Myo-inositol is a ubiquitous metabolite of plants. It is synthesized by a highly conserved enzyme L-myo-inositol phosphate synthase . Myo-inositol is well characterized during abiotic stress tolerance but its role during growth and development is unclear. In this study, we demonstrate that the apical hook maintenance and hypocotyl growth depend on myo-inositol. We discovered the myo-inositol role during hook formation and its maintenance via ethylene pathway in Arabidopsis by supplementation assays and qPCR. Our results suggest an essential requirement of myo-inositol for mediating the ethylene response and its interaction with brassinosteroid to regulate the skotomorphogenesis. A model is proposed outlining how MIPS regulates apical hook formation and hypocotyl growth. Apical hook formation occurs soon after germination and is maintained while seedlings makes their way through the soil and terminates upon exposure to light. The apical hook formation is orchestrated by a variety of hormones which leads to differential cell elongation in the hypocotyl1. Apical hook formation goes through three consecutive growth phases i.e., formation, maintenance and opening41. The plant hormones, auxin and ethylene interaction leads to differential growth in the formation of apical hook34. They have been involved in differential growth in the apical hook34. However, the mechanism by which ethylene triggers differential growth in the hypocotyl still far from understood.The apical hook formation and maintenance is one of crucial developmental process in higher plants as it protects the shoot apical meristem (SAM) till seedling emergence out of the soil19 in constitutive triple response1 (ctr1) mutant30 and the ethylene overproducer (eto) mutants55. Auxin and ethylene are involved in differential growth in apical hook, in which auxin results in cell expansion and hypostyle growth, while ethylene has an antagonistic effect34. Moreover, ethylene has a stimulatory effect on the auxin biosynthetic pathway51 which suggests another mode of interaction at the hormone level. As both auxin and ethylene are involved in the regulation of apical hook development, their activities are mutually coordinated.Ethylene enhances apical hook curvature as observed upon application of exogenous ethylene35. Arabidopsis mutants of auxin response such as TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and AFB members15 and over-accumulating free auxin34, exhibit hookless phenotype. Exogenous treatment with auxin and of polar auxin transport inhibitors affects hook curvature49, which suggests that optimal auxin transport is critical for differential growth in the apical hook and is regulated by dedicated influx and efflux carriers like AUX/LAX family of auxin influx carriers56, the PIN family of auxin efflux carriers38 and by the B-type ATP-binding cassette transporters (ABC transporters)45.Auxin gradient is one of critical factor for apical hook formation9. BR mutants defective in BR synthesis like det2, cbb1, cpd shows no hook formation during skotomorphogenesis52 and at lower concentration BR stimulates stem growth21. Exogenous ethylene treatment results in altered auxin gradient either directly or change in BR biosynthesis19. All the three hormones, BR, ethylene and auxin affect each other and are necessary for apical hook formation.Brassinosteroid (BR) is a steroid hormone which binds to receptor kinase BRI1 to initiate the signal transduction, e.g. inactivation of GSK3-like kinase; BIN2, dephosphorylation of BRASSINAZOLE RESISTANT 1 (BZR1) family transcription factor; accumulation of unphosphorylated BZR1 in the nucleus; and regulation of BR target genes36, auxin storage2, phytic acid synthesis3 and oligosaccharides synthesis28. MIPS is also required for PIN protein localization, polar auxin transport and auxin-regulated embryogenesis37. Its over-expression results in resistance towards cold, drought and salt stress in several plants along with immunity towards stem nematodes in transgenic sweet potato59. Programmed cell death (PCD) was observed in AtMIPS1 mutant which is light dependent and results in enhanced basal immunity43. Interestingly, Ma and coworkers in 2016 indicated the role of light signaling protein i.e. FHY3 and FAR1 in maintenance of optimal level of myo-inositol via directly binding to the promoter of MIPS1 and activating its expression39. Recent study of MIPS has revealed its critical role in growth and immunity via ethylene50.Previous reports showed that MIPS has been involved in cell wall biogenesisArabidopsis show that apical hook and hypocotyl development also depends on the myo-inositol level. Myo-inositol induced hook maintenance and subsequent stimulation of ethylene biosynthetic genes and auxin transporters suggest that the ethylene effect might be mediated by myo-inositol. Furthermore, myo-inositol (MI) antagonizes brassinosteroid (BR) effects during hook formation which demonstrate an important step of regulation of BR-mediated growth and development. Thus, we conclude that differential MIPS levels are required for optimal hook formation, maintenance and hypocotyl growth.Our investigation on etiolated seedlings of myo-inositol phosphate synthase (MIPS) suggest its role in ethylene response50. To further assess the role of myo-inositol (MI) in ethylene response, we analyzed the effect of myo-inositol on etiolated seedlings of Arabidopsis Col-0. Exogenous MI supplementation resulted in decrease in length of the hypocotyls and hook angle  treatment i.e. randomized growth of hypocotyls. Maximum randomization was observed at 100 nM concentration of EBL Fig. . MI suppArabidopsis seeds on media containing a combination of EBL and AgNO3. We found acute randomized growth of etiolated seedlings at different combination of EBL and AgNO3, which was increasing with increase in EBL and AgNO3 levels  followed by AgNO3 and LiCl (53%) when compared to AgNO3 (12%), BRZ (50%) and LiCl (0%)  Fig. E,F,O. A 0%) Fig. K–N. Perc0%) Fig. O, howeve0%) Fig. K.myo-inositol, we carried out the quantitative RT-PCR of AtACO3  was observed upon MI and ACC treatment  activity in MI 47. Inactivation of BIN2 by brassinosteroid signaling might lead to decrease in myo-inositol synthesis in dark resulting in randomized growth which is antagonized by supplementation of MI. In support of the later, we further investigated the effect of EBL in combination of LiCl on etiolated seedling and found excessive randomization even on 100 nM EBL with 10 mM of LiCl and similar result was observed combinatorial assay with EBL and AgNO3. Enhanced randomization of etiolated seedling with treatment with AgNO3 has also been reported by Gupta et al22. This study indicates that the brassinosteroid signaling decreases the ethylene response via inactivating the MIPS and results in increase in hypocotyl growth. However, treatment with saturating concentration of EBL results in antagonism probably due to a feedback mechanism40.Involvement of brassinosteroid in hook formation directed us to investigate the effect of myo-inositol phosphate synthase to brassinosteroid in hook formation substantiating the role of MIPS in ethylene synthesis. This indicates the existence of a cross talk between ethylene, brassinosteroid and auxin at the MIPS protein level leading to myo-inositol being crucial for hook as well as proper hypocotyl development.We checked the response of etiolated seedling in combination of BRZ and MI and found hook formation in etiolated seedlings compared to etiolated seedlings grown on only BRZ which suggests the upstream role of Atmips1 and Ataux1 mutant along with altered trafficking of PIN2 protein in Atmips111 led us to investigate the relationship of myo-inositol and auxin during hook formation. We carried out the combinatorial assay and found agravitropic behavior of etiolated seedlings with increase in IAA concentration. However, MI supplementation resulted in distinct decrease in hook angle and gravitropic growth. Hookless phenotype is observed upon over-accumulation of the active auxin (IAA), mutation of auxin polar transporter and treatment with inhibitor of auxin polar transport8. Previous report also indicates a crosstalk between ethylene and auxin in hook formation44 as inhibition of hook formation occur upon NPA treatment in eto1 and ctr1 mutant34 plus the restoration of hook occur in ethylene-insensitive mutant by auxin25. In the present study, we observe that myo-inositol was able to antagonize the effect of high level of IAA at 1 μM and 10 μM concentration. We thus conclude that myo-inositol phosphate synthase is involved in maintaining the optimal levels of auxin in the hook region via proper localization of PIN protein and ethylene synthesis which results in differential accumulation of auxin. TIBA is an auxin transport inhibitor which perturbs the auxin efflux, therefore plants treated with TIBA show agravitropic phenotype38. As expected hookless phenotype was observed when seedlings were grown in dark on media supplemented with TIBA along with agravitropic etiolated seedlings. Subsequent MI supplementation could not evoke hook formation but results in shortening of hypocotyl length. This suggests that differential auxin distribution is extreme downstream of MIPS in hook formation19.Due to importance of differential auxin accumulation in hook formation, similar behavior of myo-inositol are necessary for hook formation and their action are additive in nature as we could see additive effect of inhibitors in combinations. Another observation made was on hypocotyl length i.e. brassinosteroid is required for the hypocotyl growth as decrease in hypocotyl length was seen in BRZ treated etiolated seedlings in combination of AgNO3 and TIBA whereas the MI was antagonizing the effect of brassinosteroid as increase in hypocotyl length was observed in LiCl and BRZ treated etiolated seedling. Previous reports also suggest that hypocotyl growth is associated with brassinosteroid and ethylene antagonizes brassinosteroid effect in hypocotyl growth22. It is therefore MI is one of the important regulators of apical hook formation and hypocotyl growth. A model based on findings and published data have been proposed  seeds were sterilized with sodium hypochlorite for 10 min and three time washed with RO water. Seedlings were grown on half strength MS media (DUCHEFA BIOCHEMIE) without sucrose supplemented different concentration of MI, EBL, IAA, ACC, LiCl, BRZ, TIBA, AgNO3 as specified. Seeds were then cold stratified and exposed to 12 h light stimulate uniform germination. Plates were wrapped with aluminum foil and then transferred to growth chamber for 5 days at 22 ± 1 °C.48. Apical hook angle was measured by taking hypocotyl as a reference. When hook opens up, it creates a straight line. We considered it as 180° and opening of hook as an increase in hook angle. We measured the acute angle formed between the cotyledon and hypocotyl, i.e. inner edge of the apical hook. The photographs are of the 5-day old etiolated seedlings.Apical hook angle and hypocotyl length was measured using the ImageJ softwareAtmips1  was confirmed using left primer (LP), right primer (RP) and T-DNA-specific primers (LBb1.3) listed in Supplementary Table RNA was isolated from the different plant tissues (control and treated sample) using RNeasy plant mini kit . 5-day old control and treated etiolated seedlings were ground with liquid nitrogen and further proceeded according to the kit manual. In-column DNase treatment was done to remove the genomic DNA contamination. Quality and quantity of RNA samples were done by gel electrophoresis and nanodrop. 2 μg of RNA was used to make the cDNA using High-Capacity cDNA Reverse Transcription Kit (THERMOFISHER SCIENTIFIC) and SuperScript III First-Strand Synthesis System (THERMOFISHER SCIENTIFIC) for Full length cDNA synthesis. SYBR green PCR master mix (THERMOFISHER SCIENTIFIC) was used for qPCR analysis using primers listed in Supplementary Table Arabidopsis etiolated seedlings harboring MIPS1 promoters fused with Egfp:uidA gene were checked according to the protocol described by Jefferson et al.26. 5-days old Arabidopsis seedling were harvested and dipped in GUS staining buffer for 24 h at 37 °C and plant samples were then rinsed with 70% ethanol to remove chlorophyll from the stained tissue. 5-day old etiolated seedlings grown on media supplemented with MI, ACC, AgNO3, EBL, and BRZ were GUS stained and staining was observed using stereo microscope Leica M205 A .β-Glucuronidase activity in 5-days old All values reported in this work are the average of at least two to three independent biological replicates having at least 15 seedlings each. Error bars represent SE. Statistical differences between control and each treatment were analyzed using Student’s t test with paired two-tailed distribution.Supplementary Information


Do damaging gene variants account for increased cancer risk in patients with congenital heart disease (CHD)?In this case-control study, loss-of-function variants in cancer risk genes were increased approximately 1.3-fold in 4443 patients with CHD compared with 9808 control participants. This burden was highest in cancer risk genes previously associated with CHD (7.2-fold) or that regulate gene expression (1.9-fold); patients with CHD and extracardiac anomalies and/or neurodevelopmental delay had the highest rates of damaging variants in cancer risk genes.Genetic analyses of patients with CHD may identify precise causes of heart malformations and also patients with CHD and increased cancer risks. This case-control study compares the frequency of damaging cancer risk gene variants in patients with congenital heart disease vs control participants and identifies associated clinical variables. Patients with congenital heart disease (CHD), the most common birth defect, have increased risks for cancer. Identification of the variables that contribute to cancer risk is essential for recognizing patients with CHD who warrant longitudinal surveillance and early interventions.To compare the frequency of damaging variants in cancer risk genes among patients with CHD and control participants and identify associated clinical variables in patients with CHD who have cancer risk variants.This multicenter case-control study included participants with CHD who had previously been recruited to the Pediatric Cardiac Genomics Consortium based on presence of structural cardiac anomaly without genetic diagnosis at the time of enrollment. Permission to use published sequencing data from unaffected adult participants was obtained from 2 parent studies. Data were collected for this study from December 2010 to April 2019.−5) loss-of-function (LoF) variants in cancer risk genes.Presence of rare , were statistically assessed by binomial tests in patients with CHD and control participants.P = 1.38 × 10−12), and among CHD genes previously associated with cancer risk . The LoF variants were also nominally increased in 14 constrained cancer risk genes with high expression in the developing heart. Seven of these genes  harbored LoF variants in multiple patients with unexplained CHD. The highest rates for LoF variants in cancer risk genes occurred in patients with CHD and extracardiac anomalies  and/or neurodevelopmental delay .A total of 4443 individuals with CHD  and 9808 control participants  were included. The frequency of LoF variants in regulatory cancer risk genes was significantly higher in patients with CHD than control participants (143 of 4443 [3.2%] vs 166 of 9808 [1.7%]; odds ratio [OR], 1.93 ; Genotypes of CHD may account for increased cancer risks. In this cohort, damaging variants were prominent in the 216 genes that predominantly encode regulatory proteins. Consistent with their fundamental developmental functions, patients with CHD and damaging variants in these genes often had extracardiac manifestations. These data may also implicate cancer risk genes that are repeatedly varied in patients with unexplained CHD as CHD genes. This model is supported by the increased prevalence of damaging variants in CR genes among children with developmental delays,9 including autism and intellectual disabilities, which occur in some patients with CHD. To explore potential molecular relationships, we analyzed rare loss-of-function (LoF) variants in CR genes among a large CHD cohort and defined accompanying clinical features. These analyses identify patients with CHD and the highest CR gene burden, who may warrant longitudinal cancer screening.Damaging gene variants contribute to both CHD7 with undefined causes for CHD at the time of enrollment and unaffected control participants in studies of autism7 and schizophrenia10 , 216 CR genes that regulate RNA transcription or processing, and 227 CR genes with LoF mechanisms, including 107 genes with LoF germline mechanisms.The Catalogue of Somatic Mutations in Cancer–Cancer Gene Consensus database defines 723 CR genes  heterozygous LoF variants. All P values reflect binomial tests after Bonferroni correction with a P value threshold of 1.67 × 10−3 (10 gene lists and 3 comparisons). A false discovery rate P < .05 was used as the significant threshold throughout. The eMethods in the Whole-exome sequences from patients with CHD and control participants were processed using established pipelinesInitial analyses of LoF variants in CR genes . The presence of LoF variants was also increased among CR genes with regulatory functions , a prominent feature of many CR and CHD genes. Genes found in both categories  had the highest frequency of LoF variants in individuals with CHD . The CR genes with dominant variants that cause cancer by haploinsufficiency (n = 46) were also enriched in those with CHD , neurodevelopmental defects , both ECA and NDD (n = 878), and neither ECA or NDD . The LoF variants in CR genes were highest among patients with CHD and ECA , while the LoF in CR genes in patients with isolated CHD was comparable with that of control participants. Notably, 10 genes had LoF variants in 3 or more patients with CHD and NDD or CHD and ECA , including 2 CR regulatory genes  and transcription factor 12 ; eTable 10 in the Damaging CR variants are increased in individuals with developmental delays.P = 1.2 × 10−6; OR, 1.24 ; eTable 9 in the ARHGEF12], CTNNB1, lipoma-preferred partner [LPP], afadin [MLLT4], phosphatase and tensin homolog [PTEN], TCF12, and transferrin receptor [TFRC] genes; Given the dual role of some genes in both CHD and cancer, we considered if some CR genes might contribute to CHD. When considering only patients with CHD without pathogenic variants in OMIM CHD genes (n = 4293), we identified significantly more LoF variants in affected individuals (576 in 4293 [13.4%]) than control participants  gene, which encodes a chromatin modifier with broad tissue expression. Dominant de novo LoF variants in MLL2/KMT2D cause Kabuki syndrome with CHD,12 while somatic variants cause lung and colon adenocarcinomas.8 Our data extend these recognized linkages by defining more CR genes in more patients with CHD and indicating potentially shared disease mechanisms. For example, dysregulated vascular endothelial growth factor signaling from damaging variants in the kinase insert domain receptor (KDR) gene, which encodes a vascular endothelial growth factor receptor, could account for malformations of the great vessels7 and cancer-associated angiogenesis.Patients with CHD have common CR factors found in the general population, while our data indicate that CHD genotypes can contribute additional CR. Prior studies defined this association in a few CHD genes, including the lysine methyltransferase 2D  and other RASopathy genes.14This study has limitations, including the young ages of the patients with CHD and the need for longitudinal cancer surveillance to benchmark the clinical relevance of CR variants in patients with CHD and help determine causal associations, as well as whether associations are modified by nongenetic factors. Control participants were considerably older, potentially introducing survivor bias that would enhance the burden of CR variants in patients with CHD. Depth of sequencing precluded differentiation of variants as germline or high-level mosaic. We analyzed only LoF variants since defining missense variants as damaging requires detailed functional assessments, and thus our data provide only a conservative estimate of CR variants in patients with CHD. This also precluded assessment of burden for CR genes that operate by gain-of-function mechanisms in cancer, such as protein tyrosine phosphatase non-receptor type 11 (1 The recognition that CHD genotypes influence CR can promote clinical surveillance and early interventions and further promote lifelong health in adult patients with CHD. Additionally, we suggest that mechanistic studies into the molecular and cellular processes that are disrupted by damaging variants in CHD and cancer genes may uncover insights that inform new treatment strategies for both disorders.Decades of therapeutic progress enable long-term survival for newborns with CHD, and current estimates indicate 6 in 1000 adults are survivors of CHD.


To prospectively investigate retinal vascular changes in patients undergoing epiretinal membrane (ERM) and internal limiting membrane (ILM) peeling using swept source optical coherence tomography angiography (SSOCTA).Consecutive patients were grouped based on ERM severity and followed using SSOCTA up to month 3 after surgical intervention. Superficial and deep foveal avascular zone (s/dFAZ) as well as foveal and parafoveal vessel density (VD) were correlated with ERM severity and visual acuity. Differences between groups were evaluated.Significant correlations were found between ERM severity and baseline sFAZ, dFAZ and best corrected visual acuity (BCVA), central retinal subfield thickness (CST) and ΔCST . Vascular flow parameters did not correlate with age, peeling size, pseudophakia or CST, but correlated with intraretinal cysts presence. No associations of BCVA with any of the OCTA parameters across time were found.Significant differences between ERM severity groups 1 and 2 were found for sFAZ at baseline (p = 0.005) and at the 3-month follow-up (p = 0.014), and for dFAZ at baseline (p = 0.017). Superficial foveal and parafoveal VD were not significantly different between groups .This study clearly shows that ERM severity based on ERM staging has to be taken into account when undertaking studies in patients with idiopathic ERM using SSOCTA. Further, specific changes in the superficial and deep retinal vasculature in eyes undergoing ERM and ILM peeling were found. However, the clinical usefulness and prognostic value for post-surgical treatment BCVA of the SSOCTA-derived variables  used remains questionable. Idiopathic epiretinal membrane (ERM) is one of the most common macular diseases in the elderly with an incidence of 4–6%, increasing with age –4. It caThe most frequent surgical approach in patients with symptomatic ERM is pars plana vitrectomy, followed by peeling of the membrane and the ILM aimed at restoring normal retinal structure and function ,9. DespiA myriad of morphologic and functional markers have been studied in an attempt to predict postoperative outcome. , 10–14 SFluorescein angiography was used in previous studies to assess the foveal avascular zone (FAZ) area. Perifoveal leakage and decreased blood flow , with paThis prospective, clinical study was approved by the Ethics Committee of the Medical University of Vienna (EK1020/2014) and followed the tenets of the Declaration of Helsinki. All participants signed an informed consent form after receiving an in-depth explanation of the study and before being included in the study.The consecutive patients included had to be over 18 years of age and have an idiopathic epiretinal membrane scheduled to undergo pars plana vitrectomy and ERM and ILM peeling without endotamponade. Patients with cataract underwent a combined procedure with phacoemulsification and intraocular lens implantation. Exclusion criteria were any other eye disease or abnormalities that prevented reliable measurements, e.g. severe cataract or ametropia exceeding +/- 3 diopters spherical equivalent. Consequently, an image quality score below 40 was also considered an exclusion criterion . In caseAll patients underwent a comprehensive ophthalmic examination, including BCVA testing using ETDRS charts , intraocA cube comprising the central 6 x 6 mm of the posterior pole with a resolution of 1,024 x 49 (A-scans x B-scans) centered on the fovea was acquired. We ensured that the same retinal area was scanned at each visit by using the follow-up mode of the device. The software automatically calculated the central retinal subfield thickness (CST) within the central millimeter. Care was taken that the automated segmentations were correct and manual adjustments made if needed.ERM was staged based on OCT B-scans as described . In shorThe SSOCTA en face images obtained for the superficial and deep FAZ covered an area of 3x3mm and those for the VD 6x6mm. The built-in software  was used to delineate the superficial capillary plexus (SCP) and the deep capillary plexus (DCP). The DCP delineated by this software was 15.6 μm below the junction between the inner plexiform and inner nuclear layer (IPL/INL) to 70.2 μm below the IPL/INL. Segmentations were corrected manually if necessary. The FAZ area was delineated manually using the software included on the device. VD evaluations were also performed with the system’s own software and exported based on the ETDRS grid. Consequently, we report foveal VD as VD1 and parafoveal VD as the mean of the ETDRS segments 2 to 5 (VD2-5), representing the inner ETDRS ring . This apAll affected eyes underwent 23-gauge pars plana vitrectomy with ERM and ILM peeling under general anesthesia. Expert retinal surgeons  at the Department of Ophthalmology and Optometry, Medical University of Vienna, Austria performed the operations. In short, a 3-port pars plana vitrectomy with ERM and ILM peeling after staining with View ILM  was performed. A combined procedure of ERM and ILM peeling with phacoemulsification and intraocular lens implantation was chosen for eyes with cataracts. The optic nerve head size was used as a measure to record the size of the peeling area. Standardized post-operative treatment comprised gentamycin with dexamethasone eye drops (Dexagenta-POS eye drops) as well as gentamycin with dexamethasone ointment  for the night.Statistical analyses were performed using SPSS version 22.0 . Descriptive analyses are reported as the mean ± standard deviation (SD). BCVA was converted to the logarithm of the minimum angle of resolution (logMAR) equivalents for statistical evaluation. The Shapiro-Wilk test was used to test for normal distribution. A repeated measurements ANOVA was used to test for statistically significant differences over time. Each timepoint was tested with a post-hoc analysis using the least significant difference (LSD) test for the correction of multiple testing. We calculated Pearson correlation coefficients to assess associations between ERM severity and morphologic and vascular OCT/A variables. The point biserial correlation coefficient was calculated for correlations with dichotomous variables. A p-value of less than 0.05 was accepted as statistically significant.After excluding patients whose images were of insufficient quality (N = 2) and patients lost in follow-up (N = 3), 32 eyes of 32 patients were included in this study. The mean age was 71 ± 7 years, 45.6% were women.At baseline 21 patients were phakic and 11 pseudophakic. Fourteen eyes received an ERM and ILM peeling combined with phacoemulsification and intraocular lens implantation. Baseline characteristics and data grouped based on clinical severity stage are summarized in The mean superficial retinal plexus FAZ was statistically significantly reduced at day 1 after surgery (p = 0.043), whereas the change in mean deep retinal plexus FAZ did not reach the level of significance . The mean VD2-5 showed a statistically significant change during the first 3 months after surgical treatment  as well as VD2-5 . Consequently, the foveal and parafoveal VD of Groups 1 and 2 showed an almost identical course see .Statistically significant and negative correlations were found between ERM severity (Groups 1 and 2) and baseline sFAZ , dFAZ , as well as BCVA , the difference from baseline to month 3 in deep FAZ area (ΔdFAZ), baseline VD1 and VD2-5, the difference from baseline to month 3 in VD1 (ΔVD1), the difference from baseline to month 3 in VD2-5 (ΔVD2-5), the difference from baseline to month 3 in BCVA (ΔBCVA), peeling size, and age, showed non-significant correlations  with ERM severity.A subgroup analysis of group 1 and 2, correlating best corrected visual acuity (BCVA) and vascular parameters , revealed non-significant correlations in both groups over time, except for VD1-BCVA at baseline  in group 1 and sFAZ-BCVA at week one  in group 2.The SSOCTA-derived vascular variables showed statistically significant correlations between ΔsFAZ and ΔVD2-5 , but not ΔVD1 . Further, statistically significant correlations were found between ΔdFAZ and ΔVD1 , as well as ΔVD2-5 . Point-biserial correlations between ΔsFAZ , as well as ΔdFAZ  with the presence of intraretinal cysts were statistically significant.The correlation of ΔsFAZ and ΔdFAZ showed non-significant correlations  with baseline BCVA , ΔBCVA , age , peeling size , and ΔCST . ΔVD1 as well as ΔVD2-5  showed non-significant correlations with ΔBCVA.Point-biserial correlations between pseudophakia and superficial FAZ  as well as deep FAZ  were not statistically significant. Correlations between pseudophakia and BCVA were not statistically significant  at baseline or 3 months after surgical intervention . However, a statistically significant correlation was found between ΔBCVA and ΔCST . No correlations for subretinal fluid were calculated as only three patients presented with subretinal fluid at baseline.With the availability of high-resolution SSOCTA technology, a non-invasive imaging tool has become available raising hopes for new biomarkers that can predict the visual outcome and therapeutic response in retinal and choroidal disease, including idiopathic ERM. The recent literature on OCT/OCTA and ERM highlights changes during the disease process and after surgical intervention, with vascular changes occurring predominantly in the superficial and deep retinal plexus. With this study, we tried to take current knowledge a step forward by describing the natural course of SSOCTA variables in patients following ERM and ILM peeling and further by correlating SSOCTA variables with ERM severity based on OCT staging, functional and morphologic features. This is of interest as idiopathic ERM is a common retinal disease that critically affects visual function  and progThis study shows a clear influence of ERM severity derived from ERM stage on pre-surgical treatment factors. Statistically significant correlations were found between ERM severity (Groups 1 and 2) and baseline sFAZ, dFAZ as well as BCVA, CST and ΔCST. Further, at baseline as well as at the 3-month follow-up superficial and deep retinal FAZ areas decreased with ERM severity increase. Foveal VD increased with ERM severity at baseline. This effect diminished 3 months after ERM and ILM peeling. Although currently clinically not relevant, the association between SSOCTA factors and ERM stage may aide in the process of taking ERM severity assessment to a more uniform and automated analysis in future.Statistically significant differences between ERM severity groups were found for sFAZ and dFAZ, both at baseline and the 3-month follow-up. The differences between groups in foveal and parafoveal VD were not statistically significant. Also, a subgroup analysis of groups 1 and 2, correlating BCVA and vascular parameters , revealed non-significant correlations in both groups over time, except for VD1-BCVA at baseline  in group 1 and sFAZ-BCVA at week one  in group 2. We consider these findings to be by chance, as out of 40 correlations performed only two were statistically significant and as all other time points showed no statistically significant correlations.This study revealed statistically significant correlations between ΔsFAZ and ΔVD2-5, and between ΔdFAZ and ΔVD1 as well as ΔVD2-5. All variables were positively correlated, indicating that the bigger the change in FAZ area, the bigger is the change in VD during the first 3 months. This shows, that FAZ and VD in patients with ERM are variables dependent on each other; a finding interesting for future studies.Age, peeling size, pseudophakia, and ΔCST were not statistically significantly correlated with ΔsFAZ and ΔdFAZ, but the presence of intraretinal cysts was statistically significantly correlated. Regarding BCVA, no statistically significant correlations of ΔBCVA with the SSOCTA variables ΔsFAZ, ΔdFAZ, ΔVD1 or ΔVD2-5 could be found during the first 3 months after surgical intervention. The correlation with ΔCST, however, was statistically significant.These findings are of special interest as up until now the ERM severity or stage have not been considered in studies using OCTA reporting FAZ or VD. This may explain the variation in earlier results, which we refer to in the following.Our findings contradict previous studies on the correlation between sFAZ area and BCVA that showed the smaller the preoperative FAZ area, the poorer was the postoperative BCVA ,24. TheyCommon findings reported in various studies are smaller baseline FAZ area and fluctuant floveal, para- and perifoveal VD of both the superficial and deep retinal plexus compared with healthy contralateral control eyes ,24,28–30The above-summarized findings are not surprising as previous studies showed retinal vessel displacement and macular constriction in ERM eyes, which can be attributed to a centripetal and tangential traction during ERM and ILM peeling compressing or curling retinal vessels in one place and straightening vessels in other areas of the retina , explainPreliminary results indicate that mechanical forces might affect the blood flow in the DCP more than in the SCP, with the flow in both improving one month after ERM peeling . This fiFor VD interpretations caution is advised as different ways of computation are used. The most basic definition of vessel or vascular density is the ratio of pixels representing vessels over the total area investigated. Image processing before VD calculation varies greatly and is sometimes not known to the user. Also previously shown, retinal perfusion indices in different-sized OCTA volumes are not interchangeable .The associations found between ΔBCVA and ΔCST indicate that the bigger the effect of ERM + ILM peeling on CST, the great the gain in BCVA.Some limitations have to be considered. As consecutive patients were included in this study, the distribution among ERM stages is very heterogeneous, therefore patients were grouped based on ERM severity. Future studies will have to evaluate the influence of each individual ERM stage in more detail. Various ERM studies report a 6-month follow-up period, which we omitted as statistically significant morphological changes have been shown to occur within month one . BaselinThe influence of axial eye length on OCT/A measurements could be omitted as we excluded patients exceeding +/- 3 diopters of spherical equivalent. Although, we could not show statistically significant correlations between pseudophakia and BCVA, the influence of a combined procedure on visual acuity cannot be waived. To ensure comparability in future studies a consensus on automated VD calculations or a manual approach would be helpful. Also, averaging of multiple OCTA images  could heIn conclusion, we could not prove that the hemodynamic SSOCTA variables sFAZ and dFAZ area, as well as foveal and parafoveal VD are of prognostic value for post-surgical treatment BCVA. Despite the obvious advantages OCTA-derived variables would have in the prognosis of BCVA, the clinical usefulness of OCTA in eyes with idiopathic ERM remains questionable. However, this study clearly shows ERM severity or staging should be taken into account when investigating patients with idiopathic ERM using SSOCTA.


Brachial artery flow-mediated dilatation (FMD) is used as a surrogate for invasive assessment of coronary artery endothelial function, concordance with invasive assessment of coronary endothelial function is controversial.We assessed both brachial FMD and coronary CPT noninvasively at the same sitting using cardiac MRI (CMR) and compared the two in a population with and without coronary risk factors.Endothelium-dependent vasodilator function was assessed in the brachial and coronary arteries in 39 subjects. Using a Siemens 1.5 T scanner, the left anterior descending (LAD) coronary artery was localized using a navigator angiographic sequence and a proximal cross-section imaged using TrueFISP bright blood imaging and T2 weighted turbo spin echo imaging. Images were obtained at rest and during cold pressor testing(CPT) and evaluated in ARGUS (Siemens). The % change in lumen cross-sectional area in mid diastole was determined. FMD was assessed by inflating a blood pressure cuff to a reading 50 mmHg higher than the subject's resting systolic pressure, and deflating after 1 min. Imaging was done using TrueFISP cine bright blood imaging at rest, inflation and at 5 sequential time points during deflation. Images were evaluated in ARGUS (Siemens). The % change in lumen cross-sectional area pre and post inflation was determined.Of 39 subjects, 16 were females and the mean age was 52 ± 8 yrs. Coronary risk factors  were present in 19 while 20 had no risk factors.The mean brachial FMD of the pooled cohort was 13% and the mean coronary response to CPT was 16%. There was no significant relationship between the brachial FMD and coronary CPT response .When determined with similar noninvasive methods at the same sitting, brachial FMD does not correlate closely with coronary endothelial response to CPT in a population with and without coronary risk factors. Use of brachial FMD as a surrogate for coronary endothelial function should be reconsidered.


Molecular and culture-based methods together detect more organisms than either method alone.Optimal clinical decision-making depends on identification of clinically relevant organisms present in a sample. Standard microbiological culture may fail to identify unusual or fastidious organisms and can misrepresent relative abundance of sample constituents. Culture-independent methods have improved our ability to deconvolute polymicrobial patient samples. We used next-generation 16S rRNA gene sequencing (NGS16S) to determine how often cultivatable organisms in complex polymicrobial samples are not reported by standard culture. Twenty consecutive bronchoalveolar lavage (BAL) samples were plated to standard and additional media; bacteria were identified by NGS16S analysis of DNA extracted directly from samples or from washed culture plates. 96% of organisms identified were cultivable, but only 21% were reported by standard culture, indicating that standard work-up provides an incomplete assessment of microbial constituents. Direct NGS16S correlated well with standard culture, identifying the same predominant organism in 50% of samples. When predominant organisms differed, NGS16S most often detected anaerobes, whose growth is unsupported by standard culture conditions for this specimen. NGS16S  As the mainstay of the modern clinical microbiology laboratory, isolated growth of individual organisms is required for antimicrobial susceptibility and virulence testing, epidemiological investigations, and genome sequencing. Nevertheless, standard culture often fails to identify a causative pathogen when unusual or fastidious organisms are present, or after antimicrobial therapy has been initiated9. The developing field of culturomics has enabled the isolation of hundreds of new microorganisms, previously considered uncultivable, using a variety of growth conditions and extended incubation times12. For example, the addition of the antioxidant uric acid enables the aerobic growth of many organisms thought to be strictly anaerobic13. Therefore, we hypothesized that simple changes to routine culture conditions  could expand the repertoire of recoverable organisms in the clinical laboratory.Culture is a “complex and difficult art”14. However, this technology is expensive and requires technical expertise, limiting its routine use in the clinical laboratory. Here, we used NGS16S analysis of BAL samples, a readily accessible polymicrobial sample type, to evaluate the ability of culture to accurately catalog the microbial constituents of complex clinical samples. First, we analyzed DNA extracted directly from patient samples to determine the identity and prevalence of organisms for which current culture conditions are sub-optimal. In addition, we expanded standard culture by including four additional culture conditions, and evaluated washes of culture media plates by NGS16S analysis to determine the frequency with which cultivable organisms are not reported after standard work-up. We found that NGS16S identifies more organisms per sample and allows identification of fastidious organisms, while culture is better at capturing organisms when bacterial load is low and allows incidental recovery of non-bacterial pathogens such as yeast or molds. Both methods together detected more organisms in clinical samples than either method alone.Next generation 16S rRNA gene sequencing (NGS16S) can be utilized for deconvolution of polymicrobial clinical samples that are difficult or impossible to resolve by standard molecular methods. Using synthetic polymicrobial samples of defined composition, we have shown that NGS16S analysis more accurately catalogs the bacterial contents of polymicrobial samples than standard culture). An equal volume of each specimen was also plated to study culture media  and incubated anaerobically for seven days. Standard and study culture plates were washed with PBS and organisms present were identified by NGS16S analysis  were from the same patient; all other samples were collected from different individuals. Specimens were analyzed as outlined in Fig. S1Enterococcus species and Pseudomonas species. This is likely due to the small sample size of our study, and sampling restricted to transplant and oncology patients.Thirty-nine clinically relevant classifications (CRCs) were identified in this study either by identification of colonies in standard clinical culture workup or by NGS16S sequencing as described in the methods Table . CRCs coBacteria were defined as present in any given specimen if any one of the following criteria was met: (1) reported in standard culture; (2) read mass detected in one or more culture plate washes above filtering threshold (see methods); (3) reportable by direct NGS16S analysis. If either of the former two criteria were met, the organism was considered cultivable. Eleven of the 39 CRCs identified in this study (28%) were reported from standard culture at least once and 35 (90%) were recovered on at least one culture plate  were cultivable Fig. . Only 39Aspergillus, recovered from BAL10, is not detectable by NGS16S analysis , and Tropheryma whipplei, detected by direct NGS analysis in BAL17, requires very specialized growth conditions. Twenty-one assignments of viridans streptococci, Staphylococcus aureus, Neisseria species and Bacillus species constituted a group for which current culture conditions were sufficient for detection and reporting.The CRC assignments fell into four categories based on comparisons of culture report, direct NGS16S analysis, and NGS16S plate wash analysis Table . Two orgThe largest category comprised a total of 121 assignments of 26 CRCs for which current culture conditions were insufficient for detection . This category represents a majority (26/39) of all CRCs and 6 of the 10 most prevalent classification groups Tables  and 5. FAspergillus, making isolation of bacterial colonies difficult. In two specimens (BAL02 and BAL14) the predominant organism was Staphylococcus aureus; the presence of a known pathogen with a distinct colony morphology may have resulted in a less rigorous examination of the plates for additional colony types. Rothia species were frequently reported by standard culture and identified from plate washes of standard culture media. However, this classification was infrequently considered reportable by direct NGS16S (Table 4). An alignment of the universal NGS16S primer sequence against the 16S sequences of Rothia mucilaginosa type strains revealed a single nucleotide mismatch, which could affect relative amplification and account for the lower than expected reporting rate of this species from direct specimens.A final category includes a total of 44 assignments of seven CRCs for which there was poor correlation between the CRCs report from standard culture and growth on standard media as measured by NGS16S plate wash analysis . Specimens were fully de-identified after being aliquoted from the material submitted for clinical testing, and as such this study does not constitute human subjects research according to University of Washington Institutional Review Board criteria. All experiments were performed in accordance with relevant guidelines and regulations. A 2 mL aliquot of each BAL sample was frozen immediately after culturing and stored at −80 °C until DNA extraction.48. Briefly, samples were plated on 5% sheep blood (BA), MacConkey (MAC) and chocolate (CA), agar plates (standard media), and incubated aerobically at 37 °C for 72 h. An internal review of all organisms reported from BAL by clinical NGS16S analysis in our institution identified organisms for which standard culture conditions may be inadequate; additional media was selected for ability to support the growth of these organisms (study media). Brucella agar  is a general purpose anaerobic medium. Laked Sheep Blood with Kanamycin and Vancomycin Agar  is used for the selective isolation of fastidious and slow growing Gram-negative obligately anaerobic bacteria. Selective Strep Agar  is designed to inhibit Gram-negative bacilli and Staphylococci, thereby allowing the isolation and identification of pathogenic streptococci, including beta-hemolytic streptococci and Streptococcus pneumoniae. Columbia CNA Agar  was designed to suppress the growth of most Gram-negative bacteria, thus enriching for Gram-positive bacteria. In addition to standard media, 0.1 mL of each specimen was plated to study media and incubated anaerobically at 37 °C for seven days. Photographs were taken of all plates at the end of incubation.Standard microbiological culture was performed by the University of Washington Clinical Microbiology Laboratory, as previously described14. Briefly, the 16S v1–v2 region was amplified using custom primers incorporating Illumina-compatible sequencing adaptors and a sample-specific 8-bp barcode sequence; paired-end sequencing was performed on an Illumina Miseq using a 500-cycle sequencing kit (version 2) to a minimum read depth of 50,000 reads per sample. Sequence data generated for this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession no. PRJNA555084.All culture plates except MAC  were washed with 3 mL sterile PBS and bacterial colonies were released by gently scraping agar surface with a sterile cell scraper. Bacteria from 1 mL of the resulting suspension were collected by centrifugation, resuspended in 0.2 mL MagNA Pure DNA Tissue Lysis Buffer (Roche) and stored at −80 °C until extraction. DNA was extracted from patient samples and plate washes using the QIAamp UCP Pathogen Mini Kit (Qiagen) with mechanical disruption of samples with 1.4-mm ceramic beads followed by enzymatic lysis via proteinase K. Next generation sequencing libraries were prepared and DNA sequencing was performed as previously described49. SVs were identified as 16S rRNA by multiple sequence alignment using cmsearch50 using the default settings and a covariance model available from the Infernal web site . SVs were then passed through the decontam software package51 to identify and remove contaminants. To reduce the effects of possible DNA carry over between runs or samples, SVs corresponding to 100 reads or fewer in each sample were excluded. The remaining SVs were used as blast queries against a curated set of 16S rRNA records retrieved from NCBI. Alignments of at least 90% query coverage were grouped taxonomically and classified as previously described14. An Acinetobacter species SV present in 100% of samples analyzed was used as an internal standard to calculate the number of templates for each classification. All reads were classified, and classifications >1% of the total specimen read mass were considered reportable. In three cases, biologically relevant organisms were only slightly below this threshold and were also included: Actinomycyes odontolyticus was included for BAL01 and BAL02  and Solobacterium moorei for BAL13 (0.95% raw reads). Plate wash reads were processed as described above, with additional filtering steps: (1) all sequences with fewer templates than the internal standard were removed from each sample as likely reagent background, and (2) the number of templates expected to produce a visible colony was empirically determined (2500 templates) and SVs below this threshold were excluded from further analysis. Five classification assignments that were not excluded by these filtering criteria were manually excluded from analysis as contaminating DNA; these were recovered from plates without corresponding colonies and were near the filtering threshold. On average, 98% of total reads from plates with bacterial growth were analyzed (range 83–99.9%); all reads from no-growth plates were excluded, confirming that filtering was appropriately removing irrelevant sequences. To compare standard clinical lab culture and NGS16S results, organisms were manually combined into clinically relevant classifications (CRCs) at genus level or based on similar taxonomy and/or colonial morphology . Refer to Supplementary Table NGS16S analysis of DNA extracted from patient samples (direct NGS16S) was performed by the University of Washington Clinical Microbiology Laboratory. Sequence analysis was performed without knowledge of culture results. Briefly, sample sequences were demultiplexed into paired end sequence fastq files using the Illumina on-board software with barcodes and adapters removed. Sequence variants (SVs) were generated from the paired end sequence fastq files using DADA2Supplementary Table S1Supplementary Figure S1


Fetal growth restriction defined as the failure to achieve the fetal genetic growth potential is a major cause of perinatal morbidity and mortality. The role of maternal adaptations to placental insufficiency in this disorder is still not fully understood. We aimed to investigate the biological processes and protein–protein interactions involved in late-onset fetal growth restriction in particular. We applied 2D nano LC–MS/MS proteomics analysis on maternal blood samples collected at the time of delivery from 5 singleton pregnancies with late-onset fetal growth restriction and 5 uncomplicated pregnancies. Data were analyzed using R package “limma” and Ingenuity Pathway Analysis. 25 proteins showed significant changes in their relative abundance in late-onset fetal growth restriction . Direct protein–protein interactions network demonstrated that Neurogenic locus notch homolog protein 1 (NOTCH1) was the most significant putative upstream regulator of the observed profile. Gene ontology analysis of these proteins revealed the involvement of 14 canonical pathways. The most significant biological processes were efflux of cholesterol, efflux of phospholipids, adhesion of blood cells, fatty acid metabolism and dyslipidemia. Future studies are warranted to validate the potential role of the detected altered proteins as potential therapeutic targets in the late-onset form of fetal growth restriction. This condition is also associated with cardiovascular, metabolic and neurodevelopmental changes in the offspring that persist into adulthood5. Late-onset form of FGR, usually diagnosed after 32 weeks of gestation and delivered at term6, is the most common clinical presentation of this condition encompassing more than 90% of FGR cases7 and constituting a major contributing factor to adverse perinatal outcome8. Placental dysfunction is the main culprit in FGR causing an impairment in the transfer of nutrients and oxygen from the mother to the developing fetus9. Maternal adaptations to placental insufficiency may also play a role in the pathophysiology of FGR10. Thus, exploring biological pathways in the maternal blood in pregnancies complicated by late-onset FGR may help in identifying the involved etiological mechanisms and detecting potential therapeutic targets for this disorder with the aim of preventing its short and long-term consequences.Fetal growth restriction (FGR) is one of the established pregnancy complications that involves fetal distress due to the failure in achieving the fetal genetic growth potential. FGR, usually defined as a birthweight less than 10th centile, affects 7–10% of pregnancies and implicates an increased risk of perinatal morbidity and mortality11. Its application in pregnancy-related disorders has been implemented to improve the understanding of their pathophysiology12. However, a handful number of previous studies have exploreed the maternal proteomic fingerprint of FGR15. Moreover, none of them investigated separately late-onset FGR, indeed the studied population in the literature was principally formed by early-onset FGR cases since it’s the most severe phenotype. This approach carries a certain bias given that late-onset FGR might have a different pathogenesis than its early-onset counterpart with similar long-term consecuences7.Proteomic profiling exemplify the study of the global set of proteins in a particular biosampleOur objective in this study was to focus on late-onset FGR and to analyze the maternal blood proteome in pregnancies complicated by this disorder compared to healthy pregnancies in order to determine the biological processes and protein–protein interactions involved in late-onset FGR.Maternal baseline characteristics were similar between the study groups with the exception of lower maternal age in FGR cases compared to controls, as shown in Table In terms of perinatal outcomes, all the included gestations were delivered at term (> 37 weeks) with no difference between cases and controls (p = 0.24). In accordance with the study design, FGR newborns had significantly lower birthweights (p = 0.01) with birthweight centiles < 3rd centile in all the cases (p = 0.01) compared to controls. No cases of perinatal mortality were observed in the study population.16, p = 0.003; (2) lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), an autocrine regulator of cell growth mediated by growth regulators17, p = 0.005; (3) Lactotransferrin precursor (LTF), a stimulator of endothelial cell migration and proliferation which has a possible role in the regulation of bone growth18, p = 0.010; (4) Galectin-7 (LGALS7), p = 0.010; (5) Phospholipid transfer protein (PLTP), p = 0.013. Some pregnancy specific beta-1-glycoproteins  were also altered in late-onset FGR mothers, these proteins are mainly secreted by the placenta. In addition, our results uncover the high abundance of many lipoproteins in FGR mothers such as Apolipoprotein C2, Apolipoprotein C3 and Apolipoprotein E as well as fatty acid-binding protein 5. These lipoproteins play a pivotal role in the pathomechanisms of atherosclerosis by the regulation of triglyceride levels19. Among the other proteins that are differentially expressed in FGR galectin-3-binding protein, proteoglycan 4 and transgelin-2 which promote cell adhesion21; epidermal growth factor receptor which may play a role in membrane ruffling and remodeling of the actin cytoskeleton22; THAP domain-containing protein 4, platelet glyprotein Ib alpha chain and fibrinogen alpha chain which regulate endothelial cell proliferation and hemostasis23; beta-defensin 103 that has an antimicrobial activity; Di-N-acetylchitobiase, involved in the degradation of asparagine-linked glycoproteins and other degradational proteins like ATP synthase subunit g, mitochondrial and Olfactomedin-like-protein 2B. Individual values of the 25 differentially expressed proteins are displayed in Supplementary Table A total of 688 proteins were identified in our proteomics analysis, 25 proteins of them were differentially expressed  between cases and controls Fig. . Out of Direct protein–protein interactions among the different components of the network were established by the Ingenuity database Fig. . NeurogeGene ontology analysis of the corresponding proteins that were statistically different in FGR revealed the involvement of 14 canonical pathways, the top 5 canonical pathways canonical pathways are shown in Table This is the first study that focuses on maternal proteomic profile in pregnancies complicated by the late-onset form of FGR. The results of the present study elucidate that lipid metabolism is disturbed in mothers from pregnancies complicated by late-onset FGR compared to healthy pregnancies. Furthermore, our results indicate that NOTCH1 could be an important regulator of the observed profile.15 or mixed up early and late-onset cases14. A vast majority of the identified proteins and biological processes are related to lipid metabolism which is in line with prior studies25. Among the differentially expressed proteins in late-onset FGR, the highest magnitude of change was observed in Adiponectin that was underexpressed in FGR mothers. This observation might reflect a failure in the physiological response to pregnancy demands since Adiponectin concentrations are usually elevated in healthy pregnancies due to pregnancy related “Adiponectin resistance”26.The findings of our study demonstrate that late-onset FGR has a proteomic signature in maternal plasma similarly to the previous observations that focused on the early-onset form of this disorder27, the regulation of trophoblast invasion and the expression and activity of placental amino acid transporters (STAT3)28 or the differentiation of estrogen-dependent cells (ESR)29. Indeed, previous studies have demonstrated alterations in the immunoreactivity and localization of NOTCH proteins as well as decreased STAT3 in placentas from pregnancies complicated by FGR suggesting a contribution of these disruptions in trophoblast differentiation and function31. In the present study, NOTCH1 was the most featured regulator in the spotted network linking the disturbed profile of lipid metabolism with placental growth and being a potential target for future therapeutic agents.In light of the protein–protein interactions observed in late-onset FGR, we can hypothesize that different biological processes related to lipid metabolism and homeostasis have an impact on other regulator proteins. These proteins may play an essential role in the pathogenesis of late-onset FGR due to their close relationship with the development of the extravillous trophoblast lineage in the human placenta (NOTCH1)18 and known to have a protective function against dysregulated fetoplacental lipid homeostasis32. In addition, FXR/RXR activation and LPS/IL-1 Mediated Inhibition of RXR Function were also initiated, which might affect several functions since FXR is a metabolic regulator and cell protector against oxidative stress33. The same lipoproteins are involved in atherosclerosis and IL-12 signaling replicating the link between inflammation, lipid dysregulation and endothelial cell dysfunction. In fact, oxidative stress, inflammation and placental thrombosis are likely to interrupt the placental ability to transfer the necessary nutrients and oxygen to the fetus and therefore impede the normal fetal growth37.Furthermore, our gene ontology analysis has revealed that the top canonical pathways and biological processes involved in late-onset FGR are mostly related to the efflux of cholesterol and phospholipids. Indeed, multiple pathways share similar identified molecules where the lipoproteins Apolipoprotein C2, Apolipoprotein C3 and Apolipoprotein E are central. Thus, co-activation of parallel pathways seems to have occurred in FGR mothers. The most significant pathway was LXR/RXR activation, a fundamental pathway in the balance of cholesterol levels38. It is widely accepted that placental insufficiency is the main culprit in FGR resulting from shallow trophoblast invasion during the early stages of gestation7. Indeed, many placental enzymes involved in the supply of fatty acids to the growing fetus, like endothelial lipase and lipoprotein lipase, have been described to be dysregulated in FGR pregnancies39. Thus, it seems plausible that maternal poor response to pregnancy demands of lipids and fatty acids may contribute to the placental dysfunction and suboptimal fetal growth. In addition, we observed a favored proinflammatory status in the studied cases of late-onset FGR supporting the existence of an inflammatory bias in this disorder40.The current study focused on the late-onset form of this disorder revealing specific pathways and key player proteins that can provide further insights into the pathophysiology of late-onset FGR. In fact, lipid metabolism during the normal pregnancy is essential to provide the necessary fatty acids for fetal growthThis study has some strengths and limitations that merit a comment. All the pregnancies included in this study were recruited prospectively, well selected and characterized to constitute homogenous groups of late-onset FGR cases and controls. Cases and controls were matched by gestational age at maternal blood sampling. Moreover, this was a comprehensive proteomics study not only revealing the different proteins in late-onset FGR but also the protein–protein interactions and the involved biological processes. The center of our analysis was to identify the pathophysiological pathways that may play a role in this form of FGR, thus we opted for a wider look at the results without applying a statistical correction for multiple comparisons. On the other hand, we acknowledge the small sample size of our study and the importance of future validation of our findings in larger cohorts.In conclusion, the present study indicates that lipid metabolism dysregulation plays a vital role in pregnancies complicated by late-onset FGR. Importantly, our findings highlight the central regulator of the observed profile being NOTCH1. These results enhance our understanding of the pathophysiology of late-onset FGR which remains poorly defined. Furthermore, they may constitute a starting point for future studies to investigate the potential therapeutic targets of the involved pathways.2. Late-onset refers to delivery occurring after 37 weeks of gestation. Uncomplicated pregnancies with appropriate fetal growth for gestational age-defined as estimated fetal weight and birthweight above the 10th centile were randomly selected from our general population to be included as controls and frequency paired with cases by gestational age at maternal blood sampling (± 2 weeks). In all pregnancies, gestational age was calculated based on crown–rump length measurement on first-trimester ultrasound41 and weight centiles were assigned according to local standards42. Pregnancies with congenital malformations, chromosomal abnormalities or intrauterine infection were excluded. The study was conducted in accordance with the principles of the Helsinki declaration. The study protocol has been approved by the local ethics committee  number HCB/2016/0253. Participating patients provided their written informed consent.We conducted a prospective case–control study in the Departments of Maternal–Fetal Medicine at BCNatal  between July and October 2016. The study population included 5 singleton pregnancies diagnosed with late-onset FGR which was defined as an estimated fetal weight and birthweight below the 10th centile43, the umbilical artery44, and the fetal middle cerebral artery pulsatility indices44, with the calculation of the cerebroplacental ratio45. These values were normalized into z scores accordingly and considered abnormal if > 95th centile for uterine arteries mean and umbilical artery pulsatility indices and < 5th centile for the middle cerebral artery pulsatiliy index and the cerebroplacental ratio45. At the time of delivery, gestational age, birthweight, birthweight centile, Apgar scores, umbilical artery pH and perinatal mortality were recorded. In addition, maternal blood samples were collected for subsequent proteomic analysis.The following data were recorded upon enrollment: maternal age, ethnicity, known chronic disease , parity, obstetric history, mode of conception and smoking status. Feto-placental Doppler parameters were obtained in the last 2 weeks of pregnancy, including the uterine arteriesg for 10 min at 4 °C, and samples were immediately stored at − 80 °C until analyzed.Maternal blood samples were drawn from peripheral veins within 2 h after delivery and collected in EDTA-treated tubes. Plasma was separated by centrifugation at 1500Before proteomic analysis, the depletion of fourteen highly and medium abundant proteins was performed using Seppro IgY14 and Seppro SuperMix columns following the manufacturer’s instructions. Afterwards, samples were processed for tandem mass tag (TMT) before acquisition on a nanoscale liquid chromatography coupled to tandem mass spectrometry (2D nano LC–MS/MS) analysis from Thermo Fisher. Protein identification/quantification was performed on Proteome Discoverer software v.1.4.0.288 (Thermo Fisher) by Multidimensional Protein Identification Technology. On initial proteomic analysis, readers were blinded to each patient’s status. Detailed methodology is provided as supplementary information.Clinical characteristics of the study population were summarized as median (interquartile range) or percentages for continuous and categorical variables respectively. The analysis was performed using STATA 14.2  including the use of Mann Whitney U test and Fisher exact test for continuous and categorical variables respectively. All reported p values are two-sided. Differences were considered significant when p < 0.05.46. Data were preprocessed, normalized and a moderated t-test was applied (p < 0.05). Network analysis was generated through the use of QIAGEN’s Ingenuity Pathway Analysis (IPA), QIAGEN Inc., . Networks combined focused proteins that correspond to differentially expressed proteins which were detected by “limma” analysis and non-focused proteins that were added by IPA, using knowledge derived data from their own database. To gain a further insight into the potential mechanisms involved, the identified proteins were mapped to IPA database.For proteomics data, differentially expressed proteins were determined using R package “limma”Supplementary Information S1.Supplementary Information S2.


Correction to: BMC Psychiatry 21, 36 (2021)https://doi.org/10.1186/s12888-020-03033-wFollowing the publication of the original article , some wobold typeface.The changes have been highlighted in Statistical analysis:checked using Shapiro–Wilk test (P > 0.05).The normal distribution of the numeric variables was Discussion:nine regular medications (35).The residents with a diagnosis of dementia in Australia are exposed to polypharmacy, with an average exposure of [35], while it was 19.4% at pre-intervention in the current study.Concomitant use of antipsychotics and antidepressants is considered to be of inappropriate practice, was prescribed to 15.7% of participants in an earlier study [45].A total of 65% of participants were using antipsychotics for > 3 months in an earlier study [46]. Our findings showed evidence of long term use of psychotropic and anti-dementia medications for ≥6 months, although current guidelines stipulate review and withdrawal within 12 weeks [47]. Our study was conducted in rural nursing homes which are experiencing substantial challenges in recruitment and retention of skilled staff as additionally, there are limited resources available for professional development compared to metropolitan areas [48]. In a previous study, nursing staff working in rural settings demonstrated that that NPI management of BPSD in PwD do not fall under their responsibility [49].Prolonged use of benzodiazepines along with antidepressants has also been reported previously in 40% of the study participants [50].Nevertheless, in clinical practice, a non-significant outcome does not always mean the treatment was not clinically effective as small sample sizes had a substantial impact The author group has been updated above and the original article  has been


Acute decompensated heart failure (ADHF) can occur early after transcatheter aortic valve implantation (TAVI), but the risk factors or mechanisms associated with it have not been fully determined. This hypothesis-generating study aimed to investigate the clinical indices associated with the development of ADHF within 72 hours after TAVI and to improve procedural approaches for TAVI.In this single-centre hypothesis generating prospective observational study, we enrolled 156 consecutive patients with severe aortic stenosis who underwent TAVI between January 2016 and February 2018 at our institution. We set the primary endpoint as the new development of ADHF within 72 hours after TAVI, and clinical indices associated with it were evaluated using a multivariable logistic model. The median age of the patients was 83 (quartile range 80–86) years, 48 (30.8%) were men and the median Society of Thoracic Surgery-Predicted Risk of Mortality was 7.1 (range 5.2–10.4). Mitral stenosis (MS), defined as mean transmitral valve pressure gradient ≥5 mm Hg, was present in 15 (9.6%) patients. After TAVI, the invasive mean transaortic valve pressure gradient (mAVPG) decreased from 48 (36–66) to 7 (5–11) mm Hg, and 12 (7.7%) patients developed ADHF within 72 hours after TAVI. Multivariable logistic regression analysis showed that MS  and greater decreases in mAVPG  were associated with ADHF.MS and drastic improvement of mAVPG were associated with new development of ADHF within 72 hours after TAVI. Patients with mitral stenosis (MS) who underwent transcatheter aortic valve implantation (TAVI) have increased in-hospital death and 1-year mortality.Patients with MS have increased risk of acute decompensated heart failure (ADHF) early after TAVI.We should take careful management for patients with MS to avoid the development of ADHF early after TAVI.Aortic stenosis (AS) causes left ventricular outflow impairment, and the subsequent pressure overload with or without left ventricular systolic dysfunction can lead to heart failure (HF).2 (or an effective orifice area index <0.6 cm2/m2) by transthoracic echocardiography (TTE), according to the guideline for valvular heart disease of the European Society of Cardiology and the European Association for Cardio-Thoracic Surgery.This single-centre prospective observational study included 156 consecutive patients with symptomatic severe AS who underwent TAVI at Osaka City University Hospital between January 2016 and February 2018 . TAVI atWe chose the transfemoral approach as the first option when patients did not have an excessively narrow access route for insertion of the sheath or aortic arch atheroma. We performed TAVI under general anaesthesia in a hybrid operating room, except for two patients who underwent conscious sedation due to pulmonary dysfunction. Transcatheter heart valves were classified as balloon-expandable  or self-expandable . Balloon-expandable valves were the first choice, and self-expandable valves were reserved for patients with a narrow aortic annulus. We performed simultaneous catheter measurement to evaluate the invasive mAVPG as well as a measurement of left ventricular end-diastolic pressure before and after TAVI. After successful TAVI, patients were given a saline solution by intravenous infusion (1 mL/kg/hour) until the start of ingestion. The brachial blood pressure was maintained at less than 130 mm Hg using calcium channel blocker, if necessary.All data shown in the tables and figures were collected prospectively from patient records. Because this study aimed to generate hypothesis, preprocedural TTE evaluated AS-related indices as well as mitral stenosis (MS)-related findings such as the transmitral valve pressure gradient and presence or absence of mitral annular calcification (MAC). We defined significant MS as a mean mitral pressure gradient ≧5 mm Hg and defined MAC as a structure producing intense echocardiographic signals located at the junction of the atrioventricular groove and posterior mitral leaflet, although severe AS could result in low-flow, low-gradient MS with prolonged pressure half time which is related to impaired left ventricular relaxation.21We set the primary endpoint as the new development of ADHF within 72 hours after the procedure and the objective of the present study was to investigate its risk factors. We defined the new development of ADHF after TAVI as worsening of HF symptoms, such as dyspnoea with objective signs of congestion like pulmonary oedema or hypoperfusion requiring treatments such as intravenous administration of diuretics, vasodilator, inotropes or respiratory supports based on recommendations by international guidelines.222 test, respectively. Then, the prespecified analysis included the evaluation of the risk factors of the new development of AHDF in the early phase after TAVI using univariable logistic regression analysis with its 95% CI. Since the absolute number of primary endpoint was estimated to be low, ad hoc adjustments of confounding factors by multivariable regression model was admitted only as a reference in the prespecified statistical protocol. In the multivariable model, we determined the confounders statistically based on the Akaike information criteria (AIC) and also performed conventional multivariable model employing variables, which showed statistical significance in the univariable logistic regression model. To avoid the problem of multicollinearity, New York Heart Association (NYHA) functional classification and urgency of the procedure were not used because these indices are included in the STS-PROM score, and invasive mAVPG improvement was selected as a representative value for preprocedural and postprocedural severity of AS in a multivariable model. In addition, we employed classification and regression tree (CART) analysis to reveal the most important risks among these variables.Continuous variables were summarised using medians and IQR (quartiles 1–3), and categorical variables were summarised using means of counts and percentages. We first divided our patients into two groups depending on the presence or absence of ADHF within 72 hours after TAVI and compared patient backgrounds because this is the hypothesis-generating study. Differences in continuous and categorical variables between groups were compared using the Wilcoxon rank-sum test and the χPatient characteristics are shown in 10.1136/openhrt-2020-001348.supp1Supplementary dataIn this hypothesis-generating single-centre prospective observational study, the incidence of ADHF after TAVI was 7.7%. In addition, both univariable and multivariable logistic regression analyses revealed that the presence of significant MS (mean pressure gradient ≧5 mm Hg) and improvement of mAVPG were associated with new development of ADHF within 72 hours after TAVI, with the adjusted OR of MS much higher than that of mAVPG which also indicated its importance in the CART analysis. Since few studies have focused on the epidemiology and risk factors of the new development of ADHF within 72 hours after TAVI, our study could provide physicians with new insights into the management strategies of TAVI in patients with severe AS complicated with MS.242 to 3.1±0.7 after TAVI.In general, the pathophysiology of developing HF is considered multifactorial, and numerous disparate aetiologies are indicated in previous reports such as left ventricular dysfunction, coronary artery diseases and valvular diseases.et al reported that severe MS was an independent predictor of in-hospital death and indicated that severe MS was an independent predictor of 1-year mortality and HF-related hospitalisation.et al reported that patients with MS had an increased risk of cardiovascular death both at 30 days (adjusted HR 4.05: 95% CI 2.10 to 7.82) and 1 year (adjusted HR 3.64: 95% CI 2.38 to 5.56).We believe that our results remind us one important and two possible clinical implications. First, it is noteworthy that the OR of significant MS was much greater than those of other variables and that CART analysis suggested that the most predictive index of new development of ADHF early after TAVI was significant MS. Since the high incidence of comorbid MS (>10%) due to degenerative MAC (>45%) in patients who undergo TAVI, it is important to know the potential risk of MS.This study has several limitations due to the nature of single-centre design. First, the small study population (n=156) and low incidence of primary endpoint underpowered the statistical analysis, and there are some differences regarding baseline characteristics, such as AS severity and HF condition, between study groups even though we tried to minimise these differences using multivariable models. The management of perioperative HF in the ADHF group was insufficient partly because we could not help but manage AS patients complicated with MS in a wet volume condition, in order to avoid low-output HF associated with AS and MS. Second, although we defined significant MS as a mean mitral pressure gradient ≧5 mm Hg according to the guidelines of the American and European Society of Echocardiography, severe AS could underestimate the mitral valve pressure gradient.18Also, the planimetry data using three-dimensional transoesophageal echocardiography were not available although it is prioritised for the diagnosis of MS complicated with AS in the current guidelines, because we were unaware of the guidelines when we planned the study in 2016.In conclusion, MS and drastic improvement of mAVPG were associated with the new development of ADHF within 72 hours after TAVI.In-out balance was calculated as a volume of infusion and blood transfusion minus urine output.