Vasculature plays a key role in bone development and the maintenance of bone tissue throughout life. The two organ systems are not only linked in normal physiology, but also in pathophysiological conditions. The chronic kidney disease–mineral and bone disorder (CKD-MBD) is still the most serious complication to CKD, resulting in increased morbidity and mortality. Current treatment therapies aimed at the phosphate retention and parathyroid hormone disturbances fail to reduce the high cardiovascular mortality in CKD patients, underlining the importance of other factors in the complex syndrome. This review will focus on vascular disease and its interplay with bone disorders in CKD. It will present the very late data showing a direct effect of vascular calcification on bone metabolism, indicating a vascular-bone tissue crosstalk in CKD. The calcified vasculature not only suffers from the systemic effects of CKD but seems to be an active player in the CKD-MBD syndrome impairing bone metabolism and might be a novel target for treatment and prevention. What were once seen as merely structural components in the human body, the view on the vascular and skeletal systems has changed to represent organs with multiple regulatory functions. In addition to the core tasks of delivering oxygen and nutrients, and removal of waste, as well as providing posture and protection of internal organs, these two tissues secrete several factors with local and systemic effects. Research has identified vascular and bone cells as important actors in the homeostasis of the cardiovascular system, inflammation, immune system, energy balance, and mineral handling ,2. This The close relation between the vasculature and bone is illustrated by the highly vascularized bone tissue, and the important role of the microcirculation in bone development (modeling) and in the continuous cycle of remodeling, where bone is replaced by balanced bone resorption followed by bone formation . A conneCardiovascular disease is still the most serious complication to CKD, causing increased morbidity and mortality ,18,19. TDuring embryogenesis, bone develops simultaneously with blood vessels as a result of a complex interplay of bone-derived factors (osteokines) and vascular-derived factors (angiokines) in the microenvironment . Still, It is well known that the endothelium regulates the tonus of the vascular smooth muscle cell by nitric oxide (NO), prostaglandins, and endothelin-1 ,25. In bIn addition, the bone vessel endothelial cells secrete several factors demonstrated to have direct effect on bone metabolism. The tumor necrosis factor (TNF) superfamily includes receptor activator of nuclear factor-κβ (RANK), RANK ligand (RANKL), and the endogenous antagonist receptor osteoprotegerin (OPG). The RANKL-RANK-OPG regulatory system is a fundamental part of bone homeostasis and represents osteoblast/osteocyte regulation of osteoclast activity ,35. AddiOsteolinage cells  secrete osteokines with paracrine effect on the vasculature. In bone modeling and remodeling, increased blood supply is observed. VEGF-A is secreted by hypertrophic chondrocytes and osteoblast-lineage cells and stimulates angiogenesis ,42. CrosThe complex crosstalk between bone vascular cells and bone cells in the microcirculation of bone is essential for normal bone development, bone repair after fracture, and maintenance of bone tissue throughout life. The bone microcirculation and cell communication are most likely altered across the stages of CKD. However, this has been less studied ,54.CKD has deleterious systemic effects on the viability and function of several organs namely the cardio-vascular system, bone metabolism, immune system, muscle strength, energy balance, fertility, and cognitive functions. Furthermore, increasing evidence has shown that CKD is a condition of accelerated aging . Of all 33PO4) and found a great difference in buffering capacity and distribution of phosphate in adenine treated rat (CKD model). All the larger arteries had significantly higher phosphate uptake, even above the distribution in bone of CKD rats with severe vascular calcification . Th. Th66]. We also found several osteokines altered in the calcified vessel  66]. Ag. Ag66]. Calcification of the medial layer of arteries is characteristic for metabolic diseases and especially CKD. Unlike focal calcification of atherosclerotic plaques, the medial calcification runs longitudinal through the vessels impairing the elastic properties as well as the dynamics of vessel’s tonus. The vascular medial calcification is mainly seen in the elastic and larger arteries, resulting in increased workload for the heart, which eventually develops left ventricular hypertrophy, reduced cardiac perfusion, and heart failure. Heart failure is commonly seen in CKD patients . InductiThe classical understanding of the multiple systemic effects of CKD has been explained by a reduction of glomerular filtration, resulting in an accumulation of uremic toxins and waste products, the low grade of chronic inflammation, and the hormone disturbances related to erythropoiesis, blood pressure, mineral balance, and bone turnover. However, a new paradigm for CKD-MBD was recently proposed by Hruska et al. that kidney injury-derived factors have systemic effects on the vasculature and bone in CKD . The novAnother system activated in kidney disease is the TGF-β superfamily, well known for its potent regulation of extracellular matrix production and the importance of the fine balance of its activity . One of More lines of evidence are supporting the notion of an important role of these developmental factors in CKD-MBD ,101,104.Even though established vascular calcification persists after normalization of kidney function, some of the changes in CKD vascular disease are dynamic and may represent a target for treatment. Treatment with BMP7 has been shown to reduce plasma phosphate and aorta calcium content in an animal model of low turnover renal osteodystrophy (combined background of metabolic disturbance and CKD) ,107. OurActivation and changes in the Wnt signaling pathway are central to the process of uremic vascular disease. In our molecular pathways and gene ontology cluster analysis of the genetic shift in the calcified aorta from 5/6 nephrectomized rats, we found large changes in genes related to Wnt and TGF-β pathways, remodeling of extracellular matrix and bone formation . AnotherSince several signal molecules induced in the vasculature in the process of calcification are also found at higher levels in plasma in CKD, we speculated whether these factors were secreted by the vessel and had endocrine effect on bone, thereby linking the dysfunction of the two tissues. In order to examine our hypothesis of a vascular–bone tissue crosstalk, we developed a novel model of aorta transplantation where the calcified aorta from 5/6 nephrectomized rats  was transplanted into a normal recipient rat  80]. In. In80]. We have explored the hypothesis on secretion of signal factors and a direct signaling from the vasculature to bone further in elaborated in vitro experiments. The aorta from normal rats and the calcified aorta from CKD rats were incubated ex vivo to detect possible secretions of signal molecules. We found that the calcified aorta secreted high amounts of sclerostin, illustrating that the activation of Wnt pathway in the calcifying vessel also results in secretion of sclerostin into the circulation  80]. We. We80]. In ex vivo incubation of normal and uremic calcified aortas, we have also detected high secretion of activin A by the calcified aorta rings (unpublished data). As previously mentioned, this member of the TGF-β superfamily is widely expressed in the organism and has multiple functions in normal physiology and pathology. It is thought to exacerbate the fibrosis process in uremic vasculopathy and calcification . In boneThese early data do support the notion that factors secreted by the calcified aorta affect bone cells and the existence of a direct signal from the calcified vasculature to bone tissue resulting in disturbed bone metabolism. Clarification of this vascular–bone tissue crosstalk may improve the understanding of the CKD-MBD syndrome and the common finding of vascular calcification and lower BMD in several clinical conditions.One cannot help to speculate over the divergent processes of demineralization of bone and mineralization of vessel, the so-called ‘calcification paradox’ . AlthougBone disorders in CKD include a wide range of disturbances, from low to high turnover diseases and mineralization defects. The CKD bone disease is classified according to the histological appearance by the TMV system  . The patIn our studies on the vascular–bone tissue crosstalk, we found an upregulation of bone’s expression of sclerostin and downregulation of classical Wnt target genes in normal rats transplanted with uremic calcified aortas . These rjck mouse). Already at 5 weeks, where kidney parameters of creatinine and BUN are similar to wild type, the bone of the jck mice showed increased sclerostin and phosphorylated β-catenin . At 5 weeks, jck mice have higher plasma levels of phosphate, FGF23, and 1,25 vitamin D, but bone-derived markers of bone resorption and RANK/RANKL/OPG system are similar to wild type [The clinical studies are backed up by experimental studies also supporting the notion of disturbances of Wnt pathway may play an important role in renal osteodystrophy. Sabbagh et al. used a model of progressive renal disease (juvenile cystic kidneys/ild type . Taken tild type . Dkk1 isild type . There iild type . The impild type . The calThe vascular and skeletal systems are closely connected in normal homeostasis and pathological conditions. CKD-MBD continues to be the most serious complication to CKD regarding morbidity and mortality. The identification of the direct negative impact of vascular calcification on bone metabolism adds to the complexity of the pathological processes in CKD-MBD, and so these secreted factors from the calcified vasculature may represent a treatment target related to the vascular–bone tissue crosstalk. This novel concept of pathological vascular–bone tissue crosstalk expands the understanding of vascular and bone disease in CKD and might lead to new strategies for prevention and treatment of CKD-MBD.


The latter is particularly thought to produce reactive oxygen species to elicit neurotoxicity under excessive levels.In this issue of the Journal, Xia et al. address the question of the possible role of iatrogenic iron from metal implants on the development of neurological diseases.,Eventually with aging, iron accumulates in the human brain, exhibiting an anatomical selective vulnerability demonstrated by a pronounced accumulation, particularly in the basal ganglia.This studyInvestigating the underlying mechanism of exogenous iron effects on neuronal function, mice hypodermically injected with iron dextran were examined and the study demonstrated increased serum iron levels and brain iron deposition. Astrocytic and microglial up-regulation of messenger ribonucleic acid (mRNA) and protein expression of a major cellular iron import protein, divalent metal transporter 1 (DMT1), was demonstrated with a contrasting down-regulation in neurons which showed increased mRNA levels of Nedd4 family interacting protein (Ndfip1), a key player in DMT1 degradation. Furthermore, the study demonstrated reactive astrogliosis, microglial activation, and apoptosis in mouse brains and behavioral changes in mice in response to the iron dextran treatment. Based on these findings, the study concludes that excess iron, including that from surgical implants in humans, should be considered a risk factor for neurodegenerative disorders (NDDs).,Iron involvement in the pathogenesis of NDDs is increasingly recognized by the emerging studies demonstrating the direct interaction between iron and the disease-hallmark proteins including alpha-synuclein in PD. Fundamentally, iron and alpha-synuclein are seemed to be mutually involved in their physiological and pathological cellular homeostasis.in vivo, the authors suggest the consideration of surgical metal implants as a risk factor for NDDs. Establishment of the suggested link merits future studies with a wider selection of disease subjects, including patients of other iron-associated NDDs such as AD, PSP, and MSA. Demonstration of protein pathology, or at least dysfunction of cellular protein processing systems characteristic of PD in the mice models could further provide a significant support of the proposed risk for NDDs.With a combination of clinical patient data analysis and experiments Let us step back and get the bird’s eye view on the findings of this study. Despite the demonstrated mechanism of neuroprotection (1) apoptosis is still prevalent in iron-treated mouse brains; and (2) PD occurrence still seems to be higher in individuals with histories of metal implantations. This discrepancy suggests that the iron-induced neuroprotective mechanisms might be limited or unsuccessful. To tackle this question, the ultimate fate of iron-packed glia and their effects on the CNS, and the cellular expression changes of other iron homeostatic proteins in response to elevated iron levels need to be further investigated. Iron homeostasis is mediated by a number of import, storage, and export proteins in each cell typeIn summary, this article addresses a critical relationship of external iron to NDD. Of particular interest is whether exogenous iron from a yet underestimated external source can act as a deal-breaker driving the brain into neurodegeneration, or the brain is able to mobilize a successful protective mechanism. The study by Xia et al.This work was supported by the Rossy Foundation.The authors declare no conflict of interest.


Nasib Alchoikani J Pediatr Urol. 2021 Apr;17(2):192.e1-192.e3.In this interesting paper the authors studied the intra-operative findings of patients underwent orchidopexy for ascending testes and concluded that the patients with ascending testes seem to share similar intra-operative findings with patients who have true undescended testes. The authors show the presence of abnormal attachment of the gubernaculum and epididymal anomalies in the groups studied. The gubernaculum seems to be the most important anatomical structure in the testicular migration process, by means of contraction and shortening, thus imposing traction strength on the testis and facilitates the transition of the testis by through the inguinal canal . Undesce


Dengue is one of the major public health problems in Sri Lanka. Its outbreak pattern depends on a multitude of drivers, including human mobility. Here we evaluate the impact of COVID–19 related mobility restriction (lockdown) on the risk of dengue in Sri Lanka.Two-stage hierarchical models were fitted using an interrupted time-series design based on the notified dengue cases, January 2015 to July 2020. In the first stage model, the district level impact was estimated using quasi-Poisson regression models while accounting for temporal trends. Estimates were pooled at zonal and national levels in the second stage model using meta-analysis. The influence of the extended period of school closure on dengue in children in the western province was compared to adults.Statistically significant and homogeneous reduction of dengue risk was observed at all levels during the lockdown. Overall an 88% reduction in risk  was observed at the national level. The highest impact was observed among children aged less than 19 years showing a 92% reduction . We observed higher impact in the dry zone having 91% reduction  compared to wet zone showing 83% reduction . There was no indication that the overall health-seeking behaviour for dengue had a substantial influence on these estimates.This study offers a broad understanding of the change in risk of dengue during the COVID–19 pandemic and associated mobility restrictions in Sri Lanka. The analysis using the mobility restrictions as a natural experiment suggests mobility patterns to be a very important driver of dengue transmission. Every year about 100 million symptomatic dengue cases occur with the highest-burden reported in Southeast Asia followed by Latin America. The burden is increasing and considered driven by population densities, human mobility, vector proliferation, and climate variability. Evaluation of the impact of human mobility on dengue transmission is methodologically challenging, but an important aspect in public health research.Aedes vectors and the spread of the dengue virus. The mobility restrictions due to the COVID-19 response in Sri Lanka provided a natural experiment to understand the importance of human mobility on dengue in an endemic setting.The ongoing COVID-19 pandemic has resulted in dramatic reductions in human mobility due to social distancing measures. These population-level interventions in most countries have changed the exposure patterns to Using interrupted time series design, we evaluated the impact of mobility restriction on dengue transmission in all 25 administrative districts, climate zones, and age groups in Sri Lanka. The results of this empirical study provided evidence of an overall statistically significant reduction of dengue during the period of mobility restriction in the country, which appears not to be related to changes in health seeking behaviour. Our study provides a methodological framework to quantify the impact of lockdown interventions on dengue transmission and its heterogeneous nature at different geographical, climatic, and socio-demographic levels within and between countries. Dengue epidemics have become a serious concern with increasing morbidity and mortality in tropical and sub-tropical regions globally . It is eSri Lanka, a tropical island in the Indian Ocean, with a population of 21 million, has reported dengue cases since the early 1960s ,8. From However, in 2019, the mid-year peak in reported cases was shifted to the latter half of the year with most of the cases being reported during November and December. This trend continued into early 2020 with a total of 11,607 cases reported in January 2020 which is higher than the reported case 10,927) for the same period during the epidemic year 2017 [,927 for th March, declaring public holidays and imposing quarantine curfew throughout the entire island since 20th March [th March closing airports. After achieving acceptable control of COVID-19 transmission in the country, on 11th May, the government decided to restore normalcy in life while maintaining quarantine curfew. The curfew was completely lifted from 28th June 2020. Even during the curfew period, all health care institutions remained open and accessible to those who were in need.The government of Sri Lanka responded proactively to the global COVID-19 pandemic by imposing strict mobility restrictions throughout the country from March 2020 ,15. Thisth March . People The population-wide mobility restriction imposed as a response to the COVID-19 epidemic created an opportunity to evaluate the dengue epidemic before and after mobility restriction and estimate the counterfactual effect on human mobility on dengue transmission. Here we compared epidemiological characteristics of dengue transmission during the first (January to March) and second (April to June) quarters of 2020 with that of the averaged reporting of the preceding 5 years (2015–2019) in all districts in Sri Lanka. Further, we evaluated the impact of mobility restriction on dengue transmission patterns in all districts and dry zone, wet zone and national levels using the two-staged interrupted time series (ITS) method . The impMonthly dengue surveillance data for the study period January 2015 to July 2020 for each of the 25 administrative districts in the country were obtained from the National Communicable Disease Surveillance System . The sysThe mode and the timeframe of COVID-19 preventive interventions were obtained from the press release published on the official website of the Department of Government Information which was identified as the competent authority for circulation of COVID-19 related information to the public . A time Analysis of the impact of lockdown on dengue across Sri Lanka was first assessed descriptively and then analytically using an ITS design. Stratified analysis was conducted to capture different seasonal transmission dynamics observed in the wet zone and the dry zone. The Western Province situated within the wet zone was considered as a separate entity due to the historically highest prevalence of dengue. The western province was further divided into two age strata i.e., age groups 0 to 19 years (children) and more than 20 years (adults) to capture the potential influence on the extended period of school closure on dengue transmission among children. Each stratum was then divided into a pre and post-lockdown period. The first quarter of the year 2020  was considered as pre-lockdown period and the second quarter  was considered as post-lockdown period. This assumed any potential lockdown effects were delayed by two weeks from its onset in mid-March.In the descriptive analysis, the five-year average (from 2015 to 2019) of the first and second quarters were taken as the comparator. The quarterly average of reported dengue cases per 100,000 population in each district was calculated to obtain quarterly expected (five-year average) and observed (the year 2020) values. Standardized incidence ratios (SIR) were calculated by dividing observed by expected dengue case counts in each quarter.ITS analysis is a rigorous quasi-experimental research design for studying the effectiveness of such large-scale programs ,19,20. Wt is time in months from January 2015 to July 2020; Dit is the aggregated monthly dengue cases from 2015 to 2020 in each i district at time t. αi is the model intercept in each i division. Yearit represents each year from 2015 to 2020. Monthit represents months from January to December in each year. The model was specified by fixed-effect variable for the year (Yearit), and month (Monthit) with the corresponding parameters (β1i) and (β2i) respectively to account for yearly variability, seasonality in risk and general time trends in dengue cases over the 67 months of the study period. The indicator variable for mobility, (Lockdownit) marked the onset of the lockdown leading to reduced mobility and was introduced as a binary dummy variable which set to zero during the pre-intervention period and to one during the lockdown period, i.e., April 2020 onwards. Estimates for the counterfactual scenario was obtained by setting the variable Lockdownit to zero during the lockdown period. Relative risk along with a 95% confidence interval was calculated by exponentiation of the model coefficient (β3i) of the intervention (Lockdownit) variable. We developed the models by a rigorous investigation of all variables, including lagged effects, by model fit statistics, and model residual diagnostics  level and the second stage meta-analysis at the district level. We used weekly dengue and climate data from 2010 for the analysis. The model was specified by a spline function for rainfall lag strata up to 12 weeks (Rain–lag 5 to 8 and Rain–lag 9 to 12), temperature lag up to 12 weeks (Temperature–lag 5 to 8 and Temperature–lag 9 to 12). The selected lag dimensions for climate variables in the models were based on our findings for the same study area using distributed lag non–linear models in a previous study conducted in the same district .The sensitivity analysis was further extended to evaluate the potential impact of lockdown interventions on the health care seeking behaviour in the Kalutara district. We calculated the SIR for total hospital admissions  and for other main communicable diseases in the district for the same study period. Data on non-severe dengue (dengue fever) and severe dengue (dengue haemorrhagic fever) was extracted from one of the hospitals in Kalutara district to investigate the potential change in seeking health care for milder dengue. The ratio between dengue fever and dengue haemorrhagic fever for each year from 2015 to 2020 was calculated and compared as a proxy for the direct impact of lockdown on dengue admissions. A detailed description of the climate adjustment and the evaluation of the impact of lockdown intervention on health care seeking behaviour is given in the supporting information .A total of 451,299 suspected dengue cases were reported for the study period. The wet zone which includes nine districts, Colombo, Gampaha, Kalutara, Kandy, Kegalla, Nuwara-Eliya, Ratnapura, Galle and Matara reported 301,944 (66.9%) cases. The dry zone which has thirteen districts  reported 110,834 (24.6%) cases. The rest of the cases  were reported from the intermediate zone . Out of nine provinces in the country, the majority  of dengue cases were reported from the Western province which is the most populous province. The Western province is situated in the wet zone and has three districts; Colombo (the main metropolitan area in Sri Lanka), Gampaha, and Kalutara.st and 2nd quarters from 2015 to 2019. In the first quarter of 2020, more than the average number of dengue cases were reported predominantly among several districts in the dry zone. However, following the COVID-19 lockdown from mid-March, there was a ubiquitous island-wide reduction of dengue cases in the second quarter of 2020 . The estimate was not statistically different from the first stage model estimate of 0.25  obtained for Kalutara through the main analysis  reduction of dengue risk across the nation involving both wet and dry zones.th March by closing schools and universities, banning public gatherings and imposing a curfew. As evident by the Google community mobility patterns, these social distancing measures dramatically reduced the mobility of the people outside the residence [The simultaneous reduction of dengue risk in two climate zones with different transmission patterns coincided with the proactive COVID-19 preventive interventions. The government encouraged the people to stay at home from as early as 12esidence . In Sri esidence –27.Aedes vectors [During the lockdown period, the dengue outbreak control program conducted with civil–military partnership was interrupted. The public health staff and the military were heavily involved with COVID-19 mitigation activities with less emphasis on dengue source reduction . However vectors . TherefoThe dry zone of Sri Lanka, with a larger landmass and having a lower population density showed a higher impact of mobility restriction  compared to the wet zone  and to the more urban western province which is a part of the wet zone . It has been previously observed that secondary dengue outbreaks were initiated when infected people from endemic wet zone cross the district boundaries and travel into the dry zone areas. This phenomenon was significantly restricted during the 2020 strict lockdown period (e.g. migrant construction and apparel workers were restricted to their boarding places with no access to their home-towns). Low population densities, differences in the climate factors, distribution and type of vector breeding places, heard immunity to circulating virus and human mobility patterns in the dry zone compared to the wet zone may have contributed to observed sharp decline in the model predicted dengue cases with intervention in the dry zone. The impact of the community mobilization campaign as a continued behavioural response to a large dengue outbreak that occurred during the latter part of 2019 and extending to early 2020 in the dry zone may also have contributed. The local communities might have been motivated to actively remove common breeding places in and around dwellings during the extended homestay.th March to 6th July has had nearly 1.5 times the higher impact on the dengue incidence among children when compared to adults.No public health intervention in the past had previously achieved a reduction of the dengue cases below 40 per 100,000 per quarter in the western province [Aedes vectors during day time [In Singapore, in 2020, the social distancing measures had an opposite impact causing an increased of over 37.2% in the dengue cases from the baseline . It was day time . Accordiday time . The hetday time –36.In Sri Lanka, an integrated surveillance system of communicable diseases includes dengue and has an Island wide coverage through trained and dedicated clinical and public health staff. National surveillance data are based on timely reports with a high yield capturing symptomatic dengue patients seeking medical care based on standard surveillance classification. Once detected cases are reported to the respective public health medical officer of the area where the patient resides. Dengue is a notifiable disease through the surveillance system since 1996. More recently, in 2010, a web-based early-warning sentinel surveillance system was established in the major hospitals across the country to augment the existing system. Despite the lock-down measures, both systems were operating with a weekly feedback mechanism in place as part of the regular monitoring and evaluation process. During the lockdown period, outpatient departments and inpatient care in all curative institutions were functioning without disruption. Community awareness on dengue prevention continued and early health-seeking for febrile patients was encouraged. Despite quarantine curfew in place, the sick people were allowed access to the hospitals round the clock. Mobile clinics were conducted particularly in strict lockdown areas with active COVID-19 clusters to attend to the acute health needs of people. National guidelines were issued by the Ministry of Health on managing dengue during COVID–19 epidemic . If any The non-availability of the climate information for all 25 districts at the time of this analysis could be one of the limitations. Due to the absence of climate information in the models, the confounding effect of climate may not be captured completely. However, our sensitivity analysis conducted with rainfall and temperature data in ten sub-district locations in Kalutara showed that the meta estimate  which was not significantly different from the district estimate obtained for Kalutara in the first stage models  without climate information. Furthermore, it was difficult to verify the vector abundance in relation to the observed reduction in the dengue cases as the door-to-door vector surveillance activities in and around high-risk localities also have been disrupted to some extent during the lock-down period in Sri Lanka.Considering the findings of this unique natural experimental situation, we emphasize the importance of vector control in schools, workplaces, industrial zones, hospitals, and public places where people tend to gather more during the daytime when the vector is also active. Better understanding human mobility is an important contributor, early identification of the clusters and explores the possibility of identifying those who have frequent travel requirements to and from high-risk areas for selective vector and fever surveillance would be important. Promoting usage of the newly introduced “Dengue Free Child” app  enables Our study offers a broader understanding of the epidemiology of dengue as a response to mobility restriction during the initial months of the COVID–19 pandemic in Sri Lanka. The finding highlights human mobility is a strong driver of the incidence and nationwide spread of dengue. The methodology can be used to explain heterogeneous outcomes of mobility restriction on dengue across regions and subgroups and its public health implications on prevention and control.S1 Text(DOCX)Click here for additional data file.S2 Text(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


In esthetic restorations of anterior teeth the clinician has to manage several aspects in order to have a predictable outcome. A deep knowledge of the anatomy as well as the adhesive procedures and the optical properties of resin-based composites are mandatory to achieve esthetic results. Contemporary restorative materials present either several shades and different translucency properties and therefore they are able to mimic teeth’s optical behavior thus providing a natural aspect to anterior restorations. The wrong thickness of different composite layers may provide unpleasant results such as low value (grayish) restorations that often requires reintervention. A precise step-by-step procedure is therefore mandatory to provide the proper shade at the correct place. There is therefore the need of some corrections and adjustments during the layer procedure in order to avoid errors in shade positioning that could affect final result. The authors present a case series  treated with the proposed technique with up to five years follow-up. Resin based composite (RBC) materials show good clinical performance for Class III, IV, shape modification and diastema closure providing excellent results with minimal biological costs ,2,3,4,5.Common errors are generally wrong translucent (enamel) thickness that could result in a low value (grayish) outcome or the revealing of the opaque dentinal layer during the finishing of the restoration ,15. ThesThe proposed technique is described in detail in A healthy 10-year boy presented to the dental office after traumatic accident during sport activity. Upper central incisor presented horizontal fracture with no pulp exposure . Tooth f2  for 20 s. A thin layer of composite enamel  was applied on the silicone index in order to reproduce the palatal wall. The silicone index was then repositioned in the mouth and the palatal composite was adapted to the palatal margin of the preparation and light cured. Another increment was then applied to reproduce the incisal margin. The silicone index was then removed : Once the frame was completed, a very meticulous analysis was performed on the accuracy of the three components of the frame: palatal wall, incisal margin and interproximal walls. Before placing in fact, the dentinal/opaque material, several aspects must be analyzed about the frame: the correct thickness of the palatal wall, the correct position, design and thickness of the incisal margin, the correct emergence, position, thickness and height of the inter-proximal walls. Every deviation from the ideal thickness, position and emergence of the abovementioned elements could affect the result if not corrected in advance.Step 2 (modification): The frame therefore is adjusted adding or removing composite material before beginning the internal dentinal body layering.Burs ;Discs  ;Abrasive stripes ;EVA points .Excesses external to the frame can be reduced with:The internal ones can be reduced with diamond burs  .Step 3 (adhesive procedure): After frame modification, dust was removed with strong air pressure and internal surface was treated with a silane coupling agent  applied with a microbrush in a thin layer and allowed to react for 60 s. An adhesive  was applied following manufacturer’s instructions, and photoactivated for 20 s .A single shade of dentin  was then applied placing separate increments  to buildFinishing and polishing procedures were performed with a diamond bur , silicone points  brushes , and diamond pastes  . SatisfaA 42-year old woman presented to the clinic with esthetic concerns related to her upper left lateral incisor . Pre-opeA 27-years lady presented to the dental office after traumatic accident . Tooth #Once completed, the two frames  showed internal and external excesses that were reduced using diamond burs and discs  strictlyA 53-years old man presented to the dental office asking for the restoration of the mesial incisal angles of both central incisors . After pA healthy 38-year-old man referred to the dental office for the esthetic rehabilitation of left central maxillary incisor . Shade wA healthy 23-year-old man referred to the dental office for the esthetic rehabilitation of right central maxillary incisor . Shade wExcesses were removed from the frame  and silaEsthetic direct restorations in anterior teeth show median overall success rate of 95% after 10 years for Class III and of 90% for Class IV . In respNatural teeth are characterized both from shades and from opaque and translucent areas. Contemporary composites can emulate both characteristics, providing therefore, the clinician powerful tools to face esthetic challenges. The preliminary color analysis of a tooth generally involves a basic shade selection and the detection of opaque and translucent areas . CommercA wise balance of these opaque and translucent shades represents the key factor in obtaining reliable esthetic outcomes and great attention should be paid for their correct positioning ,27. NeveIn this article, a step-by-step procedure aimed at solving this issue is therefore proposed. There are several layering techniques proposed for anterior esthetic restorations. The one adopted from the authors of the present article is based on the completion of a frame before applying the dentinal body and finally the superficial outer enamel layer. The frame is defined by palatal wall, incisal margin, and interproximal wall/s. Despite the preliminary color and opacity chart can help the clinician in applying correct shades in proper place, some discrepancies in thickness, outline and position can occur. A too thick incisal opaque margin could for example affect the incisal third of a restoration compromising the incisal halo effect. Discrepancies in interproximal walls can also affect final result while they could reduce the volume to be left for dentinal anatomy. Not only the thickness of the abovementioned portions is important. The correct shape, height and emergence of the interproximal walls can provide the clinician many references to take advantage of in managing the thickness of the outer layer that is crucial for the value of the restorations as supported by several authors ,29. The Composite repair is a reliable procedure that is generally carried out either immediately  or on aged composite restorations (in case of secondary caries or fracture) ,32,33.Dieckmann et al.  reported2 sandblasting. This is supported by Dieckmann et al. and by Kupiec KA and Barkmeier WW that reported no statistical differences in repair performed with or without sandblasting [Regarding the surface treatment, the proposed technique involves the use of abrasive instruments  and do not advocate the use of AlOblasting ,41. Regablasting . Conversblasting ,43.In the proposed procedure, a coupling agent was employed for the following reasons: creation of covalent bonds of exposed fillers; copolymerization with methacrylate groups of repair material ; increasThe proposed technique, strictly applied in all cases, resulted in clinical acceptable results despite of the different composite used. This technique aims to allow the clinician to precisely manage opacities and translucencies of anterior composite restorations therefore could help the clinician in achieving reliable esthetic results. Among the limitations of the proposed technique there is the absence of clinical trials that could validate the procedure and the use of only one adhesive type (E&R three steps).The proposed technique is an easy procedure aimed to reduce esthetic unpleasant results in direct anterior restorations. The precise execution of the frame and its eventual modification allows the clinician to get more predictable results in terms of shade and translucency management.Modification of composite frame during layering procedures, can be, therefore, considered a safe and reliable approach to enhance the esthetic outcome of anterior composite restorations.


Escherichia coli. This work builds upon a transformer-based neural network framework designed for prokaryotic genome annotation purposes. We find that the majority of subunits (attention heads) of the model are specialized towards identifying transcription factors and are able to successfully characterize both their binding sites and consensus sequences, uncovering both well-known and potentially novel elements involved in the initiation of the transcription process. With the specialization of the attention heads occurring automatically, we believe transformer models to be of high interest towards the creation of explainable neural networks in this field.The effectiveness of deep learning methods can be largely attributed to the automated extraction of relevant features from raw data. In the field of functional genomics, this generally concerns the automatic selection of relevant nucleotide motifs from DNA sequences. To benefit from automated learning methods, new strategies are required that unveil the decision-making process of trained models. In this paper, we present a new approach that has been successful in gathering insights on the transcription process in  Deep learning techniques are increasingly obtaining state-of-the-art performances for a multitude of prediction tasks in genomics . CapableDeep learning has offered solutions for functional genomics where the current understanding of biological processes is insufficient , 2, 6. AToday, the main contributions towards dissecting the workings of neural networks are achieved by mapping the importance of input features towards the model output, either through investigation of the gradient  or permuIn a previous study, we introduced a model for performing annotation tasks on the genomic sequences of prokaryotes, achieving state-of-the-art results for the identification of transcription start sites (TSSs), translation initiation sites and methylation sites. This model is based on the transformer-XL architecture, first introduced for natural language processing . Vaswanicument}.  found thin vivo methodologies for the detection of TSSs applied on Escherichia coli. Based on four independent data sets, we curate an improved set of annotations; (ii) we lay out our approach to characterize the function of attention heads. Many of these are specialized towards detecting the presence of regulatory elements based on sequence and positional information; (iii) using this information, we offer a comparison with existing knowledge on the mechanisms involved during the transcription process. As such, we link existing findings to literature and discuss their relevance; and (iv) using the model output predictions along the full genome sequence, we get a better understanding into the model characteristics and potential flaws. Based on these results, we conclude that the analysis of the discussed models for genome annotation tasks offer singular potential for extracting biological meaning, where the discussed techniques are applicable for any genome annotation task.This work offers several contributions: (i) we evaluate and compare the annotations of a variety of recent, mostly sequencing based, The creation of methodologies to facilitate the interpretation and role of nucleotide sequences started with mathematical formulas for consensus sequences , weight Despite efforts towards creating meaningful descriptors of nucleotide sequences, the understanding of how these influence biological processes is still lacking. Even with the help of early machine learning methods, the role of DNA for many biological processes is not properly understood. Today, the branch of deep learning is increasingly returning state-of-the-art performances for a variety of tasks in the field of functional genomics . By inteToday, two popular strategies exist that aim to offer insights into the workings of neural networks by evaluating the influence of the input features towards the output prediction. The 1st group achieves this by targeted permutation of the input features, first described by Zeiler cument}. . A multiocument}. and Zintcument}. . The othcument}.  in 2014 cument}. , 36. In cument}.  permute cument}. .in vivo experimental methods were compared for the detection and annotation of TSSs in prokaryotes: Cappable-seq by Etwiller ′- and 3′-end sequencing (SEnd-seq), by Ju To reduce the potential influence of noise, the annotations from a variety of recent, high-precision ument}., , retrievcument}. , retrievGiven its importance to the community, TSS listed by RegulonDB has also been included. RegulonDB features an up-to-date collection of manually curated and automated  TSSs from a plethora of independent sources . It has The model is built from the transformer-XL  architecThe full genome sequence is processed in consecutive segments of length mentclass2pt{minimThe multi-head attention applied in each residual block is methodologically identical. From each input hidden state In a previous study, we found that the contextual information embedded within the hidden states derived from single nucleotides is limited. Motifs formed from multiple neighboring nucleotides are generally deemed of greater importance towards biological processes. The addition of a convolutional layer allows the Positional information is required within the vectors ormances , 38.A model architecture was optimized for the detection of TSSs in a previous study, resulting in a model with a segment length of 512 nucleotides, six layers  and six attention heads within each residual block . To inclThe transformer-based model was trained using the full genome sequence, resulting in a total sample size of 9 283 204. The sizes of the positive and negative samples are listed in Table The Area Under the Receiver Operating Characteristics Curve (ROC AUC) and Area Under the Precision Recall Curve (PR AUC) are the used evaluation metrics. ROC AUC performances represent the area under the curve created by connecting the true positive  the TSS is detected by at least two separate data sets (maximum distance of five nucleotides); (2) the exact position of a shared TSS is determined by the majority vote. In case of a tie, the position is selected based on the novelty of the technique (SEnd-seq > SMRT-Cappable-Seq > Cappable-seq > RegulonDB); (3) finally, given the novelty of SEnd-seq and its high overlap with other data sets, all TSSs detected from this technique have been added. The resulting data set has a total of 6580 positively labeled positions, denoted as the curated set henceforth.In order to offer a means of validation about the quality of the annotations and the various steps followed to create the curated set, the performances of different models on the variety of sets have been trained and evaluated. The validation and test sets span identical regions of the genome. Therefore, performances of trained models on the test set can be easily evaluated for different annotations. Table The ROC/PR AUC scores show that the model trained on the curated annotations has the best overall performances on each of the test sets. The highest ROC AUC score on the SMRT-Cappable-seq and SEnd-seq test set is returned by the model trained on the curated TSS annotations. A possible explanation lies in a lower occurrence of false negative predictions in the model’s output due to a reduced number of missing annotations in the training data. False negative predictions come at a higher cost than false positive predictions, as the true positive rate , featuring a distance of 11 base pairs [For each attention head, sequence motifs were created based on the 50 highest scoring samples at the position with the highest averaged score  and S6.  complex . The motr Figure A. Anothen factor  binds. Tocument} , capturecument}.  report ton (TGn) , spanninse pairs . The 2ndDue to the complexity of the subject and the vast amount of literature, it is difficult to verify the function of attention heads without doing extensive research for all potential elements. In addition, not all mechanisms related to the transcription process are identified. Attention head 1 of layer 6 is highly specific for position –82, which is generally associated with the binding box of the Lac Repressor . AlternaTo further investigate the importance of each attention head with respect to the final model prediction, two strategies were applied. First, the correlation between the attention scores at each position with the model output is quantified using the Spearman’s rank correlation . As the A second strategy can compare the distribution of attention scores of two groups of samples, those that result both in the highest and lowest model predictions towards the presence of a TSS. An initial analysis highlights the importance of the attention heads that are specialized towards detecting ositions  and S4.The output of the transformer-based model results in a continuous output probability profile along the genome and is unlike previous studies , 27, 39.Increased model outputs are generally concurring on both the forward and reverse strand. Figure Transcription termination sites were obtained from Ju cument}.  and showThe advancements in the field of functional genomics and deep learning offer new opportunities towards gaining understanding of biological processes. Today, several methods exist that analyze the sensitivity of the input features with the model output and are applicable irrespective of the model architecture , 45. HowIn the case of functional genomics, the training of machine learning models on genome annotation tasks generally raises several challenges: (i) the imbalance between output classes, a negative set, which is several orders of magnitude larger than the positive set; (ii) the lack of high-quality annotations, where a high amount of noise is present; (iii) the dimensions of the input feature space, a multi-million length nucleotide sequence; and (iv) the biological variation of the annotation landscape, which often depends on, among other factors, the (sub)-species, the growth phase and the growth conditions of the organism.E. coli [We recently introduced a transformer architecture specialized for processing full genome sequences that proved to be well equipped to address the issue of class imbalance and dimensionality of the input space. State-of-the-art performances were returned on the annotation of TSSs, translation initiation sites and methylation sites . FollowiE. coli , the detin vivo expression profiles of Cappable-seq, SMRT-Cappable-seq and SEnd-seq were obtained from E. coli under optimal growth conditions, the detection of TSSs activated by specialized A curated set of TSSs was created and was shown to result in better performances than any individual set of annotations available. Potentially, the selection of TSSs from multiple data sets can prevent the model to fit on systematic noise of individual data sets, and make way with missing annotations. Discrepancies occurring between the different annotations might point to an influence of growth conditions on the expression profile. Given that the ar genes , are bouentclass1pt{minimaar genes , 27, 39,′ untranslated regions. To evaluate the impact of these multi-nucleotide peaks on the reported PR AUC scores, we averaged the output probabilities over larger The shifts in annotated TSSs might also be the result of a transcription process that is not fixed and allows a more flexible start site for 5Our findings establish the specialization of the attention heads, which reveals new opportunities towards explainable deep learning in this field. Focusing on the function of individual attention heads by evaluating the attention scores given to the upstream hidden states, we were able to characterize its functioning by answering a variety of questions, such as ‘From which positions within the promoter region does the attention head generally extract its information?’ and ‘What is the optimal nucleotide composition to obtain the best attention scores?’. Based on this information, we identified attention heads specialized towards well-known transcription factors Figure  and relaWe were able to characterize many important elements related to the biological process the model was trained for. As shown, the effectiveness of the transformer model to specialize certain attention heads towards identifying important promoter regions is enhanced by the convolutional layer that incorporates information from seven neighboring hidden states. This length works well for the binding boxes of the on heads  and S6 hIn addition to characterizing the individual attention heads, future approaches can be outlined that aim to map the relatedness between these attention heads and the model output. Higher-order interactions of the attention score profiles between different attention heads might uncover another source of information that offers understanding towards the biological process. Attention heads can, for example, be correlated in order to map transcription factors with binding sites larger than 7 nucleotides. Independent mechanisms for transcription might also be revealed this way. For example, this might be applicable for attention heads picking up alternative  element  or the e element . SupplemIn a previous study, it was urged that attention scores do not necessarily offer transparency into the neural network, as only a weak relation between attention scores and feature importance was found on natural language processing tasks . SimilarProcessing of the full genome is unique for this type of study, as the negative set on which the model is trained is normally subsampled , 27, 39.in vivo methodologies. To further improve model performances on TSSs, possible options include the incorporation of both sense and antisense up– and downstream of the evaluated position.The increased probabilities along the sense and antisense of noncoding regions corroborate mechanisms of interference as described in literature . SeveralThe low-transcriptional activity common for pseudogenes  could bein vivo validation methods, thereby fueling the discovery of new mechanisms.To conclude, the study reveals that the attention mechanism of the transformer-based models offers new opportunities for explainable deep learning in genomics. The availability of both sequence and positional information towards determining attention scores reveals the influence of both factors. Combined with the observation that attention heads perform distinct functions, we were able to determine both the motif and position related to several essential factors involved in the process. In addition, this property might result in correlation studies between the attention scores of different attention heads, potentially unveiling the relatedness between regulatory elements or mechanisms. Using all this information, it is possible that further analysis might be aided by In this work, we focus on exploring advantages that transformer networks could provide towards interpretable machine learning for genome annotation tasks.For detection of TSSs, we are able to characterize the function of attention heads within the model and find these to be specialized towards the detection of transcription factor-binding sites. Without any manual input, we can pinpoint the location of several essential promoter elements, their optimal sequence motifs and their importance.Although we provide this analysis for TSSs specifically, the methods outlined are generic and applicable for any genome annotation task.supplementary_files_bbab060Click here for additional data file.


This study examined whether and how pileup of insufficient sleep is associated with day-to-day trajectories of affective and physical well-being. Participants from the Midlife in the United States Study  provided diary data for eight days. Pileup of insufficient sleep was operationalized as the number of consecutive nights with <6 hours of sleep. Multilevel models evaluated the linear, quadratic, and cubic effects of pileup of insufficient sleep on daily well-being, adjusting for sociodemographic covariates. Daily negative affect increased and positive affect decreased in curvilinear fashion as the pileup of insufficient sleep increased. For example, daily negative affect increased, but the rate of increase decelerated as the pileup of insufficient sleep increased. In the days most distal to baseline, the rate of increase in negative affect accelerated again. Results were consistent for physical symptoms. Findings suggest that making efforts to break the vicious cycle of insufficient sleep may protect daily well-being.


Over-the-counter analgesics (OTCA) such as Paracetamol and Ibuprofen are frequently used by adolescents, and the route of administration and access at home allows unsupervised use. Psychological distress and pain occur simultaneously and are more common among females than among males. There is a dynamic interplay between on-label pain indications and psychological distress, and frequent OTCA use or misuse can exacerbate symptoms. No studies have to date provided an overview of frequent OTCA use in a larger population-based study. The current study used survey data to explore associations between and the relative predictive value of on-label pain indication and measures of psychological distress, together with sex differences for weekly OTCA use.This study included 349,528 adolescents aged 13–19. The data was collected annually between January 2014 and December 2018 as part of the Norwegian Young Data survey. Performance analysis was conducted to explore the relative roles and associations between on-label pain indication and psychological distress in weekly OTCA use. A mixed-effects logistic regression model was used to explore the unique contributions from four domains of on-label pain indication and psychological distress as measured by a combined measure of anxiety and depression (HSCL-10) and peer-bullying involvement as victims or bullies.Thirty percent of females and 13 % of males use OTCA weekly. Headache is the strongest on-label pain predictor of weekly OTCA use, followed by abdominal pain. Depression and anxiety are the strongest psychological predictor of weekly OTCA use, and higher symptom levels and being female increase the strength of this association. Anxiety and depression also predict weekly OTCA use after controlling for physiological pain.Sex, pain and anxiety and depression are inter-correlated and strong predictors of frequent OTCA use. Frequent OTCA use in the context of psychological distress may be a form of self-medication that can exacerbate symptoms and decrease psychosocial function. Longitudinal studies that explore causal trajectories between frequent on-label OTCA use and psychological distress are required. OTCA use among adolescents, and particularly among females, with anxiety and depression should be administered with caution and closely monitored. Acetaminophen is the major metabolite of acetanilide and phenacetin responsible for the analgesic effects . Predicting OCA*Gender by psychological distress was superior to the random effect model . The on-label model performed relatively better than the psychological distress model .Predicting OTCA*Sex by on-label indication (M1) performed better that the random effects only model [M0| M1;  |  and  for severe degrees of symptoms across sexes, and was  for moderate degrees of symptoms and  for severe degrees of symptoms in females (R2 = .356)  is a subtype of chronic daily headache caused by overuse of one or more analgesics. MOH prevalence is estimated in about 1–2% of the general population , and is Hereunder, a recent review concludes that parents are the most important source of information regarding the use of OTCA in adolescence and are also the main supplier of the medicine .The current study has several imitations that should be mentioned. A wide definition of OTCA is employed as no specific questions in the survey ask for the type of OTCA. The definition of OTCA does not take prescriptions from health services into account. Analgesics sold over the counter can also be introduced as treatment by primary and specialist healthcare services, which are also allowed to prescribe higher doses. The survey was conducted within school hours and chronic forms of pain may affect the degree of school attendance, and therefore also influence compliance in the current study. The results may have relevance to other forms of self-medication in different cultural settings, like the use of cannabinoids, in countries where these drugs are legal and widely available. Longitudinal studies will help explain causal trajectories that underlie associations between psychological distress and frequent OTCA use between sexes and should also include an assessment of the female menstrual cycle. The study was conducted in a large sample, and it addressed and revealed unobserved sex differences, and used performance analysis prior to conducting mixed-effects logistic regression modelling which is a strength.Headache is the dominant on-label indication related to weekly OTCA use in adolescence followed by abdominal pain. Females more often use OTCA at least weekly when they experience headache and abdominal pain. Anxiety and depression are associated with pain, and adolescents with a more severe degree of symptoms more often use OTCA at least weekly. This relative proportion is also larger in females with more severe degrees of symptoms who use OTCA at least weekly ten times more often than those with minimal symptoms. The current study provides evidence that requires health professionals to be careful when assessing OTCA use in adolescents with anxiety and depression.


The aim of this medical education case report was to outline the development and outcomes of a reverse-mentorship project that enabled cross-generational collaborative learning. The project took the shape of a philosophy of psychiatry journal club facilitated by a psychiatry core trainee in west London, UK.Reverse-mentorship reverses traditional roles of mentor and mentee. It is an increasingly fashionable concept in medical education. The junior mentors the senior clinician. The implicit learning outcomes include provision of a two-way learning process, development of mentoring skills for the more junior clinician and collaboration that builds social capital within the workplace. Reverse-mentorship is effective when the junior mentor is recognised for their expertise in a particular area. In this instance, the junior mentor has a special interest in the philosophy of psychiatry.Junior mentor and senior mentees formed a monthly journal club. The club tracked arguments from anti- and biological psychiatry on the meaning of mental illness. The debate offered insight into a semantic analysis of mental illness and a deeper conceptual understanding of medicine. The learning material derived from the core concepts of philosophy and mental health . The role of the mentor was to facilitate group discussion around arguments from relevant papers. A survey, adapted from a recent reverse-mentorship review article, measured the quality of educational experience for mentor and mentees.Overall, mentees (senior clinicians) agreed that the mentor (junior clinician) displayed attributes and behaviours for effective mentoring across most domains, including enthusiasm, effective communication, respect for mentee expertise and active listening to the needs of the mentee. The mentor was particularly impressed with the mentees’ openness to learn new concepts and respect shown. General reflections on the experience of reverse-mentorship were positive overall. A thematic review highlighted particular aspects, including: a good way to learn a new skill and great opportunity to develop professional skills of mentoring.The importance of mentoring in medical education is well established. Reverse-mentorship is a new concept that looks to harness the unique qualities of millennials, including their aptitudes for empowerment, innovation and collaboration. This medical education case report shows that an enthusiastic junior clinician can successfully pilot an educational-mentoring scheme aimed at senior clinicians. To make more explicit the intuitive benefits of reverse-mentorship, longitudinal reviews are needed. However, this case report contributes important insights into this burgeoning field of medical education.


Post-coercion review is increasingly regarded as a mean to reduce the negative consequences of coercive interventions, including the development of posttraumatic symptoms. However, the efficacy of this intervention in preventing posttraumatic symptoms or PTSD has not been sufficiently studied. The objective of this study is to examine the influence of a single, standardized post-coercion review session on the development or exacerbation of PTSD symptoms in patients with psychotic disorders.In a multi-center, two-armed, randomized controlled trial, patients who experienced coercive interventions during current hospitalization were either randomized to standard treatment or an intervention group receiving a guideline-based, standardized reflecting review session. Factorial MANCOVA and subsequent ANCOVAs investigated the effects of the post-coercion reflecting review session on post-traumatic symptoms as measured by the subscales of the Impact of Events Scale-Revised (IES-R). Similarly, the effect of the intervention on the intensity of the peritraumatic reactions measured by the Peritraumatic Distress Inventory (PDI) was analyzed by conducting a factorial ANCOVA.N = 82 patients were included in an intention-to-treat analysis. MANCOVA and post hoc ANCOVAs revealed a significant main effect of the intervention for the IES-R subscales intrusion and hyperarousal, when controlling for levels of peritraumatic distress, whereby intervention group participants presented lower respective mean scores. There was no significant difference regarding the intensity of the peritraumatic reaction.Standardized post-coercion review contributes to a reduction of the burden of PTSD symptoms in patients with psychotic disorders experiencing coercive interventions in acute settings and shall be recommended as a measure of trauma-informed care.The trial was registered at ClinicalTrials.gov (ID NCT03512925) on 01/30/2018 (retrospectively registered). Coercion in psychiatric care has been increasingly the focus of clinical and scientific attention, mainly due to legal, ethical and clinical issues raised by the use of coercive measures such as mechanical restraint or seclusion. Although their life-saving potential is undisputed in emergency scenarios such as a delirium tremens, their use should be restricted to situations in which other alternatives have been exhausted . MoreoveConcurrently, the presence and management of trauma experiences and related post-traumatic stress disorder (PTSD) among patients suffering from severe mental disorders such as psychosis has raised much attention over the last decades. Previous works showed very high rates of traumatic experiences such as sexual abuse and experience of violence among patients suffering from psychoses , 4. In a..The performed multivariate analysis (MANCOVA) across all three IES-R subscales with intervention and gender as independent factors and the mean PDI score as covariate showed a significant effect of the intervention at the multivariate level, Pillai’s trace = 0.109, Subsequent univariate ANCOVAs using the different IES-R subscales as dependent variables, intervention and gender as independent variables and mean PDI score as a covariate were performed. Results are summarized in Table X2 (1) = 3.614, p = 0.057.When analyzing the clinical probability of PTSD across the studied sample using the formula proposed by Maercker et al., results highlight that 13 patients (28.3%) in the control group and 4 (11.1%) in the intervention group showed a high diagnostic probability of having a PTSD . This diThe results of this RCT suggest for the first time a beneficial effect of post-coercion review sessions on the development of certain symptoms that might be indicators of the development of PTSD after coercive interventions in patients with psychotic disorders. The performed analysis indicated that patients who underwent a standardized post-coercion review showed significantly lower levels of intrusion and hyperarousal symptoms as measured by the IES-R. Accordingly, a lower proportion of probable PTSD was found among patients who received the intervention compared to the control group. This difference was, however, only marginally significant, which is most probably linked to the fact that avoidance symptoms were not affected by the intervention and to a lack of statistical power. These findings, thus, highlight that post-coercion reviews might be a means of counteracting the negative effect of coercive measures on these symptoms known to be invalidating and pervasive in some patients.This result is not in keeping with the single previous study on this issue . HoweverTherefore, it can be discussed that the effect of the present intervention relates to its particular setting and its psychotherapeutic character. The close involvement of patients and the encouraged dialog with the staff members facilitates differentiation of emotions and exchange of subjective perceptions of the coercive situation. The given opportunity to repair the potentially damaged therapeutic relationship and to restore trust and respect being essential for self-worth and -efficacy (post coercion review denotes the option of joint crisis plans) in spite of the coercive intervention might be additional factors contributing to the reduction of PTSD symptoms.Beyond these results, this study confirmed the highly traumatic potential of coercive measures. Overall, about 70% of the included patients presented distinct peritraumatic reactions rendering them at risk of developing a PTSD. Accordingly, about 20% of the participants showed a high clinical probability of PTSD. These results are in line with previous works investigating the deleterious effect of coercive measures and traumatic experiences made within psychiatric settings , 9. TheyA number of limitations might have influenced our results. The study design did not encompass the assessment of previous traumatic experiences that might have been made outside of the psychiatric context, during previous inpatient hospitalizations or through the experience of psychotic states. The possible association between these previous experiences and the severity of the reaction to coercive interventions should be studied in further works. The retrospective assessment of the peritraumatic reaction and PTSD symptoms weeks after the coercive intervention took place could also be considered as potential bias, as events that followed the coercive measure and that took place during the hospital stay might have influenced responses. However, the findings of the present study regarding the prevalence of PTSD are in line with previous works and it can, thus, be assumed that this bias did not significantly affect the results .Another limitation refers to the inclusion rate of patients which did not allow the research team to meet the expected inclusion goals during the planed recruitment period and, thus, resulted in a loss of power. Unfortunately, a relevant number of patients were not reached by the research team prior to their prompt or unplanned discharges. Willingness to participate in this study may have been associated with younger age as a potential indicator of lower chronicity. As selection bias is not fully avoidable in this and similar investigations, the studied sample must not be considered entirely representative of the inpatient population experiencing coercive measures. Future evaluations of post-coercion review should ensure that briefly hospitalized patients are receiving the foreseen intervention. A stronger focus on staff training or a stable team of moderating staff members might be useful to achieve this goal. The assessment of long-term effects of post-coercion review on PTSD symptoms and the development of manifest PTSD itself shall be focused on in future research.In summary, the developed standardized post-coercion review can be seen as an intervention that might contribute to the reduction of the burden of PTSD symptoms in severely ill patients subjected to coercive interventions. It can be implemented without greater effort and serves as an important tool to strengthen trauma-informed care in inpatient settings.


The genetic signature of modern Europeans is the cumulated result of millennia of discrete small-scale exchanges between multiple distinct population groups that performed a repeated cycle of movement, settlement, and interactions with each other. In this study we aimed to highlight one such minute genetic cycle in a sea of genetic interactions by reconstructing part of the genetic story of the migration, settlement, interaction, and legacy of what is today the Transylvanian Saxon. The analysis of the mitochondrial DNA control region of 13 medieval individuals from Feldioara necropolis  reveals a genetically heterogeneous group where all identified haplotypes are different. Most of the perceived maternal lineages are of Western Eurasian origin, except for the Central Asiatic haplogroup C seen in only one sample. Comparisons with historical and modern populations describe the contribution of the investigated Saxon settlers to the genetic history of this part of Europe. The present-day territory of Romania reunites four distinct historical regions , divided by the Carpathian Mountains Range that arches through the middle of the country. Until their unification into a single country in the 20th century, these provinces evolved rather independently, sometimes at odds with each other, for more than five centuries under different political and religious influences, being alternately challenged by distinct foreign powers . This taRepresenting, today, the central-western region of Romania, Transylvania was, in the Middle Ages, a province with a special identity within the Kingdom of Hungary, whose structure was gradually integrated during the 10th–13th centuries. This integration was carried out in stages and involved mainly two populations, known in historiography as the Szeklers and the Saxons. The first group, already existing in the kingdom since the 10th century, had a predominantly military role in defending the borders . The secThese colonization events imprinted the demographic history of populations from the current territory of Transylvania creating a cultural complex space, but the extent of these influences and other ancestral fine details remain largely unknown. Few studies have explored the genetic features of ethnic groups presently living in Romania in the hope of a better understanding of their biological and cultural identity, connections to other populations, and genetic legacy ,9,10. MoThe purpose of this study is to investigate the maternal  genetic diversity of medieval individuals discovered in Feldioara necropolis, potential members or descendants of a group that attained the farthest German colonization limit , and to The human archaeological remains analyzed in this study were discovered at Feldioara necropolis, also known as Marienburg (Brașov County). The cemetery was located in a square between the evangelical church and the parsonage. The excavations in this area were conducted between 1991 and 1995 , in 1998The routine funerary ritual in this necropolis involved the direct deposition of the adult bodies, without coffins, in graves with a step. This type of grave consists of a first larger rectangular hole continued by a smaller inferior pit outlining the shape and size of the human body, including a niche for the head. The funeral inventory is very poor, consisting of reused Roman coins, six Hungarian anonymous denars, and three lockrings with “S” -shaped ends . While tAll DNA extractions and amplifications of the selected archaeological samples were performed in a sterile environment in laboratories exclusively dedicated to ancient DNA research at the Interdisciplinary Research Institute on Bio-Nano-Sciences, “Babeș-Bolyai” University, Romania. All steps were conducted following precise guidelines and appropriate workflow protocols for aDNA processing previously described . Even thThe biological material used for DNA isolation was represented by dental samples with a very good preservation status for each tested historical individual. When available, multirooted teeth were selected. Prior to DNA extraction, each sample was decontaminated by mechanical removal of the external layer of the sample using a dental micro-drill . Then, the samples were UV-irradiated (254 nm) for 45 min. Routinely, 100 mg of powder was collected from the dentine of the tooth root and then used for aDNA isolation. A negative control  was processed for every batch of three samples during the extraction and DNA amplification steps to monitor potential contamination with exogenous DNA.The DNA extraction was performed in a designated pre-PCR area following a published silica-based protocol , but we The amplification of both hypervariable regions (HVR-I and HVR-II) of the human mitochondrial genome was performed using a set of eight mini-primer pairs, specially designed for highly degraded samples . BesidesThe mtDNA data generated for the individuals sampled in this study were compared to ancient and modern mtDNA sequences deposited in online databases to identify their genetic relatedness. This comparison aims to provide traces of evidence on past migratory routes and geographic origin of the analyzed population. The human mitochondrial sequences retrieved from publicly available data, detailed below, were first manually grouped based on their age , and then according to their geographic origin. The resulting datasets, one consisting of 21 historical populations (including the population considered in this study) and one, including 35 modern Eurasian populations, were used for haplogroup-based analysis, as well as for sequence-based analysis as detailed below. The population from Feldioara were compared to an ancient dataset consisting of 747 sequences of European populations and a Byzantine group : Lombardp-values) for each cluster using 10,000 bootstrap replications. The significance of each cluster in the resulting dendrogram was given as an approximately unbiased (AU) p-value shown at branches.Principal component analysis (PCA) was carried out based on mtDNA haplogroup frequencies of medieval and modern datasets. In total, 33 mitochondrial haplogroups were considered in the PCA of 21 ancient populations , whereasp-values of the historical populations were graphically represented as a level plot . The haplogroup affiliations were corroborated by diagnostic coding region sites which are all detailed in The maternal genetic composition of the analyzed group consisting of 13 samples indicates a high level of heterogeneity since it is comprised of 13 distinct haplotypes. The fact that these individuals do not share the same mtDNA profiles suggests that there are no strong maternal relationships among the analyzed group. The lack of identical maternal lineages might be a consequence of the small sample size, randomly selected from different parts of the necropolis, hardly any from adjacent burials . HoweverThe mitochondrial haplogroup architecture of the analyzed medieval group includes predominantly common west Eurasian clades , but not exclusively, as one of the sequences belonging to a middle-aged female (M26) was classified into haplogroup C, an east Eurasian lineage . The genThe sequence of the M26 sample showed a transition at 16357, characteristic of the C4a2 subclade. This variant is commonly found in the mtDNA pool of modern inhabitants of Siberia (particularly Evenks and Yakuts), while in Eastern Europe it is generally poorly represented ,54. ThisHaplogroup H, the prevailing lineage in the modern gene pool of Europeans, accounts for nearly 40% of the mtDNA variation in the investigated group, being represented by different clades . For most of them, further classification into derivative sub-lineages might have been possible with the aid of whole coding region data, but not for H7f, which is precisely identified due to the presence of the diagnostic mutation 16168T in the reconstructed sequence of the M42a sample. This particular haplotype seems rather scarce in historical samples as it has been revealed only by the mitochondrial signature of a female from Isola Sacra associated with the Imperial Rome . The HV9To gain more clues regarding the genetic affinities between the sampled individuals as a group and other historical and modern populations reported in literature, we conducted haplogroup-based and sequenced-based analyses. The PCA of 21 ancient populations was constructed based on mtDNA haplogroup frequencies , and visp values were visualized on a level plot  . The prep < 0.05) from two modern populations located in Central Asia (Kazakhstan and Kyrgyzstan), while the lowest genetic distances were observed relative to contemporary Slavic populations, mainly from Eastern Europe . The calculated Fst values between pairs of populations and their corresponding p-values, along with reference population information are given in The medieval Feldioara population showed significant differences  dominate among the sampled individuals, the exception being haplogroup C. Based on available published data, the C4a2 variant identified in one Feldioara sample, can be ultimately traced to the Eastern Asian Bronze Age. A possible scenario of the influx of this lineage into Feldioara might be related to the arrival of Hungarian conquerors in the Carpathian Basin at the end of the 9th century. The population genetic comparisons performed in this study potentially reveal some connections to other historical or modern Eurasian population, but these relations should be treated with care due to the low resolution of the emerging pattern shaped by the low samples size and the use of a partial mtDNA genomic marker. A much more complex and accurate picture of ancestry for the Transylvanian region might be provided by future genome-wide studies and increased number of samples.


The photosynthetic performance of crop plants under a variety of environmental factors and stress conditions, at the fundamental level, depends largely on the organization and structural flexibility of thylakoid membranes. These highly organized membranes accommodate virtually all protein complexes and additional compounds carrying out the light reactions of photosynthesis. Most regulatory mechanisms fine-tuning the photosynthetic functions affect the organization of thylakoid membranes at different levels of the structural complexity. In order to monitor these reorganizations, non-invasive techniques are of special value. On the mesoscopic scale, small-angle neutron scattering (SANS) has been shown to deliver statistically and spatially averaged information on the periodic organization of the thylakoid membranes in vivo and/or, in isolated thylakoids, under physiologically relevant conditions, without fixation or staining. More importantly, SANS investigations have revealed rapid reversible reorganizations on the timescale of several seconds and minutes. In this paper, we give a short introduction into the basics of SANS technique, advantages and limitations, and briefly overview recent advances and potential applications of this technique in the physiology and biotechnology of crop plants. We also discuss future perspectives of neutron crystallography and different neutron scattering techniques, which are anticipated to become more accessible and of more use in photosynthesis research at new facilities with higher fluxes and innovative instrumentation. Neutron scattering (NS) techniques are widely used for the structural and dynamical characterization of condensed matter. Neutrons—due to their electric neutrality—can penetrate deeply into most samples. Due to the nature of their interaction with the nuclei, they are especially sensitive to light atoms while exhibiting different scattering cross-sections for different isotopes of the same atom. Of particular importance for biology, the large difference in scattering length between hydrogen and deuterium allows contrast variation experiments, highlighting and hiding different constituents, e.g. lipids and proteins. Considering also their non-invasive nature, neutron scattering techniques are ideally suited for structural  scale. It offers information about the shape and size of macromolecules and molecular assemblies in solution, as well as about the morphology of fibrillar and lamellar structures Neylon , includia and c studied in benzene-d6 solutions  demonstrated the benefits of contrast matching allowing the separation of the scattering signals from that of the protein complex and the attached predeuterated detergent  and revealed its structural similarity to a stable mutant version of the protein, rendering the latter a potential structural analogue for the light-activated OCP , are in reasonable agreement with each other  of thylakoids cf. Fig. . This inF(Q). The form factor is the Fourier transform of the scattering length density distribution of the unit cell . The lattice formation of thylakoid membranes is described by the so-called structure factor, S(Q), and the scattering intensity is given by the product of |F(Q)|2S(Q)  for cyanobacteria . In this model, the thylakoids are represented as a bilayer containing intrinsic proteins, and inner  and outer aqueous phases, also containing proteins and protruding polypeptide sections. The presented advanced mathematical model, which uses different stacked layers with variable layer number and membrane rigidity (as the structure factor), demonstrated that the periodic order of the thylakoid membranes in different cyanobacterial cells could be simulated with reasonable precision  cannot be observed—probably due to the relatively poor periodicity of these thylakoids.The full mathematical model of the scattering profile from a stacked double bilayer membrane system of thylakoids  studies on reconstituted thylakoid lipid systems, also advanced our understanding on the self-assembly of thylakoid membranes. Experiments using different combinations of the four lipid constituents of thylakoid membranes deposited on silicon wafers and measured at varying relative humidity conditions highlighted the importance of bilayer and non-bilayer lipids and lipid phases  LHCII, coupled with absorption and fluorescence measurements, indicated correlation between the presence of conformational dynamics and the position of the excited electronic states in the antenna complex -binding niche of the D1 protein of Photosystem II (PSII)—resulting in impaired electron transport—influence the dynamics of the thylakoid membranes. Specific amino acids in the PQ-binding niche appeared to be essential for preserving a more rigid environment, required for efficient electron transport from QA to QB  in determining the mechanical properties of thylakoid membranes. -1,1-dimethylurea).)Neutron spin echo (NSE) experiments also provided a unique insight into the dynamics of thylakoid membranes in live cyanobacteria. Stingaciu and coworkers carried out the first in vivo measurements using NSE on Phaeodactylum tricornutum, in which DCMU did not block the light-induced swelling of thylakoid membranes but prevented the recovery of the membrane reorganizations upon dark-readaptation  in the protein structure previously determined by complementary techniques. Unfortunately, to this date, the number of protein structures determined via neutron crystallography is very limited, mostly due to flux limitations at existing neutron sources and due to the small number of neutron crystallography beamlines and the limited beam-time availability. Collecting neutron crystallography data from membrane proteins is especially challenging, due to the large crystal size required for the experiments—challenging to be grown from membrane proteins, which poses extra limitation on the study of proteins relevant in the light reactions of photosynthesis—almost exclusively taking place in the thylakoid membranes.Nevertheless, recent advancements in neutron macromolecular instrumentation—see e.g. (Meilleur et al. Apparently, the state-of-the-art technology is at the brink of addressing substantial questions in photosynthesis research, thus the next-generation instruments proposed (Coates and Robertson In this mini-review, our major aim was to show how neutron scattering techniques have advanced our knowledge about the structure, flexibility and dynamics of thylakoid membranes and other photosynthetic preparations. These techniques provide unique information on in vitro and in vivo systems and—when combined with complementary techniques of structural biology and functional measurements—will most certainly help testing the physiological performance of crop plants, and by this means, help designing and constructing or breeding plants with improved stress tolerance and/or productivity.


The endothelium is the single-cell monolayer that lines the entire vasculature. The endothelium has a barrier function to separate blood from organs and tissues but also has an increasingly appreciated role in anti-coagulation, vascular senescence, endocrine secretion, suppression of inflammation and beyond. In modern times, endothelial cells have been identified as the source of major endocrine and vaso-regulatory factors principally the dissolved lipophilic vosodilating gas, nitric oxide and the potent vascular constricting G protein receptor agonists, the peptide endothelin. The role of the endothelium can be conveniently conceptualized. Continued investigations of the mechanism of endothelial dysfunction will lead to novel therapies for cardiovascular disease. In this review, we discuss the impact of endothelial dysfunction on cardiovascular disease and assess the clinical relevance of endothelial dysfunction. The defining characteristics of biological systems are a set of functional organs and hemodynamic systems that facilitate the functioning of organs. A major feature is the manner in which these compartments are separated and in mammalian systems, this is via the single cell lining of blood vessels known as the endothelium ,2,3,4. AThe endothelium and endothelial cells have been the source of some of the major discoveries in human biology. The phenomenon of “endothelial dysfunction” has been in the literature for at least 25 years. Endothelial dysfunction (ED) can be narrowly defined as the vasoactive property or reduced vasodilatory capacity and more broadly as any changes that impact the vasoprotective homeostatic function of the endothelium. Endothelial cells were found to be the source of vasoconstricting 21 amino acid peptides known as endothelins which were discovered in 1987 by the Japanese group of Masaki and colleagues who were subsequently awarded the prestigious Tsukuba Prize ,21,22. TSeveral clinical studies speculate that ED leads to accelerated atherosclerosis. The two driving forces of ED that lead to atherosclerosis include the impact on vaso-regulation and chronic unresolving inflammation. In response to vascular injury, a plethora of pro-inflammatory cytokines and chemokines are released. Inflammatory cascades are complex involving pro- and anti-inflammatory molecules and cells. We previously described the multiple possible inflammatory targets, which represent therapeutic targets in this area ,24.In this review, we referred to the two aspects of ED—the narrow application related to vasodilatation and the broader context of inflammation. The functional and therapeutic difference in these two designations are exemplified by the actions of newer anti-diabetes drugs—these drugs including sodium-glucose transport protein 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) agonists have beneficial effects in large clinical trials where cardiovascular events and deaths are reduced by treatment with the index agents. It has however been very difficult to show consistent favorable actions on endothelial function assessed as enhanced vasodilation ,26. ThisOne area that very explicitly demonstrates the role of the endothelium is that of erectile function and dysfunction ,28. ErecA normal endothelium clearly regulates the biologically desirable quiescence of the vasculature and protects against atherosclerosis. The effects on the endothelium can be a very difficult area to study in vitro, especially in the context of blood pressure or inflammation. In vitro data typically arise from studies on isolated vascular cells, however, the impact of other factors such as NO, endothelin (ET)-1 and angiotensin (Ang)-II has multiple actions in driving the development of atherosclerosis. The role of the immune system and inflammation is very complex involving multiple biochemical and cellular mediators and, although the knowledge on the immune system is rapidly evolving, more insights are required before an amenable therapeutic intervention can be described. Human trials of anti-inflammatory strategies such as the use of anti-interleukin (IL)-1β antibodies in the CANTOS trial have shown promising results but the initial data arise in high-risk patients and secondary cardiovascular disease rather than the desirable targets occurring in younger people with early atherosclerosis ,32.SGLT2 inhibitors (SGLT2i) block the renal reabsorption of glucose and have a major effect on increasing glucose excretion and decreasing hyperglycaemia. SGLT2i reduce cardiovascular events and death in clinical trials . EmerginThe critical factor in this area is the relationship between ED and the initiation, development, progression and clinical manifestation being the rupture of an atherosclerotic plaque and a heart attack or stroke ,37. TherOne of the specific mechanisms that shows some promise is the role of the sub-endothelial retention of atherogenic lipoproteins being the initiating step in atherogenesis ,43,44. TClinical evaluation of ED corresponds to two expressions, impaired vasoactive regulation and inflammation and thrombotic response  Figure . While EFlow-mediated dilation (FMD) of the brachial artery is a frequently used method in clinical research that was introduced as a non-invasive measure of endothelial function in 1992 by Celemejer et al. . While tDespite its widespread use in research, FMD has not been adopted in routine practice as a clinical assessment tool. This is likely due to the technical requirements of FMD measurement, including access to suitable ultrasound devices and well-trained sonographers, as well as the time and expertise required for image analysis. Alternative methods that may be better suited to clinical settings have been developed and tested, including passive leg-movement hyperaemia ,75, whicMany traditional risk factors only account for around 50–60 percent of cardiovascular events . The uptIn conclusion, the study of ED has provided great insights into the initiation and progression of cardiovascular disease in patients. Many of the data from cardiovascular disease clinical trials can be explained by actions of drugs, directly or indirectly through their actions on risk factors, by their actions on the endothelium. Although the original concepts focused mostly on the role of the endothelium in vascular tone and the regulation of blood flow and pressure, it has more recently been recognized that the endothelium has a profound role in inflammation which underlies many disease states. The expression of pro-inflammatory molecules on the surface of endothelial cells and the secretion of pro-inflammatory, pro-atherosclerotic and pro-thrombotic substances indicate a role of the endothelium beyond its barrier function and role in vascular tone. It is likely that studies of endothelial function will become more common in the clinic and provide early insights into the development of atherosclerosis in patients also leading to the basis of early interventions to treat cardiovascular risk factors which will prevent or delay the occurrence of cardiovascular events in susceptible patients.The clinical assessment of ED may be useful in some circumstances, but as the mechanistic links between ED and atherosclerosis remain mostly speculative, this does not directly point to a mechanism-based therapeutic intervention and treatment remains empirical. Treatment to prevent atherosclerosis and cardiovascular disease should focus on traditional established cardiovascular risk factors whilst continuing studies delve into the mechanism of ED and the relationship between ED and the initiation and progression of atherosclerosis.