De novo genetic events seem to play a larger role in sporadic cases.Schizophrenia is a heterogeneous disorder with strong genetic vulnerability. Family history of schizophrenia has been considered in genetic studies under several models. This study used the familial–sporadic distinction with the aim of identifying a more homogeneous phenotype to delineate the genetic and clinical complexity of schizophrenia.The study was conducted at Weskoppies Hospital, Pretoria, South Africa.The study included 384 participants with schizophrenia or schizoaffective disorder from the Afrikaner founder population in South Africa who are considered comparable to Caucasian patients from the United States. A comprehensive data capturing sheet was completed.When schizophrenia and schizoaffective disorder diagnoses were considered jointly, we found no significant differences between the sporadic and the familial groups for age at disease onset, season of birth, comorbid diagnoses, clinical symptomatology, history of suicide or marital status. When the diagnoses were examined separately, however, the sporadic schizoaffective disorder, bipolar type, was found to have a significantly lower age at onset (mean 20.6 vs. 25.3 years).The sporadic schizoaffective disorder, bipolar type, forms a more homogeneous subgroup for genetic studies. However, despite the difficulties, it is a variable of fundamental importance in genetic studies, as already demonstrated. Specifically, significantly increased rates of 6 and suicidality,7 amongst others.Numerous attempts to parse the complicated, heterogeneous clinical presentation of schizophrenia into homogeneous groups that may be suitable for genetic research have taken place without much success. A number of clinical as well as environmental factors have been considered, such as gender, birth and perinatal insults, season of birth, early childhood pre-psychotic deviant behaviour, substance abuse, several comorbid conditions, specific clinical symptoms, age at the onset of primary illness8 and Parkinson’s disease.9 Gene identification efforts in schizophrenia may also benefit through the definition of more homogeneous phenotypes that allow a meaningful sub-grouping of individuals with the disease.5Phenotype refinement has been beneficial in elucidating the genetic architecture of other neuropsychiatric disorders, such as Alzheimer’s diseaseTo our knowledge, a phenotype using a familial versus sporadic sub-grouping has not been characterised. The aim of this study was to use this sub-grouping to determine a more homogeneous phenotype for genetic research via comparison of various clinical, socio-demographic as well as environmental variables in familial versus sporadic schizophrenia.10 Participants were drawn from the Afrikaner founder population in South Africa and were initially recruited from inpatient, outpatient and follow-up clinics at Weskoppies Hospital in Pretoria. Previous work has shown that the Afrikaner sample is clinically comparable to samples recruited from outbred populations, including the United States.10 The Afrikaner sample, however, being derived from a founder population, offers additional advantages for mapping genes caused by reduced genetic heterogeneity and a more uniform environment.10 The close-knit nature of Afrikaner families and the presence of long-term care mental health facilities in the country allow for a relatively reliable ascertainment of family history of mental illness and subsequent distinction between sporadic and familial groups.This study included participants from the collaborative study on Genetics of Schizophrenia led by Dr Karayiorgou since 1997.10 From this extensive database, we selected a subsample of 384 participants for this study. Our criteria for this selection were: (1) subjects met full Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria for schizophrenia or schizoaffective disorder , and (2) subjects in whom family history of schizophrenia or schizoaffective disorder had been ascertained and was available. Any subject with a first-degree, second-degree or third-degree relative diagnosed with schizophrenia or schizoaffective disorder was considered as a familial case for the analyses presented here.This study made use of a cross-sectional observational design. We used data from the database that has been assembled for the ongoing genetic research on this population. The database contains detailed information obtained by administering in-person the Diagnostic Interview for Genetic Studies (DIGS) to all subjects, medical intake forms, questionnaires on early development, family history, medication history, as well as information derived from witnesses and medical records when necessary.12 marital status, employment status and the highest level of education.All data recorded upon the initial recruitment of subjects, as well as any available follow-up data, were reviewed and captured using a data capturing sheet. We assessed basic socio-demographic variables, such as gender, month of birth ,We also assessed several clinical variables, including age at onset of the primary psychiatric diagnosis (defined as the age when full DSM criteria were first met), suicide ideation and number of suicide attempts, as well as the presence of comorbid diagnoses. Amongst the comorbidities, we considered the presence of intellectual disability (ID) or borderline ID, obsessive compulsive disorder or symptoms, major depressive disorder, alcohol abuse, cannabis abuse or use of more than one illicit substance. An illicit substance was defined as any substance with abuse potential and not legal under South African law. Notable legal substances include tobacco and alcohol, with cannabis defined as an illegal substance under current legislation. Several individual clinical symptoms were also considered. These included delusions, hallucinations , disorganised behaviour, disordered thought and alogia.13 was also obtained from the database where available. The following seven early deviant behaviours were captured and analysed: social difficulties, extreme aggression, odd behaviour, extreme fears, chronic sadness, learning disability and impairment in attention.14Information about history of early childhood, pre-psychotic and deviant behaviour before the age of 10, which has been previously linked to the development of schizophrenia,Finally, birth and perinatal insults were also considered.p > 0.15 were retained in the model. In the reporting of results, p-values were given for relationships with p < 0.05 and estimates of odds ratios together with 95% confidence intervals (95% CI). Where more than one covariate was retained in the model, the p-value for the overall model based on the Wald chi-square test statistic was also given.Logit models were fitted to test whether there is a relationship between a binary outcome variable and covariates of interest . Patients for whom the response to the outcome variable was unknown were excluded from the analysis. The binary outcome variables considered in the different models were comorbid diagnosis, cannabis use, illicit substance abuse, a patient being single, perinatal insults, hallucinations, early deviant behaviour and suicidality. Descriptive data is provided in p-values given in the table with 0.025 instead of 0.05. General linear models (GLM) were fitted to determine whether any of the covariates (same as for the logit model) are significantly related to the mean age at onset. The normality assumption was validated and Least Squares Mean (LSMeans) was calculated to adjust for the effect of other covariates in the model and differences in group sizes. The results are given in p-values given with 0.05/3 = 0.0167. Two main logit models were considered to account for the large differences in group sizes of primary diagnosis. In the first model  and Research Ethics Committees of the Faculty of Health Sciences of the University of Pretoria. Written approval was obtained from both committees .Of the 384 participants, 279 (73%) had a diagnosis of schizophrenia, 66 (17%) had a diagnosis of schizoaffective disorder, bipolar type, and 39 (10%) had a diagnosis of schizoaffective disorder, depressive type; 242 (63%) were male and 142 (37%) were female participants. The mean age at onset of the primary diagnosis was 23 years (standard deviation [SD] = 7); 238 (62%) participants had at least one comorbid diagnosis; 45 participants (12%) had suicidal ideation and 86 (22%) had suicide attempts; 115 participants (38%) had birth or perinatal complications; 89 (23%) were born in the winter or spring months (July to September in the southern hemisphere); and 261 participants (79%) had one or more of the seven early childhood deviant behaviours that were assessed. Specifically, 166 participants (51%) had early social difficulties: 25 (8%) had extreme aggression, 44 (14%) had odd behaviour, 89 (28%) had extreme fears, 41 (13%) had chronic sadness, 97 (30%) had a learning disability and 168 (53%) had impairment in attention.p < 0.0001), an association driven mostly by the schizoaffective disorder bipolar type group, where the corresponding odds ratio was 4.6 . Another difference in the combined schizophrenia and schizoaffective group was that male participants had higher odds for a comorbid diagnosis than female participants  and were also more likely to have abused cannabis than female participants . This association held true when prior abuse of any substance (not just cannabis) was considered .Initial comparisons across the sample, controlling for familiality and diagnosis, mainly identified some gender differences. In the combined schizophrenia and schizoaffective group, when controlling for diagnosis, the odds of being a male subject and single was four times higher compared to female participants . The adjusted or LSMeans that was calculated also accounted for differences in group sizes. The same was true for schizoaffective disorder, depressive type . However, there was a statistically significant difference in schizoaffective disorder bipolar type, where disease onset was at 20.6 years of age in sporadic cases versus 25.3 years of age in familial cases . The difference was also gender-dependent .From a general linear model, the mean age at onset for sporadic schizophrenia versus familial schizophrenia when controlling for sex did not differ significantly  when controlling for sex .In the combined schizophrenia and schizoaffective disorder group, the odds for the presence of any early deviant behaviour was 1.9 times higher in the familial compared to the sporadic group , but there was no significant difference based on familiality.Suicidal ideation and suicide attempts were significantly higher (2.3 times) in the schizoaffective disorder group compared to the schizophrenia group , and the findings may therefore not be applicable to other races. Although appropriate statistical corrections were made, the diagnostic, gender and familial–sporadic groups were not equally represented. The duration of illness may have also influenced severity and thus presentation at recruitment. The database of information was not complete for all the covariates that were assessed.The size of the schizoaffective disorder subset also limited the analyses that could be performed. Accurate recording of family history was challenging, and although it was much more accurate in the Afrikaner, and perhaps other founder populations, than outbred populations, there might still be some errors.1The sporadic bipolar type of the schizoaffective diagnosis seems to form a more homogeneous group with lower age at onset. The age at onset has prognostic value with a possible genetic origin. An earlier age at onset has also been linked to a higher genetic load in Alzheimer’s disease, systemic lupus erythematosus and cancer.3 Specifically, we have previously shown that a genetic linkage to the 13q locus was 4.16 times more common in schizoaffective disorder compared with linkage to the 1p locus.17In addition to this study, genetic linkage analysis also appears to support a relatively specific genetic component in schizoaffective disorder.Therefore, the sporadic bipolar type of the schizoaffective diagnosis may be helpful in elucidating the genetic complexity of schizophrenia and should be studied as a separate diagnostic entity in genetic studies.18 and, in particular, the distinction between the bipolar and the depressive types. Equally challenging is the accurate assessment of the sporadic versus the familial nature of psychiatric illness. Founder populations offer certain advantages on that front.Unfortunately, there is low diagnostic reliability for the schizoaffective disorder diagnosisGiven these results, it appears that the efforts required for both correct diagnostic assessments and family history assessments are warranted, as the bipolar type of the schizoaffective diagnosis in its sporadic form is a clinical entity with potential utility in genetic research.


This work compares the metabolic profiles of plasma and the cerebrospinal fluid (CSF) of the patients with high-grade (III and IV) gliomas and the conditionally healthy controls using the wide-range targeted screening of low molecular metabolites by HPLC-MS/MS. The obtained data were analyzed using robust linear regression with Huber’s M-estimates, and a number of metabolites with correlated content in plasma and CSF was identified. The statistical analysis shows a significant correlation of metabolite content in plasma and CSF samples for the majority of metabolites. Several metabolites were shown to have high correlation in the control samples, but not in the glioma patients. This can be due to the specific metabolic processes in the glioma patients or to the damaged integrity of blood-brain barrier. The results of our study may be useful for the understanding of molecular mechanisms underlying the development of gliomas, as well as for the search of potential biomarkers for the minimally invasive diagnostic procedures of gliomas. Gliomas, which represent the majority 81%) of malignant brain tumors, are classified using WHO grade criteria into 4 grades based on increasing degree of anaplasia and aggressiveness, with malignant gliomas comprising grades III and IV tumors. The most prevalent glioma types include astrocytoma (WHO grade I–IV), oligodendroglioma (WHO grade II–III), and oligoastrocytomas (WHO grade II–III). High-grade gliomas include glioblastoma (WHO Grade IV astrocytoma), anaplastic (WHO Grade III) astrocytoma, anaplastic (WHO Grade III) oligodendroglioma and mixed (WHO Grade III) oligoastrocytomas % of mali.The most common type of glioma is glioblastoma, which accounts for 82% of malignant gliomas and has the worst survival prognosis, with only 5% of patients surviving 5 years after diagnosis ,2. The 5Although the clinical point of view exists that early diagnosis (and treatment) do not improve the outcomes in glioblastoma, an alternative opinion suggests that an early diagnosis and accurate tumor classification must become a cornerstone of an efficient personalized therapy . In factAn early diagnosis of a brain tumor is, thus, an important task for the improvement of treatment efficiency and survival of the patients. The development of minimally invasive approaches for the early detection and classification of tumors is indispensable for choosing a more efficient therapeutic strategy.The malignant cells including those of brain tumors have been long known to possess metabolic alterations ,5,6. TheThe cerebrospinal fluid (CSF), which is secreted by the choroids plexus and the meninges, and circulates between brain ventricles and spinal subarachnoid space, is involved in a number of protection, metabolic and drainage functions. Consisting of ~80% of blood-derived and 20% of brain-derived components and being permanently renewed, it maintains the electrolytic and acid-base balance of the brain and supplies the nutrients and signal molecules to the neuronal and glial cells. Meanwhile, it serves as a lymphatic system for the CNS by draining off the wastes and neurotoxic products of brain metabolism. It also provides mechanical support and protection against brain traumas and is involved in intracranial pressure maintenance. Filtration of blood-derived immune molecules, such as the immunoglobulins and cytokines through the blood-brain barrier (BBB) into the CSF ensures the immunological function of the CSF. Finally, the CSF is directly involved in the circadian cycle control via its prostaglandin D2 (PGD2) and prostaglandin-D-synthase (PGDS) system .The CSF is an important diagnostic tool for the search of biomarkers of brain diseases and dysfunctions. A number of published studies is dedicated to the identification of biomarkers of Parkinson’s and Alzheimer’s diseases, multiple sclerosis, etc. ,12,13,14A number of published studies presents the metabolomic screening for glioma biomarkers in individual CSF ,16 or plThe plasma and CSF samples were obtained from each participant and analyzed in three replicates. As much as 289 metabolites  were scrThe projection of metabolic profiles onto a 2-D space using the Uniform Manifold Approximation and Projection (UMAP)  was perfThe metabolic profiles of each sample were further averaged across the three replicate measurements. A comparison of mean CSF and plasma metabolic profiles in the combined group of glioma patients and healthy controls shows their high degree of similarity with the correlation coefficient R = 0.85 . The samThus, herewith we show that the mean plasma and CSF metabolite profiles are highly correlated to each other across the combined group of glioma patients and healthy controls. However, this analysis does not provide information on the correlation between the content of individual metabolites in plasma and CSF samples.p < 0.05) were observed for 75, 68 and 23 out of 101 studied metabolites in the combined group of glioma patients and healthy controls, or in the patients or the controls groups, separately  revealed that the most significant overrepresented pathways with more than one metabolite found were: Metabolism of nucleotides (R-HSA-15869) and Metabolism (R-HSA-1430728).Almost 70 pathways were found to be overrepresented with 53 metabolites showing plasma-CSF correlation, which is lost in control compared to patients  ,33, as wThe application of the described approach made it possible to detect, for example, Alzheimer’s disease at early stages ,37. AnotThe use of targeted metabolomic screening by HPLC-MS/MS searches for a large number of metabolites with high sensitivity, so we believe that such studies will provide a more detailed description of the metabolic changes associated with this disease, which may be useful for disease management. The clinical significance of our study lies in the approach to the development of diagnostic methods based on the measurement of metabolites with correlated plasma and CSF content. All of the above suggests the possibility of predicting the diseases of the CNS using plasma metabolome analysis of patients. This will allow future assessment of the metabolomic profile of CSF from plasma sample data, paving the way for the creation of minimally invasive diagnostic methods without CSF sampling.Study subjects  were enrThe study was reviewed and all experimental protocols were approved by the Ethics Committee of the Novosibirsk Research Institute of Traumatology and Orthopedics named after Ya. L. Tsiviyan (No 050/17 of 11.09.2017). The study was registered on ClinicalTrials.gov, Identifier No NCT03865355 (accessed on 21 January 2021) . All pro® KEDTA tubes containing potassium EDTA as an anticoagulant. Plasma was separated from blood cells by 15 min centrifugation at 2000× g and 4 °C, aliquoted and kept frozen at −80 °C until further use.The matched blood and CSF samples were collected from fasting subjects on the first day of admission before taking any medications (~7:30–9:30 a.m.). Venous blood was collected into 10 mL BD VacutainerCSF withdrawal was performed by a lumbar puncture at L3–L5 levels using a 19-gauge atraumatic needle. As much as 3 mL of CSF was withdrawn into a 15 mL polypropylene tube, the CSF cells were removed by a 10 min centrifugation, and the CSF samples were aliquoted and kept frozen at −80 °C until further use.g for 15 min. Supernatant was transferred into a new polypropylene tube and dried in a SpeedVac concentrator centrifuge . Reconstitution was performed in 100 µL of water/methanol (80:20) and subjected to a modified targeted metabolomics analysis with relative quantification. Each sample was analyzed in three replicates.All samples were processed at the same time according to the protocol described by Yuan et al. . BrieflySamples were analyzed using a Shimadzu LC-20AD Prominence chromatograph  equipped with SIL-20AC autosampler  thermostated at 10 °C. Sample (10 μL) was injected onto a Prontosil 120-5-Amino column (2.1 × 75 mm) . The mobile phase consisted of HPLC buffer A  and HPLC eluent B (100% acetonitrile), the flow rate during analysis was 0.25 mL/min. The HPLC elution gradient was as follows: from 0 to 3 min, the mobile phase B was decreased from 97% B to 85%; from 3 to 4 min, the percentage of solvent B was decreased from 85% to 30%; from 4 to 10 min, the mobile phase B was decreased to 2% and was kept at 2% for an additional 4.5 min. At minute 14.5, solvent B was increased back to 97% and the column was equilibrated for additional 2.5 min at the flow rate of 0.5 mL/min.n = 289) were analyzed in MRM mode. Data acquisition was performed on API 6500 QTRAP mass spectrometer  equipped with an electrospray ionization source operating in the positive/negative switch mode. The main mass spectrometric parameters were as follows. The IS (ion spray) voltages were set at 5500 V and −4500 V for positive and negative modes, respectively. The ion source temperature was set at 475 °C, CAD gas was set as “medium”, Gas1, Gas2 and curtain gas were 35, 35 and 30 psi, respectively. Declustering potential was at ±93V, entrance potential at 10V, and collision cell exit potential at 20 V for positive and negative ion modes. In addition, the polarity switching (settling) time was set to 5 ms, and dwell time was 3 ms for each MRM transition. The precursor ion and fragment ion transitions, the metabolite names, dwell times, and the appropriate collision energies for both positive and negative ion modes were adapted from [Metabolites . Gaussian smooth width was 1.0 point, the minimum peak height was 300 cps, and retention time half window was 30 s. After automated integration, the chromatograms were controlled visually and then the integration results were exported to Microsoft Excel spreadsheet.The Uniform Manifold Approximation and Projection (UMAP) method was used to reduce the dimension and projection of metabolomic profiles into two-dimensional space . MetabolTo assess the relationship between the level of metabolite in plasma and CSF samples, both conventional least squares linear regression and robust regression with Huber’s M-estimates were applied . CompariStatistical analysis was done using Python and its packages: numpy, scipy, pandas, statsmodels, seaborn, and matplotlib. Graph visualization was done using graph-tool  and Cyto


Many biological surfaces with the multi-scale microstructure show obvious anisotropic wetting characteristics, which have many potential applications in microfluidic systems, biomedicine, and biological excitation systems. However, it is still a challenge to accurately prepare a metal microstructured surface with multidirectional anisotropy using a simple but effective method. In this paper, inspired by the microstructures of rice leaves and butterfly wings, wire electrical discharge machining was used to build dual-level (submillimeter/micrometer) periodic groove structures on the surface of titanium alloy, and then a nanometer structure was obtained after alkali-hydrothermal reaction, forming a three-level (submillimeter/micrometer/nanometer) structure. The surface shows the obvious difference of bidirectional superhydrophobic and tridirectional anisotropic sliding after modification, and the special wettability is easily adjusted by changing the spacing and angle of the inclined groove. In addition, the results indicate that the ability of water droplets to spread along parallel and perpendicular directions on the submillimeter groove structure and the different resistances generated by the inclined groove surface are the main reasons for the multi-anisotropic wettability. The research gives insights into the potential applications of metal materials with multidirectional anisotropic wetting properties. Many biological surfaces in nature exhibit directional wettability, which comes from the fact that nature provides a variety of submillimeter/micron/nanocomposite multi-scale microstructural surfaces, and the different arrangements and combinations of multi-scale structures directly affect the motion direction of water droplets ,2,3,4,5.Controlling the anisotropic wettability of the surface is of great significance for basic research. Liquids can be driven along the preferred direction according to the designed structure, which has a broad application prospect in many engineering fields, including self-cleaning, water collection, anti-icing, microfluidic transmission, catalysis, and drag reduction ,17,18,19Inspired by rice leaves and butterfly wings, this paper uses WEDM to prepare a two-level combination structure on the surface of Ti-6Al-4V with submillimeter inclined grooves and micron-level pits and bulges, and then a tertiary structure with nanoscale is finally obtained after alkali-hydrothermal (AH) reaction. In addition, systematically, studies have been conducted on the surface hydrophobicity and anisotropic sliding. As the asymmetric submillimeter level inclined groove structure is the key factor to form the surface hetero-wetting, while the micro/nano structure is an effective way to reduce the adhesion of the solid surface, in this work the degree of asymmetry of the groove structure is properly adjusted by designing the spacing of the inclined groove and the size of the inclination angle. As a result, a surface with bidirectional hydrophobicity and tridirectionally anisotropic sliding superhydrophobicity has been fabricated for the first time through WEDM on the Ti-6Al-4V surface. This work is expected to help improve the understanding of anisotropic wettability and provide new insights into the preparation of functional metal surfaces and the further development of microfluidic systems.3OH), acetone (C3H6O), alcohol (C2H6O), and sodium hydroxide (NaOH) used in the experiment were produced by Beijing Chemical Works . The deionized water used in the AH reaction was supplied by Jiangsu Xizhimeng Trading Co., Ltd. . 1H,1H,2H,2H-perfluorodecyltri-chlorosilane modifier (C13H13F17O3Si) was produced by Shanghai Macklin Biochemical Co., Ltd. .The Ti-6Al-4V workpiece was purchased from Changchun Heng Feng Metal Materials Co., Ltd. ; the chemical composition of the Ti-6Al-4V alloy is shown in L represents the width of the groove, L1 refers to the interval of the processing area, H is the height of the parallel groove structure, and A is the inclination angle of the groove and the parallel plane. The anisotropy of the groove structure surface was obtained by varying L and A , and L1 and H remain unchanged . The WEDM processed samples were ultrasonically cleaned in acetone, absolute ethanol and deionized water, respectively, and then dried in the air.In this research, a WEDM machine  was used to prepare the groove structure with periodic inclination. In this process, the electrode wire is connected to the cathode, the Ti-6Al-4V workpiece is connected to the anode, and deionized water is used as the working medium for electrical discharge machining. The WEDM machining process is shown in F), and reverse the direction perpendicular to the inclined groove (⊥R).Next, the nanostructure is fabricated on the dual-level structure surface by AH reaction. In this regard, the dual-level structured samples were immersed into a polytetrafluoroethylene container with NaOH aqueous solution (30 mL of 10 mol/L). The container was properly sealed and put in an autoclave to heat at 180 °C for 24 h. After that, the samples were taken out and rinsed repeatedly with deionized water to flush away residual NaOH, and then the samples were put in a drying oven to dry at 100 °C for 1 h, the nanostructure embedded on the surface of dual-level structures was obtained, the construction of three-level  combined microstructures were completed. Finally, the prepared sample with three-level structure was immersed into the fluorosilane methanol solution for 2 h to reduce the surface energy of the sample and obtain excellent hydrophobic properties. Three directions are defined in A scanning electron microscope  was used to obtain the details of the morphology of the tertiary composite microstructure on the Ti-6Al-4V surface. The surface chemical composition was analyzed using X-ray energy dispersive spectroscopy ). The static contact angles, sliding angles and water droplets sliding process were measured using an optical contact angle measuring instrument . In each measurement, 7 μL of deionized water was used to measure three different locations on the surface of each sample at room temperature, and the average value was taken as the final test result. During the measurement process, the solid–liquid contact area in the water drop profile picture taken at each position is enlarged to the same multiple, and the actual solid–liquid contact line position is marked to obtain the contact angle of the water droplet. Measurements were recorded five times to average to ensure the correctness of the results.L is 300 μm, and A is 90°, 75°, 60°, 45°, and 30°), and A of 30° and L of 200 μm. Thus, the precise construction of periodic groove structure with different inclinations and spacing values has been realized by WEDM. By further amplifying the WEDM processed surface, as shown in A three-level combined bionic groove structure was prepared by using WEDM combined with AH reaction, and the surface morphology of the structure is shown in EDS was used to analyze the chemical composition of the sample surfaces treated by different methods, as shown in L is 300 μm, A is 45°) and composite micro-nano structures superimposed on the surface. The appearance of the structure caused the surface of the sample to exhibit anisotropic wettability. In the direction parallel to the groove, the contact angle of the water droplet reached 160.7°, while in the direction perpendicular to the groove, the contact angle of the water droplet was 147.6°, evidencing a difference between the contact angles in both directions to reach 13.1°. From the above analysis and comparison, it can be seen that the combination of multi-level microstructures makes the Ti-6Al-4V surface exhibit good superhydrophobicity, and the submillimeter-level inclined groove structure is a key factor generating anisotropic wettability. Therefore, the three-level combined bionic structure surface prepared by the combination of WEDM and AH is one of the ideal processing methods to form an anisotropic superhydrophobic surface.In order to study the influence of surface microstructure characteristics on the wetting property of the Ti-6Al-4V surface, the wettability of different microstructure surfaces was compared and analyzed. A of the water droplet on the groove of 45° and spacing L of 200 μm, 250 μm, 300 μm, and 350 μm, respectively. As shown in L was 250 μm in the direction parallel to the grooves, the contact angle of water droplets on its surface was 148.4°, showing a similar superhydrophobic effect. If the spacing was 300 μm, the contact angle of the water droplets on its surface reached 160.7°, exhibiting the superhydrophobic property, as shown in h is the height of the water drop and l is the length of the TPCL. From the formula, it can be known that when the height of the water drop is almost the same, the shorter the length of the TPCL, the greater the contact angle. It can be seen in L was 300 μm, the number of grooves in contact with water droplets was reduced to one. After measurement, the length of TPCL was about 1.27 mm when L was 250 μm, and the length of TPCL was about 0.91 mm when L was 300 μm. It can be concluded that the shorter the TPCL, the greater the contact angle. However, by comparing panels (a) and (b), and (c) and (d) in L increased; at the same time, as can be seen in L. L of 300 μm and an inclination angle A of 90°, 75°, 60°, 45°, and 30°, respectively. Under the same groove L, a decrease in A caused the equivalent TPCL of the parallel contact between the water droplet and the groove end surface to increase. Therefore, the contact angle of the groove in both directions has the same change rule as the groove A decreases and L increases.To further investigate the influence of the submillimeter scale groove structure on the wettability of water droplets, the contact angle of the surface of the inclined groove structure was measured and analyzed. L and A, the contact angles of water droplets on the groove surface in parallel and perpendicular directions with different structural parameters were measured. It can be seen in L, it can be clearly seen in L and decreasing A except that the contact angle in the parallel direction of the groove showed a significant change with the groove A of 45°. This is because an increase in L caused the number of water droplets immersed in the groove to increase gradually, resulting in an increased TPCL, and therefore the contact angles in both directions decreased with increasing L; when L was constant, the parallel distance of the end surface of the groove supporting the water droplets gradually increased as the inclination angle A of groove decreased, and thus the contact angles in perpendicular directions decreased stepwise, showing the same trend with the situation where L was increased. For the anomalous CAs values at 45° and 30° presented in L increases, the water droplet contact angle in the parallel direction decreases as L increases, as shown in To further explore the relationship between the contact angles in two directions and the sub-millimeter groove structures L = 300μm, A = 30°) were photographed in three directions. It is clear that the water droplet had the smallest sliding angle in the direction parallel to the inclined groove. As shown in F was 22.8° when sliding perpendicular to the inclined groove due to the influence of the asymmetric heterostructure; R was 15.6°when sliding perpendicular to the inclined groove reversely. It can be seen that the Ti-6Al-4V surface of the three-level combined and inclined groove structure displayed obvious tridirectionally anisotropic sliding characteristics. In order to further explore the influence of the structural parameters of the inclined groove on the droplet sliding performance, we further measured the sliding angles of the groove surface under different structural parameters. L. At a 90° angle, the upper end of the groove was parallel to the parallel plane. The water droplets were subject to the same resistance when sliding in both directions of the perpendicular groove, exhibiting only one kind of sliding characteristics. As can be seen in A was 75°, 60° and 45°, the sliding angles in three directions on the surface can be expressed as SA⊥R > SA⊥F > SA//; if A was 30° and L was greater than 200 μm, the sliding angles in three directions on the groove structure surface can be described as SA⊥F > SA⊥R > SA//. Due to the influence of the groove pitch L and the inclination angle A on the sliding process of water droplets, when the inclination angle A of the groove was 90° and 75°, respectively, the sliding angles in the directions parallel and perpendicular to the groove surface decreased with increasing L, as shown in A was 60°, 45°, and 30°, respectively, the tridirectionally anisotropic sliding angles increased as L increased, as shown in The three-level combined and inclined groove structure prepared by the method of WEDM and AH realizes the three-dimensional anisotropic sliding of water droplets. As shown in Aθ is the advancing angle, Rθ is the receding angle, m is the mass of the droplet, R is the contact radius of the droplet and the solid surface, and LGγ is the interfacial tension between the liquid and the gas. The left part of the equation is the driving force of the water droplet on the surface under the influence of gravity, and the right part is the sliding resistance caused by the surface adhesion. It can be seen from the formula that when the water droplet quality was constant, the smaller the adhesion resistance of the water droplet, the smaller the sliding angle of the surface. The adhesion of sliding water droplets was closely related to the morphology of the surface microstructure. A decrease in R caused the contact area between the water droplets and the solid surface to decrease, leading to a smaller adhesion resistance of the water droplets on the surface. R is mainly affected by large-scale grooves. Specifically, the diffusion degree of water droplets on the surface was different with a change of L and A of the groove, resulting in varied R. On the other hand, for the surface with microstructures, the combination of micro- and nanostructures established discrete contact between the solid and the liquid to avoid the intrusion of external water droplets, which effectively reduced the contact area between the liquid and the solid, thereby effectively reducing the adhesion resistance of the surface [This section studied and analyzed the anisotropic sliding behavior of water droplets on the surface of the prepared three-level inclination groove structure so as to understand the influence of groove structure parameters on the anisotropic sliding performance of water droplets. As a matter of fact, there are several factors affecting the sliding angle when water droplets slide on a solid surface. According to the theory of Furmidge and Frenkel, the sliding angle α of the solid plane can be described as follows ,(2)mg.To study the effect of the designed tridirectionally anisotropic superhydrophobic titanium alloy surface on the condensation of water droplets, a water droplet condensation test was carried out. First, we used thermal grease to fix the test piece on the DSA100 refrigeration plate inclined 10°, the temperature was controlled at 1 °C, and then during the test, the indoor temperature and humidity were kept at 25 ± 2 °C, 85 ± 10%. As shown in L, the greater the parallel directions contact angle; while the contact angle in the direction perpendicular to grooves decreases with increasing L and decreasing A. When the inclined groove A is large, during water droplets sliding, the adhesion and resistance of the water droplets against the inclined end surface of the groove are small, and the difference in anisotropic wettability is small; as the inclination A of the groove decreases, the external water droplets can gradually contact the groove wall of the microgroove and the water droplets experience greater resistance when sliding, making the bidirectional hydrophobicity and tridirectionally anisotropy sliding angle shows a more significant differences. Therefore, it is a feasible solution to use WEDM and AH for processing a large number of micro-nano structures on the Ti-6Al-4V surface to reduce the adhesion of the solid-liquid contact area, and then to adjust the hydrophobicity and anisotropic sliding in different directions by considering the submillimeter inclined groove structure, which provides a simple but effective processing method for the anisotropic wetting of commercial metal materials.A periodic sloped groove structure with a submillimeter/micro/nano three-level combination was prepared on the surface of Ti-6Al-4V by the combined process of WEDM and AH. The surface of the Ti-6Al-4V exhibited obvious bidirectional superhydrophobicity and tridirectionally anisotropic sliding properties. The results show that the smaller the number of grooves and the smaller the groove


To leverage benefits in larger enterprises, Agile development methods have to be scaled. Scaled Agile Framework (SAFe) is the most commonly used scaling framework. Performing of the Product Owner role has been identified as crucial in project success in large-scale environments. Staffing the right Product Owner is one of the challenges of adopting SAFe. [Motivation] Research papers focused on Product Owner in SAFe are scarce. Our study outcomes help enterprises to understand the Product Owner role in SAFe and therefore contribute to the removal of challenges with finding the right Product Owners. Additionally, we aim to improve the research community’s understanding of the Product Owner role within the context of SAFe. [Method] Qualitative data were collected through three semi-structured interviews and analyzed using deductive content analysis. [Results] This paper presents the initial results of a single case study. We found out that many activities identified for Product Owners in previous research are not carried out by Product Owners in this particular SAFe implementation. Agile, as a set of iterative and incremental software engineering methods , becomesThe paper is organized as follows. Following Introduction, Sect. In this section, we provide a literature review and present a taxonomy of PO functions. We use the term “function” to refer to a designated group of coupled activities.Scaling Agile was identified as an important topic . Dikert Originally, Scrum defined the PO role relatively simply as the sole person responsible for managing the Product Backlog . Yet, thSAFe provides prescriptive guidelines for implementing enterprise-scale Lean-Agile development, and a number of companies that have applied SAFe have reported significant benefits from it . InteresDespite some existing research on SAFe  , papers SAFe adopted the PO role but, in contrast to Scrum, assumes that a single person cannot handle product and market strategy while also being dedicated to agile teams. It was confirmed in , 10 thatTo understand the fragmentation of PO activities, we extracted the taxonomy of the PO functions from the previous research on POs outside the context of SAFe , 10–14. The mapping in Table The single-case study has been conducted in a division of multinational enterprise that delivers mainframe software. The examined value stream specifically focuses on workload automation software, written mostly in low-level programming languages. For newer components (e.g. modern user interfaces), higher languages like C, Java, or JavaScript are used. The division follows the Portfolio SAFe configuration  and claiThree POs have participated in the initial phase of the study. Table https://rebrand.ly/4ylvsbo. The interviews consisted of 54 questions, 3 to 4 per each function, and took from 65 to 85 min to complete. Our aim was to identify if the activity of the function had been performed, to what extent, how often, typically followed by a request to provide an example to validate the previous answers. All recordings and transcripts were given codes and stored separately from any names or other direct identification of participants.The interviews were done in person, audio-recorded, and transcribed. The interview guide is available at A deductive content analysis  was usedIn this section, we provide the preliminary results from the examined SAFe implementation. The comparison of each PO function from the taxonomy introduced in Table “Which of the activities described for Product Owners in previous research are performed by the Product Owners in the examined enterprise using SAFe?”. We found out that many activities identified for PO in previous research are not carried out by POs in this SAFe implementation. Specifically, our preliminary research findings demonstrated that PO in this particular SAFe implementation is *not*: leading the teams; mitigating identified risks; designing, implementing and disseminating technical architecture; providing governance frameworks; ensuring compliance with corporate guidelines; communicating with senior executives; nor providing support and customer training. This is surprising because previous research conducted outside the context of SAFe assumed that PO responsibilities are very broad. Below, we offer a hypothesis about the reason for the inconsistency.Examining the Scaled Agile Framework  and its The mapping provided in Table de-facto leads to a new form of the management-driven top-down approach We offer a possible explanation. SAFe provides rigorous descriptions for the implementation of Agile processes on a large-scale and put the emphasis on the process . This deapproach  with theThe PO role in the examined SAFe implementation deviates from the previous understanding of the role outside the context of SAFe, as the range of PO activities is narrowed. We attribute it to the top-down approach criticized by practitioners and confirmed during our single-case study. We identified PM as the person accountable for, and steering, the product.The presented results were obtained in one division of a large multinational enterprise and, therefore, might be influenced by the local context. Achieving a validity in a single case study is a known challenge, thus plausible, rival explanations or triangulation methods should be used in further research to strengthen validity of our findings . The stuAs this research is ongoing, another 6–7 interviews will follow to validate the preliminary findings. Our next steps will be to gather, analyze, and incorporate more data from POs with different experience and from different parts of the organization, which will help to understand the real-life PO functions in SAFe implementations. More case studies exploring further enterprise SAFe adoptions are needed to confirm the findings presented in this study.


Stimulation of A2AR inhibits and blockade of A2AR enhances D2R-mediated locomotor activation and goal-directed behavior in rodents. In striatal membrane preparations, adenosine decreases both the affinity and the signal transduction of D2R via its interaction with A2AR. Reciprocal A2AR/D2R interactions occur mainly in striatopallidal GABAergic medium spiny neurons (MSNs) of the indirect pathway that are involved in motor control, and in striatal astrocytes. In the nucleus accumbens, they also take place in MSNs involved in reward-related behavior. A2AR and D2R co-aggregate, co-internalize, and co-desensitize. They are at very close distance in biomembranes and form heteromers. Antagonistic interactions between adenosine and dopamine are  caused by allosteric receptor–receptor interactions within A2AR/D2R heteromeric complexes. Such interactions may be exploited in novel strategies for the treatment of Parkinson’s disease, schizophrenia, substance abuse, and perhaps also attention deficit-hyperactivity disorder. Little is known about shifting A2AR/D2R heteromer/homodimer equilibria in the brain. Positron emission tomography with suitable ligands may provide in vivo information about receptor crosstalk in the living organism. Some experimental approaches, and strategies for the design of novel imaging agents  are proposed in this review.Adenosine and dopamine interact antagonistically in living mammals. These interactions are mediated via adenosine A Adenosine, a purine nucleoside, plays several behavioral and physiological roles throughout the central nervous system (CNS). Adenosine is generated in the living brain from adenine nucleotides such as adenosine triphosphate (ATP) and adenosine monophosphate (AMP). A much less important, other source of adenosine is S-adenosylhomocysteine, that originates from S-adenosylmethionine after physiological transmethylation . Increas1, A2A, A2B and A3, which belong to the P1 receptor family. A1 and A2A receptors have a high affinity for adenosine (10–100 nM range), whereas A2B and A3 receptors are only activated when extracellular adenosine reach very high (micromolar) levels, after tissue damage . Physiological levels of adenosine will stimulate the A1 and A2A receptors. It is unlikely that adenosine exerts major physiological functions via A2B and A3 receptors in the brain, since physiological levels of adenosine are too low to activate these proteins, and A2B and A3 receptors are mainly expressed in peripheral organs rather than in the CNS -5’-N-ethylcarboxamido-adenosine (CGS21680) was shown to induce catalepsy in the rat, and this effect was counteracted by systemic administration of the adenosine antagonist theophylline or the dopamine D2 agonist 5,6,7,8-Tetrahydro-6-(2-propen-1-yl)-4H-thiazoloazepin-2-amine dihydrochloride (BHT-920) propylnorapomorphine without changing the Bmax  [d and Bmax for binding of the dopamine D2 antagonist [3H]raclopride were not affected i to quinpirole [2R [2R in the cells to dopamine receptor agonists [D of dopamine at high-affinity D2R sites after administration of CGS21680 was noted in Chinese hamster ovary (CHO) cells that were co-transfected with A2A and D2 receptors [2 receptors for [3H]dopamine but not the number of dopamine binding sites quinpirole but also the D2R antagonist [3H]raclopride. Yet, CGS21680 and caffeine canceled out each other’s effect on D2R affinity when they were administered together [In CHO cells transiently transfected with Atogether . These a2A and D2 receptors could also be demonstrated in cryostat sections of rat and human brain. CGS21680 significantly increased the IC50 values of competition between the D2/3R ligand [125I]iodosulpiride and dopamine in the striatal region of such preparations SCH58261 suggested that A2AR in the striatum of the rat are not enriched in synaptosomes iodosulpiride from D2R in the nucleus accumbens. Infusion of the A2AR agonist CGS21680 in the nucleus accumbens had the same effect as infusion of the D2R antagonist raclopride , and the stimulation of GABA release by an A2AR agonist and a D2R antagonist were found to be synergistic MRS5425, an analogue of the A2AR antagonist SCH442416, was used to image A2AR in the brain of rats that had been unilaterally lesioned with 6-hydroxydopamine. In this animal model of Parkinson’s disease, the authors observed an increased binding of the tracer in the ipsilateral (lesioned) striatum with respect to the contralateral  striatum. The increase of [18F]MRS5425 in the lesioned hemisphere suggests that loss of dopaminergic neurons can cause upregulation of postsynaptic D2 and A2A receptors, and binding of the PET ligand [18F]MRS5425 may be used as a biomarker to monitor Parkinson’s disease. Some animals were subsequently treated with the dopamine D2R agonist, quinpirole. A significant (15–20%) decrease of the striatal uptake of [18F]MRS5425 was observed after acute administration of quinpirole. The decreased binding of the A2AR ligand after a dopaminergic challenge indicates that interactions between D2R and A2AR can be monitored in living animals with PET raclopride to D2R in the brain. A small but significant increase in the binding potential of [11C]raclopride was detected in the putamen and ventral striatum (5 to 6%), but not in the caudate nucleus. The rise in the ventral striatum was associated with an increase of alertness caused by caffeine [11C]raclopride binding in the ventral striatum was also noted, but this did not reach statistical significance raclopride. Significant decreases of [11C]raclopride binding potential were detected, which were strong (>50%) after intraperitoneal administration of CGS21680 and moderate (30%) after administration of istradefylline raclopride binding potential, since the PET studies employed an acute drug challenge and measured radioligand binding shortly after the challenge. The increased binding potential of [11C]raclopride that was noticed in the ventral striatum after administration of caffeine cannot reflect a decrease of extracellular dopamine, since increased alertness was noticed under these conditions. Increased alertness is normally related to augmented release of dopamine in the striatum, whereas reductions of extracellular dopamine are accompanied by increased tiredness and sleepiness [11C] raclopride binding after caffeine intake may reflect an altered affinity of D2R for the radioligand or a reduced internalization of D2R in the presence of caffeine. However, these studies also highlighted the complexity of interactions in the living brain and difficulties in pinpointing the exact mechanisms underlying the observed changes. Altered binding potentials in PET imaging may indicate: (i) An altered size of the total receptor population . (ii) An altered affinity of existing receptors for the radioligand . Both Aagonists ,129.  Indopamine  and may dopamine . The fireepiness . Thus, tThe PET studies mentioned above ,382,383 2A and dopamine D2 receptors at the MSNs of the striatum occur at physiological levels of receptor occupancy in the living brain. The D2R antagonist haloperidol is widely used as an antipsychotic, but can induce extrapyramidal symptoms ). In non-human primates, the duration of the cataleptic posture induced by haloperidol  was reduced when animals were treated with the A2AR antagonist ASP5854 . A PET study with the A2AR ligand [11C]SCH442416 showed that the anti-cataleptic effect of ASP5854  was reached at an A2AR occupancy of 85% n-Lys-Glu (n = 0–7) linker  linker . In comp2A/D2 heteromer complex. The points of attachment of the linker to the pharmacophore units are another crucial aspect of bivalent ligand design. During the development of a bivalent ligand with A2AR antagonist and D2R agonist action, it was found that the [COOH] position of the adenosine antagonist XCC and the N-terminal position of dopamine D2R agonist were most suitable for the attachment of a linker PE2I or [18F]-FE-PE2I, or by visualizing neuroinflammation with radiolabeled TSPO ligands, in longitudinal studies. An exciting final possibility is to compare changes of the binding of a radioligand for presynaptic A2A receptors, such as [11C]SCH442416, with changes of the binding of a radioligand for postsynaptic A2A receptors in disease models .PET may not only be used to visualize and quantify Aotective ,402,403.2A/D2 interactions is important in unravelling basal ganglia physiology. Initial observation of antagonistic interactions between adenosine and dopamine in membrane preparations, intact cells and living animals was followed by proof of direct interactions between A2A and D2 receptors supplied by various biophysical techniques and demonstration of the molecular mechanisms involved in these protein-protein interactions. Such biochemical and biophysical findings facilitated further studies of basal ganglia disorders.Understanding the complexities of A2AR and dopamine D2R are an important aspect of striatal function and appear to be altered in Parkinson’s disease, schizophrenia, substance abuse, and ADHD. A2AR and D2R have been proposed as therapeutic targets. Decreasing A2A signaling by selective A2A antagonists may result in a recovery of GPe activity in Parkinson’s disease, thereby reinstating the thalamocortical motor stimulatory activity. A2A agonists, either alone or in combination with D2R antagonists, have been proposed for the treatment of schizophrenia. Such combination treatments reduce overactivity of the D2R protomer in the A2A/D2 receptor complex. Drugs that are known to increase the levels of extracellular adenosine  may be used as a replacement for A2A agonists in A2AR-D2R based combination therapies. Stimulation of A2AR has also been postulated as a possible strategy to treat substance abuse, in particular addiction to cocaine. A2AR antagonists, on the other hand, could have a beneficial effect in combination therapies for ADHD. Thus, heteromers of A2AR and D2R are potential targets for the treatment of several human disorders.Several neurodegenerative and neuropsychiatric disorders are associated with a dysregulation of corticostriatal and nigrostriatal afferents that leads to aberrant neurotransmission. Membrane interactions between adenosine A2AR/D2R heteromers in the physiology of the healthy and diseased brain. PET studies with radioligands for A2AR or D2R before and after a pharmacological challenge to the other protomer in the A2AR-D2R complex may be used to assess the strength of A2A/D2 receptor interactions. Bivalent ligands that bind simultaneously to A2A and D2 receptors if the receptor proteins are at close proximity may be used as molecular probes to assess the regional abundance of A2AR-D2R heteromers. The physiological consequences of pharmacological targeting of A2AR-D2R heteromers in patients can be assessed by PET imaging with tracers visualizing cerebral energy metabolism, cerebral perfusion, neuroinflammation, or dopamine transporter expression.PET imaging may provide significant in vivo information that leads to greater understanding of the role of A


Background and objectives: Organ shortage is considered to be a major limitation for increasing transplantation rates. Brain-dead donors (DBDs) are an important source of organs, but up to 50% of potential DBDs might not be identified. An active brain-dead donor search could potentially increase a deceased donor pool. The aim of this study was to evaluate the effectiveness of an active potential DBD identification program and to evaluate one year impact on the potential organ donor pool in Lithuania‘s biggest medical institution. Materials and Methods: An organ donor coordinator service was established and active DBD search strategy was implemented in the hospital of LSMU Kauno Klinikos, and retrospective data analysis was performed between December 2016 and December 2017. Collected data was compared to the available data of the previous year in the same center and to the donation dynamics of the whole country. Results: A total of 6734 patients were treated in all intensive care units (ICU), and 234 (3.5%) of them were identified as possible donors. No increase in potential donor’s number was observed in study year (n = 34) compared to remote year (n = 37). No significant difference in potential donor’s demographic data, cause of death, family refusals and medical contraindication rates. Cerebral angiography (CA) repeated in 20% of potential donors in order to confirm brain death diagnosis. More potential donors for whom CA was repeated had decompressive craniectomy done . Decompressive craniectomy statistically significantly increases the rate of repeated CA . Active search strategy increased length of hospital stay of potential donors comparing to previous year . An optimal time of the first four days of hospitalization to identify a potential donor was observed during our study . Conclusions: We were not able to demonstrate active donor identification strategy superiority over the passive strategy during a short one year period; nevertheless, valuable knowledge was gained in brain death diagnostics, new terminology was implemented, and the stability of actual donor numbers was observed in the experimental donor center in the light of decreasing national results. Long-term strategy is required to achieve sustainable results in organ donation. Organ transplantation became the optimal treatment method for terminal organ failure in the last 50 years, as it improves quality of life and is cost-effective ,2,3,4. ODeceased donors, either brain-dead donors (DBDs) or donors after circulatory death (DCD), are considered to be the main organ source for transplantation, but the tendency of reduction of potential DBD donors is being reported ,7. This There have been both living and deceased donation programs with ranging results 10–21 donors per 1 million population in the Lithuania within last 15 years. The vast majority of organs are procured after declaration of brain death. Living donation and recently introduced DCD programs have a limited impact; however, their potential is great . AccordiAlthough identification is the first and most important step of donation process, up to 50% of potential donors still might not be identified ,23,24,25The aim of this study was to evaluate the effectiveness of an active potential DBD identification program and to evaluate the one-year impact on the potential organ donor pool in Lithuania‘s largest medical institution.The donor coordination team was implemented in the Hospital of Lithuanian University of Health Sciences (LSMU) Kauno klinikos between December 2016 and December 2017. Retrospective analysis of donor coordination team data was performed. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee .The donor coordination team, composed of five ICU physicians with neuro ICU backgrounds, was set up prior our study period. No system of active possible DBD search was available before study period in the hospital. A donor coordinator visited five specialized ICUs on a daily basis—neurosurgical, cardiosurgical, general, trauma, and cardiologic ICUs with total bed number of 81. Patients with severe neurological damage were referred by the treating physician to the donor coordinator. Neurological assessment was carried out for every selected patient by coordinator and patients who met possible brain-dead donor criteria—1) severe brain injury and 2) GCS ≤ 5—were identified. All data was registered in an original color-coded follow-up system according to the patient status. Three main colors were used—1) green was used for active follow-up with recorded trend of GCS; 2) yellow was used when the patients were put on hold because of sedation; 3) red color marked the completion of the follow-up. The result of the completion was circulatory death, recovery, or brain death . CollectDefinitions of the organ donor groups are based on national protocols published by the Lithuanian Health Ministry. A potential DBD donor is a patient who fulfils clinical brain death criteria and whose protocol for brain death is started. An actual DBD donor is a potential donor from whom at least one organ was recovered for the purpose of transplantation. The definition of possible donor is not mentioned in the Lithuanian legislation. According to the WHO, a possible DBD donor is considered a patient in coma with devastating brain injury or lesion and apparently medically suitable for organ donation. A possible DBD definition was included in our study. In Lithuania, brain-death protocol is initiated when all six brainstem reflexes  and spontaneous breathing are clinically absent. Brain death is determined clinically by three physicians (two intensivists and a neurologist or neurosurgeon) every 12 h for three times. Brain death diagnosis can be determined at any time after the first brain death confirmation is clinically determined by performing ancillary tests , electroencephalogram (EEG)) if available in hospital. In our study, cerebral angiography was performed in every case to confirm brain death. Clinical and demographic data was recorded for all potential brain-dead donors on the registry.p < 0.05 was considered statistically significant. Statistical analysis used the chi-squared test or Fisher’s exact test for categorical variables and the Mann–Whitney test for comparisons of means.Statistical analysis was performed with the SPSS Version 23.0 statistic software package . A value of During the study period, a total of 6734 patients were treated in all ICUs; 234 (3.5%) of them were identified as possible donors (met our criteria: were GCS ≤ 5 and had severe brain injury). In addition, 117 of 125 GCS 3 patients died during the hospitalization period, and three survived ICU stay and were transferred to wards having been tracheotomized and with spontaneous breathing. Most of deceased GCS 3 patients (83 out of 117) died after cardiac arrest not fulfilling BD criteria (with preserved spontaneous breathing or brain stem reflexes), and 34 were identified as potential DBDs. A flowchart of possible donor data is presented in p = 0.728). Males with spontaneous intracerebral hemorrhage (ICH) as the main cause of brain death were dominant in remote and study years (43.2% and 29.4%), and the other causes remained subdural hematoma (SDH) (21.6% vs. 23.5%), subarachnoid hemorrhage (SAH) (21.6% vs. 23.5%), and postanoxic encephalopathy (PE) (8.1% vs. 8.8%), p = 0.627. Demographic data and cause of death are compared in No increase in the number of potential donors was observed in the study year compared to the remote year (34 vs. 37). There was no significant difference in potential donor demographic data. The mean age was 52 ± 13.34 in the remote year and 53.41 ± 16.17 in the study year (p = 0.018) (p = 0.023).Brain death was confirmed for 33 out of 34 potential donors in the study year. Cerebral angiography was repeated twice in six (17.64%) out of 34 cases due to a presented circulation during the first scan and once angiography was repeated for the third time (2.94%). For one of them, cerebral angiography was not repeated because blood flow was found performing control transcranial doppler, and cardiac arrest was registered later before confirming brain death by angiography. The mean time of first cerebral angiography for all potential donors was 138.32 ± 62.51 min after clinical examination confirming brain death. Time of angiography between those with confirmed brain death diagnosis the first time and those requiring second investigation was similar (137.07 ± 64.46 min vs. 144.17 ± 57.48 min). The increased number of potential donors for whom cerebral angiography was repeated had decompressive craniectomy done and was statistically significant  . Decomprp = 0.355). Almost one third (29.6%) of all GCS 3 patients had neurosurgical intervention. The surgical intervention rate was higher in the PD group compared to GCS 3 patients with no brain death signs, although no statistical significance was detected . The difference in family refusals between the study and previous years was not significant . There was also no statistically significant difference between medical contraindications  and other causes . Nevertheless, a trend toward negative impact on organ donation was obvious in the study year—the potential donor number decreased by 13% in Lithuania and 8.1% in our center. However, the actual donor number decreased by 27% in Lithuania and by 15% in our center. Comparison of the donation results is shown in Potential donor conversion to actual donor was similar in both the study and remote years . There is an increased possibility of reporting a potential donor during the first four days of a hospital stay in the study year .The length of hospital stay of potential donors was significantly longer in the study period compared to the previous year  after the clinical signs for brain death have been confirmed. CA repeated for 20% of all potential donors with a first test performed in average time just above two hours after clinical examination. Decompressive craniectomy significantly increased the probability of repeated CA in our study, perhaps causing even longer waiting times for brain death confirmation than for the potential donors with intact skulls. It is reported that decompressive craniectomy may lead to false negative results in cerebral angiograms showing normal-appearing blood flow in at least some intracranial blood vessels due to lowered intracranial pressure . Salih eWe found that time from admission to brain death diagnosis was significantly longer in the study period comparing to the previous year, and this could be related to the increased attitude and awareness of personnel of the donation process. These findings could potentially lead to better identification rates of potential donors. We were not able to find any literature reports on this topic.We would like to highlight a few important findings based on our study that could be implemented at national or regional level. First, there is an urgent need in expanding organ donation terminology according to WHO recommendations by means of the critical pathway . Second,We could not analyze how active search impacts the possible donors pool because, before this study, there were no statistical data for possible donors in our hospital. The definition of possible, eligible, or utilized donors was never used in Lithuania.An active search of brain-dead donors neither increased the total number of the potential donors nor increased the conversion rates or changed a donor profile in our donor center in a one-year period. A longer observational period and more sophisticated follow-up system might be required. Local protocols were changed in brain death diagnostics, and new terminology was implemented based on the study results. A national long-term strategy is required to strengthen all the components of organ donation.


Subcellular localization and function of L-type calcium channels (LTCCs) play an important role in regulating contraction of cardiomyocytes. Understanding how this is affected by the disruption of transverse tubules during heart failure could lead to new insights into the disease.Cardiomyocytes were isolated from healthy donor hearts, as well as from patients with cardiomyopathies and with left ventricular assist devices. Scanning ion conductance and confocal microscopy was used to study membrane structures in the cells. Super-resolution scanning patch-clamp was used to examine LTCC function in different microdomains. Computational modeling predicted the impact of these changes to arrhythmogenesis at the whole-heart level.We showed that loss of structural organization in failing myocytes leads to re-distribution of functional LTCCs from the T-tubules to the sarcolemma. In ischemic cardiomyopathy, the increased LTCC open probability in the T-tubules depends on the phosphorylation by protein kinase A, whereas in dilated cardiomyopathy, the increased LTCC opening probability in the sarcolemma results from enhanced phosphorylation by calcium-calmodulin kinase II. LVAD implantation corrected LTCCs pathophysiological activity, although it did not improve their distribution. Using computational modeling in a 3D anatomically-realistic human ventricular model, we showed how LTCC location and activity can trigger heart rhythm disorders of different severity.Our findings demonstrate that LTCC redistribution and function differentiate between disease aetiologies. The subcellular changes observed in specific microdomains could be the consequence of the action of distinct protein kinases.10.13039/100000002NIH grant (ROI-HL 126802 to NT-JG) and British Heart Foundation . Funders had no role in study design, data collection, data analysis, interpretation, writing of the reportThis work was supported by  Although cardiomyocyte L-type calcium channels, essentials for the contraction of the heart, have been the focus of studies of human heart failure in the last decades, no emphasis has been placed on their regional variation within the cell. The localization of these channels within cell membrane, has been found to be altered by the remodeling that cells undergo during the progression of heart failure.In this work, using a combination of microscopy and electrophysiological techniques, we study human cardiomyocytes from ischemic and dilated cardiomyopathy patients, and the impact that the left ventricular assistant devices have of them. Extensive remodeling of the cellular structures is stablish, which leads to redistribution of L-type calcium channels. Furthermore, the spatial location and function of L-type calcium channels is found to be different between ischemic and dilated cardiomyopathies. Changes that we found in each of the diseases are connected to a specific and different intracellular signaling pathway. Using computational simulations of the whole heart, we reveal the implications that these findings could have in heart rhythm dysfunction.Ischemic and dilated cardiomyopathies are two pathologies that are different in origin but share the same endpoint, which is the need for a heart transplant. This study found that despite the same end-stage time point, major differences exist at the sub-cellular level between aetiologies. These findings describe new targets specific to each disease, and this could open the door to new pharmacological treatments.Alt-text: Unlabelled box1The progression of structural heart diseases from an early form towards advanced heart failure (HF) is accompanied by an increased risk of arrhythmia and sudden cardiac death ,2. As prCa,L) in the progression of HF remains, however, not well understood. Although CaV1.2 channel density has been shown to be decreased on the surface of failing human cardiomyocytes Ca,L has been widely reported to not be altered o) in human HF, which could be linked to a higher phosphorylation of the channel.Calcium signaling plays a critical role in the pathogenesis of heart failure. L-type calcium channels (LTCCs) are the main mediators of calcium influx into cardiac cells and are essential in determining the electrical and mechanical properties of cardiac muscle 2+-handling microdomains and their integrity underlies the normal contractile function of the human myocardium In adult ventricular myocytes, a critical subpopulation of LTCCs, which participates in excitation-contraction coupling, is located in the specialized transverse tubules  microdomains. These structures represent highly branched invaginations of the cardiomyocyte sarcolemma In the current study, conducted exclusively on control and failing human cardiomyocytes, we analysed and compared nanoscale LTCC distributions from healthy donor hearts, failing ischemic cardiomyopathy (ICM) hearts, and failing dilated cardiomyopathy (DCM) hearts, as well as from failing hearts after the implantation of left ventricular assist device (LVAD). We used a combination of electrophysiological and optical techniques and a multi-scale computational model to understand how changes at the cellular level affect the behavior of the whole organ and how different aetiologies may be distinguished.22.1Experiments on isolated human cardiomyocytes were approved by Imperial College Institutional Review Board. Informed consent was taken from each patient. Samples from failing hearts of patients with end-stage heart failure were used with the approval from Brompton Harefield & NHLI Ethics Committee under Biobank REC approval reference 09/H0504/104+5. Samples from donor hearts unsuitable for transplantation were used with the approval of NHS BT with REC approval reference: 16/LO/1568.2.2n = 31 patients, average age 49.6 ± 1.78 years, 6 females and 25 males). Clinical characteristics of the heart failure patients used in this work can be found in n = 5, average age 69±2 years, two females and three males), with their consent during valve replacement procedure at Hammersmith Hospital, Imperial College London, London, UK. The second one was obtained from samples of left ventricles taken from donor hearts unsuitable for transplantation . None of the donors had a history of hypertension, cardiothoracic disease, or diabetes. Two of them presented a history of smoking. The reasons for these hearts being rejected for transplantations were: degeneration on the organ care system prior to transplantation; heart size/logistics; and increased lactate and K+.Failing ventricular myocytes were prepared from hearts of patients with end-stage heart failure caused by ischemic or non-ischemic cardiomyopathies who were undergoing transplantation . No differences in the parameters checked in this work were observed between these 2 populations of control cells, apart from the number of cells available after the isolation, which was drastically lower from the biopsy samples, due to the intrinsic small size of the sample.2.33). The cubes were transferred to fresh calcium free Krebs-Ringer solution at 37 °C and washed 3 times for 3 min each. After this, the tissue samples were incubated in 10 ml of Krebs-Ringer solution (in g/L): NaCl 7.012, KCl 0.402, MgSO4 1.332, Pyruvate 0.55, Glucose 3.603, Taurine 2.502, HEPES 2.383; pH = 7.4, adding 200 nM CaCl[2] and Proteinase type XXIV  under mechanical agitation. After 25 min, the partially digested tissue was transferred to 10 ml of fresh Krebs-Ringer in which proteinase was substituted by collagenase type XIV (1 mg/ml Sigma-Aldrich) continuing at 37 °C under agitation. Every 10–15 min the tissue was transferred to a fresh solution while the remaining solution contained single isolated cardiomyocytes. Single rod-shape cardiomyocytes were visible under light microscopy after the first 10–15 min, with the biggest density of cells being obtained after another 10–15 min. Supernatants were centrifuged for 3 min at 600 rpm after each incubation step, and the pellets were re-suspended in 2–3 mL of Krebs-Ringer solution without enzymes. Cardiomyocytes were plated on dishes coated with laminin and left to stick to bottom for at least 45 min before experiments. Cardiomyocytes were used on the same day of isolation for an average time of 7 h, with a maximum time of 12 h.Ventricular cardiomyocytes from failing and donor hearts were isolated as previously described ,14. Due n = 10), dilated (n = 21), and myocarditis (n = 1) cardiomyopathy. Among these patients, 4 from the ischemic group, 8 from the dilated group, and the patient with myocarditis were implanted with LVAD. As control, 3 isolations were done in donor hearts not suitable for transplantation and 5 isolations were performed on left ventricular biopsies samples from papillary muscles. In total, 40 isolations were done for this study. Data extracted from 11 of these isolations has already been published in Circulation Research End-stage heart failure was caused by ischemic  using a single dimension Fourier transformation from a custom-written macro for Matlab  as described before Z-groove index was characterized by scanning ion conductance microscopy (SICM) which produced a 3D topographical map of the surface To maximize the number of measures, cell length and width were obtained from bright field and confocal images. Only cells with clear cross-stations and rod-shaped were selected.2.5Cell-attached patch-clamp recordings of single L-type calcium channels were obtained and classified based of the specific location of the pipette: on T-Tubule or crest microdomain. T-Tubules are defined in the SICM images as deep rounded or semi-rounded invaginations, connected by less deeper striations which define the Z-grooves. Crest areas are the spaces between the Z-groove lines, occupying most of the topographical surface in some images from heart failure cardiomyocytes. The use of SICM with pipette clipping modification Experiments were performed at room temperature using the following solutions; external solution containing in (mmol/L): 120 K-gluconate, 25 KCl, 2 MgCl2, 1 CaCl2, 2 EGTA, 10 Glucose, 10 HEPES, pH 7.4 with NaOH, ∼290 mOsm; internal recording solution containing in (mmol/L): 90 BaCl2, 10 HEPES, 10 Sucrose, pH 7.4 with TEA-OH, ∼250 mOsm. For the experiments with the inhibitors H-89 (10 µM) and KN-93 (5 µM), cells were incubated with the blocker for a minimum 5 min before the first cell was patched, and each dish was used for a maximum of one hour. The pipette used for cell attached recordings after clipping had an average resistance of 27.78 ± 0.26 MΩ. Currents were recorded using Axopatch 200A amplifier , controlled and monitored using pClamp software version 10 (Axon Instruments). Liquid junction potential was calculated as −16.7 mV and corrected for all the data shown in this work. Single LTCCs were identified and characterized by their voltage dependent properties. After performing a seal, the holding membrane potential was held at −96.7 mV. A protocol of incremental step of 10 mV with pulses from −36.7 to +23.3 mV was applied a minimum of 3 times. A current-voltage (I-V) relationship was generated form this data, when a channel was present. For the study of the Po the −6.7 mV step voltage was choose and a protocol of 50 consecutives pulses at this voltage was applied a minimum of 3 times. Single channels were sampled at 10 kHz and filtered at 2 kHz . Analysis was performed using Clampfit version 10.2 All the sweeps were checked for the presence of LTCCs.Occurrence of LTCCs was calculated as the percentage of recording showing activity versus the total number of recordings. Channel density was calculated for each group, as the relation between the total area sealed  and the total number of channels. The area sealed under the pipette for each recording was estimated from the resistance of the pipette for each experiment as described by Novak et al. The Po was averaged from a minimum of 20 sweeps at −6.7 mV for each cell. Each cell was recorded only once. The total number of channels in the recording was input into pClamp software to calculate the single Po of one single channel. To estimate the number of channels per seal a carefully examination of all the recordings per cell was done. The level of the spikes determined the number of channels present in the seal. When only a single level of spikes was observed that seal was considered to contain only one channel.The conductance was determined by plotting the average amplitude of all the openings in the recording against the test potential for every single experiment. The slopes from the linear relationships of these I/V plots were calculated as the conductance for that single channel.2.6The O'Hara-Rudy ionic model Then, two new parameters encoding for the distribution of LTCCs between the TT and the Crest domains were introduced. Thus, the ratio between the channels located in the TT domain to the channels located in the Crest was found to be 0.8 using the equation:Therefore, a fraction of approximately 0.45 of LTCCs within a cell were located on the TT membrane, while the remaining 0.55 were relocated to the Crest.As the experimental evidence from human myocytes indicated that remodeling has no effect on the peak of the LTCC current Following the experimental evidence here, PKA was incorporated into the ICM cardiomyocyte model and allowed to interact with the LTCCs present in the TT domain. Similar to the O'Hara-Rudy human ventricular model combined with the electrophysiological module of the Heijman beta adrenergic model Apart from the remodeling observed in the LTCC current, all the other ionic changes described in Sanchez-Alonso et al. Finally, electrophysiological differences between epi‑ and endocardial cells in each etiology were represented. As in the previous study Using the three models described above , for both endocardial and epicardial myocytes, single-cell simulations were performed using both a voltage-clamp protocol to analyze the whole-cell LTCC current behavior, and a pacing protocol in order to observe differences in action potentials and the possible emergence of cellular-level triggers of arrhythmias such as EADs.2.7To evaluate if the EADs observed in the cell-level simulations would develop into reentrant arrhythmias in a human heart, the ionic models for both endocardial and epicardial cardiomyocytes were implemented in an MRI-derived, anatomically realistic, healthy human ventricular model. The same model was utilized in our previous study 2.7.1Experimental datasheet for the figures is available:https://data.mendeley.com/datasets/9mbxk5mcpp/draft?a=a77df96a-59e4-4602-8af1-96e649f8a31d2.7.2P<0.05 was considered statistically significant. All data and statistical tests used in this work is available in the supplemental datasheet.All statistical analysis and graphs were performed using GraphPad prism. To test normality a Kolmogorov-Smirnov test was used. To test for statistical differences on normal distribution data, One-Way ANOVA was used. When data failed the normality test, the nonparametric Kruskal-Wallis test was used. All data are expressed as mean ± standard error of the mean (SEM). A value of 33.1p<0.01). LVAD implantation reduced the size of failing cells significantly, as described previously A well-known characteristic phenotype of human failing cardiomyocytes is their increased size ,19, whicp<0.05; decrease of 31.3% for DCM TT density, p<0.05; decrease of 68.6% for ICM TT regularity, p<0.001; decrease of 61.77% for DCM TT regularity, p<0.001), consistent with our previous findings n = 96, versus ICM: 5.3 ± 0.3, n = 68, p<0.05; ICM+LVAD: 4 ± 0.2, n = 70, p<0.001; DCM: 4 ± 0.2, n = 116, p<0.001; DCM+LVAD: 4.4 ± 0.2, n = 80, p<0.001; Supp. Figure S1).These changes in cell shape were accompanied by deeper cytostructural changes. In human failing myocytes from both ICM and DCM patients, we observed a significant decrease in regularity and internal density of T-tubules as compared to controls  versus crest (2.14 channels/µm2) in control cells, with a ratio of 1.84. However, in failing cells, this ratio is reduced , which can be attributed to the loss of channel density in TT in failing cells, suggesting that the loss of TT structure in HF is also linked to a loss of active LTCCs in the remaining structurally-intact TTs.An estimation of active channels density in each microdomain was also done (see 2 in TT and 5.05 channels/µm2 in crest) confirming that in TT the density of LTCCs is higher than in crest .LTCC agonist Bay K 8644 was used on control cells to record the maximum density of channels available in the surface of the cardiomyocytes (2). In fact, observed clusters of functional channels in the crest of control cells were lost in failing cells. The 3 recordings showing LTCC activity in the control crest microdomain present multiple channels . In contrast, in failing cells, from 32 recordings showing LTCC activity in the crest, only 12 present multiple channels .Surprisingly, the higher chance of finding LTCCs on the crest of failing cells (control occurrence 10.3% vs failing 28.7%) was not found to be linked to a higher density of LTCCs (control: 2.14 channels/µm vs failing: 2.36 channels/µm3.3p>0.05) or in any of the failing groups . The low conductance reported here may be lower than expected due to amplitude underestimation, because of lack of fully resolved openings for some of the voltages studied. To facilitate full openings at all test potentials, the LTCC agonist Bay K 8644 (5uM) was applied in the pipette in a sub-set of experiments in control cells. Under stimulation, the slopes of the linear I/V relationships were 24.94±0.4 for TT LTCCs and 23.72±0.95 for crest LTCCs (p<0.001 vs control TT and p<0.01 vs control crest), consistent with data from other authorsSingle LTCC activity from control and failing human cardiomyocytes was elicited by 1 s depolarizing pulses from a holding potential of −96.7 mV to test potentials between −36.7 to 23.3 mV . The average amplitude from all the openings during the pulse was plotted against the test potential for every experiment, to determine the conductance as the mean of the slopes of this linear relationship. No significant statistical differences were found between TT and crest LTCCs in control cells , without changes in Po of the DCM TT LTCCs, as it has been recently shown o , without a statistically significant increase in Po of the crest channels, indicating a different pathological substrate between DCM and ICM patients.The P −6.7 mV c and 3d.o for all the channels analyzed. Specifically, the channels found pathologically active in DCM patients in the crest and in ICM patients in the TT showed a significant reduction in Po following LVAD implantation . However, in ICM myocytes, blocking CaMKII did not reduce the Po of the hyperactive LTCC located in the TT (from 0.102±0.0046 for ICM TT to 0.101±0.025 for KN-93 treated ICM TT). PKA is also a well-established regulator of LTCC function o , without affecting DCM crest channels . This suggests that two different mechanism are responsible for phenotype observed: in DCM CaMKII in the crest microdomain is pathologically active, whereas in ICM PKA is over-active in the TT domain.It has been shown that CaMKII can phosphorylate LTCCs in cardiomyocytes nction o e and 3f.Next, we used computational modeling to provide mechanistic insight into how the sub-cellular microstructure changes in HF can lead to electrical dysfunction at the organ level, and specifically, how the phosphorylation state of LTCCs in different microdomains can trigger arrhythmogenic events in the whole heart.3.52+ ions into the cell similar in magnitude across all the three cases  and the location of these phosphorylated channels (TT vs. crest) causes the emergence of arrhythmogenic triggers in both cases but leads to reentrant arrhythmia only in DCM.4Altered calcium signaling contributes to the pathology of human HF, and thus its signaling pathways are typically targeted in current therapies for HF 4.1Cell hypertrophy could be regarded as a compensatory mechanism during the progression of HF, but it may further impair cardiac performance in the long term. A number of studies have shown that isolated cardiomyocytes from HF patients are larger compared to healthy cells ,26,27, wIt is well known that the cardiomyocyte TT system is remodeled during HF progression V1.2  are mainly concentrated in the TT membrane Many studies have found an extensive loss of TT structure in HF Taking all together, we conclude that failing myocytes hypertrophy leads to remodeling of the TT system, which manifests in both reduced TT density and fewer TT opening on the sarcolemma, which in turn causes an imbalance in LTCC distribution. Interestingly, LVAD implantation can significantly reduce the size of the cells but is unable to fully recover the TT structures and the balance of the LTCC distribution.4.2o in failing hearts  in cardiomyocytes PKA and CaMKII are two of the main kinases known to modulate LTCC  expression can be also considered to interpret the results. The enhancement of calcium current by βARs could happen through phosphorylation of several residues along the C terminus of the LTCC pore subunit by PKA 1 and β2 adrenergic receptors play in the regulation of the LTCC will be the next step for a better understanding of the role of LTCC on human cardiomyopathies.β4.4Our single-cell simulations showed how the loss of TT and the subsequent redistribution of LTCCs alters cellular action potential. EADs have been widely described in the literature CaL compared to control, with a higher magnitude and a slower decay . If this is accompanied by an increase in Po (DCM simulations), electrophysiological disturbances will progress to reentrant arrhythmias.Whole heart simulations demonstrated that LTCC alterations in DCM have worse consequences at the organ level (reentrant arrhythmias) than LTCC alterations in ICM . This po4.5K+ currents The number of experiments that could be performed on human cardiomyocytes in different aetiologies is limited by several factors. The widely used inhibitors, H-89 and KN-93, were selected for this project to explore the involvement of PKA and CaMKII in the regulation of LTCC. We cannot disregard the potential involvement of other signaling pathways that could be indirectly affected by these inhibitors. H-89 is described as a blocker of PKC as well, and at higher concentration it had been suggested to block 4.6The prognosis and survival of patients with HF may benefit from an early diagnosis of the etiology of the disease, thus allowing specific targeted treatment informed by the different sub-cellular pathologies described here. This work presents new insights into the end-stage phenotype of human HF and suggests how different pathologies could result from changes in microdomains at the single cell level. Our modeling approach spanning from stochastic LTCC gating to arrhythmogenesis at the organ level enabled us to understand how subcellular changes can influence the development of arrhythmias. These outcomes may be useful in informing choice of anti-arrhythmic therapies and strategies specific to the patient's pathophysiology and probability of the type of underlying arrhythmic substrate. Historically most calcium-channel blockers in clinical trials had been more detrimental than beneficial in patients with advanced left ventricular dysfunctions The authors declare no competing interests.