While telemedicine has been utilized with more frequency over the past two decades, there remained significant barriers to its broad implementation. The COVID-19 global pandemic served as a stimulus for rapid expansion and implementation of telemedicine services across medical institutions worldwide in order to maximize patient care delivery, minimize exposure risk among healthcare providers and patients alike, and avoid overcrowding of patient care facilities. In this experience report, we highlight the teledermatology initiatives executed by the Dermatology Service at Memorial Sloan Kettering Cancer Center in New York City, with particular emphasis on image ingestion and potential for future automation and improvement. Virtual physician-patient visits provide significant opportunity to advance delivery of care in a visual specialty such as dermatology, however, prior to March 2020, were significantly underutilized . In partThe Dermatology Service at Memorial Sloan Kettering Cancer Center (MSK) provides dermatology services for patients undergoing oncologic care  and manaWe outline how, with the support of administration, clinicians, information technology, and nursing, we were able to rapidly implement teledermatology services for patients at multiple locations in order to continue providing critical patient care. The expansion of reimbursement for telemedicine by the Centers for Medicare and Medicaid Services (CMS), while beyond the scope of this work, also supported our efforts to implement store-and-forward static image-based consultations, live face-to-face teledermatology, and telephone encounters.We addressed three patient scenarios: patient at home, patient in an office that was not a dermatology office, and patient admitted to the hospital (inpatient), for which teledermatology services were desirable. We also implemented three types of dermatology responses: live telemedicine visit, store-and-forward dermatology consultation, and a traditional in-person visit for urgent/emergent cases to these scenarios, which are highlighted in Fig. In this report, we describe the implementation for teledermatology services by the Dermatology Service at Memorial Sloan Kettering Cancer Center (MSK) in response to the COVID-19 global pandemic.We evaluated the expansion of teledermatology from March 13, 2020, until June 13, 2020. These dates were selected as schools closed in New York Statewide on March 16, a closure that reflected a significant decrease in the in-person volume in our dermatology clinic, and as New York City began phase I reopening on June 8, after which clinic volume has gradually begun to normalize.Imaging: In order to triage the patients that would be appropriate for different virtual or in person visit types, photographs were requested from patients requiring dermatologic evaluation as shown in Fig. Several patient education resources were created for at home imaging while internal resources with instructions were also generated for healthcare team members requesting dermatologic consultation –15.Patients at home uploaded their photos to a secure patient portal, while healthcare team members were encouraged to send photos to the appropriate (outpatient or inpatient) on-call dermatology consult team created for this purpose in a Health Insurance Portability and Accountability (HIPAA)-compliant Voalte™  application.Once photos were received, administrative and nursing team members fielded the messages, and photographs with clinical history were reviewed by the physician via email or in the Voalte™ system directly in order to determine the appropriate visit type.Triage: Dermatologists at MSK based their decision on the type of visit to perform primarily based on images supplied by patients or referring providers, as well as clinical information. Due to the rapidity with which teledermatology was implemented, triage was primarily performed by physicians directly; however, ongoing efforts are being put into place to improve efficiency and automation of patient triage based on types of dermatologic requests. For example, our dermatologic surgeons are frequently performing initial consultations for known cutaneous malignancies via telemedicine visits to triage low-risk and high-risk lesions and determine the appropriate timing of surgical treatment. Store-and-forward images were reviewed in advance to assess for site identification and lesion assessment. Post-surgical follow-up and scar images were further triaged by nursing, and physicians would then determine which patients would benefit from a live telemedicine call.Image Ingestion: All images reviewed were ultimately uploaded into our secure image database, Vectra™  imaging software, by a member of the office or clinical staff. For patients at home, portal messages were directly retrieved, saved to an intermediate on-campus secure desktop, and uploaded to the imaging database. The lack of imaging standards for dermatology to guide interoperability, metadata, color, and scale, required upload to proprietary software be performed manually for this initial deployment ; howeverTeledermatology Consultation: Dermatologists provided both synchronous  and asynchronous  patient-provider interactions and provider-to-provider virtual consultations. Due to the expansion of potential telemedicine platforms and relaxation of HIPAA protections by CMS, clinicians were able to use Cisco Jabber , Doximity, and a variety of other face-to-face methods to communicate with patients . We also enabled direct telephone consultation for patients who sent in photographs via portal message.Figure Figure Figure Initiation of workflows that relied on secure messaging and patient portal upload allowed dermatologists at MSK provide care to patients while minimizing in-person patient volume to address the impact of COVID-19.We found that triage of patients via asynchronous (store-and-forward) images provided by patients or healthcare team members as shown in Fig. Over the course of the 3-month observation period, dermatologists at MSK received more than 10,000 photographs acquired by patients or their families via smartphone applications or other mobile devices. The image quality, despite patient education materials, was highly variable. Further development of image standards such as Digital Imaging and Communications in Medicine (DICOM) for dermatology imaging could also improve interoperability and security of image ingestion . An autoDermatologists at MSK reviewed images through secure messaging, email, and portal messages that were uploaded manually to our image viewing software. Future automation efforts could be devoted to triage approaches and image ingestion. Clinicians observed that they were frequently reviewing patient cases twice: first during triage and subsequently during a televisit. Automated approaches to quantify body surface area affected by skin disease, flag abnormal vital signs, and visit types or patient questions could improve the efficiency and perhaps largely automate the process of patient triage.One can envision a scenario in which images are automatically ingested to the image database through those three streams of receipt and matched to patient identification and anatomic site. At that point, clinicians could simply review images in one master database rather than monitoring multiple streams of communication, and physician observations would be documented in the electronic medical record (EMR). This could apply for store-and-forward teledermatology visits as well with the option to document directly in the image storage platform that seamlessly links the images and the EMR. As shown in Fig. At the end of the three-month observation period, outpatient visits were still being performed via virtual consultation 20–30% of the time, which likely reflects ongoing enthusiasm for teledermatology as well as a lack of complete return to clinic volume. Further efforts toward automation, image standards, and efficiency will support continued development and response to potential future crises.We successfully implemented teledermatology workflows that provided continued access to dermatologic care during the COVID-19 global pandemic. We achieved this using a variety of methods (both store-and-forward teledermatology and live telemedicine platforms), in various settings (inpatient and outpatient), as well as with different key players . Forward triage telemedicine offers a patient-centered approach that in a public health crisis such as SARS-CoV-2 protects patients, healthcare providers, and the community while supporting uninterrupted patient care . Despite


Early diagnosis of inborn errors of metabolism (IEM)—a large group of congenital disorders—is critical, given that many respond well to targeted therapy. Newborn screening programs successfully capture a proportion of patients enabling early recognition and prompt initiation of therapy. For others, the heterogeneity in clinical presentation often confuses diagnosis with more common conditions. In the absence of family history and following clinical suspicion, the laboratory diagnosis typically begins with broad screening tests to circumscribe specialised metabolite and/or enzyme assays to identify the specific IEM. Confirmation of the biochemical diagnosis is usually achieved by identifying pathogenic genetic variants that will also enable cascade testing for family members. Unsurprisingly, this diagnostic trajectory is too often a protracted and lengthy process resulting in delays in diagnosis and, importantly, therapeutic intervention for these rare conditions is also postponed. Implementation of mass spectrometry technologies coupled with the expanding field of metabolomics is changing the landscape of diagnosing IEM as numerous metabolites, as well as enzymes, can now be measured collectively on a single mass spectrometry-based platform. As the biochemical consequences of impaired metabolism continue to be elucidated, the measurement of secondary metabolites common across groups of IEM will facilitate algorithms to further increase the efficiency of diagnosis. Inborn errors of metabolism (IEM) are a group of about 1000 relatively rare inherited disorders arising due to genetic variants that perturb the proper functioning of proteins requisite for cellular metabolism . While mThis article focuses on the improved diagnostic efficiency of IEM inherent in metabolomics, which comes from advances in technology scalability, streamlined operations management and the potential for greater ‘democratisation’ of the metabolomic screening approach.A clinician’s index of suspicion for a metabolic disorder is dependent on a variety of factors. There is the consideration of disease incidence with the oft-touted adage of “when one hears hoofbeats, think horses, not zebras” subverting efforts at early diagnoses of rare yet treatable disorders . Other cAs an example, while a clinical geneticist, metabolic physician or paediatrician may consider mucopolysaccharidosis (MPS) in a coarse-appearing child with short stature and developmental delay, this diagnosis may never be considered by the midwife or general practitioner. A paediatrician may know to order urine glycosaminoglycans (GAG) in this case of iduronate-2-sulfatase deficiency causing Hunter syndrome. However, the differential diagnosis in an infant with these phenotypic features may be quite large and include many untreatable disorders. This example illustrates the complexity of IEM diagnosis and how it requires multiple tiers/iterations to identify convincing evidence of the underlying cause. In response, clinicians have become accustomed to ordering a ‘metabolic screen’ with multiple assays increasingly available to cover many conditions. This reflex of ordering a panel works well for a busy clinician trying to diagnostically solve an undifferentiated patient in a timely fashion and to ensure they do not miss the opportunity to offer early treatment.Multi-disorder panels have become cemented in the workflow of clinicians in recent years, with multi-gene next-generation sequencing panels and genome-wide sequencing approaches. However, often the ‘test requestors’ are unfamiliar with functional validation of findings made on these panels, utilising biochemical investigations. Thus, multiplex metabolomics-based workflows solve the clinical problem of not being entirely sure of what is going on with the patient in front of them. It also assists with interpreting molecular findings from genomic testing, whilst being able to have confidence that a wide range of treatable conditions have been tested.The choice of first-tier laboratory tests is somewhat limited and is dependent on the nature of the symptoms and signs (phenotype) being investigated . The potA treatment should be available that changes the outcome of patients based on early diagnosis.There should be a good understanding of the natural history of the condition.There should be a good understanding of which patients should be treatedA screening test should be available that is valid, reliable and acceptable to the public.NBS involves screening neonates early in life to detect life-threatening or long-term health conditions to reduce morbidity and mortality. NBS began with phenylketonuria (PKU), once it was shown that early treatment spared intellectual disability, by a bacterial inhibition assay on dried filter paper blood spots (DBS) in the 1960s . PrincipPKU is a condition which, when left untreated, results in intellectual disability, motor dysfunction and mental health issues . TreatmeShifting NBS for PKU from a single-test method to a multiplexed mass-spectrometry method allowed for considerable expansion of the number of IEM that could be screened for at birth. Many of these had clear benefits from screening, as early diagnosis before the onset of symptoms enabled therapeutic intervention, improving health outcomes for infants . HoweverNBS has advanced substantially over the past ten years. Numerous applications of metabolomic-based NBS have been piloted, introduced or proposed in recent years ,16. The New York State’s introduction of NBS for Krabbe disease  has followed a similar course with higher numbers of individuals being detected than are clinically affected . Of thosA child presents with paroxysmal events, such as seizures or abnormal movements. Initial laboratory assessment may show reduced blood sugar, abnormalities on blood gas, such as metabolic acidosis with an increased anion gap due to lactic acidosis. Non-specific markers may also be abnormal, such as liver enzymes or other features, such as coagulopathy, being present. Somewhat more specific testing such as ketones or ammonium may be elevated or ‘inappropriately normal’ , which allows for directed attention to specific metabolites in further testing .In this example of an acute metabolic acidosis, the approach could consider undertaking metabolic screening with plasma amino acids and urine organic acids. Specific metabolites could also be requested by a metabolic physician, such as urine orotate or succinylacetone. These could be arranged in tandem, however in most situations, these reflex pending results from the initial ‘screen’. Some centres have moved away from employing untargeted gas chromatography (GC)-based urine organic acid analysis towards semi-targeted liquid chromatography–electrospray ionisation (LC-ESI)-MS/MS analysis. This approach, first published by Pitt et al. , has beeClinical phenotypes assist in selecting the appropriate laboratory investigations when ruling out IEM. Determining whether the condition is likely due to defects in small molecule metabolism  or defects of organelle metabolism  will assist in the diagnostic pathway , as currIn the case of acutely presenting conditions, the approach to screening begins with plasma amino acids, plasma acylcarnitine profile and urine metabolic screen . Turnaround time for these tests varies from a day to weeks, significantly influencing management and parallel investigations. Some conditions present with a characteristic pattern on these initial tests, for instance, raised plasma alloisoleucine and branched-chain oxoacids in the urine of maple-syrup urine disease. Other conditions present with less specific patterns, where experienced and well-established laboratories may detect the presence of an atypically elevated metabolite. This may not be obvious to others. Examples include the presence of elevated 3-methylglutaconic acid in Barth syndrome or metabolites related to tyrosinemia type I in a urine organic acid screen. Urine organic acid analyses have utilised GC-MS-based approaches in an untargeted fashion with post hoc filtering of well-known metabolites . These tLysosomal enzymology performed on leukocytes is a first-tier line of investigation for phenotypes such as cardiomyopathy and young-onset dementia . This reThere are additional specific first-tier tests which are requested less frequently due to regional laboratory availability. Disorders of creatine metabolism present predominantly with developmental and neurological disorders and are often treatable . DisordeN-glycosylation [N-glycosylation [The final significant group of conditions involve protein glycosylation. Transferrin isoform analysis can be performed by numerous methods and is used to screen for congenital disorders of sylation ,29,30. Usylation . Apoliposylation ,33,34. TSecond-tier testing typically follows a positive first-tier test and is nominally based on refining the exact IEM. For the majority of IEM arising due to an enzyme deficiency, the standard is to measure the enzyme activity in cells, blood or biopsy tissue. The gold standard of enzyme confirmation from a biopsy is often impractical or unethical when reliable biomarkers or molecular testing offer an adequate alternative. For many metabolic conditions, specific enzyme assays exist to validate either molecular- or metabolite-based diagnoses. Examples include the diagnosis of Lesch–Nyhan syndrome (hypoxanthine-guanine phosphoribosyltransferase) reflexed from urine purine and pyrimidines, congenital disorder of glycosylation-1a (phosphomannomutase) from transferrin isoforms and neuraminidase and β-galactosidase activity to differentiate galactosialidosis and sialidosis following an abnormal oligosaccharide pattern. Multiplex enzymology performed by mass spectrometry and digital microfluidics has been described, both of which are being used for NBS programs ,36. AlthChallenges remain in the goal of developing whole metabolome screening methods. For instance, disorders with no specific biomarker, such as Niemann–Pick type C disease. Plasma oxysterols represent useful screening metabolites for this group of conditions ,40, but Molecular and cytogenetic testing has a complicated relationship with IEM. The earliest diagnostic genetic methods for diagnosing genetic conditions were biochemical, such as for alkaptonuria and PKU ,43. BiocCFTR variants following positive immunoreactive trypsinogen for cystic fibrosis from NBS, which can detect up to 90 variants within the gene [Molecular testing strategies depend somewhat on environment and funding arrangements, often taking a tiered approach and are dependent on the conditions tested. In the context of a known biochemical diagnosis with one candidate gene, a single gene is usually interrogated, e.g., Fabry disease, a small gene of seven exons. In some conditions, there are common variants, either causing the condition generally or as a founder variant in a specific geographical region, ethnicity or family. In this context, genotyping technologies are still useful, such as Sanger sequencing, allele-specific amplification and restriction enzyme digestion. A variety of high-throughput genotyping technologies for conditions with common variants also exist. To this end, MassArray is a technology employing a mass spectrometry (MALDI-TOF) platform coupled with termination primer amplification and bioinformatic techniques . An examthe gene .In conditions for which the causative gene from the biochemical phenotype is poorly defined or in metabolic disorders that are not able to be biochemically sub-typed , an NGS panel undertaking post hoc filtering for genes of interest (a ‘panel’) can be informative. An example is maple-syrup urine disease, which has multiple genes linked with an indistinguishable metabolite profile . In pracGenomic diagnoses create the inherent need for biochemical validation. One of the earliest examples followed the introduction of chromosomal microarray, which allowed the identification of many patients with X-linked steroid sulfatase deficiency. The diagnosis in these individuals could be corroborated by measuring enzyme activity and determining that this was below normal levels . More reIncreasingly, acute, complex and undiagnosed diseases are referred for untargeted genomic testing rather than iterative molecular and biochemical testing . Thus thEvidence is pointing towards three principal utilities in clinical practice for metabolomic methods. Firstly, orthogonal  validation of molecular discoveries, mainly VUS. Secondly, profiles can be utilised in a multivariate model for diagnostic classification. Thirdly is screening for IEM, either in isolation or in tandem with molecular methods . NGS heiWith all of these technologies, it is possible to glean additional information and thus move away from a single analyte per diagnosis, to a ‘profile’ or classification model approach ,54,55,56Untargeted methods are being employed to increase the number of conditions screened for in the clinic, based on DBS, urine, plasma and CSF samples ,58,59. TMany classic metabolic disorders are thought to be ultra-rare, with examples such as Farber disease being reported in about 100 patients worldwide. Although enzymology is available for this condition, this would usually be performed in the context of family history, a confirmatory context or in extremely uncommon cases where the condition is suspected. Attenuated cases are yet to be described, possibly linked with the lack of clinical specificity of the presentation. Metabolite diagnosis is now possible while being multiplexed with other sphingolipids , which iIndeed, the problem that some conditions are too rare to sustain highly specialised biochemical tests in a clinical diagnostic laboratory has led to many tests only being able to be offered in a research environment. Multiplexing of diagnostic metabolites and enzymology using metabolomics, will hopefully reverse this trend and re-enable access to diagnostic biochemical testing for these ultra-rare conditions and contexts.Mass spectrometry is being increasingly utilised for metabolomics, beginning with screening for a select group of organic/amino acidemias and fatty acid oxidation disorders in the late 1990s, followed by the introduction of GAG, oxysterols and oligosaccharides ,64. AdopThe high-dimensional nature of the data being dealt with requires scalable pre-processing platforms, sophisticated multivariate analysis methods and a shift from the ‘one analyte per diagnosis’ approach of old to the metabolome-wide profiling of new . These tWith more substantial amounts of data being produced by these multiplex assays, machine learning tools facilitate reproducible and understandable models of prediction (classification and regression) . These tA biomarker is defined as an objective measure of the presence or severity of a non-physiological state, which can be measured accurately and reproducibly . For manIDH1 cause hydroxyglutaric aciduria, whereas mono-allelic hypermorphic mutations cause a druggable form of brainstem glioma [While many of the methods in this space have been developed for diagnosis and monitoring of constitutional errors of metabolism, the same techniques lend themselves to the measurement of perturbations in acquired (somatic) errors of metabolism seen in disease states such as cancer and in differentiating features of tumours. Indeed, some of these conditions even involve the same genes, an example being that biallelic mutations in m glioma ,86. Recem glioma . PatientIn practice, the subsequent reuse of metabolomic data collected for primary diagnostic purposes has already yielded benefits. Urine, plasma and CSF metabolomic assays have been employed, revealing significant findings on clinically analysed samples ,86,87,88Limitations of current technologies mean that metabolomic testing is not truly pan-system, but it is progressing, and many laboratories are cognisant of the approach. Undoubtedly, conditions and diagnostic metabolites/profiles are yet to be determined, and on the contrary, others are well-defined. Conditions such as PKU still have incremental value, added through expanded metabolite screens, and new disorders continue to be discovered. Bioinformatics workflows remain non-standard and often operate with only quasi-reference to either analysis of other samples but also other diagnostic methods, such as nucleic acids and proteins. The strength of the metabolomic approach is not only discriminating a condition but also reflecting on the primary-, second- and third-order effects of a metabolic perturbation, and the resulting response to treatment. The future of both metabolomic- and genomic-based diagnoses involves integrating these technologies to make and validate diagnoses, to prognosticate as well as monitor disease progression and response to therapy.


Crop genomics has seen dramatic advances in recent years due to improvements in sequencing technology, assembly methods, and computational resources. These advances have led to the development of new tools to facilitate crop improvement. The study of structural variation within species and the characterization of the pan-genome has revealed extensive genome content variation among individuals within a species that is paradigm shifting to crop genomics and improvement. Here, we review advances in crop genomics and how utilization of these tools is shifting in light of pan-genomes that are becoming available for many crop species.The online version contains supplementary material available at 10.1186/s13059-020-02224-8. Crop improvement is needed now more than ever with challenges associated with feeding an ever-expanding population under increasingly variable growth conditions. The ability to produce crops that meet societal needs is enhanced by a thorough understanding of the genome of a species. Genomic resources expand the toolbox available for plant breeding and crop improvement efforts. Various tools have risen in popularity for plant breeding, in some cases as short-lived bandwagons and others as paradigm shifts in crop improvement , 2. WithAccess to plant genome assemblies in the early 2000s revolutionized thinking about the biology of crops and plant breeding –5. TheseThere are many mechanisms that can generate a structural variation. For example, transposable elements (TEs) can replicate themselves in a genome and can also capture and carry gene sequences to new genomic locations –16. Thistb1 gene played an important role in changing maize architecture during its domestication [The generation of multiple, reference-quality genome assemblies per crop species is now a reality –24. Our tication . In facttication , 37. GivCrop genomics has transitioned from the era of a single reference genome to a time when we now have access to tens or hundreds of reference-quality genome assemblies within a species Table . This arOver the last two decades, advances in sequencing technology and assembly algorithms have profoundly affected our understanding of the complexity and structure of genomes. Crops were among the first species with assembled genomes given their economic importance and the relevance of genomic information to breeding. The earliest model crop genomes were assembled with Sanger sequencing, BAC-by-BAC approaches, and overlap-layout-consensus (OLC) assemblers . EaAs short-read resequencing decreased in cost and became commonplace, whole-genome sequencing (WGS) approaches were more frequently used to characterize CNV/PAV in crops –79. ThesThe use of whole-genome sequencing allowed for characterization of a greater breadth of variants than hybridization arrays, but this approach suffers similar limitations: (1) sequence from loci that are missing in the reference genome due to either incomplete assembly or true biological absence does not map and remains uncharacterized, (2) divergent reads map less efficiently, and (3) uneven coverage bias of short-read sequencing can result in inaccuracies . These sNew approaches have rapidly developed for CNV/PAV characterization that leverage recently developed library preparation techniques and the maturation of single-molecule, long-read sequencing (comprehensively reviewed in ). For exAccess to multiple reference-quality genome assemblies within a species provides opportunities to identify SVs in a non-reference-biased manner. However, a number of challenges arise in such an approach. First, several crop species have large, complex genomes, making numerous assemblies per taxon cost-prohibitive. To overcome this limitation, a small number of breeding program founder individuals, which capture the majority of segregating haplotypes, can be targeted for genome assembly and identification of relevant SVs. Second, while multiple assemblies will reduce reference bias, assembly errors can lead to the detection of false SVs and compromise downstream analysis, particularly when de novo assemblies are generated using different data types or assembly algorithms. A third challenge is the consolidation of pan-genome variation into a single reference or coordinate system, a useful step for the analyses of the biological significance of SVs in crop species including QTL analysis, GWAS, and genomic prediction.Several methods exist for summarizing SV information in a pan-genome context. One approach is to map resequencing reads to a reference genome, de novo assemble unmapped reads, and add the assembled contigs to the reference assembly (known as the map-to-pan approach) , 86. ThiZmCCT9 gene to promote flowering under long-day conditions [OsFRDL4 gene and promote aluminum tolerance [Ruby gene, resulting in its enhanced expression and driving convergent evolution of the blood orange trait [Rider has created polymorphism in the SUN locus resulting in the oval shape typical of the Roma tomato variety [As pan-genomes become widely available for crop species, TEs, a driver of structural variation, will receive increasing attention in crop improvement. Plant genomes (including crop species) are particularly rife with TEs , and thenditions . In riceolerance . Two Copge trait . Finally variety , 104. DeTEs create the majority of insertions and deletions in many crop genomes. For example, > 75% of large InDels  in both tomato  and soybOnce TE sequences are captured in de novo genome assemblies, a critical remaining challenge is an accurate annotation to the family level. Three general approaches are used. The first is homology-based using existing TE databases such as Repbase  and P-MITwo main approaches are used to identify genomic regions associated with a desired phenotype: QTL mapping with biparental populations and GWAS with panels of diverse individuals. Early crop reference genome assemblies facilitated the development of platforms  that allow for rapid, cost-effective genotyping of thousands or millions of SNPs across large sets of individuals. This increase in marker density dramatically increased resolution in mapping studies, which aided in the identification and cloning of QTLs associated with disease resistance, drought tolerance, yield, plant architecture, and other important agronomic traits . With thOne major concern in QTL mapping or GWAS based on a single reference genome is reference bias . If variQTL and GWAS studies have primarily relied on SNP data to date, but other markers have been useful in linking different types of variation to phenotype. For example, GWAS performed with both read depth variants , and SNPs in maize demonstrated that RDVs were enriched for significant GWAS results relative to SNPs for traits such as leaf development and disease resistance . SimilarBrassica napus, GWAS was performed with PAVs identified from eight whole-genome assemblies, and causal associations between SVs and silique length, seed weight, and flowering time were discovered that were not captured by SNP-GWAS. Likewise, GWAS based on the graph soybean pan-genome identified a PAV associated with variation in seed luster [As the crop improvement paradigm shifts to a pan-genome perspective, the contribution of SVs to trait variation is becoming clear. Recently in d luster . In peacd luster . HoweverA number of important traits for crop improvement are controlled by many QTLs with small effect . A complex genetic architecture makes it difficult to identify all QTLs underlying a trait, correctly estimate their effects, and introgress them into elite lines using methods such as marker-assisted selection –124. GenTraditionally, SNPs identified relative to a single reference genome have been used for genomic selection. However, as described above, there are a number of limitations and biases that are introduced with the use of a single reference for such applications. New approaches for identifying markers within a pan-genome framework are needed to improve prediction accuracy. The Practical Haplotype Graph (PHG) is one such method that successfully deals with the complexity of a species’ pan-genome at the scale necessary for complex traits and plant breeding programs . In the A major issue in genomic prediction is that genotype by environment (G×E) interactions decrease the prediction accuracy for individuals grown in novel environments. Statistical models that account for G×E have been designed to attempt to overcome this limitation –137. IncBeyond the promise that recent genomic advances offer for characterizing diversity in model crop systems and for improvement of trait mapping and prediction, they also present opportunities to tackle difficult and understudied crop genomes and could potentially enable novel, gene-editing approaches to breeding.Allopolyploidy (the result of interspecific or intergeneric hybridization and chromosome doubling) and autopolyploidy (the result of whole-genome duplication) are particularly common in plant species , 141. InWhile polyploid crops are vitally important to sustain human life, genomic studies in these species have traditionally been very challenging for a number of reasons. High-quality genome assembly of polyploid species has been difficult to achieve due to their inclusion of multiple, closely related subgenomes and the associated challenges in discriminating homeologous loci and creating non-mosaic subgenome scaffolds Fig. a. Many hAdvances in sequencing technologies and assembly algorithms are already addressing technical challenges in crop genomic research in polyploids . Long-reSilphium integrifolium, a species with a large genome size  is another species that is currently being domesticated for use as an oil crop [Lolium perenne) has a fragmented draft genome [Festuca brevipila), long-read sequencing of the transcriptome has been used as a proxy of the reference genome, but it remains difficult to distinguish homeologs using this approach [For understudied crops, pan-genome-assisted breeding efforts remain limited due to the small size of the research communities for these species and, in some cases, due to the challenges associated with genome complexity. For the majority of understudied crop species, transcriptome assemblies are currently used as a proxy to the genome for improvement efforts. One such example is f ~ 9 Gb ;) that ientified . While Soil crop . While ioil crop  to a fuloil crop , access t genome , which mapproach .Capsicum (pepper) and Juglans  species [While pan-genomic studies may be in their infancy in non-model crops, it is anticipated that rapid advances in sequencing, assembly algorithms, and analysis pipelines in model systems and diminishing costs will very quickly enable this research. The time from publication of the first rice genome assembly to release of the first rice pan-genome was over a decade ; Table 11. We ant species , 51, andfw3.2 is caused by tandem duplication of the cytochrome P450 gene SlKLUH [SKILUH gene successfully altered fruit weight, a crop domestication phenotype [TomLoxC promoter region was also discovered that modifies fruit flavor [The recent availability of high-quality genomes and pan-genomes has enabled a new era of crop domestication. With pan-genome information, breeders can more effectively identify causal genetic variants  underlying domestication traits and apply gene-editing tools to rapidly achieve desirable agronomic traits in wild plants. For example, the tomato pan-genome has revealed that variation at the fruit weight QTL e SlKLUH  rather te SlKLUH . CRISPR/henotype . Similarhenotype . Furtherhenotype . A non-rt flavor . These vSolanum pimpinellifolium) and significantly increased its yield, productivity, and nutritional value resulting in de novo domestication of tomato [Domestication has greatly reduced the genetic diversity of crops compared to their wild relatives . Identiff tomato .In summary, the complete catalog of variation that has been made possible by recent genomic technology and a pan-genome approach presents a substantial opportunity for crop improvement. We can, not only move beyond single-reference-based resequencing in model crops to a full understanding of structural variation and its link to phenotype, but also tackle complex, polyploid genomes, rapidly move understudied crops into the genomic era, and bring down barriers between crops and their wild relatives so that breeders can more easily expand their tool kit to include exotic germplasm. While further infrastructure and method development is necessary to fully realize this potential, there is a paradigm shift in the making.Additional file 1. Review history.


This study aims to explore the impact of healthcare digitalization on smart hospital project financing (PF) fostered by pay-for-performance (P4P) incentives. Digital platforms are a technology-enabled business model that facilitates exchanges between interacting agents. They represent a bridging link among disconnected nodes, improving the scalable value of networks. Application to healthcare public–private partnerships (PPPs) is significant due to the consistency of digital platforms with health issues and the complexity of the stakeholder’s interaction. In infrastructural PPPs, public and private players cooperate, usually following PF patterns. This relationship is complemented by digitized supply chains and is increasingly patient-centric. This paper reviews the literature, analyzes some supply chain bottlenecks, addresses solutions concerning the networking effects of platforms to improve PPP interactions, and investigates the cost–benefit analysis of digital health with an empirical case. Whereas diagnostic or infrastructural technology is an expensive investment with long-term payback, leapfrogging digital applications reduce contingent costs. “Digital” savings can be shared by key stakeholders with P4P schemes, incentivizing value co-creation patterns. Efficient sharing may apply network theory to a comprehensive PPP ecosystem where stakeholding nodes are digitally connected. This innovative approach improves stakeholder relationships, which are re-engineered around digital platforms that enhance patient-centered satisfaction and sustainability. Digital technologies are useful even for infectious disease surveillance, like that of the coronavirus pandemic, for supporting massive healthcare intervention, decongesting hospitals, and providing timely big data. This study aims to explore the impact of healthcare digitalization on smart hospital project financing (PF) fostered by Pay-for-Performance (P4P) incentives. The background is represented by a consolidated trend of aging patients and population growth, which contributes importantly to the rise in healthcare costs. The main drivers for growth in healthcare costs, besides an aging population, are represented by health insurance, and—mainly—technological progress .As Deloitte indicates, financial sustainability, care delivery, patient centricity, digital transformation, and regulatory compliance are at the top of the agenda . Additiost century is increasingly dependent on technology. Unlike in many other areas, the cost of medical technology is not declining, and its increasing use contributes to spiraling healthcare costs. Many medical professionals equate progress in medicine to the growing use of sophisticated technology, which is often expensive and beyond the reach of the average citizen 81]. An increasingly patient-centric vision, consistent with personalized medicine;A closer interaction between actors that are traditionally part of the healthcare supply and value chain ;Augmented use of digital technologies that make healthcare services cheaper, and more readily available, consistently improving Value for Money in PPP agreements;The entry of disruptive and non-conventional competitors ;The demand for more sophisticated care delivery services  and siteBig data that are continuously created by wearables, etc., and fuel eHealth or mHealth applications, fostering value co-creation and easing patient-centricity;revamped payment and public funding models, increasingly following P4P/RBF patterns and trying to optimize the trade-off between Traditional Procurement (TP) and PPP;a digitally networked reinterpretation of analogic stakeholder interactions.Disrupting trends impact the healthcare ecosystem and concern:This study has shown that many healthcare bottlenecks can be softened with digital investments that foster long-term sustainability. New technologies and telemedicine/mHealth are useful even for infectious disease surveillance . They miThe empirical example, with its sensitivity analysis, has shown that important savings can be achieved thanks to digitalization, supporting the research question. This can generate extra profits that produce incremental economic and financial margins. Additional marginality can then be shared between the main stakeholders that participate in the PPP initiative . Higher margins reduce risk (represented by the WACC) and ease the bankability of traditionally capital-intensive PF healthcare investments. Sharing of these benefits may conveniently occur via P4P/RBF, incentivizing mechanisms that ignite value co-creation patterns. Whereas P4P applications have long been applied to healthcare issues, the peculiarities of this case, considering the scalable impact of digitalization , are stiThe analogic extension of this template to other cases is possible, as illustrated in the discussion. Whereas the interpretation of smart healthcare PPP investments ,16,19 shThis sophisticated framework requires multilevel analysis interpretation, and its evolving boundaries are too wide to be holistically considered in this study. There is room for further interdisciplinary research on this popular topic, remembering that complex issues require lively interpretation and unconventional scrutiny.


Breast cancer is the most common cause of cancer-related deaths in women worldwide. Identification of reliable prognostic indicators and therapeutic targets is critical for improving patient outcome. Cancer in companion animals often strongly resembles human cancers and a comparative approach to identify prognostic markers can improve clinical care across species. Feline mammary tumors (FMT) serve as models for extremely aggressive triple negative breast cancer (TNBC) in humans, with high rates of local and distant recurrence after resection. Despite the aggressive clinical behavior of most FMT, current prognostic indicators are insufficient for accurately predicting outcome, similar to human patients. Given significant heterogeneity of mammary tumors, there has been a recent focus on identification of universal tumor-permissive stromal features that can predict biologic behavior and provide therapeutic targets to improve outcome. As in human and canine patients, collagen signatures appear to play a key role in directing mammary tumor behavior in feline patients. We find that patients bearing FMTs with denser collagen, as well as longer, thicker and straighter fibers and less identifiable tumor-stromal boundaries had poorer outcomes, independent of the clinical variables grade and surgical margins. Most importantly, including the collagen parameters increased the predictive power of the clinical model. Thus, our data suggest that similarities with respect to the stromal microenvironment between species may allow this model to predict outcome and develop novel therapeutic targets within the tumor stroma that would benefit both veterinary and human patients with aggressive mammary tumors. Breast cancer is a significant cause of morbidity and mortality in both human and veterinary patients. As such, identification of reliable prognostic markers that can accurately predict patient risk will greatly reduce overtreatment of patients with less aggressive tumors and identify patients at higher risk for recurrence who would benefit from more intensive treatment. Cancer in companion animals, particularly dogs and cats, resembles cancer in humans in various ways, including: 1) its multifactorial nature, including both genetic and environmental risk factors; (2) its latency, clinical manifestation and metastatic potential; (3) its histopathologic features, including tumor cell heterogeneity and permissive microenvironment; and (4) shared prognostic markers and response, as well as resistance to therapeutics. Thus, a comparative approach to studying spontaneously occurring mammary cancer has advantages to improving clinical care across species [ its multrd most common neoplasm in female cats, they account for 12% of all neoplasms and 17% of those in female cats [Breast cancer is the most common cancer in both women and female dogs worldwide . Althougale cats . Notablyale cats –8. Despiale cats , FMT remale cats . FMTs haale cats –13. FMT ale cats . The metale cats . As in hale cats –17. Due ale cats .Although FMTs are generally considered aggressive, as in humans with hormone-independent cancers, survival times can vary significantly . TraditUntil relatively recently, investigations of cancer development and progression have focused on mechanisms within neoplastic cells that drive uncontrolled growth and metastasis. In a paradigm shift, we now realize the growth and spread of cancer also depends on the support of non-malignant cells and the extracellular matrix (ECM), particularly collagen, in the surrounding tumor stroma collectively referred to as the tumor microenvironment (TME). Overall collagen abundance correlates with both an increased breast cancer risk –24 and pIn our recently published study , we idenThirty female cats that underwent surgical excision for histopathologically confirmed mammary adenocarcinoma between November 11, 2002, and June 21, 2016, diagnosed through the section of Pathology at the Veterinary Hospital of the University of Pennsylvania (PennVet) and for which medical records from PennVet or referring veterinarians could be obtained, were included in this study. Biopsy samples were collected during standard of care procedures for the diagnosis and treatment of mammary carcinomas. All data in this study were obtained from materials collected in the course of routine clinical care, including residual FFPE tumor samples and medical records. Medical records from PennVet or referring veterinarians were used to obtain clinical information. Paraffin embedded tissues were obtained from PennVet for sectioning and imaging. Retrospective studies are exempt from review by the University of Pennsylvania’s Institutional Animal Care and Use Committee and the Veterinary School’s Privately Owned Animal Protocol Committee. Information collected from the medical records of cats included in the study consisted of age at the time of surgery, breed, whether an ovariohysterectomy (OHE) had been performed prior to or at the time of surgery, and the date of surgery. The number, location , if indicated) and largest diameter of the tumor was recorded. Staging  was recorded. Cats were excluded if there was documented previous FMT (if the biopsy represented recurrence). Although the majority of cats (24/30) had thoracic radiographs to rule out metastatic disease prior to surgical excision, thoracic radiographs were not obtained in six cats prior to surgery. Cats with distant metastases were not eligible/were not included in this study. However, data regarding regional lymph node status was collected and included in the analysis.Feline mammary gland carcinoma biopsies were acquired from the biopsy service archives of the PennVet Diagnostic Laboratory at the University of Pennsylvania School of Veterinary Medicine. To avoid inter-observer variability in histological interpretation that has been documented in recent veterinary studies , 42, allInformation on the cats regarding the use of post-operative chemotherapy, subsequent local or distant metastasis, and cause of death was obtained through review of medical records. Disease-free survival (DFS) was established as the time interval in days from the date of the surgery to the date of any event associated with their cancer or if no cancer-associated event, then to the date of death for any reason. This included documented lymph node metastasis, new regional lymphadenopathy, documented distant metastasis or the development of new or recurrent mammary tumors. Mammary tumor recurrence was defined as regrowth of a tumor in the same region as the original resection site  or as a new tumor(s) when subsequent masses were noted in a region distant from the original tumor(s). Survival time (ST) was established as the time in days from the date of the surgery to the date of death for any reason. Only two cats in the study were still alive at their last known veterinary visit . Tumor-related death was defined as euthanasia or death resulting from local tumor recurrence, new tumor development, or regional or distant metastasis. Additionally, death or euthanasia not associated with new tumors or documented local or distant recurrence was classified as suspect tumor-associated death based on the clinical signs and the results of the diagnostics suggestive of metastatic disease performed at the final veterinary visit. The clinical and survival details are summarized in SHG imaging of the fibrillar collagen was acquired using a Leica SP8 confocal/multiphoton microscope , as previously described . BrieflyWe evaluated collagen density from SHG images using Fiji Image Analysis software which measured the integrated density of each SHG image, which includes intensity of each pixel and % area of positive SHG signal. For tumor-stromal boundary quantification, the SHG images were evaluated by six individuals as to whether the predominant pattern in the image was characterized by distinct tumor-stoma boundaries or a lack of discernable boundaries. Image order was randomized and evaluators were blinded with respect to tumor identity. Each evaluator had the option to score each image as either a 1, 0 or N. A score of 1 indicated that discrete tumor-stroma boundaries were the principal pattern observed. A score of 0 indicated that distinct divisions between the tumor cells and stromal collagen were not observed in the majority of the image. An “N” was reserved for images in which there was not enough collagen present to score it appropriately. Images were excluded from analysis if >50% of reviewers scored the image with an “N.” This resulted in the exclusion of 18 out of the total 286 images. The scores for each image were averaged together and the average score for each tumor was then calculated. To analyze the collagen fiber qualities  CT-FIRE was used, as previously described . The CT-Descriptive analyses included computation of means and standard deviation for normally distributed continuous variables and tabulation of categorical variables. Shapiro-Wilk test was used to assess normality of data. Skewed data were summarized using median and interquartile range (IQR). Frequency counts and percentages were used for categorical variables such as signalment and others. Besides performing subsequent analysis with continuous data, collagen parameters were converted into categorical data, and the data sets were divided into two groups: lower and higher than the mean . All desInference statistical analysis was conducted in three steps. First, an exploratory univariate cox regression was used to assess the association between the outcomes (ST and DFS) and the independent variables. The assumption of proportional hazards was tested based on Schoenfeld residuals. Independent variables showing association with the outcome with p<0.2 were included in the subsequent analysis. Second, each combination of collagen signature variables was investigated in a well-specified base ST model that contained the significant clinical non-collagen signature variables  in a step-wise fashion. Akaike information criterion, Bayesian information criterion, Harrell’s C concordance statistic and Somer’s D concordance statistic were evaluated to identify a best-fit model containing both clinical and collagen fiber parameters. Third and final, the best-fit models were used for reporting the inference findings. All inference analyses were conducted using either Stata 14.1MP, StataCorp, State College TX, with two-sided tests of hypotheses and a p-value < 0.05 as the criterion for statistical significance.The cohort comprised 30 female cats with surgically excised, histologically confirmed grade I (N = 2), grade II (N = 6) or grade III (N = 22) mammary carcinomas. Clinical, histopathological, and outcome information for the cats in this study can be found in To investigate which clinical parameters obtained from medical records at the time of diagnosis could predict survival in our cohort of patients with FMT, we examined whether Grade category (I/II vs III), mitotic count (continuous variable), lymphatic invasion (yes vs no), tumor diameter (continuous variable based on largest diameter), lymph node metastases at diagnosis (yes vs no), or surgical margins (clean-wide vs clean-narrow or incomplete) were associated with the outcomes, specifically ST or DFS using Cox regression univariate analysis . Grade, To examine whether fibrillar collagen characteristics in FMT biopsy samples could predict survival outcome, SHG imaging was performed on histologic biopsy samples from 30 cats with mammary carcinoma Figs –4. GivenIn our previous study examining the regulatory role of collagen on mammary tumor behavior in dogs, a new prognostic variable, boundary score, was developed, which we showed positively correlated with survival in canine malignant mammary tumors independently of clinical parameters , thus weAs in our canine study , collageTo test if the collagen parameters were independently driving the differences in outcome, multivariable analysis was used. First, a base mode was identified by testing all clinical parameters in a step-wise fashion. After all combinations were considered, grade and surgical margins were included in the final model. Next, the collagen parameters  were individually tested in a cox multivariable regression model controlling for the base model of clinical parameters . CollageFurther, we aimed to discover if the collagen parameters could help predict outcome better than the clinical model alone, and therefore uncover the best-fit model including clinical and collagen parameters. For this, Akaike information criterion , Bayesian information criterion , Harrell’s C concordance statistic and Somer’s D concordance statistic  were evaluated to identify a best-fit model containing both clinical and collagen fiber parameters . We testCompanion animals serve as useful models of corresponding diseases in humans and also improve understanding of these diseases in veterinary patients. After previously defining collagen characteristics that could predict outcome in canine mammary tumor patients, we hypothesized that collagen plays a critical role in directing the biologic behavior of FMT and certain collagen signatures could predict clinical outcomes in these feline patients. Additionally, as mammary tumors in cats are nearly always malignant and typically more aggressive than in dogs, this may in part explain the difference in the distribution across grades between cats and dogs. Notably, only 2 cats in our study had grade I tumors and the majority had grade III tumors, this is the opposite of what is seen in dogs where grade I, hormone receptor positive carcinomas are the most common and grade III tumors represent 20–30% of the cases , 45. ThuPredicting the course of disease for patients diagnosed with FMT is crucial for planning care and treatment for the patient. Currently, veterinarians and pathologists take tumor size, grade and associated mitotic count, lymphovascular invasion, surgical margins, as well as available information on lymph node involvement and distant metastasis into account when predicting outcome and recommending treatment. In our study, tumor size did not predict outcome. Given that previous studies had identified that tumor size is only predictive of outcome when the tumor is greater than 3cm , 46 and While the human breast cancer field is identifying new prognostic markers for human patients at a fast pace, the discovery of useful biomarkers in FMT has lagged behind, although several candidates have been considered. Recently the receptor CXCR4 and its ligand CXCL12 have been studied in FMT, and decreased CXCL12 was associated with poor prognosis, but only in tumors highly expressing HER2 . StaininAlthough human breast cancer research has looked at breast density, determined in part by collagen density, as a marker of poor outcome for patients for decades , it was Our canine study was the first to link tumor-stromal (collagen) boundary score to clinical outcome . This pain vivo imaging could predict response to chemotherapy [While our study confirms that collagen fiber length, straightness, number, width, density, and lack of a tumor-stromal boundary are all associated with poor outcomes in FMT patients, it is probable that additional prognostic information could be acquired from collagen based on recently described imaging techniques and analytics. Collagen quantity, uniformity and organization measured with SHG and ImageJ software in both the forward and backwards directions can identify human mammary tumor subgroups  and collotherapy , which cotherapy , 62.Although this initial study supports further investigation and validation of the use of SHG imaging to improve prognostication in FMT patients, it should be acknowledged that this imaging modality is not readily available to all Veterinary pathologists and oncologists. Assessment of collagen fiber features has also been performed using picrosirius red imaging with polarization, a technique with wider accessibility. The two staining techniques have been directly compared , and whiin vitro gels that mimic tumor-associated collagen architectures with large, aligned collagen bundles, which elicited cancer cell contact guidance and enhanced their directional migration [From our findings, we conclude that the density and organization of the collagen in FMT directs tumor aggressiveness and therefore clinical outcomes. Certainly, several mechanisms by which collagen density and stiffness promote metastasis and shorten survival and the mechanisms contributing to their formation and cellular responses have been suggested, including changes in collagen type proportions , 65 and igration . While tigration –77. Actiigration , 79. Subigration . Future We propose that several features of collagen in the tumor microenvironment, specifically collagen density, organization, and fiber characteristics affect biologic behavior of mammary tumors and can predict outcome in FMT. In this relatively small study, the most predictive multivariable model for overall survival included the clinical parameters grade and surgical margins, as well as the collagen parameters boundary score, collagen fiber length and collagen fiber straightness. Thus, validation of these findings in a larger study will help to improve accurate prediction of the clinical course for cats with mammary cancer. Importantly, this may improve delivery of clinical care in individual patients by determining which patients are most at risk for recurrence of disease. Finally, a better understanding of the mechanisms by which these tumor-permissive and restrictive collagen signatures develop and regulate cancer cell activities and fate will improve development of novel therapies for both human and veterinary patients using a “One Health” approach.S1 TableClinical variables of cats enrolled in this study. DFS, Disease-Free Survival; ST, Survival Time; OHE, ovariohysterectomy; FS, female ovariohysterectomized (spayed); *, ovariohysterectomy performed at time of tumor excision; FU, female unknown OHE status; NA, not able to assess; DHSA, domestic shorthair; DHLA, domestic longhair; RGDL, ragdoll; PERS, Persian.(DOCX)Click here for additional data file.


During spaceflight, the central nervous system (CNS) is exposed to a complex array of environmental stressors. However, the effects of long-duration spaceflight on the CNS and the resulting impact to crew health and operational performance remain largely unknown. In this review, we summarize the current knowledge regarding spaceflight-associated changes to the brain as measured by magnetic resonance imaging, particularly as they relate to mission duration. Numerous studies have reported macrostructural changes to the brain after spaceflight, including alterations in brain position, tissue volumes and cerebrospinal fluid distribution and dynamics. Changes in brain tissue microstructure and connectivity were also described, involving regions related to vestibular, cerebellar, visual, motor, somatosensory and cognitive function. Several alterations were also associated with exposure to analogs of spaceflight, providing evidence that brain changes likely result from cumulative exposure to multiple independent environmental stressors. Whereas several studies noted that changes to the brain become more pronounced with increasing mission duration, it remains unclear if these changes represent compensatory phenomena or maladaptive dysregulations. Future work is needed to understand how spaceflight-associated changes to the brain affect crew health and performance, with the goal of developing comprehensive monitoring and countermeasure strategies for future long-duration space exploration. Recent advances and renewed investment in human spaceflight have accelerated the timeline for long-duration space exploration missions, including crewed missions to Mars. The success of these endeavors is contingent on our ability to monitor and maintain human health and performance during the mission. Maintaining the integrity of the central nervous system (CNS) and the brain during long-duration space exploration is a high priority, because high-level sensorimotor and cognitive processes are essential to many mission critical tasks.1. Crewmembers of the short-duration Apollo era missions reported altered driving performance on lunar excursions2, and during the first 100 Space Shuttle Missions 20% of orbiter landings fell outside acceptable limits3. In-flight performance decrements have been noted during missions aboard Mir and the International Space Station (ISS), with reports of several close calls and one collision between a vehicle and space station components4. Although current and future generations of NASA spacecraft have been designed for autonomous flight, the crew must be capable of manually operating the vehicle in case the automatic control fails5.Decrements in operational performance have been reported throughout the history of spaceflight5. In particular, sensorimotor dysfunction has been implicated as a major potential cause of performance decrements during spaceflight7. Sensorimotor deficits reported during and after spaceflight include impaired gaze control, reduced fine motor control, spatial disorientation, impaired coordination, postural ataxia, and loss of motor efference4. Of note, whereas these sensorimotor changes are greatest immediately after gravitational transitions, the extent and duration of some of these alterations have been associated with increased mission length9. As an example, poorer landing accuracy in Space Shuttle pilots was associated with longer mission duration and greater vestibular dysfunction, as determined by postflight assessment7.Previous studies have demonstrated changes across multiple neurologic domains after spaceflight including changes to sensation, movement, coordination, and cognition10. However, a growing body of evidence suggests that other spaceflight-associated factors may also impact the brain, including space radiation, isolation and confinement, circadian disruption, and chronic hypercapnia  distribution and dynamics, tissue microstructure, and functional connectivity21. In this review, we aim to summarize current knowledge regarding neuroimaging-detected changes to the structure and connectivity of the brain after spaceflight, particularly as they relate to mission duration.Magnetic resonance imaging (MRI) is a high-resolution neuroimaging technique and powerful supplementary tool that allows detailed assessment of the structure and functional connectivity of the brain. Past reviews of spaceflight-associated changes to the brain have highlighted results from neuroimaging studies conducted in animal models and in humans who are exposed to analogs of spaceflight22. Several studies have also used functional MRI (fMRI) to examine the connectivity within or between different brain regions during rest or activated states23. A summary of the findings from currently available MRI-based studies examining changes to the brain after spaceflight are summarized in Table To date, multiple MRI modalities have been used to assess the structure and function of the brains of crewmembers before and after spaceflight at both the anatomical level and the microstructural level 18. A follow-up study demonstrated an increase in total ventricular volumes in crewmembers of long-duration ISS missions (10.7%) that was absent in crewmembers of short-duration Space Shuttle missions (0%)19. Within the cohort of long-duration fliers, a continuous linear association (r = 0.72) was seen between increasing mission duration and greater total ventricular volume19. Of note, volume increases were primarily observed in the left lateral ventricle , right lateral ventricle , and third ventricle  with relative sparing of the fourth ventricle 19. Van Ombergen et al. reported similar findings, with significant increases in the volume of the LV (13.3%) and 3 V (10.4%) after long-duration spaceflight and minimal change in the 4 V21. On 7-month follow up-scans, the ventricular volumes had decreased from the initial postflight measurements but still remained significantly elevated from preflight measurements21. Kramer et al. reported increased ventricular volumes in astronauts 1 day after they returned from long-duration spaceflight, which were persistently elevated at 12 months post-flight22.In 2017, Roberts et al. reported widespread structural change within the brain after spaceflight, including narrowing of the central sulcus, upward shift of the brain, twisting of the cerebral aqueduct, and increased ventricular volumes24. The authors found greater fluid shifts in astronauts following 12 months in space compared to 6 months, which returned to baseline by 6 months post-flight24. Ventricular volumes were significantly increased in all astronauts in the 6-month mission group . Interestingly, 1 of 2 subjects studied following a 12-month mission demonstrated significantly increased ventricle volumes  while the second subject showed smaller percent increases , potentially due to larger baseline ventricular size24. On 6-month follow-up scan, approximately half of the 6-month mission astronauts demonstrated post-flight resolution of ventricular volume changes, while the other subset showed continued ventricular enlargement post-flight24. Subsequent analysis demonstrated that LV volumes were correlated with number of post flight days, number of previous missions and less time between missions; no effect of age was seen on pre-flight ventricular volume24.A recent study by Hupfield et al. examined fluid shifts and ventricular volume changes within a group of long-duration ISS astronautsThe brain itself is composed of two primary tissue types, known as gray matter (GM) and white matter (WM). GM, composed of neuron cell bodies and glial cells, is responsible for modifying and collecting information transmitted from local neurons and from distant regions via bundles of long, myelinated axons known as WM tracts.20. However, Kramer et al. recently reported a significant increase in the global WM volume of long-duration astronauts immediately after spaceflight (5.5%) that persisted out to 12 months (4.6%), with no significant changes to the global GM on post-flight scans22. Kramer et al. also reported increased summated mean brain and CSF volumes post-flight, which were not seen in previous studies22. The difference in these findings may be attributable to alternative volumetric quantification methods; total pre-flight WM volume in prior studies averaged 540–580 ml, while in the recent Kramer study reported pre-flight WM volumes between 417–470 ml. This discrepancy highlights an important point; a standardized method of quantification for neuroimaging in spaceflight is key to improved accuracy and reproducibility of future studies.Several studies have demonstrated no significant change in global GM or WM volume following spaceflight in astronauts or cosmonauts20. Koppelmans et al. has previously noted increased GM in sensorimotor and motor areas of the brain13, a finding echoed by recent work by Hupfield et al. showing increased GM in the SMA, pre-central and post-central gyrus24. It is important to note that the majority of these GM alterations overlapped significantly with alterations in CSF and FW, as well as shifting of brain position in the skull. In addition, GM volume changes were largely resolved approximately 6-months post-flight24.Given the high regional specificity of neurologic functions, examining focal alterations within brain tissue, rather than global tissue shifts, may yield greater insight into the neurologic regions affected during long-duration spaceflight. Several studies have reported focal GM changes in the brain following spaceflight. Decreases in GM volume of the orbitofrontal and temporal poles have been reported in both astronauts and cosmonauts following spaceflight25. They demonstrated that the vast majority of GM changes were due to volume shifts; these GM changes were largely reversed on 7-month follow up, in agreement with previous studies25. Interestingly, by their method, a net gain of GM tissue was noted in the basal ganglia, an area involved in voluntary movement25. In addition, Jillings et al. noted increased WM volume in the cerebellum, corticospinal tract and primary motor cortex in cosmonauts immediately following spaceflight, which were partially resolved on 7 month follow-up scan. Taken together, these results may suggest that spaceflight induces WM volume increases in the motor and coordination regions of the brain, which partially resolves upon return to earth25.Jillings et al. has recently published an MRI study in cosmonauts utilizing a technique called multi-tissue spherical deconvolution with the goal of differentiating GM changes driven by local volume shifts  versus genuine tissue loss such as that seen in neurodegeneration16 have shown focal changes in WM microstructure after spaceflight within multiple sensory regions, including regions important to vestibular and proprioceptive processing16. These areas included the inferior cerebellar peduncle, which transmits information between the vestibular receptors and the cerebellum, and the right inferior and posterior parietal lobe, which integrate vestibular and proprioceptive information and play an important role in perception of the spatial representation of the body and upright perception16. Lee et al. also noted WM changes in regions related to vision and visual processing, including the superior longitudinal fasiculus (SLF), which plays an important role in visuospatial processing, visual attention, and visuomotor control26. Interestingly, greater changes in the SLF in astronauts was found to be correlated with larger postflight balance disruptions16. WM changes were also noted in the inferior longitudinal fasiculus (ILF) and inferior fronto-occipital fasiculus (IFOF), which run along the lateral ventricle in close proximity to the optic regions and are believed to play a role in visual recognition , visual processing, visually-guided decision-making and language comphrension16.Several studies have also utilized diffusion-weighted MRI to examine the microstructure of WM by fractional anisotropy. Lee et al.17. WM was altered in the posterior thalamic radiations, which contain the optic radiations and help carry visual information between the thalamus and occipital cortex. Interestingly, investigators also noted GM changes to both the thalamus and occipital cortex were also noted, which were hypothesized by the authors to occur secondary to fluid accumulation in the visual pathway or to the downstream effects of changes within the optic nerve itself17.Riascos et al. recently reported spaceflight-associated WM changes within areas associated with vision and visual processing16. WM changes were also detected in the middle cerebellar peduncle, a region that contains the cortico-ponto-cerebellar tract that facilitates initiation, timing and planning of movement27. Interestingly, astronauts had greater baseline WM organization in the corticospinal tract and cerebellar peduncles compared to control subjects16. Because all astronauts included in this study had previous spaceflight exposure, this may demonstrate an adaptive change in motor and balance control induced by spaceflight, and/or induced by the spaceflight simulations and other countermeasures used to prepare crewmembers for spaceflight exposure16. In addition, Lee et al. reported that that crewmembers of long-duration missions had greater postflight WM organization in the cerebellar white matter than did crewmembers of short-duration missions, indicating this adaptation may accumulate with increasing time in space16.Alteration of the microstructure of WM tracts involved in motor function was also described by Lee et al., including decreased organization in the corticospinal tract and the WM underlying the primary motor cortex, theorized to occur secondary to the decreased use of the lower limbs in space15. The investigators found decreased connectivity within the right insula, which is involved in vestibular processing and cognitive control, and altered connectivity between the motor cortex and cerebellum15. A second, larger study by Pechenkova et al. also demonstrated that spaceflight decreased functional connectivity of the cerebellum to regions with proprioception, visual, motor, and somatosensory functions23. The cerebellum is important for coordination and fine-motor control, and is believed to play a significant role in sensorimotor adaptation to microgravity29. Pechenkova et al. found decreased connectivity between the vestibular nuclei and sensory and motor regions of the brain after flight, which is believed to result from central adaptation that down-regulates vestibular input during spaceflight, reducing sensory conflict and helping to ameliorate space motion sickness30.The first study of altered brain connectivity after spaceflight was conducted by Demertzi et al. in a single Russian cosmonautSpaceflight analogs are useful for studying the individual contributions of spaceflight-associated stressors on the CNS. Similarly, shared features of spaceflight-associated neurologic decrements and features of terrestrial human disease may help identify biomarkers and inform future countermeasure strategies.0 tilt likely represents one of our greatest assets in assessing the influence of spaceflight-analog conditions on the brain, as it exposes subjects to cephalic fluid shift, hypokinesia, unloading of the axial body and environmental depriviation31. Several common neuroimaging changes are induced in HDBR and in spaceflight, including rotation of the brain within the skull, crowding of tissue at the vertex, alterations in free water, and decreases in front-orbital and temporal GM35.Head-down bed rest (HDBR) at a 636. In addition, performance of sensorimotor and spatial working memory tasks changes, balance and locomotor function declines, neural efficiency during vestibular stimulation decreases, and neurocognitive reserve during dual tasking decreases during spaceflight and HDBR38. These data suggest that alterations in the thalamus, parietal cortex, and motor and sensory cortices with their associated declines in sensorimotor, balance, and dual task performance may be related to altered mobility and axial body unloading.HDBR and spaceflight also induce similar changes in brain connectivity, including altered connectivity of motor, somatosensory and vestibular areas of the brain39. Interestingly, recent work has demonstrated that strict adherence to HDBR reproduces SANS-like pathology in an analog setting, suggesting that these equivocal results may be the result of methodological differences in study design and protocol adherence, and that hypercapnia may play a role in the development of SANS pathology40. However, no HDBR study to date has been able to reproduce the full spectrum of ocular changes seen in SANS, as the globe flattening and visual acuity changes seen in long-duration microgravity were absent in terrestrial studies.Spaceflight analogs have been able to recapitulate some of the findings from spaceflight studies, including changes in connectivity, GM volumes, CSF dynamics and ventricular volumes. However, evidence of WM changes during HDBR remain equivocal. Koppelmans et al. reported no WM changes after 70 days of HDBR, whereas Li et al. reported widespread alterations in WM after 30 days of HDBR without adjustment for multiple comparisons in the statistical tests used. Similarly, studies have demonstrated mixed results regarding ventricular volume changes after HDBR2)40. Previous research suggests that chronic hypercapnia itself may have detrimental effects on the CNS41. Crewmembers on the ISS are chronically exposed to levels of CO2 that are 10-fold higher than those experienced on Earth, and some reports document increased sensitivity to elevated CO2 during spaceflight42. Kramer et al. demonstrated that combined HDBR and CO2 results in increased ventricular volumes and altered CSF hydrodynamics39. Subsequent work by the authors demonstrated that CSF hydrodynamics were altered immediately after spaceflight itself, in conjunction with increased postflight ventricular volumes22. Elevations in arterial CO2 can decrease CSF absorption by 50% in an animal model, offering one potential mechanism for the additive effect of HDBR and CO2 on the ventricular system43. Further experiments are required to determine if both the cephalic fluid shift during long-term HDBR and the elevated CO2 levels are both necessary and sufficient for increased ventricular volume.Interestingly, the subjects who developed SANS-like pathology in the aforementioned HDBR experiment by Laurie et al. were also exposed to chronic mild hypercapnia as part of the study design  of subjects after they spent 520 days in isolation; changes were thought to occur secondary to exposure to reduced space and low environmental enrichmentMany spaceflight-related CNS alterations likely represent beneficial adaptations to the novel environment of spaceflight. However, given both the unprecedented duration and magnitude of the spaceflight-related stressors, it is important to examine whether changes within the CNS have the potential to become pathologically dysregulated during future long-duration space exploration.51. Studies that support this theory have demonstrated dual-tasking of sensorimotor and cognitive behaviors is impaired during both short and long-duration space missions54. However, data from majority of studies examining changes in cognitive performance during long-duration spaceflight and analog environments have been largely inconclusive55. A novel neurocognitive assessment tool, Cognition, was developed to address some of the gaps in the current cognitive test battery used to assess US astronauts56. Recently published work from the NASA Twins Study demonstrated that one subject had significant decrements in performance as determined by the Cognition battery after 340 days of spaceflight as compared to both his own baseline and that of his twin, who served as a terrestrial control57. A longitudinal study is ongoing to examine the effects of long-duration spaceflight on neurocognitive performance paired with neuroimaging results from anatomical and functional MRI58.In addition to alterations to the peripheral input and output pathways, the sensorimotor changes that occur during spaceflight may result in extensive central adaptation extending to the cortical level. For example, the neurovestibular system includes peripheral sensory organs that relay visual, proprioceptive and vestibular information to the brain stem for processing and integration with signals from the cerebellum and cerebral cortex. It has been suggested that long-term deprivation of vestibular senses may result in adaptation of the entire thalamocortical vestibular system, including areas involved in cognitive functions such as sensory integration14. Smooth WMH, as seen by Alperin et al. in crewmembers after spaceflight, have previously been associated with subependymal gliosis and trans-ependymal CSF diapedesis, and are not believed to be ischemic in nature59. Given the association between increased ventricular volumes and the smooth periventricular WMH and the absent deep WMH after spaceflight, it is possible that these alterations may represent trans-ependymal CSF diapedesis that are at least partially reversible after spaceflight.21 Alperin et al. conclude that the absence of deep WMH in astronauts suggest that cognitive changes after spaceflight are likely unrelated to WMH burden, unlike the hypoxia-related deep WMH pattern seen in high-altitude pilots14.In response to reports of an association between white matter hyperintensities (WMH) and cognitive alterations in high-altitude pilots, Alperin et al. demonstrated that increased WMH were also seen in astronauts after long-duration flight, and these increases were associated with increased ventricular volumes. However, WMH in astronauts were restricted to the periventricular region and had partially reversed by one month after return to Earth60, and Roberts et al. found that crewmembers who developed SANS had smaller changes in ventricular volume than crewmembers without SANS symptoms19. While preliminary, this data supports one existing theory that CSF entry into the optic nerve may contribute to SANS, and may be more likely in crewmembers who have insufficient ventricular expansion62. However, it must be noted that a more comprehensive understanding of SANS pathogenesis is still developing, and a single driving mechanism has yet to be fully proven63. For a comprehensive review of SANS pathophysiology and current research, we direct readers to a recent review by Lee et al.63.Cephalic fluid shift and CSF redistribution have also been implicated in the etiology of spaceflight-associated neuro-ocular syndrome (SANS), a disorder characterized by ocular changes in crewmembers during long-duration spaceflight, including optic disk edema, globe flattening, choroidal folds, cotton wool spots and a hyperopic refractive change. Van Ombergen et al. noted postflight increases in CSF spaces near the optic nerve22, a condition characterized by an enlargement of cerebral ventricles without increased intracranial pressure, resulting in progressive gait apraxia, cognitive impairment and late-stage urinary incontinence19. Subsequent work reported an association between expanded ventricular volume and periventricular tissue displacement, with a trend towards poorer postural control in crewmembers with greater changes in ventricular volume19. Crowding at the vertex, enlarged ventricles, periventricular WMH, and a similar pattern of decreased GM volumes occur after both long-duration spaceflight and in NPH19. Interestingly, several tracts identified in our review of postflight neuroimaging have also been reported to be affected in NPH, including the IFOF, thalamic radiations and the ILF64. In contrast, Williams et al. posit that the enlargement of the ventricles in crewmembers does not meet the clinical criteria for hydrocephalus, and that the comparison to NPH has limited utility given the absence of NPH-like symptoms in crewmembers after flight65. The controversial nature of this topic represents an important gap in knowledge, and underscores the importance of further research into the clinical and operational significance of ventricular volume changes after spaceflight.Similarly, the pathogenesis and clinical implications of spaceflight-associated ventricular changes to the brain itself remain largely unknown. It is important to note that several hypotheses exist regarding the clinical implications of spaceflight-associated ventricular changes, and that the topic remains a highly controversial one within the field. Recently, several investigators have hypothesized that the effects of spaceflight on brain structure are similar to those seen in normal pressure hydrocephalus (NPH)18. Increases in total ventricular volume were greater in long-duration fliers than in short-duration fliers, and within the cohort of long-duration fliers, increases in ventricular volume were positively associated with mission duration21. Potential sequelae of ventricular expansion, including periventricular WMH and displacement of tissue surrounding the ventricles, were observed only in long-duration fliers19. Given that these studies demonstrate only partial resolution of these changes 6 to 12-months post flight, and that elevated pre-flight ventricular volumes are seen in astronauts with cumulatively higher mission numbers and flight days, the alterations of ventricular volumes in spaceflight appear to result in sustained ventricular expansion beyond the rate expected with normal aging24. Importantly, prolonged spaceflight exposure may also have operationally beneficial adaptations; Lee et al. noted that crewmembers of long-duration missions had greater WM coherence in the corticospinal tract and cerebellar peduncles, a finding they postulated may reflect motor adaptation secondary to repeated gravitational exposure16.Although the exact pathogenesis and clinical impact of spaceflight-associated alterations in brain structure and function remains unknown, several studies reviewed here provide evidence for an association between mission duration and the extent of changes as determined by neuroimaging. Greater rates of brain structure changes were recorded after long-duration missions, including greater increases in CSF space narrowing, upward brain shift, and pituitary stalk stretchingTaken together, these results indicate that some brain alterations become more pronounced with increasing mission duration. Current long-duration neuroimaging studies have recorded minor brain alterations after an average of 6 months of spaceflight; further progression of these symptoms during longer periods of spaceflight exposure, such as during a multi-year interplanetary expedition, could significantly affect a mission. Further study is required to determine whether individual alterations represent adaptive or maladaptive processes, based on their impact to crew health and operational performance during a given mission phase.33. In addition, the timing of the scans between subjects was not uniform due to the retrospective nature of the studies, and much of the MRI data available was of low-resolution, increasing the risk of measurement variance and volumetric error33. Lack of an age or sex matched longitudinal health control group in many of the studies also limits the ability to interpret which brain changes are directly related to spaceflight exposure vs. baseline or training related alterations that may be inherently present in the elite subset of the population our crewmembers represent22. Notably, the data on female astronauts or sex differences in neuroimaging after spaceflight is very sparse across all studies, an unfortunate pattern that is present throughout much of medical literature to date and must be addressed moving forward given the plans to send both men and women on future long-duration space missions. As noted above, perhaps the largest limitation is the extraordinarily small sample size, which significantly limits the ability to use the robust statistical methods typically applied to this type of research. While a definitive interpretation of the current data is not currently possible due to these study limitations, the body of work to date represents a Herculean scientific effort and has proven to be exceedingly important to our developing understanding of the impact of spaceflight on the brain. These results have helped to inform and refine further experimental planning, including prospective studies with uniform neuroimaging testing across all crewmembers that are currently underway16. As highlighted by Roberts et al.,66 the development of standardized protocols for pre- and post-flight studies across nations and space agencies also represents an opportunity to optimize the power and validity of future neuroimaging studies.It is important to note that the interpretation of virtually all spaceflight neuroimaging studies is limited by significant study limitations reflective of the exceedingly small n of their astronaut and cosmonaut subjects. As MRI can only be performed terrestrially, there remains no current ability for high resolution neuro-imaging in flight, limiting our knowledge of spaceflight-associated brain alterations to those with residual effects that last post-landing. In addition, many studies are limited by the time delay between pre and post-flight MRI assessments; changes during this time could also be reflective of training effects during the intensive preparation crewmembers perform leading up to their flightThis review summarizes the current findings of neuroimaging-evaluated changes to the brain after spaceflight, and includes comparison to data from spaceflight analog studies. We have also reviewed the evidence indicating that mission duration is a key factor in spaceflight-associated brain alterations, and have highlighted potential sources of CNS-related risk to crew performance during spaceflight. A summary of the CNS regions with altered structure or functional connectivity, their physiologic function, and the potential consequences of pathologic changes within these brain regions is given in Supplementary Table In conclusion, current data suggest that multiple CNS regions are impacted during spaceflight, resulting in structural and functional changes within both the primary regions and in the integration of those regions during higher-level functions Fig. . These cSupplementary Material