Psychiatric disorders are complex brain disorders with a high degree of genetic heterogeneity, affecting millions of people worldwide. Despite advances in psychiatric genetics, the underlying pathogenic mechanisms of psychiatric disorders are still largely elusive, which impedes the development of novel rational therapies. There has been accumulating evidence suggesting that the genetics of complex disorders can be viewed through an omnigenic lens, which involves contextualizing genes in highly interconnected networks. Thus, applying network-based multi-omics integration methods could cast new light on the pathophysiology of psychiatric disorders. In this review, we first provide an overview of the recent advances in psychiatric genetics and highlight gaps in translating molecular associations into mechanistic insights. We then present an overview of network methodologies and review previous applications of network methods in the study of psychiatric disorders. Lastly, we describe the potential of such methodologies within a multi-tissue, multi-omics approach, and summarize the future directions in adopting diverse network approaches. Psychiatric disorders, a group of prevalent brain disorders involving complex disturbances in socio-cognitive functioning, are a leading cause of disability . SurpassKey unanswered questions include: How do polygenetic contributions interact to affect molecular signaling and endophenotypes? Do different combinations of common and rare variants produce distinct manifestations of psychiatric symptoms? How do we meaningfully expand the understanding of these genetic interactions, and how do we leverage such knowledge to promote precision medicine a?One promising approach is to broaden the analysis to noncoding regulatory elements and consider their effects within the complete architecture of a functional genome ,4. CurreIn this review, we discuss recent advances in network approaches that may address this complexity. Broadly, for example, network approaches that define the superstructures of interactions and probabilistic models that localize key nodes in this structure could compress the genetic search space to the most important elements. We will firstly introduce the prevailing view on the polygenetic architecture of psychiatric disorders and review genetic studies that have linked rare and common genomic loci to different conditions. We will then discuss several challenges in dissecting the full architecture of psychiatric disorders with this view. We will present advances in multi-omics approaches and network methodologies that could address these challenges in the context of an omnigenic model. We will introduce emerging network tools that are underutilized but promising. Lastly, we will summarize potential future directions of developing network approaches.We surveyed six common psychiatric disorders with various levels of heritability (twin heritability ranging from 0.37 to 0.85) and distinct domains of manifestation and etiology: alcohol use disorder, autism spectrum disorder (ASD), bipolar affective disorder, major depressive disorder, post-traumatic stress disorder (PTSD), and schizophrenia. Details regarding disorder characteristics, heritability, and impacts are outside the scope of this review but can be found in previous reviews, such as Sullivan and Geschwind, 2019 [Over the past decade, genetic studies have linked both rare and common genomic loci to different disorders and traits ,10,11,12By contrast, recent GWAS studies have uncovered common genetic loci with relatively high frequencies in the population but each with small effect sizes, making common variants challenging to identify unless studied in large populations comprised of as many as 100,000 subjects ,20. DespThere are several challenges in investigating the polygenic architecture and mechanisms of psychiatric disorders. First, large population samples are needed to overcome statistical hurdles to identify common variants with small effect sizes. Second, interpreting the biological roles of common variants is challenging because 93% of the common variant loci located in non-coding areas of the genome can regulate gene expression in a subtle or indirect way . Third, Multi-omics and network approaches can address these levels of complexity by exploiting the structure of physiological contextualization and connectivity to generate otherwise inaccessible insights. Multi-omics approaches integrate genetics, functional genomics, transcriptomics, proteomics, and epigenetics. Integrating multiple levels of analysis in this manner can provide unique windows into key driving elements and hypothesized biological functions of genes that would otherwise be opaque to a single-level analysis. One way to integrate multi-omics data is through network approaches and we will discuss the biological and pragmatic motivations for transitioning from a polygenic model to an omnigenic network model of psychiatric genetics.A polygenic model views disorder risk from a multitude of common and rare variants as combinatorial contributions. More recent inferences from the polygenic model and advances in technology and biology have promoted the recognition of an omnigenic model, which views genetic architecture from a network perspective ,23. NetwIn the omnigenic model, core genes and peripheral genes contribute differentially to the heritability of complex traits . The smaThe omnigenic network model calls for systems biology tools to make inferences about pathogenic mechanisms; however, variants alone are insufficient to construct disorder-related networks—additional molecular data that help establish or infer functional relationships are needed. Multiple levels of data from gene expression to protein interactions can be integrated to facilitate the construction of disorder-related networks . We descIntegrating and embedding multi-tissue, multi-omics data into network architectures offers unprecedented relational insights while anchored to physiologic contexts. Common omics data for network construction include genetics, transcriptomics, proteomics, and epigenomics . TranscrAs mentioned, the vast majority of heritability involves common variants that are often in non-coding areas, which cannot be directly mapped onto a gene network. To connect these loci with molecular networks in disease-relevant tissues, functional genomics serves as a bridge between genetics and other omics. Intermediate phenotype quantitative trait loci (iQTL) are mostly used for this purpose, which are genetic loci associated with specific quantitative traits such as gene expression or protein levels, which are intermediate traits between genetics and clinical phenotypes . In termAs efforts devoting to large-scale omics profiling proceed, there has been an accumulation of databases of different data modalities that can be used for psychiatric research . GWAS caNetworks commonly used in systems biology include gene regulatory networks, protein-protein interaction networks (PPI), literature-curated networks, and hybrid networks . These nBelow we discuss each network method and its application in psychiatric disorders. We also highlight approaches that are not yet widely adopted in psychiatric research.Three main kinds of gene regulatory networks are commonly adopted: gene co-expression networks, causal relationship networks, and regulator-target pair networks.Gene co-expression networks are correlation-based networks in which highly co-regulated genes are clustered into modules, illustrating the functional clustering of genes and pinpoint core genes based on connectivity. Commonly used methods to generate gene co-expression networks include WGCNA  and MEGEUsing WGCNA or MEGENA, one can identify modules associated with certain conditions based on the transcriptomic profiles in both cases and controls using the module-trait correlation analysis a. By annAnother common application of co-expression networks is first to construct co-expression networks based on the transcriptomic profiles from control subjects and then to examine the module enrichment level of genes affected in a specific disorder a, as exeGene co-expression networks are powerful tools in determining co-regulatory relationships of genes involved in distinct functions and how these modules connect to psychiatric disorders. However, co-expression networks are not directional, and thus are unable to provide causal relationships between genes, an important aspect to retrieve upstream regulators. This limitation can be addressed by causal regulatory networks described in the following sections.BNs are directed acyclic graphs summarizing causal regulatory relationships between genes. BNs can be generated with transcriptomic data alone, but the incorporation of prior information capturing regulatory information can offer higher prediction accuracy for regulatory relationships ,58. cis-Arc was found as a key driver gene of the clock/IEG network, which may link depression and sleep loss.Integrating genetics, transcriptomic, functional genomic inputs, and more, BN can capture causal regulatory relationships in a given tissue and can be used as a ‘roadmap’ in pinpointing key regulatory genes  Figure b. There PPP4C and MAZ as potential top driver genes in the most common ASD-related CNV 16p11.2.Protein-protein interaction and tissue-specific gene expression patterns have also been used to construct BNs, as in GIANT BNs . GIANT BAlthough not yet widely adopted in psychiatric research, BN has been applied to study many other diseases such as Alzheimer’s disease , Type IIDysregulation of transcription factors and non-coding RNAs have been indicated in psychiatric disorders ,65,66,67Nr3c1, the gene encoding the glucocorticoid receptor, as a master regulator across many brain regions in alcohol-dependent rats [One piece of software that infers transcription factor regulons based on transcriptomic information is the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe). Repunte-Canonigo et al., applied ARACNe to a rat model of alcoholism and identified ent rats ,69. The ent rats , was appent rats . Five reIFNG and IL-12, which exhibit increased expression levels in PTSD patients [hsa-miR-193a-5p could decrease the high expression level of IL-12, which may help reduce the excessive inflammation response in PTSD patients.The Ingenuity Pathway Analysis (IPA), a commercial tool constructed based on a comprehensive curation of different networks from experimental datasets, text-mining literature, and other databases, also contains transcriptional factor and miRNA-target pair networks . Using tpatients . They alhsa-miR-21-3p, targets neuronal/synaptic genes down-regulated in ASD, which may play an essential role in pathogenesis.Another software tool, TargetScan, predicts mRNA targets of miRNA based on conserved sequences in mRNAs . Wu et aIn a PPI network, the nodes are proteins, and the edges depict the physical interaction relationship between proteins based on experimental datasets or computational simulation. The edges are undirected, and the weights of the edges indicate the reliability of the interaction. StringDB is the most commonly used PPI database, with the latest version v11 covering around 25 million proteins from 5099 organisms . StringDPPIs have been extensively used in psychiatric research to identify hub genes. Commonly used methods of identifying subnetworks and hubs include DAPPLE, DMS, MCODE, and PINA ,75,76,77PPIs are also broadly used in combination with other networks, such as the co-expression network, to identify disorder-related networks. Gulsuner et al., profiled schizophrenia-related de novo mutations and leveraged a combination of the PPI network and co-expression network to examine the functional relevance of these de novo mutations and identify their enrichment in pathways and tissues . By mappTBR1, NFIA, and KDM6B.Since proteins interacting with each other may be co-regulated by the same upstream regulatory signal, they may also exist in the same co-expression network. Thus, genes that are both co-regulated and show protein interaction may be of higher relevance to a specific condition and should be prioritized for further study as driver genes b. PariksThe comprehensive PPI databases provide abundant resources for reliable PPI networks. However, one main limitation of the PPI network is that it does not reflect causality or regulatory relationships. In addition, the PPI network is not tissue-specific and may bring in interactions that are not relevant to the disease tissues.Literature-based networks are curated based on integrating general knowledge such as pathway/function annotation databases and text-mining existing experimental data and literature to capture genes contributing to specific biological functions. Although gene ontology terms can be constructed into a network in terms of the relatedness of biological pathways, genes in this type of network are not interconnected and do not reflect the topological properties of gene-gene relationships . Thus, pLeveraging a knowledge-based and data-driven gene-phenotype likelihood network, Gilman et al., developed the NETwork-Based Analysis of Genetic Associations (NETBAG) . The genHTR4 and MCHR1, encoding serotonin and melatonin receptors, respectively, and have been indicated in depression and schizophrenia.Similar to the NETBAG, Ward et al., constructed phenotypic-linkage networks (PLN) to identify nervous system gene sets related to the GWAS loci of mood instability . The autApart from the individual usage of the networks mentioned above, constructing a hybrid network consisting of two or more kinds of networks is also a common practice. As different networks cover different aspects of gene interaction, a hybrid network can leverage the strengths and overcome the disadvantages and limitations of each network type. For example, combining PPIs with BNs integrates both causal gene regulatory information and protein physical interaction, which covers distinct aspects of gene interactions and promotes the identification of hub genes that play regulatory roles at the gene and/or protein levels.Gazestani et al., constructed hybrid networks incorporating knowledge-based, functional, and experiment-derived co-expression networks to identify transcriptional perturbation patterns in leukocytes from ASD cases and control children . The autIn addition to constructing literature-curated networks or hybrid networks from scratch, there are numerous existing hybrid network resources available. Huang et al., benchmarked 21 popular human gene or protein networks, including StringDB, GeneMania, and GIANT, based on a disease gene set recovery test and found that networks with a larger size have better performance in retrieving known disease genes . They thDue to the genetic correlation and comorbidity across psychiatric disorders, studying pathogenesis mechanisms across disorders may yield therapeutic targets for several disorders or specific endophenotypes. A recent study by Gandal et al., leveraged WGCNA gene co-expression networks constructed based on case-control human brain transcriptomic profiles across five psychiatric disorders: schizophrenia, major depressive disorder, bipolar affective disorder, ASD, and alcohol use disorder . In addiThe conclusions from the Gandal et al., studies are consistent with previous pathway analyses based on genetic risks and studied leveraging of a similar network approach. Using pathway analysis, a study by PGC found that common genetic risks of schizophrenia, major depressive disorder, and bipolar affective disorder converge on neural, immune, and histone modification pathways . Kim et Besides network applications in studying pathogenesis, networks are also powerful tools to identify driver genes in treatment response. Although many psychopharmacological drugs are available and have been applied for more than five decades, their mechanisms of action remain mostly elusive. Moreover, drug responses in individuals differ tremendously. Elucidating the mechanisms of action and identifying biomarkers that predict individual responses to drugs can greatly aid precision medicine in psychiatry and the development of novel therapeutics.Lithium (Li) is a first-line mood stabilizer for bipolar disorder, although its mode of action is not fully elucidated. To reveal the mechanism of Li in treating bipolar disorder and the differential responses across patients, Breen et al., characterized the transcriptomic profiles of subject-derived lymphoblastoid cell lines from Li responders and non-responders . WGCNA gNetwork methodologies provide us with a perspective beyond the identified disorder-associated variants. For instance, candidate genes in the network which show high connectivity to many previously identified genes can be prioritized as hub genes or driver genes. Due to their high connectivity, these hub genes may act as the converging points of disorder-related variants and pathways, making them potential targets for therapeutics even if they have not been implicated by genetic evidence yet. Besides revealing hidden novel genes in disorder etiology, network methodologies can elucidate regulatory relationships and coherent biological functions between disorder genes. For example, co-expression networks can identify covariation across modules of genes and differentially regulated modules, while one may fail to identify significant differentially expressed genes.Many fruitful findings have been made through network methods, which are summarized in Schizophrenia also engages the immune system, synaptic functions, and neurodevelopmental processes, which fall into the same pathway category as ASD ,113. HowMAP4 and ILF2 were also suggested, but due to fewer study numbers and a lack of validation, a consensus cannot be reached [The polygenic component of bipolar affective disorder overlaps with schizophrenia significantly, but network application in bipolar affective disorder is very limited. The role of postsynaptic density in bipolar affective disorder pathogenesis has been indicated by independent studies ,116. Hub reached ,100,102.Dkkl1, Neurod2, and Sdk1 were validated in vivo, indicating the reliability of network predictions and implicating synaptic transmission, cell-cell signaling, and oxidative phosphorylation pathways in depression pathogenesis [Major depressive disorder and PTSD are stress-related disorders that share neuronal and immune dysregulations based on network studies ,118,119.ogenesis .Nr3c1, predicted by transcription factor network was validated in vivo in an alcohol-dependent mouse model [Lastly, alcohol use disorder is a disorder combining features of addiction and neurotoxicity. Network studies of alcoholism have revealed processes related to mitochondrial dysfunction, synaptic transmission, neuroplasticity, calcium signaling, and immune functions ,125,126.se model . More stIn summary, we have introduced and illustrated the main network approaches, their strengths and limitations, and how they can complement one another by highlighting relevant studies. Despite the aforementioned recent discoveries, network applications in psychiatric research are still in their infancy. Networks such as the WGCNA co-expression network and PPI have been extensively applied, while other networks such as BN are rarely adopted despite indications as being powerful tools in other research fields. Integrating these network approaches may reveal hidden pathogenic mechanisms by capturing underappreciated information from the data. We recommend the adoption of diverse types of network approaches in each study to derive comprehensive molecular insights.Besides leveraging complementary network methods, another future direction would be to have an integrated framework followed by the field to apply a set of benchmarked and well-performing networking methodologies systematically. Such a framework would eliminate technical bias caused by different methods to enable systematic comparison of psychiatric disorders at a network level.Benchmarked and standardized network methodologies are applicable regardless of disorder types. However, in order to better elucidate trait-specific biology, we recommend careful collection of multi-omic data types that reflect the unique aspects of a certain disorder, including specific causal factors  and the corresponding omics data types , and related brain regions and circuits at the relevant developmental stages. In addition to collecting relevant types of data, tissue heterogeneity needs to be addressed as a future direction. As a highly complex organ, the brain consists of numerous subregions and nuclei, each containing various cell subtypes. Previous network application studies in psychiatric disorders were mostly performed at the brain region level using bulk tissue transcriptome. Obviously, with the advancement of single cell omics technologies, exploring cell-level networks becomes an urgent need. The abundance of single-cell transcriptomic datasets enables researchers to further dissect the pathogenic mechanisms of psychiatric disorders at an increasing granularity at the cell type or subtype level. Thus, it is possible to identify cell subtypes related to a specific condition and pinpoint key driver genes in different cell subtypes.Appealing as it is, network methods for single-cell datasets are still limited. Due to the challenge of data sparsity, methods applicable to bulk tissue transcriptomics do not perform well on single-cell datasets . HoweverBesides using single cell omics as a resource providing pathophysiological insights with increasing granularity, single-cell and bulk tissue transcriptomic profiling can also be applied as an approach to validating in silico predictions. Experimental validations have been limited despite the current progress of in silico findings in key drivers and pathways. More experimental validations should be performed to facilitate the transition from in silico predictions to the bench and eventually to the bedside. Current experimental validation methods include RT-PCR and transcriptomics for evaluating possible expression alteration of the key drivers from samples with the disorder ,52,60; hIn conclusion, approaching psychiatric genetics from a network perspective enables researchers to identify the converging pathways in genetic architecture and leverage the abundance of the omics databases to yield a better understanding of the pathophysiology and predictions for therapeutic targets. With this review, we hope to provide a systematic overview of network methodologies, previous network applications, and their findings in psychiatric research. Much remains to be explored—including adopting network approaches from other fields, standardizing a benchmarked and integrated framework, developing single-cell network construction methods, and performing corresponding experimental validations.


Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly; however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 individual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results. Circulating cell-free DNA (cirDNA) in blood has been extensively researched due to its potential as a biomarker across multiple settings including oncology, prenatal testing, toxicology, organ transplantation, and cardiovascular and autoimmune diseases. In cancer alone, cirDNA-related publications have increased exponentially  with neaThe release of cirDNA from cells into the blood is thought to be driven by apoptosis, which is closely linked to inflammation and cell damage. In this review, we aim to gather and summarise all published studies that have investigated an association between biological and lifestyle factors and cirDNA. By ‘biological factor‘, we refer to intrinsic physiological variables such as body mass index (BMI), menstruation, hypertension, circadian rhythm, stress, biological sex and age. By ‘lifestyle factor’, we refer to all external variables that involve individual choice, such as exercise, alcohol consumption, occupation and smoking. We have chosen to include only studies conducted on plasma and exclude all studies on serum as serum has been shown to be the less preferred substrate due to its propensity for leukocyte DNA contamination and dependence on processing time . The useIn total, this review summarises the results from 66 individual studies published between January 2000 and January 2021 . It is i2), categorises an individual as underweight (<18.5), normal weight (18.5–24.9), overweight (25–29.9) or obese (>30). BMI, however, does not distinguish whether the weight is fat or muscle mass, hence its usage is not accurate for groups of people such as body builders and athletes.BMI, calculated by dividing weight (kg) by the square of height  and six cohorts that included individuals with various diseases and women undergoing hormone replacement therapy (HRT). Five of the studies conducted on healthy individuals, totalling to 1098 subjects, reported no relationship between cirDNA and BMI. They contained relatively large, combined numbers of healthy male and female subjects , but most did not sex-disaggregate their data for analysis. A single study reported significantly higher cirDNA level with increasing BMI in a group of 366 healthy women . AlthougA positive correlation between BMI and cirDNA was found in 62 hepatitis-related liver fibrosis patients  . HoweverAs discussed in Section 4, accurate cirDNA quantification is strongly dependent on extraction and measurement methods. Limiting discussion to studies that either measured cirDNA directly from plasma or used a dedicated extraction kit, two studies from the same group reported no relationship between BMI and cirDNA concentration in a total of 402 nonagenarians , while a2), as determined by computed tomography, positively correlated with plasma single-stranded DNA concentration , as having diastolic blood pressure (DBP) and systolic blood pressure (SBP) of ≥140 mm Hg and ≥90 mm Hg, respectively, can lead to stroke, kidney failure, blindness, rupture of blood vessels and cognitive impairment. The elevated pressure can cause endothelial cell DNA damage, inflammation and oxidative stress , all of Five individual studies on cirDNA and hypertension were identified, containing four healthy cohorts (n = 1247) and five cohorts that included patients with various diseases and women undergoing HRT. Overall, conflicting findings were reported. No correlation between cirDNA levels and hypertension was found in 258 nonagenarians , 218 womDue to its global prevalence, the effects of hypertension on cirDNA are of interest. Future studies should include hypertensive people both with and without medication, as well as healthy controls. The studies should be disaggregated by sex and include subjects selected for the absence of other comorbidities and lifestyle variables that can potentially be confounding factors.The circadian rhythm, driven by the suprachiasmatic nucleus of the anterior hypothalamus of the brain, influences physiological profiles such as temperature, hormone levels and urine volume, as well as behaviour such as activity and sleep. Daytime is characterised by high body temperature, high urine volume, low melatonin and high cortisol, while the opposite occurs during night-time . DisturbSeven individual studies on cirDNA and circadian rhythm were identified, containing seven healthy cohorts (n = 73) and two cancer patient cohorts . Overall, there is mixed evidence regarding circadian rhythm influence on cirDNA. No differences in cirDNA as a function of time were observed in a group of 11 healthy subjects measured at 7 AM, 12 PM and 5 PM  or in 4 Two studies implemented cirDNA tailored pre-analytical practice , as well as limiting physical activity of study participants to minimise changes due to exercise. As described above, no circadian pattern was observed in four healthy subjects measured five times between 8:30 AM and 7:30 PM, with the experiment repeated on two separate days . HoweverTwo studies measured cirDNA fluctuations over short time intervals, specifically over 1 hr  and everA relationship between cirDNA levels and circadian rhythm can be expected as multiple studies have shown cirDNA is primarily haematopoietic in origin  and haemThe circadian rhythm studies do not report the time interval between waking and blood collection from subjects. Given the rapid clearance of cirDNA following exercise-induced increase, it is possible that cirDNA does change during sleep but returns to normal by the time a blood sample is taken for measurement. Furthermore, only two studies report what activity the participants were involved in between blood draws. In those studies, participants were told to minimise physical activity before blood draws  or givenPsychological stress burden has been linked to cirDNA changes through inflammation mechanisms  and DNA Within a longer timeframe, Twenty-five individual studies on cirDNA and biological sex were identified. These included 12 cohorts of healthy subjects and 24 cohorts of patients with diagnosed disease . In the In patient cohorts and workers exposed to occupational hazards, most studies (21/24) report no differences in cirDNA between males and females. A single study of 72 greenhouse workers reported higher cirDNA level in males , but theConsidering only studies in healthy volunteers that either measured cirDNA directly from plasma or used a dedicated extraction kit, higher cirDNA levels in males relative to females were measured in three cohorts  , while nTwenty-nine individual studies on cirDNA and age were identified. These contained 45 cohorts, with 19 groups of healthy individuals and 26 of patients with diagnosed disease and women undergoing HRT . AmongstConsidering only studies with healthy cohorts and cirDNA-tailored methodology, two studies with a total of 197 subjects observed a relationship between cirDNA levels and age , while fIn a comprehensive study of the tissue sources of cirDNA conducted by In the patient cohorts, 23/26 observed no age effect, while in three studies comprising 100 thyroid-related pathology patients , 30 gastComparing the effect of age across different studies is difficult for several reasons. Firstly, the way the subjects are categorised, and therefore analysed, varies between the studies. Some studies use specific age cutoffs (younger than or older than a certain age) to split their cohorts into two or three continuous groups. For example, Of all variables reviewed, exercise holds the most evidence to support its effect on cirDNA levels. A large and robust increase in cirDNA levels is associated with acute exercise, while less consistent changes are reported with chronic exercise  of the endurance exercises reported cirDNA increases ≥9-fold, and the maximum reported increase was 18.6-fold . In contThere is some evidence for a positive relationship between duration of exercise and fold change in cirDNA. Mavropalias and colleagues investigated the effect of exercise intensity by splitting their cohort of 20 men into low- or high-intensity eccentric cycling groups. High-intensity cycling induced a significantly higher cirDNA increase in plasma than low-intensity cycling . FurtherThe kinetics of cirDNA during exercise indicate rapid initiation of clearance, with cirDNA peaking either immediately , 15 min Grouping participant results in kinetic studies might not be appropriate due to interindividual differences in cirDNA clearance. Since 55% of cirDNA comes from leukocytes , it is pIndividual fitness levels appear to have little impact on cirDNA increase and clearance rates. It is difficult to conclude if there are any differences in the way cirDNA levels respond to exercise in men compared to women. Half of the research studies conducted (11/21) involved male subjects only. Eight studies had a mix of males and females with equal or near equal representation, while two consisted predominantly of male subjects . The lacSix individual studies on cirDNA and chronic exercise were identified, far fewer studies compared to acute exercise. Overall, it appears to have less robust effects than acute exercise. The chronic exercise studies ranged in duration from 8 days  to 6 monNo change in cirDNA levels was observed following a chronic intense running programme in male endurance runners , resistaInconsistent effects of chronic exercise intensity are also reported. The increase in cirDNA during exercise has been attributed to apoptosis due to the ladder pattern of DNA fragments . This paSex-mismatched haematopoietic stem cell transplantation provides a means to determine whether the exercise-induced cirDNA increase originates from haematopoietic lineage cells. In one study, male and female patients with sex-mismatched transplant donors performed incremental treadmill exercise until exhaustion and their total and Y-chromosome cirDNA levels were measured. The results showed that most of the increase in cirDNA is from haematopoietic cells and not from the liver . FurtherAnother clear candidate for a cirDNA source during exercise is skeletal muscle; however, only one study to date has specifically measured muscle-derived cirDNA in this setting. Surprisingly, no increase in skeletal muscle-derived cirDNA, as identified via the methylated HOXD4 promoter, was observed after exercise. The authors only analysed 5 out of 20 men recruited in this tissue-origin investigation . Muscle There is a strong association between alcohol consumption and increased risk of cancers such as liver, breast, oesophageal and pancreatic cancer . Due to Two studies measured association between alcohol consumption and plasma cirDNA levels  and founIn both studies, alcohol consumption was recorded as a part of investigating plasma cirDNA in the context of cancer. A more focused study that utilises protocols tailored to cirDNA measurements to confirm the effect of alcohol on cirDNA is needed. Both acute and chronic effects of alcohol should be investigated with clear information on alcohol intake amounts and duration. Recall bias is also a major issue in self-count-based studies which leads to under-reporting particularly for heavy and non-routine drinking .Two studies on cirDNA and occupational hazard exposure were identified. One study examined ionising radiation in 176 people working at nuclear sites  and the Cigarette smoke has potent DNA-damaging constituents ; therefoTen individual studies on cirDNA and smoking were identified, containing 6 healthy cohorts and 13 cohorts that include cancer patients, women undergoing IVF treatment and greenhouse workers. Four out of six studies that included healthy cohorts reported no effect of smoking on cirDNA levels in a combined sample size of 444 . The remAmongst the cancer studies, four lung cancer , one oesThere were no cirDNA differences between former and current smokers versus non-smokers in a group of 37 women undergoing IVF-embryo transfer treatment  or in T2One limitation across the published studies is the imbalance of non-smoking and smoking cohorts. For example, in the study conducted by Many other variables have been investigated in smaller individual studies. No effect on cirDNA has been noted in studies examining height , spring There is evidence that meal-derived DNA is present in the circulation. This DNA is mostly intact (>10 kbp) . HoweverWhile there is general agreement that standardised methodology and complete reporting of experimental parameters is desirable, consensus for a standardised set of protocols for cirDNA analysis has not been developed. There are numerous steps in cirDNA analysis, starting from blood collection all the way through to quantification, and variation at each step creates difficulty in comparing studies and a hindrance in translating biomarkers to the clinic. Here, we highlight some of the differences in the technical aspects of the 66 publications reviewed .A range of tubes for blood collection and plasma separation are available, including tubes stabilised to avoid leukocyte lysis. Most studies used EDTA tubes; however, 3 studies used heparin-based additives , 15 studIt is important that the time between blood collection and plasma processing is considered and reported as prolonged blood storage allows leukocytes to lyse and hence create genomic DNA contamination . DiffereA key step in blood processing for cirDNA extraction is centrifugation to separate the plasma from the red blood cells and leukocytes. Thirty-eight out of 66 studies (58%) centrifuged the blood samples twice, 16 studies (24%) did so once and 12 studies (18%) did not specify . The rolSixty-four studies used a single extraction method while three utilised multiple methods to analyse the same cohort, including quantifying the cirDNA directly from plasma. In total, there are 11 studies that omitted the extraction step and quantitated cirDNA directly .Most studies used commercial kits to purify cirDNA. There are several kits available to specifically extract cirDNA such as QIAamp Circulating Nucleic Acid (CNA) Kit, NucleoSpin Plasma XS Kit, MagMAX Cell-free DNA Isolation Kit and Circulating DNA Purification Mini Kit. Only 37% of the 46 studies that involved a DNA extraction step used a kit specifically formulated for cirDNA. Using non-specialised kits results in substantially reduced cirDNA yields . For exaOut of these studies that did not use a dedicated kit, nearly half used QIAGEN kits (mostly QIAamp DNA Blood Mini Kit) and the remaining used a variety of other kits such as MagNA Pure LC DNA Isolation Kit-Large Volume. Some studies used various non-kit methods such as phenol-chloroform-based extraction  and in-hBoth more  and lessThe quantitation methods were predominantly qPCR, which remains the ‘gold standard’ for cirDNA quantitation (just over 50% of studies), followed by Qubit. Other means of quantitation were PicoGreen, cell death detection ELISA, SYBR nucleic acid gel stain, DNA DipStick TM Kit and GB genetic human DNA assay.It is apparent in this analysis of 66 studies that there remain major inconsistences in the methods used to extract and analyse cirDNA. This not only raises questions about the validity of individual results but makes comparing studies and evaluating results difficult.The effects of lifestyle and biological factors on cirDNA are summarised in Alcohol use and menstruation appear to have a negligible effect on cirDNA levels; however, the number of studies is small. Most other biological and lifestyle factors have been reported to both increase and decrease cirDNA levels. Better-designed and disaggregated studies are required to confidently rule in or out their effect.Addressing each of these factors to accurately answer how they impact cirDNA level would require a very large study with a substantial number of participants and multidiscipline expertise to ensure that each factor is investigated and controlled properly. If this type of study was to be conducted, the best practice should be applied. Blood should be collected in a single-tube type, with short and consistent time between blood collection and centrifugation. Double centrifugation and specialised cirDNA kit for extraction should also be used.A step towards obtaining more reliable data is choosing methods appropriate for cirDNA analysis, as well as careful sample handling and detailed reporting of protocols. Transparency in disclosing the way samples are processed and analysed may be aided by the development of reporting guidelines analogous to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines .There remain large knowledge gaps in how common biological and lifestyle factors impact cirDNA. An understanding of cirDNA dynamics outside of a disease context is important for establishing accurate baseline levels and developing sensitive clinical tests that minimise the confounding effect of healthy variation.


Epithelial ovarian cancer is predominantly diagnosed at advanced stages which creates significant therapeutic challenges. As a result, the 5-year survival rate is low. Within ovarian cancer, significant tumor heterogeneity exists, and the tumor microenvironment is diverse. Tumor heterogeneity leads to diversity in therapy response within the tumor, which can lead to resistance or recurrence. Advancements in therapy development and tumor profiling have initiated a shift from a “one-size-fits-all” approach towards precision patient-based therapies. Here, we review aspects of ovarian tumor heterogeneity that facilitate tumorigenesis and contribute to treatment failure. These tumor characteristics should be considered when designing novel therapies or characterizing mechanisms of treatment resistance. Individual patients vary considerably in terms of age, fertility and contraceptive use which innately affects the endocrine milieu in the ovary. Similarly, individual tumors differ significantly in their immune profile, which can impact the efficacy of immunotherapies. Tumor size, presence of malignant ascites and vascular density further alters the tumor microenvironment, creating areas of significant hypoxia that is notorious for increasing tumorigenesis, resistance to standard of care therapies and promoting stemness and metastases. We further expand on strategies aimed at improving oxygenation status in tumors to dampen downstream effects of hypoxia and set the stage for better response to therapy. Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer, and it is the fifth leading cause of cancer related deaths in women . A lack Ovarian cancer most commonly presents in post-menopausal women at which point there are other age-related physiologic changes. Contributing risk factors include a familial history of EOC, increased lifetime ovulatory events due to nulliparity, undergoing hormone replacement therapy (HRT) and comorbidity factors such as diabetes and obesity , 6. ManyCurrently, the common treatment protocol for ovarian cancer includes cytoreductive surgical debulking accompanied with chemotherapy, typically carboplatin and paclitaxel . AlthougThe ovary is surrounded by a single layer of epithelial cells called the ovarian surface epithelium (OSE). The activity of the OSE is hormone-dependent; the ovary is a primary endocrine organ where both peptide and steroid hormones act on the OSE cells to mediate their activity throughout various reproductive processes. These hormones have an influential role on proliferation and differentiation of OSE cells and aberrant endocrine signaling can inflate the risk of EOC and contribute to its tumorigenesis , 12.Gonadotropin-releasing hormone (GnRH) is a peptide hormone secreted by the hypothalamus. Its primary function is to regulate the production and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary . These hThe involvement of steroid hormones in ovarian cancer carcinogenesis is supported by both experimental and epidemiological findings , 22. ProWith the growing body of epidemiological and experimental evidence, an association between the endocrine status of the patient and ovarian cancer tumorigenesis is well-supported as summarized in Angiogenesis is a naturally occurring process to develop new blood vessels from pre-existing vasculature. Angiogenesis is involved in a number of homeostatic processes including vascular repair in wound healing . In resp3  appear to favour hypoxic sites within tumors . CSCs arA unique feature of peritoneal tumors, including ovarian cancer, is the presence of ascites fluid. This complex mixture of soluble factors accumulates due to leaky tumor vasculature as well as disrupted lymphatic patency . Given tThe tumor immune microenvironment (TIME), which encompasses not only malignant transformed cells, but also normal cells such as epithelial cells, fibroblasts, endothelial cells, muscle cells and immune cells in EOC has been described as ‘highly permissive’ to tumor growth, metastasis and therapy resistance –112. WhiLow oxygen partial pressure within the tumor activates hypoxia-dependent signaling, where HIFs are stabilized. HIF-1 maintains elevated myeloid-derived suppressor cell levels and regulates their function and maturation . MyeloidThe fundamental basis of successful immunotherapy to treat cancer is positive immunogenicity of the specific tumor subtype, which considers presence of tumor associated antigens and effective presentation of these antigens. The immunosuppressive TIME as well as low mutation rate, which impairs neo-antigen formation, challenges the use of immune-based therapies in ovarian cancer –132. FinHypoxia has been associated with the development of chemoresistance through a variety of mechanisms. One of the mechanisms by which hypoxia induces chemoresistance is through alteration of cancer cell metabolism. In response to hypoxia, ovarian cancer cells undergo a metabolic switch, with changes in the glycolytic pathway that promotes resistance to carboplatin –144. As in vitro . MiRNAs are small, approximately 19-25 nucleotides endogenous non-coding RNAs that regulate gene expression  in humanin vitro , 164. Inin vitro , suggestin vitro . Hypoxiain vitro . MiRNA eHIF-1 expression alone is not an indicative prognostic marker onRESA the disease progression in ovarian cancer, however, overexpression of HIF-1 in combination with the presence of non-functional p53 is associated with a more aggressive phenotype and a poorer prognosis . A gain-Hypoxia-activated prodrugs were designed to specifically target the hypoxic tumor cells within a solid tumor. Their mechanism of action work by their enzymatic reduction to reactive oxygen species under hypoxic conditions, with capacity to re-oxidize in oxygenated environments. In pre-clinical and clinical settings, hypoxia activated prodrugs have shown promising efficacy in successfully attacking hypoxic cells of solid tumors –188. ThiAnti-angiogenic therapies can normalize tumor vasculature, uniformly increase oxygen delivery and reverse the hypoxic areas of the tumor. This allows for better uptake and distribution of therapeutic drugs to the tumor . Many anEstrogen receptors are specifically located on smooth muscle pericytes within blood vessels and estrogen is known to inhibit proliferation of vascular smooth muscle cells , 218. AdReversing hypoxia within the TME will not directly contribute to tumor cell death, however, we can utilize this as an advantage to create more personalized combinational therapeutic approaches. The presence of hypoxia and poor tumor vasculature is characteristic to many solid tumors including EOC . In lighHypoxia-induced changes in the endocrine environment and immune status of the tumor play an influential role in promoting tumorigenesis in ovarian cancer. By understanding the changes that occur in the ovarian TME, we can develop novel therapies that target these changes to improve efficacy and reduce therapy resistance. An approach to personalized treatment strategies on a patient-by-patient basis may ultimately improve the way we treat women with ovarian cancer.The article was conceptualized by JP. MP, KM, CJ, and JP contributed to the writing and editing of the review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Narcissism is a Janusian personality construct, associated with both grandiose self-assuredness and dominance, as well as vulnerable insecurity and reactivity. Central questions of intra- and interpersonal functioning in narcissism are still a matter of debate. Neuroscience could help to understand the paradoxical patterns of experience and behavior beyond the limitations of self-reports. We provide a systematic review of 34 neuroscience studies on grandiose, vulnerable, pathological narcissism, and Narcissistic Personality Disorder (NPD), spanning experimental investigations of intra- and interpersonal mechanisms, research on neurophysiological and neuroendocrine aspects of baseline function, and brain structural correlates. While neuroscience has scarcely directly studied vulnerable narcissism, grandiose narcissism is associated with heightened vigilance to ego threat and stress responses following ego threat, as well as heightened stress indicators in baseline measures. Such responses are not commonly observed in self-reports, highlighting the potential of neuroscience to augment our understanding of self-regulatory dynamics in narcissism. Interpersonal functioning is characterized by deficits in social–affective processes. Both involve altered activity within the salience network, pointing to a double dissociation regarding the expression of narcissism and self/other oriented situational focus. Findings are summarized in an integrative model providing testable hypotheses for future research along with methodological recommendations. In this myth, the beautiful young hunter Narcissus rejected the love of the nymph Echo, which is why he was deemed by the gods to fall in love with his own mirror image. His self-centeredness ultimately led Narcissus to the fate of a tragic death. Thus, on a metaphorical level, this early myth already reflects two aspects of narcissism that are currently being extensively studied, namely grandiose and vulnerable narcissism. The ancient concept of narcissism was picked up and refined by psychodynamic theorists, who regarded narcissism as both a self-regulatory mechanism and a personality disposition Freud, , and wasIt was not until the 2000s, however, that research on narcissism attracted a broader scientific and public interest, which is presumably due to cultural changes in the new millennium. Popular writings such as Jean Twenge’s  “GeneratThere is now an emerging consensus that narcissism is multifaceted in nature and different expressions of the phenomenon can be discerned  in the third edition of the Diagnostic and Statistical Manual of Mental Disorders , exploitativeness , grandiose fantasy (imagining grandiose self), and self-sacrificing self-enhancement , as well as facets related more to narcissistic vulnerability, which are contingent self-esteem (dependence upon others’ regard), hiding the self (not showing own needs and weaknesses), and devaluing . The grandiose and vulnerable factors are intrinsically related in the PNI  narcissism mostly focused on adaptive qualities, and maladaptive as well as vulnerable aspects might have been underrepresented  intrapersonal, self-related characteristics of narcissism and (2) interpersonal, other related characteristics of narcissism.(1) It has long been hypothesized that narcissism, particularly grandiose narcissism and NPD as its clinical expression, entail vulnerable aspects, which need not necessarily be overtly expressed  circumscribes the capacity to cognitively represent others’ mental states  Another controversial aspect of narcissism concerns the exact nature of altered interpersonal functioning observed in different expressions of narcissism. Interpersonal functioning includes – alongside more complex constructs such as attachment  or interpersonal (other related paradigms) in focus. This classification is in line with social/personality models of narcissism , or status threat induce stronger stress responses  or aggressive behavior (administering noise blasts) in individuals high in grandiose narcissism. One study, however, did not find such an effect using RSA , turning then to more low-level paradigms , and finally to social–psychological accounts .Fan and colleagues  conducteTurning now to more low-level emotion processing paradigms, Marcoux and colleagues  used a sTurning now to the social–psychological line of research on narcissism, Lobbestael and colleagues  investig2.4We identified 10 studies that investigated different biophysiological measures of baseline functioning in narcissism: six studies used measures of neuroendocrine markers including cortisol, other neuroendocrine markers targeting habitual stress levels, or testosterone, three investigated resting-state fMRI . The effects were stronger for the entitlement/exploitativeness factor than for other factors of the NPI, and were also evident in females when considering only this factor. This shows that particularly those aspects of grandiose narcissism that are most maladaptive  of narcissism and salivary testosterone in a large sample of adult men, but not of its neighboring personality dimensions psychopathy or Machiavellianism.Studies targeting neuroendocrine markers of habitual stress mostly speak to elevated stress in relation to narcissism, partially moderated by other variables: Reinhard and colleagues  carried Two more studies investigated cortisol and further neuroendocrine markers in narcissism: Cheng and colleagues  studied Three studies investigated resting-state functional brain connectivity by means of fMRI. The first of these , with the sole exception of Yang and colleagues  and neuroendocrine (cortisol) indicators. Stressful responses to stimuli occur only for self-relevant material; reactions to aversive stimuli that are self-related but not self-relevant can be “cool”.Grandiose narcissism and NPD are related to altered baseline functioning. This manifests in higher cortisol or 8-OH-DG levels, and distinguishable resting-state connectivity patterns among regions that are also implicated in experimental research  as well as the mPFC (default mode network) and the amygdala (limbic system).Grandiose narcissism, pathological narcissism, and NPD are related to brain structural changes, which almost exclusively manifest in negative associations. The most consistent finding is lower cortical volume/gray matter volume in the mPFC for pathological narcissism/NPD. White matter in the frontostriatal pathway, connecting the ventral striatum and the mPFC, displays lower integrity related to narcissistic grandiosity. Prefrontal white matter tracts are also weakened in NPD.When considering all of the available evidence of functional, baseline, and structural studies together, the following main points can be summarized from the available evidence:3.2The study of interpersonal functions in narcissism mostly targets the long-lasting notion of empathy deficits in narcissism, as included in the diagnostic criteria for NPD since its first mention in the DSM-III  and the default mode network (mPFC). Activity within the salience network points to a double dissociation of narcissism and self- versus other related processes.self-relatedness and self-relevance, putting forward the hypothesis that increased salience network up-regulation or stress responses are only observed when both conditions are met. Potentially aversive stimuli or situations that are self-related, but not self-relevant might lead to unaffected responses, or as Coleman and colleagues , which allows for a comprehensive assessment of different aspects of narcissism grounded in the Five-Factor Model dimensions  on well-established ego threat paradigms, such as, for instance, the TSST or the Cyberball paradigm, and well-established social affect and cognition paradigms. Quasi-experimental studies using measures of brain structure or baseline functions could contribute most effectively to acquiring a robust base of knowledge by using data analysis methods corresponding exactly to those of previous studies  in large datasets. While such studies have recently been carried out for the Five-Factor Model dimensions and yield a rather pessimistic picture regarding the consistency of personality neuroscience findings (Avinun et al., Finally, it needs to be stated that many neuroscience studies of narcissism used small or very small samples to investigate genuinely subtle associations between personality and physiological or biological indicators, which sometimes include complex relationships such as moderation. This may naturally raise doubt about the robustness and replicability of the reported findings, and we think that such doubt is vital for state-of-the-art planning of future research. However, despite the use of small samples – which is a common problem particularly in earlier neuroscience work – the relatively high degree of consistency across different studies still allows to deduce testable hypotheses, which can inform future confirmatory research using state-of-the-art designs. The model displayed in Figure Though existing neuroscience research could already contribute significantly to the understanding of narcissism, there are several ways in which future studies could corroborate and extend the present findings. Here, we provide suggestions on how this might be accomplished:


Vision and hearing disorders comprise the most common sensory disorders found in people. Many forms of vision and hearing loss are inherited and current treatments only provide patients with temporary or partial relief. As a result, developing genetic therapies for any of the several hundred known causative genes underlying inherited retinal and cochlear disorders has been of great interest. Recent exciting advances in gene therapy have shown promise for the clinical treatment of inherited retinal diseases, and while clinical gene therapies for cochlear disease are not yet available, research in the last several years has resulted in significant advancement in preclinical development for gene delivery to the cochlea. Furthermore, the development of somatic targeted genome editing using CRISPR/Cas9 has brought new possibilities for the treatment of dominant or gain-of-function disease. Here we discuss the current state of gene therapy for inherited diseases of the retina and cochlea with an eye toward areas that still need additional development. Many forms of vision and hearing loss are inherited and current treatments only provide patients with temporary or incomplete relief. Because many inherited sensory defects have identified disease genes, developing effective genetic therapies has been a primary research goal for several decades. Recent exciting advances in gene therapy have shown promise for the treatment of inherited retinal and cochlear diseases, both in the area of gene replacement and in the area of targeted genome editing  to correct underlying genetic defects. However, there are still substantial limitations hampering widespread clinical use of genetic therapies in the retina and cochlea, including issues of distribution and uptake of the vectors by the target cells, a limited window of opportunity for treatment in degenerative disorders, attenuation of gene expression after a period of time, and efficiency/off-target concerns with novel genome editing strategies.Over 2.2 billion people worldwide experience vision impairment or irreversible vision loss3 , that can target various cells within the retina. Virtually all inheritance patterns are represented in IRD genes including autosomal dominant, autosomal recessive, X-linked, digenic, polygenic, and even mitochondrial disorders. IRD genes expressed in photoreceptors can lead to various macular degenerations/dystrophies and retinitis pigmentosa (RP) and are involved in ocular development, phototransduction, cellular metabolism, and in the development and maintenance of photoreceptor structure. Mutations in retinal pigment epithelium (RPE) genes include those involved in retinoid recycling, cellular metabolism, and cellular homeostasis and can lead to macular dystrophies as well as Leber’s Congenital Amaurosis (LCA). Mutations in retinal ganglion cell IRD genes are largely associated with mitochondrial function, but also include regulators of intracellular transport and transcription, and can lead to optic atrophy. A summary of known IRD genes can be found at: Depending on the gene, the onset of disease varies from birth to adulthood. Photoreceptor dystrophies are the most common within the retina and can be classified by the primary affected cell type and whether central or peripheral vision is lost first. RP and rod-cone dystrophies typically affect rods first, while macular dystrophies/degenerations and cone-rod dystrophies often affect cones first . HoweverCurrent treatment options to correct the more mild symptoms of vision loss include corrective lenses or contacts and more permanent laser correction surgery. In more severe cases where progressive vision loss occurs due to retinal degeneration, a more invasive approach is necessary. These advanced therapeutic options are all still in development or in early clinical use and include gene therapy, gene editing, reprogramming, cell therapy, optogenetic therapies, and retinal prostheses. The time at which these therapies are useful varies greatly. For maximum efficacy, gene therapy needs to be delivered in the early stage of disease to prevent further degeneration. In contrast, other options have a wider therapeutic window, but have had less success thus far, both in development and in clinical use .The inner ear is a small, fluid-filled compartment  encased 4 and , defined as the presence of a deafness-causing etiology in the cochlea or auditory nerve, accounts for more than 90% of hearing loss in adult patients. Approximately 50–60% of all cases of congenital deafness are directly linked to genetic factors, with higher numbers in developed countries . Current4 and  with man4 and . As in tCurrent treatment options focus on amplifying sound through hearing aids or electrical stimulation of auditory neurons via cochlear implants for individuals with severe deafness. Cochlear implants are currently the most successful sensory prosthetics available, allowing deaf individuals to understand speech . HoweverIn addition to inherited disease genes affecting a single tissue , there are a large variety of disease genes that are syndromic, affecting multiple tissues. Structural parallels in photoreceptors and cochlear hair cells mean that there are also a large number of disease genes that cause dual syndromic deafness and blindness. These genes often induce photoreceptor degeneration rather than optic atrophy and are frequently classed under the larger umbrella of ciliopathies. The most common form of dual deafness and irreversible vision loss involves the many genes associated with Usher syndrome , 2019. UMECR and ACO2 which are also associated with cerebellar defects and ataxias  without concern about dilution of non-integrated vectors by cell division. Similarly, non-integrated vectors are not lost over time due to cell proliferation. In contrast, post-mitotic cells can be harder to transfect and target than dividing ones, making it more difficult to achieve cellular uptake of therapeutic vector. In addition, the non-proliferating nature of retinal and cochlear sensory cells means that in general once lost, cells are gone forever. Though research effort has been focused on combatting this by the use of stem cells or by regenerating cochlear hair cells, in general the degenerative nature of disease when post-mitotic cells are affected means the therapeutic window is narrow, as treatments delivered after too many cells have died will not be effective.From a therapeutic development standpoint, the bilateral nature of the retina and cochlea is also beneficial. Treating only one eye or cochlea allows the contralateral eye/cochlea to serve as an internal control for analysis, since the treatment rarely escapes the treated eye/cochlea. However, leakage from one ear to the other after scala tympani injections has been reported , so restIn vivo imaging in the retina includes spectral-domain optical coherence tomography (OCT) and fundus imaging/angiography to assess distribution of tagged genetic vectors and retinal structure. Non-invasive functional testing in the retina include electroretinography (ERG) to measure retinal neuronal responses, as well as optokinetic tracking (OKT) to measure visual acuity  and distortion product otoacoustic emissions (DPOAE) to evaluate inner ear function. Imaging modalities can be used in parallel, including computed tomography (CT) and magnetic resonance imaging (MRI) to evaluate cochlear structure  optimizing vector content, including promoter/enhance choice, gene sequence, inclusion of intronic and untranslated region (UTR) sequences, etc.; (2) evaluating methods to prevent gene silencing, such as elimination of CpG islands, the inclusion of insulators, etc.; (3) testing methods to promote or prevent genomic integration, such as the inclusion of transposases or S/MARs; (4) evaluating vector packaging, including the use or development of various viral compaction methods, engineering novel viral serotypes to enhance tissue tropism, engineering novel non-viral vectors; (5) assessing delivery methods; (6) assessing cellular uptake and targeting of vectors, including vector/particle optimizations to enhance cellular uptake; and (7) evaluating gene expression levels, knockdown efficiency, or duration of expression. The length of this list highlights how much development and testing often occurs before genetic therapies can even be evaluated for their ability to elicit functional improvement in the target disease.Animal studies are necessary to fully understand the effects of gene therapy in a true organ microenvironment and to investigate the toxicities involved in long-term studies. The majority of preclinical retinal and cochlear studies have used rodents as preferred animal models. Mice have well characterized genomes, with genetic, biological, and behavioral characteristics similar to humans, and their genomes can be easily manipulated which is a boon for studies of inherited diseases. Guinea pigs have also been used for evaluation of auditory dysfunction as they are susceptible to noise induced hearing loss, making them a commonly used model for optimizing gene delivery approaches and to assess the efficacy of gene-based treatments not targeted to a specific disease gene  . Final pThere is an extensive body of literature using mouse models for retinal gene therapy  and for the study of IRD disease mechanisms e.g., . HoweverCNGB3-associated achromatopsia has been generated in monkeys using AAV9-mediated delivery of a CRISPR/Cas9-based construct , retinal ganglion cells , Müller cells , bipolar cells, (Cln3), and the RPE,   . Notable5, Lrat) .cap gene variants, harvesting the retina, performing PCR to enrich cap gene variants present  and reformulating virus. This selection process is repeated several times with additional steps included in subsequent rounds to increase diversity in cap genes. The exciting end result was the identification of novel AAV variants capable of efficiently transducing non-human primate cones after intravitreal injection, a key therapeutic goal that had not been achieved previously , and shown to promote long-term protection of retinal structure and function , Guca1a (cone-rod dystrophy), and Pde6b (RP) with varying levels of success  homologous region is supplied, and the whole expression cassette  fit into a single AAV. A study using this approach to target a Gnat1 model reported improvement in light-sensitivity and visual acuity (Opn1mw) with an Opn1mw-specific gRNA where it activates gene transcription , Cep290-associated LCA (further discussed below), and Gucy2d-associated cone-rod dystrophy , promotor found no significant side-effects after 12 months but reported no significant improvement in vision  in patients with vision loss associated with RPE65. Bilateral subretinal injections of the highest dose tested in the prior trial found improved vision with no adverse effects or immune response after 1 year . The trial, which targets CEP290-associated LCA is not only a milestone in retinal gene therapy, but also represents the first time CRISPR-based treatments will be delivered directly into the body in any trial, and results are eagerly anticipated. This study uses an AAV5 vector to deliver Cas9 and CEP290 specific gRNAs and was effective in both a humanized CEP290 mouse model and in a non-human primate model  after delivery at P10-12 (Ush1c) leading to functional improvements in low-frequency hearing  mice at P2.5 and found to preserve hair cell function near wild-type levels  than later delivery (P10) (Delivering AAV to older/adult mice (compared to neonates) results in a drop in efficiency and duration of expression. For example, AAV-mediated delivery of ry (P10) , althougry (P10) . Howeverry (P10) . AlthougAs in the retina, there has been great interest in designing optimized AAVs for cochlear delivery. This includes several new synthetic AAVs such as Anc80L65, which has shown very high efficiency in transducing both inner and outer hair cells after injections at 7 weeks of age through the posterior semicircular canal . As in tA number of non-viral gene therapy strategies have also been tested in the cochlea. One of the earliest was a study in which cationic liposomes were microinjected into the guinea pig cochlea, and short-term reporter gene expression was observed in the organ of Corti . Lipid cin vivo cochlear genome editing . The study involved intralabyrinthine delivery of recombinant adenovirus carrying the Hath1/Atoh1 transcription factor . This trWhile progress is being made for both retinal and cochlear gene therapy, it is necessary to remember one of the key limiting factors of gene therapy for inherited dystrophies: time. Due to the nature of degenerative hereditary diseases, unless intervention is applied early, the optimal therapeutic window will be missed. In many cases patients may not even know they have a genetic disorder until symptoms are severe enough to see their physician, at which point, the progression of the disease is often far enough along that affected cells are dead or dying. This is particularly challenging in the case of degenerations that affect neuronal cells, since there is, as yet, no effective way to regenerate or replace lost neurons . To a certain degree, this is an area where cochlear gene therapy is ahead of retinal gene therapy. There have been extensive preclinical studies focused on regeneration of hair cells in deafened mice (largely in non-genetic models) by delivery of the transcription factor Math1  , and sevIn addition to delivery at the appropriate time in the course of the disease pathology, an additional concern is the potential need for repeat dosing. The ideal gene therapy would be effective long-term after delivery of a single dose. However, generating and maintaining sufficient levels of gene expression remains a challenge. Improving duration of expression at levels high enough to maintain functional rescue is essential .In the case of the retina, the current measures of therapeutic longevity come from the early LCA trials, where results have been variable. Some groups reported functional visual improvement up to 3 years despite cell degeneration still taking place , while oBoth the eye and cochlea are organs which have favorable anatomy for gene therapy with small sizes that can be easily treated with small doses of therapeutics, a contralateral control and easy non-invasive analytical techniques. They are also sites of many different forms of inherited hearing loss and irreversible vision loss caused by several hundred known genes, and thus they are prime targets for novel therapies. The monogenic nature of most inherited diseases of the eye and ear make gene therapy an attractive method to potentially correct causative genetic defects and cure disease, rather than just treating symptoms. There are various limitations and challenges still to face including (1) the limited number of patients carrying any given mutation, although allele-independent approaches are being developed, (2) limited carrying capacity of common viral-delivery methods, (3) the rapid progression of degeneration in some pathologies, (4) limited distribution and longevity of gene expression after single-dose treatments, (5) difficulty transfecting/transducing post-mitotic cells, and (6) the need for delivery methods that are less invasive. Yet all these areas are being actively studied, and outcomes with genetic therapies are likely to keep improving. Another area that has seen rapid progress in the last decade is genetic diagnosis. The accessibility and accuracy of genetic testing in the past few years has improved drastically . As geneThe commonalities between the eye and the ear, coupled with the numerous syndromic genetic disorders with both an auditory and visual component mean that coordination between the fields is likely to be beneficial. Collaboration and resource sharing between groups studying both the retina and the cochlea may help advance the development of clinically relevant therapies for both vision and hearing loss, and lead to significantly improved quality of life in patients.RC wrote initial draft and created the figures. SMC, MRA, and MIN were editors of final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neuroimaging research suggests that alcohol dependency is associated with impairments in anticipating monetary rewards, but not aversive or alcohol-related cues.We sought to investigate if reinforcement sensitivity is altered in young adults, who regularly consume harmful levels of alcohol, using a monetary incentive reinforcement (MIR) task. In light of previous research suggesting reduced motivation to obtain reward, we hypothesized that young alcohol users would show reduced motivation for monetary gain, but unimpaired loss avoidance behaviour.We recruited 46 volunteers from the local community in Cambridge (UK), half of whom reported consuming alcohol at harmful levels, as reflected by the Alcohol Use Disorder Test. Participants completed a number of personality questionnaires, including the Barratt Impulsivity Scale (BIS-11) and Sensation-Seeking-Scale (SSS-V) and performed the MIR task, which measures participants’ efforts in avoiding punishment and gaining rewards. Data were analysed using Statistical Package for Social Sciences (SPSS) version 25 . Analysis of co-variance (ANOVA) were used to explore group differences in demographics, personality traits and task performance; age and gender were included as co-variates.The groups were well-matched in terms of socioeconomic status and education levels. As the alcohol group was significantly younger than the control group and dominated by females, age and gender were statistically controlled for. Alcohol users reported significantly higher levels of impulsivity  and sensation-seeking traits  and demonstrated normal sensitivity to monetary value . However, when challenged to on the MIR task to gain reward or avoid punishment, alcohol users were as equally motivated as control volunteers to take action to avoid financial loss  but showed less motivation to work towards financial reward . Especially for small rewards, alcohol users exerted significantly less efforts, as reflected by a reduced accuracy rate  and a significant increase in late responses . The lack of motivation to work for reward was negatively associated with the severity of alcohol use, as reflected by the AUDIT score .We observed reduced motivation to obtain financial reward, but not avoid loss in a community sample of heavy drinkers. As the observed effect was directly related to alcohol use severity, it may suggest changes in reinforcement sensitivity occur at an early stage of chronic alcohol use. Future research may want to monitor reward motivation in alcohol users longitudinally to evaluate whether it would be a suitable target for early intervention.


Nuclear fusion potentially offers a clean, environmentally friendly and intrinsically safe energy source with an abundant fuel supply. Magnetic fusion energy research is approaching a new era of fusion power reactor design and cons-truction planning. New physics understanding and powerful predictive tools have become available for improving fusion performance, developing and optimizing various magnetic confinement concepts. Emerging transformative enabling technologies can potentially mitigate and transform some present physics challenges from the possibly insurmountable to the potentially solvable.physics and technology may increase absolute fusion performance at smaller scales, thus offering exciting opportunities to accelerate progress towards an economically viable fusion power reactor.These recent innovations and advances in, as well as synergy between, From nearly seven decades of worldwide effort to develop fusion energy, a vast knowledge base has been established of science and technology for creating and controlling high-temperature plasmas, ranging from relatively low-density magnetic-field confinement to extremely high-density laser-driven inertial confinement. The highest-performance approach towards steady-state fusion is a doughnut-shaped magnetic confinement configuration known as the tokamak, as evidenced by the joint international effort to build a power-plant-scale experiment, ITER (Latin for ‘the way’) . ITER wiAnticipating the scientific and technological success of ITER, demonstration of the economic viability, ease of maintenance and safety of fusion systems will then be at the forefront of next-step fusion energy efforts leading to ultimate commercial use. As indicated by Fig. Advanced magnetic confinement, relying on the toroidal field magnet system to increase the plasma stability, including the tokamak, stellarator and reversed field pinch (RFP) that has a smaller but substantial toroidal field;Simplified magnetic topology, which is a singly connected magnetic configuration having no toroidal field coils, such as the field reversed configuration (FRC) and the spheromak compact toroids, as well as hybridization of magnetic and inertial fusion (MIF).The challenge of the first route is not least an engineering one—to reduce the cost for construction and operation, and to make the magnetic confinement system efficient, sustained and more compact. The major challenge today for the simpler magnetic configurations is more on the physics side, as they are much less mature and require significant improvements in their ability to confine high-temperature plasmas for the necessary durations relative to tokamaks or stellarators. Such a high-scientific-risk approach is currently largely driven by private enterprise, such as Tri Alpha Energy, Lockheed Martin and General Fusion, motivated by their highly desirable engineering features and potential for smaller and cheaper fusion power plants, if breakthroughs can be made in achieving fusion conditions.alternative fusion concepts have highly desirable engineering features for a compact, economic fusion reactor, but have not yet been proven viable and so require further exploration.On the physics front, the tokamak offers solutions in which good performance potentially aligns with configurations that are self-sustaining. Advances such as the recent discovery of the super-H mode of energy confinement in the DIII-D tokamak may enable still higher fusion performance , potentiOn the technology front, a number of promising transformative enabling capabilities provide enormous opportunities to accelerate fusion science and technology towards power production , includiIn summary, a confluence of physics and technology innovations in recent years show great promise for accelerating fusion energy development in the next few decades and lowering its cost. For the mainline tokamak approach, innovations gained in new physics understanding may enable access to and sustainment of enhanced confinement beyond ITER baseline scenarios, which, coupled with high magnetic confinement fields, potentially allows for high fusion power density at reduced size and cost. Exciting progress has also been made in the stellarator approach, achieving high temperatures up to ∼10 keV. The physics is less mature for the other alternative fusion concepts, and breakthroughs are needed to reach a similar technical readiness level to that for tokamaks, in particular for those with simplified magnetic topology, as exemplified by compact toroids. A number of emergent enabling technological capabilities, heavily funded by major non-fusion applications, may help solve long-standing challenges for the development of a fusion power plant, such as fusion materials, power handling and tritium production.


While normal-pressure hydrocephalus (NPH) is most commonly diagnosed in older adulthood, a significant body of literature has accumulated over half a century documenting the clinical phenomenon of an NPH-like syndrome in pediatric patients. As in adult NPH, it is likely that pediatric NPH occurs due to a heterogeneous array of developmental, structural, and neurodegenerative pathologies, ultimately resulting in aberrant cerebrospinal fluid (CSF) flow and distribution within and around the brain. In this review, we aimed to systematically survey the existing clinical evidence supporting the existence of a pediatric form of NPH, dating back to the original recognition of NPH as a clinically significant subtype of communicating hydrocephalus. Leveraging emergent trends from the old and more recent published literature, we then present a modern characterization of pediatric NPH as a disorder firmly within the same disease spectrum as adult NPH, likely with overlapping etiology and pathophysiological mechanisms. Exemplary cases consistent with the diagnosis of pediatric NPH selected from the senior author’s neurosurgical practice are then presented alongside the systematic review to aid in discussion of the typical clinical and radiographic manifestations of pediatric NPH. Common co-morbidities and modern surgical treatment options are also described. The existence of a syndrome in children and young adults with presentation and clinical findings similar to normal-pressure hydrocephalus (NPH) has been acknowledged in the pediatric population ,13,14,15Despite decades of recognition in the published literature, classification and diagnosis of NPH in the pediatric population remains controversial. The term “compensated ventriculomegaly” has been a misnomer applied in associated conditions which are linked to the development of chronic hydrocephalus which may or may not necessitate shunt treatment. Such conditions include prematurity, post-traumatic hydrocephalus, spina bifida, Chiari malformation, suboccipital pathologies , benign extra-axial fluid collections in infancy, craniosynostosis, Down Syndrome or other syndromic conditions, storage diseases, meningitis, vascular malformation  or other vascular complications ,13,15,22If one considers that the clinical hallmarks of NPH include the clinical triad  with symptomatic progression in the presence of chronic ventriculomegaly, or progressive cognitive and motor decline with or without urinary incontinence but in the absence of progressive ventriculomegaly, the literature collectively supports an NPH-equivalent diagnosis in the pediatric population. Most such situations where a similar clinical pattern is seen among children, often including failure to meet developmental milestones in lieu of marked deterioration of function per se, are associated with the above chronic conditions. Among both adults and children, presentation of cognitive dysfunction as well as treatment responsivity is widely variable, perhaps due to generally high prevalence of comorbidity with other brain pathology. Indeed, the possible importance of comorbid neurodegenerative pathology has recently emerged in the adult NPH literature ,24,25,26In this article, we aimed to systematically survey the literature evidence of pediatric NPH as a unique diagnostic entity which has emerged over the past five decades. Trends regarding etiology and diagnosis are qualitatively analyzed and discussed. Literature findings are coupled with a series of representative case presentations from the senior author’s clinical practice to support the concept of pediatric NPH applied in a modern cohort. Special consideration is paid to etiological circumstances or comorbidities of NPH in the pediatric population, and implications for clinical diagnosis and surgical management are highlighted.We conducted a systematic search of the PubMed database  to identify pertinent literature on the topic of pediatric NPH. Search terms included variants of “pediatric,” “child,” “infant,” or “newborn” in combination with “normal pressure hydrocephalus” or “idiopathic hydrocephalus,” contained within the title and/or abstract of the article. The citation lists of relevant articles identified by these criteria were additionally reviewed for studies which may have been excluded based on search criteria alone. This search methodology yielded a total of 75 PubMed-indexed articles for further examination. Abstracts and available full texts of identified articles were then individually reviewed for possible inclusion in this systematic review according to predefined criteria . ArticleOur single-institutional case series spans eleven years of the senior author’s neurosurgical practice in pediatric hydrocephalus, spanning from 2009 to 2020. Electronic medical records were reviewed for cases consistent with the diagnosis of pediatric NPH, as described in the summarized literature. Nine cases were identified as illustrative examples of pediatric NPH, and key details including suspected or confirmed etiology, clinical presentation, radiographic findings, and treatment outcomes were tabulated. Six cases were selected for narrative case presentation vignettes (‘Case Presentations’).Review of the literature revealed 15 clinical studies describing unique cases of pediatric NPH, comprising a total of 146 patients ranging in age from infancy to teenage years. Included articles, along with key methodological details and results, are tabulated in Etiologically, pediatric NPH has been identified by the existing literature as emerging from a diverse range of degenerative, congenital, traumatic, and genetic conditions . Common The literature suggests that the presentation of children with NPH is similar to that of adults with NPH, frequently demonstrating at least two of the three of the classic triad of symptoms: progressive cognitive impairment, motor/gait disturbance, and urinary sphincter dysfunction. In children, progressive cognitive decline associated with NPH is frequently seen as failure to meet developmental milestones, sometimes such that overall progression lags behind verbal development if detailed neuropsychological testing is performed ,10,14,15Nine illustrative cases from the senior author’s practice demonstrating the etiologic diversity and typical clinical presentation of NPH are tabulated in A 6-year-old female patient with a known history of Chiari I malformation (CM1) with inferior displacement of the cerebellar tonsils presented with recent, progressive deterioration of school performance, increased distraction and irritability, and frequent falling, as well as recent worsening of occipital headaches attributed to CM1. The patient had also experienced recent occasional episodes of incontinence. MRI demonstrated ventriculomegaly . On examA 17-month-old male patient with cerebral palsy presented with a life-long history of developmental delay secondary to chronic lung disease and tracheobronchomalacia after premature birth at 24 weeks gestation. During a six-month stay in the neonatal ICU, the patient struggled with respiratory failure requiring months of respiratory support and was believed to have suffered anoxic brain injury early in life. The patient also had a history of episodic breath holding with cyanosis. The patient did not have any associated intraventricular hemorrhage based on serial CT evaluation. However, by 18 months, there was concern for incongruent motor and cognitive developmental delays, so VPS was offered in the setting of ventriculomegaly, and webbing of the cerebral aqueduct visualized on MRI. The patient had an uncomplicated procedure but experienced an inpatient course complicated by continued breath holding spells with episodes of bradycardia and was eventually discharged home after 1 week. The patient experienced continued psychomotor developmental delay and unfortunately was diagnosed with shunt infection and underwent VPS removal at 18 months following placement. The patient’s parents opted not to pursue further shunt placement given that the patient did not have any improvement. The patient required extensive support for ongoing cerebral palsy with associated spasticity and dystonia over the subsequent decade, but hydrocephalus remained stable with mild ventriculomegaly persistent through most recent MRI follow-up .An otherwise healthy 18-month-old female patient with history of premature birth (36 weeks) and being the smaller fraternal twin presented with delayed motor development relative to the twin first noticed as frequent tripping, falling, and unsteady gait. Mild spastic diplegia was noted on exam. Ultrasound and MRI studies both demonstrated ventricular enlargement. MRI additionally confirmed diagnosis of communicating hydrocephalus with possible partial aqueductal webbing . After dTwo male siblings with history of Hunter’s syndrome (including a third sibling who was diagnosed and passed away) presented at 10 and 12 years of age, respectively with progressive ventriculomegaly, cerebral atrophy consistent with neurodegeneration, and functional and motor deterioration. VPS was recommended in both cases. Both surgeries were completed uneventfully; in one, a Rickham reservoir was placed, and in the other, a Codman cylindrical valve. Both patients had post-operative difficulty with airway secretions and management but improved gradually and were each discharged within 3 days of surgery. The 12-year-old patient (case #7) was re-admitted two months later for headache, during which time he was diagnosed with an elbow abscess positive for staph aureus. Both patients experienced continued progressive decline as expected with Hunter’s syndrome over subsequent years but did not experience acute worsening of hydrocephalus or require shunt revision .An 18-month-old male patient with history of intrauterine left-sided hemispheric stroke with residual cerebral ischemia and porencephalic cyst presented with interval history of slowing of motor development but continued verbal development. MRI demonstrated communicating hydrocephalus with massive left lateral ventricular enlargement with marked porencephaly, possibly dysplastic left-sided basal ganglia, and left-to-right midline shift . VPS wasTaken together, the results of the systematic literature review and institutional series comprise 155 total patients under the age of 18 years who underwent diagnosis and treatment of NPH-like syndromes. The findings of this systematic review help to establish the clinical phenomenology of a pediatric NPH-like syndrome. The 155 patients found through this review likely represent an under-estimation of the true number of pediatric cases published to date which are consistent with a diagnosis of NPH, as NPH diagnosis in this age group remains poorly defined and likely not often explicitly mentioned. Literature on this subject has also interestingly waned over the last two decades. These trends further underscore the need for additional study in support of evidence-based recommendations for this population. Based on published articles, the diagnosis and treatment of both secondary and idiopathic pediatric NPH is, in practice, comparable to that for adults, though without the same degree of standardization. That individual case series exhibit between-article variability in both etiology distribution and specific diagnostic criteria for NPH also highlights lack of standardization as far as when to suspect NPH in children. Establishment of pediatric NPH guidelines is warranted, similar to the guidelines that have been previously proposed for the adult NPH population ,21.While pediatric NPH is less likely to be mentioned explicitly in the more recent literature, some of the articles published in the last decade have provided novel mechanistic insights into potential genetic and circulatory pathophysiology present in children, as well as in adults, which may contribute to the development of NPH across ages. Two pediatric articles have implicated genes pertinent to ependymal ciliary structure and function; coupled with a third recent study by Yang et al. 2021) which observed a separate genetic defect also related to ependymal cilia function in 8/53 (15%) of adult idiopathic NPH patients who underwent full exosome DNA sequencing, this relatively new line of research provides insight into a potential mechanism of NPH related to cerebrospinal fluid production and circulation dynamics  which ob. SeparatBiomechanically, the impact of altered brain structure and neurodegeneration due to underlying comorbidities may also significantly predispose patients to developing NPH early in life, perhaps conceptually related to the high comorbidity of Alzheimer’s Disease-like pathology which has been appreciated in adult (but not pediatric) NPH ,38,39,40As in adults, variation in CSF pressure dynamic profile across pediatric hydrocephalus etiologies has been documented previously ,8. ContiContinuous recordings have been previously applied to model and categorize communicating hydrocephalus in young adult patients, including those with congenital structural abnormalities underlying altered CSF dynamics such as myelomeningocele and leading to so called “arrested hydrocephalus” ,45. GiveClinical and surgical decision making should be driven primarily by symptomatology, with clear evidence of deterioration or failure to meet milestones coupled with ventriculomegaly taken as a sign that surgical intervention may be indicated. In this series, all patients underwent VPS due to presentation including deterioration in some symptom domain consistent with NPH. Appropriately diagnosed and treated infants and young toddlers may be more likely to present chiefly with reduced global activity and progressive macrocephaly  while slightly older toddlers and children older than 4 may present with motor development outpacing verbal development, developmental slowing, or worsening of baseline neurologic symptomatology . Older children are more likely to have more classic NPH-like symptomatology, such as in our 6-year-old female patient with Chiari I malformation who had deterioration of school grades (suggestive of cognitive slowing), balance, and urinary continence over a period of months (case #1). This patient was also diagnosed with ADHD and, leading up to VPS, reportedly became increasingly irritable. These symptoms were present at baseline and originally attributed to her Chiari I malformation but seemed to be exacerbated by progressing NPH. Somewhat similarly, case #7, a 12-year-old male with Hunter syndrome and baseline seizure activity, experienced an increase in seizure frequency prior to VPS. Such patients may benefit from close attention to progressive symptomatic worsening, and even mild early ventriculomegaly should be tracked closely with serial neuroimaging so as to discern more subtle but clinically significant presentations of NPH alongside existing neurologic comorbidities. Both of these patients experienced improvement after surgery, which consisted of ventriculoperitoneal shunt placement. It should also be noted that, as seen in adult hydrocephalus due to the neurodegenerative nature of NPH and frequently associated pathologies, patient symptoms can sometimes worsen despite VPS. This was unfortunately seen in two of our presented patients with baseline genetic comorbidities (cases #3 and #5), also with associated morbidity and mortality. While all nine pediatric NPH cases in this institutional series, and most cited from the literature, underwent VPS, endoscopic third ventriculostomy (ETV) is a viable evidence-based alternative in obstructive pediatric hydrocephalus, though evidence specifically in pediatric NPH is quite limited.Normal-pressure hydrocephalus (NPH) remains a pertinent clinical diagnosis which is likely heterogeneous in pathophysiology and can occur in patients across the life cycle, including both older adults and children. The diagnosis of pediatric NPH is backed by less substantial clinical evidence than adult NPH. However, diagnosis and treatment across over 150 known patients, including those presented in this case series, has substantiated presenting clinical features similar to those in adult NPH including delay of cognitive, motor, and urinary developmental milestones during childhood. Ongoing study of genetic risk factors and neurodegenerative pathologies associated with NPH-like syndromes in children and adults may produce near-term mechanistic insight into this relatively common but likely underdiagnosed disorder. Ventriculoperitoneal shunting is a viable treatment option for patients with symptomatic pediatric NPH and the impact of endoscopic third ventriculostomy should be studied.