Dyslipidemia in pregnancy is associated with adverse pregnancy outcomes as elevated triglycerides might be considered as a risk factor for hyperglycemia and gestational diabetes. As only a few studies have addressed the association between maternal triglycerides and glucose metabolism, we aimed to explore the pathophysiologic associations of moderate hypertriglyceridemia and maternal glucose metabolism in pregnancy.Sixty-seven pregnant women received a detailed metabolic characterization at 12+0–22+6 weeks of gestation by an extended 2h-75g OGTT ; with measurements of glucose, insulin and C-peptide at fasting and every 30 min after ingestion and assessment of triglycerides at fasting state. All examinations were repeated at 24+0–27+6 weeks of gestation.p=0.022 for an increase of 10 mg/dl).Elevated triglycerides in early gestation were associated with insulin resistance and β-cell dysfunction. Mean glucose concentrations during the OGTT in early pregnancy were already higher in women with hypertriglyceridemia as compared to women with triglycerides in the normal range. A higher degree of insulin resistance and increased OGTT glucose levels were also observed when metabolic assessments were repeated between 24 and 28 weeks of gestation. Of note, elevated triglycerides at early gestation were associated with development of gestational diabetes by logistic regression  contains supplementary material, which is available to authorised users. Gestational diabetes mellitus (GDM) is widely accepted as a major reason for adverse pregnancy outcomes including fetal macrosomia, cesarean section rate, neonatal hypoglycemia or the development of obesity in the newborns’ later life –3. In adβ-cell function) are described, and their possible association with the consecutive development of GDM is assessed as a secondary objective.Therefore, this study aims to assess the pathophysiologic characteristics of subclinical hyperlipidemia in early pregnancy. In particular, associations between maternal triglycerides and parameters of glucose metabolism . The study was approved by the Ethics Committee of the Medical University of Vienna and performed in accordance with the Declaration of Helsinki. All participants gave written informed consent.The present explorative study aimed at identifying risk factors for glucometabolic disorders in pregnancy as published in detail elsewhere . In shorβ-cells can adapt to impaired insulin sensitivity was examined by the ISSI-2 index  as ISI-comp × AUC-insulin/AUC-glucose 0–120. In addition, basal insulin secretion (BIS) and total insulin secretion rate (TIS) were assessed by mathematical modelling from insulin and C-peptide data according to 19].[19].18][.[19].18]patients . This iset al. assessed differences in maternal lipid content in women who developed GDM as compared to normal glucose tolerant control women and observed elevated levels of triglycerides in women with GDM in the first trimester, already before GDM was diagnosed 29].[29].28][.[29].28]Distinguishing independent contributions of pre-pregnancy obesity, pre-pregnancy insulin resistance or maternal lipid levels to impaired insulin resistance are challenging. We conclude that even moderately elevated triglycerides at the beginning of pregnancy are associated with impaired insulin action and β-cell dysfunction and hence with a potential risk factor for GDM development.This study investigated pathophysiological patterns of maternal glucose metabolism during pregnancy in association with fasting triglyceride levels. Hypertriglyceridemia at the start of pregnancy was closely related to impaired insulin action and β-cell function. Women with hypertriglyceridemia had higher mean glucose levels in early- and mid-gestation. Consequently, increased serum triglycerides were associated with a higher risk for the development of GDM.Supplementary file1 (DOCX 12 kb)Below is the link to the electronic supplementary material.


Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use.We previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform.Comparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥  ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma  and significantly correlated  to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis.Our study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.The online version contains supplementary material available at 10.1186/s12931-021-01860-3. Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic and fatal lung disease with a huge unmet medical need –3. DespiPast studies have profiled gene expression in lung tissues, peripheral blood and isolated cells through microarray and bulk/single cell RNA-sequencing analyses, identifying aberrant cell populations as well as molecular signatures of progressive IPF –12. ReceWe hypothesized that a combined analysis of lung and plasma gene/protein signatures will identify robust biomarkers with potential clinical utility. Here, we have performed unbiased proteomic analyses of matching plasma from the advanced IPF cohort through the Somascan-1300 aptamer platform and compared the plasma proteome signature to the previously reported lung transcriptome signature. Our data reveal a striking enrichment of pathways involved in chemotaxis/Th2 chemokine and T-cell signaling, Wnt signaling, mast cell migration and activation, and extracellular matrix degradation in both tissue and plasma of advanced IPF. Notably, the Th2 chemokines CCL17 and CCL22 as well as mucosal chemokines CCL25 and CCL28 were robustly upregulated in IPF and correlated within subjects. The mast cell maturation chemokine CXCL12 was also increased in IPF together with a concomitant increase in the mast cell protease, TPSB2. Neoepitopes of collagen type III and VI degradation (C3M and C6M) were strongly downregulated and highly correlated in advanced IPF subjects. Our data provide a comprehensive signature of IPF tissue and plasma that could be potentially validated and utilized for clinical assessment of advanced IPF.All human subject sample acquisitions and experiments were conducted with the appropriate approval from the Institutional Review Board . The clinical profile and demographics of IPF, ALI and control subjects used in this study have been previously described . The IPFDetails on the RNA-sequencing method and analyses have been described previously in detail .Plasma samples were collected in Citrate EDTA tubes in operating room prior to explant, centrifuged at 4 ℃ at 3521 RPM for 10 min, and stored at − 80℃. Plasma samples were analyzed on the SOMAscan V2 multiplex proteomic assay —an aptamer-based quantitative proteomic biomarker discovery platform which measures 1033 analytes , 19. TheEach sample in the study was normalized by aligning the mean to a common reference. Inter-plate and inter-run calibration were achieved by applying a multiplicative scaling coefficient to each SOMAmer. These scaling factors were calculated using the eight reference calibrators on each plate. Sample data were first normalized to remove hybridization variation within a run followed by median normalization across all samples to remove other assay biases within the run and finally calibrated to remove assay differences between runs. Log transformed RFU values were used to analyze differential expression of biomarkers across cohorts by using the Limma linear modeling framework for differential expression , 21. PatNeoepitopes of collagen III and VI degradation (C3M and C6M) respectively were analyzed by previously described ELISA methods , 23.Statistical analyses of differential gene expression data using the R package has been described previously . SomaLogPlasma samples from the IPF, ALI and healthy control cohorts were analyzed using the Somascan 1300-plex aptamer platform. Normalized log-transformed data were used to generate protein expression intensities. Expression data were visualized using t-scholastic neighborhood enrichment (t-SNE), that showed a robust separation of the IPF samples from the control and ALI samples , we identified 53 genes commonly regulated in lung and plasma, with 34 of them moving in the same direction Fig. a and plaSince pathway analyses of IPF plasma revealed a strong dysregulation of proteins involved in ECM homeostasis and remodeling, we measured C3M and C6M, neoepitopes of Collagen III and Collagen VI degradation respectively. Interestingly, we found a marked decrease in the levels of both C3M and C6M in IPF compared to healthy controls Fig. a, b. AddWe describe here for the first time a simultaneous comprehensive analyses of plasma proteome and lung transcriptome of a unique cohort of advanced IPF patients in comparison to that of normal healthy donors. Our studies suggest a strong dysregulation of T-cell activation, chemokine signaling, mast cell maturation, Wnt signaling and ECM homeostasis pathways in lung tissue as well as peripheral blood of these patients and identify new biomarkers that could have clinical utility. Although numerous profiling studies in the past have identified dysregulated genes, proteins and pathways in early and progressive IPF, there are currently no diagnostic or prognostic biomarkers in clinical practice , 24–26. Unbiased hierarchical clustering of protein expression across the cohorts shows that the IPF samples were clearly separated from control and ALI samples. Consistent with this separation, we did not find major differences between the ALI and control cohorts, although our previously published RNA-seq data identified significant differences between the cohorts at the gene expression level. In the ALI samples, the degree of diffuse alveolar damage varied with the majority having extensive areas of pathology, while some had more focal areas. This degree of sample heterogeneity is expected and may have potentially contributed to the similarities seen between the ALI and donor sample groups in our study.Plasma data from the IPF cohort not only confirmed and extended our previous lung transcriptome findings in the same cohort , but alsAnother key finding from our study was the upregulation of the mast cell chemokines, CCL5, CCL21 and CXCL12, and the mast cell protease tryptase-B2. Prior studies have shown increased infiltrating mast cell numbers and tryptase activity in human IPF , 36, andAberrant reactivation of developmental pathways including that of Wnt signaling is known to play a role in the pathogenesis of IPF . In our Consistent with an advanced fibrotic state, we found ECM remodeling and proteolysis pathways strongly dysregulated in the IPF cohort. The matricellular protein, SPARC, plays a key role in promoting collagen assembly into the ECM, and implicated as a biomarker in previous studies , 45. SPAAlthough RNA-seq and SomaLogics represent distinctly different platforms that limit robust comparison, our analyses clearly confirmed many analytes dysregulated at gene level to be differentially regulated in plasma as well. It is possible that many other markers of interest may be missed due to the targeted 1300-plex analyses. Future studies with the currently available expanded Somascan platform  could help further identify and validate additional biomarkers of disease. Interestingly, new emerging data indicates that the use of a multi-omic approach such as ours could be valuable in identifying molecular disease signatures and biomarkers of IPF . AnotherIn summary, we have presented a unique comparative transcriptome-proteome signature of advanced IPF and identified key tissue and circulating biomarkers that could be predictive of progressive/worsening IPF. Further validation of these findings in larger cohorts will help develop a comprehensive panel of biomarkers with clinical utility to address the current unmet need in the diagnosis and management of IPF.Additional file 1. Differential protein expression in plasma of IPF patients compared to healthy controls.


The eye and the brain have limited capacities for regeneration and as such, immune-mediated inflammation can produce devastating consequences in the form of neurodegenerative diseases of the central nervous system or blindness as a result of ocular inflammatory diseases such as uveitis. Accordingly, both the eye and the brain are designed to limit immune responses and inflammation – a condition known as “immune privilege”. Immune privilege is sustained by physiological, anatomical, and regulatory processes that conspire to restrict both adaptive and innate immune responses. A striking example of immune privilege was reported by Abel who treated an 8-year old boy who had a 10 week history of ocular pain Figure 1th century when it was discovered that although lymphatic vessels were invisible to most anatomical observations, antigens introduced into the AC could reach the ipsilateral cervical lymph nodes  of the eye is devoid of lymphatic drainage, which presumably isolates the interior of the eye from the systemic immune apparatus. Although corneal transplants enjoy a survival that is unrivaled in the field of transplantation, they can occasionally undergo immune rejection, which remains the leading cause of corneal graft failure. The notion that the AC of the eye is devoid of lymphatic drainage was proposed by Medawar over a half-century ago  and persph nodes , however pathway .Over the past 50 years it has become clear that immune privilege in the AC and the success of corneal allografts are due to a combination of anatomical, physiological, and dynamic immunoregulatory processes that restrict the expression of both innate and adaptive immune responses , 9. In aThe aqueous humor that fills the AC of the eye is a potpourri of anti-inflammatory and immunosuppressive molecules including: a) transforming growth factor-β(TGF-β), b) α-melanocyte stimulating hormone (α-MSH), c) complement regulatory proteins (CRP), d) calcitonin gene-related protein (CGRP), e) somatostatin (SOM), f) macrophage migration inhibitory factor (MIF), and g) vasoactive intestinal peptide (VIP) –16. ThesIn addition to the anatomical and physiological properties of the eye, dynamic immunoregulatory processes contribute to ocular immune privilege. Antigens entering the eye including histocompatibility antigens sloughed from the corneal endothelium of corneal transplants induce a dynamic form of immune deviation in which cell-mediated immune responses such as delayed-type hypersensitive (DTH) and cytotoxic T lymphocyte (CTL) responses are suppressed by T regulatory cells (Tregs) – a phenomenon termed anterior chamber-associated immune deviation (ACAID) . Not onlThe induction of ACAID is an extraordinarily complex process that involves four organ systems: a) eye, b) spleen, c) thymus, and d) sympathetic nervous system. Removal of the eye, thymus, or spleen within three days of introducing antigens into the AC abrogates the induction of ACAID. Likewise chemical sympathectomy prior to AC injection of antigens blocks the induction of ACAID. The contributions of these organ systems in the induction of ACAID are complex and are discussed in detail elsewhere  , which under the influence of soluble molecules in the AC, induce the generation of Tregs. Antigens are captured by resident F4/80ce ACAID . Ocular ce ACAID . The rol KO mice .ACAID is intimately associated with corneal allograft survival , 24 ManeThe remarkable success of corneal allografts defies the laws of transplantation immunology. Studies in animal models of penetrating keratoplasty demonstrate that in the absence of any form of immunosuppressive agents corneal allografts mismatched with the recipient at all known MHC and minor histocompatibility gene loci routinely enjoy a 50% acceptance rate . By contThe leading risk factor for corneal allograft failure in human subjects is the rejection of a previous graft. The incidence of corneal allograft rejection rises three-fold in patients receiving a second corneal transplant . On firsIn an attempt to unravel this paradox we employed a mouse model of penetrating keratoplasty and recapitulated experiments in which patients receive two corneal transplants from unrelated donors. For these experiments we employed a widely utilized mouse model in which C57BL/6 corneal allografts are transplanted to BALB/c recipients. These two mouse strains differ at all known major and minor histocompatibility gene loci, yet only 50% of the corneal allografts undergo immune rejection even though no immunosuppressive drugs are employed. To evaluate the effect of a first corneal transplant on the survival of subsequent corneal grafts we transplanted corneas from C3H mice to the right eyes of BALB/c recipients. Sixty days later we transplanted C57BL/6 corneas to the opposite eye  of these mice. Instead of the expected 50% incidence of rejection for initial C57BL/6 corneal allografts in naïve hosts, we observed 100% graft rejection . ImportaIt is important to note that SLIP should not be confused with a similar condition called sympathetic ophthalmia (SO) that sometimes occurs in individuals following a penetrating traumatic injury to one eye that is subsequently followed by intense inflammation in the opposite eye . The infThe impact of neuropeptides on immune responses has long been recognized. Neuropeptides exert an important effect on immune privilege in the eye . InteresSevering corneal nerves also adversely affects ACAID. Placing a shallow corneal incision in one eye prevents the induction of ACAID in both eyes , 32. The+ ocular surface DCs to the area where we placed circular incisions in the cornea. When stimulated via their NK1-R, CD11c+ DCs inhibit IL-10 production and promote the generation of Th1 immune responses  and adoptively transferring them to naïve recipients. ACAID could not be induced in these recipients but was induced in recipients of CD11c+ DCs from donors that had not been subjected to corneal nerve injury  cells based on their capacity to suppress Tregs and their similarity to “contrasuppressor” cells described over four decades ago  CD4+CD25+ corneal allograft-induced Tregs. In both cases the CS cells exert their effect in an antigen-non-specific manner. The molecular mechanisms involved in Treg suppression are still under investigation, but at least one molecule has emerged as a crucial player. Microarray analysis on 12,000 genes expressed on CD8+ ACAID T regs revealed an 85-fold increase in the expression of CD103  CD8of CD103 . The finD T regs . It bear+ T regs –45. WithD T regs . The dowin vitro . CD103 iin vitro , 48. TGFin vitro . These i+ CS cells yet “X” shaped corneal incisions of similar linear dimensions do not adversely affect corneal allograft survival or ocular immune privilege  are closely associated with the release of SP and the termination of ocular immune privilege  and other pathogen recognition receptors (PRRs). Although TLRs are expressed on cells comprising the ocular surface, other danger signals also originate at the ocular surface. Corneal nerve injury, alkali burns, and infectious agents can terminate immune privilege by a process that involves the release of SP, which represents another “danger signal” . It is nrivilege –55. It ilococcus . Pain siThe author confirms being the sole contributor of this work and has approved it for publication.Supported in part by P30-EY030413 and an unrestricted grant from Research to Prevent Blindness.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Desulfovibrio vulgaris (Dv) with Methanococcus maripaludis (Mm) by longitudinally monitoring mutations accumulated over 1000 generations of nine independently evolved communities with analysis of the genotypic structure of one community down to the single-cell level. We discovered extensive parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance within many evolution lines of a rare lineage of Dv that retained sulfate-respiration (SR+) capability, which is not required for metabolic cross-feeding. An in-depth investigation revealed that synergistic epistasis across pairings of Dv and Mm genotypes had enhanced cooperativity within SR− and SR+ assemblages, enabling their coexistence within the same community. Thus, our findings demonstrate that cooperativity of a mutualism can improve through synergistic epistasis between genomes of the interacting species, enabling the coexistence of mutualistic assemblages of generalists and their specialized variants.Early evolution of mutualism is characterized by big and predictable adaptive changes, including the specialization of interacting partners, such as through deleterious mutations in genes not required for metabolic cross-feeding. We sought to investigate whether these early mutations improve cooperativity by manifesting in synergistic epistasis between genomes of the mutually interacting species. Specifically, we have characterized evolutionary trajectories of syntrophic interactions of  The slower growing SR+ lineages (K-strategists) are favored in later stages of growth when the resources are limited but stable, and cell density is high [r/K tradeoffs might explain why SR+ populations are retained in the absence of sulfate. In the natural world, where sulfate availability varies over time, persistence of SR+ genotypes in the absence of sulfate may stabilize sulfate-reducing populations overall. In other words, it may be a bet-hedging strategy  thaDv and Mm mutations within each EPD reflect coevolution will require additional experiments, including pairing with evolved populations from preceding generations [Dv and Mm to synergistically improve growth characteristics lends credibility to the claim that complementary genetic changes  enhanced metabolic coupling and cross-feeding between the two interacting organisms, significantly increasing their cooperativity.It was significant that each of the two EPDs segregated a subset of high G-score mutations into simplified assemblages but retained growth rate and carrying capacity of the parental evolved population. This result demonstrated that multiple independent evolutionary strategies can coexist in the same population, albeit in vastly different proportions. Whether the distinct sets of erations . NotwithDv and Mm would be aligned and exploitation unlikely [Dv and the only energy source available for Mm. Efficient transfer of electrons through hydrogen is in the best interests of both species [Mm (MMP1511) could reflect the evolution of a new costly dependency. Alanine was earlier shown to be exchanged between the two interacting partners during syntrophic growth, likely at a cost to the producer (Dv) and of energetic advantage to Mm [Dv provides a mechanism to re-oxidize reduced internal cofactors during syntrophic growth, but at the cost of a high-energy phosphate bond. In turn, alanine taken up and converted to pyruvate and ammonia by Mm serves as both a carbon and a nitrogen source, alleviating complete dependency on energetically costly autotrophic growth with hydrogen. A cheater population, e.g., one with a loss of function mutation in MMP1511, might consume additional alanine through passive transport and therefore consume less hydrogen to maintain lactate consumption by Dv. Indeed, mutations in MMP1511 rose to fixation in six out of nine lines, and we cannot rule out if minor MMP1511 mutant populations also exist in low frequency in the other lines, including UE3.The nature of cooperation in this syntrophic mutualism is unclear. On the surface, it seems like the fitness of unlikely –63 becau species . However species , 65. Onege to Mm . AlanineDv and Mm, invoking the possibility of partner choice and partner fidelity feedback [Dv and Mm. In conclusion, the multiscale dissection of independent laboratory evolution lines has demonstrated that selection of complementary mutations across Dv and Mm synergistically increased the cooperativity and productivity of syntrophic interactions within both SR− and SR+ communities, while supporting their coexistence in vastly different proportions as r- and K-strategists, respectively.The observation that interactions among some genotypes were more productive than other pairings suggests that the enhanced cooperativity of evolved communities could have occurred through the selection of complementary mutations across feedback . FurtherAll the strains, culture conditions, and the setup of the laboratory evolution experiment were the same as described before , 5. , processed for sequencing using Nextera DNA library preparation kit  and sequenced on Illumina Hiseq with 100 bp paired-end sequencing or on Illumina MiSeq in the paired-end mode producing 2 × 250 bp long reads as described before [https://github.com/sturkarslan/evolution-of-syntrophy)  . VariantDv and Mm from mid-log phase EPD cultures were sorted into wells of a 96-well plate and lysed by using a freeze-and-thaw cycle. Whole Genome Amplification from single cells was performed using REPLI-G Single-Cell kit . We screened single amplified genomes (SAGs) with 16S universal primers for Dv and Mm, and used AmpPure XP magnetic beads  to clean and purify confirmed SAGs; QC was performed with Bioanalyzer. DNA concentrations were determined using Quant-iT PicoGreen dsDNA assay kit . Sequencing was performed as described above.Single cells of Variants detected in at least two cells at >80% frequency were converted into a matrix  with unique mutation in rows and single cells in columns. Mutation histories of single cells were determined using the SCITE algorithm  with parZ-score as described in G-score (“goodness-of-fit”) for each gene was calculated based on the frequency of observed nonsynonymous mutations  across 13 evolved lines, as described before . G-score2:CO2 headspace at 30 °C without shaking. All stationary phase cultures were diluted to the same OD600 value and subjected to a 1.5-fold dilution series in 96-well plates. Blank media were included in the first column of each 96-well plate to rule out contamination. All steps were performed in a Coy Anaerobic Chamber with 95%:5% N2:H2. Growth measurements were performed in a plate reader . See Ancestral cocultures and EPDs were grown anaerobically in balch tubes containing coculture medium A (CCMA)  with 80%Dv clonal isolates were revived anaerobically in balch tubes containing CCMA flushed with 80%:20% N2:CO2. Mm isolates were revived in balch tubes pressurized to 30 psig with 80%:20% H2:CO2. Aliquots (0.1–0.2 ml) of stationary phase cultures (after two transfers) of Dv and Mm were combined in 20 ml of CCMA containing 30 mM sodium lactate in balch tubes flushed with 80%:20% N2:CO2, and incubated at 37 °C with shaking at 300 rpm. Growth parameters were estimated by analyzing changes in cell density (OD600nm) using grofit [All g grofit .Dv (fDv) and Mm (fMm) was determined by pairing evolved clonal isolates with ancestral clones. The expected fractional effect on growth rate and yield fDvMm, induced by the combined effect of evolved isolates was calculated as:Synergistic, additive, and antagonistic effects of clonal isolate pairings was determined using the Bliss Independence model . The expfz, and the expected fractional inhibition, fDvMm.Excess over Bliss (EOB) was determined by computing the difference in fractional improvement of growth rate or yield induced by combination, A clonal isolate pair combination for which EOB ≈ 0 has an additive behavior, whereas a pair with positive or negative EOB values has synergistic or antagonistic behavior, respectively. Error was computed by propagating the standard deviation of fractional effects.P value of an experimental observation as P = (s + 1)/(n + 1), where s is the number of the simulation instances with equal or stronger association as observed experimentally and n is the number of simulated mutational trajectory experiments (n = 1 × 106).To quantify the likelihood of observing a mutated gene given another one mutated, we generated a background distribution assuming a mutation in a particular gene as an independent event. We simulated a large random set of mutational trajectory experiments maintaining the number of mutations per evolutionary line and the mutational frequency observed for each gene. We computed an empirical Supplemental Material


Orthogonal projections to latent structures discriminant analysis (OPLS-DA) followed by pathway and enrichment analysis generated classification models and revealed significant metabolites that were associated with altered pathways. A clear association between maternal and cord blood altered metabolomic profile was evidenced in IUGR pregnancies. Increased levels of the amino acids alanine, leucine, valine, isoleucine and phenylalanine were prominent in IUGR pregnancies indicating a connection with impaired amino acid metabolism and transplacental flux. Tryptophan was individually connected with cord blood discrimination while 3-hydroxybutyrate assisted only maternal blood discrimination. Lower glycerol levels in IUGR samples ascribed to imbalance between gluconeogenesis and glycolysis pathways, suggesting poor glycolysis. The elevated levels of branched chain amino acids  in intrauterine growth restricted pregnancies were linked with increased insulin resistance.Intrauterine growth restriction (IUGR) is a fetal adverse condition, ascribed by limited oxygen and nutrient supply from the mother to the fetus. Management of IUGR is an ongoing challenge because of its connection with increased fetal mortality, preterm delivery and postnatal pathologies. Untargeted nuclear magnetic resonance ( More specifically, intrauterine growth restriction (IUGR) is associated with chronic hypoxia and stress, leading to adverse endocrine axis reprogramming5. The aforementioned alterations predispose IUGR infants to impaired glucose homeostasis, insulin sensitivity and adipose tissue development, factors that are associated with coronary vascular disease, hypertension and diabetes mellitus in adulthood7.An emerging body of literature indicates that abnormal fetal growth is associated with increased risk of perinatal morbidity and mortality8. The main cause of IUGR is placental insufficiency9, causing impaired transfer of nutrients to the fetus due to limited blood flow10.IUGR results from the failure of the fetus to reach its intrinsic growth potential, due to maternal, fetal, or placental pathology11. Different rates have been recorded in the literature depending on fetal intrinsic factors, as well as prenatal follow-up and optimal perinatal care12.The incidence of IUGR is approximately 8% in total population13, it is critical to elucidate the underlying pathogenesis of IUGR and the respective implicated metabolic pathways.Considering that fetal growth along with gestational age at birth determine neonatal survival, as well as short and long-term complications14.Prenatal diagnosis of IUGR mainly relies on abnormal umbilical and uterine Doppler measurements that enable real time estimation of placental/fetal circulation, but do not permit early intervention. Diagnosis is also assisted by an ultrasound-estimated fetal weight below the corresponding 10th percentile for the gestational week18.Thus, it is essential to determine biomarkers framing the time of insult or maternal–fetal metabolic interaction suggesting early intervention. Although several metabolites have been suggested in literature as putative biomarkers for IUGR screening, still none is generally applied in clinical practiceThe current study presents a nuclear magnetic resonance (NMR) based untargeted metabolomic approach focusing on IUGR pathology with a twofold aim; (a) to provide explanatory models for IUGR pathogenesis and (b) to identify putative markers of prediction which can be validated at the early stages of pregnancy. Both umbilical cord and maternal blood from well-defined IUGR and appropriate for gestational age (AGA) cases were investigated in one of the biggest sample sizes compared to relevant studies.1H-NMR spectra with characteristic annotations is presented in Fig. Assignment for 56 individual metabolites excluding the lipid and sugar regions is presented in Supplementary Table Principal component analysis (PCA) on NMR data was implemented in each of the four studied groups  to assess sample homogeneity and pinpoint outliers. Several characteristics including maternal pathology, smoking habits, gestational age, maternal age, maternal BMI, mode of delivery and gender were examined in each of the produced statistical models. Maternal pathology testing is shown in Supplementary Figs. Six IUGR participants with gestational diabetes mellitus who presented increased glucose levels and one participant with thrombophilia with a distinct profile were excluded from further investigation from the IUGR group . Furthermore, in umbilical cord blood samples (41 IUGR and 36 AGA), a discrimination trend was observed among IUGR and AGA samples based on the mode of delivery  after the removal of lactate and methanol regions is presented in Fig. As depicted in Fig. A similar data analysis process was followed among maternal samples. Figure The metabolites assigned to the discriminatory bins of the loadings plot Fig. b along wTable Pearson correlation testing between the peak integral areas of the remaining lipids at 0.82 and 1.16 ppm with the BCAAs and the 3-hydroxybutyrate areas for 10 representative samples of each group  probed to no significant correlation   for cord blood discrimination and isoleucine (AUROC = 0.812) for maternal discrimination.Enrichment analysis provided the deregulated pathways due to the IUGR pathology. Supplementary Tables A similar metabolic pattern was evident for the first time between maternal and umbilical cord blood in IUGR pregnancies. This finding may indicate common mechanisms underlying the IUGR condition which connected between mothers and fetuses and can pave the way for the identification of putative biomarkers in maternal blood targeting early IUGR screening.19.Controversial information is evidenced in the literature concerning candidate metabolites for IUGR-AGA discrimination, with abnormal amino acid metabolism, glucose intolerance and limited insulin resistance being an important finding20 which may result in a reduced utilization rate of the BCAAs and alanine, leading to a consequent increase of their levels. This increase could also be explained by the observation that IUGR fetuses experience the same metabolic derangements with certain diseases, with urea cycle defects being the most relevant21.IUGR off springs have altered protein expression profiles2 which facilitate the energy production22. IUGR is linked to reduced oxygen and nutrients supply through the placenta23 which could partly explain the observed BCAAs increase. Nutrient deficiency has also been proposed to describe the up-regulation of valine and isoleucine in the IUGR vs AGA groups16.Starving conditions promote protein degradation leading to the consequent increase of BCAAs. These are degraded in tissues, other than liver, yielding reduced NADH and FADH26. Dessi et al.18 suggested that IUGR fetuses stimulate a number of adaptive mechanisms in order to save glucose and promote nutrition of the vital organs, resulting in down-regulation of insulin secretion. Herein, the observed increase of BCAAs in the IUGR group could be associated with insulin resistance of the fetus. Glucose intolerance or limited insulin adequacy in IUGR, may account for altered tricarboxylic acid (TCA) metabolic intermediates, such as alanine and leucine27.Moreover, increased levels of BCAAs have been associated with insulin resistance and future type 2 diabetes26, the branched-chain keto acid (BCKA) 3-methyl-2-oxovalerate, has been identified as one of the strongest predictors of impaired fasting glucose (IFG). This BCKA arises from the incomplete catabolism of BCAAs28 which primarily occurs in the mitochondria. High levels of the corresponding metabolite are also linked with the metabolic disorder of maple syrup urine disease that is caused by the deficiency of the branched-chain alpha-keto acid dehydrogenase complex, leading to the accumulation of BCAAs28.In a previous type 2 diabetes metabolomic study22. Higher alanine levels were also found for IUGR having a birth weight between 3rd and 10th percentile29.The glucose-alanine cycle describes the synthesis of glucose from alanine in the liver and the transport of glucose back to the muscles. Alanine serves as a precursor or regulator of glucose metabolism that is of critical importance in the nutrient-deprived IUGR, thus explaining the increased alanine levels found in the IUGR group30. In the liver, glycerol may be converted into glucose, meeting the required energy needs for cellular metabolism31. Glycolysis, a compensatory pathway that breakdowns glucose, is deregulated in conditions of limited insulin adequacy or resistance32. IUGR infants have insulin deficiency18 which partly explains poor glycolysis together with a continuous conversion of glycerol to glucose through gluconeogenesis. Also, lower glycerol levels in the IUGR groups could indicate an imbalance between triacylglycerol breakdown and re-synthesis during several metabolic reactions in a continuous flux between adipose tissue and liver.In our research, lower glycerol levels were observed in the IUGR group, signifying glycerol deprivation. Gluconeogenesis is a pathway describing glucose synthesis from lactate, pyruvate, glycerol and glucogenic amino acids33, resulting in the accumulation of 3-hydroxybutyrate. Furthermore, the observed increase of the ketogenic essential amino acids leucine and isoleucine, may also account for increased 3-hydroxybutyrate synthesis33. The metabolomic study of Powel et al.34 suggested 3-hydroxybutyrate is a predictor of IUGR complication in maternal serum as well, however, with low predictive power.In pregnant women with glucose intolerance, ketone body formation is favored against carbohydrate consumption16. Cosmi et al.35 in line with our observations indicated tryptophan as a candidate marker of IUGR pathology, but reported findings are different to our results regarding our observed up-regulation of valine and isoleucine and the down-regulation of phenylalanine. Of note, such outcomes refer to twin neonates and are deprived by limited sampling.Different brain serotonin synthesis has been associated with altered tryptophan levels.36 found a lower fetal/maternal enrichment ratio for phenylalanine and leucine in IUGR compared to normal pregnancies, suggesting an impaired transplacental flux. An altered placental metabolism or transport alongside with a higher catabolic condition has been also suggested to explain the lower phenylalanine levels in the IUGR samples37. The fetomaternal ratio for the essential amino acids is reduced in cases of small for gestational age (SGA) compared to AGA, as a result of fetal hypoxemia38. Finally, increased levels of BCAAs and alanine in the maternal blood of IUGR vs AGA groups, is possibly explained due to the altered adaptation mechanisms of IUGR pregnancy with incomplete hormone production39.Paolini et alIn our study we interpreted the generated metabolomic data and attempted to correlate a fetal adverse condition with alterations in the metabolic profile as putative biomarkers. A similar metabolic profile was evident between maternal and umbilical cord blood in IUGR pregnancies, with seven out of 56 identified metabolites being responsible for discrimination. As an exception, tryptophan contributed only to cord blood discrimination while 3-hydroxybutyrate only to maternal blood discrimination.Higher levels of the BCAAs, together with alanine were observed in the IUGR groups, in contrast to increased phenylalanine and glycerol levels for the AGA groups, indicating the crucial roles of amino acid metabolism, insulin resistance and glycolysis pathway. The elevated levels of BCAAs in intrauterine growth restricted pregnancies were linked with increased insulin resistance. Although a number of clinical studies have facilitated metabolomics to elaborate on the IUGR condition, biomarkers that determine this condition early on, are still lacking. From our findings, the role of previously reported metabolites has been strengthened together with the significance of 3-hydroxybutyrate in IUGR maternal blood. The proposed metabolic signatures have to be validated at an earlier stage of pregnancy in order to obtain clinical impact as diagnostic and prognostic biomarkers for IUGR.An approval (No. EE-2/26/06-06-2017) from the Aretaieio Hospital Review Board along with the Ethics committee was received before the experiments were started. All experiments were performed in accordance relevant guidelines and regulations. Signed informed consent was obtained from the participating mothers prior to enrollment.The study comprised 84 parturients giving consecutively birth to 48 IUGR (≤ 10th customized centile) and 36 AGA (between 10 and 90th customized centile) full-term infants , vortexed and placed at − 20 °C for 20 min. Samples were centrifuged  for 20 min to allow protein parts to precipitate. Supernatants placed to new eppendorf tubes and evaporated to dryness. Samples were reconstituted to 400 μL D2O and 150 μL phosphate buffer . Then, 500 μL of each mixture with 50 μL of internal standard trimethylsilyl propionic acid sodium salt (TSP) (2.75 mM) were transferred to 5 mm NMR tubes for 1H-NMR analysis.Serum samples were thawed at room temperature and extracted according to Nagana Gowda et al1H-NMR spectra were acquired using a Varian 600 MHz spectrometer equipped with a triple resonance probe , at room temperature (25 °C). The CPMG pulse sequence following water suppression was applied in all experiments since it provided better spectra in terms of S/N ratio compared to 1DNOE pulse sequence. In total, 128 transients were collected with 64 K data points and a relaxation delay of 5 s. Receiver gain was auto set 60 for all acquisitions. All 1H NMR spectra were referenced at TSP chemical shift (0.00 ppm) and processed at 0.3 exponential line broadening.All 1H-NMR spectra were preprocessed with MestreNova (v. 10.1) software. Manual phase correction, automatic baseline correction and sinc apodization were applied to improve spectra resolution. Total area normalization and binning of 0.001 ppm were selected. Superimposed spectrum was constructed and peaks were manually aligned. The water region (4.68–5.00 ppm) was excluded together with the peak at 3.36 ppm attributed to methanol from protein precipitation procedure.All 41 and the online NMR databases HMDB42 and BMRB43. The metabolites 3-hydroxybutyrate, isoleucine as well as the 3-methyl-2-oxovalerate that presented low signal to noise ratio were further elucidated by spiking the reference pool sample and performing 2D gHMBCad NMR experiment  on a representative pool sample. Assignment of spectral peaks was assisted by Chenomx database , an in-house software (Metaboneer)2Y (goodness of fit) and Q2 (goodness of prediction) values. Validation of discrimination was made through response permutation testing (999 permutations), analysis of variance (CV-ANOVA) and extraction of ROC curves.Multivariate statistical analysis was employed to aligned spectra after Pareto scaling, using SIMCA software . At first, PCA was applied to provide a general insight  of samples. OPLS-DA was applied next, to generate classification models. Model performance assessed through the R46 was applied next to find significant metabolites discriminating umbilical cord blood and maternal groups. Biomarker analysis between IUGR-AGA pairs revealed candidate biomarkers  and used for enrichment analysis to provide altered metabolic pathways.Color coded loadings plots (s-line) attributed certain metabolites responsible for the discrimination pattern. MetaboAnalyst 4.0Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector  Gene drives have the potential to be applied as a novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism—most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance. Anopheles gambiae and Anopheles stephensi [CRISPR/Cas9 homing gene drives have shown much promise in providing an effective drive mechanism for novel genetic population modification and suppression strategies against insect vectors of major diseases, in particular the mosquitoes tephensi –4. In getephensi –7. UnderSelection of mutations at the target site, or pre-existing alleles in the population, however, can hinder this type of gene drive strategy and prevent further spreading of the drive element . MutatioA. gambiae doublesex (dsx) gene, complete suppression of wild-type laboratory populations was achieved, while no alleles resistant to the gene drive were selected [As a proof of principle, this approach has been used to mitigate resistance in small cage/scale testing observed in previous applications that targeted female fertility genes . By targselected . AlthougAnopheles genus, spanning over 100 million years on an evolutionary timescale, as well as wild caught individuals of the closely related Anopheles gambiae and Anopheles coluzzii species [Anopheles species revealed over 8000 ultra-conserved sites (100% identity) of sufficient length  to be targetable by homing-based gene drives [Our ability to identify novel genes containing gene drive targets that show high functional constraint has been greatly enhanced by genome resequencing efforts that have covered species across the  species –11. A ree drives . It is tIn this study, we focussed our analysis on ultra-conserved sites within putative haplosufficient essential genes as potential target sites for robust gene drives aimed at population suppression. Having generated an active gene drive targeting one of these essential genes, as proof of principle we subjected it to a maximal selection pressure to test its resilience to the selection of gene drive resistant alleles and its overall suitability for vector control.Anopheles species and search of functional annotations of genes containing UCEs by O’Loughlin et al [AGAP029113 gene is of interest as it contains multiple ultra-conserved sites with a total of 140 invariant sites. Its apparent orthologue in Drosophila melanogaster is Smrter (Smr), several null alleles of which lead to a recessive lethal phenotype, consistent with this gene being haplosufficient.A recent analysis of ultra-conserved elements (UCEs) from 21 different in et al  identifiAGAP029113 gene is located on Chromosome 2L: 2895973–2931030 and was recently reassigned as a fusion of AGAP004734 and AGAP004732 (Vectorbase.org). It consists of 12 exons  (An. gambiae and Anopheles coluzzii populations (2784 wild Anopheles mosquitoes sampled across Africa), this particular target site showed only negligible variations of less than 0.6% frequency [We chose a target site within AGAP029113 that is ultra-conserved across Culicidae  but not in  m.y.a.) . Even wirequency  S1 Fig)DrosophilhdrGFP’) into the target site within exon 5, which is expected to prevent the generation of a functional AGAP029113 transcript and likely represents a null allele. The insertion of this cassette was performed by CRISPR-mediated HDR (hdrGFP/+) and homozygous (29113hdrGFP/hdrGFP) individuals at the expected Mendelian ratio  was inserted into the locus as described previously [CRISPRh construct contained Cas9 with expression controlled by the zero population growth (zpg) germline promoter [RFP marker gene to assist in tracking the inheritance of the gene drive (CRISPRh cassette. This gene drive construct showed high rates of biased inheritance from heterozygous (29113CRISPRh/+) female (92.7% ± 2.9) and male mosquitoes (91.3% ± 3.2)  .CRISPRh/+) males and heterozygous (29113CRISPRh/+) females were crossed to wild-type G3 mosquitoes crossed repeatedly over multiple generations .Impaired fecundity and fertility of heterozygotes has been observed in other gene drive strains . We performed fecundity assays where heterozygous  and theCRISPRh/+ individuals into a wild-type caged population  at a starting frequency of 20%, in triplicate, and monitored the frequency of gene drive individuals over time. The same experiment was performed in parallel with the non-driving 29113 hdrGFP/+ mosquitoes as control.Despite the fitness costs apparent with this gene drive in heterozygous carrier individuals, the extent of biased inheritance was still expected to cause the element to increase in frequency in a population ,2. To teCRISPRh/+ females and reduced larvae to adult eclosion rate of 37.8% and 46.2% for 29113CRISPRh/+ males and females) (light red dashed line) and c) further fitness costs  that were not measured and were chosen arbitrarily to signify a very high genetic load (dark red dashed line). Overall, the data fitted the model c) assuming high additional fitness costs associated with the gene drive construct. This could include, for example, fitness costs such as a prolonged larval developmental time or a reduced adult mating success, which we did not assess.For releases with the control strain the frequency of individuals with the control allele decreased slowly over 3 generations to an average of 10.6%, consistent with the expectation for a non-driving null allele . HoweverWe also investigated the possibility of the generation and subsequent selection of a functional resistant allele to the gene drive that could have led to loss of the transgene. Such alleles can be formed when Cas9-mediated cuts are repaired by error-prone end joining DNA repair pathways producing small insertions or deletions or substitutions . Our modAGAP029113 gene between larval and adult transition, the distribution of in-frame and out-of-frame alleles was markedly different between the larval stage (where a complete null genotype is tolerated) and the adult stage (where it is not): pooled sequencing of the target site in L1 instar larvae revealed that 26.5% of non-drive alleles contained a target site mutation, of which 10.8% were in-frame ) of progeny would be expected to be homozygous for the gene drive and so would die during larval development. Of the progeny that survive to form the following generation, the majority would be heterozygous and destined to produce predominantly non-viable offspring. Under these conditions a functional resistance allele will therefore have a high selective advantage since it will confer viability on any genotype that inherits it.In order to investigate our assumption that these mutations could be strongly selected, we mimicked a scenario of genotype fixation , where selection pressure for functional resistance alleles is expected to be highest. We crossed individuals heterozygous for the gene drive construct, allowing all offspring to survive and reproduce for multiple generations. Given our previous estimate of gene drive transmission rate (93%), under this scenario the vast majority (87% (0.93hdrGFP/+ controls (in green), consistent with the majority of the offspring lacking a functional copy of the AGAP029113 gene. However, by generations 6 and 7 we observed a rise in frequency of larvae as well as the number of adults and eggs produced by the gene drive cage. Together, these observations are consistent with a breakthrough of functional resistance alleles that are refractory to gene drive invasion and restore the carrying capacity of the population. Indeed, sequencing of adults from the G7 generation  was constantly suppressed , producineration  showed oneration . Since tAny strategy, from antibiotics to insecticides, that aims to impose a fitness load on a population inevitably leads to strong selection for resistance. For gene drives, the easiest pathway for resistance to occur is for an allele to arise that codes for a functioning gene that lacks the endonuclease recognition site, thereby blocking the biased inheritance of the gene drive element. These alleles may pre-exist in the population, arise through spontaneous point mutation, or arise through error-prone (non-HDR) DNA repair pathways that introduce small mutations in the repair of the double stranded break generated by the gene drive.AGAP0029113 and showed that this gene is essential for development of both male and female mosquitoes. However, the invasive potential of the gene drive construct was hindered by high fitness costs in the heterozygous 29113CRISPRh/+ individuals compared to the wild-type strain. Fitness costs in gene drive carriers are not unusual and have been reported for other gene drive strains [zpg promoter has revealed a paternal [We developed a CRISPR homing gene drive targeting an ultra-conserved site in the viability gene  strains ,20,21. Apaternal  dependinAn. gambiae, An. coluzzii, Anopheles arabiensis and hybrid individuals, collected from 19 countries in Africa as part of Phase 3 of the Anopheles gambiae 1000 Genomes Project [Potentially such effects are more problematic for essential genes that are required during development in both sexes, compared to female fertility genes, since in the latter case heterozygous carrier males are unaffected. This is important for the phenotypic assessment of the suitability of future gene drive candidate genes required for viability in males and females and more emphasis should be placed on the restriction of gene drive activity to the germline. To look at the potential for gene drive resistance to arise at our chosen ultra-conserved target site, we devised both a single generation resistance assay and a population experiment designed to mimic the highest selection pressure. In both scenarios we recovered the same single point mutation at the target site. Possibly, the resistant allele we isolated might carry a small fitness cost which would be selected against in natural mosquito populations, which is why it was not detected within variation data from the 2784  Project . However Project . ConsequAs a general rule, for a population suppression gene drive, the largest load is imparted on a population when the target is a gene essential for female fertility or viability, yet a considerable, though reduced, level of suppression can be achieved when the gene produces a recessive lethal phenotype that manifests in both sexes . Given tOur mimicking of genotype fixation could be a useful experimental approach for swiftly assessing target site susceptibility, since it isolates resistant alleles faster than possible in population experiments of gene drive spread into wild-type populations—the selective advantage to a resistant allele over a wild type allele is highest when the likelihood of either allele finding themselves balanced against a gene drive allele is highest. A similar rationale applies in the resistance assay balancing the gene drive against a marked null allele ,24 that doublesex (1). In that specific case the gene drive targets a conserved site at an intron-exon boundary and it may be that the sequence is important at the RNA level and thus has even less degeneracy. Considerations such as this, or choosing target sites where the cleavage site lies in non-degenerate codons, or the non-wobble positions of degenerate codons, may add additional levels of resilience to a gene drive. However, at scale, in a field setting and considering the vast population sizes of mosquitoes, it is likely that such drives will need to be augmented by targeting multiple sites in the same gene, in order to greatly reduce the likelihood of multi-resistant alleles arising on the same haplotype [It will be essential to build on successes to date that have shown gene drives that can suppress populations robustly, without obvious selection for resistance, for example when targeting the female isoform of the sex determination gene aplotype –29. A siaplotype ,30. MoreAll animal work was conducted according to UK Home Office Regulations and approved under Home Office License PPL 70/8914.Cas9 coding sequence (hCas9) under the control of the zpg promoter and U6::gRNA spacer cloning cassette was utilized as described previously and modified using Golden Gate cloning to generate a PolIII transcription unit containing the AGAP029113-specific gRNA [GFP transcription unit under the control of the 3xP3 promoter enclosed within two reversible ϕC31 attP recombination sequences flanked both 5′ and 3′ by 2 kb sequence amplified using primer 29113 ex5 B1 f (CAACCAAGTAGTTACTGTGCTC) and 29113 ex5 B4 r (GTCTTTTGTTGTTGTTCACGT) and primer 29113 ex5 B3 f (TGTAGGCCGTGATCGTGC) and 29113 ex5 B2 r (GCGACACCATACTCCGATG) of the exon 5 target site in AGAP029113. To generate the 29113CRISPRh allele a gRNA spacer (GAACAACAACAAAAGACTGTAGG) bearing complete homology to the intended target sequence was inserted by Golden Gate cloning into a CRISPR construct (p17410) as described previously [A CRISPR construct p16510) containing a human-codon-optimized 6510 contAnopheles gambiae G3 strain with the 29113gRNA-modified p16510 plasmid (300ng/μl) and donor plasmid (300ng/μl) and obtained 7 transients that led to 6 transgenic individuals. Site-specific Integration was confirmed by PCR using primers binding the docking construct, 5′GFP-R (TGAACAGCTCCTCGCCCTTG) and a primer binding the genome outside of the homology arms: DL_29113 Ex5_F2 (TTCCACCTCTCGCTCGTAGT) and sequencing of the flanking sites. For the recombinase-mediated cassette exchange reactions a mix containing the CRISPR plasmid (200 ng/μl) and 400 ng/μl vasa2::integrase helper plasmid [hdrGFP docking lines. Progeny from the outcross of surviving G0 individuals to WT were screened for the presence of RFP and the absence of GFP that should be indicative of a successful cassette-exchange event.In order to generate the docking line, we injected 253 eggs of the  plasmid  was injeCRISPRh construct to home at super-Mendelian rates, individuals heterozygous (29113CRISPRh/+) for the construct were crossed with wild type individuals and the progeny scored for inheritance of the CRISPR construct (via the proxy of RFP) from individual lays (N = 15 per cross).To assess the ability of this 29113hdrGFP allele were crossed with each other and 60 L1-L2 larvae were analysed by Multiplex PCR using primer 3’-GFP-F (GCCCTGAGCAAAGACCCCAA), 5’-GFP R (TGAACAGCTCCTCGCCCTTG) and the primer DL_29113 Ex5_F2 (TTCCACCTCTCGCTCGTAGT) flanking the transgenic construct. The same PCR was repeated for 60 adult mosquitoes from the same cross.Males and females heterozygote for the 29113CRISPRh strain was maintained in 2 batches by crossing either transgenic males with wild type females and by crossing transgenic females with wildtype males. This was done to account for any parental effects caused by deposition of the Cas9. The 29113hdrGFP strain was maintained by crossing transgenic females with wildtype males. G roups of a minimum of 40 virgin heterozygotes carrying the 29113hdrGFP/+ or 29113CRISPRh/+ genotype respectively were mated to an equal number of virgin wildtype mosquitoes for 5 days and the number of eggs and offspring was counted from individuals lays (N≥12). Females that failed to lay eggs and did not contain sperm in their spermathecal were excluded from the analysis. Raw data including list of test statistics and sample sizes, as well all raw experimental data points, are included in S1_Data as an Excel file.The 29113hdrGFP/+ or 29113CRISPRh/+ genotype respectively were mated to an equal number of virgin wildtype mosquitoes for 5 days. 100 transgenic L1 larvae were selected and reared to adulthood for each experiment (N≥3).Groups of a minimum of 40 virgin heterozygotes carrying the 29113CRISPRh allele were mixed within 12 h of eclosion with age-matched WT larvae at a ratio of 1:5 in rearing trays at a density of 200 per tray (in ∼1 litre rearing water). The mixed population was used to seed three cages (36cm3) with 400 adult mosquitoes each. For three generations, each cage was fed after allowing 5 days for mating, and an egg bowl placed in the cage 48 h after a blood meal to allow overnight oviposition. A random sample of 450 eggs was selected. After allowing full eclosion, offspring were scored under fluorescence microscopy for the presence or absence of the RFP-linked CRISPRh allele, then reared together in the same trays and then used to populate the next generation.First instar mosquito larvae heterozygous for the 29113CRISPRh transgene, cleavage of the W allele occurs in W/T individuals, followed by either homology directed repair, converting W to T, or non-homologous end joining, converting W to R. Cleavage and end joining rates were calculated from the proportion of progeny inheriting the 29113CRISPRh allele (d) (F) = 0.927, (M) = 0.913 (u) = 0.266 u, (F) = 0.893, (M) = 0.872 and end joining (given cleavage) = p) of non-homologous end joining products to be functional (r) and 1−p to be non-functional (R). To incorporate the heterozygote fitness costs identified from the phenotypic assays we consider female heterozygotes carrying the transgene to lay fewer viable eggs compared to wildtype: fecundity cost (1—relative hatching rate) (F) = 0.216 , two sexes and discrete non-overlapping generations, following the structure of Beaghton et al , without = 0.913  and the  = 0.216 , and mal = 0.216 . We alsoCRISPRh allele were placed in a small cage (25cm3). Females were blood-fed after 5 days and an egg bowl was placed in the cage 48h post blood meal. After allowing full eclosion the number of eggs and RFP+ and RFP- larvae were recorded. All offspring were reared to adulthood and crossed with each other. This was continued for 7 generations. The same procedure was repeated for adults heterozygous for the 29113hdrGFP allele for 2 generations. Offspring was screened for GFP+.20 male and 20 virgin female adults heterozygous for the 29113CRISPRh allele (RFP+) were crossed to 400 females heterozygous for the 29113hdrGFP allele (GFP+). To collect L1 for DNA extractions, 7000 individuals were screened for GFP using COPAS. Larvae that were GFP+ only or RFP/GFP+ (RFP+ only and larvae with no fluorescent marker were discarded) were pooled and frozen in ethanol at -20°C. To collect adults for DNA extraction, large number of larvae were needed (as the majority would die before reaching adulthood). 10,000 L1 larvae were screened for GFP+, and the sorted larvae were split into 20 trays of 500 individuals. A single tray was then reassessed for frequency of GFP+ and RFP/GFP+. This tray was then rescreened at L4 stage for frequency of GFP+, RFP/GFP+ and the rate of larval mortality. Surviving larvae were collected from all 20 trays and screened manually for GFP+. These were allowed to emerge as adults before collection and storage at -20°C for later DNA extraction.400 males heterozygous for the 29113TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGCTTGCGGGAACACTGTTAG) and 29113-R (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTTAAGGAAGTCCCGACAGCG). PCR reactions with KAPA HiFi HotStart ReadyMix PCR Kit (Kapa Biosystems) were setup with 80ng genomic DNA. These PCR reactions, library preparation and deep sequencing were performed as previously described [Pooled DNA extractions (minimum 90 adult mosquitoes or 3500 L1 larvae) were performed using the Wizard Genomic DNA Purification kit (Promega). A 349 bp locus containing the predicted on-target cleavage site was amplified with primers containing Illumina Nextera Transposase adapters (underlined), 29113-F  was fitted to the egg counts, with stains  being the main effect. Quasi-binomial was used to explore the difference in the hatching rates across. The quasi family was adopted as the data exhibits overdispersion. A mixed-effect binomial GLM was run to study the eclosion rates for the various strains and across generations. A random effect was included for experiments with replicates.S1 Fig(A) Schematic representation of the 12 exons of AGAP029113 and location of the target site in exon 5 (white arrowhead). (B) Nucleotide alignment of exon 4 and 5 sections of AGAP029113 (Chromosome 2L: 2921441–2921488) belonging to Anopheles gambiae/coluzzii , An. merus (AmerM1), An. arabiensis (AaraD1), Anopheles quadriannulatus (AquaS1), An. melas (AmelC1), Anopheles christyi (AchrA1), Anophles epiroticus (AepiE1), Anopheles farauti (AfarF1), Anopheles dirus (AdirW1), Anopheles darlingi (AdarC2), Anopheles albimanus , Anopheles atroparvus (AatrE1), Anopheles minimus (AminM1), Anopheles culicifacies (AculA1), Anopheles funestus (AfunF1), Anopheles stephensi , Anopheles maculatus (AmacM1), Anopheles sinensis (AsinS1), Ae. Albopictus , Aedes aegypti (Aeaeg), Culex quinquefasciatus (Cqui) and Drosophila melanogaster (Dmel). The CRISPR gRNA target site is underlined in red and the CRISPR-Cas9 cut site is indicated by a black arrowhead. The PAM sequence is indicated in bold letters. (C) Ag1000g phase 3 SNP data and frequency at the AGAP029113 target site sampled from 2784 wild Anopheles mosquitoes across Africa and 297 parents and progeny from 15 lab crosses. Anopheles gambiae (Agam), Anopheles coluzzii (Acol).(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigCRISPRh/+ individuals to 29113hdrGFP/+ individuals, and subsequent screening of the offspring for GFP+, provided mosquitoes with a deficient 29113 allele, with the other allele being wild-type or containing indels generated by NHEJ repair. (A) L1 larvae (7000). (B) Adults (90). The gRNA binding site is underlined, with the PAM highlighted in bold. Horizontal dashes represent deletions whilst vertical dashes show the predicted cleavage site. Bases highlighted in red show substitutions, while bases in a red box represent insertions.Crossing of heterozygous 29113(TIF)Click here for additional data file.S1 DataIncludes all raw data points, lists of test statistics and sample sizes.(XLSX)Click here for additional data file.


This review highlights current knowledge on the expression and function of connexins and pannexins, transmembrane channel proteins that play an important role in intercellular communication, in both the developing and mature lymphatic vasculature. A particular focus is given to the involvement of these proteins in functions of the healthy lymphatic system. We describe their influence on the maintenance of extracellular fluid homeostasis, immune cell trafficking to draining lymph nodes and dietary nutrient absorption by intestinal villi. Moreover, new insights into connexin mutations in primary and secondary lymphedema as well as on the implication of lymphatic connexins and pannexins in acquired cardiovascular diseases are discussed, allowing for a better understanding of the role of these proteins in pathologies linked to dysfunctions in the lymphatic system. GJA–GJE) with numbering according to their chronology of discovery. The topology of Cxs consists of four α-helical transmembrane (TM) domains, two extracellular loops (ELs), an intracellular loop (IL) and a cytosolic amino- and a carboxyl-terminal (NT and CT) domain. Cxs form hexamers, called connexons, in the endoplasmic reticulum or Golgi apparatus, which traffic thereafter to the cell membrane where they may function as hemi-channels participating in exchanges between the cell and its environment [Twenty one gap junction proteins, called connexins (Cxs), are found in humans, compared to twenty in mice ,2. Cx prironment . Gap junironment ,5. Anothironment . One priironment . Reportsironment ,9. All tironment . Cxs andironment ,11,12. IThe lymphatic system is a network of capillaries and collecting lymphatic vessels allowing for the unidirectional transport of interstitial fluids to the venous system . It is pLymphatic vessels actively regulate tissue fluid homeostasis, trafficking of immune cells to draining lymph nodes (LNs) and absorption of dietary fats . Under pAnother important function of the lymphatic system is to allow absorption of dietary nutrients from the gut, which depends critically on villi in the small intestine . These sLECs of lymphatic capillaries are attached by anchoring filaments to the extracellular matrix (ECM), which prevents vessel collapse under conditions of increased interstitial pressure. Lymphatic capillaries lack a basement membrane and have discontinuous intercellular junctions, favorable conditions for the uptake of interstitial fluid , left. LSince Compton and Raviola showed in 1985 by electron microscopy that LECs of the marginal sinus of the rabbit popliteal lymph node contain gap junctions , severalMore recently, three Cxs have been consistently found in LECs of collecting lymphatic vessels, i.e., Cx37, Cx43 and Cx47 ,29,30. C−/−Cx37 and −/−Cx43−/−Cx37 mice but not in −/−Cx43 mice. Furthermore, −/−Cx43−/−Cx37 mice exhibit abnormal thoracic duct development, defective lymphatic valve formation and lymphedema at E18.5 and at birth [−/−Cx43+/−Cx37 mice had a chylothorax at birth and exhibited reduced postnatal viability. Chylothorax was not observed in +/−Cx43 mice and was extremely rare in −/−Cx37 mice. As signs of retrograde lymph flow were also observed in −/−Cx43+/−Cx37 mice, the authors proposed that the chylothorax may be due to a failure to move lymph in an unidirectional (anterograde) manner [−/−Cx37 mice resulted in rapid regression of valve leaflets and severe valve dysfunction [Cxs play an essential role in the homeostasis of blood and lymphatic vessels . Cxs andat birth . Markedl) manner . Finallyfunction .−/−Cx37 mice appeared normal, most lymphatic vessels in −/−Cx37 mice displayed a severely reduced number of lymphatic valves, resulting in perturbed lymphatic drainage in embryos and adult mice [+/−Cx37−/−Foxc2 mice and observed that these mice typically die perinatally and suffer from generalized lymphedema in utero, craniofacial abnormalities, severe dilation of intestinal lymphatics, abnormal lacteal development and lack of lymphatic valves [+/−Cx37−/−Foxc2 mice further confirmed that Foxc2 and Cx37 are elements in a common molecular pathway directing lymphangiogenesis. Mechanical forces, including shear stress and matrix stiffness, are increasingly recognized as key regulators of cell behavior and fate during development, homeostasis and disease. It is therefore of particular interest that culturing LECs on a soft matrix of 0.2 kPa, which mimics the stiffness of embryonic tissue allowing LEC progenitor migration, led to GATA-binding factor 2 (GATA2)-dependent upregulation of many genes controlling valve morphogenesis, including Cx37 [The formation of lymphatic valves is initiated around E16 and involves a four step process . The firult mice ,30,35. Mult mice ,37. Kanac valves . The woring Cx37 .−/−Cx43 mice die perinatally due to cardiac outflow tract malformation [Ubiquitous knock-out of Cx43 in mouse embryos leads to complete lack of mesenteric lymphatic valves at E18.5 . As Cx43ormation , the posormation . The absormation . Of noteormation .GJC2), the gene encoding Cx47, were the first shown to be implicated in lymphedema, Cx47 was only observed in a subset of LECs at upstream sides of lymphatic valves of embryonic mice [−/−Cx47 mice. Although Cx47 appeared also dispensable for long-chain fatty acid absorption from the gut, it seemed to promote serum lipid handling as prolonged elevated triglyceride levels were observed in −/−Cx47 mice after OLTT [Although mutations in gap junction protein gamma-2 , a hypomyelinating leukoencephalopathy, in which lymphedema does not occur [GJC2 promoter, and generally segregate from the mutations found in lymphedema patients with only one exception for G146S [GJC2 mutations in detail, it should be noted that the literature regarding Cx47 mutations has been somewhat confusing as amino acid sequences were originally based on the first ATG start site for human GJC2. Over the years, increasing evidence pointed to the second ATG site for initiation of translation for human GJC2 and a progressive change was made by authors to using this site as a basis for numbering the amino acid sequence. We will use a nomenclature based on the second ATG site throughout this review, resulting in some of the amino acid substitutions in this review being numbered three amino acids less than in their original publications.Missense mutations in mphedema ,46. Althot occur . Mutatioor G146S . Before The reported mutations for primary lymphedema are missense mutations and result in amino acid substitutions in various domains of the Cx47 protein, i.e., NT (H16P), ELs (S45L and R257C), IL (R122Q and G146S) and CT   Table 1Table 1. Xenopus laevis oocytes using a two-electrode voltage clamp. Resting potentials of oocytes expressing WT and mutant Cx47 were not different; however, transmembrane currents observed for Cx47-G146S mutants were reduced, suggesting a loss-of-function in the mutant hemi-channels [The cellular and molecular consequences of the Cx47-G146S mutation have been studied after transfection in communication-incompetent cell lines. Gap junction plaques were detected at the plasma membrane in mutant-expressing cells. The mutated protein appeared enriched in the endoplasmic reticulum as compared to cells transfected with the WT Cx47. Next, resting membrane potentials and non-junctional transmembrane currents were studied in single channels . UnfortuGJC2 [−/−Cx47 mice [GJC2 missense mutations lead to dominant negative effects on WT Cx47 or on other Cx channels, which may then be detrimental for gap junction communication and homeostasis in LECs. The creation of transgenic animals carrying specific missense mutations would be of tremendous help to resolve these ongoing questions.Cx47 mutations have also been associated with an increased risk for secondary lymphedema following breast cancer treatment. In a case-control study Finegold and colleagues sequenced 81 breast cancer patients with secondary lymphedema and 108 control breast cancer patients for mutations in GJC2 . Four heGJC2 . Two othGJC2  and one GJC2 . After e−/− mice ,54, show−/−Cx37 mice, recently the hypothesis was tested that mutations or polymorphisms in gap junction protein alpha 4 (GJA4), the gene encoding Cx37, are associated with secondary lymphedema following breast cancer surgery [GJA4 gene (rs3543 and rs705193), which is associated with an increased risk of secondary lymphedema in these patients. As the 3′-UTR of genes is often regulating RNA stability, the authors hypothesized that these SNPs may reduce the amount of Cx37 RNA resulting in decreased expression of the protein. Although this molecular mechanism was not further investigated, the authors proposed the use of these SNPs as novel genetic biomarkers for assessing the predisposition to secondary lymphedema in human breast cancer patients.Following the studies describing impaired lymphatic drainage in  surgery . A totalGJA1 gene encoding for Cx43 [GJA1 missense mutation leading to an amino acid substitution (K206R) in EL2 of Cx43  in TM1 of Cx43  is an inherited multisystem developmental disorder caused by around 80 distinct mutations in the for Cx43 ,57,58. Tfor Cx43 . Lymphos of Cx43 . Two oth of Cx43 . On the Cxs and Panxs have been shown to be involved in different disease and repair processes with links to the lymphatic system, such as atherosclerosis and cardiac edema after myocardial infarction (MI), for instance.−/−Apoe) mice. As described above, Cx47-deficient mice had prolonged elevated triglyceride levels after OLTT [−/−Apoe−/−Cx47 mice was increased as compared to −/−Apoe mice. In agreement with these altered serum lipids, advanced atherosclerotic lesions tended to be larger in old −/−Apoe−/−Cx47 mice [Tg Panx1fl/fl Apoe−/−Tie2-Cre) displayed increased atherogenesis, pointing to a protective role for Panx1 in these cells. On the other hand, atherogenesis was not changed in mice with ubiquitous Panx1 deletion, but −/−Apoe−/−Panx1  mice had reduced body weight, serum cholesterol, triglycerides and free fatty acids, suggesting altered lipid metabolism in these mice [−/−Apoe−/−Panx1 mice after gavage with an olive oil bolus. Secondly, injection of Evans blue in the footpad of −/−Apoe−/−Panx1 mice showed decreased drainage of interstitial fluids in −/−Apoe−/−Panx1 mice, which was further confirmed by an increased tail diameter in these mice. Finally, an increased content of CD68-positive cells was observed in atherosclerotic plaques of −/−Apoe−/−Panx1 mice as compared to −/−Apoe mice, which may be due to a lower efflux of these cells from the atherosclerotic plaques via lymphatic vessels. Taking all results together, the authors concluded that the detrimental effect of Panx1 deletion in endothelial and monocytic cells during atherogenesis was counterbalanced by an opposite effect resulting from impaired lymphatic function in ubiquitous Panx1-deficient mice [The role of Cxs in atherosclerosis has been extensively studied for many years, while a potential involvement of Panx1 has only recently been highlighted . Atheroster OLTT . Moreove−/− mice . Secondlese mice . Thus, aent mice . AlthougAn important complication of atherosclerosis is MI. The heart has a unique system of lymphatic vessels covering the epicardial surface, which is most dense at the level of the ventricles . In the GJC2 and GJA1 genes, encoding for Cx47 and Cx43, respectively, have been linked to primary and secondary lymphedema in various families and populations, which is of major importance for the stratification of lymphedema patients. Although the symptoms of human disease cannot be directly recapitulated in knockout mouse models, the development and function of the lymphatic system is severely perturbed in these mice. Still, it shows that the effects of specific gene mutations are likely more complex than a straightforward loss or gain of function. Whether we will be able to target Cxs for the treatment of lymphatic disorders will very much depend on the unraveling of the effects of the GJC2 and GJA1 gene mutations  in relevant in vitro, ex vivo and in vivo models. In this respect, it is important to keep in mind that mounting evidence suggests that Cxs, and in particular Cx43, also have non-canonical channel-independent functions, including the modulation of cell differentiation, adhesion and gene transcription, which are believed to depend largely on their interacting partners. As protein–protein interactions may also be influenced by missense mutations leading to amino acid substitutions, we still have a long and exciting way to go in understanding all aspects of Cxs in lymphatic disease.Cx-built gap junction channels have been considered for over 50 years as integral components for cell-to-cell coupling within the cardiovascular system, and Panx channels have been more recently added to this repertoire of proteins for intercellular communication. While studies on Cxs and Panxs in the lymphatic system are at an infant stage, it is clear that these structures play an essential role in the normal functioning of this system in humans. Indeed, mutations in the