Glaucoma is a chronic disease that shares many similarities with other neurodegenerative disorders of the central nervous system. This study was designed to evaluate the association between glaucoma and other neurodegenerative disorders by investigating glaucoma-associated protein changes in the retina and vitreous humour. The multiplexed Tandem Mass Tag based proteomics (TMT-MS3) was carried out on retinal tissue and vitreous humour fluid collected from glaucoma patients and age-matched controls followed by functional pathway and protein network interaction analysis. About 5000 proteins were quantified from retinal tissue and vitreous fluid of glaucoma and control eyes. Of the differentially regulated proteins, 122 were found linked with pathophysiology of Alzheimer’s disease (AD). Pathway analyses of differentially regulated proteins indicate defects in mitochondrial oxidative phosphorylation machinery. The classical complement pathway associated proteins were activated in the glaucoma samples suggesting an innate inflammatory response. The majority of common differentially regulated proteins in both tissues were members of functional protein networks associated brain changes in AD and other chronic degenerative conditions. Identification of previously reported and novel pathways in glaucoma that overlap with other CNS neurodegenerative disorders promises to provide renewed understanding of the aetiology and pathogenesis of age related neurodegenerative diseases. The axons of RGCs form the core component of the optic nerve, relaying visual information detected by the retina to the brain. Therefore, a loss of structural and functional integrity of RGSs plays a major role in the development and progression of visual deficits reported in common optic neuropathies and particularly in glaucoma2. While several risk factors have been identified that are associated with premature loss of RGCs, high intraocular pressure is currently the most significant risk factor in glaucoma3. The molecular mechanism(s) that result in RGC dysfunction in various optic neuropathies however remain ill-defined. Greater understanding of the underlying neurodegenerative processes is crucial for the development of effective therapeutic strategies for glaucoma.Glaucoma represents one of the major causes of irreversible blindness in the elderly. This complex degenerative disorder is characterised by progressive and selective loss of retinal ganglion cells (RGCs)6. AD is characterized by the presence of extracellular plaques containing abnormal amyloid β (Aβ) aggregates as well as intracellular neurofibrillary tangles composed of hyper-phosphorylated tau, a microtubule-associated protein localised in axons. Clinically, patients exhibit a progressive decline in memory, cognition and learning, which are accompanied by visual and ocular manifestations, similar to glaucoma7. These pathological features may explain the impaired contrast sensitivity and motion perception often reported in AD patients. There is evidence for a positive association of glaucoma in patients with AD and the involvement of Aβ and hyper-phosphorylated tau in ocular degeneration in glaucoma patients9.It is increasingly recognized that glaucoma and AD show overlap of several molecular pathological features including β-amyloid accumulation in the retina10. Similar anatomical and functional deficits predominantly localised to the inner retina have also been reported in clinical studies in AD subjects11. However, the molecular bases of these links remain obscure mainly due to the limited availability of data concerning biochemical and “omics” related retinal changes either in glaucoma and/or AD. Importantly, the lack of sufficient information about which molecular changes are involved in either glaucoma or AD pathology precludes either an early and accurate disease diagnosis, or the development of critical target-based therapeutic development.We have previously demonstrated that AD is associated with ocular deficits including inner retinal thinning and impaired retinal electrophysiological response in animal models of ADIn this study, we investigated the molecular basis of glaucoma pathogenesis by taking a systems level perspective of the retina and vitreous proteome using unbiased quantitative proteomics approaches. Large-scale proteome profiling of human post-mortem tissues were conducted, comparing protein abundances in vitreous and retinal tissues from ‘control’ and glaucoma eyes. This study provides significant mechanistic advancements into the common molecular pathways implicated in the pathogenesis of glaucoma and other neurological disorders with a particular focus on AD as a representative neurodegenerative condition.Frozen human post-mortem eye tissues (20 donors) from open angle glaucoma  and age matched control  subjects who had consented to the use of their tissues for research purposes were obtained from the Sydney Eye Bank, NSW (Lions NSW Eye Bank), Australia. Human ethics approval was obtained from the Macquarie University Human Research Ethics committee. Research was carried out in accordance with the principles outlined in the declaration of Helsinki. Both the retinal and vitreous tissues were examined . Tissues were obtained within 6 hours after death. A history of glaucoma had been obtained from the donors’ medical records. None of the subjects had a known history of AD, Parkinson’s disease, ocular surgery, macular degeneration or other ocular disorders affecting visual function. Frozen eyes were allowed to thaw and washed with PBS; retina, optic nerve head and vitreous tissues were carefully removed from the eyecups without the retinal pigment epithelium (RPE) layer contamination under the surgical microscope .3, 100 mM NaF, 1 mM Na2MoO4 and 10 mM Na4P2O7 and sonicated using a probe sonicator (3 pulses/15 s/50 Hz with 20 s between each pulse). Insoluble materials were removed by centrifugation at 15,000 g for 10 min at 4 °C. Extracted proteins were reduced with 5 mM DTT for 15 minutes at room temperature, and then alkylated with 10 mM iodoacetamide for 30 minutes in the dark at room at 4 °C. The alkylation reaction was then quenched with addition of 5 mM DTT for 15 minutes in the dark.Retina and vitreous tissues were lysed in lysis buffer  containing 10 μg/ml aprotinin, 10 μM leupeptin, 1 mM PMSF and 1 mM NaVO12. The protein pellet was resuspended in 200 μl 8 M Urea in 50 mM Tris (pH 8.8). Protein concentration was determined by BCA assay kit  using bovine serum albumin (BSA) as a standard. Dual digestion was carried out on 150ug protein, initially with Lys-C  at a 1:100 enzyme: protein ratio overnight at room temperature, followed by Trypsin  at a 1:100 enzyme: protein ratio for at least 4 hours at 37 °C. Samples were then acidified with TFA to a final concentration of 1% (pH 2 to 3) and desalted using SDB-RPS (3M- Empore) Stage Tips13.In order to remove the interfering detergents and contaminants, proteins were precipitated using the chloroform methanol precipitation protocolTo accommodate the 40 biological samples (10 control and 10 glaucoma for retina and vitreous tissues each), four separate 10plex TMT experiments were carried out (two TMT experiments for retina and two for vitreous tissues). A detailed experimental design and TMT workflow is illustrated in Fig. z and an AGC of 2 × 105). MS3 was performed using HCD with 55% collision energy and reporter ion detection in the Orbitrap with an AGC of 150,000 ions, a resolution of 60,000 and a maximum ion accumulation time of 150 ms. Peptide fragmentation and collection of reporter ion spectra were performed using the synchronous precursor selection (SPS-MS3 method)14. In this method, first MS2 analysis was conducted using CID fragmentation on the top 10 most intense ions with following settings: normalized collision energy of 35%, AGC 4 × 103, isolation window 0.5 Da, maximum ion accumulation time 150 ms with 40 seconds of dynamic exclusion. Following each MS2 scan, for the MS3 analyses, precursor isolation was performed using a 2.5 Da window and fragmented in the ion trap using CID as above, except with an AGC setting of 8,000. Multiple fragment ions (SPS ions) were co-isolated and further fragmented by HCD at normalized collision energy (NCE) of 37.5%. Selection of fragment ions was based on the previous MS2 scan and the MS2-MS3 was conducted using recently described sequential precursor selection (SPS) methodology14.Fractionated peptide samples were reconstituted in 30 μl of 0.1% formic acid and 10 μl of samples were analysed using an Orbitrap Fusion Tribrid-MS  equipped with an ultra-high pressure liquid chromatography system (Proxeon). Peptides were separated for 3-hours on a reverse phase column with a gradient of 6–30% acetonitrile in 0.125% formic acid at a flow rate of ~400 nl/min. In each data collection cycle, one full MS scan (400–1400 m/z) was acquired in the Orbitrap . Sequest matches were filtered using linear discriminant analysis to a false discovery rate (FDR) of 1% at the peptide level based on matches to reversed sequences, as previously reported18. The final peptide-level FDR fell well below 1% (~0.2% peptide level). A reductionist model was used for assignment of peptides to protein matches, where all peptides were explained using the least number of proteins. Protein rankings were generated by multiplying peptide probabilities and the dataset was finally filtered to 1% protein FDR.In-house software tools were used to convert RAW file to the mzxml format19. Briefly, a 0.003 Th window centred on the theoretical m/z value of each reporter ion was recorded for each of the 10 reporter ions, and the intensity of the signal closest to the theoretical m/z value was recorded. TMT signals were also corrected for isotope impurities as per manufacturer’s documentation. Peptides were only considered quantifiable if the total signal-to-noise (S/N) for all channels was >200 and a precursor isolation specificity of >0.75. Within each TMT experiment, reporter intensities values were normalized by summing the values across all peptides within each channel and then each channel was corrected so that each channel had the same summed value. Protein quantitation was performed by summing the normalized S/N values for all peptides assigned to a given protein. The protein quantitations from the two TMT experiments for each respective sample type (vitreous and retina) were aggregated into a single report. The proteins were regarded as differentially expressed based on a two sample t-test p-value (p ≤ 0.05) and a fold change threshold (≥1.3 for up-regulation or ≤0.76 for down-regulation). The reproducibility of all four TMT experiments output was evaluated by further statistical analysis such as overall data quality, un-supervised analyses such as clustering and PCA analysis, all implemented using our in-house ‘TMTPrepPro’ software20.Quantitation of peptides using TMT reporter ions was performed as previously publishedversus control samples and also previously reported to be altered in AD were analysed using a highly sensitive electrochemiluminescence (ECL) based MSD platform. Serum amyloid A (SAA), C-reactive protein (CRP), soluble vascular adhesion molecule 1 (sVCAM1), soluble intercellular adhesion molecule 1 (sICAM1), alpha-2-macroglobulin (A2M), beta-2 microglobulin (B2M), Factor VII (FVII), adiponectin, clusterin and Tenascin (TNC) were quantified in human retinal and vitreous samples from control and glaucoma subjects. Aliquots from tissue lysates were prepared according to the manufacturer’s instructions for each panel of assays. Assay plates were washed and blocked using the supplied buffers. Samples and standards were then loaded in duplicate into each assay plate, sealed and incubated at room temperature for 2 hours. Plates were washed using PBST (pH 7.4), then secondary detection antibodies added and plates were re-sealed and incubated again for 1 hour. Plates were finally washed 3 times with PBST, read solution added according to the assay instructions and read using a Sector Imager 1200 plate reader . The supplied software was used to determine standard curve and sample concentration, according to 5 parameter logistic curve-fitting techniques.Selected proteins identified by quantitative mass spectrometry as being differentially abundant in glaucoma Western blot analyses were carried out on selected proteins  to confirm the proteomic data. 25 μg of proteins from each sample was separated by 4–12% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked by Tris-buffered saline containing 5% milk for 1 hour, and primary antibodies added at a final concentration of 1–2 μg/ml. After incubation with the primary antibody overnight at 4 °C, blots were washed with Tris-buffered saline and then incubated with anti-IgG antibody linked to horseradish peroxidase for 2 hours. Signals indicative of protein levels were detected using an enhanced chemiluminescent substrate  and Bio-Rad ChemiDoc MP detection system and band intensities quantified in the linear range of detection.www.ingenuity.com), Reactome Functional Interaction (FI) network (http://www.reactome.org/) and ClueGO. For Ingenuity, the gene identifiers and their fold change value (glaucoma vs. control) for each tissue were uploaded separately to the software. Identified proteins were correlated to corresponding gene using Ingenuity Pathway Knowledge Base (IPKB). Significant interaction networks (p < 0.05) and molecular and cellular functions were identified based on known protein–protein interactions in the published literature (knowledge base). Networks were “named” on the most common functional group(s) present. Canonical pathway analysis allowed us to identify the function-specific genes significantly present within the networks. For specific functional protein ontologies that were differentially regulated, the list of differentially expressed proteins for both retina and vitreous were analysed by the Reactome Functional Interaction (FI) network Cytoscape plug-in. All relevant data are available in the paper and its Supporting Information files.Pathways enrichment analysis was carried out on differentially expressed proteins using Ingenuity Pathway software  statistically significant  using a student t-test and (2) differed by at least ± 30%. This two-step differential analysis of glaucoma versus control for retina tissue yielded 252 up regulated proteins  and 133 down regulated  and retinal tissues (355 proteins).orange bars), while the coagulation system, gluconeogenesis I, and G protein and EphrinB signalling pathways were enriched more significantly in the vitreous .Analysis of the differentially regulated proteins using Ingenuity Pathway Analysis (IPA) revealed that the top canonical pathways associated with immune and inflammatory pathways  were similarly over-represented in the vitreous and retina Fig. . In contTo visualize the functional connectivity among these differential proteins, we assembled together retinal and vitreous protein networks using the Reactome analysis tool. A high degree of connectivity, with 136 of the 357 differential proteins in the retina and 568 of the 1153 differential proteins in vitreous, forming interconnected networks Fig.  was idenIPA analysis was performed to investigate the top disease-related biological functions commonly associated with the differential proteins in the retina and vitreous. This analysis highlighted networks associated with neuropathological diseases such as amyloidosis, tauopathy, and dementia Fig. . Specifi21. The energy required for cell growth, development and differentiation is provided by mitochondria in the form of ATP produced via oxidative phosphorylation (OXPHOS)22. The mitochondrial OXPHOS machinery comprises five key enzyme complexes consisting of 80 proteins, of which 13 are coded by the mitochondrial DNA. The mRNAs for these proteins are translated on mitochondrial ribosomes. The malfunction of ribosomal machinery is associated with decreasing the capacity for protein synthesis, followed by a significant reduction in ribosomal RNA and tRNA levels and subsequently up-regulation of RNA oxidation23. This dysregulation in protein synthesis lead to OXPHOS malfunction affecting all complexes, except the complex II. The electron transport chain is major source of reactive oxygen species (ROS) that accelerates oxidative damage followed by lipid peroxidation that eventually leads to OXPHOS damage and culminates in cell death due to energy restriction24. These processes are considered converging features of neurodegenerative diseases, and have been implicated in both glaucoma25 and AD28.Mitochondria play a central role in regulating neuronal cell survival and its impairment is associated with cell death since it governs both energy metabolism and apoptosisIn our dataset, we identified 32 retinal and 16 vitreous proteins associated with mitochondrial dysfunction and oxidative phosphorylation, plus seven proteins linked to the mitochondrial ribosomal machinery. Of the 32 retinal proteins, 24 were identified as down regulated, and belonged to outer and inner membranes and ETC complexes I–V, including 13 subunits of complex I (NADH: ubiquinone oxidoreductase), 4 subunits of complex III (Ubiquinol cytochrome c-oxidoreductase), 4 subunits of complex IV (cytochrome c oxidase complex) and 3 subunits of complex V (ATP synthase). In contrast, in the vitreous, proteins in complex III and V were not affected, and 3 subunits of complex I  were up regulated although these were down regulated in the retinal tissue. Similarly, four subunits of complex IV  were downregulated in the vitreous but not the retina  assembly endogenous inhibitors, vitronectin and clusterin, were also found to be upregulated in glaucoma  were identified in both vitreous and retinal tissues, however a greater abundance related differences were observed in the vitreous. Four other members were specific to the retina  and three were detected uniquely in the vitreous . Interestingly, of the 12 identified apolipoproteins, eight have previously been reported to be associated with neuropathological progression in AD50 : GSTM1, GSTM2, GSTM3 and GSTM5 from the mu family, GSTT1 and GSTT2 from the theta family, and two microsomal GSTs, MGST1 and MGST2. Interestingly, all eight GSTs were identified as down-regulated in glaucoma eyes compared to the control samples Fig. . Five of51 and AD53. Accordingly, we identified 29 protein associated with mRNA processing as significantly up regulated in vitreous along with SRSF9 and HNRNPM proteins, which were upregulated in both the retina and vitreous. From this network, 8 members of the serine/arginine-rich splicing factors (SR proteins) were found, which are involved in alternative mRNA splicing via the spliceosome, including; SRSF1, SRSF2, SRSF3, SRSF4, SRSF7, SRSF9, SRSF10 and SRSF11. Additionally, 5 members of hnRNPs complexes (Heterogeneous nuclear ribonucleoproteins) HNRNPC, HNRNPH3, HNRNPF, HNRNPM, HNRNPU and 7 members of small nuclear ribonucleoproteins  were upregulated exclusively in the vitreous tissue . The Reactome pathway analysis revealed that crystallins are clustered with proteins associated with cholesterol transport and apoptosis , which were identified in our proteomics datasets. The association of these markers with AD is extensively reported in the literatureics Fig. . This ma62. For instance, Funke et al.61 has previously reported identifying 600 proteins from human retinal tissues, of which about 10% showed proteome alterations in the glaucomatous tissues. In the current study, our approach analysed both retinal and vitreous tissue proteomes to a significant depth of about 5000 proteins, providing the first comprehensive elucidation of proteome changes in the these tissues from glaucoma patients. We report differential regulation of 122 proteins in retinal and vitreous tissues, which overlap with functional networks associated with neurodegenerative conditions of CNS with a particular focus on AD pathology. We believe that the elucidation of the complex proteome alterations occurring in the vitreous and retinas of glaucoma subjects will serve as a key source for the identification of glaucoma specific biological targets for diagnostic biomarkers, disease prevention, and therapeutic strategies. Our in-depth computational analysis highlights several unique biochemical pathways that are also known to be impaired in AD conditions. The proteomics overlap highlighting similarities and differences between glaucoma and AD will further help to elucidate converging pathological mechanisms underlying AD and various other neurodegenerative disorders.This study used a combination of multiplexed mass spectrometry-based proteomics (TMT labelling), functional protein network and pathways analyses, ECL analysis, and western blotting to delineate global and specific proteomics changes in human retinal and vitreous glaucoma tissues. Only a limited number of proteomics studies have been performed on human glaucoma tissues64.More specifically, our data analysis revealed that a significant proportion of annotated proteins belonged to categories related to neuronal components and neurological disorders. Pathway enrichment analysis followed by examining functional networks supported the notion that pathways linked to cellular energy regulation, complement pathway activation, antioxidant defence mechanisms and acute phase response signalling were predominantly affected. Our findings also implicate impairment in mitochondrial machinery, cholesterol metabolism, inflammatory responses, regulation of cytoskeletal proteins, receptor mediated endocytosis and retinoid nuclear receptor alterations in glaucoma conditions. Interestingly, we observed significant downregulation of several crystallin proteins in the vitreous of glaucoma subjects. Crystallins are a class of heat shock protein which are suggested to protect neurons by suppressing signals linked to stress mediated apoptosis; therefore, reduced crystallin levels in glaucoma vitreous could make the retinal neurons more vulnerable to cytotoxic injury and cell death. Downregulation of various crystallin proteins and mRNA has previously been reported in a rat model of experimental glaucomain vivo and in vitro studies have demonstrated the potential neuroprotective role of crystallins  in survival of injured retinal ganglion cells in rat65 and axonal regeneration in the injured optic nerve and photo receptors in animal experimental model66. Interestingly the most recent retinal proteomics study of the experimental glaucoma model64 reported down regulation of several crystalline proteins 3 weeks after IOP elevation, however, their expression trend was reversed after the 7th week. Furthermore the authors showed that an intravitreal injection of β-crystallin B2 during the IOP elevation is responsible for increasing the retinal ganglion cell survival and protecting against the loss of the retinal nerve fiber layer and optic nerve injury64.Several 67.Conversely, while AD brains show increased αB-crystallin expression, changes in retinal expression of various crystallins in AD remains unclear58 to evaluate protein expression of selected AD candidate markers. Consistent with mass spectrometry findings, we observed that A2M, B2M, Clusterin, TNC, FVII, Adiponectin, CRP, SAA, ICAM-1 and VCAM-1 were differentially affected in the retinas and vitreous of glaucoma subjects. Importantly, of these A2M, B2M, Clusterin, TNC, FVII, Adiponectin represent metabolic markers that are modulated in AD58. Altered expression of AD associated vascular markers like CRP, SAA, ICAM-1 and VCAM-1 also correlated well with our proteomics analysis. These observations support the hypothesis that glaucoma is associated with both neuronal and vascular stress.Proteome characterization of the retina and vitreous tissues, which are primarily associated with glaucoma disease process, represent the first step towards enhancing our molecular understanding of this complex neurodegenerative disorder and explain the development of specific features of the disease. Functional network analysis of the differentially regulated proteins in glaucoma may hold the key to pathognomonic processes associated with other CNS neurodegenerative disorders that have increasingly been shown to affect molecular, anatomical and functional networks in the retina. We also validated our proteomics findings using highly sensitive electro-chemiluminescence assay on MSD platformThe biochemical interactions identified in several signalling networks elucidated in this study could lead to neurodegeneration through cell apoptosis. The impact of altered metabolic and biochemical function on complex biomolecules can lead to conformational alterations and covalent modifications. It is difficult to determine whether proteome alterations in glaucoma are a consequence or cause of the disease. Our analysis highlighted a significant decrease in proteins associated with regulating glutathione metabolism that directly modulates oxidative stress response, which may reflect an attempt by the retinal and vitreous tissues to neutralize the increased oxidative stress associated with glaucoma pathogenesis. Similarly, we observed a down regulation of proteins involved in mitochondrial function and ATP generation, which may suggest an inability of the retinal tissue to meet the energy requirements leading to shifting of equilibrium to pro-apoptotic pathways. Increased expression of some of the components of complex 1 in vitreous may represent a compensatory effect or efflux of these proteins from dying retinal cells.68. C1q along with synaptic markers such as PSD95 has been implicated in early glaucoma pathogenesis, suggesting complement activation in synaptic regulation in glaucoma. Increased C1q expression was also reported in neurons in AD animal models69. Differential effects on complement pathways is known in other neurodegenerative disorders and amyloid β accumulation in particular is an activator of complement pathway70.Apart from oxidative stress, our study suggests complement cascade involvement in glaucoma. The complement pathway not only plays a critical role in the inflammatory response, but is also involved in synaptic development and pruning. Defective synaptic elimination has been associated with limited refinement of the retinogeniculate pathway. Preferential over-expression of several components of the complement pathway were identified in glaucoma samples. Our study provides a highly inclusive data set regarding alterations in complement proteins. Together with previous observations, our findings corroborate the reported upregulation of C1q complement protein in glaucoma71. Similarly, plaque formation in AD is influenced by cholesterol, chronic oxidative stress, complement activation and neuroinflammation. The inflammatory responses, such as the activation of complement pathways play a fundamental role in synaptic development and pruning, cell death and ultimately in pathophysiology of numerous neurodegenerative diseases, including glaucoma73. The complement system is activated by three separate pathways; the classical, alternative and lectin74. However, among the three pathways, the classical pathway has been shown to be predominantly affected in both AD33 and glaucoma30. The classical complement pathway is comprised of more than 20 proteins, comprising several serine proteinases linked as an amplifying cascade. There is evidence for the bidirectional cross-talk between the complement and coagulation cascades76. Since some complement proteins act as substrates for coagulation factors, enhanced levels of coagulation cascade proteins could potentially promote the complement cascade activation. Alternatively, complement activation might also lead to coagulation and fibrin deposition77.Aβ deposits are well known to accumulate in the retinas in both AD and glaucoma conditionsAPOE allele variant is a significant risk factor for sporadic type of late onset AD47. The APOE allele is involved in moderating Aβ proteolysis and clearance, and is associated with both glaucoma and AD effects on the retina10. This study establishes the changes in expression of various members of the apolipoprotein family, indicating significant alterations in apolipoprotein metabolism. It is thus highly likely that apolipoproteins may form potential biomarkers to predict the effects of glaucoma in the future.We identified for the first time a direct link of apolipoprotein expression in glaucoma. Our study found significant upregulation of various apolipoproteins in the retinas and vitreous of glaucoma subjects. Apo A-I plays a crucial role in cholesterol transport and metabolism and we observed increased Apo A-I concentrations in glaucoma samples. The regulatory effects of Apo-A1 on reverse cholesterol transport pathway have also been suggested in AD. ApoE4 changes were primarily localised to the retina and no overexpression of ApoE4 was identified in the vitreous. Importantly the 27. Association between the production of oxidant/free radical species, dysfunction of the antioxidant system, and the onset and progression of neurodegenerative diseases has been increasingly well established79. For instance, glutathione (GSH), a thiol-containing tripeptide, and GSH-related enzymes (GSTs) are important endogenous antioxidant components that are involved in mediating protection of cells against oxidative stress by neutralising highly reactive free radical species80. GSTs catalyse the conjugation of reduced GSH to xenobiotic compounds, which are less reactive and more soluble, and hence facilitate clearance from the body. GSH synthetases regulate the GSH system in both cytoplasm and endoplasmic reticulum and dysregulation of the system is linked with neurodegeneration81. GST has also been described as a glaucoma associated stress marker, and increased serum GST immunoreactivity was reported in glaucoma patients. Furthermore, polymorphisms of selected GSTs such as GSTM1 and GSTT1 were shown to be associated with the onset and development of glaucoma83, cataract85, PD87 and AD88.Increased ROS production underlies oxidative damage to macromolecules in the cells, including lipids, proteins and nucleic acids. These changes have been directly associated with aging and neurodegenerationbona fide vitreous proteome alterations. Further, although a great deal of reproducibility was observed between the samples suggesting differential regulation of proteins in the disease state, detailed information from the post-mortem donor samples including the drug treatments etc. will help for in-depth analysis of the data. Nevertheless, this study enables us to understand the complex nature of disease-associated pathology in retinal and vitreous tissues although it does not suggest whether it is a cause or effect of the chronic disease condition. Furthermore, the downregulation of several proteins in the retina may be caused by loss of neuronal cells in glaucoma leading to reduced protein quantity from particular retinal cells. This may reflect reduced expression at the tissue level and not at individual neuron levels.A limitation of our approach is that we analysed whole retinal tissues, and thus cannot distinguish the individual contribution of distinct cell types that comprise the retina. This may lead to changes in proteins in the inner retinal region, which are particularly affected in glaucoma to be masked, or lead to significantly diminished fold alterations. Future studies may use techniques like laser dissection microscopy in the unfixed tissues to identify region and layer specific proteomics changes in the retina. Immunostaining of the retinal sections with selected markers will further help to elucidate the layer/region specific protein expression differences in the retina under glaucoma conditions. The protein expression changes in vitreous may similarly be caused by efflux of proteins from retinas or other ocular tissues and may not reflect Overall, very few studies have tried to substantiate the molecular similarities and differences between open angle glaucoma and more generalised CNS neurodegenerative disorders such as AD. The comprehensive profile of the eye proteome changes identified in this study provides novel insights into the pathobiology of glaucoma. Similarities and differences between various proteins in glaucoma and other neurodegenerative conditions may lead to the development of novel drug targets and disease specific biomarker identification. Our results provide a significant advancement towards a better understanding of the mechanistic basis of glaucoma and its association with other neurodegenerative diseases.Supplementary FiguresSupplementary Dataset S1Supplementary Dataset S2


MYLK. We tested the hypothesis that thoracic aortic smooth muscle contraction may also be susceptible to variations in the smooth muscle-specific isoform of the motor protein myosin where inactivation of one Myh11 allele or the presence of one Myh11 missense variant associated with an increased risk of human aortic disease may result in a reduced force development response. Additionally, other kinds of smooth muscles may be less sensitive to the effects of mutations in one smooth muscle myosin allele, similar to results obtained with Mylk. Force development responses were reduced in aortic tissue from a conditional knockout of smooth muscle myosin heavy chain in adult mice (Myh11+/- or Myh11-/-) with a greater reduction with homozygous vs heterozygous tissues. Similar reductions in force responses were obtained with tissues containing either a heterozygous or homozygous knockin mutation in smooth muscle myosin heavy chain (Myh11+/R247C or Myh11R247C/R247C mutations that cause human aortic disease) with no significant changes in RLC phosphorylation. Agonist-dependent force responses were not reduced significantly in urinary bladder, ileal, or tracheal tissues from Myh11+/- mice while only ileal tissue showed a reduced force response in Myh11R247C/R247C mice. Thus, heterozygous mutations in Myh11 associated with reduced myosin function result in compromised contractile function primarily in aortic smooth muscle.Aortic smooth muscle contains limiting amounts of myosin light chain kinase (MLCK) for myosin regulatory light chain (RLC) phosphorylation and contraction that predisposes to thoracic aortic disease in humans containing heterozygous loss-of-function mutations in  ACTA2 (smooth muscle α-actin), MYH11 (smooth muscle myosin heavy chain), MYLK (myosin light chain kinase), and PRKG1 (PKG type-1) i. Ca2+ binding to calmodulin leads to MLCK activation and RLC phosphorylation to initiate smooth muscle contraction [2+]i returns MLCK to an inactive state i in bladder tissues [2+/calmodulin, was limiting. Thus, the aortic smooth muscle cell appears particularly sensitive to the partial loss of MLCK.Different cellular mechanisms may be associated with aortic force reduction responses. Reduction of MLCK content by half did not significantly reduce forces in bladder, tracheal, or ileal smooth muscles , 18. The tissues , 24, 37  tissues . The norOther smooth muscle tissues containing half of the normal amount of myosin or a mutated myosin R247C had no or little reduction in force development compared to responses from aorta. Thus, the aortic smooth muscle cell also appears particularly sensitive to the disrupting effects of mutations in myosin. Force development is not only dependent on smooth muscle myosin and RLC phosphorylation, but also requires the polymerization of submembranous cytoskeletal actin filaments to strengthen membrane adhesion complexes involved in transmitting force generated in actin-myosin filaments to extracellular structures , 38. TheMyh11R247C/R247C mice exhibited normal growth, reproduction, and aortic histology but decreased aortic contraction. In response to vascular injury, Myh11R247C/R247C mice showed significantly increased neointimal formation due to increased SMC proliferation when compared with the wild-type mice. Although additional studies have not been performed on Myh11+/- or Myh11R247C/+ mice we note that they live for at least six months and appear normal. In the absence of other disease modifiers, these mutations may be tolerated because the smooth muscle cells can compensate physiologically in vivo for the force output necessary to maintain aorta structure and function. In terms of induction of aortic disease in mice, additional studies are needed to identify additional genetic or environmental modifiers.Mutations that reduce aortic force production in mice do not produce aortic aneurysms or dissections similar to human disease for up to 10 months of age , 14, 15.Myh11 associated with reduced myosin function without changing the extent of RLC phosphorylation result in compromised contractile function in aortic smooth muscle, similar to reported results for decreased MLCK function which acts to reduce myosin function by reducing the extent of RLC phosphorylation. The aorta is particularly sensitive to compromised contractile function compared to other kinds of smooth muscles which may disrupt mechanosensing mechanisms and predispose to thoracic aorta disease.In summary, studies with genetically modified mice show that modifications of MLCK and myosin heavy chain independently converge to disrupt the ability of the aortic smooth muscle cell to generate force. Heterozygous mutations in


There was no difference in plasma/CSF C1q or C3 between older adults and youth. In 18–24-year-old youth, a nearly significant (p = 0.052) elevation of CSF C1q expression was observed in cognitively impaired subjects compared to cognitively normal subjects. Further investigation into the role of the CNS complement system in the neuropathogenesis of HIV is warranted and should be considered in a developmentally specific context.The complement system (C1q/C3) is a key mediator of synaptic pruning during normal development. HIV inappropriately induces C1q and C3 production in the brain, and reduces neuronal complement inhibition. HIV may thus alter neural connectivity in the developing brain by excessively targeting synapses for elimination. The resultant pattern of neuronal injury may fundamentally alter neurodevelopmental and cognitive processes differentially across ages. This study aimed to (1) measure the association between the cerebrospinal fluid (CSF) complement factors (C1q/C3) and a marker of neuronal injury (NFL) in HIV+ subjects; (2) quantify the differences in CSF C1q/C3 between HIV+ youth and older adults; and (3) define the relationship between CSF C1q/C3 and cognitive impairment in each age group. We performed a retrospective cross-sectional study of 20 HIV+ 18–24-year-old youth and 20 HIV+ 40–46-year-old adults with varying levels of cognitive impairment enrolled in the CNS Antiretroviral Therapy Effects Research study. We quantified C3, C1q, and NFL by ELISA in paired CSF/plasma specimens. We found that CSF C1q correlates with NFL in all subjects not receiving antiretroviral therapy ( The complement system is a key mediator of synaptic pruning during normal development  is a structural protein specific to neurons that is released into the CSF and blood following axonal disruption Julien . In HIV In order to begin to examine the role of the CNS complement system as one mechanistic player in HIV neuropathogenesis, we sought to elucidate the relationship between complement factors and neuronal damage in HIV infection across the developmental age span. Specifically, we measured the correlations among CSF and plasma complement factors (C1q and C3) and NFL as a marker of neuronal injury. We then examined the differences in complement protein concentrations in youth and older adults, and finally explored the relationship between these markers and cognitive function in each age group.We performed a retrospective cross-sectional study using biological samples  and data from 40 HIV+ subjects enrolled in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort of the NIMH/NINDS/NIH. Characteristics of the CHARTER cohort are described elsewhere  certified, or CLIA equivalent, medical center laboratory. Plasma HIV RNA measurements  were quantified by a RT-PCR ultrasensitive assay  in a central lab . Paired CSF/plasma samples were assayed for C3  and C1q . CSF samples were assayed for NFL . Previous work by our group did not demonstrate measurable quantities of NFL in plasma  and an instrumental activities of daily living (IADL) questionnaire . Non-parametric analysis methods were used given small sample sizes to improve accuracy of statistical estimates and reduce sensitivity of analyses to any statistical outliers. Continuous variables were described using median and intraquartile range (IQR), and intergroup differences were evaluated using the Wilcoxan rank-sum tests and Kruskal-Wallis tests. Categorical variables were described using counts and percents, and intergroup differences were compared using the Chi-square test. Spearman’s correlation coefficients were used for correlations between biomarkers. Statistical significance was determined a priori as a two-tailed p = 0.005), and a subsequent lower nadir CD4+ T lymphocyte count . Other markers of systemic infection  were similar between the different age groups. Older adults trended towards more frequent current ART use , likely related to duration of time linked into appropriate care and counseling. Younger adults had higher Wide Range Achievement Test (WRAT) scores , suggesting a higher level of premorbid cognitive functioning despite comparable total years of education. It is unclear if this difference is simply reflective of the population currently being infected with HIV, or if it is age-related. However, all neurocognitive data are adjusted for age and demographic factors, making the latter possibility less likely. HAND diagnoses were comparable between the two age groups. Finally, no subjects were using illicit drugs at the time of this study.Cohort demographics, clinical, and laboratory characteristics of everyone and of each specific age group are summarized in Table n = 17) did approach statistical significance . We subsequently performed an exploratory analysis eliminating outlier NFL values  that demonstrated a significant relationship between CSF C1q and NFL    with the entire cohort, and 2) restricted to subjects not receiving ART in order to avoid any potential confounding effects of ART on expression of biomarkers of neuroimmune dysregulation or neuronal injury. There were no significant correlations between NFL and either CSF complement marker in the overall cohort. However, the correlation between CSF C1q and NFL in subjects off ART (p = 0.052) elevation of CSF C1q expression was observed in cognitively impaired subjects compared with cognitively normal subjects  . Of note, four of the 40 total subjects were not classified for HAND so were therefore excluded from this analysis. In 18–24 year-old youth, a nearly significant (p = 0.001) , demonstrating a consistent relationship between systemic immune status and risk of CNS neuronal injury previously demonstrated in studies of older-aged HIV cohorts  between older adults and youth, on or off ART (data not shown). NFL was significantly lower in youth compared to older adults across the cohort  Fig. . FurtherThis is the first study to examine CSF complement protein expression as a novel immunologic mediator of HIV-associated neuronal damage in human subjects. It is also the first study to address the possibility of an age-specific ontogenetic neuropathogenesis of acquired HIV infection. We have demonstrated a potential positive correlation in this small cohort between CSF expression of C1q and NFL in subjects of all ages not receiving ART. These data suggest that C1q in CSF may be a correlate or biomarker for neuronal injury across the age span in HIV. Furthermore, in 18–24-year-old youth we found a nearly significant elevation of CSF C1q expression in cognitively impaired subjects to cognitively normal subjects. This second observation may reflect an age-specific vulnerability to complement-mediated cognitive dysfunction. Finally, we have confirmed a positive correlation between CSF expression of C1q and C3, suggesting our data are likely demonstrating true differences in activation of the complement system, rather than anomalous assay results. Borderline statistical significance may thus reflect actual biological significance but too small of a sample size for definitive results.C1q is a 460 kDa hexameric glycoprotein that serves as a pattern recognition molecule and the first recognition subcomponent of the complement classical pathway. It binds HIV, IgG, IgM, and many other ligands and receptors largely via a gC1q domain  and CD4+ T lymphocyte count. This observation supports the idea that at least part of HIV neuropathophysiology occurs independently of peripheral disease and is clinically consistent with the fact that mild forms of HAND persist in the antiretroviral era in subjects with good systemic viral control.There have been several recent compelling studies examining the presence of systemic inflammatory markers in youth with different developmental synaptopathies, including perinatally acquired HIV. One such study demonstrated an inverse correlation between serum markers of three combined pro-inflammatory markers  and processing speed, suggesting that residual inflammation may contribute to neurocognitive deficits despite well-controlled HIV disease in the periphery , because there are fewer specimens available from younger adults in the CHARTER cohort. Thus, overall analyses and sub-analyses with even smaller numbers  may be underpowered, and truly significant associations may therefore be underestimated. Second, while 19/36 subjects did have cognitive impairment (four were not classified), most of these (17/19) were mild (ANI). Therefore, linear correlations of complement markers with degree of impairment are limited. This is why we chose to use cognitive impairment as a binary variable in the majority of our analyses. Third, because of these small numbers and the cross-sectional nature of this correlation study, factors confounding the relationship between complement proteins and NFL are not accounted for in statistical associations. However, univariate analysis did not suggest relationships between complement proteins and systemic markers of disease, or peripheral complement levels, so not including these variables in subsequent models likely had little effect on the associations examined. In addition, causality cannot be inferred in a cross-sectional study. Finally, in our statistical analysis, we had several outlier values for NFL that we were not able to biologically explain why they otherwise differed from the remainder of the cohort. While the non-parametric analyses we used with our data should minimize the impact of outliers on statistical associations, a larger cohort would allow us in the future to determine if these subjects have a different specific biological reason for elevations in NFL, or if they were indeed spurious.Our study had several limitations. First, because we wanted to specifically include a comparable number of 18–22-year-old youth compared to older adults, the overall cohort size was relatively small (We chose to investigate the role of the complement system in HIV neuropathogenesis based on several recent articles by Stevens et al.  and Step


Anopheles coluzzii. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of Enterobacteriaceae within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract.Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector  When a female mosquito takes a blood meal from a human, the bacteria residing within its gut grow significantly. Following a blood meal, female mosquitoes produce a barrier within their gut, known as the peritrophic matrix, which physically separates the blood meal from the cells of the epithelium. Here, we show that the presence of bacteria in the gut is required for the synthesis of the peritrophic matrix. By experimentally disrupting this barrier, we find that this structure plays a role in limiting the extent to which bacteria of one particular family are able to grow and persist in the mosquito gut. We also find that the peritrophic matrix ensures that bacteria remain within the gut, preventing them from invading the mosquito body cavity. These results will be useful in designing disease control strategies that depend on the ability of bacteria to colonize and persist in relevant tissues in the mosquito host. Anopheles genus are responsible for the transmission of Plasmodium parasites, the causative agents of malaria. The study of the Anopheles gut microbiota has recently emerged as an important field in an effort to characterize mosquito-parasite interactions in greater depth and to develop new methods to stop disease transmission. The microbiota have been shown to trigger a constitutive immune response in the mosquito gut epithelium that enhances resistance to parasite infection [Mosquitoes of the nfection ,2. Furthnfection ,4. Finalnfection . The sucDrosophila, commensals are controlled largely by the production of reactive oxygen species (ROS) by the dual oxidase (DUOX) enzyme [Drosophila gut, the Imd pathway, is under strong negative regulation to prevent its stimulation by commensals [A. gambiae, commensals are known to induce the Imd pathway, and suppression of the Imd pathway receptor PGRPLC and its transcription factor REL2 causes microbiota overgrowth [The balance between immune resistance and tolerance is key to bacterial persistence within the gut environment. Resistance refers to bacterial killing or the prevention of bacterial growth, whilst tolerance encompasses the prevention or repair of host tissue damage caused by pathogens or immune responses . In Dros) enzyme ,8, whilsmmensals –11. In mmmensals ,13 and immensals . In A. gergrowth .A. gambiae heme peroxidase is induced by blood feeding and, together with DUOX, forms a network of dityrosine bonds that is thought to protect the gut epithelium from immune elicitors, thus mediating bacterial persistence [Serratia bacteria with a protective niche in which they can proliferate without inducing a gut immune response, increasing susceptibility to infection [Ixodes scapularis, the gut microbiota induces the formation of a peritrophic matrix whose presence facilitates colonization of the spirochete bacterium Borrelia burgdorferi, possibly by protecting the pathogen from blood meal pro-oxidants and cellular immunity [Drosophila, oral bacterial infection induces the expression of genes encoding proteins with chitin binding domains (CBDs) [Some tolerance mechanisms are based on the strengthening of physical barriers between the microbiota and the host. Notably, an sistence . The pernfection . In the immunity . In Dross (CBDs) , and a ps (CBDs) ,20.Plasmodium effectively facilitates traversal of the peritrophic matrix [P. falciparum invasion [Aedes aegypti peritrophic matrix is thought to play a role in blood meal detoxification, sequestering large quantities of heme released during blood bolus digestion [The mosquito peritrophic matrix is often considered as a barrier to parasite infection, though one that parasites have evolved to overcome. Secretion of chitinase by c matrix –25. A coinvasion . More geigestion . The rolA. coluzzii mosquito (until recently known as A. gambiae M form). We find that the gut microbiota induce the expression of several components of the peritrophic matrix, and that the microbiota are necessary for the synthesis of a structurally complete peritrophic matrix. We also show that the peritrophic matrix plays a role in resistance to the Enterobacteriaceae bacteria present in the gut microbiota, both reducing the extent to which this family of bacteria grows and persists within the gut, and precluding this family of bacteria from seeding a systemic infection.Here, we use RNA sequencing to explore the microbiota-dependent gene expression in the midgut of the A. coluzzii midguts at five time points: 2–3 day old mosquitoes that had accessed only fructose since emergence , 5h, 24h and 72h after a human blood meal and 24h (96h) after a second human blood meal that was given at the 72h time point. This time course was performed with conventionally-reared mosquitoes that harbored their native microbiota, and a cohort of mosquitoes that were fed an antibiotic cocktail  in both sugar and blood meals. This antibiotic treatment was effective in substantially depleting mosquito guts of bacteria as detected by qRT-PCR against 16S rRNA (A. gambiae PEST genome (AgamP4). A total of 6753 genes  had non-zero counts in all forty samples. Principal component analysis (PCA) indicated that the samples clustered according to their blood feeding status, with no obvious outliers  that is known to be transcriptionally induced by both P. berghei and bacterial infections and encodes two CBDs and a proline-rich domain that may be involved in protein-protein aggregation [As observed previously , we noteomponent  and was omponent . We alsoregation  Fig 2A)AgAPER1  and CHS2 (AGAP001205), of which CHS2 is expressed in the midgut and responsible for the synthesis of peritrophic matrix-associated chitin [In addition to the protein components of the peritrophic matrix, the main structural constituent is chitin, a polymer of N-acetylglucosamine. Insects are able to synthesize chitin from glucose in a multistep reaction ; fructosd chitin . Here, wAedes aegypti mosquitoes [We speculated that this transcriptional response in the production of both chitin and peritrophic matrix proteins could be bacterially induced either directly, through detection of bacterial elicitors, or indirectly as a result of bacteria causing thinning of the peritrophic matrix (e.g. through chitinases) that results in compensatory transcription to produce a peritrophic matrix of normal thickness. We used thin abdominal sections 24h post blood meal stained with hematoxylin and eosin (H&E) to observe the effect of oral antibiotic treatment on the structure of the gut tissue . In the squitoes ,33,34. ICEC1, CEC2 and CEC3), one defensin (DEF1), gambicin (GAM1) and two C-type lysozymes (LYSC1 and LYSC7). We therefore sought to explore whether the peritrophic matrix plays a role in mediating the mosquito immune response to the microbiota. We supplemented the blood meal with 100μM polyoxin D, a chitin synthase inhibitor that has previously been demonstrated to abolish synthesis of the A. gambiae type I peritrophic matrix [The RNAseq data indicated that the microbiota play a significant role in regulating antimicrobial peptide (AMP) expression in the gut, with seven characterized immune effector-encoding genes being upregulated at one or more time points by the presence of the microbiota . These Ac matrix . StaininCEC1 and GAM1, which showed strong microbiota-dependent expression at all time points examined  has been shown to induce the expression of genes encoding proteins with CBDs, with the induction of a proportion of these genes being dependent on the Imd pathway, one of the main immune signalling pathways in the fly gut [Drosophila peritrophic matrix, Drosocrystallin, is induced by oral bacterial infection [I. scapularis has also been shown to be necessary for production of a peritrophic matrix of proper thickness, with this being dependent on STAT signalling [Sodalis glossinidius, suggesting similar interaction of this bacterium with the peritrophic matrix [The type I peritrophic matrix is specifically produced by adult female midgut cells upon blood feeding and physically surrounds the blood bolus where blood digestion takes place. We show that synthesis of a structurally complete type I peritrophic matrix is dependent on the presence of the gut bacteria. Similar observations have been reported in other insects. In adult  fly gut . Similarnfection . The pregnalling . Finallyc matrix .A. coluzzii peritrophic matrix, though it is noteworthy that a number of regulated genes have canonical STAT binding sites in their upstream regions , 70–80% humidity with a 12h light/dark cycle.The Human blood for mosquito feeding was acquired from the NHS blood service. During feeding, blood was maintained at 37°C on a membrane-feeding device or in a parafilm-covered Petri dish warmed with a handwarmer. Mosquitoes were allowed to feed for 1h and non-engorged mosquitoes were removed within 24h. Mosquitoes were offered egg dishes for oviposition the night before each subsequent blood meal. Antibiotics  or an equal volume of water were supplemented in the sugar solution offered from emergence, in the blood meal and in the egg dish provided for oviposition. For polyoxin D feeding, 0.01M stock solution was prepared from powder in water and added to human blood at a final concentration of 100μM immediately before feeding. An equal volume of water was added to the control blood meal.in vivo knock down of target genes by RNAi. The target region was amplified from total A. gambiae cDNA using primers flanked with the T7 RNA polymerase promoter sequence (sequences are listed in lac operon (LACZ), not present in the A. gambiae genome, was injected as a control for the injection process.Double stranded RNA (dsRNA) was used for transient APER1 experiments) or 8–10 , and stored at -20°C until processing. For carcass dissections, the abdominal carcass and attached fat body tissue was dissected, ensuring that all other organs  were removed. Carcasses were frozen immediately on dry ice in pools of 3–5 (for APER1 experiments) or 8–10 (for polyoxin D experiments) then stored at -20°C until processing.Prior to dissection mosquitoes were ‘surface sterilized’ by immersion in 75% ethanol for 3–5 min and washed three times in phosphate buffered saline (PBS) to minimize environmental contamination from cuticle bacteria into dissected midgut samples. Midguts were removed under a dissecting microscope, frozen immediately on dry ice in pools of 20 (for RNA sequencing), 3–5 (for excretion experiment and Frozen tissues were homogenized in TRIzol (Invitrogen) and chloroform using a Precellys24 tissue homogenizer with bead beating (Bertin). RNA was precipitated from the aqueous phase with isopropanol, washed twice in 70% ethanol and resuspended in water. For RNA sequencing experiments, samples underwent a further column purification using the Qiagen RNeasy kit. For qRT-PCR experiments, cDNA was synthesized from up to 500 ng RNA using the Takara reverse transcriptase kit, according to the manufacturer’s instructions.Libraries for sequencing were prepared in accordance with the Illumina TruSeq RNA sample preparation v2 guide  for Illumina Paired-End Indexed Sequencing. PolyA mRNA first underwent two rounds of purification using Illumina poly-T oligo-attached magnetic beads. During the second elution, the polyA mRNA was fragmented and primed with random hexamers for cDNA synthesis. After first strand cDNA synthesis, the RNA template was removed and a replacement strand was synthesized to generate double stranded cDNA. Ends were then repaired, dA base added and Illumina indexing adapters ligated. cDNA fragments with adapters on both ends underwent 15 cycles of PCR. Libraries were validated with the Agilent 2100 bioanalyzer to check size distribution. Samples were quantified by qRT-PCR, the concentrations normalized and samples pooled according to biological replicate. Pools were loaded at 10 pM onto four lanes of an Illumina flowcell v3 and sequenced using the Illumina HiSeq 1500, 2 X 100 bp paired-end run. Sequences are deposited in the NCBI Sequence Read Archive under the BioProject ID PRJNA385903.A. gambiae in the SILVA rRNA database (release 119) [A. gambiae tRNAs and mitochondrial rRNAs in the AgamP4.2 geneset in Vectorbase. The splice aware aligner Tophat2 v2.0.9 was used to align paired-end reads to the A. gambiae PEST genome AgamP4. The mean inner distance between mate pairs was set to -25 with a standard deviation of 60. Default settings were used for alignment with the following exceptions: the maximum number of mismatches allowed between a read and the reference sequence was 5 to allow for the highly polymorphic nature of the A. gambiae genome, and the minimum intron length was set to 30 bp. The accepted hits were filtered such that only reads that were uniquely mapped were accepted for downstream analysis.Quality control, filtering and alignment were conducted in the Galaxy platform –65. Grooase 119) , plus alAligned reads were converted to gene count data using HTSeq, specifying the union mode . The inpa priori gene filtering, is noise robust and allows genes to be placed in more than one cluster, making it ideal for time-course data. The fuzzifier m was chosen with the mestimate function, and the optimal number of 12 clusters was selected based on when the minimum distance between cluster centroids (Dmin) declines at a reduced rate.Variance stabilizing transformation of count data was performed in DESeq2 prior to clustering. A median-transformed value of the four replicates was calculated for each condition and soft clustering performed in Mfuzz . Soft clKEGG and GO term enrichment analysis for differentially expressed genes were performed in g:Profiler . Genes wA. gambiae mRNA levels, as well as bacterial load by amplification of the 16S rRNA gene, employing primers that anneal to a region of the sequence that is common to all eubacteria or specific to bacterial families examined. Primer sequences are listed in A. coluzzii ribosomal protein encoding gene S7 (AGAP010592) was used as an internal control of the quantity of input RNA. Expression ratios were calculated using primer efficiencies that were determined by amplification of serially diluted targets. qRT-PCR amplifications were performed in duplicate using the SYBR premix ex Taq kit (Takara) in a total volume of 10μl on a 7500 Fast Real Time PCR machine (Applied Biosystems).qRT-PCR was used to quantify Aedes saline , and embedded and sectioned as described. For Gram staining, sections were dewaxed and stained according to the Gram/Twort protocol. Slides were observed under a Leica DMR microscope. For immunostaining, sections were stained with E. coli polyclonal antibody at 1:400 (bs-2351R Bioss Antibodies) and a goat anti-rabbit antibody bound to Alexa 647 (Thermofisher A21245) was used as secondary (1:1000). Slides were mounted in Prolong Gold antipode (Invitrogen) and observed under a Zeiss Widefield Axio Observer Microscope. For calcofluor white staining, sections were dewaxed and rinsed in distilled water before being stained for 2h in calcofluor white solution in the dark (Sigma 18909). Sections were observed under a Zeiss Widefield Axio Observer Microscope.For hematoxylin and eosin (H&E) staining, whole mosquito abdomens were dissected 24h post blood feeding, fixed overnight at 4°C in Duboscq’s-Brasil fixative  and washed in 70% ethanol. Abdomens were then processed to paraffin wax and sections cut to 4μm onto Superfrost Plus slides (VWR). For Gram staining, immunostaining and calcofluor white staining, whole mosquito abdomens were dissected 24h post blood feeding, fixed overnight at 4°C in 4% formalin, washed in qRT-PCR data  were analyzed by generalized linear mixed models (GLMMs) in R (version 3.1.2). GLMMs fit both fixed-effect parameters and random effects in a linear predictor via maximum likelihood. Mixed effect models were used to account for the use of multiple sample pools per condition within each independent replicate, avoiding issues of pseudoreplication. Statistical significance was assessed by an ANOVA test on a linear mixed effect regression model . Correlation was analyzed by a Spearman rank-order correlation test.S1 TableTerms were identified in g:Profiler. Shaded entries indicate terms that are enriched in genes downregulated by antibiotic treatment. Non-shaded entries indicate terms that are enriched in genes upregulated by antibiotic treatment. Only terms with two or more genes are listed. Statistical significance was assessed with a Fisher’s one tail test with Bonferroni correction for multiple testing. ‘n’ indicates the number of genes associated with that term that are significantly regulated at the time point indicated.(DOCX)Click here for additional data file.S2 TableAmong genes identified in  as top candidates for peritrophic matrix components, entries highlighted in grey are identified in this study as being transcriptionally regulated by the presence of the microbiota. Abundance ranking refers to the relative abundance of the protein in the mass spectrometric analysis of Dinglasan et al. Fold change given in the final column is the transcriptional log2 fold change identified here upon antibiotic treatment. CBD = chitin binding domain.(DOCX)Click here for additional data file.S3 TableLog2 fold changes of immune effector encoding genes (AMPs and C-type lysozymes) upon antibiotic treatment. Highlighted entries indicate statistical significance .(DOCX)Click here for additional data file.S4 TableSequences highlighted in red are consensus STAT binding motifs (TTCNNN(N)GAA). Sequences were taken from Vectorbase (AgamP4.5 gene set).(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 FigClusters were generated in the Mfuzz package. Each line represents one gene in the cluster, with line colour indicating strength of membership in the cluster (red being the strongest membership and green the weakest). C = control, Ab = antibiotics.(TIF)Click here for additional data file.S2 Fig(A) and calcofluor white (B) stained thin sections of engorged midguts 24 h post blood feeding with the addition of 100μM polyoxin D to the blood meal. In (B), arrowheads indicate staining of the cuticle (grey arrows) and the fragmentary peritrophic matrix (white arrows).H&E (TIF)Click here for additional data file.S3 Fig(A)CEC1 expression, relative to AgS7, in the midgut 24h after feeding with a blood meal supplemented with 100μM polyoxin D or an equal volume of water (control), plus or minus antibiotic treatment, as determined by qRT-PCR. (B)CEC1 and GAM1 expression in the midgut of APER1 and LACZ (control) knock down mosquitoes 24h after blood feeding, plus or minus antibiotic treatment, as determined by qRT-PCR. (C) Total bacteria load, Flavobacteriaceae load, and Acetobacteraceae load 24h after feeding with 100μM polyoxin D or a control blood meal, relative to AgS7, as determined by qRT-PCR with family specific or universal 16S primers. (D)Enterobacteraceae load in APER1 or LACZ (control) knock down mosquitoes 24h after blood feeding. A-D: Each dot represents a pool of 8–10 (polyoxin D experiments) or 3–5 (APER1 experiments) guts, derived from 4–5 independent experiments. Ratios are normalized within biological replicates to the mean of the control pools. Mean plus/minus standard error is indicated. Statistical significance was assessed by an ANOVA on a linear mixed effect regression model. ‘*’ p<0.05.(TIF)Click here for additional data file.S4 FigFlavobacteriaceae load in the midgut 72 h after feeding with a blood meal supplemented with 100μM polyoxin D or an equal volume of water (control), as determined by qRT-PCR with universal of family-specific 16S primers. Each dot represents a pool of 8–10 guts, derived from 4 independent experiments. Ratios are normalized within biological replicates to the mean of the control pools. Mean plus/minus standard error is indicated.Total bacteria load and (TIF)Click here for additional data file.S5 Fig(A)GAM1 expression in the carcass 72 h after feeding with a blood meal supplemented with 100μM polyoxin D or water as a control, plus or minus antibiotic treatment, as determined by qRT-PCR. (B)GAM1 and LYSC1 expression in the carcass of APER1 and LACZ (control) knock down mosquitoes, 24 h after a human blood meal. A-B: Each dot represents a pool of 8–10 (A) or 3–5 (B) carcasses, derived from 4 independent experiments. Ratios are normalized within biological replicates to the mean of the control pools. Mean plus/minus standard error is indicated.(TIF)Click here for additional data file.S6 Fig(A)Flavobacteriaceae and Acetobacteraceae load in the carcass 72h after feeding with a blood meal supplemented with 100μM polyoxin D or water as a control. (B) Scatter plots of relative Flavobacteriaceae and Acetobacteraceae load against CEC1 expression in the carcass of polyoxin D fed mosquitoes at 72h post blood feeding. A-B: Ratios are normalized within each biological replicate to the highest value across all conditions (‘100%’). Each dot represents a pool of 8–10 Click here for additional data file.S1 FileTab 1 (‘Significant genes’) lists all genes that were found to be differentially expressed in antibiotic treated versus control guts at one or more time points, together with their adjusted p values at each time point. Subsequent tabs contain the full output of DESeq2 differential expression analysis at each time point.(XLSX)Click here for additional data file.


We examined whether UCP1-oe impacts reproductive tissues and seed production by monitoring the progress of flower development, focusing on the early stages of pollen formation. UCP1-oe lines induced the expression of mitochondrial genes and increased mtDNA content in reproductive tissues, which increased the consumption of carbohydrates and reduced H2O2 content and pollen disturbances. Finally, the beneficial impact of UCP1-oe on the source and sink organs resulted in an increased seed size and number under both control conditions and drought stress.Mitochondrial uncoupling proteins (UCPs) sustain mitochondrial respiration independent of intracellular ATP concentration. Uncoupled respiration is particularly beneficial under stress conditions, during which both photosynthesis and respiration may be impaired. Sustaining carbon fixation during the reproductive phase is essential for plants to develop viable pollen grains and for seed setting. Here, we examined whether UCP1 overexpression (UCP1-oe) would help tobacco plants cope with drought stress during reproductive development. We observed that WT and UCP1-oe plants lost water at the same rate under moderate drought stress, but that UCP1-oe lines regained water faster upon rewatering. UCP1-oe plants maintained higher levels of respiration and photosynthesis and decreased H Plants face a range of adverse conditions that impose metabolic constraints during their lifecycle. Among these stresses, water deficit is perhaps the most important because it affects both cell energy supply and demand  (WT) and three transgenic lines  overexpressing A. thaliana UCP1   and 15 days after drought stress imposition (T = 15). At T = 15, plants were again irrigated and visually monitored for recovery, and leaf and flower bud samples were collected 24 h after rewatering on day 16 (T = 16). Flower buds and the youngest expanded leaf were sampled for all plants, frozen in liquid nitrogen and stored at −80°C. We defined the first expanded leaf as the younger mature leaf that was fully developed before the beginning of stress. As large variations are expected when plants develop outside the growth chamber, the whole experiment was repeated twice under the same conditions. This additional experiment was designed to confirm the results by using important proxies, such as physiological parameters, RWC, and seed yield. All the data presented in this manuscript were obtained from a single trial.The experimental design consisted of three conditions , each composed of 20 plants for a total five biological replicates for each genotype  was determined for evaluating plant water status  content was measured in flower buds and leaves using the potassium iodide (KI) method  and the youngest expanded leaves from WT and UCP1-oe plants grown under control, drought and recovery conditions were evaluated. Five biological replicates were analyzed for each genotype and condition.Hydrogen peroxide (H−2 s−1). Stomatal conductance (GS), net photosynthetic rate (AN), respiration in the light (RL), and respiration in the dark (RD) were measured for each plant/treatment. RL was estimated by the Kok method as described . The measurements resulted in a Kok break point at 20 μmol m−2 s−1 PPFD. RD was evaluated after 3 h of acclimation in the dark. A photosynthetic CO2 response curve was estimated by measuring AN at saturating irradiance  during which CO2 was supplied at a range of concentrations . The GS values obtained from this curve were used to calculate the GS inhibition by CO2 by comparing the values obtained at 400 and 1,000 μmol CO2 mol−1. We estimated the maximal velocity of Rubisco carboxylation (Vcmax) as the slope of the linear phase of the CO2 response curve .Gas exchange was measured in the youngest completely expanded leaf using an infrared gas analyzer  at growth light intensity , maximum PSII fluorescence in the dark-adapted state (Fm), maximum PSII fluorescence in the light-adapted state (s), and initial (minimum) PSII fluorescence in the light-adapted state (II (Fv/Fm) was estimated as [(Fm – F0)/Fm] and the effective PSII quantum yield during illumination YII as ΔF/m – qP) was estimated as (s)/(II reaction centers (1 – qP).Chlorophyll fluorescence was measured using a photosynthetic yield analyzer . When necessary, a dark-adaptation period of 40 min was imposed. Initial (minimum) PSMetabolites were isolated from flower buds as described  was applied to each anther. Slides were kept in the dark for 3 h before visualization in a fluorescence microscope . Three biological replicates for each genotype were analyzed for each flower bud size.Flower buds were collected and fixed in a 3:1 ethanol:acetic acid solution. Anthers were removed from flower buds, placed on a glass slide and gently crushed to release pollen grains. Thirty microliters of DAPI staining solution  according to the manufacturer's instructions. For each experiment, a positive control was prepared by treating the sections with 1 U μl−1 DNase I for 10 min at 37°C before labeling as above. The negative controls were labeled in parallel, except for the absence of the enzyme terminal deoxynucleotidyl transferase (TdT). The reaction solution was removed by rinsing the slides 3 times with PBS (pH 7.2). Samples were analyzed under a fluorescence confocal scanning microscope . The fluorescence filter was set to view the green fluorescence of fluorescein at 520 nm.For the TUNEL assay, fixed anthers were removed from the flower buds and embedded in paraffin as described previously .Total RNA was isolated from flower buds using a Spectrum Plant Total RNA Kit (Sigma-Aldrich). The first-strand cDNA was synthesized using a RevertAid First Strand cDNA Synthesis kit  according to the manufacturer's protocol. Mitochondrial DNA was quantified from total DNA isolated from flower buds using the Plant DNAzol reagent . Real-time PCR was performed using the ABI PRISM 7500 system  with SYBR Green dye . At least triplicate reactions were performed with four biological replicates, and the results were expressed relative to the expression level of the Total soluble proteins were isolated from WT and transgenic lines by grinding frozen flower tissue in protein extraction buffer . A cOmplete™, EDTA-free Protease Inhibitor Cocktail  was added to the protein extraction buffer before use. The proteins were separated on 12% SDS-PAGE gels, transferred to nylon membranes, and blotted using UCP1  and Actin11  polyclonal antibodies raised against Arabidopsis . Bands were detected using an ImageQuant LAS500  system with the SuperSignal West Pico chemiluminescent substrate .Five flower buds from each plant were tagged at the end of the drought stress treatment. The tagged buds were kept for seed set, the mature pods were collected at the same time for all genotypes, and their entire seed content weighed. The yield was estimated as the mass of seeds per pod. For seed size analysis, an image field containing ~100 seeds was imaged, and seed area was measured using ImageJ software. Six siliques were analyzed for each plant from five biological replicates. Approximately 2,500 seeds were imaged to estimate the average seed size for each condition.ANOVA with Tukey-Kramer test was used to assess the significance of changes in this research. The analyses were conducted using PRISM 6.0 . The only exception was for principal component (PCA) and correlation analysis, on which we used the Metaboanalyst webportal  were grown until the beginning of the flowering stage Figure , when thA Figure  and UCP1A Figure  in floweD) did not differ between WT and UCP1-oe lines under control conditions   and during recovery (25%)  Figure . Althougy Figure . In cont) Figure .N) under control conditions and water limitation as well as during the recovery period   Figure  was alsoII. Excitation pressure (1 – qP)    Figure , photosy) Figure , the max) Figure  and nonp) Figure  were simN   and MALE STERILITY 1 (MS1) that act during tapetum maturation    Figure  and mito) Figure  was more2O2 content in UCP1-oe flower buds corresponded to 72% of the level in those of WT  than in WT (155%).Due to their higher mitochondrial content, flower tissues are much more prone to oxidative stress than are leaf tissues  to regulate the expression of its targets in Arabidopsis . During drought stress, AMS expression was 50% lower in WT but was practically unaltered in UCP1-oe lines  there is an increase in photosynthesis in UCP1-oe plants, and consequently, GS is not the only responsible for the increased AN  regeneration  but low ATP production. This means that it is not only respiration that is increased but the pattern of respiration is modified (coupled to uncoupled). We observe that a hypoxic response is triggered in these conditions, which leads to an accumulation of metabolites in the leaves that are markers of hypoxic adaptation such as glucose, sucrose, starch, fumarate, and succinate . A more in-depth look at how both proteins influence this reduction in efficiency reveals how different they are and how they could complement each other. Increased AOX activity will decrease both the ratio of NADH consumption and generation of the proton gradient, indicating that AOX would have to consume more substrate to produce the same amount of proton gradient because AOX bypasses proton-pumping complexes III and IV (Vanlerberghe, The expressions of these proteins are at least partially correlated when either UCP1 is knocked out (Sweetlove et al., N, in sink tissues this correlation is negative (Figure N in both leaves (Sivamani et al., 2O2 in UCP1-oe lines. We also observed that compatible osmolytes that are broadly associated with increased drought tolerance, such as proline, did not differentially accumulate between WT and UCP1-oe plants.The other side of the plant energy balance relies on sink demand for energy, which is known as sink strength or sink dominance (Lemoine et al., e Figure . ApparenThe impact of drought stress on tapetum development, which is a high energy-demanding process in plants for sustaining pollen formation, was analyzed by profiling the expression of DYT1, TDF1, bHLH89, AMS, MS1, and MYB26. We found that DYT1, bHLH89, AMS, and MS1 were less inhibited under drought stress in UCP1-oe lines than in WT plants. Some of these TFs were affected under control conditions, which indicates that UCP1-oe impacts tapetum metabolism. There were no apparent differences in tapetum PCD between UCP1-oe and WT plants when drought stress conditions were compared to control conditions. Thus, it is not clear how UCP1-oe affects the expression of these TFs and what the consequence of the downstream processes are during tapetum development.The positive effect of UCP1-oe on leaf photosynthesis and flower bud metabolism resulted in a higher yield compared to that of WT under both control and drought stress conditions. Drought stress affects reproductive tissue development by affecting pollen grain viability, ovary development, and turgor maintenance of floral organs (Alqudah et al., per se is beneficial to mitochondrial metabolism itself as well as to the whole cell because it reduces ROS production by stimulating respiration when needed (Borecký et al., Nevertheless, from the observations made in this study, it is important to note that the effect of UCP1 was not drought specific because it also improved plants in well-watered conditions. At this point, it's hard to establish the basis of the resistance of UCP1-oe plants to biotic and abiotic stresses. UCP1 activity PB, JY, ZW, and PA designed the experiments. PB and JY performed experiments. PB, JY, ZW, and PA analyzed the data. PB and PA wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fourteen single nucleotide polymorphisms were replicated, of which nine were associated with skin colour at genome-wide significance in a meta-analysis across the two studies. These results validated the association of two previously known skin pigmentation genes, SLC24A5  and SLC45A2 , and revealed the intergenic region of BEND7 and PRPF18 as a novel locus associated with this trait . The most significant variant within this region is common among African-descent populations but not among Europeans or Native Americans. Our findings support the advantages of analyzing African-admixed populations to discover new genes influencing skin pigmentation.Skin pigmentation is a complex trait that varies largely among populations. Most genome-wide association studies of this trait have been performed in Europeans and Asians. We aimed to uncover genes influencing skin colour in African-admixed individuals. We performed a genome-wide association study of melanin levels in 285 Hispanic/Latino individuals from Puerto Rico, analyzing 14 million genetic variants. A total of 82 variants with  Skin pigmentation is essential in the protection against ultraviolet (UV) radiation1Skin colour varies among different populations and is strongly correlated with latitude due to the variation in UV radiation intensity2The colour of unexposed skin (constitutive skin pigmentation) is a complex trait12214161819142122Hispanics/Latinos from Puerto Rico are the result of the admixture of European, African, and Native American ancestry. Specifically, the Native American component derives from the Taínos, the native population of Puerto Rico, which was highly reduced by slavery trade, warfare and diseases2627This study has been approved by the institutional review boards of University of California San Francisco and all participant centres. Written informed consent was obtained from all subjects or from their appropriate surrogates for participants under 18 years old. All methods were performed in accordance with the relevant guidelines and regulations for human subject research, in accordance with the Declaration of Helsinki.Samples from the Genes-environment & Admixture in Latino Americans (GALA II) Study and the Study of African Americans, Asthma, Genes & Environments (SAGE II) were used for the discovery of genetic variants associated with skin colour and replication of results, respectively. The GALA II and SAGE II studies are two independent case-control studies initially conceived for the study of genetic and environmental factors involved in asthma. Both studies used the same protocol and questionnaires to recruit unrelated children aged 8 to 21 years old, but focused on two different racial/ethnic groups: Hispanics/Latinos in GALA II and African Americans in SAGE II. All recruited subjects must have reported that all four grandparents self-identified as Hispanics/Latinos (GALA II) or African Americans (SAGE II). Participants from GALA II with skin colour measurements included in this study were recruited in Puerto Rico, while SAGE II participants were recruited from the San Francisco Bay Area29We used the DSM II ColorMeter  to measure skin pigmentation in triplicate for each participant along the inner side of each upper arm. Melanin was measured using the melanin index, defined as the inverse of the melanin reflectance measured at 650 nmGenome-wide genotyping data from participants of both studies were obtained by using the Axiom LAT1 array , and quality control procedures were performed as described elsewhere29Genetic ancestry was initially assessed by performing a principal components analysis (PCA) using EIGENSOFTGenetic variants located in autosomal chromosomes were imputed by means of the Michigan Imputation Serverp ≤ 1 × 10−5) were followed up for replication in SAGE II African Americans. Association testing was performed similarly in the replication sample, with the exception that only African ancestry was used to adjust for genetic ancestry.Association testing with skin colour was performed in Hispanics/Latinos from Puerto Rico by means of the linear Wald test implemented in the software EPACTS 3.2.6p-value ≤ 0.05) and fixed effects models for those SNPs without evidence of heterogeneity p-value ≤ 5 × 10−8.Results from the discovery and replication samples were meta-analyzed using METASOFT. Random-effects models were applied for single nucleotide polymorphisms (SNPs) showing heterogeneity of effects between studies .Our analysis of the ancestral composition using PCA revealed that no individuals were outliers regarding their ancestry composition . As expep > 0.05) and therefore they were not included as covariates in the GWAS. Additionally, Hispanics/Latinos from Puerto Rico had lighter skin (45.8 ± 6.8) than African Americans (71.9 ± 13.5) (p < 0.001). Actual distributions of the melanin index for the two populations are shown in A summary of the descriptive data of the individuals from our study is shown in p-value ≤ 1 × 10−5) (GC = 1.02) was observed in the Q-Q plot .Association analyses of the 14 million imputed variants with MAF ≥1% in the discovery sample revealed a total of 82 SNPs associated with skin colour at a suggestive significance level ( × 10−5) . No majoQ-Q plot  and the Q-Q plot . The topp-value < 0.05) and effect sizes were all in the same direction and of similar magnitude as the discovery sample  or had a MAF <1% (two SNPs). Out of the 77 SNPs, 14 replicated in the African American sample (y sample .p < 5 × 10−8)  . These SSLC24A5 showed the strongest meta-analysis association signals: rs1426654 , rs2675345 , and rs2470102 . We also detected two SNPs near SLC24A5 that were genome-wide significant, one located within DUT  and the other within MYEF2 . After including all five SNPs located within or near SLC24A5 in one common regression model, we determined that the association signal among Hispanics/Latinos from Puerto Rico was driven by the top SNP (rs2675345), as the regression coefficients for the other SNPs were not significant in the common model  and rs35397 . These two SNPs had high LD (r2 ≥ 0.82) in both populations and their association with skin pigmentation was driven by rs16891982 (a SNP associated with skin colour by previous studies)p = 0.945)  .BEND7 and PRPF18 (p = 6.14 × 10−9) and rs6602666 , which showed strong LD in the discovery and replication samples (r2 = 0.99). These SNPs were more significantly associated with skin colour in Hispanics/Latinos from Puerto Rico  than in African Americans . As expected by the high LD between the two SNPs, they represented one association signal in regression analysis when both SNPs were incorporated into the same model (p = 0.788 for rs6602666).Moreover, two SNPs located in the intergenic region of d PRPF18  were assThe frequency distribution of the G allele of rs6602666 in 1KGP Phase III  showed tBEND7/PRPF18 intergenic region. We also validated the association of five genes already known to contribute to skin colour identified primarily in European populations: two loci with genome-wide significance (SLC24A5 and SLC45A2), and three at a suggestive level . In addition to replicating previously described SNPs18SLC24A5).In this study, we performed the first GWAS of skin colour in Hispanics/Latinos from Puerto Rico from the GALA II study. After performing genotype imputation and subsequent association testing, we detected 82 suggestive association signals in Hispanics/Latinos, 14 of which replicated at nominal significance in an independent African American sample from the SAGE II study. We identified novel, genome-wide significant associations between skin colour and variants from the SLC24A5 showed the strongest association signals. This gene is located in the 15q21.1 chromosomal band and encodes the NCKX5 protein , an intracellular membrane protein whose function has been associated with skin colour and diseases related to skin pigmentation211819202324Among the three most significantly associated gene regions, variants near or within SLC45A2 with skin colour in Hispanics/Latinos from Puerto Rico and African Americans. This gene encodes SLC45A2, which is a transporter highly expressed in the melanosomal membrane of melanocytic cell lines, where it is overexpressed in melanoma cellsSLC45A2 has been associated with different pigmentary traits 24We also validated the association of BEND7 and PRPF18 with skin colour. Both variants are located closer to PRPF18 (approximately 23 kb) than to BEND7 (approximately 83 kb). The function of the intracellular protein encoded by BEND7 (BEN domain-containing protein 7) is not extensively known. Nevertheless, it contains the BEN domain, which is involved in transcription regulation throughout recruitment of chromatin remodelling factors and DNA-protein interactionsPRPF18 (pre-mRNA processing factor 18), encodes a splicing factor implicated in pre-mRNA splicing by means of protein-protein interactionsPRPF18Notably, we detected two novel genome-wide significant associations (rs6602665 and rs6602666) in the intergenic region of Interestingly, based on 1KGP data, the rs6602666 G allele (which is associated with darker skin colour) is present in African, South Asian, and admixed American populations, rare in Europeans, and completely absent in Native Americans. Therefore, differences in the proportion of African genetic ancestry may provide a simple explanation as to why this locus has not been detected in previous GWAS, since they were predominantly focused on populations of European descent. This observation underscores the importance and scientific benefit of studying admixed populations, as the inclusion of genetically diverse groups improves statistical power, particularly when genetic variants are rareSLC45A2, have also been associated with protection against basal cell carcinoma, squamous cell carcinomaASIP, has been previously implicated in basal cell carcinoma65Identification of genes implicated in human skin pigmentation has high anthropological, forensic and biomedical interest56BEND7 and PRPF18) with any of these skin diseases are lacking; future studies should therefore investigate the association of the novel locus with these skin diseases.In addition to skin cancer, other diseases of the skin  could be affected by the genetic variants identified in the current study. To date, association studies linking either of the genome-wide significant SNPs (rs6602665 and rs6602666) or their flanking genes (EIF2S2ASIPATP8B4SST and RTP2) and deserve further attention in future studies.Some of the SNPs identified as suggestively associated with skin pigmentation in the current study are located in gene regions previously associated with skin pigmentation, such as Our study has several advantages that should be highlighted: a) skin colour was assessed using skin reflectance spectrometry obtaining a quantitative measure of skin pigmentation, as opposed to many previous studies based on self-reported skin colour; b) skin pigmentation measures were obtained using the same instrument in different recruitment centres participating in the GALA II and SAGE II to reduce possible biases; c) the analysed samples were genotyped with a specific array for African-admixed populations, providing good representation of their genomic variationThe current study also has some limitations that should be considered. Sample size was relatively limited, yet we had sufficient statistical power in the discovery sample (83%) to detect the association of genetic variants with allele frequencies ≥25% and effects sizes (β) ≥ 3.5. Statistical power was limited for variants with lower allele frequencies and modest effect sizes. Moreover, the differing proportions of African admixture and distinct distributions of skin colour between our study populations point to differences in the genetic architecture of skin colour between our two populations. In fact, a proportion of the SNPs associated with skin colour in Hispanic/Latinos was monomorphic or had a low frequency in African Americans, precluding replication attempts of those variants among African Americans. Given that Hispanics/Latinos from Puerto Rico have lower Native American proportions than other Hispanic/Latino subgroupsBEND7-PRPF18, with skin pigmentation has not yet been described. Given the involvement of these genes in pre-mRNA processing and transcription regulation, these loci could be related to melanocytic proliferation. Unfortunately, skin biopsies from the patients in this study are unavailable for performing histologic studies or in vitro experiments using epidermal cell cultures. Therefore, the functional role of associated variants will need to be assessed by future studies.We measured skin pigmentation using the melanin index. It is possible that our GWAS could have yielded different results had we used alternative methods for measuring melanin, such as pyrrole-2,3,5-trycarboxilic acid (PTCA), aminohydroxyphenylalanine (AHP), or electron paramagnetic resonance spectroscopy (EPR)SLC24A5 and SLC45A2 with skin melanin levels in Hispanics/Latinos from Puerto Rico and African Americans, and identified a novel association of variants in the intergenic region of BEND7 and PRPF18 with this trait. Therefore, this study reinforces the advantages and the necessity of analyzing African-admixed populations to identify new loci involved in complex traits.In summary, this GWAS validated the role of How to cite this article: Hernandez-Pacheco, N. et al. Identification of a novel locus associated with skin colour in African-admixed populations. Sci. Rep.7, 44548; doi: 10.1038/srep44548 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.