophila melanogaster have the capacity to regenerate during the larval stages and provide a particularly well-characterised model system for analysing this phenomenon . The im .  recover –13. One  recover . When th recover . Apoptos recover  and in w recover –17.in vivo culture conditions increases the number of dead cells throughout the discs, even in control non-amputated discs . Mo. MoDrosoOur results indicate that regeneration promotes cell death not only in the region adjacent to the wound edge, but also in regions that were away from it. This effect suggests the existence of one or more signals that are activated near the damaged epithelia and that spread throughout the rest of the disc. Different reports have shown that the Jun-N terminal Kinase (JNK) signalling pathway plays an important role during regeneration and its function is also involved in the induction of apoptosis , 27–31. eiger loss of function mutant discs. To this end we amputated a fragment of eiger3/eiger1; Hh-dsRed Ci-Gal4 UAS-GFP/+ wing discs and analysed the apoptotic pattern. We found that in these discs cell death was strongly reduced at 20 hrs AC compared to control regenerating discs  , in eiger3/eiger1 mutant regenerating discs cell proliferation is only slightly increased  , which controls the activity of JNK signalling by a negative feedback loop [puc is over-expressed in regenerating discs that were amputated using our method  . However p<0.01) . We also p<0.01) . In addiol discs . Finallyneration . All theack loop , inhibitack loop . We founr method .eiger plays an important role in activating autonomous and non-autonomous apoptosis during disc regeneration. However, the presence of numerous dead cells in regenerating eiger mutant discs, in contrast with the complete suppression of apoptosis in these mutant discs after cell death induction [eiger3/eiger1 regenerating discs. To this end we used a LacZ insertion in the gene puckered (puc). As previously reported, we observed that in regenerating control discs this reporter was activated firstly at the wound edge, but later expanded to most of the cells of the wing blade [puc-LacZ is strongly down-regulated in regenerating eiger3/eiger1 discs compared to control regenerating discs, we observed some cells at the wound edge that express high levels of this reporter    Figs. Theiger mutant discs far away from the wound edges suggest that the induction of apoptosis during disc regeneration does not only depend on the apoptosis-induced apoptosis mechanism triggered by Eiger. Consistent with this hypothesis, we found that the temporary ectopic expression of eiger using the Gal4/Gal80Ts system   [hepCA vs 0,00119 ±0,00042,n = 8 in the anterior compartment of control regenerating discs)  . Thus, cboundary . We have (hepCA)  cause sig discs) . Altogeten-Gal4 nub–Gal4 UAS-p35/+ discs and analysed cell death 20 hrs AC. Previously it has been shown that in in vivo cultivated regenerating discs, the ectopic expression of p35 almost completely suppressed apoptosis [en-Gal4 nub–Gal4 UAS-p35 discs compared to control regenerating discs  higher than in the non-amputated compartment  of the en-Gal4 nub–Gal4 UAS-p35 regenerating discs the expression of Wg disappeared from part of the D/V boundary in the region close to the wound edge. In all these cases the down-regulation of Wg expands more than 15 cell rows from the wound site [en-Gal4 nub–Gal4 UAS-p35/+ regenerating discs the vein/intervein pattern is disrupted during regeneration  Fig 8)en-Gal4 nen-Gal4 nub–Gal4 UAS-p35/+ larvae amputated at different times during development. As observed in control regenerated wings, dependent on the time when the amputation was performed, we found different categories of regenerated adult wings. When the cut was produced late in development , we find that as in controls, 100% of the adult regenerated wings displayed lack of wing tissue  , gave rise to adult wings that contain small or large nicks , and 14% of the wing analysed have completed the regeneration process and were normal compared with the control contra-lateral wing. These results are similar to those observed in control regenerated wings amputated at that stage, thus 76% of these wings contain nicks, whereas 23% have completed the regeneration process (n = 220)  . Most ofral wing ).p35 in regenerating discs has a minor effect in the process of regeneration.Altogether our data indicate that the partial suppression of apoptosis produced by the ectopic expression of Regeneration involves multiple cellular processes to restore the damaged organs or tissues. One of the mechanisms that has been proposed to play a key role as a source of signals required for regeneration in different organisms is apoptosis –13. ApopOne of the limitations of our system is that the earliest time in which we can eliminate a fragment of the discs is between 96-120h AEL. We have previously shown that during this interval of time the amputated discs can fully regenerate and give rise to a completely regenerated adult wing . In addieiger might be involved in promoting cell death in regenerating discs, other signals and mechanisms are also required. Thus, we have found that cell death is not completely abolished in regenerating eiger mutant discs, as occurs after cell death induction [eiger is not sufficient to induce the high number of “non autonomous”dead cells found in regenerating discs. These data suggest that during regeneration, either there are apoptotic signals that are independent of JNK signalling, or there are additional mechanisms, independent of eiger, that trigger JNK signaling. Interestingly, we found that in eiger mutant discs, JNK signaling is active, though mostly in the cells around the wound edge. This result implies that during regeneration, the JNK pathway is activated by an alternative way to Eiger, at least in some cells. Previous studies have demonstrated that loss of epithelial integrity induces JNK signaling. This might be mediated by proteins like the small GTPase, Rho1, that is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton, and can promote JNK pathway activity through an interaction with Slpr, an upstream component of the JNK pathway [eiger, and therefore induce cell death, even in absence of eiger. However, the fact that the activity of JNK signalling in eiger mutant regenerating discs is mostly restricted to the wound edges, though we found apoptotic cells far away from this site, and that the ectopic activation of JNK signalling is not sufficient to reproduce the strong increase “non-autonomous” cell death observed during disc regeneration, suggests that apoptotic signals independent of JNK signaling are triggered during regeneration. Recently, it has been reported that after inducing cell death or as consequence of the mechanical stress generated during wounding, imaginal disc cells produce reactive oxygen species (ROS) [eiger would not be sufficient to block all the apoptosis during discs regeneration.The pattern of cell death that we have observed in regenerating discs is consistent with a model in which dead cells generated at the wound edge can induce cell death throughout the entire disc by an apoptosis-induced apoptosis mechanism. It has been shown that this process relies on the production of Eiger by the dying cells, which activates the JNK pathway in neighbouring cells, inducing them to die . The evonduction . In addi pathway . Thus, tes (ROS) . ROS, eies (ROS) . Thus, RThis process seems best suited to aid complete regeneration, since dying cells generate after amputation might promote cell proliferation of the surrounding tissue to restore the tissue lost after damage. The nature of these signaling molecules differs depending on the tissue . DiffereIn summary our results suggest that apoptosis does not seem to play an important role inducing cell proliferation during regeneration, since partial suppression of cell death does not affect cell proliferation at early stages of regeneration. However, as we have not completely inhibited cell death in regenerating discs, we cannot rule out that programmed cell death contributes to effectively complete disc regeneration.The variety of processes that take place during regeneration, such as remodelling, migration and, reorganization of epithelia, likely requires the elimination of “unwanted cells”. This could be achieved by the action of different cell death-induced mechanisms that might co-operate during regeneration, increasing the robustness of the process. Then it is possible that the mechanical stress and tissue tensions generated during amputation, in addition to the activation of signaling pathways previously mentioned, also might trigger new unidentified signals to promote apoptosis and/or other mechanisms of programmed cell death, such as autophagy and necroptosis. Functional redundancy of some of these mechanisms could explain the difficulty in completely eliminating cell death during regeneration. This would be consistent with our observations which indicate that the inhibition of only one pro-apoptotic pathway is not sufficient to block cell death during regeneration.S1 Figen-Gal4 UAS-GFP discs stained for the apoptotic marker anti-cleaved Caspase-3 . (A-A’) Control discs. (B-B’) Regenerating discs at 3 hrs after cut (AC). We only observed dead cells in the wound edge or in the region adjacent. (C-C’) Regenerating discs at 6 hrs AC; we observed a significant increase in the number of dead cells in the posterior as well as the anterior compartments. (D-D’) 20 hrs AC, we observed a high number of apoptotic cells in the region near to the wound edge, as well as in the anterior compartment. Note the cluster of dead cells in the anterior compartment. Schematic illustrations on the left indicate the cutting lines and the regions eliminated in each disc.(A-D’) Third instar wing (TIF)Click here for additional data file.S2 Figen-Gal4 UAS-GFP regenerating discs double staining for the apoptotic marker anti-cleaved Caspase-3  and DAPI  at 20hrs AC. (A-B) Regenerating discs at 20 hrs AC; we observed dead cells in the posterior as well as in the anterior compartments. (C-D) Higher magnification of the panels (A-B), note the cluster of dead cells in the anterior compartment. These apoptotic anterior cells, to difference to posterior apoptotic cells, do not express GFP (Arrows). (E-F”) Y-Z projections show a cross-section at the position of the white line in the posterior compartment (E-E”) or the anterior compartment green line (F-F”). We observe Caspase-3 positive cells in the anterior compartment that are integrated in the columnar epithelium (arrowhead in F). Posterior apoptotic cells express GFP (arrow in E).(A-F”) Third instar wing (TIF)Click here for additional data file.S3 Figen-Gal4 UAS-GFP nub-Gal4; UAS-DIAP1/+ control non-amputated contra-lateral discs. (B-B”) en-Gal4 UAS-GFP nub-Gal4; UAS-DIAP1/+ regenerating disc at 20 hrs AC. Cell proliferation increases in the posterior compartment of these discs. (C) Bar charts show the average fold change in the mitotic index of control regenerating discs (control), and en-Gal4 UAS-GFP nub-Gal4; UAS-DIAP1/+ regenerating discs (nubG4 dIAP1 reg) at 20 hrs AC, compared to control non-regenerating discs. The error bars represent the standard deviation. Schematic illustrations on the left indicate the cutting lines and the regions eliminated in each disc.(A-B”) Third instar wing discs stained for the mitotic marker Phospho-Histone H3  and anti-Wg . (A-A”) (TIF)Click here for additional data file.S4 Figeiger3/eiger1; pucLacZ/+ control non-amputated discs. (B-B”) Third instar pucLacZ/+ amputated discs. (C-C”) eiger3/eiger1; puc-LacZ /+ amputated discs. The discs were cultivated during 6 hrs after amputation (see puc-lacZ. In eiger3/eiger1mutant discs the expression of the reporter is mostly restricted to the wound edges (compared C-C” with B-B”). Optical z-sections below the panels showed a cross-section at the position of the white line. Note that to difference to control discs, that have completed the wound healing process  and the epithelial integrity is restored, in eiger3/eiger1mutants the wound is unhealed, and the epithelial is disrupted at the wound site .(A- A”) Third instar tion see . The dis(TIF)Click here for additional data file.S5 FigHh-dsRed Ci-Gal4 UAS-GFP/ puc-LacZ non-amputated control discs. (B-B’) Third instar en-Gal4 UAS-GFP/ puc-LacZ amputated discs. (C-C’) eiger3/eiger1; Hh-dsRed Ci-Gal4 UAS-GFP/puc-LacZ /+ amputated discs. The discs were analysed 20 hrs AC. The discs were stained with anti-ß-Galactosidase (red in A-C and grey in A’-C’) to reveal the pattern of expression of JNK reporter puc-lacZ. In eiger3/eiger1mutant discs the expression of the reporter is mostly restricted to the wound edges (compared C with B).(A- A’) Third instar (TIF)Click here for additional data file.S6 Figeiger3/eiger1; Hh-dsRed Ci-Gal4 UAS-GFP/+ discs wing stained for the apoptotic marker anti-cleaved Caspase-3  and anti-Wg (red in A and A”), at 20 hrs AC.(A-A”) Third instar (TIF)Click here for additional data file.S7 Figen-Gal4 UAS-puc UAS-GFP/+ regenerating discs at 20 hrs AC (C-C”). (C-C”) In en-Gal4 UAS-puc UAS-GFP/+ regenerating discs 20 hrs AC the expression of Wg does not disappears at the d/v boundary as it occurs in control regenerating discs (B-B”).(A-C”) Expression of Wg, stained with anti-Wg  in control third instar non amputated discs (A-A”), control amputated discs (B-B”) and (TIF)Click here for additional data file.S8 Figen-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+ non-amputated discs. (B-B’) en-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+ regenerating discs at 20 hrs AC. We observed that cell dead in reduced compared to control regenerating discs (compared to (A-B’) Discs stained for the apoptotic marker anti-cleaved Caspase-3 . (A-A’) Control (TIF)Click here for additional data file.S9 Figen-Gal4 nub-Gal4 UAS-p35 UAS-GFP/+ wings, and control contralateral wings . The discs were amputated at different times during development. Depending on the size of the fragment amputated and time passed BPF (left) we observed a range of regenerated adult wing phenotypes.(A) Different examples of adult regenerated (TIF)Click here for additional data file.


The calculations show that by introducing ferroelectric state in BTO, SCMO transforms from an antiferromagnetic semiconductor to a half-metal. Specially, altering the polarization direction not only controls the interfacial magnetic moment, but also changes the orbital occupancy of the Co-3d state. This novel multiple magnetoelectric coupling opens possibilities for designing new type of spintronic and microelectronic devices with controllable degree of freedom of interfacial electrons in the heterostructures.Due to the demand of controlling magnetism by electric fields for future storage devices, materials with magnetoelectric coupling are of great interests. Based on first-principles calculations, we study the electronic and magnetic properties of a double perovskite Sr These superior characteristics possess wide range of possible novel applications, such as ME actuators, sensors, and high density nonvolatile memory devices7. Because of the relatively high ME operation temperatures and remarkable performance among the multiferroic family, composite multiferroic heterostructures, which are composed of magnetic materials and ferroelectric oxides, have been widely studied11. In general, three types of physical mechanism of ME coupling in such structures have been proposed12, including strain mediated17, charge mediated20, and exchange interaction mediated mechanisms22. Under such theoretical guidance, many efforts have been focused on searching new multiferroic systems with novel properties and high performance25.Multiferroics has attracted extensive research interests because magnetoelectric (ME) coupling effect enables control of magnetic properties via electric fields, and vice versa27. Usually, the orbital occupancy and shape can be modulated through introducing strain, but it is hardly controllable and inevitably introduces defects to materials. Very recently, Song et al. found in their experiment that the interfacial orbital occupancy can be partially changed under electric field in BaTiO3/La2/3Sr1/3MnO3 multiferroic heterostructures29, providing an orbital mediated new ME coupling mechanism. However, theoretical study on this new type of ME coupling is rare, and new materials with better performance are vital for future applications.The orbital degree of freedom plays an important role in electronic structure and magnetic ordering and hence has a significant effect on physical properties3 (BTO) and antiferromagnetic (AFM) double perovskite Sr2CoMoO6 (SCMO) with different BTO polarization states. SCMO has a conventional double perovskite structure: the alternated CoO6 and MoO6 octahedrons with opposite rotations along [001] are arranged as a checkerboard configuration. Its antiferromagnetic order is along [101] direction and the Neel temperature is about 34 K31. When introducing electric polarization in the BTO-SCMO heterostructure, SCMO transforms from AFM semiconductor to a ferromagnetic (FM) half metal. Via changing the direction of the dipole moments in BTO, a considerable variation of interfacial magnetization has been achieved, corresponding to a strong ME coupling. Particularly, we find that the orbital occupancy of interfacial Co2+ ions can also be controlled by the polarization. This new degree of freedom of interfacial electrons together with magnetism, conductivity and spin polarization, are all coupled with the electric polarization, leading to a multiple ME coupling effect. We believe that such coupling opens new perspectives in applications of composite multiferroic heterostructures for magnetic storage, microelectronic and spintronic devices.In this paper, by means of first-principles calculations, we have studied three types of multiferroic heterostructures composed by ferroelectric material BaTiOc-direction with different BTO polarization states: Nonpolar system (NP)  Fig. ; Type 1 NP) Fig.  and TypeNP) Fig. . More deAFM−FM and atomic magnetic moments on Co and Mo atoms are almost the same as in bulk SCMO, indicating that the SCMO slab is almost undisturbed and keeps the AFM state even in very thin film (~7.9 Å) and nonpolar BTO shows negligible effect on SCMO’s magnetic state. An interesting phenomenon arises when BTO generates ferroelectric instability in the P1 and P2 configurations, in which both of them turn into FM states from the bulk AFM solution. A considerable change in the atomic magnetic moment is observed (See Table B to 2.69 µB), while MMo varied from 0.05 µB to −0.42 µB, corresponding to a large change of about 0.47 µB. On the contrary, in P2, MCo has a big change from 2.73 µB to 3.10 µB, while MMo remains nearly zero as in NP system.For NP heterostructure in Fig. EF, so we only show the local DOS of atoms in SCMO; the local DOS from Sr atom is not shown in Fig. d and O-2p electron states while the conduction band minimum (CBM) mainly contains Mo-4d and O-2p states. There is no big difference in the DOS between the NP configuration  of bulk SCMO and heterostructures of BTO-SCMO-BTO in Fig. ion Fig.  and bulk P1 Fig.  and P2  that two t2g electrons occupy the minority spin state. The EF in Fig. Co, turning this system into half-metallic state. In P2  of the minority spin Co-3 NP Fig. , P1  with a dramatic increase of conductivity32. This is also clear evidence that conducting electrons may induce FM magnetic coupling between Co2+ ions via the double-exchange mechanism in SCMO. Unfortunately, as the Neel temperature of SCMO is only about 34 K, it is therefore difficult to achieve the AFM−FM transition at room temperature. However, it is possible that double perovskites that have higher Neel temperature can be found through rational material design, and our approach provides a possible way to realize controllable AFM−FM transition though magnetoelectric coupling.The polarization induced half-metallic property may be responsible for the magnetism transition between the AFM and FM states. When SCMO turns into the half-metallic state due to the polarization from BTO, hopping interactions between the localized electrons and the conduction electrons cause a large spin splitting of the effective Mo Co equals to 3.06 µB near the interface; while for the Co atom in the center of SCMO, MCo is reduced to 2.96 µB, indicating that the moment near the interface is mostly affected. Furthermore, we perform a calculation of a new BTO-SCMO polar state where both BTO polarizations point to the same direction . The magnetic response near the interface resembles the characters in P1 and P2 depending on whether the BTO polarization pointing to or away from SCMO . By switching electric fields between SCMO and BTO, one can change the polarization state between P1, P2 and this new polar state and thereby control the system magnetization. The resulting surface ME coefficient αs can be estimated by formula38μ0ΔM = αsE, where ΔM is the range of magnetization change, E is the strength of applied electric field and μ0 is the vacuum permeability. Taking ΔM = 0.84 μB/a2 (a is the lattice constant of SCMO) and assuming the coercive field of BTO Ec = 100 kV/cm, we obtain a ME coefficient αs ≈ 3.1 × 10−10 Gcm2/V which is comparable to that in Fe/PbTiO3 system as reported by Lee et al.39.We have also tested a structure with thicker SCMO layers (1.5 u.c. SCMO + SrO) in P2 type of BTO polarization. We find that Md states drastically, which is originated from the distortion of the CoO6 octahedron in addition to the bound charges at the interface. Our results not only provide a novel ME system with tunable multiple magnetoelectric effects, but also present a broad opportunity to realize applications of orbital degree of freedom in spintronic and microelectronic devices.In summary, we have demonstrated multiple magnetoelectric effects in the BTO/SCMO/BTO heterostructures based on first-principles calculations. It is shown that the polarization state in BTO can be used to tailor the magnetic ground state, interfacial magnetic moments and conductivity of SCMO. More importantly, switching polarization direction changes the orbital occupancy of the Co-33/Sr2CoMoO6 [001] multilayer heterostructures  method generated pseudopotentials43. For all types of multilayers, a vacuum layer of 15 Å were used with a BaO surface termination as shown in Fig. 30 which is slightly smaller than 3’s lattice (3.991 Å in experiment)44, the induced compressive strain in BTO can therefore suppress the in-plane ferroelectricity instability45. Several optimizing processes have been made to eliminate possible artificial strains along c-direction. Details about the BTO-SCMO interface construction are shown in the supplementary material (SM). The convergence criterion was set to 10−5 eV for the self-consistent electronic minimization and 0.03 eV/Å for forces during the ionic relaxations. Since Co has 3d valence electrons, the on-site Coulomb interaction has been taken into account by using the LSDA+U approach46. We have used U = 5.0 eV and J = 0.89 eV for Co atom because they well reproduced the AFM semiconductor state in SCMO33. After careful convergence tests, we have chosen a cutoff energy of 530 eV for the plane wave basis and a k-mesh grid of 13 × 13 × 1 for Brillouin zone sampling. The tetrahedron method was used for the Brillouin zone integration. More calculation details can be found in the supplementary material (SM).We have performed first-principles calculations of the electronic structures for three different BaTiOCoMoO6 00 multilayCoMoO6 00 multilaySupplementary information.


Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients.screen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test.In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 – 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests.The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics.Among the parameters evaluated, aPTT-LA The aPTT-LAscreen appears to display higher sensitivity and the dRVVT, described as being the most robust assay for LAC testing, might possess a higher specificity for detecting LAC (The detection of lupus anticoagulants (LAC) is based on interference testing of the coagulation cascade and therefore testing it proves challenging (There is no agreement between the guidelines regarding LAC testing for patients taking vitamin K antagonists (VKA) or heparin. Generally, factor deficiency in patients on VKA with an international normalized ratio (INR) above 1.5 can affect LAC testing and the results have to be taken with caution. While CLSI and ISTH suggest caution with respect to the results of LAC testing of patients under heparin therapy, the BCSH recommends no LAC testing of such samples at all.Due to high demands on laboratory facilities, the incurred costs and the wide availability of integrated confirmation testing, the need for mixing tests has to be carefully evaluated. However, there is hardly any data for a suspected LAC patient cohort with representative pre-test probability of having LAC. Hence we conducted a retrospective cohort study to evaluate the predictive capacity of various LAC screening tests and derived mixing tests in detecting a positive confirmation test result within the same blood sample in 5832 patients without any factor deficiency or anticoagulation therapy (dataset A), in patients with heparin therapy (dataset B) and in patients on VKA therapy (dataset C). Whether or not positive results of confirmation testing could be confirmed at a later time point  was not the goal of this assessment.This retrospective cohort study was performed between 2010 and 2014 at the Vienna General Hospital, Medical University of Vienna, Austria, a tertiary teaching hospital with 2145 beds. The study was approved by the local ethics committee (EC-Number: 1197/2013) and was conducted in accordance with the ethical principles of the Declaration of Helsinki.screen or dRVVTscreen, and one derived mixing test as well as the aPTT-LA- and dRVVT- confirmation tests (see Supplementary figure 1). No patients were included more than once, as only the first LAC request for a patient was included during the study period.Due to the retrospective design of the study, informed consent was not obtained from study participants. All consecutive patients older than 18 years of age with clinical suspicion of having LAC from whom the treating physician requested LAC testing were included. Before requesting coagulation testing, clinicians were obligated to document any anticoagulant therapy. Based on this information, INR measurements (for patients on VKA) or anti-Xa-measurements (for patients on heparin therapy) were performed. Patients without a complete LAC testing panel, patients with known coagulation interfering antibodies other than LAC or known haemophilia A, B or contact factor deficiency, patients under direct oral anticoagulants or heparin therapy with anti-Xa activities above 1 IU/mL were excluded. A complete LAC panel included the following assessment: prothrombin time (PT) according to Owren and Quick, thrombin clotting time (TCT), fibrinogen, LAC screening tests including aPTT-A (using STA–PTTA reagent), aPTT-FS (using Actin FS reagent), aPTT-LATo increase the within-group homogeneity and to address the discrepancy between available literature and recommendations with the clinical daily practice, three datasets  were estBetween January 2010 and December 2014, LAC testing was conducted in 9328 patients. In 597 patients, no full LAC testing panel was available. In further 178 patients, the anti-Xa activity for monitoring heparin therapy was above 1 IU/mL . For dat9/L). Except LAC confirmatory testing, all coagulation analyses were conducted by using freshly drawn samples within 4 hours after blood taking. Thawed plasma specimens, stored below - 70°C upon analysis, were used for LAC confirmatory testing. Reagents, analysers and established reference ranges are summarized in Supplementary table 1 and quality control materials as well as their coefficient of variation are presented in Supplementary table 2. All test procedures were conducted according to the manufacturer´s recommendations. Using a coagulometric method, PT Owren , PT Quick , TCT , fibrinogen , activated partial thromboplastin time , aPTT-LAscreen , factor sensitive activated partial thromboplastin time , dRVVT  and dRVVT confirmation test  were assessed on the STA-R Evolution . The aPTT-LA confirmation test  was applied on the MC10 PLUS .Blood samples were collected in siliconized tubes  with 0.023 M pre-filled sodium citrate . In accordance to the H-60-A CLSI guideline, all samples were first centrifuged at 2500xg for 15 minutes at 15 °C , transferred to a secondary polypropylene tube  and again centrifuged under the same conditions to obtain platelet-poor plasma  analyser, the coagulation factors VIII (FVIII), IX (FIX), XI (FXI) and XII (FXII) were determined by one-stage coagulometric methods in a multidilution procedure using Actin FS reagent  and appropriate factor deficient plasmas . For the LAC screening procedure the aPTT-A, aPTT-FS, aPTT-LAconfirm  assessment, patient plasma is mixed with reagent buffer 1 (tube 1) or with reagent buffer 2  and incubated for 9 minutes at 37 °C. Afterwards human plasma (reagent 3) was added to both tubes, and incubated for one minute and finally lyophilized PTT-LS reagent (reagent 4) was added and incubated for another five minutes. CTs were measured after adding CaCl2 . The dRVVTconfirm  assessment was again carried out in two separate reactions. Patient plasma was mixed either with DRVV-Screen reagent or with the DRVV-Confirm reagent containing excess phospholipid concentration . After adding the pre-incubated starting reagent, both CTs were measured and the ratio between CTscreen and CTconfirm was calculated.For the LAconfirm decreased CT (CTtube1 – CTtube2) for more than 3 s or the ratio of dRVVTscreen to the dRVVTconfirm was above 1.25 within the same sample. The LAC confirmatory cut-offs were established by in-house evaluations. According to our standard operation procedures, the dRVVTscreen was not assessed in patients with hereditary or acquired FX deficiency. All analyses were performed under standardized conditions at the Department of Laboratory Medicine, which maintains a certified (according to ISO 9001:2008) and accredited (according to ISO 15189:2008) quality management system.Both confirmation tests, which are widely used, were applied in all patients. Confirmation testing and occurrence of LAC was considered positive when either LAMetric data are given as median  and occurrence of normal distribution was analysed using the Shapiro-Wilk test. Group-differences of metric data were compared by applying Mann-Whitney U tests. The diagnostic performance of parameters was analysed using the area under the receiver operating characteristics curve (ROC-AUC). Since no perfect LAC screening test exists and the majority of relevant guidelines recommend the utilization of at least two screening tests, the correlation between LAC screening parameters was assessed by the Spearman correlation coefficients and graphically presented as a correlogram . Calculation of ICA significantly decreased the performance of the ∆Mix–PNP parameters in the aPTT-A, aPTT-LA, and dRVVT . The mixing test was performed only in the test system showing the largest absolute deviance from the upper reference range, which are presented as subgroups in In 1390 patients of dataset B (LAC positivity rate: 16.3%), 319 patients presented with heparin therapy (up to 1 IU/mL anti-Xa activity). In these patients, a tendency for lower ROC-AUCs to detect LAC was found in all aPTT-A- and aPTT-LA-derived parameters (see Supplementary table 3). Due to the considerably low sample size of patients with heparin therapy, statistical significance was achieved in none of the ROC-AUC differences with the exception of the aPTT-FS .range < 0.001 to 0.969).In patients with VKA , the LAC positivity rate ranged between 11.1% and 16.7% (see Supplementary table 3). While the accuracy of the aPTT-A mixing test was lower in patients with INR > 3.51, this effect was not seen in the aPTT-LA mixing test or their derived parameters. Patients with VKA therapy were statistically compared to those patients without evidence of factor deficiency and anticoagulation therapy  , the LAC positivity rate was 36%. Within dataset A patients, the highest ROC-AUCs for detecting LAC were found by using the aPTT-LAscreen was higher than those ROC-AUCs of dRVVT-derived mixing parameters. This effect was not observed in aPTT-LA derived parameters. Although these differences were not statistically significant, the use of the dRVVT mixing test should be carefully evaluated. A clear benefit of the dRVVT mixing test was not observed in this cohort. This finding adds to the controversial discussion regarding the clinical need for the mixing test, especially in the context of integrated test systems  whose difference to the reference value was the highest in the aPTT-FS among the LAC screening parameters. Typically this occurred in patients with factor deficiency. These patients were presented in the results section, since their deletion would have altered the pre-test probability of a positive confirmation test, and since it is important to make clear the inappropriateness of such an algorithm. Even with an optimal threshold of only 1.75 s, the aPTT-FS ∆Mix–PNP presented with only 66.7% sensitivity, which clearly restricts its usefulness for LAC testing. To prevent a potential impact of the inappropriate aPTT-Actin FS on the overall study results, the diagnostic accuracy analysis of screening tests were split and statistical comparison of aPTT-Actin FS to LAC-sensitive screening tests was avoided.Interestingly, the FVIII, FIX, FXI, FXII activities and fibrinogen concentrations had some predictive capacity for identifying patients with LAC. For all five parameters, patients with LAC had significantly higher levels than patients without evidence of LAC. This might be based on an acute phase reaction or an inflammatory response reaction, which might be accompanied with the expression of LAC or be based on an infectious genesis of the expression of LAC  was not evaluated, since NOACs were hardly used in Austria during the study period. According to the manufacturer´s instruction sheet  the test is insensitive to heparin up to 1 IU/mL due to a heparin inhibitor and no false positive result was found in patients under VKA therapy. In the case of dRVVT confirmation test, no precise information is given in the manufacturer´s instruction sheet on testing patients with heparin or with VKA (#140 0312A). According to our data, LAC testing in patients taking VKA was not statistically impaired in comparison to those patients without VKA (dataset A).The aPTT-LA-derived parameters presented with partly better ROC-AUCs in patients with VKA than in the control cohort. This finding might indicate that the presence of LAC is more easily unmasked in patients on VKA than in patients without factor deficiency. However, the pre-test probability for having LAC or the positivity rate of confirmation tests might be altered in these selected patients. Further prospective studies are needed to assess the possibility to evaluated LAC testing in patients with VKA. Alternatively, an additional dilution step of patient’s plasma and PNP in patients with VKA might be considered. However, our results are concordant with the literature, which indicates the possibility of LAC testing in patients with VKA  assessed in all dataset A patients.For this retrospective cohort study approach, several limitations have to be addressed. Although the overall sample size is considerably large, a bigger sample size would be needed for some subgroup analyses. LAC positivity was defined as a positive result of the aPTT-LA or/and dRVVT-based confirmation test in the same plasma sample, thus false positive or false negative confirmation test results could not be ruled out. Finally, in our laboratory the established reference range of the aPTT-LAIn conclusion, the pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LA-derived parameters showed a better diagnostic accuracy for identifying LAC than dRVVT-derived parameters. Usage of the mixing test had no further advantage in the LAC diagnostics and the ICA did not improve the performance of the mixing test. According to the results of this study, no impairment was found in patients with VKA, while patients with heparin presented with considerably lower diagnostic accuracy.Supplementary figure 1. The standard operation procedure for LAC testing in our laboratory.Supplementary table 1. Overview of methods used for assessment of LACSupplementary table 2. Descriptive information about quality control materials and coefficients of variationSupplementary table 3. Coagulation parameters in patients with and without detectable anti-Xa activitiesSupplementary table 4. Coagulation parameters in patients with INR assessment


Scientists and amateur seismologists, particularly in China and Japan, have attempted over hundreds of years to use the behavior of both wild and domestic animals, disturbed by some sensory input well before a major earthquake, as a predictor of that event. The most striking case occurred in 1975, when, in addition to other precursor events, domestic and wild animals in and around the city of Haicheng, China behaved in an extremely anomalous fashion. The city was partially evacuated and many thousands of lives were saved. Re-analysis of these data, however, found it difficult to reconstruct the source, timing and impact of the actual earthquake warnings. Here we provide, for the first time, an explanation of how animals might detect, in advance, the occurrence of an earthquake and why inconsistencies are likely in such a prediction.We use recent research to provide an explanation of how animals might detect earthquakes before they occur. While the intrinsic value of such warnings is immense, we show that the complexity of the process may result in inconsistent responses of animals to the possible precursor signal. Using the results of our research, we describe a logical but complex sequence of geophysical events triggered by precursor earthquake crustal movements that ultimately result in a sound signal detectable by animals. The sound heard by animals occurs only when metal or other surfaces (glass) respond to vibrations produced by electric currents induced by distortions of the earth’s electric fields caused by the crustal movements. A combination of existing measurement systems combined with more careful monitoring of animal response could nevertheless be of value, particularly in remote locations. Attempts to use the behavior of wild and domestic animals, disturbed by some sensory input, to predict the occurrence of an earthquake, have a long history. Despite the many occasions when extreme adherent animal behavior has been observed, no consistent prediction of an earthquake, based upon animal behavior, has been reported ,2.No predictions of earthquakes have withstood careful scrutiny ,4. The mIn this paper, we present evidence that animals are capable of hearing a wide range of sounds, with frequencies ranging from ultra- to infrasound (kHz-Hz) and from biotic to abiotic sources. The sounds dealt with in this study are abiotic sounds not commonly considered within the biological acoustic literature ,6.We call upon our recent discoveries which describe sound signals, detectable by animals, that might serve as earthquake predictors ,8. BecauFinally, we will evaluate these findings and address their contribution to earthquake prediction and the potential role that can be played by documenting animal response.Animals generate and detect sounds over a wide range of frequencies and for many purposes. Bats, for example, use ultra sound (20–200 kHz) to navigate and capture prey . ElephanFar fewer studies have investigated animal use of abiotic sounds, such as thunder produced by rainstorms or noise generated prior to and during earthquakes. Kelley and Garstang  have rep−3 Pa. With the threshold of hearing of humans at a frequency of 1 kHz at 2 × 10−5 Pa, humans can hear these sounds [−5 Pa. These animals are also able to hear such sounds at much greater distances, as described below.Kelley and Garstang  estimatee sounds ,18. ElepThe Boxing Day Tsunami on 26 December 2004 was created by a magnitude 9.0 earthquake, 160 km off the northwest coast of Sumatra, one minute before 0800 local time (00:58:53 UCT). The earthquake resulted in a 15.6 m (50 ft) wave that crashed onto the coast of Sumatra. This wave impacted the shoreline, producing low-frequency sounds at and below 100 Hz with much of the energy residing in infrasonic frequencies between 1 Hz and 10 Hz. These sounds travel in the atmosphere at the speed of sound. At the tropical ocean surface this speed will be about 1260 km/h. The tsunami, in contrast, travels at a speed of 700 km/h. Elephants at locations 1000 km distant from Sumatra  detected and responded to this sound wave 38.1 min prior to the arrival of the tsunami. No elephant in either location was lost or injured. In contrast, a significant number of people on the beaches of Thailand lost their lives .Kelley and Price , argue tKelley et al.  show thaThe Slant Total Electron Content is measured by using two frequencies transmitted from the Global Positioning Signals (GPS) satellites to the earth. The time difference between these two signals is directly proportional to the number of electrons between the satellites and the ground. This time-delay gives a direct estimate of changes in the electron content of the ionosphere. The satellites are at an orbital height of approximately 40,000 km, well above the ionosphere.Changes in the line-of-sight electron content are triggered by electric fields generated by failure in the earth’s strata preceding an earthquake . These cIn this case, the simultaneous GPS signals shown in Substantial evidence exists that animals can detect a range of abiotic sounds. Crustal movements in the earth’s near-surface produce such sounds. The production of this sound, however, is not a direct product of the crustal releases. Instead, a sequence of events are set in motion, starting with crustal failure, occurring prior to an earthquake. These signals are transferred at the speed of light, guided by the earth’s magnetic field, to the ionosphere. Here the crustal signature is detected in the STEC. These STEC fluctuations of the ionosphere are transferred to the earth’s surface and are detectable by ground-based GPS. At this point, they are not in the form of sound waves but trigger vibrations in metal, glass and other surfaces. This latter process, known as electrophonics, generates audible sounds with frequencies ranging between 20 Hz and 20,000 Hz. These are considered to be the sounds that many animals respond to.The lead time between the precursor crustal movements and the earthquake is indeterminate, but can probably range from tens of minutes to many hours. The ultimate signal, however, represents definitive evidence of the imminent earthquake, an achievement long sought by humans.The sequence of events described above provides both a testable framework for detecting a precursor earthquake signal and a basis for understanding the inconsistencies observed in animal behavior prior to an earthquake. It is also likely that the severity of the earthquake, magnitude of the audible response and the behavioral reaction of animals are all related. The understanding of this process coupled with the technological ability (GPS), to monitor the magnitude and occurrence of the signals should provide the basis for earthquake prediction. Reliance upon and the use of animal responses to the potential earthquake can thus be refined and improved. Reliable response to the adherent animal behavior could still prove useful, particularly in poorly monitored remote areas.Methodology which relates the precursor crustal movements to the intensity of the earthquake remains uncertain. Nevertheless, we present a plausible predictive approach which can be subjected to both post and future analysis. Issues relating to how predictions of earthquakes are used go beyond the scope of scientific inquiry.


This issue's invited Editorial is provided by John A. Antolak, Chair, AAPM Subcommittee on the Oversight of MedPhys Match and Tim Solberg, Associate Editor of the JACMP. —Michael Mills, JACMP EIC.Following much debate regarding the appropriate course of medical physics training necessary to enter the field, the AAPM Board of Directors passed professional policy PP 19‐A in 2007: “It is the policy of the AAPM that graduation from an accredited clinical residency program should be a requirement for qualifying for board certification, with an implementation date to be negotiated with the certification boards.” This was despite the fact that few residency programs existed at that time, and counter to the willingness of the American Board of Radiology to accept completion of an approved graduate degree program as an appropriate pathway to board certification. The result was a series of actions and reactions that was in many ways predictable. Residency programs seeking the best candidates often exerted pressure to quickly accept an offer. Candidates occasionally accepted one offer only to rescind it for a later offer. A Gentlemen's Agreement was subsequently implemented but did little to improve the situation. In 2014, the AAPM Board passed professional policy PP 28‐A endorsing a national matching program for medical physics residencies, and together with the Society of Directors of Academic Medical Physics Programs (SDAMPP) made a multiyear commitment to fund the MedPhys Match (MPM).In this issue, Dr. Kristi Hendrickson and colleagues summarize the results of a postmatch survey of physics residency applicants and residency programs that participated in the first two years of the MPM. When Dr. Hendrickson first approached the AAPM Subcommittee on the Oversight of MedPhys Match (SCOMM)Somewhat disappointingly, efforts continue on the part of both program and applicants to game the system. While these behaviors might seem rather innocent, they are deceitful and are of almost no benefit to either party. In the words of the authors of the MPM matching algorithm, “both applicants and programs can be advised that trying to get a preferred match by behaving strategically is far more likely to harm than to help them.”Perhaps more disturbing, the survey results indicate that federal antidiscrimination laws are possibly being violated on a regular basis. As the authors point out, programs rarely do this out of malice, but rather because they are unaware of the illegality and how such practices affect the applicants. Programs need to be aware that federal employment laws cover recruitment for training positions. These laws are in place for very good reasons; therefore, programs should behave accordingly. These behaviors need to be eliminated if at all possible.The authors provide excellent advice on creating more objective rank lists by reducing or eliminating the influence of postinterview communication, and programs and applicants can both apply this to their rank lists. Finally, their template for interview participants is a good model for emphasizing correct legal and ethical behavior by programs. It is very important that programs model this behavior for applicants; doing so will encourage them to also behave ethically.As a closing note, it would seem that the desire to seek an advantageous position in any situation is a common characteristic of human nature. Given that, it is prudent to carefully evaluate potential ramifications of any initiative to preempt inevitable shortcomings.Opinions expressed in this editorial belong to the authors and are not intended to imply endorsement by JACMP, AAPM, SDAMPP, or the authors' employers.