Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disorder, caused by the degeneration of upper and lower motor neurons for which there is no truly effective cure. The lack of successful treatments can be well explained by the complex and heterogeneous nature of ALS, with patients displaying widely distinct clinical features and progression patterns, and distinct molecular mechanisms underlying the phenotypic heterogeneity. Thus, stratifying ALS patients into consistent and clinically relevant subgroups can be of great value for the development of new precision diagnostics and targeted therapeutics for ALS patients. In the last years, the use and integration of high-throughput “omics” approaches have dramatically changed our thinking about ALS, improving our understanding of the complex molecular architecture of ALS, distinguishing distinct patient subtypes and providing a rational foundation for the discovery of biomarkers and new individualized treatments. In this review, we discuss the most significant contributions of omics technologies in unraveling the biological heterogeneity of ALS, highlighting how these approaches are revealing diagnostic, prognostic and therapeutic targets for future personalized interventions. Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease, characterized by the progressive deterioration of cortical and spinal motor neurons (MNs), leading invariably to progressive muscle weakness and paralysis. Death, often resulting from respiratory failure due to respiratory muscle weakness, generally occurs after 3–5 years from symptom onset, with only 5–10% of patients’ survival beyond 10 years . ALS is Over the last few years, the complexity of ALS has led to the concept of a spectrum of different disorders with different pathogenic mechanisms rather than a single disease. From a clinical point of view, in addition to typical or classic ALS (characterized by the simultaneous involvement of upper and lower motor neuron (UMN and LMN) at disease onset), several different phenotypic subtypes can be recognized based on the rate of progression, survival, age of onset, site of onset  and prevalence of UMN or LMN motor signs . AdditioDifferent clinical profiles are likely to reflect molecular heterogeneity in ALS. In fact, for example, the majority (∼90%) of ALS cases are sporadic (SALS), with unknown cause, while ∼10% of ALS patients show familiarity for the disease, usually transmitted according to an autosomal dominant inheritance . HoweverAdvances in “omics” technologies  and their correlation with the clinical phenotypes of the individual patient, are enabling medicine to move from a “one-size-fits-all” approach toward a “personalized” model, helping to clarify the molecular mechanisms underlying human disease and to provide both potential biomarkers and pharmacological targets for a more detailed patient stratification and personalized treatments . In thisApplications of omics platforms range from the detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics), epigenomic factors (epigenomics), and metabolites (metabolomics). Thanks to omics technologies, it is now possible to quantify the amount of particular molecules  of a biological system, and observe massive interactomes describing their complex interconnections. For complex and multifactorial pathologies such as ALS, the analysis and integration of different omics layers are crucial for the full knowledge of the disease, opening the way to the development of personalized diagnostic and therapeutic tools. Several omics studies have suggested multiple pathologic mechanisms associated to ALS, providing new insights into molecular signatures/markers and moving toward molecular-based classifications and tailored interventions.1. After the identification of mutations in the SOD1 gene in 1993  or mutations, but also genomic alterations and chromosomal changes, with consequent protein dysfunction or differences in concentration levels. Detailed information regarding ALS-related genes is available via the Amyotrophic Lateral Sclerosis Online Database (ALSOD) in 1993 , more th disease .Thanks to the development of genome-wide association studies (GWAS) as well as the advances in massive parallel sequencing approaches, including whole-genome sequencing (WGS) and whole-exome sequencing (WES), enormous progress has been made in understanding genomics of ALS . A growiSOD1 usually have an aggressive form of ALS, whereas those with the homozygous Asp91Ala mutation tend to have a very slowly progressive disease with a generally ascending upper motor neuron phenotype), the majority of disease-causing genes show a high degree of phenotypic heterogeneity, with mutations in the same gene giving rise to different clinical entities, supporting a genetic basis for the observed clinical heterogeneity in ALS. A striking example of pleiotropy is due to C9orf72 hexanucleotide repeat expansion mutation, which is clearly linked to ALS and FTD but pathogenic expansions have been also observed in a small percentage of patients affected by Alzheimer’s (<1%), Huntington’s (1–5%), and Parkinson’s diseases (1%), as well as atypical parkinsonian syndromes, such as progressive supranuclear palsy (1–8%), corticobasal degeneration (3%), and Lewy body dementia (2%)  suggesting a potential implication of APOE genotype as a biomarker to discriminate clinical efficacy in ALS clinical trials , through a process known as repeat-associated non-ATG-initiated (RAN) translation, leading to an impairment of ribosomal biogenesis, nucleocytoplasmic transport, RNA metabolism and protein sequestration, that can cause neurodegeneration and behavioral deficits  . Anotherl domain . The posl trials . Anothersurvival . As for deficits . Disease for ALS .SOD1, EPHA4, KIFAP3, and UNC13A seem to affect the progression of ALS disease or the survival of ALS patients , is potentially informative of genomic alterations related to disease phenotype through the modulation of the expression and function of genes. Several studies have investigated the involvement of these variants in ALS, demonstrating their involvement as risk factors, with multiple rare CNVs more important than common ones . In partgression .Notwithstanding the increased knowledge of ALS from a genomic perspective, substantial dilemmas remain from a clinical perspective and large-scale NGS and GWAS projects are currently underway to fully unravel the underlying causes. Among these, of note is Project MinE, an international, large-scale research initiative devoted to discovering genetic causes of ALS by performing whole-genome sequencing of at least 15,000 ALS patients and 7,500 controls, resulting in an open-source genome database, in conjunction with the collection of skin samples to make patient induced pluripotent stem cell lines (iPSCs) . Future Changes in gene expression are widespread in ALS, as revealed by a large body of work on gene expression profiling of RNA samples from peripheral cells or post-mortem nervous tissue of ALS patients and animal models. These signature patterns of gene expression have started to provide a more detailed picture of molecular events implicated in ALS pathobiology .The advent of systems biology and development of high-throughput technologies, including RNA sequencing and high-density microarray platforms, is enabling us not only to discover and define mechanisms of pathogenesis in ALS, but also to differentiate ALS from the “ALS mimic syndromes” and healthy controls and stratify ALS patient into subgroups, facilitating the discovery of biomarkers and new individualized treatments for patients . In thisC9orf72 repeat expansion, revealing both shared and distinct transcriptome changes and pathways associated with these two subsets of ALS cases  technologies has allowed the simultaneous analysis of multiple proteins, allowing for the definition of comprehensive lists of possible candidate ALS biomarkers . In thispatients . Severalpatients . Proteompatients .As for genomics studies, systems biology-oriented approaches in proteomics play a crucial role to reveal relevant biological knowledge on pathological mechanisms that trigger the onset and progression of ALS, providing a mechanistic rationale for stratification of ALS patients based on unique molecular profiles, and identification of disease biomarkers and targets for drug efficacy measurements. In this scenario, the analysis of protein-protein interaction (PPI) networks provides the possibility to group proteins that are interacting with each other’s in functional complexes and pathways, resulting critically important in helping us to comprehend complex processes, like ALS, and identify key signaling cascades, upstream regulatory components, interactome domains, and novel disease-associated protein candidates suitable for therapeutic intervention . In thisC9ORF72, SOD1, TARDBP, and FUS), suggesting the existence of distinct neurodegenerative processes associated with different subtypes of ALS , offering exciting new possibilities for ALS research. In this context, metabolomics, the scientific study of chemical processes involving metabolites , represents the downstream of systems biology that links the genome, transcriptome and proteome to patient phenotype, providing an important key tool for discovering potential markers in health or disease . In the s of ALS . It is is of ALS . Howevers of ALS . These cAnalysis of metabolite profiles can be also used to identify metabolites and biochemical pathways in ALS patients that are modified before or after treatment exposure, giving rise to a new field called pharmacometabolomics . To thisOPTN, hypermethylation of C9orf72 expansion CpG islands in the blood of FTD/ALS patients, whereas mutant SOD1, FUS and TDP43 contribute to global epigenome alteration by inducing alterations in histone post-translational modifications and DNA methylation , small non-coding molecules of about 20–22 nucleotides, represent an additional layer of epigenetic regulation that, thanks to their capability to be highly stable in human body fluids, are considered promising biomarkers for neurodegenerative diseases, including ALS . Over thAs detailed in the previous paragraphs, omics technologies have been used to identify and/or provide functional supporting information for deciphering important players and pathways involved in ALS pathogenesis and identifying a panel of candidate therapeutic targets and biomarkers that will assist in the rapid diagnosis and prognosis assessment of the disease, and in the stratification of patients into different subgroups for specific targeted therapies. However, if considered individually, these technologies are insufficient to clarify the intricate disease mechanisms implicated in ALS. Taking a holistic molecular approach, based on the integration of multiple types of omics data with existing biological knowledge, has the potential role in improving the knowledge of the molecular basis underlying complex and heterogeneous diseases, establishing different molecular subtypes and patient stratification, thus providing a rational foundation for designing new studies to identify novel targets and clinical trials . Numerouin vivo and in vitro ALS models (An interesting example of applying integrated omics approaches to define an individual’s molecular profile useful for the development and application of personalized medicine in ALS, is represented by recent studies carried out by our research groups. As previously described, transcriptional profiling of post-mortem motor cortex samples from SALS patients has allowed to differentiate two distinct patient subgroups characterized by different deregulated genes and pathways ,b,c. To S models , 2019.In the past decade, advanced omics technologies have fostered our understanding of the complex molecular architecture of ALS, contributing in part to explain its clinical heterogeneity, and providing a basis for a molecular taxonomy that may radically change our medical approach to ALS. The identification of relevant classifiers and subgroup-specific diagnostic, prognostic and predictive biomarkers is in fact urgently needed for accelerating the development of effective and personalized treatment approaches in ALS. In this review, we discuss the most significant contributions of omics approaches in unraveling the biological complexity of ALS, highlight how holistic systems biology approaches and multi-omics data integration are ideal to provide a comprehensive characterization of patient-specific molecular signatures that could potentially guide therapeutic decisions. We strongly believe that the future research in ALS, as well as in other neurodegenerative diseases, calls a multidisciplinary holistic approach, integrating multi-layer omics data with multimodal neuroimaging and clinical data. This approach will provide a clear understanding of disease prognosis and progression and accelerate development of innovative, effective and personalized strategies for ALS.GM wrote the manuscript. SS, VD’A, and FC participated in revising the manuscript. SC conceived, directed, and supervised the project. All authors contributed to the article and approved the submitted version.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.


Coastal acidification in southeastern U.S. estuaries and coastal waters is influenced by biological activity, run-off from the land, and increasing carbon dioxide in the atmosphere. Acidification can negatively impact coastal resources such as shellfish, finfish, and coral reefs, and the communities that rely on them. Organismal responses for species located in the U.S. Southeast document large negative impacts of acidification, especially in larval stages. For example, the toxicity of pesticides increases under acidified conditions and the combination of acidification and low oxygen has profoundly negative influences on genes regulating oxygen consumption. In corals, the rate of calcification decreases with acidification and processes such as wound recovery, reproduction, and recruitment are negatively impacted. Minimizing the changes in global ocean chemistry will ultimately depend on the reduction of carbon dioxide emissions, but adaptation to these changes and mitigation of the local stressors that exacerbate global acidification can be addressed locally. The evolution of our knowledge of acidification, from basic understanding of the problem to the emergence of applied research and monitoring, has been facilitated by the development of regional Coastal Acidification Networks (CANs) across the United States. This synthesis is a product of the Southeast Coastal and Ocean Acidification Network (SOCAN). SOCAN was established to better understand acidification in the coastal waters of the U.S. Southeast and to foster communication among scientists, resource managers, businesses, and governments in the region. Here we review acidification issues in the U.S. Southeast, including the regional mechanisms of acidification and their potential impacts on biological resources and coastal communities. We recommend research and monitoring priorities and discuss the role SOCAN has in advancing acidification research and mitigation of and adaptation to these changes. The ocean serves as a giant reservoir for COy humans , resultiy humans . Becausefication .2 concentrations than the open ocean; the atmosphere is not the only carbon source associated with changes in carbonate chemistry , total alkalinity, pH and concentrations of dissolved inorganic carbon (DIC), which includes carbon dioxide, carbonic acid, bicarbonate ions, and carbonate ions. Under increasingly acidic conditions, the concentration of carbonate ions decreases, potentially harming organisms that use carbonate to produce calcium carbonate skeletons including corals, mollusks, crustaceans, and phytoplankton. The form of calcium carbonate mineral most commonly used by organisms is aragonite or calcite; the decrease in carbonate results in a lower aragonite saturation state (Ωarag) or calcite saturation state  meaning that skeletons made of aragonite or calcite will be energetically more difficult to form (+] (reduced pH) are more influential on calcification  have formed throughout the United States to build public knowledge of regional drivers and impacts of acidification and to provide a collaborative framework for stakeholders, policymakers and scientists . The Souarag 2.6–4.0;−1, which is much higher than open ocean increases   . These erganisms .2, CO2 levels in water can be influenced by (1) nutrient inputs, (2) seasonal, event-based, and long-term warming, and (3) organic carbon export from land. In oceanic waters on the middle shelf and possibly in the Gulf Stream, warming likely contributes to an increase in pCO2 and decrease in pH  are negatively influenced by acidification showing dramatic declines in survivorship, delayed metamorphosis, and size reduction, even at relatively moderate levels of CO2 (650 μatm) but more so at higher levels   in laboratory experiments   that comeriments . There msurvival  and on psurvival , and prosurvival . Oyster survival . Such exCallinectes sapidus. When the ambient pCO2 was raised from 800 to 8,000 μatm, juvenile blue crabs showed no sensitivity to the duration of the intermolt period or to food consumption rate  are also vulnerable to acidification, especially at different life-stages, slowing down embryonic development, reducing hatching success, and decreasing larval survivorship  are found along the shelf edge off the east coast of central Florida between 70 and 100 m  with elevated pCO2  or indirectly  . Suggestconomies . Laboratted pCO2 . Multipled e.g., . Moreoveed e.g.,  and to ded e.g., . Combinied e.g., .pCO2 in coastal waters primarily resulting from oxygen consumption by respiration at night when photosynthesis declines and CO2 fixation decreases  is to produce more pigment in hypoxia. Studies have shown that this adaptive response to hypoxia is muted with elevated pCO2 , and input parameters for predictive model algorithm development.Monitoring key ocean parameters across various spatial and temporal scales that will provide information on mechanistic drivers of acidification . The paired continuous monitoring locations would provide key insights into causal factors of changing chemistry  and the region has important shellfish farms that have partnered with SOCAN to help inform monitoring and research activities.Priority 2 – The second proposed site includes either a new mooring farther offshore of GRNMS or an upgraded mooring offshore of Edisto Island, SC. These locations would allow for measurement of chemical changes in the Gulf Stream, a dominant and important regional feature in determining the regional chemistry.Priority 3 – The third proposed site is Biscayne National Park, at the northern edge of the Florida Reef Tract coinciding with locations that showed seasonal net dissolution of the reef and highest rates of reef and seafloor erosion .2, land- based sources of acidification present an opportunity for state and local-level mitigation. SOCAN worked with state partners to identify those programs that monitor and regulate water quality and have indirect linkages to potential acidification, such as state programs that regulate nutrient loads and monitor hypoxia. Existing water quality monitoring programs provide an opportunity to leverage additional efforts to monitor carbonate chemistry. Though the workshops were clear evidence of the diversity of stakeholders interested in acidification issues in the U.S. Southeast, most stakeholders suggested there is not yet enough information on localized carbonate chemistry dynamics and species responses within the Southeast region to guide an actionable response.SOCAN workshops have stimulated significant discussion of the heterogeneity of acidification in the coastal region and its impact on translating available scientific information to an actionable response for stakeholders. Diffuse and inland sources of nutrients and other pollutants create an inherent problem in organizing management of coastal water quality. However, relative to regulations of global anthropogenic COAccording to North Carolina stakeholders, there are accounts of sudden and significant losses of shellfish at mariculture farms that have not been accounted for by disease or other commonly measured water parameters such as temperature and salinity . These ein situ studies to connect changing chemistry with biological impacts to shellfish in the region.While information on regional and estuarine carbonate chemistry has quickly evolved, there is a scarcity of data on potential biological and socioeconomic impacts of acidification in this region. SOCAN is currently pursuing partnerships and funding opportunities to advance biological research in association with shellfish hatcheries and industry partners. In addition to a dearth of information on the response of U.S. Southeast shellfish to acidification, there are also significant gaps in our understanding of acidification impacts on harmful algal blooms, finfish, and microalgae in the region as well as identifying areas of refugia and community level responses. Acidification must be considered in combination with warming, high precipitation events, sea level rise, eutrophication, and hypoxia as investigations of single drivers can sometimes produce misleading results. Though many studies have shown impacts of acidification in the laboratory, there has been very little research conducted on Southeast stocks of shellfish and no A long-term aim of SOCAN is to expand efforts to develop models from existing data that can be used to determine the biological, chemical, and physical drivers of acidification. Identifying the relationships between coastal acidification conditions and other water quality parameters will provide a foundation for how acidification could be considered within existing water quality regulations. Due to the current relative importance of coastal acidification mechanisms  over atmospheric inputs, the Southeast has a variety of tools to address these localized contributions to changing coastal chemistry. Existing regulations for point and non-point source pollution, wastewater treatment and initiatives to address impervious surfaces can contribute to curbing acidification. As the research evolves, SOCAN has a role in translating coastal acidification, chemical and biological impact data to a holistic understanding of potential consequences for coastal communities. Working with both scientific and natural resource management partners SOCAN can translate scientific models to operational models.In closing, SOCAN offers the following recommended emphasis on current acidification research priority foci specific to the Southeast region:Multiple and/or compounding drivers of acidification;Single organism and community investigation on impacts of acidification;Life-stage analyses of “important” organisms;Effects of acidification on harmful algae species;Buffering capacity of coastal areas to acidification;Monitoring of estuarine and riverine acidification;Social and economic impacts from acidification; andPartnering with local management and industry to understand local impacts of acidification.


Pharmacogenomics (PGx) is the knowledge of diverse drug responses and effects in people, based on their genomic profiles. Such information is considered as one of the main directions to reach personalized medicine in future clinical practices. Since the start of applying next generation sequencing (NGS) methods in drug related clinical investigations, many common medicines found their genetic data for the related metabolizing/shipping proteins in the human body. Yet, the employing of technology is accompanied by big obtained data, which most of them have no clear guidelines for consideration in routine treatment decisions for patients. This review article talks about different types of NGS derived PGx variants in clinical studies and try to display the current and newly developed approaches to deal with pharmacogenetic data with/without clear guidelines for considering in clinical settings. FGVs or actionable genetic variants are those alterations in genome, with at least one report for introducing the effects on drug safety and/or efficacy in people. Moreover, the variants found in the research area with strong potential effects on drugs could be considered as FGVs during prescription. However, the latter needs clinical evidence to be influential on treatment decisions by physicians). Furthermore, the poor knowledge and background of PGx and the different related alleles and variants for many healthcare professionals may directly affect their desire to order the tests.It has been reported for decades that different drugs show different responses and efficacy in diverse individuals or populations. Investigations proved that part of this diversity 20–30%) is because of genetic background and, more precisely, the inheritance of various alleles and variants in genes for drug-metabolizing and transporting (pharmacogenes) or drug target molecules . Pharmac0–30% is Yet, several rare and uncommon FGVs can be detected through the PGx tests in both clinical and research areas, especially when comprehensive and high capacity methods, such as NGS, have been utilized . MoreoveBoth common and rare alleles are demonstrated as the functional biomarkers in PGx clinical practice. Low frequency and rare variants have been shown by 1–5% and lower than 1% minor allele frequency (MAF), respectively, in populations. Moreover, they proved to be very population-specific and the causative elements for diverse drug responses in alternative ethnic groups ,11. NGS Currently, core web-based resources for clinical PGx annotations include Pharmacogenomics Knowledge Base (PharmGKB), the Clinical Pharmacogenetics Implementation Consortium (CPIC), the Pharmacogenomics Research Network (PGRN), and Dutch Pharmacogenetics Working Group (DPWG). These are considered as reference databases that provide information about how human genetic variations affect response to medications. All of the confirmed data about clinically actionable gene-drug associations and genotype-phenotype relationships are sorted properly and available as a guide for personalized medicine implementation by healthcare professionals. However, other modules, such as PharmVar, FINDbase, SuperCYP, SEAPharm, etc. could also be applied when a specific type of gene or drug was on the desk. Nevertheless, according to PGx reference organizations , all the diagnosed alleles and variants in a gene-drug interaction, based on the number of published studies and clinical evidence, will be classified in various types of level with clear explanations for each of them . HoweverCYP2C9 and rs9923231 in VKORC1 genes for Warfarin). (2) Annotated variants with no clinical guideline . (3) Variants with annotation or guidelines for other drugs . (4) Non-pharmacogenetically annotated variants . And (5) Variants of unknown significance (VUS). The next part will focus on different approaches for such variant interpretation and curation in clinical practice.Regarding the abovementioned level of classification for the identified variants, the utilization of NGS platforms for clinical PGx tests brings various types of alleles, which after confirmation and validation processes could be categorized as functional/potential effective variants, fall into “five groups of (1) annotated variants with the clear guideline  are the novel and unreported variations in databases , but found in a PGx test mostly through comprehensive methods, such as whole exome or whole genome sequencing (WES and WGS), with no clue for their function in causing a particular phenotype. Moreover, incidental findings (IFs) are the group of known variations, but not related to specifically investigated phenotype, and accidentally revealed during a sequencing test. Both the VUS (novel variants) and IFs will be manageable with higher accuracy by the combined usage of highly specialized bioinformatics pipelines to find any possible interaction with drug responses in patients. IFs are mostly displayed as the annotated functional drug-related variants in pharmacogenes and potentially useful markers if the appropriate genomic analysis and accurate genotype–phenotype correlations are performed subsequently . We willSIFT, PolyPhen2, MutationTaster) and PGx dedicated tools  [As the majority of revealed variants through implementation of broad range high throughput sequencing tests could be categorized in group 4 and 5 , the process of identifying clinically relevant PGx variants from complex genomic data mostly concerns about the detection of any potential FGVs in these two categories. The procedures usually start with digging the variant call format (VCF) file for filtration of variants and selection of those alterations, which come from drug-related genes. Based on the employed sequencer machine and the selected platform for PGx data clinical assessment, different types of variants are available in subsequent result analysis (SNVs and/or CNVs from coding and noncoding/regulatory parts of the genome). Routine silico analysis is considered for filtration of NGS derived pharmacovariants data at the first step . Next, the selected variants go for pathogenicity and functional annotation analysis through the utilization of prediction algorithms in both common  ,28,29. AVAT, VarAFT, ANNOVAR, etc., is inevitable. Then, the prediction of potentially damaging, deleterious, and/or functionally neutral non-synonymous variants will be performed via the algorithms as mentioned earlier. Currently, the mutual beliefs for PGx data analysis are the combined utilization of 6 to 7 of such prediction tools and choosing those variants, which are commonly introduced as pathogen/likely pathogen in all applied software, according to reliable reference guidelines, such as those given by ACMG, CAP, and CPIC [Stargazer, Astrolabe, PharmCAT, PHARMIP, etc., are increasing rapidly in a fast-developing mode. Hence, integrating them in applied algorithms seems necessary. Examples for the generation of clinical recommendations for the variants using in silico analysis of WGS PGx data were done before. The related studies showed the PGx dosage recommendations are heavily influenced by the higher availability of genotyping results, which may lead to more clinical evidence too . Yet, thand CPIC . While tACE), following environmental and epigenetic modifications on drug-related genes, working with challenging and problematic variants, in particular drug-related genes , and most important of them the lack of previous knowledge on possible phenotype modifications for many genetic changes (as PGx is a pre-emptive genotyping test in numerous cases) can potentially increase the difficulties in variant analysis and pose the clear effects on changing the drug responses in individuals. Albeit, providing more genotype to phenotype translation methods by reference organizations and guideline developers will result in more consistent genotype interpretation in both clinical and research area [NGS technologies have been used in several PGx studies in recent years. Based on the employed platforms, the acquired data analyzed through different approaches. Due to the lower amount of identified variants , finding the FGVs and phenotype prediction is usually easier when targeted sequencing for a specific set of the gene (panels) is performed as the selected method. WES and WGS, however, show a lot of obstacles when applied for a PGx analysis and this is mainly because of the huge number of functionally unknown and unreported alterations in a patient’s genetic profile . Moreoverch area .SIFT, PolyPhen2, and CAAD, and deleterious rare-novel variants in some of VIPs evaluated by in-vitro analysis to find impaired functions evidence. Moreover, additional and novel faraway variants (group 5), contributed to the alteration of estrogen receptor binding site and breast cancer risk identified in 400 patients by NGS deep sequencing and functional genomics. As the number of investigated genes was low, any novel PGx variant was confirmed through the laboratory tests, such as chromatin immunoprecipitation (ChIP), gene expression analysis, and protein degradation assays [CYP2D6, HLA-A, and HLA-B) by applying the long read sequencers (group1 and 2). All the SNVs, CNVs, and InDels were revealed through the utilization of customized long-range PCR and the subsequent NGS machine (MinION nanopore sequencer) [SIFT, and PolyPhen2 for functional effect analysis of novel non-synonymous coding SNVs, mapping the deleterious variants with PharmGKB and DrugBank, and finally PLINK and VCFTools for reaching allele frequencies and validation through 1000 genome and HapMap databases. In the end, a drug pathway map for functionally impaired pharmacogenes displayed, using identified deleterious variants [CYP2D6 CNV and UGT1A1*28 TAA repeats in promoter in addition to allele frequency and homozygosity in 235 patients. Common in-vitro and bioinformatics tools used for both known and novel variant detection rate accuracy and sensitivity  [Despite the challenges, the number of publications for NGS derived PGx data analysis are still significant. Gordon et al. successfully identified common, rare, and novel variants in 84 clinically actionable drug-related genes in more than 280 individuals through a targeted resequencing custom panel. They used deep coverage of the known genes to follow both previously recognized and possible novel variants. New potentially deleterious nonsense and missense variants across some VIPs were selected for more genotype-phenotype association studies to find any relation with particular traits . Moreover, actionable plus rare unreported variants in absorption, distribution, metabolism, and excretion (ADME) core genes revealed in 114 drug genes in 376 people by Han and colleagues. The number of variants in each gene , MAF, and novelty assessed and compared to open genotyping datasets . In silico functional assessments performed by the prediction tools, such as n assays ,73,74. Oquencer) . Moreovequencer) . In anotvariants . Even th, and 5) .PharmCAT) and tried to reveal which patients in a clinical dataset include the variants of interest [Two important points could be mentioned from the above investigations as well. As the majority of functional prediction tools and algorithms are relying on evolutionary conservation and therefore will not be completely fit with the pharmacogenes (poorly conserved) and show low predictive accuracy as the conventional algorithms (up to 50%), most of the studies emphasize combined utilization of such tools in in silico phenotype prediction for novel variants and introduced various software in each report. This may remind the necessity of the attitude for new PGx data in high throughput sequencing methods, as they are not observable in many cases (pre-emptive genotyping). Recent efforts, however, have been focused on developing new pharmacogene optimized frameworks with more relation to PGx data assessment through the integration of specific algorithms or presenting the allele dedicated for pharmacovariant calling and showed to be more compatible with ADME genes with a higher rate of sensitivity and specificity (90–99%) ,63. Otheinterest .The second point is the ability of NGS technologies to the detection of any kinds of PGx variants in clinical practice. They have introduced several novel PGx markers successfully and the fact may indicate the faster incorporation of PGx test results into the future precision medicine as well. However, there are still essential issues with high importance in the field, which need to be addressed properly. For example, if the particular novel variant causes a loss of function or gain of function effects on the related protein(s) (making a poor or rapid metabolizer) in tested individuals and also possible misinterpreting of VUS in the result, which may lead to ignore or miss the functional variants in pharmacogenes. Such complexities must be followed by the in-vitro assessments in addition to appropriate pre and post-test counseling for individuals ,78.The intricacies are not limited to the detection of variants, but the nature of drug actions according to particular alleles too. Investigations displayed the dual or multiple impacts of some specific pharmacovariants toward the different diseases and/or drugs . FurtherThe field of pharmacogenomics faces several challenges throughout the process of the identification of pharmacogenomic variants and their implementation in clinical practices. Many of these challenges arise at the genomics level, including the statistical considerations associated with the design of the clinical trial and genome-wide association studies (GWAS), a large number of candidate variants compared to available samples (p > n), the lack of reproducibility in independent studies and determining the functional impact of variants on drug response. In the age of PGx and personalized drug therapy, using the high throughput sequencing approaches will assist the translation of different pharmacovariants into clinical care. As mentioned before, for moving genomic medicine toward personalized drug therapy, there should be a genetic screening test, which fits all ethnicities . NGS, as


To this end, we first review the roles of the lung epithelium and vasculature, with focus on the alveolar-capillary membrane, and then discuss the available and emerging technologies for ex vivo bioengineering of the lung by decellularization and recellularization. While there have been many meritorious advances in these technologies for recovering marginal quality lungs to the levels needed to meet the standards for transplantation – many challenges remain, motivating further studies of the extended ex vivo support and interventions in the lung. We propose that the repair of injured epithelium with preservation of quiescent vasculature will be critical for the immediate blood supply to the lung and the lung survival and function following transplantation.The shortage of transplantable donor organs directly affects patients with end-stage lung disease, for which transplantation remains the only definitive treatment. With the current acceptance rate of donor lungs of only 20%, rescuing even one half of the rejected donor lungs would increase the number of transplantable lungs threefold, to 60%. We review recent advances in lung bioengineering that have potential to repair the epithelial and vascular compartments of the lung. Our focus is on the long-term support and recovery of the lung  The inde < 4 h) . Howevere < 4 h) . Unfortue < 4 h) . Some exac death . Neverth2) and removes carbon dioxide (CO2) from circulating blood, through the process called gas exchange, the main function of the lung that occurs in the alveolar spaces. In addition to the skin, the lung is the only organ in direct contact with the external environment. Before reaching the alveoli, air passes through the conductive airways, where it gets filtered by the host physical barriers and cleared by the immune system.The lung is an extremely complex organ, featuring intricate 3-D architecture, diversity of cellular composition (with more than 40 cell types) , a highl2 and release CO2. In the alveoli, ATI cells – the thinnest cells in the human body – cover approximately 95% of the total alveolar surface  comprises about 90% of its volume; the other 10% consists of conducting airways and larger vessels. The air that reaches the alveoli is separated from the blood perfusing the lung by a three-layer structure: (a) the alveolar epithelium lining , (b) connective tissue , and (c) the endothelial lining of the alveolar capillaries, where venous blood coming from the pulmonary arteries flows to capture O surface . AT1 cel surface .2, but they can also be reached by airborne particles, toxins, and microbes that bypass the conductive airways – these insults can severely injure the epithelial cells. Notably, most lung diseases affect the epithelium  regeneration of injured or diseased lungs; and (2) bioengineering of the lung as a platform for modeling lung regeneration, disease and drug discovery. Together, these two areas aim to reduce the burden of end-stage lung disease.In this review, we focus on recent advances in lung bioengineering aiming to repair the epithelial and vascular compartments of the lung, and in particular on bioengineering methodologies that allow preservation of the integrity and function of lung vasculature.2 and nutrients to the organs, and regulates coagulation cascade and metabolism between organs. In addition, the endothelial cells are responsible for vital physiological activities involved in the homeostasis and metabolism of the surrounding tissue during lung development, repair and regeneration. During development but also in adulthood, endothelial cells are regulated by organotypic transcription factors that inform the transition into organ-specific endothelial cells.Blood vessels are extremely important in the architecture of every organ of the body, and especially in organs as highly vascularized as the lungs. The lung vascular network represents the largest microvascular network in the body, with a capillary network of endothelial cells that connects to the large arteries and veins. The capillary network is a barrier-forming cell lining and a conduit that delivers Oin vitro different types of tissue-specific vasculature  of the capillary network are in proximity with the alveolar epithelial cells , with thPCECs have a distinct phenotypic signature (VEGFR2 + FGGR1 + CD34 + VE-cadherin+) that makes them distinguishable from all other cell subtypes . During The importance of VEGF during embryogenesis has been documented in experiments with genetic inactivation of VEGF. VEGF isoforms 120, 164, and 188 are highly expressed in ATII cells during mouse lung development, especially during the canalicular stage when the angiogenesis occurs . Mice wiAnother set of factors shown to play a role in lung alveolarization and angiogenesis is the set of Hypoxia Inducible Factors (HIFs) . HIF-1, Vegfr2 and Fgfr1 after pneumonectomy impaired alveolarization and propagation of ATII cells  differentiation. BASCs are a small cell population localized at the bronchioalveolar duct junction (BADJ) in the mouse and capable of repopulating both the airway and the alveolar compartments after injury . In a 3-The alveolar-capillary barrier consists of the bipolar layer of epithelial and endothelial cells, separated by a thin membrane of specialized extracellular matrix called ACM. On the airway side, there is a continuous alveolar epithelium made of ATI and ATII cells; on the capillary side, there is a continuous PCECs lining. The continuity and tightness of the alveolar epithelium and the capillary endothelium are essential for fluid and ion transport between the alveolar and vascular spaces. The ACM is not only a fundamental structural component of the respiratory zone of the lung but also regulates – together with the adjacent cells – gas exchange, fluid homeostasis, microenvironment sensing and cell-cell interaction and migration .The ACM consists of large insoluble molecules that form a sheet-like mesh, and are regulated by anchor proteins and receptors . LookingMass spectrometry proteomics has allowed characterization of the matrisome, or the total amount of proteins in the ECM . The corType IV collagen, a key structural factor of the ACM, provides barrier function between epithelial and endothelial cells . FibroneTwo characteristics of the ACM that are fundamental for bioengineering of all organs are the matrix topography and stiffness . Matrix 2. During lung development and growth – from late fetal life to somatic maturity – respiratory movements are essential for lung maturation together with forces coming from blood flow. The balance between the inward and outward forces of the rib cage impose mechanical stretch on lung parenchymal cells, allowing tissue growth and remodeling to relieve stress and strain  in isolated and perfused rat lungs, where constant alveolar PO2 (∼100 mmHg) was maintained by ventilation (ATP channels (+2 channels with increased intracellular Ca + 2 and activation of nitric oxide (NO) synthase, increase in NO, and consequent vasodilation  and lung parenchyma (secondary target) contributed to the declining lung function. Once lungs were again re-perfused, the return of blood flow led to further oxidant production, and the damage due to reperfusion exceeded that resulting from ischemia . The onsSeveral events characterize I/R injury: epithelial injury/dysfunction, release of cytokines and damage-associated molecular patterns (DAMPs), and vigorous innate immune responses including activation of alveolar macrophages, invariant natural killer T cells and neutrophils. Most of these responses are initiated by rapid and robust generation of ROS that leads to cell/tissue injury, activation of multiple cell types, lipid membrane peroxidation and secretion of inflammatory cytokines and DAMPs . The actin vivo implantation.On the basis of these findings, it is clear that the altered cell signaling from abnormal blood flow (cessation of blood flow or reperfusion) affects the viability and overall quality of the lung. These factors are imperative to account for in the preparation of biological lung scaffolds for lung bioengineering or transplantation. The magnitude and distribution of mechano-transduction signals are critical for facilitating cell repopulation and engraftment, matrix remodeling, and re-establishment of the integrity of ACMs prior to ex vivo, it was observed that a drop in shear of >90% of the physiological flow was triggering the ischemia mechanosome (ex vivo (see next section), may be a maneuver that prevent ischemia-reperfusion signaling and injury, significantly improving the quality of the lung grafts, the number of suitable organs for transplantation and reducing their failure post-transplantation.An approach to prevent lung I/R injury may be to maintain lung grafts within physiological conditions during procurement, preparation and storage to minimize the mechanotransduction signaling cascade initiated by ROS. In the rat lungs isolated hanosome . Thus agex vivo lung perfusion (EVLP)   and crosn (EVLP) . In bothEx vivo lung perfusion is the most common current approach. Hypothermic preservation that maintains organ viability by reducing cellular metabolism, does not facilitate lung tissue repair following the ischemic injury post-explant. Instead, EVLP support in normothermic conditions could provide prolonged preservation, but mostly, ameliorate the effects of ischemic injury and induce lung repair. Since its introduction by 2 and electrolytes. This technology has been improved over time and was applied to human lungs by the Toronto Group in 2011, where 20 of 23 lungs initially deemed not suitable for transplantation were reconditioned on EVLP and transplanted into patients  followed by 12 h of EVLP support versus 24 h of CPS, showed less interstitial edema, hemorrhage, cell infiltrate and lower lung injury score (LIS)  Cypel e. A seminpatients . Althougpatients . The mospatients .ex vivo is limited by the inability to provide the metabolic clearance and homeostatic physiologic environment of the lung, with its innate reparative mechanisms.Over the years, EVLP used in research settings has enabled therapeutic cell delivery, airway lavage and surfactant replacement , and eveseverely injured lungs (most importantly those injured by gastric aspiration). Development of pulmonary edema, diminished gas exchange capability, and deterioration of the endothelial-epithelial barrier remain ubiquitous and formidable challenges that hinder the effectiveness of EVLP in providing long-term support. To address these limitations, our group recently introduced a novel XC platform for extracorporeal lung support using a large animal model (swine)  support is required for advanced bioengineering interventions  that can recover  (swine) . In XC s (swine)  (O’Neill (swine) . Importa2/FiO2 ratio, and tight-junction protein ZO-1 expression  decreased over the first 12 h, but then returned to the baseline by 24–36 h on cross-circulation. Notably, ischemic lungs recovered both functionally and morphologically, had decreased interstitial edema, restored the barrier function, and attained PaO2/FiO2 ratios measured in healthy lungs.Initial studies demonstrated the use of cross-circulation with an anesthetized swine host for support and recovery of injured donor lungs with a perfusion time of 36 h and a total preservation time greater than 60 h. In one study , two expex vivo can be implemented for the functional recovery and regeneration of severely damaged lungs  lung regeneration over 36 h of XC with monitoring of the lung at the molecular, cellular, and tissue levels. The prolonged lung support enabled us to determine the effects of multiple therapeutic interventions: bronchoalveolar lavage, surfactant delivery, and alveolar recruitment.Based on the recovery of ischemic lungs on 36 h of XC, we investigated whether normothermic perfusion/ventilation of whole lungs ed lungs . To thised lungs . We inve2/FiO2 of injured and control lungs, demonstrating that even severely injured lungs can be recovered using this methodology , and recipient swine were hemodynamically stable . Radiography confirmed extracorporeal lungs remained aerated, and bronchoscopy revealed normal airways without edema or secretions. Analysis of bronchoalveolar lavage fluid revealed no significant increases in inflammatory cytokines from baseline to day 4. Histologic evaluation showed an intact blood-gas barrier and full preservation of the airway and alveolar architecture as well as cellular viability and metabolism over 4 days of normothermic support. While future studies will need to investigate and confirm that this approach is robust and reproducible across large numbers of lungs with different types and stages of injury, and a wider variety of experimental conditions, we anticipate that this model can be extended into a human model of awake extracorporeal lung recovery. The use of a multi-day XC platform could enable recovery of damaged lungs not currently salvageable using EVLP systems with the ultimate goal to recover the functionality of the lung and reduce the risks of primary graft dysfunction often due to lung injuries that occurred from the time of brain death to reperfusion in the recipient  could not be contained within an average adult body size. (ii) Limited-time performance as they last only for days to weeks; (iii) Thrombogenicity, as these membranes are made of synthetic material and easily activate the recipient immune system and the intravascular coagulation, requiring life-long anticoagulation. Several groups are investigating the optimal strategies to cover those membranes with autologous endothelial cells to improve their haemocompatibility and build a “bio-hydrid” system, but the clinical reality of a long term lung substitute remains far at the moment  <50 ng double-stranded DNA (dsDNA) per mg of decellularized material; (ii) less than 200 bp length of DNA fragments; (iii) preservation of structural proteins of ECM; and (iv) retention of mechanical properties . Particuin vivo for the whole 3 days , and mutations in the ABC subfamily 3 (ABCA3)  stress and apoptosis  . These d (ABCA3) . Expresspoptosis . SFTPC a in vivo . EpithelExtensive epithelial damage is often involved in the loss of the epithelial-mesenchymal homeostasis, with rearrangement of ECM and lung architecture leading into fibrosis. Reconstitution of functional epithelium  is crucial for preventing pathological lung remodeling and for recovering the most important lung function: gas exchange. Hypothetically, regeneration with healthy epithelial cells could promote local proliferation of the remaining undamaged epithelium, activated by the local lung progenitor or use of exogenous stem cells .2, much effort has been invested into finding the best strategies to repair lung epithelium while keeping the vascular network intact and functional. The derivation and use of vascularized lung scaffolds has been pioneered by our group  the challenging hurdle to properly recellularize an organ consisting of 40 distinct cell types ; (ii) thex vivo. In both cases, lungs were maintained on air ventilation and vascular perfusion. We demonstrated that this method could be used for decellularization and lavage needed to remove epithelial cells, and also for delivering therapeutic cells to the denuded locations in the airway.To enable replacement of the epithelium in specific local areas of the lung (rather than in the entire lung) while maintaining intact vascular perfusion and endothelial lining , our group developed a methodology for targeted delivery of microliter volumes of decellularization fluid or cell suspension into the lung from the upper airway all the way to the most distal alveolar spaces , 2017. I2, nutrients, growth factors, and signaling molecules, (ii) providing biophysical and mechanical signals via perfusion  and ventilation (strain), and (iii) maintaining the ECM  and the interstitial and support cells . De-epithelialized lung scaffolds could be repopulated with embryonic, fetal, adult primary or induced pluripotent stem cells, while maintaining functional vascular and interstitial compartments, and provide a valuable physiological model for investigating: (i) lung development, (ii) etiology and pathogenesis of lung diseases involving the pulmonary epithelium, (iii) acute lung injury and repair, and (iv) drug and cell therapies  enabling the delivery of Oherapies . Lung scThe shortage of transplantable donor lungs for patients with end-stage lung disease has motivated scientists to investigate alternative therapeutic strategies to traditional transplantation. Cell-based therapy has dominated the lung field in the last two decades. The main strategy has been to generate a functional lung graft by combining a natural lung scaffold with appropriate cell types to create a transplantable bioengineered lung.ex vivo, demonstrated the capability of a decellularized lung graft to support engraftment of multiple cell types, despite the fact that alveolar edema and thrombosis caused lungs to fail after only a few hours following transplant  the possibility to intervene with cell therapy in lung grafts supported ex vivo. However, these advances also posed a major challenge to researchers: to create a graft with the functional capability of the lung, an extremely complex organ containing more than 40 different cell types  in 2006  cells are removed and subsequently replaced with healthy, engineered lung progenitors. Cell type, number, engraftment and delivery strategies (airway versus intravenous delivery) are important factors that have been explored in recent years.Methods for optimization of lung scaffolds have radically improved over time to promote cell engraftment . Additioher EVLP  or the Xher EVLP . Recogni in 2006 , there hin vitro for differentiation to mature cell types  prior to delivery in the lung  was used to induce epithelial injury in the airways and remove endogenous cells to make room for new cells to engraft. Several other groups have shown engraftment of murine or human lung stem cells in different lung injury models in mouse, including naphthalene, radiation, detergent, and virus mediated injury  epithelial cells and replacement with normal human bronchial epithelial cells or iPSCs where CFTR mutation is corrected  type I and lack MHC type II and T-cell costimulatory molecules, making them highly non-immunogenic. Additionally, MSCs can be rapidly expanded the lung . These cLP or XC . Lee et neumonia . In ARDSneumonia , as wellneumonia .E. coli toxin injured human lungs on EVLP reduced lung weight gain, improved lung endothelial barrier permeability, and restored alveolar fluid clearance in a Fibroblast Growth Factor (FGF) dependent manner  intact perfusable and viable vascular network, and (iii) regional specificity to allow seeded lung progenitors to migrate to their specific natural niches and differentiate into site-specific lineages.In the stem cell field, a challenge is to identify the key ACM factors that could modulate alveolar stem cell function and regulate lung scaffold repopulation, including, for example, different subtypes of metalloproteases, serine proteases, and matrisome proteins that could act through integrins and augment lung progenitors’ regeneration. Vascularized lung scaffolds with intact ACM could be applied to identify the proper ACM signals and define the “ACM regenerative profile.” Cocktails of factors  could also be used to specifically enrich decellularized lung scaffolds prior to seeding with lung progenitor cells, with the goal to achieve lung regeneration and repair under increasingly physiological conditions. An “ideal” scaffold for lung tissue engineering, obtained by de-epithelialization of the lung, would maintain: (i) functional ACM for lung progenitor cells to adhere, engraft, and differentiate, (The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.Guide for the Care and Use of Laboratory Animals, 8th Edition.The studies conducted by our group complied with the relevant ethical regulations for animal testing and research. Approval for this study was received from the Institutional Animal Care and Use Committee at Columbia University. Animal care and procedures were conducted in accordance with the US National Research Council of the National Academies ND and GV-N jointly planned and wrote the 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.


Objectives: We explore the association between different patterns of change in depressive symptoms and memory trajectories in US and European Mediterranean  and non-Mediterranean  older adults. Methods: Samples consisted of 3,466 participants from the Health Retirement Study (HRS) and 3,940 participants from the Survey of Health, Aging and Retirement (SHARE). Individuals were grouped as follows: non-case depression (NO DEP), persistent depression (DEP), depression onset (ONSET), depression recovery (RECOV), and fluctuating (FLUCT). Memory was measured using immediate and delayed recall tests. Linear mixed models were used. Results: DEP and RECOV had significantly lower baseline memory scores compared to NO DEP, at intercept level. At slope level, ONSET had a significantly faster decline in both tasks compared to NO DEP. Discussion: Cross-cohort robust and consistent new empirical evidence on the association between depression onset and faster decline in memory scores is provided. The association between cognitive function and depression in older adults has been extensively studied . Cross-sMost of the aforementioned studies include depression at baseline, assuming that depressive symptomatology in older adults follows a stable pattern. However, a number of studies have provided evidence regarding changes in depression over time , though Within this context, the general aim of the present study is to explore the association between different patterns of change in depressive symptoms and trajectories of immediate and delayed recall in U.S. and European older adults.The U.S. sample consisted of a subsample of 3,466 respondents  of the Health Retirement Study (HRS) and the European sample consisted of a subsample of 3,940 respondents  of the Survey of Health, Aging and Retirement (SHARE). Both studies are biannual, longitudinal, and nationally representative surveys that focus on adults aged 50 and over. Data from five consecutive waves of each study have been used: Wave 4 (1998) to Wave 8 (2006) of HRS, and Wave 1 (2004) to Wave 5 (2013) of SHARE (Wave 3 on SHARE was not included as it did not have available data on the relevant variables for the present study). Wave 4 of HRS was selected as “baseline” as 1998 is when HRS and AHEAD were fully integrated and when HRS assessment procedure matched other sister studies as SHARE. Considering previous research , the couFor the purpose of this study, the first wave considered will be labeled as baseline and the subsequent ones follow-ups. Only respondents who were between 65 and 75 years old at the first wave were considered (baseline) and who had at least 3 waves of complete data in depressive symptoms and immediate and delayed recall were included, according to Both HRS and SHARE assessed memory using tests of immediate and delayed recall of 10 common nouns. Respondents were asked to recall as many words as possible immediately after the list was read and after a short delay of 5 min, during which they completed other cognitive tests. In both studies, different randomly assigned word lists were used at each occasion. Total scores are the number of words recalled in each test and range between 0 and 10, with higher scores indicative of better memory.symptom not present) or 1 (symptom present), and item scores are summed to produce a scale with a minimum score of zero and a maximum of 12. The psychometric properties of the EURO-D have been extensively investigated and criterion validity demonstrated in the cross-cultural context. Criterion validity for this measure was demonstrated with an optimal cut-off point of a score of 4 or above against a variety of criteria for clinically significant depression , which is a questionnaire developed as a depressive symptoms screening scale for epidemiological investigation . Its valpression . Previoupression .To identify the different patterns of change described in previous research , we follBaseline age, sex, education and self-rated health (SRH). Education was coded in three categories . SRH reflects the general perception of an individual’s health status, and it is one of the most inclusive and informative indicators of health status . SRH, orPreliminary descriptive and exploratory analysis examined differences at baseline between groups within each study. Differences were examined using chi-square and Kruskal–Wallis tests. To examine the trajectories of immediate and delayed recall over time, in each group, linear mixed models with random coefficients  were estDescriptive statistics for depressive symptoms, immediate and delayed recall and covariates at baseline in the study samples are shown in Despite the variation of prevalence rates for each of the observed patterns of change in depressive symptoms in HRS and SHARE Mediterranean and non-Mediterranean countries, when we order the prevalence from highest to lowest, we find the same order . Most ofIn HRS and SHARE Mediterranean and non-Mediterranean countries, we found an association between the pattern of change in depressive symptoms and immediate and delayed recall scores at intercept level  and 4. IWith regard to change over time, a significant overall linear decline in immediate recall was found in HRS and SHARE-Non Mediterranean samples. The immediate recall trajectories in SHARE Mediterranean sample were better described by a quadratic model. Individuals who reported a pattern of increasing clinically significant depressive symptoms over time (ONSET) showed a slightly faster decline in their immediate recall scores when compared to those who had reported less than four clinically significant depressive symptoms across all waves (NO DEP) in all the samples. Moreover, in the SHARE non-Mediterranean sample, individuals whose clinically significant depressive symptoms decreased over time (RECOV) also showed a slower decline in immediate recall compared to NO DEP. No associations with rate of change were found for any of the covariates except that HRS women who showed a slower decline over time compared to men.For delayed recall, linear models were appropriate for the trajectories in HRS, whereas quadratic models showed a better fit in SHARE. Similar to immediate recall, individuals reporting a pattern of increasing clinically significant depressive symptoms over time (ONSET) showed a slightly faster decline in their delayed recall scores when compared to those who had reported less than four clinically significant depressive symptoms across all waves (NO DEP) in HRS and SHARE Mediterranean and non-Mediterranean samples. In addition, those who endorsed depressive symptoms persistently over time (DEP) and those who had a fluctuating pattern with no trend (FLUCT) also showed a slightly faster decline in their delayed recall scores when compared with NO DEP. Differences in the rate of change were not found for any of the covariates except sex and education level in HRS. Consistent results were found for sensitivity analyses which included baseline depression in the model.The main aim of the present study was to explore the association between different patterns of change in depression and memory trajectories in U.S. and European participants of the HRS and SHARE, respectively. This study is the first, to our knowledge, that goes beyond patterns of persistent versus not persistent depression described in previous literature on cognitive decline . SpecifiWith regard to the rates of change in immediate and delayed recall scores and their association with the above mentioned patterns of change in depression, it was found, consistently across studies, that individuals who had an onset of depression across waves showed a faster decline in cognitive performance compared to those with no depression over time. It should be noted that although the individuals identified with persistent depression over time also showed, in general, a faster decline when compared with those individuals with no depression, the estimate was not significant.Our results cannot be directly compared with previous research that only considered persistent and not persistent patterns with shorter follow-ups of 2 or 3 years , 4 yearsThe main advantage of the present study is that its design and statistical analysis facilitates comparability across samples and future replication studies. Replication studies are essential to build scientific knowledge, and recent publications have highlighted the need to promote systematic replication efforts , especiaThe results of the present study not only support the well-known association between depressive symptomatology and cognitive performance in older adults, but provide new empirical evidence on the association between depression onset and faster decline in memory scores in U.S. and European adults over 65 years old. Our findings suggest that not simply duration, but the timing of onset depression, might be the key to understanding the association between depression and cognitive decline.


To quantify the link between four different types of social ties and objective measures of abdominal and general obesity, and to explore their inter-relationships in association with obesity using a gender-sensitive analysis.A cross-sectional analysis of 28,238 adults (45–85 years) from the baseline Canadian Longitudinal Study on Aging Comprehensive cohort (2012–15). Social ties  and measured anthropometry  were analyzed using linear and logistic regression models with interaction terms conditioned on known confounders.We found that being single, widowed or divorced/separated was associated with worse anthropometric outcomes in women, including higher odds of both abdominal and general obesity, and that associations were enhanced when combined with limited social participation, lone-living and greater social network size. Few clear associations were observed in men. Limited social participation  among women was associated with larger waist circumference  and higher odds of both abdominal and general obesity. By contrast, associations appeared to be reversed in men: lone-living and smaller social networks were associated with lower odds of obesity, compared to co-living and larger social networks. We also found that more regular social participation can potentially mitigate the adverse associations between non-partnership  and obesity in women. Overall, the combined influence of two types of social tie deficits on excess weight measures was more pronounced in women than men.Results highlight the importance of considering how the role of social ties for obesity prevention may differ for women and men. Frequent social participation and number of social contacts may matter for assessing whether divorced, single or lone-living older women are at risk of obesity while living arrangement and social contacts may matter for obesity in men. Healthy aging across the lifespan is an important public health and policy issue globally, including in Canada . In factDiscrimination (particularly gender) and poor social resources are both important upstream determinants of health , yet litObesity has been shown to be linked to social connections, and could be one of the biological pathways for the established link between social connections and mortality ,9,22. HoIn addition, the many different types of relationships that might influence obesity have not been examined for their joint effects ,27. The Using a new population-based aging cohort in Canada, we assessed whether four different types of social ties were related to two measured anthropometric outcomes in gender-specific ways, and whether different social connections interacted to produce a combined effect on obesity. We hypothesised that each social tie variable would be linked to obesity, with stronger effects of marital status and living arrangement in men and social engagement in women. We further hypothesised that frequent social activities or co-living might mitigate the negative association of being single or widowed or having rare social contact with obesity. Similarly, frequent social contact may lessen the negative association of living alone with obesity.We used the baseline data (2012–15) of the population-based Canadian Longitudinal Study on Aging (CLSA) Comprehensive cohort that provided information on physical, social and psychological factors as well as objective measures of abdominal and general obesity from approximately 30,000 respondents aged 45–85 years living in the community . Exclude2 was considered for being generally obese in both women and men. Both continuous  and binary  outcomes were analysed.Clinically measured anthropometry included height (m), weight (Kg) and waist circumference (WC), measured from halfway between the last rib and the iliac crest bone . We usedStructural aspects of social ties were assessed by marital status (partnered (as reference), single, widowed, divorced/separated), living arrangement ), social network size  and social participation  . Social Self-reported confounders were age and age squared, smoking status (ever/never), province and education . Our selection of covariables was informed by existing literature on factors known to correlate with our exposure and outcome. As known confounders, each is associated with social relationships and independently with obesity ,45–47.Sensitivity analyses included other factors that may be additional confounders or are sex-specific biological determinants of obesity ,24,33: IMeans (SD) and frequencies were used to describe the characteristics of the cohort across levels of four structural social ties. The CLSA Comprehensive cohort only included the Canadian population that resided within 25 km of the Data Collection Sites, hence survey weights were not used as this sample was not nationally representative . We usedObjective one aimed to examine the gender-specific main and independent (of other ties) associations between each social tie and each anthropometric outcome. For this objective we ran a series of multivariable linear (WC and BMI as outcomes) and logistic  regression models. First, we assessed main associations with mutual adjustment for age, age squared, education, smoking and province. Second, we additionally adjusted for all other social tie variables so that the regression results could be interpreted as the independent association of each social tie with each outcome. Each model included an interaction term between the social exposure and self-reported female/male, giving gender-specific results. We used reverse coding of the interaction term  to provide estimates for each reference group from the same model. We reported results as post-estimated adjusted means, or odds ratios (OR), and 95% confidence intervals (95% CI). Sensitivity analyses tested the robustness of the independent associations to additional confounding.Objective two considered whether the association between each social tie and obesity changed when a second social tie was considered (joint association). For example, the association of marital status and obesity may be stronger or weaker for those that have no social participation compared to those that have the most social participation. For Objective two, WC and BMI were analysed using gender-stratified multivariable linear regression models conditioned on key confounders with interaction terms for each pair  and reported as adjusted means and 95% CI. All analyses were performed using Stata/SE 14 .The mean age of participants was 63 (SD 10) years with 50.6% women . A higheIn multivariable-adjusted models, we found significant independent associations between each social tie and adjusted mean WC in both women and men, but with gender-specific patterns . Women wUsing clinically meaningful outcomes, we found that all non-partnered categories and limited social activities (≤2) were each independently linked to higher odds of abdominal obesity (OR range: 1.25–1.53) and general obesity (OR range: 1.36–1.69) in women, compared to partnered or socially active counterparts . Women wSensitivity analysis including health behaviours, psychological factors and chronic conditions minimally attenuated most results and amplified some . Two associations with WC in men lost statistical significance , and adjusting for physical activity reversed and strengthened the association of social participation with WC in men.Figs All marital status categories had higher mean levels of WC with increasing size of social networks for women, with the exception of divorced women who did not differ in mean WC across quartiles of social network size. Also, single and widowed women had higher WC compared to partnered women across all social network size levels, especially at greater sizes, and differences in WC between divorced and partnered women were only observed in the lowest quartile , panel AWe found a consistent pattern for women in the joint association of marital status and social participation with WC. Non-partnered women who participated in two or fewer social activities had much higher mean levels of WC than partnered women with limited social participation, but these differences in marital status were reduced or removed for divorced women with the most social participation , panel AOverall, we found that the independent association between social network size and WC varied by levels of social participation with gender-specific patterning. In women, mean levels of WC were inversely associated with social network size when social participation was limited or absent, but were positively associated when social participation involved five or more activities . We founThe independent inverse association between social participation and WC was similar for co-living and lone-living women and men . With thThis population-based, cross-sectional study of nearly 30,000 adult Canadians used a gender-sensitive analysis to assess the relative contribution, and inter-relations, of different types of social ties on several anthropometric outcomes for women and men. We showed clear gender differences in the magnitude and direction of association, depending on the exposure and outcome measure. Generally, we found that non-partnership and less social participation were positively associated with WC in women, and smaller social network size and lone-living were inversely associated with WC in men.Notably, stronger associations were seen for marital status and social participation in women and for social network size and living arrangement in men. These results only partly supported our first hypothesis that social engagement would be stronger for women and living arrangement stronger for men. We also found that larger social network size and more regular social participation can potentially mitigate the adverse relationship between non-partnership (single and/or widowed), and measures of obesity in women, which we hypothesized. Overall, the joint associations were more pronounced in women than men. Contrary to our hypothesis, co-living did not appear to mitigate the adverse relationship of limited social participation or lack of social contacts and obesity.The existing literature provides mixed results for the link between social ties and obesity. Similar to our work, previous studies in US , Sweden Separate research examining social network size has shown a protective effect on mortality in older men, but that research used indices combining multiple ties such as marital status  and sociOf significance, the inter-relations between different types of social ties revealed an important source of heterogeneity and unique associations with obesity in women and men. We found that social network and social participation can potentially mitigate the association between non-partnership and obesity outcomes in women. Little research has explored the joint association of social ties and health outcomes or behaviours, and there are none examining obesity outcomes. A 20-year study of 11,000 participants (aged 10 years and older) investigated marital status and living arrangement in women and men. The study found a higher risk of mortality in unmarried women who were co-living with dependent children compared to married women . InterreOur findings revealed remarkable differences between women and men in the links between social ties and obesity, thus reinforcing how gender, a social-cultural factor, shapes how social ties impact health through physiological pathways . In womeWe acknowledge a number of limitations of this study. The cross-sectional design limits causal inference although it is informative of potentially relevant relationships for prospective examination. Self-reported measures may introduce recall or survey effect bias in results. The social network size variable may have less variance in the sample than the population as the total number of social contacts for each setting could not be specified beyond 400. Moreover, social relationship measures were assessed as a state and so recent transitions such as widowhood or separation could have a greater influence on obesity than those marital states that occurred longer in the past. Despite not measuring gender self-identity, the self-reported male/female variable provides a highly correlated proxy to produce estimates for women and men that could reveal potential gendered power relations ; notablyThis study had several strengths including the large sample size, adjustment for multiple known confounders and lifestyle variables, gender-specific estimation, effect modification and clinical measured anthropometric outcomes. We considered the main, independent and joint associations of separate measures of different relationships rather than using a summary index that masks the relative contribution of each type of connection. A particular strength of our study is in its consideration of the more complete picture of both gender and interactions of social relationships in relation to adiposity which has added nuances to the literature and helps to better inform more effective, and equitable, obesity prevention and healthy aging strategies.This study provides a more comprehensive and accurate picture of the intersecting and mutually reinforcing upstream determinants of obesity. We found a clear link between different types of social ties and obesity in Canadian adults, and the influence of social connections and their interactions on adiposity showed a differential pattern by gender. Future work needs to use longitudinal study designs to support causal inference and help inform potential structural interventions to tackle the growing burden of chronic disease and health inequities.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 FigAdjusted mean body mass index with A, marital status, B, living arrangement, C, social participation and D, social network size among older women and men in the Canadian Longitudinal Study on Aging CLSA (2012–15). Women, solid line; men, dashed line. Partnered was married or living as married; divorced includes separated. Social network size (1–573) was a sum of responses to eight questions about the number of social contacts the respondent knows , with network size increasing from smallest (Q1) to largest (Q4). Social participation was a sum of responses to eight questions about regular (≥ once per month) participation in different social activities, that was re-classified into four levels of social participation.(DOCX)Click here for additional data file.S2 FigAdjusted mean waist circumference  and body mass index  across levels of social participation, by living arrangement, among older women and men in the Canadian Longitudinal Study on Aging (2012–15). Social participation was a sum of responses to eight questions about regular (≥ once per month) participation in different social activities that was re-classified into four levels of social participation.(DOCX)Click here for additional data file.