From 18 to 22 June 2017, the fifth biennial meeting of the International Society for Zinc Biology was held in conjunction with the final dissemination meeting of the Network for the Biology of Zinc (Zinc-Net) at the University of Central Lancashire, Cyprus campus. The meeting attracted over 160 participants, had 17 scientific symposia, 4 plenary speakers and 2 poster discussion sessions. In this report, we give an overview of the key themes of the meeting and some of the highlights from the scientific programme. The meeting was jointly hosted as a partnership between the International Society for Zinc Biology (ISZB) and the Network for the Biology of Zinc (Zinc-Net), a European Union Framework Programme, Horizon 2020-funded Collaboration in Science and Technology (COST) Action. It was the fifth biennial meeting of the ISZB, which is now celebrating a decade of achievements. The society was established to allow unique interactions between scientists interested in zinc biology and has been successful in fostering links between the chemical, biological and clinical fields of zinc biology . Zinc-NeThe meeting was the final major event of the 4 year COST Action, Zinc-Net. Therefore, it began with a symposium that celebrated the achievements of Zinc-Net, with presentations from each of the theme leaders in Chemical Biology, Biomarker Discovery and Clinical Coordination. A key note lecture by Professor Janet King , and Professor Nicola Lowe , with comments by Professor Mukhtiar Zaman , placed zinc research into a global context, highlighting the extent of zinc deficiency worldwide, with the highest prevalence of over 40% of the population in low- and middle-income countries. A display of posters showcasing some of the short-term scientific missions (laboratory exchange visits) that had been undertaken by early career researchers in the network were available to view .The first keynote lecture of the main conference was given by the Founding President of the ISZB, Professor Glen K. Andrews . He was introduced by the current president, Dr. Kathryn Taylor . Professor Andrews’s lecture recognised the contributions of key scientists whose research was of fundamental importance to the advancement of the field of zinc. These included the discovery of the metallothionein (MT) and the recognition that these proteins have a wide species distribution and are inducible by metals, the elucidation of a zinc-sensing mechanism of transcriptional regulation of MT genes, and the discovery of the Slc30a (ZnT) and Slc39a (ZIP) families of zinc transporters. These findings informed further studies of the mechanisms of zinc-dependent regulation of the ZIP proteins and their important physiological role. Each of these discoveries led to thousands of subsequent research publications and provided the foundation for much of the current research into the biology of zinc .The second keynote lecture, by Professor Hidenori Ichijo  discussed the physiological and pathophysiological roles of copper/zinc superoxide dismutase (SOD1) under conditions of zinc deficiency. The importance of understanding the molecular mechanism of zinc homeostasis in living organisms is highlighted by zinc’s essentiality in a wide variety of biological processes and the consequences of zinc deficiency in human health and disease. Ichijo’s group recently reported that SOD1 is one of the key factors to regulate cellular zinc homeostasis under zinc-deficient conditions. In zinc deficiency, SOD1 adopts an abnormal conformation and evokes endoplasmic reticulum (ER) stress through specific interaction with Derlin-1, a component of ER-associated degradation (ERAD) machinery, leading to the restoration of cellular homeostasis. Intriguingly, they found that wild-type SOD1 under zinc-deficient conditions and over 100 types of amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants share the common aberrant conformation, suggesting that wild-type SOD1 has a potential to exert neuronal toxicity under stress conditions. Professor Ichijo’s group have performed genome-wide siRNA screening to identify mediators of the conformational alteration in wild-type SOD1 under conditions of zinc deficiency, and he discussed the physiological and pathophysiological implications .2+; these serve as signaling agents in a number of biological processes, including neurotransmission. A new class of hybrid fluorescent sensors that facilitate the tunability of small molecule probes and the targetability of protein-based sensors was described, which can detect exogenous Zn2+ and endogenous mobile Zn2+ in response to reactive nitrogen species in live cells. The functional role of Zn2+ in the olfactory system was discussed, including results obtained from fluorescence imaging and electrophysiology recordings of live animals exposed to a variety of odours. Synaptically released mobile Zn2+ attenuates excitatory postsynaptic currents carried by N-methyl-d-aspartate (NMDA) receptors in the olfactory bulb, thus attenuating sensory input gain [Professor Stephen J. Lippard’s  keynote discussed the role of zinc probes as tools in the study of mobile Znput gain .Despite increasing access to sufficient food for all and significant achievements in reducing global hunger, micronutrient deficiencies (“hidden hunger”), especially zinc deficiency, still remain a major public health problem in the world. An inadequate daily intake of zinc is the major reason for the problem, particularly in the developing world, where extensive amounts of cereals are consumed with very low concentrations of bioavailable zinc. In the final keynote lecture, Professor Ismail Cakmak  described several agricultural strategies that are known to improve grain-zinc concentration, including conventional plant breeding, genetic engineering, and plant nutrition-based agronomy. In recent years, there has been an increase in the number of published reports showing that the maintenance of the high pool of zinc in the leaf tissue during the reproductive growth stage is required to achieve desirable concentrations of zinc in grains for human nutrition. Field experiments conducted in different countries under the HarvestZinc project  on maizeThe 17 scientific symposia that comprised the meeting can be grouped into three main themes: zinc in health and disease, zinc signalling, and zinc proteins and transporters. A summary of each is reported below and further up-to-date information on key topics in zinc research has been published by Rink  and zincCampylobacter jejuni (Cj), a common cause of acute human gastroenteritis, as an example of an important foodborne pathogen that targets different host niches with different metal challenges, how Cj adapts to different metal stresses within its different hosts was described. Exploring the functions and mechanisms of the zinc handling systems in Campylobacter will expand our understanding of how they contribute to infections. Zinc deficiency is linked to an increased susceptibility to bacterial infection, such as Streptococcus pyogenes (Group A Streptococcus—GAS), a Gram-positive human pathogen responsible for a wide spectrum of diseases ranging from pharyngitis and impetigo, to severe invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome. It was demonstrated that zinc homeostasis is an important contributor to GAS pathogenesis and innate immune defence against infection. Strategies to manipulate zinc homeostasis in order to reduce GAS infection were discussed.The role of zinc in immunity and infectious disease was explored and debated. Zinc is recognized as an important metal ion in relation to nutritional immunity, a process by which the immune system withholds micronutrients from potential invaders. An understanding of the underlying mechanisms by which host immune defences manipulate metal levels to attack invading microbes by metal-restriction and/or exposure to metal-excess may have considerable clinical significance. Giving Zinc-based therapeutics were explored in relation to cognitive disorders, cancer and cardiovascular disease. One of the critical cell processes that becomes dysregulated with age and also in disease, and which participates both directly and indirectly in cognitive function, is metal homeostasis and the neurochemistry of metalloproteins. This is particularly true for zinc, for which 10–15% of brain zinc exists in a chelatable form, primarily within synaptic vesicles at glutamatergic synapses, highlighting its potential importance in synaptic plasticity/cognition. Zinc dyshomeostasis has been implicated in dementia and autism spectrum disorders. Taken together with other supporting data in the literature, this demonstrates a critical role for zinc in cognitive function, and that it may be a therapeutic target for improving functional outcomes in health and disease.A significant body of evidence has shown that zinc plays important roles in metabolism and the development of metabolic disease. Zinc has a role in insulin secretion, insulin signalling and subsequent glucose metabolism. Low zinc status also promotes inflammatory stress, and using mouse models of atherosclerosis, vascular inflammation and plaque formation have been shown to be enhanced by marginal zinc deficiency. Increased intestinal permeability plays an important role in the onset of a variety of chronic inflammatory conditions and metabolic diseases, and zinc has been found to improve gut barrier integrity in vitro. In humans, a low-zinc diet is associated with a decrease in fatty acid desaturase enzyme 1 (FADS1) activity, lowered arachidonic acid incorporation into lipid subclasses, and an increase in DNA strand breaks, suggesting that FADS1 activity and DNA strand breaks respond to small changes in dietary zinc that may be provided by food fortification programmes.2+ homeostasis in chronic heart failure is linked to dysregulated intracellular Ca2+ responses, resulting in leakage of Ca2+ from the sarcoplasmic reticulum in cardiac tissue. Research describing the pathophysiological role of zinc in neurological disorders provided insights into possible novel therapeutic approaches. Examples included the role of zinc in triggering neuronal apoptosis and blocking optic nerve regeneration after injury, as well as the role of extracellular zinc in the modulation of the cytokine-induced pro-inflammatory response following brain ischaemia, which may contribute to impaired memory function. New research examining the pathophysiological role of extracellular Zn2+ in cognitive decline with aging was received with interest. Highlights within this theme also included new understandings of the molecular mechanisms for maintaining cellular zinc homeostasis and the use of novel zinc sensors, which can be activated by UV light or enzymes for the study of the dynamics of cellular “free” zinc.The roles of zinc in modulating cellular function in various disease states emerged as a key theme of this meeting. These included new advances in understanding how disrupted ZnOne of the most exciting areas of research over the last decade has been the discovery of the zinc transporter families ZIP and ZnT and the elucidation of their role in the control of cellular zinc homeostasis. Within this theme, the relationship between the loss of ZnT2 function within paneth cells and intestinal dysbiosis was discussed. This research revealed that genetic polymorphisms that influence the ZnT2 transporter function might lead to clinically relevant shifts in the intestinal microbiome of preterm infants, which is a fascinating new area of research relating to infant nutrition. Similarly, ZIP7 plays a critical role in ER function within connective tissue cells, such that a loss of the function of this transporter results in inhibited cell proliferation, preventing proper dermis formation. Highlights within this theme included potential new therapies for the treatment of cancer, linked to the inhibition of mitosis through the selective blocking of ZIP transporters. Additionally, the unexpected association between genetic mutations in ZIP13, zinc homeostasis and beige adipocyte biogenesis may contribute to new therapies for obesity and metabolic syndrome.Other zinc binding proteins also shared the limelight in this theme. Notably, the influence of zinc binding on protein folding and aggregation may have deleterious consequences if intracellular zinc homeostasis becomes imbalanced. Also discussed was the mechanism of the activation of the zinc-requiring ectoenzymes, defined as secretory, membrane bound, and organelle-resident enzymes, which play pivotal roles in numerous biological responses. One such example is tissue non-specific alkaline phosphatase, which is activated in a two-step process involving ZnT transporters.The conference was attended by over 160 scientists, including early career researchers (ECRs), many of whom presented posters in one of the two evening poster sessions. Both Zinc-Net and ISZB place a strong emphasis on providing training opportunities, capacity building and support for the next generation of zinc biologists. In a competitive process, ECRs were invited to present their research in two special symposia showcasing the work of these up and coming young scientists in this exciting field.Much of the research presented at this meeting had never been presented or published before. The meeting had a strict embargo on the photographing of slides without the presenters’ permission, and the abstracts, although made available to all participants, were not to be published in proceedings of the meeting. However, it was gratifying to observe that the atmosphere within the meeting was extremely open and collegiate, with new collaborations initiated and many animated discussions during the social, networking and poster events. The zinc research community is clearly thriving, and it is exciting to be a part of it.


Poor micro-scale environmental features, such as graffiti and broken windows, have been associated with crime and signs of social disorder with a potential impact on mental health. The aim of this study is to investigate the association between micro-scale environment and mental health problems in later life, including cognitive (cognitive impairment and dementia) and common mental disorders (depressive and anxiety symptoms).The method of visual image audits was used to collect micro-scale environmental data for 3590 participants in the Cognitive Function and Ageing Study II, a population-based multicentre cohort of people aged 65 or above in England. Multilevel logistic regression was used to examine the associations between the quality of micro-scale environment and mental health problems taking into account urban/rural difference.Poor quality of micro-scale environment was associated with nearly 20% increased odds of depressive  and anxiety symptoms  while the direction of association for cognitive disorders differed across urban and rural settings. Although higher odds of cognitive disorders were found in rural settings, living in a poor quality environment was associated with nearly twice higher odds of cognitive impairment  in urban conurbations but 20% lower odds in rural areas .The causal direction could not be fully determined due to the cross-sectional nature of the data. The visual nature of the environmental assessment tool means it likely does not fully capture features related to the availability of local support services, or opportunities for social participation and interaction.The quality of micro-scale environment appears to be important to mental health in older people. Interventions may incorporate the environmental aspect to reduce cognitive and common mental disorders. •Poor micro-scale features were related to depression and anxiety in later life.•The association for cognitive disorders differed across urban and rural settings.•Improvement of micro-scale environment may support mental health in older people. PhysicaIn addition to common mental disorders, recent studies have also identified environmental factors that may be related to cognitive disorders in later life. Evidence from epidemiological research supports the presence of urban/rural differences in dementia prevalence and cognitive function . Area deIn the recent five years, some studies have used visual streetscape images, including Google Street View, Bing Maps or omnidirectional imagery, to observe these micro-scale features and show good inter-method reliability between visual image audits and physical audits . In orde22.1This study was based on the Cognitive Function and Ageing Study II (CFAS II), a longitudinal population-based cohort of people aged 65 or above across three areas in England . Details of CFAS II have been described elsewhere . In brieThe analysis here was based on a sub-sample of 3590 (47.8%) community-based CFAS II participants. A random sample of postcodes in Cambridgeshire and Nottingham was selected to collect environmental data, which was linked to the baseline interview data. The choice of these two centres was based on urban/rural difference and geographical variation in health status and social disadvantage . CFAS II2.2Socio-demographic factors were obtained from the CFAS II baseline interview, including age, gender and education . Self-reported information on co-morbidity was used to measure the number of chronic conditions  and divided into three levels .Cognitive and common mental disorders were assessed using a structured interview of psychiatric symptoms and standardised cognitive tests. Cognitive impairment was defined here by Mini-Mental State Examination (MMSE) score 25 or below . Dementi2.3Environmental features at the micro-scale level were measured using the Residential Environmental Assessment Tool (REAT), a validated and observational instrument designed for measuring the quality of living environment within a given UK postcode, which includes approximately 18 households on average . The REAIn this study, the REAT assessment was conducted through visual image audits, which used Google Street View to virtually ‘walk through’ streets and assess the quality of living environment in local areas. The reliability of physical and visual image audits has been validated in our earlier study . Visual 2.4). In order to increase the statistical power of the analyses, these urban and rural categories were combined into three types: Urban Conurbation (Major and Minor Conurbation), Urban City and Town and Rural areas  based on the similarity of their environmental features.Postcodes of CFAS II participants were mapped onto Lower Super Output Areas (LSOA), small geographical units in the UK with an average of 1500 people per area, and linked to corresponding rural/urban categories in the 2011 Rural/Urban Classification for Small Areas Geographies . The claOffice for National Statistics, 2013b2.5A small number of postcodes had missing REAT data due to unavailable or unclear streetscape images. Since the distributions of individual level factors and urban rural categories were different across those with complete and missing REAT data, inverse probability weighting was used to adjust for missing data. The weights were calculated by age, gender, education, cognitive impairment, depressive and anxiety symptoms and rural/urban category.The association between mental health problems and environmental measurements was investigated by multilevel logistic regression. The analysis first examined the overall association between cognitive disorders, common mental disorders and the REAT groups adjusting for individual level factors , and missing data. To further take into account rural/urban difference in this sub-sample, interaction terms between the binary REAT groups and rural/urban categories were added to regression modelling to investigate whether the association between micro-scale environmental features and mental health problems differed according to urban/rural context. A likelihood ratio test was conducted to investigate the statistical significance of the interaction terms.A sensitivity analysis was conducted to further test for potential bias due to missing data. Based on our investigation, postcodes with missing data were more likely to comprise a poor quality residential environment . The 4263The REAT assessment was applied to 3590 CFAS II participants and 3164 (88%) had complete data . Among tThe results of depressive and anxiety symptoms are presented in 44.1This study investigated the associations between the quality of micro-scale environment and mental health using a multicentre study of older people in England. Higher REAT score  was generally associated with higher odds of mental health problems in older people. Living in rural settings was associated with higher odds of cognitive disorders but the direction of association with the REAT score varied across the three rural/urban categories. For people living in a postcode with a high REAT score, the odds of cognitive impairment was nearly twice higher than their counterparts in urban conurbations but was 20% lower in rural areas. Living in a postcode with a high REAT score was associated with nearly 20% higher odds of depressive and anxiety symptoms, and the relationships did not appear to vary across rural/urban categories. The estimates did not change substantially after including the missing data in the sensitivity analysis.4.2This study was based on a large population-based cohort of contemporary older people across urban and rural areas in England. A structured interview was used to assess cognitive function and common mental disorders with consistent diagnostic standards across different areas. The assessment targets the postcode areas closest to the residences of participants and this might address the issue of mismatch between geographical boundaries and activity space. The quality of micro-scale environment was assessed objectively using a valid instrument and the method of visual image audits. This avoided the potential same-source bias of using perceived environmental measurements and improved the limitation of small sample size in the previous UK-based studies .The causal direction between micro-scale environment and mental health could not be fully determined due to the cross-sectional nature of the data. The direction of association might be reversed if people with mental health problems moved to areas with a poor quality environment or have less capacity to maintain their property and local environment. Information on recent relocations was not available in the CFAS II interview. Although the number of people with complete data was over 3000, the statistical power to test the significance of effect sizes might be still limited, particularly for dementia. This analysis did not further include area deprivation or other small area level factors as the two sets of environmental measurements were based on different levels and aspects of the living environment. Since the role of small area level factors in the association was not clear, adding all these environmental factors might be inappropriate and lead to over-adjustment bias .Although REAT is a validated instrument which provides a simple assessment of several micro-scale features, it might not be sufficient to describe the variety of property and street level characteristics. For example, flats are likely to be main type of properties in highly urban areas. The height of flats has been suggested to be related to the concept of defensible space but is not covered by the REAT . The vis4.3High REAT score was consistently associated with higher odds of common mental disorders across rural/urban settings. The findings provide supportive evidence on the potential influence of social disorder on depressive and anxiety symptoms in later life. Older people living in areas with poor micro-scale features may experience stress, insecurity and lack of control, with a potential impact on mental health and well-being . ExistinPrevious studies have reported inconsistent relationships between micro-scale features and depression in the general population after adjusting for individual factors . In part4.4The association between micro-scale environmental features and cognitive disorders was moderated by rural/urban settings. The differential associations between cognitive disorders and the REAT score might correspond to the potential non-linear relationship for features of land use at the small area level, suggesting both high and low levels of land use mix were associated with increased odds of cognitive impairment . AlthougThis analysis found that living outside urban conurbations was associated with higher odds of cognitive disorders. These results correspond to some of the evidence in the literature  as well 4.5Micro-scale environmental features seem to have different relationships with cognitive and common mental disorders in rural areas. The reasons for this warrant further investigation on the underlying mechanisms in various types of rural settlements. The interplay of multiple environmental characteristics might provide possible explanations on these differences. For example, although living in rural areas might be related to isolation and loneliness, this analysis and previous studies did not find clear urban/rural differences in the prevalence of depression at older age when taking into account socio-demographic factors . Environ4.6This study provides evidence on the potential environmental determinants related to cognitive and common mental disorders in older people. Adequate maintenance of streets and properties and removal of the physical characteristics of social disorders might have a positive influence on reducing the risk of mental health problems, particularly in conurbations and metropolitan areas. Public health interventions may incorporate the environmental aspect to improve the quality of local environment as well as support mental health in later life. In rural areas, environmental stimulation might be an important component to maintain cognitive function in later life . Local sPotential pathways from environmental factors to individual mental health need to be further investigated in longitudinal studies. To explore mechanisms, it is important to consistently measure the trajectory of physical and mental health status, including robust measurement of lifestyle factors and recording of information on relocation over time. The interaction between older people and their local environments may be different when comparing the younger and older old, and between those with and without disability . These i


Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed. The plasma membranes of mammalian cells contain many different lipid species, but the distribution of sphingolipids within the plasma membrane and the mechanisms responsible for this organization are of particular interest. Sphingolipids function as structural components in cellular membranes, and they are metabolized to signaling molecules that modulate diverse cellular processes, ranging from apoptosis  and dynamic plasma membrane domains that are enriched with cholesterol, sphingolipids, and glycosylphosphatidylinositol (GPI)-anchored proteins Pike, . FavorabCeramide-rich domains in the plasma membrane have also been the subject of many studies. These domains are produced by the hydrolysis of sphingomyelin to ceramide by sphingomyelinase in response to stimuli  technique. High-resolution SIMS performed on a commercial instrument, the Cameca NanoSIMS 50, enables visualizing the distributions of metabolically incorporated stable isotope-labeled lipids in the plasma membranes of intact cells with better than 100 nm lateral resolution . The intensities of the secondary ions detected at each pixel reveal the elemental and isotopic composition at the surface of the sample. Because elemental composition cannot be used to distinguish between lipid species, the sphingolipids must be labeled with distinct stable isotopes to allow their identification with a NanoSIMS instrument. This is achieved by metabolic labeling with isotope-labeled lipid precursors  of the sample. This shallow depth of secondary ion ejection minimizes the detection of secondary ions from intracellular membranes, thereby restricting the analysis to the plasma membrane. The high-yielding monoatomic and diatomic secondary ions are collected by a mass spectrometer that can discriminate between ions that have the same nominal mass but different isotopic or elemental compositions  preserve the laminar structure of biological membranes and prevent lipid reorganization during sample dehydration and subsequent analysis , so the cells must be dehydrated prior to analysis. However, previous studies demonstrate that chemical fixation techniques that crosslink the proteins with glutaraldehyde and the lipids with osmium tetroxide  are presented first. Next, studies that employed affinity-labeled sphingolipids and fluorescent sphingolipid analogs to visualize the distributions of specific sphingolipid subspecies in the plasma membranes of unstimulated cells are described. This includes a brief account of the insights into plasma membrane organization that were acquired with super-resolution fluorescence techniques. Then the sphingolipid distributions that have been imaged in the plasma membranes of intact mammalian cells with high-resolution SIMS are summarized. Finally, the implications of these experimental results on our view of plasma membrane organization are discussed.Antibody binding to proteins on the surfaces of lymphocytes was first reported to induce the crosslinked proteins to form clusters that eventually segregate into a large patch, or “cap” at one end of the cell in 1971  or biotin that could be crosslinked with antibodies or avidin, respectively, to ensure that the glycosphingolipid crosslinker had no affinity for endogenous proteins. The crosslinking of these exogenously incorporated gangliosides in the membranes of lynphocytes induced the formation of large patches and caps  studies indicated a lack of true co-localization between GPI-anchored proteins and cholera toxin-labeled GM1 . Immunoimaging studies established that CD95 activation induces acid sphingomyelinase translocation to the cell surface and subsequent CD95 clustering  antibodies  cells  and phosphatidylcholine (BODIPY-PC) also formed micron-scale domains in the plasma membranes of erythrocytes  were employed in these experiments because the micrometer-scale hemagglutinin clusters in their plasma membranes were hypothesized to colocalize with lipid rafts  the lack of cholesterol- or hemagglutinin-enrichment in the sphingolipid domains that were detected in the plasma membranes of fibroblast cells with high-resolution SIMS , and specific, where different sphingolipid subspecies bind to different protein partners. Such specific, inducible, and transient sphingolipid-protein interactions could direct the segregation of different glycosphingolipid species within different microdomains in the plasma membrane (Fujita et al., The sensitivity of many cellular processes, including antigen capping and apoptosis, to enzymes that induce sphingolipid catabolism or drugs that inhibit sphingolipid biosynthesis can be attributed to the established role of sphingolipids and their metabolites as second messengers in diverse signaling processes (Hannun and Obeid, MK created figures and wrote the paper.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Encouraging results in the central nervous tissue might impulse the study of GDMs towards preclinical arena. In this mini-review article, we revise the most relevant literature on the interaction of GDMs with the spinal cord. Studies involving the implantation of these materials in vivo in the injured spinal cord are first discussed, followed by models with spinal cord slides ex vivo and a final description of selected results with neural cells in vitro. A closing debate of the major conclusions of these results is presented to boost the investigation of GDMs in the field.The attractiveness of graphene-derived materials (GDMs) for neural applications has fueled their exploration as components of biomaterial interfaces contacting the brain and the spinal cord. In the last years, an increasing body of work has been published on the ability of these materials to create biocompatible and biofunctional substrates able to promote the growth and activity of neural cells  The outstanding physico-chemical properties of carbon-based materials including carbon nanotubes (CNTs), carbon nanofibers and graphene are encouraging their exploration as attractive materials to interface the damaged neural tissue as part of novel platforms with potential utility in neural repair  affect thousands of people worldwide every year, leading devastating consequences such as sensory loss, paralysis and bowel/bladder dysfunctions that cost millions of dollars in medical expenses. According to the World Health Organization, although there is no reliable estimation of global prevalence, estimated annual incidence is 40–80 cases per million worldwide. Unfortunately, less than 1% of SCI patients achieve complete neurological recovery by hospital discharge .Forthwith after SCI, a rapid activation of glial cells and pericytes occurs, forming a complex healing tissue that is simultaneously beneficial and detrimental for neural repair  human spinal motor system relies more dependently on supraspinal control than in other mammals Capaday, ; and (b)in vivo tissue response of the injured rat spinal cord to the implantation of flexible and porous 3D scaffolds composed of reduced graphene oxide (rGO). These scaffolds were fabricated by using the ice segregation-induced self-assembly (ISISA) technique. The lesion model of choice was a right hemisection of approximately 8 mm3 at the C6 level, rostral to the bulk of triceps brachii motoneurons. This is a suitable model to evaluate therapeutic strategies aimed at promoting neural plasticity and repair. In the main experimental group, rGO scaffolds were placed at the lesion site and covered with a thin gelatin hydrogel film. Animals without injury and those hemisected but not receiving scaffolds served as control groups. In order to study the subacute tissue response to these implants, both locally  and systemically , rats were sacrificed at 10 days after surgery. The results revealed that these substrates allowed the formation of a soft interface at the injury site, with no significant differences in the fibroglial scar features with respect to lesions without scaffolds. Due to its porous structure, extracellular matrix molecules  and different kinds of cells were able to infiltrate and migrate to the inner parts of the scaffolds contributing to the stabilization of both the scaffold and the lesion site. Colonizing cells were mainly positive for vimentin  and the β receptor of the platelet-derived growth factor (a regulator of blood vessels formation and early hematopoiesis). In addition, pro-regenerative M2 macrophages were present both at the interface tissue-material and within the scaffold, which could be potentially involved in the initiation of neural repair responses. Finally, neural cell populations were preserved at the perilesional areas and no toxic systemic responses were found, thus indicating the positive biocompatibility of these GDMs with both neural and non-neural tissues once implanted in vivo.López-Dolado et al.  were firFueled by these encouraging findings, the authors focused on the tissue responses at 30 days after surgery  was injected in the OB core. By a simultaneous injection of a retroviral vector expressing the enhanced green fluorescent protein, proliferative cells and their progeny were labeled in the subependymal zone/rostral migratory stream of the OB core in the presence of rGO. Major findings revealed that rGO had no deleterious effects either on the survival (measured as number of cells and detection of apoptosis) of the resident populations of neurons and astrocytes or on the newly generated neurons (measured as number of cells). By looking at the number and morphology of microglia cells (Iba1+), authors evidenced that alterations on the microglial response at the OB were related to the injection itself rather than the presence of rGO.At the brain, Defterali et al.  exploredin vitro and in vivo. Under in vitro conditions, the exposure to high concentrations of rGO (100 μg/mL) produced a lower toxicity in both rat astrocytes and endothelial cells than rGO functionalized with poly(ethylene glycol) (PEG), which induced a dose- and time-dependent toxicity. For the in vivo studies, rGO and rGO-PEG were injected intravenously and their toxic effects on BBB integrity analyzed for different times. Both materials caused a notable downregulation of astrocyte markers (GFAP and connexin-43), endothelial tight (occludin) and adherens (β-catenin) junctions and basal lamina (laminin) at 3 h after administration. Interestingly, this effect disappeared after 7 days of exposure to rGO, while in the rGO-PEG group it was permanent and increased over time. The increase in proteins related to the antioxidant system, such as catalase, in the animals treated with rGO-PEG, accompanied by high levels of intracellular reactive oxygen species, pointed out oxidative stress as one of the main causes of rGO-PEG-mediated damage.Recent studies by Mendonça et al.  focused In vitro experiments also demonstrated that the number of KCl-responsive dorsal root ganglion neurons was greater when treated with highly magnetic rGO. Additional studies showed that the activity of both voltage-gated calcium channels and NMDA receptors was altered in the presence of highly magnetic rGO. The disturbance in the neuronal magnetic field increased nociceptive responsiveness, suggesting a role for the magnetic component of the electromagnetic field in neuronal transmission.Although more rarely explored, the magnetic properties of GDMs are also attracting attention for their application in neural scenarios. Specifically, magnetic GDMs can generate static magnetic fields (SMFs) that should be taken into account when interfacing the nervous tissue because of their potential influence in cellular intrinsic magnetic fields related to neuronal transmission. As nociception responses depend on this transmission, they could be indirectly influenced by the presence of GDMs. Based on this, Manzo et al.  recentlyBoosted by the tremendous development of GDMs in the industry, Chen et al.  pioneerein vivo, graphene toxicity was studied by inoculating different concentrations of pristine graphene  dispersed in distilled water  levels were also detected, among other major findings, thus demonstrating interference with neuronal synapses functioning (Figure As the interaction between nanomaterials and cells could drive to intracellular changes due to their association with cell membranes, Rauti et al.  investigPioneer work by Fabbro et al.  exploredin vitro studies have outlined the relevance that the physic-chemical properties of GDMs play in cell behavior. Some toxicity concerns about their use and the critical influence of particular functionalizations have been also evidenced. In this sense, cellular aspects as diverse as differentiation, neurite outgrowth, oxidative stress and protein and gene expressions could be significantly modulated. The findings from in vivo work revealed the ability of GDMs such as graphene, rGO and MWCNTs shaped as 3D scaffolds to initiate beneficial tissue responses when implanted in the injured spinal cord and the brain. Angiogenesis, axon growth, immunomodulatory effects, maintenance of neurogenesis and diminished fibroglial scar formation are some of the most relevant pro-regenerative responses induced by these materials. Ex vivo models with spinal cord slices are presented as an attractive and alternative system for testing the phenomena taking place when interfacing central neural tissues with GDMs. Procedures used for testing GDMs in contact with neural tissues and cells should be standardized to diminish contradictory results and facilitate comparisons among laboratories working in the field.Thanks to research efforts, knowledge on the interaction of GDMs with central neural cells and tissues is increasing. Extensive results from In conclusion, recent advances such as those described in this mini-review are boosting multidisciplinary research in the field that will likely open the path to the exploration of GDMs in translational approaches in biomedicine. Diverse pathologies at the central nervous system including SCI and some brain disorders could eventually benefit from the promising properties of these materials when interfacing the damaged neural tissue. In the next decade, we might assist to a new generation of advanced biomaterials based on graphene with unprecedented modulatory properties in neural systems.All authors equally contributed to the elaboration of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While sepsis-related mortality decreased substantially in other developed countries, mortality of severe sepsis remained as high as 44% in Germany. A recent German cluster randomized trial was not able to improve guideline adherence and decrease sepsis-related mortality within the participating hospitals, partly based on lacking support by hospital management and lacking resources for documentation of prospective data. Thus, more pragmatic approaches are needed to improve quality of sepsis care in Germany. The primary objective of the study is to decrease sepsis-related hospital mortality within a quality collaborative relying on claims data.The German Quality Network Sepsis (GQNS) is a quality collaborative involving 75 hospitals. This study protocol describes the conduction and evaluation of the start-up period of the GQNS running from March 2016 to August 2018. Democratic structures assure participatory action, a study coordination bureau provides central support and resources, and local interdisciplinary quality improvement teams implement changes within the participating hospitals. Quarterly quality reports focusing on risk-adjusted hospital mortality in cases with sepsis based on claims data are provided. Hospitals committed to publish their individual risk-adjusted mortality compared to the German average. A complex risk-model is used to control for differences in patient-related risk factors. Hospitals are encouraged to implement a bundle of interventions, e.g., interdisciplinary case analyses, external peer-reviews, hospital-wide staff education, and implementation of rapid response teams. The effectiveness of the GQNS is evaluated in a quasi-experimental difference-in-differences design by comparing the change of hospital mortality of cases with sepsis with organ dysfunction from a retrospective baseline period (January 2014 to December 2015) and the intervention period (April 2016 to March 2018) between the participating hospitals and all other German hospitals. Structural and process quality indicators of sepsis care as well as efforts for quality improvement are monitored regularly.The GQNS is a large-scale quality collaborative using a pragmatic approach based on claims data. A complex risk-adjustment model allows valid quality comparisons between hospitals and with the German average. If this study finds the approach to be useful for improving quality of sepsis care, it may also be applied to other diseases.ClinicalTrials.gov NCT02820675. Sepsis is one of the major challenges for health care today . It is tGuidelines for treating sepsis recommend early recognition by standardized screening, early administration of antibiotics, early adequate fluid resuscitation, and source control . SeveralIn 2013, 279.530 patients were diagnosed with sepsis, severe sepsis, or septic shock in German hospitals. Comparable to other high-income countries, sepsis incidence had increased from 2007 to 2013 by 5.7% per year . HospitaClinicalTrials.gov identifier NCT01187134) was the first multi-center quality initiative for reducing mortality among inpatient cases with severe sepsis in Germany [The MEDUSA trial  was founded in February 2016. The participating hospitals committed to improve the quality of care for patients with sepsis and thereby to decrease sepsis-related hospital mortality. The start-up period of the GQNS is funded by grants from the German Federal Ministry of Education and Research (BMBF) via the integrated research and treatment Center for Sepsis Control and Care (CSCC) at the Jena University Hospital and runs from August 2015 to July 2018. The funded start-up phase and its scientific evaluation use the acronym ICOSMOS . Additional funding is obtained via an annual fee paid by the participating hospitals. After the start-up-period, the GQNS will continue its work based on additional funding and on the fees of the participating hospitals.This study protocol describes the conduction and evaluation of the start-up-period of the GQNS. Figure The GQNS fulfills the essential features of a quality collaborative  by beingMajor principles of the GQNS are as follows: (a) focusing on outcome quality by using sepsis-related hospital mortality as major quality indicator; (b) measuring quality based on claims data readily available at each participating hospital; (c) assuring validity of quality comparisons by implementing a complex risk-adjustment model; (d) aiming at hospital-wide quality improvement by formation of local multidisciplinary QI teams led by committed local champions; (e) implementing education based on Surviving Sepsis Campaign guidelines among all involved health care workers; (f) assuring full support by senior management of participating hospitals by their commitment to an annual participation fee and the publication of risk-adjusted sepsis-related hospital mortality; (g) ownership of the collaborative by the participants by establishing structures that assure participatory action; and (h) bringing the expertise available within the collaborative to action by a system of peer-reviews between the participating hospitals.The organization of the GQNS is regulated in bylaws ratified by the participating hospitals. All responsibility for the QI efforts is at the side of the individual participating hospitals. Local multidisciplinary QI teams have repeatedly been recommended for enhancing guideline implementation especially regarding sepsis , 36, 37.The central study coordinating bureau, hosted by the CSCC, supports the QI efforts of the participating hospitals and coordinates accompanying scientific studies. Means of supporting the QI teams are as follows: (a) developing the model for risk-adjustment and specifications for quality reporting; (b) hosting the study homepage providing literature, instructions, educational material, technical and status reports, and online tutorials; (c) organizing national and regional study meetings; (d) coordinating communication and information exchange within the GQNS; (e) counseling local QI teams; (f) measuring the progress of QI efforts and barriers to change and giving feedback on these data to QI teams and senior hospital managements on a regular basis; (g) organizing online lectures and discussions with clinical experts; and (h) organizing web-based discussions with champions of successful international QI initiatives on sepsis care.Structures of the GQNS guarantee participatory action and ownership of the collaboration by the member hospitals. Major decisions are made in the general assembly of representatives of all participating hospitals. A steering committee is elected among the members of the GQNS, to supervise and assist the work of the coordinating bureau. A committee of experts recruited from the participating hospitals organizes the peer-reviews. Claims data are collected and processed to make quality reports by the medical information technology service provider 3M Health Information Systems (3M). The GQNS is part of the Global Sepsis Alliance’s Quality Improvement Committee .Eligible for participation in the GQNS were acute care hospitals with at least one adult intensive care unit. Invitation letters were sent to managements of hospitals which were participating in former or ongoing sepsis-related quality initiatives or research networks, to regional and national hospital groups, and to all German university hospitals. The active recruitment process started in Aug 2015 and ended in Feb 2016 and aimed at including 30 hospitals. Forty-eight hospitals were enrolled in March 2016, when the first quality reports were distributed. Characteristics of these hospitals are presented in Table The core interventions in the GQNS are (a) reporting and publication of quality indicators; (b) case analyses within the participating hospitals; (c) peer-reviews for hospitals, which are outliers in the quality reports; and (d) hospital-wide staff education in participating hospitals.The only mandatory intervention is the reporting, benchmarking, and publication of quality indicators. All hospitals committed to provide their claims data for calculation of quality indicators and to publish their main quality indicators in comparison to the German average. The study coordination bureau provides encouragement, information, and support regarding the conduction of case analyses and staff education. It also offers peer-reviews to hospitals where quality indicators suggest low quality of care. But the responsibility for implementing case analyses and staff education is on the side of the participating hospitals and the participation in a peer-review is voluntary. Hospitals are free to develop and implement further interventions suitable to their local conditions.There is consensus that valid and scientifically rigorous measures of performance are a means to improve health care and a central aspect of successful quality initiatives –42.The primary quality indicator measuring outcome quality of sepsis care is risk-adjusted hospital mortality among cases with sepsis with organ dysfunction including septic shock. It also serves as the primary end-point of the study.To achieve inclusion of all cases diagnosed with sepsis and to make quality measurement also feasible for small hospitals without specialized research staff, risk-adjusted hospital mortality of cases with sepsis is measured based on claims data , 33. A dEvery participating hospital sends its pseudonymized DRG data to 3M on a quarterly basis Fig. . Based oStatistical methods of risk adjustment are needed to control for differences in patient-related risk-factors between hospitals and allow for valid comparisons of quality . Risk-adQuality indicators are provided to the hospitals via 3M on a quarterly base. A web-browser-based tool with numerical as well as graphical presentations is used. Incidence, observed mortality and risk-adjusted mortality within subgroups of cases with sepsis are presented for each hospital. Reports allow comparison of risk-adjusted mortality to the German average, among the hospitals of the GQNS, among hospitals of specific levels of care, and over time. Displaying confidence intervals prevents interpreting merely random differences. Figure Additionally, each hospital receives a report using Microsoft Excel© including information on incidence and risk-adjusted mortality of sepsis cases within each of its departments. This report also presents lists of pseudonymized individual cases of the respective hospital including their individually predicted probability of death based on the risk model. Based on these lists, hospitals can perform in-depth single-case analyses (see below).Public comparative reporting of health care provider performance measures is regarded a key element for improving quality of health care , 56. TheAnalysis of individual case records can reveal preventable deaths as well as underlying shortcomings in care –63. AnalWithin the GQNS, individual probabilities for death are predicted based on the risk model and provided to the participating hospitals. The study coordination bureau provided information on how to conduct case analyses. Deceased cases with a predicted low risk of in-hospital mortality are used to identify possible problems in the quality of care or ICD-10 coding. Hospitals can depseudonymize these cases and analyze individual medical records using established methods , 64, 65.Peer-review, a process by which physicians evaluate each other’s performance, has a long tradition in efforts to improve health care quality . A systeThe main focus of staff education is the implementation of strategies for increasing awareness and early recognition of sepsis as well as the implementation of key elements of the updated Surviving Sepsis Campaign guidelines among physician and nurses, as well as all other health care workers involved in care for patients with sepsis , 68, 69.http://www.nrz-hygiene.de/en/surveillance/hospital-infection-surveillance-system/).Participating hospitals will be encouraged to implement a protocol for early recognition of sepsis in the emergency department, recognition of deteriorating patients or patients at risk on regular wards (standardized screening for sepsis), and to establish rapid response teams, which are mandatory or widely implemented in some countries but not in Germany . FurtherThe effect of participating in the start-up period of the GQNS will be evaluated by comparing the participating hospitals to all other German hospitals regarding the difference between the retrospective baseline period from January 2014 to December 2015 to the intervention period from April 2016 to March 2018  . This apThe study coordination bureau of the GQNS receives a study report by 3M on a quarterly base that informs about the quality indicators of each participating hospital as well as about hospitals that failed to provide their claims data. Hospitals that do not provide their claims data despite several reminders are excluded from the quality collaborative. To assess fidelity and dose of the local implementation of interventions in the participating hospitals, the coordination bureau of the GQNS regularly surveys local QI team leaders by standardized online questionnaires. These questionnaires contain items regarding departments participating in the QI teams, extend of interdisciplinary analyses of quality indicators, extend of interdisciplinary case analyses, extend of education of staff, and implementation of further interventions like existence and scope of rapid response teams. The questionnaire also contains items regarding barriers to change like lacking resources or support by management, as well as items that evaluate the quality of tools and support provided by the coordination bureau. The questionnaire was developed based on results of qualitative interviews conducted during the MEDUSA trial . Peer-reHospital mortality of cases with sepsis with organ dysfunction (including septic shock) in the participating hospitals is the primary end-point of the study, since the newly revised consensus definitions of sepsis and septic shock (“Sepsis-3”) exclude cases without organ dysfunction . Since tThe final analysis of the intervention effect will be conducted using German national DRG data. The change in hospital mortality between the baseline period (January 2014 to December 2015) and the intervention period (April 2016 to March 2018) will be compared between the 48 hospitals initially enrolled in the GQNS and control hospitals. These are all non-participating German hospitals, whose DRG data are also available from the Federal Bureau of Statistics. Hospitals entering the GQNS after March 2016 will be excluded from this comparison. This comparison will be conducted in 2020 when the national DRG data for 2018 will be available from the Federal Bureau of Statistics. The primary analysis will evaluate the difference-in-differences in a hierarchical generalized linear model controlling for clustering of cases in hospitals by testing the statistical interaction between period (baseline vs. intervention) and group . Patient-related risk-factors will be considered as covariates. As a secondary analysis, the trajectories of change across time before and after intervention will be compared between intervention and control hospitals by a piecewise hierarchical model .A preliminary evaluation of the study effect will be performed after completion of the intervention period in March 2018 but only relying on the data of the 48 intervention hospitals (before-after-comparison). A secondary preliminary analysis will be done by a piecewise hierarchical model to investigate the change in the trajectory of hospital mortality over time between periods.Differences across time between hospitals of the GQNS and control hospitals might result from changes in coding in the intervention hospitals, e.g., improved ICD-coding of sepsis or comorbidities. Therefore, incidences of sepsis without organ dysfunction, sepsis with organ dysfunction and septic shock as well as incidences of mortality risk-factors will be compared across time between intervention and control hospitals. Analyses of the primary outcome will be repeated within these three subgroups of sepsis cases.Additionally, hospitals will be categorized based on the degree of their QI efforts during the intervention period but without inspection of their primary outcome. The categorization will be based on the results of the regularly conducted surveys of local QI team leaders. The analyses of the primary end-point will be repeated within the subgroups of hospitals with high QI effort and hospitals with low QI effort.Concerning the baseline period (January 2014 to December 2015), 45,419 cases with sepsis were included among the initially enrolled 48 hospitals, of which 27,331 (60%) were diagnosed with sepsis with organ dysfunction (including septic shock), resulting in approximately 1000 cases per hospital across both the baseline and intervention period. Comparing the intervention hospitals with all other German hospitals across a baseline and intervention period resembles a controlled before-after trial on the cluster level. Therefore, our sample size calculation used a design factor developed for cluster-randomized before-after trials . We obseThe study underwent review by the internal review board of the Jena University Hospital (IRB protocol 4536-08/15) and the data protection supervisor of the Free State of Thuringia, Germany. Since individual case data are only obtained in the form of pseudonymized claims data, the need for informed consent by patients was waived. Quality reports present only aggregated data by hospital. Hospital names are pseudonymized, and each hospital knows only its own pseudonym. Only 3M and the study coordination bureau can de-pseudonymize all hospitals. Individual case data are only reported to the hospital where the cases were treated. For all kinds of surveys of QI team members, hospital staff, or peer-reviewers, participants will be informed about aims of the surveys and methods for protection of data privacy. Informed consent will be implied by participation in the survey, survey data will be stored in pseudonymized or anonymized form only, and all public reports of survey data will contain only aggregated data allowing no identification of individual participants.The GQNS was officially started with a kick-off meeting in February 2016. The first quality reports were distributed in March 2016 to the initially enrolled 48 hospitals. Till October 2017, quality reports have been distributed five times. Based on the public awareness of the GQNS, additional hospitals entered the network after the active recruitment. In September 2017, the number of participating hospitals reached 75, clearly surpassing the initially planned number. Participating hospitals are located all over Germany and include hospitals of all levels of care, among them 18 university hospitals (of 39 in Germany). In August 2016 and August 2017, two surveys of local QI team leaders were performed to assess the current progress and barriers to change in the participating hospitals and results were reported to senior hospital managements. Between March 2017 and September 2017, the first four online lectures and between May 2017 and September 2017 the first three external peer-reviews took place.The goal of this project is to establish structures for quality improvement of care for patients with sepsis and thereby to decrease sepsis-related in-hospital mortality. The GQNS is the first quality collaborative using claims data and a complex risk-adjustment to measure and improve quality of care for patients with sepsis. Because of this pragmatic approach 75 hospitals signed contracts for participation.Previous QI initiatives to improve sepsis care relied on gathering prospective clinical data in an uncontrolled before-and-after design , 17–21. The approach of our study of course has some limitations. First, under- or over-coding of sepsis might occur in claims data . HoweverIf this study finds that the approach of using claims data in combination with a complex risk-adjustment is useful for measuring and improving quality of sepsis care, the approach may also be applied to other diseases.


Late Permian Karoo Basin tectonics in South Africa are reflected as two fining-upward megacycles in the Balfour and upper Teekloof formations. Foreland tectonics are used to explain the cyclic nature and distribution of sedimentation, caused by phases of loading and unloading in the southern source areas adjacent to the basin. New data supports this model, and identifies potential climatic effects on the tectonic regime. Diachronous second-order subaerial unconformities (SU) are identified at the base and top of the Balfour Formation. One third-order SU identified coincides with a faunal turnover which could be related to the Permo-Triassic mass extinction (PTME). The SU are traced, for the first time, to the western portion of the basin (upper Teekloof Formation). Their age determinations support the foreland basin model as they coincide with dated paroxysms. A condensed distal (northern) stratigraphic record is additional support for this tectonic regime because orogenic loading and unloading throughout the basin was not equally distributed, nor was it in-phase. This resulted in more frequent non-deposition with increased distance from the tectonically active source. Refining basin dynamics allows us to distinguish between tectonic and climatic effects and how they have influenced ancient ecosystems and sedimentation through time. Deposition and accumulation in the Karoo Basin began with the Dwyka Group (320–280 Ma)3, followed by the Ecca, Beaufort, and Stormberg groups respectively, finally terminating after more than 100 Million years of sedimentation with the igneous basaltic outpourings of the early Jurassic Drakensberg Group9. Theories on the origin of accommodation in the Karoo Basin and distance from the Paleo-Pacific subduction zone (>1500 km)10 and Gondwanan Mobile Belt (GMB) include, activation of the Southern Cape Conductive Belt, a crustal geophysical anomaly11, fault-controlled subsidence12, and continent-continent collision with south dipping subduction zone13. Despite disagreements, most workers recognize foreland basin tectonics with a north dipping subduction zone in the Karoo basin and it is only the timing of the onset of foreland tectonics that has been seriously disputed. It is now clear that by the time of deposition of the Dwyka Group, foreland tectonics were already operating16. Thus the Karoo Basin was most likely a foreland basin from its formation and these conditions continued until its termination at the end of the Stormberg Group17. Additionally, northward thinning of strata, condensed, diachronous, and missing lithostratigraphic units away from the source area reflects many characteristics of modern foreland systems22. Thus, foreland tectonics, with dynamic subsidence resulting from orogenic loading by flat-slab subduction24, is currently the most popular explanation for the generation of accommodation in the main Karoo Basin34.The Karoo Basin of South Africa represented a large depocenter situated in southern Gondwana supported by the Kaapvaal Craton in the northeast and the Namaqua-Natal Metamorphic Belt (NNMB) in the southwest4. Orogenic loading and unloading in the GMB changed the forebulge and foredeep’s positions, and this resulted in deposition in the proximal basin in the south during loading and distal regions of the Karoo Basin in the north during unloading. Where a time lag occurred between orogenic loading and basinal subsidence26 the upper and lower boundaries of lithostratigraphic units are diachronous, and have proximal and distal equivalents. Thus, many of the upper and lower boundaries of lithostratigraphic groups are diachronous or have proximal and distal equivalents35. This is reflected in the geologic record as the preservation of large-scale (second-order) fining upward depositional sequences bounded by subaerial unconformities (SU)37. Thus the SU in this study were identified by relatively abrupt changes in lithology (e.g. renewed fining upward cycles), mean change in paleocurrent direction, changes in fluvial style (e.g. from high to low sinuosity), and rarely by palaeoclimatic changes and faunal turnover38.Flexural tectonics partitioned the Karoo Basin into the foredeep, forebulge, and backbulge flexural provinces with a dominantly southerly sediment source area37. These cycles are recognized in the Beaufort Group39 by fluvial systems that are characterized by an initial pulse of high energy fluvial transport , often underlain by an SU, followed by increasingly finer grained sediment as tectonic activity ceased1 and river gradients declined. Similarly, this cyclicity is used to identify the position of SUs in Upper Cretaceous fluvial deposits in southern Utah41.Modern and ancient foreland basins like the Karoo Basin are bounded by tectonically active source areas. Episodic tectonic activity results in the rejuvenation of fluvial systems leading to deposition of coarser sediment, and cessation in tectonic activity results in an overall fining upward sequence36, which are the result of smaller-scale orogenic events. Recently, the Late Permian Beaufort Group stratigraphy has been revised43  and northwards towards the basin’s forebulge 57. This study provides some evidence that climatic changes associated with the PTME were occurring much lower in the stratigraphy than previously documented. Thus it is concluded the third-order tectonic SU could be overprinted by these palaeoclimatic changes. This is supported by a newly identified faunal turnover and abundance changes marked by the appearance of Lystrosaurus maccaigi and disappearance of Dicynodon lacerticeps, Theriognathus microps, and Procynosuchus delaharpeae in this SU. Palaeoclimatic changes at the point of the third-order SU in the Daptocephalus Assemblage Zone are identified by a decrease in the occurrence of subaqueous environments in the overbank facies42. In addition, distal attenuation of the Balfour Formation towards the north indicates there was more accommodation for sediment proximally, and point to more non-deposition distally during the Late Permian43. Although this faunal turnover is not identified at the base of the SU in the Beaufort West field site, it implies that the third-order SU is diachronous. Diachroneity has been identified at major lithostratigraphic boundaries of the Karoo Supergroup, such as the Ecca-Beaufort contact, Katberg-Burgersdorp contact, and Burgersdorp-Molteno contact60 and is evidence of tectonic influences controlling sediment distribution in the foredeep.Although some of the SU presented in this study have been observed in the Karoo Basin by previous workers36. Each second or third-order sequence correlates to an orogenic cycle of thrusting followed by unloading. New paroxysm dates61 fall within the dates of known active orogenic loading.All evidence collected indicates that cyclic, out-of-phase sedimentation in the Karoo Basin during the Late Permian resulted from a strong tectonic influence south of the basin which was also the source of clastic material. Our depositional model refines third-order detail in addition to the mostly first and second-order framework of the Karoo Basin sedimentary fill63 and the geophysically- defined “Willowmore arch”64, which is interpreted as a palaeotopographic high, was present between these two parts of the basin. This may explain some differences between the two parts of the basin but essentially, they are reflecting similar tectonic events in the foreland system defined by fairly repetitive, and roughly synchronous, depositional cycles37. This also entertains the possibility of alternative controls on subsidence that caused the basin to contain down-warped depressions and upward arches formed by the interplay of the foreland compressional tectonics and local rheology and structural integrity of the basement rocks. The Kaapvaal Craton situated below the distal basin would have been more rigid and buoyant than the Namaqua-Natal Metamorphic Belt which comprises the basement rocks of the proximal foredeep and back bulge. Structural anomalies could have controlled tectonics locally65, and may have contributed to the out-of-phase deposition created by flexural tectonics of the foreland system. This study’s basin model has four stages which explain the inferred tectonic setting of the Late Permian Karoo Basin  show similar lithostratigraphic sequences and it is clear they were influenced by similar tectonic and climatic events in the basin Fig. . The wesStage 1 Follows orogenic uplift in the Gondwanide Mobile Belt with the onset of sedimentation of the Balfour in the southeast and upper Teekloof formations in the southwest of the basin which began at the base of the first second-order SU and represents the beginning of a fining-upward cycle deposited by low sinuosity rivers (the eastern Oudeberg and western Oukloof members). Distally, there was non-deposition as no lithostratigraphic equivalent is documented close to the forebulge. Once sediment supply became less than available accommodation, the sequence began to fine-upward, depositing argillaceous material by high sinuosity rivers (the eastern Daggaboersnek and western Steenkampsvlakte members).43, the Javanerskop member appears to be slightly older than the Ripplemead member and is present only in the south east and central Karoo Basin. Coincidently, the decrease in sinuosity of the fluvial systems leads to reduced overbank preservation, and disappearance of lacustrine systems.Another minor orogenic loading event initiated stage 2, resulting in the out-of-phase deposition of the arenaceous Javanerskop member in the west and Ripplemead member in the east above the third-order SU. On current biostratigraphic evidence55 as indicated by paleoclimatic changes and a newly identified faunal turnover. Stage 4 marks the beginning of deposition of the early Triassic lower Katberg Formation during a new orogenic loading phase.Stage 3 is an unloading phase resulting in distal progradation of sediment when accommodation became available in the distal foredeep. This explains the attenuated but similar stratigraphy. The third-order sequence began to fine upwards, terminating in the argillaceous Elandsberg and Palingkloof members Figs  and 3. T63. Foreland tectonics meant that progradation of sediment northwards  during orogenic loading and unloading was not in phase with the south  which resulted in an incomplete and attenuated stratigraphic record to the north, and diachronous SU between the west and eastern parts of the basin.Flexural tectonics imposed phases of orogenic loading and unloading during the Late Permian Karoo Basin. This was the major control on the distribution of accommodation in the foredeep of the Karoo Basin as demonstrated by northward thinning wedge-shaped deposition of strata and cyclic sedimentation bounded by two second-order, and one third-order subaerial unconformities (SU)36, but tectonic and climatic signals are notoriously difficult to tease apart in the geologic record37. The revised Late Permian stratigraphy and tectonics43 indicate that there was some climatic change overprinting the tectonic signatures imposed by orogenic loading1 at least by the Upper Daptocephalus Assemblage Zone (DaAZ), and the third-order SU, as fauna began to go extinct or decrease in abundance43. This new basin model, in combination with revised Late Permian stratigraphic investigations, tells us that environmental changes may have been occurring earlier than previously documented and they could be related to the effects of the PTME55. Thus, understanding ancient ecosystems has important implications for the understanding basin dynamics, especially in the Late Permian Karoo Basin where there is much evidence for a coeval terrestrial counterpart to the global Permo-Triassic mass extinction (PTME)57. There is still much debate concerning what role tectonics and climate played in this major biotic crisis, and studies such as this contribute to understanding this important event in Earth’s history.Throughout the depositional cycle of the Late Permian Beaufort Group, little evidence for significant climatic change has been documentedDaptocephalus Assemblage Zone . Four field sites were chosen near Cradock, Nieu Bethesda, Beaufort West, and Gariep Dam (Fig. 54.The study area encompassed the stratigraphic range of the Dam Fig. . Numerou


Decision-making in mental health systems should be supported by the evidence-informed knowledge transfer of data. Since mental health systems are inherently complex, involving interactions between its structures, processes and outcomes, decision support systems (DSS) need to be developed using advanced computational methods and visual tools to allow full system analysis, whilst incorporating domain experts in the analysis process. In this study, we use a DSS model developed for interactive data mining and domain expert collaboration in the analysis of complex mental health systems to improve system knowledge and evidence-informed policy planning.We combine an interactive visual data mining approach, the self-organising map network (SOMNet), with an operational expert knowledge approach, expert-based collaborative analysis (EbCA), to develop a DSS model. The SOMNet was applied to the analysis of healthcare patterns and indicators of three different regional mental health systems in Spain, comprising 106 small catchment areas and providing healthcare for over 9 million inhabitants. Based on the EbCA, the domain experts in the development team guided and evaluated the analytical processes and results. Another group of 13 domain experts in mental health systems planning and research evaluated the model based on the analytical information of the SOMNet approach for processing information and discovering knowledge in a real-world context. Through the evaluation, the domain experts assessed the feasibility and technology readiness level (TRL) of the DSS model.The SOMNet, combined with the EbCA, effectively processed evidence-based information when analysing system outliers, explaining global and local patterns, and refining key performance indicators with their analytical interpretations. The evaluation results showed that the DSS model was feasible by the domain experts and reached level 7 of the TRL .This study supports the benefits of combining health systems engineering (SOMNet) and expert knowledge (EbCA) to analyse the complexity of health systems research. The use of the SOMNet approach contributes to the demonstration of DSS for mental health planning in practice.The online version of this article (10.1186/s12961-018-0308-y) contains supplementary material, which is available to authorized users. Planners and policy-makers face complex decisions that require a deep knowledge of health systems, which involve inherently complex interactions between its structures, processes and outcomes, among multiple agents. These systems are characterised by nonlinearity, interconnectivity, self-organisation, constant change, variability and uncertainty . Such coUnder these circumstances, DSS should incorporate all relevant sources of scientific knowledge, including observational, contextual and expert knowledge, as well as systematic reviews of experimental studies . To fit In previous systems analytics studies, small mental health catchment areas in Spain were analysed using hierarchical clustering , hierarcAn alternative is to combine artificial neuronal networks (ANNs) with advanced information visualisation techniques in a single DSS. Based on the demand, the aim of this study is to assess a new DSS model using a novel approach, a self-organising map network (SOMNet) , 22, comThis paper investigates the usability of the SOMNet approach combined with the EbCA approach in developing a DSS model for analysing complex mental health systems in a real-world context. This combined DSS model is applied to the assessment of the mental health care systems of three regions in Spain. The study aims to reduce uncertainty by increasing the expert knowledge of the systems through 1) the identification of outlier areas based on the systems analysis of key performance indicator (KPI) information; (2) the identification of the distinctive global and local patterns of healthcare across the different systems; and (3) the identification of the relationships between the KPIs in the systems. The domain experts are involved in the analytical process by interpreting and evaluating the analytical results. The study then evaluates the feasibility and technology readiness level (TRL) , 27 of t the idenn = 6) and health planners from the agencies of Catalonia (n = 3), Biscay (n = 2) and Gipuzkoa (n = 2).This study was conducted by an international core team working in collaboration with a group of Spanish domain experts. The core development team included two systems engineers (the SOMNet developer and a knowledge engineer) and two domain experts . The second evaluation group was composed of 13 domain experts who are end-users of the DSS, including health service researchers  was developed for the data analysis based on the main sources of information described below.These Atlases have been developed by the PSICOST Research Association using the DESDE-LTC standard classification system  and the Basque Country . The utilisation data of day care was not included due to missing data, lower reliability and absence of data linkage in the database at the time of the data analysis.The EbCA approach  was usedThe pre-processing phase comprised five steps involving a core team of knowledge engineers and systems researchers working with different groups of domain experts in health service research and policy planning at local, regional and national levels in Spain. The details of each step have been previously published , 20, 37.In summary, the first step developed the basic model of community mental healthcare. This model was developed by a group of health service researchers using the data from 12 representative small catchment areas in Spain . The secFrom the pre-processing of data, the domain experts of the three regions agreed on a common list of KPIs. This study’s core development team incorporated this information into a MMS for the data analysis. As seen in Table The MMS information on the four datasets of 106 small catchment areas in the three health systems  was used to assess integrated mental healthcare provision and resource utilisation. The core team developed a DSS model that uses the SOMNet and the incorporation of the experts in the mid-processing of the data based on the EbCA.A self-organising map (SOM) is an ANN, which is a parallel information process paradigm inspired by the biological nervous system , 39. ANNData formation and networking: The SOMNet facilitates the selection and normalisation of input and output datasets to be networked from a given database. These datasets are used for handling missing data, analysing outliers and exploring data patterns within a dataset and between datasets.Interactive visual data mining: The SOMNet incorporates an automated machine learning process, information visualisation and user interaction tools in analysing data and obtaining relevant information. The analytical process is iterative until knowledge-based information is obtained for decision support.Analytical interpretation: The SOMNet enables expert knowledge incorporation into the data mining for evaluating analytical processes and results. Based on the visual information processing, it also enables domain experts exploring and guiding other steps of data analysis for eliciting tacit knowledge.Based on machine learning and information visualisation capabilities of the SOM, the SOMNet is developed for interactive visual data mining between multiple datasets , 22. TheBased on the functional design of the SOMNet, expert knowledge can be incorporated in the mid-processing phase of KDD. The SOMNet approach allows expert knowledge to be combined with the EbCA from the prior knowledge base or expert-guided data analysis for result interpretation Fig. .Fig. 3ThThere are three main goals defined for the SOMNet application, namely (1) system outlier identification , (2) system pattern identification  and (3) KPI relationship identification (input and output indicator patterns). The domain experts in the core team iteratively evaluate the analytical functions and results provided by the SOMNet through the reasoning process.The information on the analytical processes and results of the SOMNet was presented to a group of 13 domain experts. They had previous expertise on health systems research and planning, on the characteristics of the reference study regions, and on the use of other tools and methods for decision-making in mental health. Therefore, they were able to evaluate the usability of the SOMNet approach compared with other approaches used in the previous analysis for DSS. The DSS model using the SOMNet approach was validated by the domain experts using a feasibility checklist and the previous experience in the use of operations research models, and visualisation tools  were trained for the four datasets categorised from the MMS by normalising the KPI values.In the AVA-SOM, three small mental health areas  in Catalonia were identified as outliers with extremely low or high values for the availability indicators. They were interpreted as remote and isolated areas having different service characteristics from the other areas in Catalonia. They are also distant from the reference service facilities (more than 100 km) with a very low population. These areas could be reasonably removed. Another three mental health areas  in Catalonia were identified as outliers in the USE-SOM with an extremely high value for the utilisation indicator, U3 (readmission) as seen in Fig. Through the pattern examination and interpretation, the system outliers were identified and processed to avoid pattern distortion by accounting for their relative impacts on the systems, particularly in relation to their connectivity with the other local systems. After processing the outliers, the four datasets were newly formed with a total of 102 small catchment areas. The mental health system patterns and indicators were then analysed using the newly trained and networked SOMs in the SOMNet.The mental health system patterns were first identified at the global level . The input SOMs for availability (AVA-SOM), placement (PLA-SOM) and workforce capacity (WOF-SOM) are represented in Fig. The small mental health area patterns were also visually analysed at the local level . The proximity of the SOM neurons indicates the areas having similar properties. As the local information was observed by the property similarity over the SOM, a neuron with more than one area was used as a unit cluster showing the closest property. For the input SOMs, the areas in Biscay show very similar properties as they are topologically very close to each other. The areas in Gipuzkoa also show very similar properties, while the areas in Catalonia have a more diverse pattern distribution over the region. Based on the topological property information, the further local clusters of Catalonia are exemplified in the AVA-SOM Fig. , which sThe domain experts visually examined and explained the integrated global and local pattern information using the indicator value or property shape planes of the SOMs. From the availability AVA-SOM Fig.  and a', Using the SOMNet, mental health systems were analysed by providing meaningful pattern information based on the system property dissimilarity in each dataset. Different patterns of mental health care provisions in the three different systems  were identified at a global level. The local patterns of the small mental health areas in the systems were also simultaneously examined by capturing the relative pattern difference between them. The small mental health areas showed distinctive pattern information between the input datasets and the output dataset. The domain experts who participated in the SOMNet analysis process extracted the integrated bottom-up pattern information from the complex system data. Such pattern information, which was difficult to capture in previous studies, was visually identified, interpreted and evidentially justified in the DSS model using the SOMNet.In the SOMNet, the input SOMs of availability, places and beds, and workforce  were networked to the output SOM of utilisation (USE-SOM), since the whole data space represents the association between the multiple datasets. Once the structure of each dataset was analysed, the structures between input and output datasets were analysed using their weight association. Using the SOMNet, the five input resource KPIs of acute hospital care were analysed for their ideal relationships to the output utilisation KPIs. The desired values of the five input KPIs (A1 (service availability), P1 (placement capacity), W1 (the number of psychiatrists), W2 (the number of psychologists and nurses) and W3 ) and the ideal values of the three output KPIs (U1 (discharge), U2 (length of stay) and U3 (readmission)) were provided by the prior expert knowledge, obtained from the previous EbCA studies  is the most likely related condition to the ideal use of acute care hospital services at the low-medium levels of discharge (U1), length of stay (U2) and re-admission (U3) across the mental health systems’. Based on the analytical interpretation in the mid-processing phase, the domain experts elicited new tacit knowledge. The evidence-based information obtained by the SOMNet with the EbCA confirmed the appropriateness of all the value ranges suggested by the domain experts except for the number of psychiatrists assigned to acute wards. This implies that reducing the value of W1 in the knowledge discovery process guides further analysis to examine the impact of W1 on service utilisation. The relevant decision-making could imply this information on the specific resource planning.The results of the model validation and usability have been measured by its feasibility and TRL   Fig. , since iHealth systems engineering has been adopted in the United States to analyse healthcare systems, to provide recommendations for reducing healthcare waste and to improve its overall efficiency. It connects systems engineering principles and best practices with clinical expertise for DSS to improve understanding of the interactions among agents , processes  and technologies , and to formulate innovations and better outcomes .To our knowledge, this is the first study that provides an integrated approach to DSS in mental health, following a systems thinking approach  and resuThe use of the SOMNet combined with the EbCA has overcome some of the problems of previous modelling techniques for analysing complex mental health systems, especially in the mid-processing of the data. The prior exclusion of domain experts in the mid-processing phase created a gap between experts and analysts in complex data analysis and made it difficult for domain experts to reach an evidence-informed decision . The intThe SOMNet approach allowed domain experts to guide data analysis based on their interests and to understand the results better. The domain experts were enabled to identify and process the catchment area outliers according to their connectivity with the rest of the system. For example, highly rural areas in Catalonia and areas with a predominantly private model of management in Gipuzkoa could be excluded from the system analysis, while other outlier urban areas in Catalonia required adjustment of their KPIs to be incorporated into the model. The SOMNet also facilitated the identification and explanation of the global and local system patterns at macro-level and meso-level analysis, respectively, across different systems. Finally, the SOMNet examined the input and output relationships of the KPIs, confirming the appropriateness of the assumptions provided by prior expert knowledge and questioning one of the KPIs assessed . These tasks are critical for knowledge-informed planning in mental health.Additionally, this study provides a detailed analysis of the external validity (feasibility) of the SOMNet and its TRL . It is iA number of limitations should be noted. First, this combined approach introduces new concepts and techniques in health planning. Even though many of them have been extensively tested in other fields of knowledge, the use of systems thinking and combined modelling are in their infancy in mental health policy. Nevertheless, many of the functional aspects of this study have been previously published e.g. , 17, 45), 4517, 4Obtaining scientific evidence by incorporating domain experts through empirical study in a real-world context is important in mental health research for knowledge discovery and decision support. Since mental health systems are highly complex, the underlying system patterns and behaviours have been hardly understood by existing data analysis approaches in the mid-processing of data . In this paper, we developed a DSS model for interactive visual data mining by combining the SOMNet approach with the EbCA approach for the mental health systems analysis in the mid-processing phase of KDD. The SOMNet was applied to the analysis of healthcare patterns and indicators of three different regional mental health systems in Spain. The relevant analytical goals defined in the study were achieved by incorporating the domain experts in the SOMNet analysis. The domain experts were able to (1) identify and process the catchment area outliers; (2) identify and explain the global and local system patterns across the different systems; and (3) identify and examine the input and output relationships of the key performance indicators, which guided further analysis for evidence-informed knowledge discovery. The domain experts also evaluated the feasibility of the SOMNet approach for DSS based on its analytical processes and outcomes. The feasibility evaluation results showed that the SOMNet approach is relevant and useful for analysing highly complex mental health systems and improving evidence-informed knowledge discovery. The technology used in this study (the system prototype) demonstrated potential for DSS implementation in the operational environment (TRL 7).This health systems engineering technique (the SOMNet) combined with expert knowledge (the EbCA) is an innovative and powerful method to address complex questions in health systems research and planning in a real-world context.Additional file 1:Information on the model validation process. This document provides information about ethics approval for the expert-based model validation process, the definition of feasibility dimensions, the feasibility checklist and the technology readiness levels (TRL). (PDF 648 kb)Additional file 2:Labelled small mental health areas in Spain. The small mental health areas in Biscay, Gipuzkoa and Catalonia systems in Spain are labelled using the system initial and Arabic numbers to be simplified in the data analysis. (PDF 211 kb)Additional file 3:Geographical maps of the mental health systems in Spain. The labelled small mental health areas in the three systems are shown on the geographical maps of the Basque Country and Catalonia in Spain. (PDF 519 kb)Additional file 4:Information on the DESDE-LTC classification system. This document provides information on the use of DESDE-LTC system for mental health service coding and mapping to develop the Atlas of Mental Health Care. (PDF 551 kb)