Photosynthetic carbon assimilation is the principal experimental observable which integrates important aspects of primary plant metabolism. It is traditionally measured through gas exchange. Despite its centrality in plant research, gas exchange performs poorly with rosette growth habits typical of Arabidopsis thaliana, mutant lines with limited biomass, and accounts poorly for leaf shading.We report a method to estimate carbon assimilation based on isotope ratio-mass spectrometry (IRMS) of 13C content and labeling time (R2 = 0.999) for 158 wild-type plants labeled for 6 to 42 min. Plants cultivated under different light regimes showed a linear response with respect to carbon assimilation, varying from 7.38 nmol 13C mg−1 leaf tissue min−1 at 80 PAR to 19.27 nmol 13C mg−1 leaf tissue min−1 at 500 PAR. We applied this method to examine the link between inhibition of the 2C-methyl-d-erythritol-4-phosphate (MEP) pathway and suppression of photosynthesis. A significant decrease in carbon assimilation was observed when metabolic activity in the MEP pathway was compromised by mutation or herbicides targeting the MEP pathway. Mutants affected in MEP pathway genes 1-DEOXY-d-XYLULOSE 5-PHOSPHATE SYNTHASE (DXS) or 1-HYDROXY-2-METHYL-2-(E)-BUTENYL 4-DIPHOSPHATE SYNTHASE (HDS) showed assimilation rates 36% and 61% lower than wild type. Similarly, wild type plants treated with the MEP pathway inhibitors clomazone or fosmidomycin showed reductions of 52% and 43%, respectively, while inhibition of the analogous mevalonic acid pathway, which supplies the same isoprenoid intermediates in the cytosol, did not, suggesting inhibition of photosynthesis was specific to disruption of the MEP pathway.IRMS-based carbon assimilation values from plants labeled at different light intensities were compared to those obtained by gas exchange, and the two methods yielded similar values. Using this method, we observed a strong correlation between Arabidopsis lines which are not amenable to gas exchange measurements due to low biomass and limited leaf surface area. It is suitable for screening large numbers of replicates simultaneously as well as post-hoc analysis of previously labeled plant tissue and is complementary to downstream detection of isotopic label in targeted metabolite pools.This method provides an alternative to gas exchange that offers several advantages: resilience to differences in leaf overlap, measurements based on tissue mass rather than leaf surface area, and compatibility with mutant The online version contains supplementary material available at 10.1186/s13007-021-00731-8. However, the inhibition of photosynthesis was even more evident in partial loss of function mutants affected in MEP pathway structural genes such as dxs-3 or 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase 3 (hds-3), where the IRMS-based carbon assimilation rate was 36%  and 61%  reduced, respectively, compared to wild-type plants (n = 10)  and second (DXR) step of the MEP pathway, respectively. Plants were subjected to the same time course labeling experiments 24 h after treatment, and the resulting lyophilized tissue was similarly analyzed by EA-IRMS. As controls, we also treated plants with MEV , NFZ (which blocks the phytoene desaturase step of carotenoid biosynthesis), CAM, which blocks plastidic protein synthesis, or water (control). We hypothesized that CLZ and FSM treatment prior to labeling would simulate the reduced availability of IDP and DMADP in the chloroplast imposed by mutation in structural genes but would not have the same developmental effects resulting from these mutations or long term effects related to reduced levels of photosynthetic pigments or co-factors available to participate in photosynthesis. Compared to wild-type plants treated only with water Fig. a, CAM trNFZ treatment also provoked a significant decline in carbon assimilation  Arabidopsis. For instance, many mutants of Arabidopsis affected in some aspect of the carbon reduction cycle may present a stunted growth phenotype which assimilate too little carbon to quantify gas exchange parameters reliably. Applying leaf clamps may not be physically feasible in this case, and Arabidopsis leaf surface area is instead estimated graphically. However, the partially overlapping leaves of Arabidopsis rosettes, whose extent increases as the plant develops, do not lend themselves well to accurate calculations of leaf surface area. In addition, gas exchange in general is a laborious process poorly suited to screening large numbers of plant lines.Despite the centrality of gas exchange measurements in elucidating the mechanisms of photosynthetic carbon assimilation, there are limitations to this technique when working with the model plant 13CO2 labeled plants to facilitate measurement of gas exchange in Arabidopsis and found that this approach provides the following advantages. First, it is based on tissue mass rather than leaf surface area, which avoids the uncertainty of partial leaf overlap and differences in individual leaf assimilation rates based on partial or total shading. Partially shaded leaves carry out photosynthesis at a lower rate which is otherwise difficult to account for. Gas exchange as a technique originated in combination with leaf clamps on large leaf species such as maize but performs poorly for small rosette leaves, where leaf surface area must be estimated graphically. These problems were largely overcome by basing carbon assimilation on mass of dry tissue, which provided a highly linear (R2 > 0.999) measure of assimilation  can nonetheless be labeled and analyzed by EA-IRMS to estimate carbon assimilation using the technique described here. Plate grown seedlings as young as 10–14 days could theoretically be labeled and analyzed with this technique if grown at a sufficient density on sterile plates to yield enough tissue for EA-IRMS analysis. Moreover, large biological replicate pools can be labeled simultaneously in a sufficiently large cuvette, increasing the throughput of labeling experiments and improving the statistical power of analysis. In this fashion, this technique can be used to screen multiple lines or treatments in parallel based on comparative carbon fixation rates under a given condition. It should be noted that, by design, the data presented here only address leaf level carbon fixation and do not account for carbohydrates transported to root tissue. However, the transport of 13C into root tissue may be monitored with exactly the same approach by harvesting root tissue separately and subjecting aliquots to EA-IRMS. Indeed, this technique may provide a powerful method for analyzing the partitioning of carbon resources by comparison of 13C detected in leaf and root tissue separately.Finally, small 2 partial pressure across stomata on isotopic discrimination during carbon assimilation [13C abundances rather than labeling, changes in carbon isotope composition of CO2 passing over the surface of a leaf were correlated to isotopic discrimination in intact leaves as the CO2 partial pressure was experimentally varied. It confirmed that 13C discrimination increased as the CO2 partial pressure gradient across stomata decreased in C3 plants but not in C4 plants. IRMS analysis has also been used to probe the source of carbon supplying isoprene emissions in myrtle, buckthorn, and velvet bean [13C differences in CO2 and isoprene emissions implicated DXS as the main isotopic discrimination step in isoprene formation. IRMS analysis was further used to identify the presence of carbon pools contributing to dark respiration not derived from recent photosynthate [13CO2 and IRMS to estimate carbon assimilation directly.The use of IRMS to investigate photosynthesis has several precedents. IRMS analysis of carbon isotopes in leaf tissue has previously been used to determine the influence of COmilation . In thissynthate . To our 13C content to traditional gas exchange measurements, we converted 13C content on a per mg basis to the units used in gas exchange (μmol CO2 m−2 s−1). The observed slope, slightly less than 1, reflects unlabeled carbon loss released during the labeling segment. This release is comprised of carbon within the photorespiratory pool, additional pools lost from the C2 cycle, and related processes termed non-photorespiratory CO2 release [2 release is included in A. However, once the atmosphere is switched to a 13CO2 containing atmosphere, the slow loss of residual, unlabeled CO2 is no longer subtracted from the apparent net assimilation of 13C (A13) since we include only 13C quantified by EA-IRMS in our analysis. Therefore, the same time points yield slightly higher values for A13 than for A, and consequently, the slope remains below 1. This slope remained consistent over all time point assayed, suggesting the slow release of unlabeled CO2 from non-photorespiratory sources continued throughout the ~ 45 min labeling period. In contrast, photorespiratory intermediates are expected to become fully labeled within minutes, and their loss as 13CO2 would then be factored into the 13C detected in lyophilized plant tissues. Our results support previous observations that non-photorespiratory CO2 loss is a significant and distinct form of respiration than photorespiratory CO2 loss [To compare assimilation rates estimated by  release , 77, 83.CO2 loss .in planta to ketoclomazone, which acts as an uncompetitive DXS inhibitor with respect to pyruvate but a mixed inhibitor with respect to d-glyceraldehyde-3-phosphate [hds-3 [DXS originally named chill sensitive 5  . However, all three mutants displayed significant declines in carbon assimilation rate  is a WD-40 RNA binding protein with multiple roles in immunity and stress tolerance. The n levels . It has n levels  and in rn levels . Due to ate Fig. , indicatArabidopsis, particularly mutant lines with reduced photosynthetic capacities and stunted growth phenotypes or in combination with herbicide treatments intended to dissect regulatory mechanisms controlling metabolism. Unambiguous assignment of carbon assimilation rates in greening seedlings and other early developmental stages, or in leaves vs. roots to address carbon partitioning between organs, can be implemented with minimal modification of this method. When combined with targeted analysis of isotopic tracer by more conventional metabolomics approaches, this method presents many avenues for furthering insights into the basic mechanisms of photosynthesis.The IRMS-based carbon assimilation measuring technique presented here represents a novel alternative to gas exchange measurements that is well suited to the investigation of central metabolism in Additional file 1: Figure S1. Preparation of Arabidopsis thaliana for whole plant 13CO2 labeling experiments. A, Photosynthetically active surface area was calculated by photographing the rosette against a white background and comparison of the leaf surface to that of size standards, as determined by using the ‘Magic wand’ function in Adobe Photoshop CS5 to quantify pixels of each standard and leaf surface. B and C, examples of single plant labeling cuvettes with light and temperature control used in this study. The switch valve which alternates between ‘normal’ air and 13CO2-containing air is located just upstream of the cuvette inlet in both cases. Air is sampled by CO2 and H2O sensors before entering and after exiting the cuvette to calculate gas exchange parameters and confirm a photosynthetic steady state prior to switch to a 13CO2-containing atmosphere. D, a thermocouple in continuous contact with the abaxial leaf surface monitors leaf temperature during the acclimation and labeling phases of each experiment. Photo credit: M. Phillips.


TSC1 and CRYZ/TYW3) and the enriched pathways and components  are related to, or have been associated with, the PI3K/AKT/mTOR pathway. Genes in this pathway have previously been associated with GTS, and mTOR signalling has been implicated in a range of neuropsychiatric disorders. It is thus possible that altered mTOR signalling plays a role in the complex pathogenesis of TSD.Tic spectrum disorder (TSD) is an umbrella term which includes Gilles de la Tourette syndrome (GTS) and chronic tic disorder (CTD). They are considered highly heritable, yet the genetic components remain largely unknown. In this study we aimed to investigate disease-associated DNA methylation differences to identify genes and pathways which may be implicated in TSD aetiology. For this purpose, we performed an exploratory analysis of the genome-wide DNA methylation patterns in whole blood samples of 16 monozygotic twin pairs, of which eight were discordant and six concordant for TSD, while two pairs were asymptomatic. Although no sites reached genome-wide significance, we identified several sites and regions with a suggestive significance, which were located within or in the vicinity of genes with biological functions associated with neuropsychiatric disorders. The two top genes identified ( Gilles de la Tourette syndrome (GTS) is characterized by the presence of at least one vocal and two motor tics, while chronic tic disorder (CTD) is characterized by the presence of either vocal or motor tics. Both GTS and CTD are neurodevelopmental disorders with childhood onset (before the age of 18). There is currently no clinical or genetic evidence suggesting that GTS and CTD are separate disorders, and the term tic spectrum disorder (TSD) has been proposed to replace GTS and CTD ,3. SLC6A3) and tic severity, while higher methylation levels of the dopamine D2 receptor gene (DRD2) were associated with GTS and positively correlated with tic severity [SLC6A4) in GTS individuals compared to controls [TSD is a complex disorder with a largely unknown aetiology, where multiple genes are hypothesized to interact with a range of environmental risk factors. The results of heritability studies suggest genetic risk factors play a substantial role in pathogenesis ,5, but iseverity . The othcontrols . Studying epigenetic changes is more challenging compared to genomic alterations. Monozygotic (MZ) twin studies can be advantageous to traditional case–control studies, due to the fact that MZ-twins are matched for not only genotype, sex, age and maternal environment, but also partially matched for early environmental influences. As these factors are shown or hypothesized to influence DNA methylation and therefore gene expression, MZ-twin design has a unique advantage . PreviouThrough cross-linking The Danish Psychiatric Central Register and The Danish Twin Register we identified 204 twin pairs, where at least one twin had GTS or CTD (Collectively referred to as TSD throughout the manuscript). All the twins or legal guardians were contacted, and 56 twin pairs accepted to participate. DNA was isolated from peripheral blood and the zygosity of the twins was determined with SNP genotyping using Infinium Global Screening Array (llumina). Fourteen twin pairs were monozygotic. Interviews were carried out with all the twins and a trained neuropediatrician made the diagnosis using the DSM-V  criteria. Eight of these pairs were discordant for TSD and six were concordant. Furthermore, two asymptomatic MZ twin pairs were included in the study. The study was approved by the Danish Institutional Review Board (2011 H-2-2010-144).p-value for quality control (QC) was calculated using the free R package minfi [p > 0.01, probes on sex chromosomes, probes harbouring single nucleotide polymorphisms (SNPs) and probes with known cross-reactivity were excluded from the analysis. After QC, 756,887 probes remained for analysis. Quantile normalization implemented in the R package minfi was used for data normalisation. β values defined by Illumina’s formula as β = M/(M + U) was used to quantify DNA methylation levels . To conduct statistical analysis methylation β values were logit transformed to M values.Genomic DNA was extracted from peripheral blood and bisulfite treatment was carried out using standard protocols. Infinium MethylationEPIC arrays  were used to analyse the bisulfite converted DNA according to manufacturer’s protocol. A detection ge minfi . Probes The DNA used in this study was extracted from whole blood, which contains several different types of blood cells. As different cell types are known to have different methylation patterns, the R package FlowSorted.Blood.EPIC  was usedp < 0.01) were submitted to the Genomic Regions Enrichment of Annotations Tool (GREAT 3.0), an online tool which annotates genomic positions to genes and cis-regulatory elements, and identifies enriched gene ontologies and pathways.A linear mixed model using the R package lmerTest , regressp-values of adjacent sites and assigning significance to the regions identified. We used the results from the linear mixed model, with an unadjusted p of <1 × 10−3 to seed a region, and a maximum distance of 500 bp between the CpG sites. The region results were corrected for multiple testing using the Šidák correction.In addition to investigating methylation differences at single sites, we also employed a region-based approach using the python library comb-p . This idWe did not observe any difference in cell-type composition between TSD individuals and controls .p-values was observed, which could indicate non-random association. Nine sites with suggestive significance (p < 1 × 10−5) were detected  which were previously implicated in with neuropsychiatric disorders. The DMP most significantly associated with TSD was located in the promoter region of TSC1.To identify differentially methylated probes in TSD individuals we employed a linear mixed model corrected for age with twin pairing as a random effect. None of the probes reached genome-wide significance defined as FDR < 0.05. As illustrated in detected . Three op < 0.01, n = 5707) were investigated for enrichment of gene ontologies and pathways using GREAT. Genomic positions of 5676 DMPs were annotated to one or more genes and/or cis-regulatory elements, and four gene ontologies and two pathways were found to be enriched . NEMF (nuclear export mediator factor) encodes a component of the ribosome quality control complex and has previously been associated with ASD [NEMF have been shown to cause neurodegeneration in mice [ADA (adenosine deaminase) encodes a protein which is important for purine metabolism, and ADA deficiency causes severe combined immunodeficiency (SCID). Reduced adenosine deaminase activity has been observed in ASD individuals [Of the nine DMPs with suggestive significance three were annotated to genes previously associated with neuropsychiatric disorders . SDK1 (The Sidekick Cell Adhesion Molecule 1) has been associated with ASD [CHD2 (Chromodomain Helicase DNA Binding Protein 2) variants has been identified in individuals with central nervous system pathologies [CLN8  has been linked to ASD through rare missense variants found in a Japanese family [Of the seven DMRs three were annotated to the regulatory regions of genes previously associated with neuropsychiatric disorders . Pathogenic TSC1 variants lead to tuberous sclerosis, which is characterized by skin abnormalities, developmental delay, epilepsy and behavioural problems such as ADHD and ASD [TBC1D7 [The DMP most significantly associated with TSD was within the promoter region of  and ASD . TSC1 an [TBC1D7 . TSC1 re [TBC1D7 ,32,33. m [TBC1D7 ,35,36. D [TBC1D7 , which h [TBC1D7 ,39. Alte [TBC1D7 ,42,43,44 [TBC1D7 . However [TBC1D7 .p < 0.01) we identified in this study  encoding a component of the mTOR2 complex [Genes involved in the phosphoinositide and the PTEN pathways, as well as insulin receptor substrate binding, was significantly enriched among the nominally significant DMPs  and TYW3 (TRNA-YW Synthesizing Protein 3 Homolog) which are transcribed from opposite strands. In a previous GWAS, a SNP near these two genes was significantly associated with resistin levels in blood and transcript levels of the gene encoding resistin in leukocytes [The top DMR in the present study was within the overlapping first exons of ukocytes . Notablyukocytes . In conclusion, the results of the present study and the findings from previous studies suggest that deregulation of the PI3K/AKT/mTOR pathway may be one of the contributing risk factors in TSD pathogenesis. Involvement of this pathway and particularly mTOR signalling are widely recognized as contributing factors to a broad range of neuropsychiatric disorders, which further support this hypothesis. As TSD is considered a multifactorial disease, the pathogenesis is likely to include a combination of genetic, epigenetic and environmental factors, and to consist of several pathways and cellular functions. However, further methylation studies using larger cohorts should be carried out to replicate the current findings, and expression studies of the components of the PI3K/AKT/mTOR pathway may give insights to the TSD pathogenesis.The present study is the first to date epigenome-wide study of methylation changes in monozygotic twins with TSD. The relatively small sample size was a limitation, but the twin-based design offers unique advantages over the classic case-control setup even with a relatively small sample size. We carried out the methylation studies using peripheral blood, and it is possible that brain regions known to be involved in TSD pathology may show different methylation patterns to the ones we observed. Epigenomic studies of neuropsychiatric disorders have largely been carried out on peripheral blood, as brain tissue is not available. However, studies suggest that blood methylation can be informative for psychiatric conditions, as factors affecting the brain have been shown to leave biomarker signatures in the blood. Furthermore, one of the significant findings of the GTS-pathway study implicated involvement of lymphocytic pathways in GTS aetiology, which supports that blood can be used to identify methylation biomarkers.


Endogenous erythropoietin (EPO) concentrations vary widely in preterm infants and may be associated with perinatal risk factors and neurological outcomes. Erythropoietin is elevated in fetal hypoxia but is also a potential neuroprotectant.In a prospective study of 27 infants ≤ 30 weeks gestation, serum erythropoietin concentrations were measured during the first month of life, on day 1 and weeks 1, 2, and 4, and related to perinatal risk factors and outcomes including retinopathy of prematurity and cerebral injury evaluated near term-equivalent post menstrual age using magnetic resonance imaging with quantitative scoring.Lower birth weight was associated with higher EPO concentrations throughout the first 2 weeks of life . Higher day 1 and week 1 EPO concentrations were associated with lower Apgar score at 1 minute (r = - 0.5) and 5 minutes (r = -0.7), respectively (p < 0.01). Higher day 1 EPO concentrations and 2-week area under the curve were associated with increased risk (p = 0.01) and severity  of retinopathy of prematurity. Higher EPO concentrations at 2 weeks were associated with increased total brain injury score .Elevated endogenous erythropoietin concentrations in the first two weeks of life are associated with lower birth weight and increased risk of adverse outcomes. Preterm birth is a major public-health challenge. Although survival has improved over the past two decades, children born preterm are at increased risk for cerebral palsy and cognitive and neurobehavioral deficits including lowered IQ, autism spectrum disorders, attention deficit hyperactivity disorder, anxiety disorders, and learning disabilities , 2. ErytThe biological role of EPO is complex and extends well beyond hematopoiesis; EPO and its receptor (EpoR) are expressed early in fetal development in the human brain  and EPO Independent of the beneficial effects of EPO, its concentration may also be a biomarker of stressful perinatal conditions. Among preterm newborns, elevated concentrations of endogenous EPO have been associated with elevated inflammation-related proteins  and withThe commonest neuropathology documented in the very preterm infant occurs in the cerebral white matter, and along with other forms of brain injury, has been shown to be best evaluated using magnetic resonance imaging (MRI) . AlthougThe factors that regulate endogenous EPO production in the preterm infant, as well as the influence of perinatal exposures, are not fully understood. This study investigates the impact of perinatal risk factors on endogenous EPO concentrations during the first month of life in preterm infants and the potential correlations between EPO concentrations and outcomes, including retinopathy of prematurity (ROP) and brain injury using MRI at term-equivalent post menstrual age.The Washington University Institutional Review Board approved this research, protocol 08–0233. Written consent was obtained.Prospective data were collected between June 2008 and March 2009 at St Louis Children’s Hospital neonatal intensive care unit (NICU) in Saint Louis, Missouri, USA  after IRB approval. Mothers and fetuses were not living at high altitude (>2500m). All preterm infants with gestational age (GA) ≤ 30 weeks expected to survive > 72 hours (as determined by the attending neonatologist) were eligible for recruitment. Exclusion criteria included infants with chromosomal abnormalities, major congenital anomalies, hemolytic blood disorders and suspected or proven congenital infections . Patients missing both cord and day 1 EPO concentration values were also excluded. During periods of active enrollment, parents or guardians of consecutive eligible infants were approached by the investigators or a trained NICU research nurse to obtain written informed consent as soon as possible after delivery. All infants were treated according to the accepted care standards of the NICU.Perinatal data were collected by review of maternal and infant charts by the investigators and a NICU research nurse. Information on perinatal risk factors and neonatal morbidities was collected. Prenatal factors included sex, race, gestational age, intrauterine growth restriction (IUGR), pregnancy-induced hypertension (PIH), preterm labor, maternal illnesses or drugs, multiple birth and antenatal steroids. Postnatal factors included birth weight, birth weight Z-score , mode oCranial ultrasound (US) was obtained according to standard clinical practice, which included a scan within the first 7–14 days of life to evaluate for intraventricular hemorrhage (IVH). Infants were scanned using MRI using a 3T Tim Trio system  without sedation near term gestational age or prior to discharge from the NICU. MR images were evaluated using a modified scoring system to assess brain injury as previously described  showed wide variation with median 6.5 (range 2.7–174) mU/ml. Erythropoietin (EPO) and hemoglobin (Hgb) concentrations at each time point, and their interrelationship, are shown in [EPO]0-2weeks  (0-2 weeks)  . EPO con < 0.01) . After a2 weeks) . EPO con2 weeks) .Higher early EPO concentrations were associated with lower Apgar scores. First EPO negatively correlated with 1 minute Apgar score , and 1 week EPO negatively correlated with 5 minute Apgar score  . These a[EPO]0–2 weeks were significantly higher for patients with any (grade 1–3) ROP than for patients without ROP   also correlated with stage of ROP (Early EPO concentrations were higher for subjects with ROP. First EPO and AUC = 0.01) . Week 1  = 0.01) . First Ee of ROP . The pose of ROP  Tables.A negative correlation between EPO and Hgb concentration was observed at 2 weeks   that wasThe number of transfusions received during NICU stay correlated with stage of ROP , all with p < 0.0001 . The numMRI imaging was completed for 24 subjects at 37 ±2  weeks post menstrual age. A positive correlation of 2 week EPO with total brain injury assessed by MRI was observed   but not Endogenous Erythropoietin (EPO) concentrations in premature infants may both be a biomarker for hypoxic stress and have an influence on the response to brain injury. This study investigated whether EPO concentrations in the first month of life were driven by individual perinatal risk factors and were associated with subsequent outcomes and brain injury as assessed by MRI prior to NICU discharge.EPO concentrations varied widely at birth, with a decline in point estimates to a nadir at about 1 to 2 weeks of life and recovery by week 4. This initial decline in EPO is consistent with early studies . EPO conSubjects with higher EPO concentrations at birth and 1 week had increased risk and severity of ROP. Higher initial and 1 week EPO concentrations were also associated with lower Apgar scores, and there was a trend towards higher initial EPO in subjects with perinatal stressors such as emergent caesarian section , IUGR , and early sepsis  . First EWe interpret these results to indicate that these associations of early elevations in EPO with adverse outcomes are due to increased EPO production in response to fetal/neonatal hypoxia. Early studies have shown that fetal hypoxemia stimulates erythropoiesis and that it is a very responsive system; plasma EPO peaked less than 12 hours after the onset of hypoxemia in late-gestation fetal sheep . EmergenTeramo et al  hypothesAt birth through the first 2 weeks of life, EPO concentrations also negatively correlated with Hgb concentrations and were higher in infants that received transfusion than those that did not. Furthermore, EPO concentrations increased between weeks 2 and 4 only in the non-transfused group and by week 4 were higher in the non-transfused group than in the transfused group. These findings are consistent with appropriate physiological regulation of EPO production in preterm infants.The number of transfusions received during NICU stay correlated with increased white matter injury by MRI. A reasonable hypothesis is a “double hit” to white matter: the hypoxia of low Hgb (leading to the transfusion in the first place) and the inflammation associated with transfusion . Increas[EPO]0–2 weeks], The number of transfusions received during NICU stay also correlated with stage of ROP . This finding may be relevant to current controversies over transfusion thresholds. Although no significant differences in severe ROP were observed between liberal and restrictive transfusion groups in the PINT randomized trial , a recenRegarding possible etiologies for the potential impact of transfusion on injury, the lower EPO at 1-month observed in RBC-transfused patients may translate into loss of a neuroprotective growth factor that attenuates inflammation . DecreasThis study is unique in that it investigated endogenous EPO concentrations throughout the first four weeks of life and subsequent brain injury as assessed by MRI with systematic scoring of grey/white matter injury near term gestation. EPO concentrations at 2 weeks were associated with increased total brain injury score . However, this association lost significance  after correcting for GA or birth weight Z-Score. In contrast, the correlation of 2 week EPO concentration with grey matter injury was present only after adjusting for birth weight Z-Score . In comparison to other studies, pre-discharge MRI findings of increased brain injury in association with elevated EPO levels at 2 weeks would be consistent with and could presage the long-term neurodevelopmental deficits reported by the ELGAN study investigators. In ELGAN study subjects, endogenous EPO concentrations at 2 weeks of life, regardless of intermittent or sustained systemic inflammation, were associated with low mental and/or psychomotor development indices and microcephaly at 2 years  and withThese results showing endogenous EPO concentration as a biomarker for hypoxic injury have no bearing on the potential protective effects of exogenous recombinant human EPO (rhEPO). Exogenous rhEPO is neuroprotective in animal experiments , and hasIndependent of the potential benefit of rhEPO, endogenous EPO elevations within the first 2 weeks correlated in this study with morbidities associated with hypoxic injury including ROP and brain injury. Of interest, this association was not observed for EPO concentrations at 1 month of age, and anecdotally the lowest 1-month EPO concentration was observed in a single subject with NEC who died on day of life 38.This study has limitations, including its small sample size with resulting low statistical power, and no adjustment was made for multiple comparisons. It is also limited to analysis of clinically discarded blood samples, which restricts both the number and volumes available for analysis. Furthermore, as in any observational study, confounding variables may complicate interpretation of findings. The MRI scans were performed at a range of postmenstrual ages, although the presence of injury should not influence the later MRI definition of injury presence. Finally, longer term clinical outcome data were not collected.Endogenous Erythropoietin (EPO) concentrations in the first month of life vary widely in preterm infants and may both be a marker for perinatal hypoxic injury and affect the healing response. Elevated EPO concentrations in the first two weeks are associated with lower birth weight, increased risk of ROP, and are suggestive of higher brain injury scores by MRI that may presage long term neurodevelopmental outcomes. RBC transfusions were associated with increased risk and severity of ROP and white matter injury. Additional studies are warranted to further asses the risks of transfusion and the potential role of early endogenous EPO as a biomarker for hypoxic injury in preterm infants.S1 TableSpearman partial correlation coefficient estimate adjusted for gestational age; p-value, and r are presented for association of ln(EPO) at each time point with continuous variables.(PDF)Click here for additional data file.S2 TableSpearman partial correlation coefficient estimate adjusted for birthweight Z-Score; p-value, and r are presented for association of ln(EPO) at each time point with continuous variables.(PDF)Click here for additional data file.S3 TableInjury scores by MRI at near term gestational age are presented as mean (SD) with two-sample t-test comparing subjects with transfusion (yes) vs. without transfusion (no) in gender subgroups.(PDF)Click here for additional data file.S4 TableSpearman correlation and partial correlation coefficients for association of number of transfusions with ROP and brain injury scores by MRI at near term-equivalent post-menstrual age, adjusted for ln[EPO] or [Hg] at week 1 or 2; coefficient estimate, p-value and r are presented.(PDF)Click here for additional data file.S5 TableLinear regression correlating ln(EPO) over time with continuous variables; coefficient estimate, p-value and R-square are presented.(PDF)Click here for additional data file.S6 TableLinear regression correlating Ln(EPO) over time with continuous variables adjusted for gestational age; coefficient estimate, p-value and r are presented.(PDF)Click here for additional data file.S7 TableLinear regression correlating Ln(EPO) over time with continuous variables adjusted for birthweight Z-Score; coefficient estimate, p-value and r are presented .(PDF)Click here for additional data file.


Information about optimal use of heparin in flush fluids during endovascular thrombectomy (EVT) for acute ischemic stroke (AIS) is lacking. Variables that determine total heparin dose entering the patient by flush fluids are mostly unknown. We aim to provide insight in these unknown but highly relevant variables.n = 79) collecting data on used concentration of heparin in infusion bags, number of infusion bags connected, timing of connecting the flush line and the dripping rate (ml/sec). We calculated potential heparin dose entering the patient per hour through flush fluids (IU/h). Twenty-eight interventionists (35%) representing 17 Dutch stroke centers completed the survey. Eight interventionists responded not to add any heparin to flush fluids (18%). The highest amount of heparin entering the patients was 13,500 IU/h, reported by 2 interventionists from the same center (4%).We performed a survey including all Dutch interventionists performing EVT (We provide insight in the use of heparinized flush during EVT in the Netherlands. Total amounts of heparin administered via flush fluids may go up to 13,500 IU/h. With this paper we intend to set a starting for future research and development of guidelines on the use of heparinized flush fluids during EVT for AIS.The online version contains supplementary material available at 10.1186/s42155-021-00264-0. During endovascular treatment (EVT) for acute ischemic stroke (AIS), catheters are continuously flushed, using saline infusion fluids, to avoid thrombus formation during the procedure and thus to reduce the risk for distal embolization. These flush fluids are often heparinized. In an earlier study, we showed wide variance in used heparin concentrations in flush fluids and investigated its potential impact on functional and technical outcome  in the Netherlands by creating a Google Form questionnaire  completed the survey.Thirteen interventionists had 1–5-years experience in neuro-intervention. Eleven had 5–10-years experience and 4 had more than 10 years experience in the field of neuro-intervention.Twenty-one participants responded to which concentration of heparin they used in the flush fluids. For the non-responders, we used heparin concentrations derived from colleague responders from the same center (if available) or from known heparin concentration from an earlier conducted center-based survey  guiding catheter and / or one on the aspiration catheter and / or one on the microcatheter), 5 interventionists use 2 bags  guiding catheter, aspiration catheter or microcatheter) and 7 use only 1 bag  guiding catheter).Twenty-six interventionists connected the infusion bag immediately at the start of the procedure. Two interventionists connected the infusion bag after catheterization of the common carotid artery. We did not include the time-delay from groin puncture to carotid artery catheterization (time of using catheter without flush) in our final calculations of total dose, because of the small number and potentially short time delay.n = 18), followed by 0,5 drip per second  (n = 10); 2 drips per second  (n = 2) and 1 drip per 5 s  (n = 1).All the participating interventionists set up their dripping speed manually. The most often set drip rate was 1 drip per second   national consensus and guidelines on the use of heparin in flush fluid bags during EVT for AIS, which translates into a large variety on center level . Furthermore, most of the respondents set up their drip rate manually, meaning that they adjusted the drip rate visually, with inherent inaccuracy.We provide an insight in the use of heparin in flush fluids in Dutch EVT centers. Total amounts of heparin administered via flush fluids may go up to 13,500 IU per hour while in other cases no heparin is administered. With this paper we intend to set a starting for future research and development of guidelines regarding the use of heparin in flush fluids during EVT for AIS.Additional file 1: Table S1. Different heparin doses (IU) per hour are depicted for the different heparin concentrations (IU/L) given a drip rate of 1 drip / sec.


Observational studies indicate that type 2 diabetes mellitus and fasting glucose levels are associated with a greater risk for hip fracture, smaller bone area and higher bone mineral density (BMD). However, these findings may be biased by residual confounding and reverse causation. Mendelian randomisation (MR) utilises genetic variants as instruments for exposures in an attempt to address these biases. Thus, we implemented MR to determine whether fasting glucose levels in individuals without diabetes are causally associated with bone area and BMD at the total hip.p < 5 × 10−8) in a non-diabetic European-descent population from the Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) . MR was used to assess the associations of genetically predicted fasting glucose concentrations with total hip bone area and BMD in 4966 men and women without diabetes from the Swedish Mammography Cohort, Prospective Investigation of Vasculature in Uppsala Seniors and Uppsala Longitudinal Study of Adult Men.We selected 35 SNPs strongly associated with fasting glucose , yet was also associated, albeit without reaching statistical significance, with a 4% higher total hip BMD .In a meta-analysis of the three cohorts, a genetically predicted 1 mmol/l increment of fasting glucose was associated with a 2% smaller total hip bone area (−0.67 cmFasting glucose may be a causal risk factor for smaller bone area at the hip, yet possibly for greater BMD. Further MR studies with larger sample sizes are required to corroborate these findings.The online version contains peer-reviewed but unedited supplementary material available at 10.1007/s00125-021-05410-w. Observational studies have shown type 2 diabetes mellitus to be associated with a greater risk for hip fracture , 2, despTo determine the causal effects on bone of type 2 diabetes or glucose levels in a non-diabetic population using traditional epidemiological approaches may be difficult due to a number of potential biases, including residual confounding and reverse causation . To condNo MR study to date has addressed the effect of fasting glucose on bone area, one potential mechanism underlying the association between type 2 diabetes and hip fracture. Several studies indicate that bone area at the hip is linked with the risk of hip fracture , 19, andAll study populations were restricted to individuals without diabetes in the three Swedish cohorts detailed below. We defined diabetes according to the ADA and WHO criteria: fasting plasma glucose concentrations ≥7.0 mmol/l and/or self-reported diabetes with or without treatment with oral hypoglycaemic agents or insulin.www.simpler4health.se).The Swedish Mammography Cohort (SMC) was established during 1987–1990. Between November 2003 and October 2009, a randomly selected sub-cohort (SMCC) of 5022 women living in the city of Uppsala, Sweden, underwent dual energy x-ray absorptiometry (DXA) measurements, provided morning fasting blood samples, had height and weight measurements taken, and completed a medical and lifestyle questionnaire  to the research community) through the Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX). A p value of <0.05 was considered statistically significant.In sensitivity analyses, we used multivariable MR analysis to adjust for genetically predicted height  and BMI The characteristics of participants in each of the three Swedish cohorts are presented in Table 2 = 0.045), 4% (adjusted r2 = 0.035) and 12% (adjusted r2 = 0.119), respectively, of the variance in fasting glucose concentrations in participants without diabetes. The association of each SNP with bone area and BMD in each cohort, along with the β estimates for the associations of the SNPs with fasting glucose concentrations, can be found in electronic supplementary material (ESM) Figs In the SMCC, PIVUS and ULSAM cohorts, the 35 SNPs used in our analyses together explained 4%  based on meta-analysis of estimates from the three cohorts using the fixed effects IVW method  in meta-analysis of estimates from the three cohorts  in a previous meta-analysis of genome-wide association studies of non-diabetic individuals [Major strengths of the MR approach are that reverse causation bias is avoided because genetic variants are fixed at conception, and confounding is reduced by the use of genetic variants as proxies for the exposure representing the lifelong exposure of fasting glucose concentrations . Our stuividuals , which wividuals  and therividuals . The insividuals , suggestMR analyses further rely on the assumption of absence of pleiotropy, which can occur when a genetic instrument (SNP) affects multiple phenotypes . DependeIGF2BP2) in an additional sensitivity analysis but this did not affect our estimates. Another potential source of bias in MR analyses is population stratification and population heterogeneity [We did not find any evidence of SNPs being outliers or effect change caused by any SNP outliers, evaluated using MR-PRESSO , althougogeneity , but thiBone size increases with advancing age via periosteal apposition to compensate for the losses in BMD in order to preserve bone strength . The ben2) was associated with an 8% higher risk of type 2 diabetes, assessed using an MR approach [The relationship between bone and energy metabolism is complex and may be bidirectional. A previous study found that each one SD increase in genetically estimated heel BMD


Persistent infection with high-risk human papillomaviruses (HPVs) is the major risk factor associated with development of anogenital and oropharyngeal cancers. Initial infection by HPVs occurs into basal epithelial cells where viral genomes are established as nuclear episomes and persist until cleared by the immune response. Productive replication or amplification occurs upon differentiation and is dependent upon activation of the ataxia-telangiectasia mutated (ATM), ataxia telangiectasia and RAD3-related (ATR) DNA damage repair (DDR) pathways. In addition to activating DDR pathways, HPVs must escape innate immune surveillance mechanisms by antagonizing sensors, adaptors, interferons and antiviral gene expression. Both DDR and innate immune pathways are key host mechanisms that crosstalk with each other to maintain homeostasis of cells persistently infected with HPVs. Interestingly, it is still not fully understood why some HPV infections get cleared while others do not. Targeting of these two processes with antiviral therapies may provide opportunities for treatment of cancers caused by high-risk HPVs. Human papillomaviruses (HPVs) are important pathogens that are the causative agents of many anogenital as well as oral cancers . Over 40HPV virions consist of an outer icosahedral shell that encapsidates its 7.9 kb circular double-stranded DNA genome that is bound by cellular histones. HPVs infect cells in the basal layer of stratified epithelia that become exposed by wounding. Following entry, viral genomes are established as nuclear episomes at about 100 copies per cell. These infected basal cells can persist until they are cleared by the immune system or progress to cancers that are often associated with the integration of viral DNAs into host chromosomes. In productive lesions, the synthesis of progeny virions, however, occurs in highly differentiated suprabasal cells ,6. HPV genomes consist of two major coding regions along with a third regulatory region referred to alternatively as the upstream regulatory region (URR) or the long control region (LCR) . The earThe E6 proteins are approximately 100 amino acids in size and are localized in both the cytoplasmic as well as nuclear compartments. The high-risk E6 proteins interact with a series of cellular factors that contribute to cellular transformation. The most prominent of these is ubiquitin ligase UBE3A (E6AP), which recruits p53 resulting in its rapid turnover ,15. HighThe E7 proteins are found primarily in the nucleus and bind to members of the Rb family of proteins. Binding of high-risk E7 protein induces degradation of Rb leading to constitutive activation of E2F family members, resulting in rapid entry into S-phase ,26,27,28The E1 proteins bind to HPV origin sequences and help to recruit cellular replication factors including DNA polymerases to viral genomes. E1 proteins also act as helicases to unwind strands of DNA ahead of replication complexes. Monomeric E1 proteins bind DNA with low affinity and form complexes with E2 proteins to facilitate their recruitment to HPV origins that are flanked by E2 binding sites ,35,36,37The E4 open reading frame is expressed as fusion proteins with a short peptide from the beginning of the E1 ORF to encode E1^E4 proteins that may facilitate virion egress ,46,47. EHPV virions infect cells in the basal layer of stratified squamous epithelia that become exposed upon some form of wounding. Following entry, viral genomes migrate to the nucleus where they are established as low-copy episomes. Early transcripts encoding E6, E7, E1 and E2 are expressed resulting in the establishment of viral genomes at copy numbers of about 50 to 100 per cell ,52. ReplHPV replication including differentiation-dependent amplification is mediated in large part by cellular enzymes whose activities have been hijacked by viral proteins. This includes polymerases and single-strand DNA binding proteins among others. Recent evidence indicates that HPV proteins activate members of the DNA damage repair pathways (DDR), and this is critical for viral replication . All celATM proteins are activated through autophosphorylation following recruitment to sites of double-strand breaks by the MRN (Mre11–Rad50–NBS1) complex of proteins . ActivatThe homologous recombination arm of DNA repair takes place in G2/M as it requires the presence of a sister chromatid as a template . FollowiReplication fork arrest can also induce DNA breaks, which can occur through the generation of bulky DNA lesions as well as collisions of RNA polymerases with DNA polymerases, resulting in formation of trimeric RNA:DNA structures called R-loops . R-loopsHPV-positive cells constitutively activate these pathways to levels significantly higher than normal cells ,71. ThisThe cohesin protein, SMC1, functions not only in facilitating pairing of sister chromatids in G2/M but is also critical in DNA repair, allowing for the recruitment of DDR factors. SMC1 also forms complexes with the insulator protein CTCF to regulate the formation of large DNA loops called topologically associating domains or TADs that regulate both transcription and replication .These doDNA topoisomerase II beta-binding protein 1 (TopBP1) is another protein involved in regulating ATR activation and HPV replication. TopBP1 binds and helps to activate ATR in addition to being a target of ATR phosphorylation. Phosphorylation of TopBP1 by ATR activates its ability to act as a transcription factor that regulates the expression of several DNA damage repair factors along with E2F1 and p73. TopBP1 levels are increased in HPV-positive cells by the immune signaling protein STAT5 through the action of E7 . In addiTo establish a persistent infection in keratinocytes, HPVs need to escape surveillance by the innate immune response, which is a critical host defense mechanism that has evolved as the first line of protection against pathogens. The innate immune response consists of a variety of sensors, referred to as pattern recognition receptors (PRRs), that detect the presence of foreign components such as bacterial lipopolysaccharides or viral nucleic acids such as cyclic nucleotides, ssRNA or dsRNA, cytosolic dsDNA and RNA-DNA hybrids . DetectiHPVs interfere with several PRRs that are either membrane bound or cytosolic. Toll-like receptors (TLRs) are membrane-bound sensors that have been shown to be important in activating both innate and adaptive immunity . HPV16 EHPVs have also been shown to target cytosolic DNA sensors of the innate immune pathways . When ovRNA sensors such as dsRNA protein kinase (PKR), retinoic-acid-inducible gene I (RIG-I) and MDA5 have also been identified as targets of HPVs. The levels of RIG-I and MDA5 transcripts in keratinocytes infected with hr-HPVs were found to be significantly reduced . RIG-I sAdaptor proteins link the signaling from cytoplasmic sensors to the nucleus to initiate an innate immune response against pathogens . The stiThe endpoint of these innate signaling pathways is ISGs expression and IFN production. IFNs are classified into three types according to their sequence homology and to the receptors they signal through. IFN production and ISG expression provide two major functions: the first is to clear the viral infection and/or eliminate the infected cell; the second is to signal neighboring cells through secreted IFNs by initiating an antiviral program. Upon binding of IFNs to receptors on the surface of neighboring cells, Janus kinases (JAKs), which are bound to the cytoplasmic tails of receptors, become activated and phosphorylate a series of inactive transcriptional activators called STATs that are found in the cytoplasm. Phosphorylated STATs (predominantly STAT1 and STAT2) translocate to the nucleus to bind ISG promoter regions to activate transcription . Given tIn contrast to STAT1, HPVs appear to activate STAT5 and STAT3, which have been implicated in the development of a number of solid cancers (reviewed in detail in ). HPV E7HPVs suppress the expression of IFN-α and IFN-β through interference with STATs and binding kinase Tyk2 . KeratinThe innate immune response is activated in response not only to foreign nucleic acids but also to “misplaced” self-DNAs that arise during periods of DNA damage. These damage-associated molecular patterns or DAMPs are markers of cellular malfunction and are detected by innate immune sensors that can lead to programed cell death. Genotoxic stress induces genomic instability that causes DNA breaks. These broken DNA fragments can leak into the cytosol, where they are recognized by the PPRs. Sensors such as cGAS, IFI16 and RIG-I but also STING have been linked to genomic instability ,132,133.As described in detail previously, HPV oncoproteins induce DNA breaks in infected cells to activate DDR mechanisms, which are important for viral amplification. A linkage between the DNA damage response and innate immunity in HPV-positive cells is suggested by studies linking ATR activation with phosphorylation of the autophagy protein p62, which in turn acts to suppress the transcription factor GATA4. This results in the suppression of cytokines such as IL-5 and CXCL2 and IFNκ expression . OngoingIn this review article, we have described the current knowledge of two major pathways used by hr-HPVs to establish long-term persistent infections. HPVs activate host DNA damage repair pathways to allow for genome amplification upon differentiation as well as to manipulate the innate immune responses to avoid clearance. Furthermore, it appears that these two pathways are interlinked, and HPVs need to target both for a successful infection. These studies describe biomarkers that may facilitate prognosis as well as novel ways to efficiently target and treat HPV-positive anogenital and oropharyngeal cancers.


Single photon quantum emitters are important building blocks of optical quantum technologies. Hexagonal boron nitride (hBN), an atomically thin wide band gap two dimensional material, hosts robust, optically active luminescent point defects, which are known to reduce phonon lifetimes, promises as a stable single-photon source at room temperature. In this Review, we present the recent advances in hBN quantum light emission, comparisons with other 2D material based quantum sources and analyze the performance of hBN quantum emitters. We also discuss state-of-the-art stable single photon emitter’s fabrication in UV, visible and near IR regions, their activation, characterization techniques, photostability towards a wide range of operating temperatures and harsh environments, Density-functional theory predictions of possible hBN defect structures for single photon emission in UV to IR regions and applications of single photon sources in quantum communication and quantum photonic circuits with associated potential obstacles. Quantum information science  is an emerging interdisciplinary research field of science and technology, which is primarily concerned with analysis, processing, storage, retrieval and secure transmission of information through the set of quantum mechanical principles. The information in quantum science is inscribed using various physical properties of quantum elementary particles such as spin of an electron (spintronics) and polarization of a single photon (photonics) etc.Integrated quantum photonics is a sub-field of QIS (quantum information science), which uses photonic integrated circuits to architect quantum states of photons for intersecting the quantum applications.In a broad perspective, a single photon is attained by two main mechanisms such as excitation and followed by spontaneous emission. The elementary particle, electron either in a molecule or in atom always tends to exist in ground state (un-excited state).When this electron in ground state is excited (absorbs energy), moves to higher energy state (excited state) as shown in Fig. This excited electron stays not more than few nanoseconds in higher energy state (excited state) and spontaneously return back to lower energy state (ground state). During this decay, the electron emits the absorbed energy in the form of radiation as shown in Fig. This subtler process is called spontaneous emission which leads to emit a single photon and the quantum level light sources which emits a single photon (elementary particle of light) per one excitation cycle (combination of one excitation and spontaneous emission) is called single photon emitters often called as quantum emitters.2 of classical computing and data communications technologies. Quantum computers enhance the processing speed and efficiency than classical computing using qubits3. For quantum information processing, in optical systems, the unit of light ‘photon’ is used to represent a qubit.The numerous scientists and engineers working in the domain of quantum photonics and Integrated quantum photonics, worldwide predicted and proved that these quantum technologies would overcome the limitations4. These computing and communication techniques employs polarize states of single photons to encode the information.Secure information transfer by quantum communication  is highly in demand for defense organizations and financial institutionsHowever, traditional single photon emitters in three dimensional materials limits the photon extraction efficiency and integration ability to photonic circuits due to high refractive index.8. These naturally passivated 2D materials, without any dangling bonds, makes it easier to integrate with photonic structures such as waveguides12 and cavities17. Another notable property of these materials is that these materials strongly interacts with light18.The discovery of 2D materials from insulating hBN , semiconducting TMDs  to semi metallic graphene exhibits exceptional properties such as Quantum confinement in the direction perpendicular to 2D plane leads to novel electronic and optical properties. These novel properties significantly different from their bulk counterpartThe single photon emission in TMDs was initiated by exciton formation  generated by either optical or electrical excitation and a single photon is emitted during recombination of this electron hole pair.2, WS2, MoSe2, and Mos2) and GaSe46, found to trap and localize the excitons at particular location, which in turn leads to localized single photon emission at cryogenic temperatures.Generally the excitons formed in TMDs are of wannier–mott kind, which tend to diffuse  across lattice. The point defects and crystal imperfections in the TMDs  are used to funnel the excitons for effective single photon emission48. Electrostatic potentials in moiré patterns leads to effective exciton trapping, which in turn leads to single photon emission in bilayer TMDs, as observed in ref53.Particularly, in case of WSe55, which founds to be more suitable for quantum applications.On the other hand, in wideband semiconductors like hBN, the point defects acts as flawless single photon emitters. These point defects tend to exhibit intermediate energy states (un-occupied and electron occupied energy levels) in between the conduction band edge and valence band edge of the energy bandgap of the material and the electrons in occupied energy levels found to exist in paramagnetic form. Due to optical excitation, these paramagnetic electrons excites to higher un-occupied energy states and results in spontaneous single photon emission at wide range of temperatures56 and via external magnetic fields in ref57. Tunable quantum emission from very recent 2D heterogeneous structures such as graphene/hBN/Wx2 or Mox2/hBN/graphene (where x = Se or S) opens the door for fascinating applications like quantum memory58, quantum imaging and metrology61.Particularly, in hBN, enhancement of single photon emission was studied via external electric fields in refAchieving bright and stable single photon emitters in a single chip, which covers the emission range from UV to IR spectrum and works over a wide range of temperatures is really significant because each range of SPS (single photon source) have their own on-demand unique applications.62, the schematic representation of three main  point defects, hosts in hBN are shown in Fig. 66.Among various luminescent point defects predicted  hBN, these layers are arranged either with AA' stacking (B atoms of one layer are positioned above the N atoms of another layer or vice versa for N atoms of one layer w.r.t B atoms of another layer) as shown in Fig. Alternating boron (B) and nitrogen (N) atoms in its crystal structure, monolayer hBN is a spThese individual layers are packed together by weak van der Waals forces. The inter layer interactions affects the band structure of material and the variation in electronic band structure of monolayer and bulk hBN is shown in Fig. The variation in bandgap of hBN and their corresponding Raman shifts essentially depends on interlayer interactions and number of individual layers stacked. Owing to larger bandgap, hBN exhibits high transparency of electromagnetic spectrum. hBN also exhibits relatively high refractive index and other electrical/optical/crystal properties of hBN were listed in Fig. 76 as shown in Fig. The single photon emitter is a non-classical light source, emits only one photon per excitation cycle, observed experimentally using a Hanbury, Brown and Twiss (HBT) interferometerAt zero delay time, the second order autocorrelation curve dips below 0.5 indicates the characteristics of a single photon emitter.2 (τ)) is the main characteristic of a quantum emitter and other important features of an ideal single photon source (quantum emitter) to be considered while developing for quantum applications is listed in Fig. The single photon purity  range of ∼550–740 nm), revealed that the absorption and emission dipoles are frequently misaligned. This dipole misalignment is framed as:The possible range of dipole misalignments, responsible mechanisms and their influence on light absorption are listed in Table The comparison of some of the quantum emitter characteristics among hBN and 2D TMDs were listed in Table The advantages and drawbacks of existing, emerging single photon sources from single atom to very recent TMDs and 2D heterostructures are represented in Table 96, emitters are found to be activated by annealing the samples for 30 min at 850 °C under 0.5 or 1 torr of argon. The argon gas is selected to prevent oxidation. Annealing at different temperatures found that fluorescence intensity is maximum at 850 °C.In bulk hBNTo date, no superior techniques were experimented to fabricate the new emitters in bulk hBN, as the emitters in bulk hBN faces the disadvantages as listed in Table 97, found to host stable quantum emitters around 200 (per 10 × 10 µm2 area) at room temperature. The growth technique was performed in quartz tube as schematic shown in Fig. hBN multilayers grown using low-pressure chemical vapor deposition (LPCVD) technique98.More than 85% of emitters exhibit ZPL around 580 nm, with single photon purity 98 and Ar plasma etching66 and their corresponding processing details were listed in Table Other robust methodologies determined to fabricate quantum emitters in CVD grown multilayer and exfoliated multilayer hBN flakes are electron beam irradiation, laser irradiation, ion implantationQuantum emitters are engineered in CVD grown multilayer hBN and exfoliated multilayer hBN flakes, by performing stable emitter fabrication techniques as described in Table 99 surface states are often unstable and modifies upon annealing. An important analysis from emitter formation and activation process are listed in Table To date, no supreme emitter fabrication techniques were experimentally demonstrated for bulk  and monolayer hBN and no annealing is performed to activate emitters in monolayer hBN as66 and cathodoluminescence analysis of exfoliated multilayer flakes found single photon emission in the UV region63 with a ZPL at around ~ 302 nm.In exfoliated multilayer hBN flakes, Ar plasma etching and subsequent annealing in argon produces emitters with emission wavelength greater than 700 nm102 structures. Due to this reason, we present the quantum emission properties of hBN material in bulk, multi and mono layer form.In traditional materials like diamond and silicon carbide, colour centres have similar spectral properties in both bulk and nano dimensional forms. But, in case of van der Waals crystals, the optical properties of 2D materials are different from their bulk96 was performed by complete optical characterization setup  setup couple to HBT interferometer) as shown in Fig. Optical characterization of quantum emitters in bulk hBNThe PL map of bulk hBN shown in Fig. a = bunching factor.The red dots in second order autocorrelation graph, shown in Fig. The emission from the defect bleaches after several minutes of excitation with 532 nm laser source. To obtain additional insight about luminescent point defect detection and its bleaching affect, the optical characterization was performed by replacing the 532 nm laser with another excitation laser of long wavelength (675 nm).As similar to the emitter highlighted in Fig. 105 but has the disadvantages as listed in Table Although, the emitters in bulk crystals are brightest and most stable102.Specifically emitters reported in 2D hBN has PL spectra, photostability and photodynamic characteristics which are significantly different from their bulk counterpartOptical characterization of emitters from CVD grown hBN multi, single layer and exfoliated multilayer flakes were analyzed and it was noted that their PL spectrum exhibiting ZPL at ~ 580 nm to 623 nm.64 and the emitter in hBN multilayer exhibits a stable fluorescence without any blinking or bleaching . But in case of hBN monolayer the emitter blink and bleach for continuous excitation.The emission lines in multilayer are narrower than monolayer as observed in ref106. The emitters detected exhibits ZPL range from 550 to 700 nm with better single photon purity.The creation and characterization of single photon emitters in a suspended, single crystal hBN film and free from substrate interactions were recently studied as in refA significant difference in brightness was observed for the emitters detected in freely suspended hBN film compared to the emitters detected in substrate supported hBN, which provides a breakthrough advances in fabricating quantum photonic circuits.107.Fluorescence measurements from traditional ensemble emitters were limited due to heterogeneity of emitters and individual defect-by defect studies are impractical. New techniques were proposed to quickly and accurately characterize the emission from ensemble emitters as experimented in ref54, capable to withstand aggressive annealing treatments in both oxidizing and reducing environments without any change in spectral properties65.Quantum emitters in hBN multilayer exhibits absolute thermal stability, optically stable even when operated upto 800 K55 of an emitter observed from cryogenic to room temperature is shown in Fig. The emitters’ stability towards temperature variation, annealing treatments in various gaseous environments is shown in Fig. 66 as listed in Table Quantum emitters in hBN multilayers exhibit a broad range of UV to near IR single photon emissionEmitters in hBN multilayer are classified into two groups based on Zero phonon line (ZPL) and phonon sideband (PSB) shapes. Emitters in group 1 have broad ZPLs along with PSBs and energies ranging from 576 nm (2.15 eV) to 652 nm (1.90 eV). Emitters in group 2 have narrow symmetric ZPLs with weak PSBs and energies ranging from 681 nm (1.82 eV) to 762 nm (1.63 eV).65.In a survey of ~ 40 emitters, 70% belongs to group 1 and rest 30% of emitters belongs to group 2. The variation in ZPL of emitters in both the groups is due to variations in local strain and dielectric environment96 of 0.1 s as shown in Fig. 112. The presence of fewer metastable states with shorter life time makes the emitter brighter than the emitter having more metastable states with long lifetimes.The autocorrelation function measurements over a long time scale However, robust, optically strong and thermally stable single photon emitters are detected in hBN, these emitters faces the limitations of moderate emitter brightness (fluorescence intensity), non-ideal single photon purity, more emitter lifetime, spectral diffusion at cryogenic temperatures and inhomogeneous emission distribution over a large spectral band. These limitations create a central problem for fabricating identical single photon emitters with ideal characteristics, which strengthen to fabricate the efficient quantum applications.Many new techniques are proposed and proved to overcome these limitations and enhance the emission characteristics of hBN quantum emitters as explained below:Excitation of hBN quantum emitters with single laser (either with longer wavelength (675 nm) and high power (300 µW) or with mid-wavelength (532 nm) and low power (10 µW)), exhibits the photoluminescence measurements having moderate or less fluorescence intensity and photon emission rate.Autocorrelation measurements of quantum emitters  revealed existence of a fast decaying intermediate state and a long-lived metastable state within the transition energy gap as shown in Fig. 113 as shown in Fig. By employing simultaneous both red laser (675 nm and 300 µW) and green laser (532 nm and 10 µW) excitationsThe intensified photo physical characteristics of emitter due to two laser excitation in comparison with single laser excitation were listed in Table Another superior technique to enhance the quantum emitter characteristics rather than simultaneous exciting with two laser beams is coupling the quantum emitters with plasmonic nanoparticles like gold, silver and platinum etc.114, enhances the excitation rate due to surface Plasmon resonance and spontaneous emission rate enhancement by Purcell effect (enhancement by environment).Overall photon emission rate enhancement is made possible by combination of both excitation enhancement and spontaneous enhancement rate. Coupling the gold nanospheres to the emitters in exfoliated flake edges as observed in refUsing AFM tip, the gold nanospheres are positioned to close proximity of emitters as shown in Fig. An effective alternate to plasmonic coupling enhancement is by inducing external magnetic field.It is a known fact that electronic structure of defects in wide band semiconductors possess both electron un-occupied and occupied states in between the conduction and valence band edges of the energy gap. These occupied electrons exists in paramagnetic form (possibly with up ↑ or down ↓ spins). By inducing external magnetic field, these occupied energy states split up due to Zeeman effect (let's say one occupied state with a paramagnetic electron split up into two higher and lower energy states with energy difference (ΔE) and this paramagnetic electron prefers to sit in lower energy state).57.When this lower energy paramagnetic electron absorbs a photon with energy = ΔE, then it jumps to higher energy state and emits the photon (with same or different energy) while relaxing back to lower energy state. This spin-dependent inter-system crossing (ISC) transition found to exhibit an anisotropic fluorescence response for applied magnetic field as experimented in refThe schematic representation of inducing external magnetic field to the hBN film and simultaneous optical excitation is shown in Fig. The variation in PL spectrum with and without magnetic field is shown in Fig. 115, which leads to erroneous ZPL measurements.At cryogenic temperatures, the ZPL shape of emitters was influenced and broadened due to spectral diffusion116 (electron is excited directly to minimum of excited state and then follows spontaneous emission).This spectral diffusion is observed in off-resonant excitation . The control of ZPL broadening  is observed in on-resonant excitationhf) is more than luminescent point defect ZPL energy (ΔEZPL) and on-resonant excitation occurs when hf = ΔEZPL. The controlled photophysical characteristics of emitters due to on resonant excitation is shown in third row of the Table This off- resonant excitation occurs when excitation energy  exhibits correlation value 78 (indicating the reduction of background fluorescence) compared to the emitters found in non-irradiated area exhibiting Emitters found in the He65 due to variations in local strain and dielectric environment, which creates a central problem for fabricating identical single photon emitters.Emitters in hBN multilayers exhibit different emission energies (ZPL) over a large spectral band Tuning the quantum emitters (having uneven ZPL emission energies), through external strain inducement or through external electric field via stark effect or electrostatic tuning through ionic liquid devices, leads to increase the probability of multiple uneven emitters (different ZPL energies) to have the same emission energy (ZPL), which further tends to fabricate identical quantum emitters with necessary emission wavelength.78 as shown in Fig. Controllable external strain can be induced to the emitters in hBN film by placing on a 1.5 mm thick bendable polycarbonate (PC) beam117 for deterministic single photon emitter formation by a combination of nanoscale strain engineering and charge trapping.Similar experimental approach was observed in ref118. The schematic representation of multilayer hBN between top and bottom graphene electrodes (for electric field inducement) is shown in Fig. Quantum emitters in hBN are expected to lie within the plane, which creates an in-plane dipole. By inducing an out-of-plane electric field through graphene gates, electrical tuning of quantum emission (via stark effect) from luminescent point defects hosts in hBN was observed in ref97 as shown in Fig. The quantum emission from luminescent point defects in hBN can also be tuned by placing the hBN sample in ionic liquid devicesBy applying positive (negative) gate voltages, the ZPL emission is found to be blue shifted (red shifted) by around 15 nm as shown in Fig. 119 (absorbs two photons of same frequency and generates a single photon of twice the frequency absorbed) in exfoliated multilayer hBN as shown in Fig. 120.Contemporary exploration on hBN integration with photonic microstructures like circular Bragg grating (CBG) was found to be eminent and rooted out the second harmonic generation3N4)121 and Al nano-antenna122, which revealed a 6-fold and 10 to 15 fold enhancement in photoluminescence measurements of a hBN quantum emitter at room temperature respectively.Novel techniques to enhance the quantum emission from hBN single photon sources is by coupling the quantum emitters with photonic crystal cavities from silicon nitride  substrate) and similar investigation was performed on other 2D TMDs and disseminate that TMDs exhibit significant difference in contrast on transparent substrates than opaque substrates.It was Serendipity, that hBN was found to enhance the contrast on transparent substratesThe simulation results also reveal that contrast of hBN is high under shorter wavelength light than longer wavelength. Finally it was negotiated that selecting an appropriate substrate for optical experimentation is very crucial.124 was observed in hBN layers.Tuning of quantum emission spectrum is found to feasible with externally applied tensile strain and present day a record tuning of 65 meV125 and Vienna Ab initio Simulation Package (VASP)126 on the basis of generalized gradient approximation by Perdew, Burke and Ernzerhof (PBE) functionals127.A list of 35 different possible luminescent point defects of hBN , as shown in Fig. Based on experimental observations, three main conditions, as shown in Table BVN, OBOBVN and CBVN are the defect structures found to satisfy the rules listed in Table Among the 35 luminescent point defects, NBVN64 defect structure and simulated (using VASP) electronic structure of NBVN defect in hBN monolayer is shown in Fig. The Emitters detected in experimental studies (emission in visible region) whose ZPL is around ~ 2 eV, predicted to have NBO2 defect (obtained using VASP simulations) in hBN monolayer is shown in Fig. 66 examined by DFT using hybrid functionals (HSE06), found that B-vacancy with two oxygen atoms (VBO2) defect structure as shown in Fig. BO2 defect  found to be consistent with experimental ZPL energy of near IR emitters fabricated using Ar plasma etching.The electronic structure of V131.Recent studies reveal that boron and nitrogen dangling bonds as shown in Fig. N defect (nitrogen atom is replaced with carbon) and CB defect132 (boron atom is replaced with carbon). Their quantum emission  was observed ~ 4.1 eV, which were consistent with experimental works as described in ref63.Numerous DFT calculations anticipate that single photon emission in UV region is due to carbon substitutional defects like C114, the enhancement of single photon source emission characteristics due to coupling with gold nanospheres was investigated as shown in Fig. By using Finite Difference Time Domain (FDTD) simulations as demonstrated in refBVN defect structure as shown in Fig. 78 to create similar effects of strain, induced using polycarbonate (PC) beam in experimental works.Strain tunable quantum emission from NBVN defect is observed ~ 2.01 eV (black peak), when no strain was applied (zero strain). When tensile strain is applied along ZZ1 direction, the emission peaks were tunable towards lower wavelengths (blue tones) and similarly, when tensile strain applied along AC2 direction, the emission peaks were tunable towards higher wavelengths (red tones).The quantum emission from Ni.Quantum computing,ii.Quantum cryptography (Quantum communication),iii.Quantum imaging and metrology andiv.Other fascinating applicationsMany applications of quantum emitters have been proposed and demonstrated in literature. Some major applications of quantum emitters are discussed here:In classical computing, a conventional bit can be either a 0 or 1. Quantum computer provides a breakthrough revolution, in which a qubit can be both 0 and 1 at the same time on the basis of superposition principle.In optical quantum computing, the single photons are considered as qubits. Initially, these single photons generated from single photon emitters can be either horizontally polarized (considered as logic 0) or vertically polarized (considered as logic 1). Then processing of qubits like creating superposition state (both logic 0 and 1 at same time) and flipping of qubits (logic 0→logic 1 or logic 1→logic 0) can be created using quantum gates like Hadamard (H) gate and Pauli-X gate etc. respectively. These quantum gates are developed using set of birefringent wave plates and the encrypted qubits finally accumulated using single photon detectors.136 are used for discussion.The schematic representation of a quantum photonic circuit in a quantum computer, constructed using single photon source, set of quantum gates and polarized beam splitters (PBS) and single photon detectors as shown in Fig. 137 setup is shown in Fig. Quantum cryptography technique is used to encrypt the key by using quantum effects (polarized photons) for secure communication and the complete quantum communicationThe quantum communication setup in which the single photon source (highlighted in red colour) is present at the transmitter, coupled to HBT setup for controlling the quality of source and EOM  to make photons polarized. The sender and receiver are separated by a distance of 50 m. The horizontal–vertical (H−V) and circular left–circular right (L−R) polarization basis photons were used for data encryption.87 (free space) and Quantum dots138  are used as single photon emitters in quantum communication systems.To date, Color centers in diamondQuantum imaging is a new sub-field of quantum optics, makes use of entangled photons to image the objects with higher resolution or other imaging criteria beyond the limitations of classical optics.140 are generated using BBO (beta barium borate) crystal.The simplified schematic of quantum imaging experiment is shown in Fig. 142 exploits entangled photons to make high sensitive measurements of assorted signal  and physical parameters in order to avoid statistical errors. The schematic representation of quantum metrology circuit is shown in Fig. Similar to quantum imaging application, quantum metrology2, MoSe2 and MoS2 etc.) were listed in Table Other fascinating quantum technology applications of single photon emitters  can be implemented by 2D materials using various phenomena like spontaneous emission associated to intermediate energy state transition in hBN, spontaneous emission associated to localized excitons trapped and funneled due to point defects and crystal imperfections in TMDs.Electrostatic potential traps due to moiré patterns and intentionally induced strain gradients can also effectively tap excitons for qubits implementation.2 material the crystal imperfections due to intentionally induced strain gradients by transferring on dielectric nanopillars and folding the material using gapped golden rods as shown in Fig. Especially, in case of WSeMoiré super-lattice of 2D heterobilayers structure as shown in Fig. 2/hBN/graphene heterostructure as shown in Fig. 2 acts as recombination layer, hBN as tunneling barrier and graphene as transparent electrodes. To verify the effect of charge trapping complete structure is divided into two: one with MoS2 layer (GBM) and without MoS2 layer (GB).Qubits can also be implemented by electrical excitation of excitons using 2D heterostructures like graphene/hBN/MoS2/hBN/graphene heterostructure is shown in Fig. 2, electrons tunnel through the hBN barrier into MoS2 layer and quasi fermi level of graphene (right contact) reaches maximum of valence band of MoS2, holes tunnel through the hBN barrier into MoS2 layer. Injection of electrons and holes in MoS2 layer (recombination layer) leads to formation of exciton recombination followed by single photon emission.Energy band structure of graphene/hBN/MoS61 using 2D heterostructures, as similar to shown in Fig. Electrical manipulation of excitonic emission to generate entangled photonsFew major challenges are noticed in fabricating hBN single photon emitters and developing towards quantum applications. The challenges associated with emitters (luminescent point defects) hosts in hBN were listed in Table One of the major challenge to be resolved is moderate single photon purity  due to residual background emission. In most of the experiments it was noted that emission intensity of emitters spans over a large spectral band  which is a central problem for developing identical single photon sources.High refractive index of the material which makes difficult to confine the light within the hBN structure is another important problem to be addressed and finally, indistinguishability of photons from the emitter is very poor.The challenges associated with optical quantum applications were listed in Table In case of quantum communication, Security loopholes or side channels leads to in-secure quantum communication. Currently, probabilistic single photon sources are used for entangle photon generation which are not reliable for on-demand applications like quantum imaging and metrology.The complete summary of hBN materials used and their synthesis, stable emitter formation techniques, defects consistent with experimental studies, important photophysical characteristics of emitters and their emission enhancement techniques and major applications of quantum emitters were discussed in Table Single photon emission is observed from single atom in a cavity to recent 2D materials, however, each of these sources have their own constraints. However, the state-of-the-art quantum emitters in 2D materials found to be an effective alternate to traditional single photon emitters in three dimensional materials due to the disadvantage of photon extraction efficiency and external circuit coupling.46 and in their Moiré patterns53 at cryogenic temperatures. Despite, inducing a stronger strain in TMDs is found to improve their operating temperatures22 and quantum yield48. It’s an auspicious that emitters observed in multilayer hBN are found to be stable at a wide range of operating temperatures, various harsh environments and found to be localized at flake edges in mechanically exfoliated flakes which enhances the device fabrication and coupling to plasmonic cavity for higher integration quantum photonic circuits.In the quest of A1 supreme quantum emitters, strong excitonic single photon emission is observed in monolayer TMDs2) in large few-layer hBN fabricated with LPCVD technique, strongly publicized that more than 85% of the emitters have a ZPL at (580 ± 10) nm, which is the confined uneven spectral distribution reported to date97.This solid quantum emitters in hBN are found to be generated by Ion implantation, laser ablation, Ar plasma etching followed by subsequent annealing and electron beam irradiation. But, the nature of emitters formed is still under debate. The cutting-edge research is fabricating stable quantum emitters in higher order 120 was discovered in hBN multilayer coupled to circular Bragg grating (CBG) photonic microstructures which, encourages the delightful applications.To enhance the performance of quantum photonic devices and converge to on-demand applications, fabricating the quantum emitters which covers the wide range of emission on a single platform is really worthy of attention. Significantly, hBN exhibits broad emission range from UV to near IR region and the first signature of Rabi oscillations and resonant fluorescence emission was observed from a resonantly drive hBN quantum emitter97 and very recently electric and magnetic field inducements57 etc., techniques found to enhance and tune the quantum emitters emission properties and control the inhomogeneous spectral distribution. To date a record tuning of quantum emission around 65 meV124 and higher rate of emission enhancement (around 6 to 15 fold) owing to coupling with photonic crystal cavities from silicon nitride (Si3N4)121 and Al nano-antenna122 was perceived. However the quantum emitters in hBN experience spectral diffusion at cryogenic temperatures, Resonant and antistokes154 excitation technique found to overcome the complication due to spectral diffusion.Although the emission characteristic measurements in hBN were not up to the mark, quantification enhancement techniques like Two laser excitation technique, plasmonic coupling using gold nanospheres, external strain engineering, electrostatic tuning through ionic liquid devices155. However employing different substrates like Al2O3156 or InGaP157 found to reduce the random spectral fluctuations, layered hBN mounts a problem of invisibility on opaque substrates under white light illumination. Recent explorations make a Serendip that hBN was found to enhance the contrast on transparent substrates123, which makes experimental works feasible on hBN.Emitters in hBN are mostly stable under illumination at 532 nm. On the other facet, emitters exhibits fluorescence instability and spectral diffusion at blue laser excitation146 at room temperature. A waveguide of proper design is needed to improve the coupling of quantum emitters and filter the propagating laser light, to enhance the feasible implementation of quantum repeaters.Efficient photon extraction and coupling to external photonic circuits and devices is at most important for practical implementation for quantum applications. Out of sort, only 10% efficiency in photon extraction using an optical fiber and coupling to an optical waveguide is achieved58. Entanglement of photons for Quantum imaging and metrology was made possible by a back-gated WSe2 monolayer, hBN and graphene heterostructure61, overcome the drawback of generating entangled photons at random process in nonlinear BBO crystal.As a quantum breakthrough, tunable excitonic emission in hybrid structures initiate the development of quantum memoriesBVN defect structure is more consistent with the emitter responsible for visible region emission, VBO2 defect for near IR region and CN defect for UV emission. Recent theoretical studies reveal boron dangling bonds125 are also the likely source of emission around 2 eV and CB defect126 as another candidate responsible for UV emission.Several computational evidences disclose that point defects responsible for single photon emission. Investigation of 35 different likely possible point defect structures by density-functional theory (DFT) provides a strong evidence that N


Volumetric and health assessment of the liver is crucial to avoid poor post-operative outcomes following liver resection surgery. No current methods allow for concurrent and accurate measurement of both Couinaud segmental volumes for future liver remnant estimation and liver health using non-invasive imaging. In this study, we demonstrate the accuracy and precision of segmental volume measurements using new medical software, Hepatica™.MRI scans from 48 volunteers from three previous studies were used in this analysis. Measurements obtained from Hepatica™ were compared with OsiriX. Time required per case with each software was also compared. The performance of technicians and experienced radiologists as well as the repeatability and reproducibility were compared using Bland–Altman plots and limits of agreement.High levels of agreement and lower inter-operator variability for liver volume measurements were shown between Hepatica™ and existing methods for liver volumetry (mean Dice score 0.947 ± 0.010). A high consistency between technicians and experienced radiologists using the device for volumetry was shown  as well as low inter-observer and intra-observer variability. Tight limits of agreement were shown between repeated Couinaud segment volume (+ 3.4% of whole liver), segmental liver fibroinflammation and segmental liver fat measurements in the same participant on the same scanner and between different scanners. An underestimation of whole-liver volume was observed between three non-reference scanners.Hepatica™ produces accurate and precise whole-liver and Couinaud segment volume and liver tissue characteristic measurements. Measurements are consistent between trained technicians and experienced radiologists.The online version contains supplementary material available at 10.1007/s00261-021-03262-x. Hepatocellular carcinoma is the fastest growing cause of cancer-related deaths in the USA , with 18It is widely understood that the estimation of the volume and health of remnant liver parenchyma , 11 is aFormal anatomical resection techniques are widely applied to patients with multiple, or deep-lying, lesions such as the extended right hepatectomy where Couinaud segments 1, 4, 5, 6, 7 and 8 are removed. This often results in an FLR of 25–30%, indicating an unsuitability for patients with an inflamed or cirrhotic liver . In suchLiver volume measurements and subsequent FLR estimations are typically performed by the radiologist using time-consuming manual techniques  witParticipants were selected amongst volunteers who had taken part in two previous ethically approved studies with informed consent. Liver MRI data of 48 volunteers from 2 different studies were used in evaluating the performance of the device. 18 participants (Group A) were used to verify the volumetry accuracy. 30 healthy volunteers (Group B) were used to evaluate the repeatability, reproducibility and intra- and inter-operator variability . These studies were approved by their respective ethics committee (IRAS IDs 241312 and 226607). Subject demographics are shown in Table Multiple scanners were used in this study in order to evaluate the reproducibility  and repeatability  of the new device. These scanners were Siemens Prisma 3T, Siemens Avanto fit 1.5T, GE Discovery MR750 3T, GE Optima MR450w 1.5T, Philips Achieva dStream 3T and Philips Ingenia 1.5T. Fat-saturated T1-weighted gradient recalled echo (GRE) images without contrast-agent were used for volumetric analysis. cT1, PDFF and T2* maps were acquired as previously described using multislice shortened MOLLI and IDEAL sequences , 32 withHepatica™ delineates the liver from a 3D T1-weighted MR image. The volume corresponding to the liver is segmented using a convolutional neural network (CNN) that automatically delineates the liver including the caudate. A technician can then refine the volumes obtained from the CNN with manual edits using paintbrush tools and liver lesions are excluded from the segmentation and quantifications. Liver volume data were also analysed by two trained radiologists (9 and 12 years training) using OsiriX software.Couinaud classification of liver anatomy divides the organ into nine segments, based on the vasculature . In the MultiScan™  has been demonstrated to be an accurate biomarker of hepatic fibroinflammation [Multislice cT1 extracted from Liverammation  in MR imcT1 maps are generated from 5 axial slices of T1 maps (shMOLLI) 8 mm thick with 12 mm gap, corrected for the presence of hepatic iron from a T2* map (DIXON) . PDFF isComparisons between pairs of measurements were assessed using Bland–Altman plots, and 95% limits of agreement (LOA) were calculated. The accuracy of Hepatica™ reporting whole-liver volumetry was evaluated in comparison with OsiriX, both operated by two trained radiologists. Additionally, the accuracy of a trained technician reporting whole-liver and Couinaud segment volumetry, PDFF and cT1 was evaluated by comparing results of a technician with the average of two experienced radiologists. Time spent per case by users in both devices was also measured.Precision is defined in terms of repeatability and reproducibility. Reproducibility is the difference of metrics of the same patient between the reference scanner  and non-reference scanners . Repeatability, performed on each of the six scanners, was measured as the difference between two acquisitions of the same patient under the same scanner, roughly 10 min apart. The patient was scanned, removed from the scanner, then returned and rescanned in order to induce realistic positional variation.Intra- and inter-operator variability was assessed by one technician examining the same dataset twice and two technicians examining the same dataset.n = 36 cases, 18 patients analysed separately by each radiologist, mean Dice score 0.947 ± 0.010), with resultant volume measurements from the two devices in strong agreement  and the associated low number of cases with underlying liver disease (n = 15). With the continued use of this device in more patients being considered for liver surgery, we intend to surveil the accuracy of liver tissue characterisation.The clinical utility of this medical software for preoperative planning is further demonstrated beyond saving time, as it quantifies liver health using cT1 and PDFF within the whole liver as well as within the remaining segments of the future liver remnant (FLR). Additionally, when the FLR of a particular operation is requested, radiologists must typically delineate each surgical option manually; in the device examined here, the volume of each Couinaud segment is generated Fig. ; thus, tIn conclusion, Hepatica™ is a new medical device that provides robust whole-liver and Couinaud segment volume and liver tissue characteristic measurements to support the treatment decision-making process. This enables surgeons to make individual assessments of a patient based on the volume and health of remnant livers prior to resection for liver cancer.Supplementary file1 (DOCX 60 kb)Below is the link to the electronic supplementary material.