Mtor in mice (mTOREKO). mTOREKO mutants are viable, but die shortly after birth due to deficits primarily during the early epidermal differentiation programme and lack of a protective barrier development. Epidermis-specific loss of Raptor, which encodes an essential component of mTORC1, confers the same skin phenotype as seen in mTOREKO mutants. In contrast, newborns with an epidermal deficiency of Rictor, an essential component of mTORC2, survive despite a hypoplastic epidermis and disruption in late stage terminal differentiation. These findings highlight a fundamental role for mTOR in epidermal morphogenesis that is regulated by distinct functions for mTORC1 and mTORC2.Mammalian target of rapamycin (mTOR), a regulator of growth in many tissues, mediates its activity through two multiprotein complexes, mTORC1 or mTORC2. The role of mTOR signalling in skin morphogenesis and epidermal development is unknown. Here we identify mTOR as an essential regulator in skin morphogenesis by epidermis-specific deletion of  mTOR regulates cell growth via a protein complex including mTORC1 and mTORC2, but their role in skin morphogenesis is unclear. Here, the authors delete mTORC1 and mTORC2 from the epidermis and see epidermal deficiencies but both mTORCs play distinct roles in skin morphogenesis. The epidermis as the outer layer of the skin serves as a primary interface between the body and its environment and protects the organism from dehydration and external insults. The epidermis is a stratified squamous epithelium that fulfils its functions through a lifelong self-renewal process that is precisely coordinated by regenerative pathways, which in part recapitulate those that are activated in epidermal morphogenesisA complex network of signalling pathways and factors contribute to skin morphogenesis, epidermal stratification and protective barrier formation including p63, Wnt, Notch and the PI3K-Akt cascade256810121314mTOR mediates its functions through the assembly of two structurally distinct multiprotein complexes, mTORC1 and mTORC2 refs , 11, 14.1617192022Mtor or genes encoding essential components of mTOR complexes, such as Rptor or Rictor leads to early embryonic lethality in various organisms2125152729Germline deletion of Here we examine the role of the mTOR pathway in skin morphogenesis in mice by inactivating mTOR or its adaptor proteins Raptor or Rictor specifically in the epidermis. The findings revealed essential and distinct functions of mTORC1 and mTORC2 in skin morphogenesis that have implications for the understanding of skin physiology.Mtor in the epidermis. Mice carrying a loxP-flanked allele encoding Mtor (Mtorfl/fl)Mtor deletion (mTOREKO) in the progeny  and immunohistochemical staining (E15.5) (EKO mice were born (P0) with a highly fragile and translucent skin morphology (EKO newborns were comparable to their littermate controls in body size and length of limbs (Mtor deficiency in the epidermis (Mtorfl/wtK14Cre mice) appeared normal and survived until adulthood without any obvious phenotype  . mTOREKOrphology . After brphology . Except of limbs . The expof limbs . Pups thhenotype .EKO mice revealed severe abnormalities , and suprabasal cells were positive for stratification and differentiation markers including keratin 10 (K10), filaggrin and loricrin . In conty absent . Keratiny absent .EKO newborns were associated with severe disturbances in barrier function. Whereas in newborn controls exogenously applied Toluidine blue dye did not penetrate the skin, in mTOREKO newborns the dye easily penetrated the skin and the mice appeared blue  and keratin 18 (K18). Expression of K8/18 was significantly increased in mutants versus controls, and co-immunostaining with K14 revealed that K8/18 was expressed in basal and suprabasal cell layers, the latter most likely representing the residual periderm  analysis. No obvious signs of apoptosis in the skin of mTOREKO embryos or controls were found . qRT–PCR and western blotting analysis of epidermal tissues showed a significant reduction of differentiation markers  at the transcriptional and also at the protein level in RapEKO mutants when compared with controls   . RicEKO termates . Althougrphology . Althougtermates .EKO newborn skin was stratified, but the epidermal thickness was reduced by 46%±4. This was primarily due to a reduced granular cell layer and thinner stratum corneum  was comparable in mutants and controls, that of differentiation markers K10, K1 and filaggrin was significantly reduced in mutants . ExpressEKO mutants, we performed western blotting analysis of the epidermis in E17.5 RicEKO embryos. Consistent with the attenuation of mTORC2 pathway phosphorylation of Akt at S473 and PKCα at S657 was significantly reduced  towards asymmetric cell division (ACD) in basal keratinocytes plays a central role in initiating the programme of epidermal stratification and differentiation45went ACD . Furtherwent ACD . In addiwent ACD , indicatwent ACD . TogetheTogether, these findings show that the function of mTOR is split between mTORC1 and mTORC2. Whereas mTORC1 mainly controls keratinocyte proliferation within the basal layer, early epidermal stratification, differentiation and epidermal–mesenchymal, mTORC2 primarily controls cell division orientation and late stage barrier formation of the interfollicular epidermis .We have shown that mTOR signalling is essential for proper skin morphogenesis and for the development of a protective epidermal barrier. Loss of epidermal mTOR in mice attenuated keratinocyte proliferation, abrogated the epidermal stratification programme and the formation of hair follicles. Both mTORC1 and mTORC2 contributed to effective epidermal morphogenesis through activation of distinct pathways that functionally cannot compensate for each other . WhereasEKO and RapEKO mice showed remarkable phenotypic similarities, and in both mutants cell proliferation in the basal layer was considerably reduced whereas no signs of apoptosis were detected. The mitogenic function of mTORC1 in the epidermis is consistent with its role in anabolic growth-promoting processes in most cell types, such as stimulation of protein synthesisThe developing epidermis in mTOREKO and RapEKO embryos, whereas it was not affected in RicEKO embryos. It is likely that reduction of those proteins that control proliferation of epidermal progenitor cells contributed to the hypoplastic epidermis in mTOREKO and RapEKO mutants, and they await identification in future studies. One such protein may be fibroblast growth factor-10 (FGF-10), a mitogenic factor for epidermal homeostasis and in carcinogenesis. It was recently shown that mTORC1-dependent phosphorylation of 4E-BPs is necessary for the activation of translation of FGF-10 mRNAS6K and 4E-BP1 are rate-limiting factors in mRNA translation8EKO mice. mTORC1-mediated activation of S6-mediated proliferation in keratinocytes might be more important during a disturbed homeostasis such as in wound healing or carcinogenesis. Consistent with this view is also the clinical observation that patients treated with Rapamycin do not develop obvious skin problems unless they are wounded.Interestingly, phosphorylation of S6, a downstream target of S6K, was primarily detected in suprabasal layers during epidermal morphogenesis, where cells have withdrawn from proliferation and are committed to terminal differentiation. Our findings are consistent with recent studies in adult mice detecting S6 phosphorylation in suprabasal layers in normal skin, whereas activation in basal layers required a stress response such as wounding or carcinogenesis27EKO or RapEKO embryos are strikingly similar to those in IGF-1 receptor (IGF-1R) or Akt1/Akt2 double null mutants. IGF-1-R null mutants display a hypoplastic epidermis and the number of hair follicles is reducedEKO and RapEKO mice appeared more pronounced compared with the phenotype in epidermal-specific IGF1R/Insulin receptor double knockout miceThe epidermal phenotypes of mTORRictor caused a hypoplastic interfollicular epidermis, which although less severely compromised compared with mTORC1-deficient epidermis, was associated with perinatal skin fragility and disturbances in the formation of a functional epidermal permeability barrier, despite the presence of a functional mTORC1 cascade. Together, these findings led us to speculate on a disturbed release of basal cells into the suprabasal layers and subsequent proper epidermal stratification and terminal differentiation. Intriguingly, here we might have identified a previously unrecognized function for mTORC2 in interfollicular epidermal stratification and barrier formation.Epidermal-specific deletion of The process how during embryogenesis, murine epidermis expands from a single layer of unspecified basal layer progenitors to a stratified, differentiated epidermis is complex and not entirely resolved2245648EKO epidermis we observed multiple cellular alterations that might account for the hypothrophic epidermal phenotype. First, we observed attenuated numbers of BrdU+ suprabasal cells at E15.5. Secondly, at E16.5 cortical immunolocalization of Par3 and LGN was altered in basal keratinocytes. And third, at E16.5 we detected reduced perpendicular spindle orientation within the stratum basale. How these cellular alterations might converge and contribute to disturbed epidermal stratification and differentiation in RicEKO epidermis remains an important question. Of equal importance is the identification of mTORC2 targets that mediate these stratification programmes and requires a detailed analysis which is however beyond the scope of this manuscript.In RicEKO mice. Our findings suggest that mTORC2 is a critical regulator of Akt-S473 phosporylation, a process that ultimately affects stratification and epidermal barrier function, independent of mTORC1. In liver, mTORC2 has been shown to be critical for Akt-pS473 signalling to FoxO1 and GSK3α/β, which is important for proper hepatic function and maintenance of whole-body metabolismEKO mice, phosphorylation of FoxO1 and GSK3α/β was comparable, and is unlikely to have contributed to the observed phenotype in RicEKO mice. Along these lines, a recent report revealed a critical role for insulin/IGF-1-dependent control of FoxO1-mediated p63 activation in coordinating ACD and stratification during morphogenesisEKO epidermis or Rictor-deficient cultured primary keratinocytes. Hence, the mechanisms underlying reduced ACD in RicEKO mice might be independent of FoxO1-pS256.Phosphorylation of Akt-S473 is so far one of the best characterized targets of mTORC2 and was clearly induced during late stage epidermal morphogenesis in controls, whereas it was almost absent in the epidermis of RicEKO epidermis and Rictor-deficient cultured keratinocytes. Intriguingly, in a previous study, downregulation of PKCα in an in vitro organotypic epidermis model resulted in a hypoplastic epidermis, comparable to the RicEKO phenotype53545322mTORC2 is also critical for phosphorylation of PKCα-S657 and controlling the stability and activity of PKC proteins21Our findings in skin development corroborate recent reports of a dysregulated mTOR pathway as a pathogenic factor in hyperplastic or inflammatory skin diseases, and hence their therapy with inhibitors of mTOR signallingMtor25, Rptor or RictorCre-transgenic strain expressing Cre recombinase under control of the human K14 promoterMtor, Rptor or Rictor allele served as controls. Genotyping was performed by PCR using specific primers on genomic DNA isolated from tail tips as previously described25To generate mice with epidermal-specific gene deletion, mice with homozygous floxed Body weight of newborns was monitored every 30 min at room temperature. Weight loss was calculated as percentage of initial weight.For Toluidine blue staining newborn mice were killed and fixed in methanol for 5 min, washed with PBS and incubated for 5 min in 0.1% Toluidine blue at room temperature; after extensive washing in PBS, images were taken and the blue stained area  was quantified by using ImageJ softwareTissues were fixed in 4% PFA and embedded in paraffin. Sections (10 μm) were stained with hematoxylin and eosin following a standard procedureFor immunohistochemical and immunofluorescence stainings cryosections from Optimal Cutting Temperature compound  embedded tissues were fixed (4% PFA or in methanol), blocked  and incubated with primary antibodies (diluted in blocking buffer) over night at 4 °C ref. . Bound pFreshly isolated skin tissue was fixed in buffer  and postfixed with ruthenium tetroxideTUNEL assay was performed on tissue sections using the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer's instructions. The nuclei were counterstained with DAPI (Sigma).−1 BrdU (BD Biosciences) 4 h before sacrificing. Tissue samples were OCT embedded and BrdU+ cells were visualized by staining cryosections with a monoclonal anti-BrdU antibody (BD). BrdU+ cells were determined within the stratum basale and suprabasal layers of embryonic back-skin over a distance of 250 μm in five representative fields/section.Pregnant mice were injected intraperitoneally with 50 μg gLGN apical localization and orientation were determined as described previously45Cell division orientation was determined as described previouslyPrimary keratinocytes were isolated from newborns and cultured in low calcium mediumΔΔCt) was used to calculate the expression level of the target gene normalized to GAPDH. Primer sequence information can be found in For Real-time PCR epidermis was separated and total RNA was extracted from tissues using RNeasy Minikit (Qiagen), reverse transcription of isolated RNA was performed using the High Capacity cDNA RT Kit (Applied Biosystems)Epidermis was separated from the dermis and dissociated with a MixerMill homogenizert-test. All data are presented as mean±s.d., a P value of≤0.05 was considered significant. The results were presented as the average of at least three independent experiments unless otherwise stated in the legends.Statistics was performed using PRISM software (Graph Pad Software). Significance of difference was analysed with a Student paired or unpaired two-tailed The authors declare that all data supporting the findings of this study are available within the article and its How to cite this article: Ding, X. et al. mTORC1 and mTORC2 regulate skin morphogenesis and epidermal barrier formation. Nat. Commun.7, 13226 doi: 10.1038/ncomms13226 (2016).Supplementary Figures 1-8, Supplementary Tables 1-3


Caribbean coral reefs have transformed into algal-dominated habitats over recent decades, but the mechanisms of change are unresolved due to a lack of quantitative ecological data before large-scale human impacts. To understand the role of reduced herbivory in recent coral declines, we produce a high-resolution 3,000 year record of reef accretion rate and herbivore (parrotfish and urchin) abundance from the analysis of sediments and fish, coral and urchin subfossils within cores from Caribbean Panama. At each site, declines in accretion rates and parrotfish abundance were initiated in the prehistorical or historical period. Statistical tests of direct cause and effect relationships using convergent cross mapping reveal that accretion rates are driven by parrotfish abundance (but not vice versa) but are not affected by total urchin abundance. These results confirm the critical role of parrotfish in maintaining coral-dominated reef habitat and the urgent need for restoration of parrotfish populations to enable reef persistence. Fishing of keystone herbivores such as parrotfish is thought to negatively impact corals. Here, Cramer and colleagues analyse the sedimentary record for the last 3,000 years from three coral reef sites in the Caribbean and show that indeed coral declines are linked to parrotfish declines. Acropora genus) are now dominated by macroalgae and low-relief corals tolerant of lower water quality (higher turbidity and nutrient) conditions34en masse the Acropora corals and the sea urchin Diadema antillarum in the early 1980s as well as coral bleaching outbreaks that became widespread in the late 1980s  of fish teeth, coral fragments and urchin spines preserved within reef sediment cores collected at three sites in Bocas del Toro, Caribbean Panama. Whereas previous studies of historical reef change in the Caribbean have had relatively poor temporal resolution due to the imprecision of radiocarbon dating for recent material42425263839At each site, declines in accretion rates and parrotfish abundance were initiated in the prehistorical or historical period. CCM analyses revealed that accretion rates were driven by parrotfish abundance (but not vice versa) but were not affected by total urchin abundance. These results confirm the critical role of parrotfish in maintaining coral-dominated reef habitat and the urgent need for management actions to maintain and restore parrotfish populations to enable reef persistence in the Caribbean.Cores were collected from three sites within the semi-enclosed lagoon Almirante Bay that span a gradient of influence from land-based runoff24230Th ages obtained from all six cores reveal that these cores span the period 1239±15 BC to 1984±3 AD. Corrected 230Th age errors (2σ) range from±3 to 15 years  within a sandy-muddy matrix of carbonate grains. The 27 ears see . Nearly se sites . Reef ac∼1925 AD . A linea∼1925 AD .The cessation of reef growth at the two fringing reefs, despite areas of active coral growth directly downslope of our coring locations and within 0.5 m of the water surface, suggests that the zones in which we cored may have transitioned from actively accreting to non-accreting environments due to changes in local hydrography related to changing sea levelPorites spp. (mainly Porites furcata), Agaricia spp., Madracis mirabilis and Acropora cervicornis, and the urchin assemblage is is composed primarily of Echinometra spp., Lytcehinus/Tripneustes spp. and Diadema antillarum . The coral assemblage is composed primarily of branching tillarum . There itillarum . Within Diadema Caribbean reefsDiadema die-off1Porites consistently dominates the coral assemblage within most cores and time periods, except for periods of dominance of staghorn coral A. cervicornis from 1000–1920 AD at Cayo Adriana, branching M. mirabilis from approximately 1500–1800 AD in both Punta Donato cores, and temporary and periodic dominance of lettuce coral Agaricia at Airport Point and Cayo Adriana , whose abundance closely tracks that of overall urchin abundance , the most important herbivores on post- Adriana . The urcbundance . TypicalLytechinus/Tripneustes has a significant positive causal effect on accretion rate, but neither Echinometra nor Diadema are causally related to accretion  positively affects reef accretion rate, reef accretion rate has no causal effect on parrotfish abundance . In contccretion . Parrotfpneustes . There ion rates . Diagnoson rates .The subfossils and sediments analysed from our reef matrix cores provide a 3,000-year continuous record of reef growth and herbivore abundance in Bocas del Toro that spans the period of intensifying human impacts to coastal marine ecosystems1819Our analyses of the causal relationship between reef herbivore abundance and accretion rates demonstrate the essential role of herbivory in coral growth and abundance. During the prehistorical and historical periods, parrotfish abundance was a positive driver of reef accretion, indicating that their positive effects on coral growth via removal of benthic macroalgae outweigh their negative effects via corallivory and bioerosion45Echinometra throughout the past three millennia) and reef accretion, likely signifying that Echinometra's positive effect on reef accretion via herbivory is of similar magnitude to their negative effect via intense coral boring activity48Lytechinus/Tripneustes group, likely indicating their positive effects on accretion via herbivory are greater than any negative effects from bioerosion50Diadema was not found to be causally related to reef accretion, likely a result of the consistent rarity of this urchin on the forereef slope zone of our coring sites. The multispatial CCM analyses reveal a positive causal effect of parrotfish abundance on total urchin abundance in general and Echinometra abundance in particular (Echinometra that may be caused by parrotfish-mediated facilitation of (1) coral-dominated habitat and/or (2) palatable benthic algal communities preferred by this urchin. Reef accretion rates are not causally affected by the relative abundance of the most common coral species, branching Porites. While accretion rates would undoubtedly be affected by changes in the relative abundance of exceptionally fast growing species such as Acropora cervicornisWe did not detect a causal relationship between overall urchin abundance . Reef accretion rates declined dramatically in the top 1 m of the Airport Point and Cayo Adriana cores and each included one age reversal (which we excluded from the age models). Therefore, age estimations near these reversals are less certain than those in other samples. Although reef accretion rates were used as a proxy of coral growth, non-coral carbonate producing organisms also contributed to sediment production. However, 83% of the weight of the undigested sediments>500 μm , is made up of coral. Accretion rates could also have been affected by the degree of bioerosion or physical erosion which could create finer sediments that might be more easily removed from the reef matrix by current or wave action. However, because accretion rates are not related to sediment grain size , accretiOur analysis of millennial-scale change in reef communities in Bocas del Toro suggests that historical fishing may have been significantly affecting Caribbean reefs for over two centuries, initiating ecosystem declines from which they have not recovered. The clear positive causal effect of parrotfish abundance on reef accretion rate indicates that modern Caribbean reefs that have largely been depleted of these herbivores may be locked into an alternative stable state of macroalgal dominanceTo prevent damaging living corals, we collected cores from rubble zones on the reef slope adjacent to living coral colonies. At each site, two replicate cores 10.1 cm in diameter and ranging from 3–5.5 m in length were extracted ∼10–30 m apart at 6–7 m water depth using using a self-contained underwater breathing apparatus (SCUBA) and a combination of push-coring and vibra-coring techniques. Cores were split lengthwise, and the working half was sliced into 5 cm increments (referred to as samples throughout the paper). Subfossils were analysed from every sample from the top 1 m of each core to produce a high-resolution record of change from the most recent period, while every third sample was analysed from deeper parts of the core to produce a moderately high-resolution record from the pre-historical period. This scheme resulted in the analysis of 36–41 samples from each core. Samples were dried and weighed, then wet sieved at 2 mm, 500, 250, 104 and 63 μm fractions to facilitate sorting and counting different taxonomic groups.http://ichthyolith.ucsd.edu/.We tracked changes in the abundance and taxonomic or functional composition of reef fish, corals and urchins through time. Reef accretion rate, a measure of carbonate production by hermatypic corals as well as other carbonate-producing organisms, was computed by determining sedimentation rates based on our age models (time/5 cm increment). Fish teeth were analysed from all five sediment size fractions. To remove small carbonate grains from acid-insoluble tooth subfossils, we dissolved the three sediment size fractions <500 μm in 10% acetic acid. Parrotfish teeth (family Labridae) were identified from gross tooth morphology and with the assistance of a tooth and jaw reference collection that we developed from dissection and photography of oral and pharyngeal jaws of 287 positively identified Caribbean reef fish species that represent all Caribbean reef families and most genera. Modern fish specimens were obtained from the Fish Collection of the Smithsonian Institution's National Museum of Natural History and fish markets in Bocas del Toro and Colón, Panama. Our photographic reference collection is at Acropora cervicornis, Agaricia spp., branching Porites spp., Madracis mirabilis). Urchin spines were picked from the 0.5–2 mm fraction and separated into two typically reef-associated taxa  and one typically seagrass- or reef-associated group (Lytechinus and Tripneustes spp.) and counted and weighed. Total urchin abundance was also computed to track effects of overall urchin herbivory on reef accretion rates.Coral and urchin subfossils were assigned to species or generic level. Coral fragments from the >2 mm sediment fraction were weighed and identified to common taxa  and to facilitate complete sample-tracer homogenization. U and Th were separated using conventional anion-exchange column chemistry using Bio-Rad AG 1-X8 resin. After stripping off the matrix from the column using double-distilled 7N HNO3 as eluent, 4N HNO3 and 2%HNO3+0.03% HF mixture was used to elute U and Th into 3.5 ml pre-cleaned test tubes, respectively. After screening U and Th concentrations in the U and Th fractions using their 1:100 dilute solutions on a quadrupole ICP-MS, the U and Th separates for the samples were then re-mixed in 2% HNO3 to make ∼3 ml solution in a pre-cleaned 3.5 ml test tube. The mixed solution for each sample contained the entire Th fraction and a small percentage of the U fraction. The amount of U fraction to be added to the mixed solution was calculated based on the screening results and the MC-ICP-MS working sensitivity, aiming to achieve ∼5 volts of 238U signal. The remixed U–Th solution was injected into the MC-ICP-MS through a DSN-100 desolvation nebulizer system with an uptake rate of ∼0.1 ml min−1. U–Th isotopic measurements were performed on the MC-ICP-MS using a detector configuration to allow simultaneous measurements of both U and Th isotopes. The 230Th/238U and 234U/238U activity ratios of the samples were calculated using the decay constants given in ref. 230Th ages were calculated using the Isoplot 3.75 Program230Th ages were corrected for non-radiogenic 230Th contributions using a modelled two-component-mixing non-radiogenic 230Th/232Th value based on the equation in ref. A high-resolution chronology of reef ecosystem change was obtained by U–Th dating coral fragments within the cores using a Nu Plasma multi-collector inductively-coupled plasma mass spectrometer (MC-ICP-MS) in the Radiogenic Isotope Facility at University of Queensland, following chemical treatment procedures and MC-ICP-MS analytical protocols described in ref. 230Th ages were obtained from the top 1 m of the core and one date from approximately every 0.5 m interval below that, yielding 6–8 ages per core  from which they were picked. Because proportional abundances of living communities are generally faithfully represented in subfossil assemblagesTemporal trends in individual community components were assessed by plotting abundance values of individual samples, with trends assessed within each core and across all cores combined using non-parametric locally weighted regression (‘loess')-smoothed trendlinesTo assess the presence and direction of causal relationships between herbivore abundance and reef accretion rates and between herbivore groups, we used CCM, a technique that compares the ability of time-lagged components of one process to estimate the dynamics of another. CCM tests for significant causal relationships by recognizing that the observed values of a forcing process should be significantly better explained by observed values of a response process than expected by chance, and the accuracy of that explanation should improve with increasing time series length3536Briefly, the algorithm proceeded in five steps: (1) using cross-validation, determine the optimal embedding dimension (E) that describes the number of time steps that best predicts the dynamics of each explored process , (2) test for nonlinearity and stochastic noise to ensure that none of the processes were purely random and that stochastic noise was not so large that causal links could not be recovered. Stochasticity and nonlinearity were tested by visually assessing whether predictive power was reasonably high for short time steps and decreased with increasing prediction time, respectively38The data that support the findings of this study are available from the corresponding author upon reasonable request.How to cite this article: Cramer, K. L. et al. Prehistorical and historical declines in Caribbean coral reef accretion rates driven by loss of parrotfish. Nat. Commun.8, 14160 doi: 10.1038/ncomms14160 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References


Our approach is useful for studying eye diseases in relation to osmolarity.Despite high relevance of tear osmolarity and eye abnormality, numerous methods for detecting tear osmolarity rely upon expensive osmometers. We report a reliable method for simply determining sodium ion-based osmolarity in artificial tears using sequential DNAzymes. When sodium ion-specific DNAzyme and peroxidase-like DNAzyme were used as a sensing and detecting probe, respectively, the concentration of Na When signal outputs were expressed as absorbance (using TMB in F/F0) was rapidly saturated with a steep initial slope. Half time at maximal values (t1/2) at different concentrations of artificial tears were determined to be 5–8 min as shown in t1/2 may be attributed to different substrate reactivities for peroxidase-like DNAzyme, but the overall range of t1/2 values was sufficiently rapid and similar to that by fluorogenic Na+-specific DNAzyme (data not shown), suggesting that the reaction of DNAzyme 1 was initially determined by the end product from DNAzyme 2. Importantly, the change in maximal equilibrium increased as the concentration of artificial tears increased, indicating that the cascade reaction by DNAzymes allows for rapid detection of sodium ions in artificial tears with versatile detection modes. Based on this result, we determined minimum reaction time (10–15 min) when the DNAzyme1/DNAzyme2 reactant produces colorimetric or fluorescent signal.To gain an insight into the time-dependent reaction of artificial tears by DNAzymes, we tested various concentrations of artificial tears using sequential DNAzymes, wherein colorimetric or fluorescent signals were monitored using different peroxidase-reactive substrates over time . This ex−1 (bar graph in R2) accounting for the strongly positive relationship between osmolarity and both measurements. This result shows that DNAzyme-based detection of sodium ions in artificial tears is useful for estimating tear osmolarity. Theoretically, tear osmolarity did not represent only the concentration of sodium ions (1 mM NaCl solution generates 2 mOsm L−1) because many other ions or molecules  in tears contributed to total tear osmolarity. However, the change in tear osmolarity is likely proportional to that in sodium ions detected by DNAzymes because the amount of NaCl accounts for approximately 90% of total tear osmolarity. Notably, elevated osmolarity in tears is primarily related to increased levels of sodium chloride  in artificial tears. Owing to high relevance of salinity, osmolarity and eye abnormality, our approach is useful for simple diagnostics of ocular diseases.We demonstrated the simple determination of sodium ion-based osmolarity in artificial tears using functional DNAzymes. By consecutively reacting two DNAzymes (Na


At 12 months of age, both male and female offspring exposed to prenatal hypoxia had elevated mean arterial pressure. Glomerular number was reduced by 25% in hypoxia-exposed male, but not female, offspring and this was associated with increased urinary albumin excretion, glomerular hypertrophy and renal fibrosis. Hypoxia-exposed offspring of both sexes were more susceptible to salt-induced cardiac fibrosis, however, renal fibrosis was exacerbated by high salt in males only. In male but not female hypoxia-exposed offspring, renal renin mRNA was increased at weaning. By 12 months, renal renin mRNA expression and concentrations were elevated in both sexes. mRNA expression of At1aR was also elevated in male hypoxia-exposed offspring at 12 months. These results demonstrate that prenatal hypoxia programs elevated blood pressure and exacerbates salt-induced cardiovascular and renal pathology in a sex specific manner. Given sex differences observed in RAS expression and nephron number, future studies may consider RAS blockade as a therapeutic target in this model.Prenatal hypoxia is associated with growth restriction and adverse cardiovascular outcomes. Here, we describe renal and cardiovascular outcomes in ageing mouse offspring prenatally exposed to hypoxia (12% O In utero insults, such as reduced oxygen and nutrient supply to the fetus, lead to growth restriction and predisposition to cardiovascular, renal and metabolic diseases in later life1. Growth restriction is frequently associated with impaired organ development2, meaning offspring may be born with organs lacking the robustness required for adequate support throughout the lifespan. Fetal hypoxia is a common pregnancy complication that arises from a wide range of circumstances including, but not limited to, placental insufficiency, high altitude living and maternal factors such as smoking and pulmonary disease4. In the chronically hypoxic fetus, blood flow is shunted towards the brain, heart and adrenal glands at the expense of peripheral organs such as the kidneys5; however, the long-term renal outcomes for these offspring are unknown.2, reduced from 21% O2) in late gestation are growth-restricted with impaired placental vascularisation7. Offspring develop endothelial dysfunction by 12 months of age8, which in the human population is associated with increased risk of cardiovascular events9. Studies in rat offspring from pregnancies complicated by hypoxia have also identified ventricular and aortic wall thickening10, susceptibility to cardiac ischemia-reperfusion injury11 and peripheral vascular dysfunction13. Less focus has been placed on renal outcomes following fetal hypoxia, which is surprising given the kidney is highly susceptible to in utero insults and is critical for long-term maintenance of blood pressure14.We have previously reported that both male and female mouse offspring exposed to prenatal hypoxia  are major contributors to impaired renal development and subsequent onset of hypertension in offspring20. It has been recently suggested that activation of the RAS occurs in a temporal and sex-specific manner, with earlier activation in males. Notably, males more are more frequently and/or severely affected compared to females19. RAS blockade is currently the first-line therapy for treating hypertension in humans, and attenuates age-related elevations in blood pressure. Furthermore, RAS blockade in growth restricted rats abolishes elevated blood pressure21, which supports a role of the RAS in programmed hypertension.A congenital nephron deficit, a common outcome following an 22, with ageing or a poor diet often unmasking or exacerbating disease outcomes. The postnatal ‘second-hit’ of a diet high in salt has been shown to exacerbate disease outcomes following maternal protein restriction18 and a congenital nephron deficit23 in rodents. Indeed, in our model, offspring exposed to prenatal hypoxia and a postnatal high salt diet had marked stiffening of the resistance vasculature and altered extracellular matrix composition in the aorta8, consistent with cardiovascular disease. However, no study has yet investigated whether cardiac and renal pathologies following prenatal hypoxia are exacerbated by a high salt diet and ageing.Offspring born from compromised pregnancies frequently display increased susceptibility to disease risk factors8 to assess blood pressure, renal function and cardiac/renal pathology in male and female offspring. We also examined if these outcomes were associated with sex-specific alterations in the renal RAS in young and aged animals. Given the postnatal environment may unmask or exacerbate disease outcomes, we also hypothesised that hypoxia-exposed offspring would have increased susceptibility to tissue damage due to high dietary salt intake.The aim of this study was to utilise our mouse model of hypoxia during late gestation8, body weight was reduced by hypoxia exposure at E18.5 and P21 ; and P21 (Table treatment = 0.2) or P21 (Table treatment = 0.98).As reported previouslyPost hoc analysis demonstrated males had more glomeruli than females, but hypoxia-exposed males had 25% less glomeruli compared to control males . No differences in At1aR or Ace mRNA expression in kidneys of P21 offspring were observed between groups  was elevated in male Fig.  and femasex = 0.02) and ∆ MAP (Psex = 0.049) during restraint. There was a tendency for increased ∆ SBP (Psex = 0.06) and ∆ DBP (Psex = 0.07) in males compared to females during restraint, although this did not reach significance.Restraint stress increased MAP above baseline levels in male and female offspring . Ace mRNA expression did not differ between groups  diet Table . Post hots Table . Potassits Table . Albumints Table . No diffts Table .Table 2OBody, kidney and heart weights (absolute and corrected for body weight) were similar among control and hypoxia groups at 12 months of age . The HS diet increased glomerular area in all animals, compared to offspring fed NS . Perivascular fibrosis area tended to be greater in male hypoxia-exposed offspring, with no effect of HS, although this did not reach statistical significance . Renal interstitial fibrosis was greater in male hypoxia-exposed offspring compared to controls, and further increased by the HS diet , perivascular fibrosis , interstitial fibrosis or mRNA expression of Kim1 and Tgfb1 26 and male offspring following uteroplacental insufficiency in the rat16. However, in the present study, female hypoxia-exposed offspring had a similar glomerular number to female controls. Although surprising, our finding is not unprecedented, as both modest protein restriction in the rat28 and birth asphyxia in the spiny mouse29 reduce glomerular number in male offspring only. Together, these studies highlight that the developing kidney of male fetuses is more vulnerable to prenatal insults than the kidney of females.In humans, fetal hypoxia results in asymmetric growth restriction and low birth weight15 and animal models2. Consequently, we hypothesised that only male offspring would develop high blood pressure. However, both sexes had elevated blood pressure at 12 months of age, highlighting dissociation between nephron number and hypertension. Endothelial dysfunction and impaired vascular structure (as previously reported in both sexes exposed to hypoxia)8 could contribute this increase in blood pressure. Furthermore, diastolic blood pressure was elevated in both male and female mice, consistent with increased peripheral vascular resistance of which microvascular endothelial dysfunction8 could be a contributing factor. In contrast to males, female hypoxia-exposed offspring also had elevated systolic pressure. Clinically, isolated diastolic hypertension is more predominant in young males compared to the population as a whole, and appears to be antecedent to systolic hypertension30.A nephron deficit is associated with hypertension in the human populationin utero perturbations20. Therefore, we hypothesised that elevated blood pressure in male and female hypoxia-exposed offspring would be associated with alterations in the renal RAS. Indeed, increased renal renin mRNA expression was present in male, but not female, hypoxia-exposed mice at just three weeks of age. By 12 months, renal renin mRNA and renin content were increased in both sexes exposed to prenatal hypoxia, with renin content directly associated with elevated diastolic blood pressures in both sexes. This may directly contribute to elevated blood pressure given persistent upregulation of renin production is associated with hypertension in growth-restricted offspring exposed to uteroplacental insufficiency21 and protein restriction31. Male  hypoxia-exposed offspring also had increased expression of renal At1aR mRNA. This combined with increased renal renin content suggests that there is early and sustained activation of the intrarenal RAS in males following hypoxia. Other models of prenatal hypoxia  have demonstrated increased renal sympathetic nerve activity32 and increased vascular tone 33 contribute to high blood pressure in young male rodents only. These changes are likely to occur secondary to changes in the RAS as proposed recently by Dasinger and colleagues19. Considering the increase in intrarenal renin content in aged males and females exposed to hypoxia, future studies should examine if RAS blockade is an effective anti-hypertensive therapy for both sexes in this model as previously shown in rat offspring following placental insufficency21.Alterations in the renin-angiotensin system (RAS) are a major contributor to hypertension, and changes to the renal RAS occur following a range of 19, the onset of hypertension in female rodents following a prenatal perturbation occurs in middle/old age (>12 months old). Estradiol is protective against cardiovascular risk in young female offspring following hypoxia due to placental insufficiency, however blood pressure elevation occurs around middle age following early reproductive senescence and RAS activation35. Similarly in the human population, hypertension is highly prevalent in postmenopausal women36. Future studies in our model should examine the time point of reproductive senescence and blood pressure elevation in offspring exposed to prenatal hypoxia.Temporal differences in intrarenal RAS activation between the sexes suggests that male hypoxia-exposed offspring may have developed elevated blood pressure at an earlier age to females in our study. As hypothesized recently37. Pathological myocardial fibrosis is associated with diastolic dysfunction, and although we did not perform echocardiography in our study, a similar model of prenatal hypoxia  showed that rat offspring had normal cardiac function at 4 months but developed left ventricular diastolic dysfunction at 12 months38. This is consistent with our morphological findings in the mouse, and suggests our hypoxia-exposed animals may be at risk of diastolic dysfunction and heart failure. Although we were unable to measure blood pressure in the animals fed the high salt diet, cardiac interstitial fibrosis was exacerbated in animals on this diet. Indeed, in females, the correlation between blood pressure and cardiac interstitial fibrosis suggests the high salt diet may exacerbate the elevations in blood pressure observed in hypoxia-exposed offspring. Furthermore, increased perivascular fibrosis, which is associated with impaired coronary blood flow in humans39, was most evident in hypoxia-exposed males fed the high salt diet. Together these findings suggest that the additional postnatal high salt diet may further increase risk of cardiovascular diseases in both sexes.A novel aspect of our study was the in-depth morphological analysis, in particular the development of renal and cardiac fibrosis, following prenatal hypoxia. Significant interstitial fibrosis was observed in the hearts of both sexes exposed to prenatal hypoxia, which is associated with reduced myocardial and arterial complianceKim-1 was evident at 12 months, which is indicative of progressive renal damage given no pathological changes were observed at 4 months of age. Our correlation analyses revealed elevated mean arterial pressure in male offspring was correlated with glomerular hypertrophy, suggesting offspring fed high salt were at increased risk of hypertension and subsequent renal injury. Albuminuria in humans is associated with cardiovascular risk factors such as vascular endothelial dysfunction40 which is present in our model at 12 months of age8. Fibrosis is also a hallmark of aging, and is present in pathological situations such as cardiovascular and kidney disease41. The AT1 receptors are known mediators of fibrosis, in part through the TGF-β1 pathway. Renin also initiates the production of pro-fibrotic factors (primarily TGF-β1) through the prorenin receptor, independent of angiotensin II42. Given renin content and At1aR and Tgfb1 mRNA expression were enhanced in male hypoxia-exposed offspring, this may be an underlying molecular mechanism for the widespread renal fibrosis in this group. Intriguingly, all female offspring had greater intrarenal renin content, greater albumin excretion and a tendency towards increased plasma cystatin C (indicative of reduced GFR) compared to control males at 12 months of age. No signs of renal impairments were observed in younger female offspring, suggesting these deficits emerged with age. This could be associated with naturally lower nephron endowment in female offspring compared to males. There was no effect of prenatal treatment, which may be due in part to the fact that glomerular number was not altered in female offspring exposed to hypoxia, allowing maintenance of renal function equivalent to control counterparts across the lifespan.The most profound finding of this study was the overt renal pathology observed in hypoxia-exposed male offspring fed normal salt, including progressive glomerular hypertrophy, glomerulosclerosis, tubulointerstitial fibrosis and perivascular fibrosis from early life until 12 months of age. Furthermore, mild albuminuria and elevated renal mRNA expression of the tubular injury marker Kim-1 and Tgfb1 in combination with increased glomerulosclerosis and fibrosis in the male offspring at 12 month of age. Consequently, we examined kidneys from males at 4 months of age to determine whether fibrosis was present at an earlier age. Strikingly, significant interstitial renal fibrosis was evident in young hypoxia-exposed male offspring within six weeks of high salt diet consumption, suggesting the combination of prenatal and postnatal insults markedly increases the rate of progression of renal damage. Given perivascular fibrosis was mainly influenced by diet but not prenatal treatment, consumption of a normal salt diet throughout life substantially attenuated renal damage in male hypoxia-exposed offspring. Glomerulosclerosis and renal fibrosis, in conjunction with hypertension, are hallmarks of chronic kidney disease. Therefore, we measured plasma cystatin C, currently the best endogenous marker of GFR in humans43, to achieve an indication of renal function in this study. In animals fed high salt, a reduction in plasma cystatin C was observed. This indicates glomerular hyperfiltration, a common response to nephron loss and suggested to promote glomerular hypertrophy and subsequent sclerosis44. Whether a sustained low salt diet from early in life could prevent the development of renal fibrosis is a pertinent clinical question given children over 5 years of age are commonly consuming >100 mmol/day of sodium, 5–10 times the physiological requirement45.The combination of prenatal hypoxia and the challenge of a chronic high salt diet in postnatal life was strongly associated with marked elevations in mRNA expression of in utero hypoxic insult; however, this protection does not extend to the cardiovascular system. Further investigation into the mechanisms underlying the sex differences in renal outcomes following prenatal hypoxia are needed and may contribute to a better understanding of renal disease progression and treatment. Temporal differences in renal RAS activation may be a contributing factor, and therefore future experiments could investigate whether RAS blockade in early life may be an effective anti-hypertensive therapy and ameliorate end-organ damage observed in these offspring. Given our findings, we suggest curtailing salt intake can minimise progressive cardiac and renal damage for those known to have suffered from an adverse in utero environment.We have shown that organ systems controlling blood pressure and fluid homeostasis, namely the kidney, heart and vasculature, are impaired by prenatal hypoxia. We conclude that elevated blood pressure and associated cardiac/renal injury observed in male offspring exposed to hypoxia is likely associated with the reduction in nephron number and activation of the renal RAS in early life. These sequelae were further exacerbated by a high salt diet. In females, the same prenatal hypoxic insult led to elevated blood pressure, an increase in intrarenal renin, and susceptibility to salt-induced cardiac injury in the absence of a nephron deficit. This suggests that the kidneys of female offspring were protected from the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Time-mated CD1 mice at embryonic day (E) 14.5 of pregnancy, were randomly allocated to normoxic room conditions (N = 19) or housed inside a hypoxic chamber continuously flushed with nitrogen gas to maintain an oxygen concentration of 12% (N = 19)8. A 12 h light/dark cycle was maintained and food and water was provided ad libitum. A subset of dams (N = 8) was culled at E18.56. Fetal body weight, kidney, heart and brain weights were recorded. Remaining dams were removed from the hypoxic chamber upon littering down. Pup weights were monitored daily from P1 until weaning at P21. At P21, a subset of male and female offspring (1–2 of each sex per litter) was euthanized by cervical dislocation. Kidneys, hearts and brains were dissected and weighed. The left kidney was fixed in 4% paraformaldehyde (PFA) for assessment of renal pathology, glomerular number and area. The remaining offspring were aged to 12 months for renal and cardiovascular studies.All experiments were approved by the University of Queensland Animal Ethics Committee and were conducted in accordance with the 8. Animals were maintained on the diet until euthanasia at 12 months of age.A subset of animals aged 10 weeks  was randomly allocated to receive a high salt (HS) diet  whilst litter mates remained on a normal salt (NS) diet  as previously described46. Briefly, kidneys were processed into paraffin blocks and exhaustively sectioned at 5 µm. Ten evenly spaced section pairs were systematically sampled and stained with lectin peanut agglutinin . Glomeruli from each section pair were counted using the physical disector/fractionator combination.A combined stereological-histochemical approach was used to determine glomerular number (N = 8 per treatment group) as previously described−1) and radiotelemetry transmitters  were implanted as described previously47. Recording of heart rate (HR), systolic (SBP) and diastolic blood pressure (DBP), and activity data commenced in conscious, unrestrained animals 10 days post-surgery (N = 5–6 per treatment group). Data were acquired for 10 seconds every 15 minutes for three days. Mean arterial pressure (MAP) and pulse pressure (PP) were calculated from these parameters.Animals at 12 months of age, fed the normal salt diet throughout life, were anaesthetised under isoflurane  for 15 min, and then released to the home cage. Data was acquired continuously during the restraint and 15 min recovery period. Recovery data was sampled for 10 s every 5 min for the next hour of recovery.+, K+, Cl−) were measured using a COBAS Integra 400 Plus analyser. Urinary excretion of albumin was determined in duplicate using commercially available mouse kits . In addition, water consumption in home cages was measured over 7 days at 12 months of age and averaged per day.Male and female offspring at 2 months , 4 months of age (N = 6–8 per treatment group) and 12 months (N = 9–11 per treatment group) fed the normal salt and high salt diets, respectively, were acclimatised to individual metabolic cages prior to urine collection. Animals were then placed in individual metabolic cages for 24 h. Urine was collected and frozen at −20 °C. Urinary electrolytes  and Masson’s Trichrome. Sections were assessed by an expert pathologist blinded to treatment groups and assigned a score based on pathology severity: 0, normal; 1, minimal change; 2, mild; 3, moderate; 4, severe. Glomerular area in PAS-stained kidney sections was quantified by tracing glomerular borders when the vascular pole was evident. Twenty to 30 glomeruli were analysed per animal and measurements averaged. To assess perivascular fibrosis in kidneys and hearts, two arterioles were selected in the Masson’s trichrome-stained sections, as described in ref. Plasma cystatin C levels at 12 months of age (1:300 dilution) were determined in duplicate using a commercially available kit .TM, Bio-Rad, Gladesville, NSW, Australia). Taqman assays on demand  were used to determine mRNA levels of Renin (Mm0234887_mh), Ace  and Kim-1 . Custom probes and primers to detect AT1aR (angioteninsin type 1a receptor) and Tgfb1 (transforming growth factor beta 1) mRNA levels were used as previously described49. The comparative cycle threshold method was used for all expression assays using the B-actin endogenous control. mRNA levels were normalised to the mean of the control male group at P21, or the mean of the control group of their own sex at 12 months of age.RNA was extracted from whole kidneys from offspring at P21 (N = 9–11 per group) and 12-month-old offspring (N = 7–11 per group) using the RNeasy minikit . All RNA was treated with deoxyribonuclease 1 and reverse transcribed into cDNA  and tissue renin, angiotensin II (Ang II) and angiotensin 1–7 (Ang 1–7) concentrations were measured by radioimmunoassay as previously describedt-test. A Pearson’s correlation coefficient was also used to determine the relationship between blood pressure radiotelemetry recordings and renal/cardiac parameters in offspring fed the normal salt diet at 12 months of age. All remaining data were analysed using two-way ANOVA with a Sidak post hoc test used where appropriate. P < 0.05 was considered significant.Statistical analyses were performed using GraphPad Prism 7 and IBM SPSS. Data are presented as mean ± standard error of the mean (SEM). Multivariate analysis of variance (MANOVA) was used to analyse blood pressure recordings over three days. Prenatal treatment, sex and light-dark periods were assigned as independent variables. Restraint stress response was calculated by quantifying the area under the curve during the stressor and comparing with an equivalent amount of time during the baseline period. Histology scoring was analysed using a Mann-Whitney test. Glomerular number was analysed using a two-tailed, unpaired Student’s The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Data 1


Drosophila melanogaster has become an important model organism to investigate metal homeostasis and human diseases. Previously we identified dZIP13 (CG7816), a member of the ZIP transporter family (SLC39A) and presumably a zinc importer, is in fact physiologically primarily responsible to move iron from the cytosol into the secretory compartments in the fly. This review will discuss the implication of this finding for the etiology of Spondylocheirodysplasia-Ehlers-Danlos Syndrome (SCD–EDS), a human disease defective in ZIP13. We propose an entirely different model in that lack of iron in the secretory compartment may underlie SCD-EDS. Altogether three different working models are discussed, supported by relevant findings made in different studies, with uncertainties, and questions remained to be solved. We speculate that the distinct ZIP13 sequence features, different from those of all other ZIP family members, may confer it special transport properties.The fruit fly  Ehlers-Danlos syndrome (EDS) is a group of clinically and genetically heterogeneous disorders with defects in connective tissue characterized by skin hyperelasticity, tissue fragility, poor wound healing, and joint hypermobility  transporter family . Most members of ZIP family transport zinc, but a few have also been found to transport other metals including iron. Most ZIP proteins have eight transmembrane domains (TMs) with a His-rich domain between TM3 and TM4, and hydrophilic residues in TM5 which may determine metal specificity  and the results indicate that the binding ability of this fragment changes in the series Bi3+ >> Cd2+ > Zn2+ > Ni2+ , indicating that two metal ions were trapped halfway through the membrane, unexpectedly forming a binuclear metal center. This work provides the first structural framework for understanding the metal transport mechanism of the ZIPs  of mammalian ZIP4 has been solved by Zhang et al., demonstrating that ZIP4-ECD is a crucial regulatory domain for optimal zinc transport  shares the highest overall homology with human ZIP13  flies a lower total amount of iron, but relatively normal amount of zinc, in their bodies , transferrin receptor (TfR1), duodenal cytochrome b (Dcytb), divalent metal-ion transporter 1 (DMT1), ferritin, mitoferrin (Mfrn), mitochondrial ferritin (MtFt), ferropotin (FPN), ceruloplasmin (Cp), hephaestin, hepcidin, and so on   or ceruloplasmin  is found in the serum. As stated above, mammalian iron is exported by ferroportin and very little iron is moved across the secretory compartment. Nevertheless, the ER/Golgi still needs iron as some iron-containing enzymes are also stationed in the secretory pathway in mammals  or dZnT7 (CG6672/ZnT86D) should also be involved in this process. Changing the expression of both these two genes by RNAi or overexpression leads to dissimilar phenotypes from those of altering dZIP13 expression, i.e., no obvious iron phenotypes in dZIP7- or dZnT7-modulated flies  models, as those in dZIP13–RNAi flies? This could be because in the fly the secretory pathway is loaded with iron, and the secretory pathway is the primary, if not the sole, iron exporting venue. In contrast in mammals, cellular iron is exported by plasma-membrane-resident ferroportin instead of the ER/Golgi path via secreted ferritin. Along this line of thinking, dZIP13 deficiency in the fly will result almost iron export shutdown, ending with overall iron absorption defect and deficiency. In mammals, iron is not exported via this avenue, so no general iron phenotype might be observed.Drosophila ZIP13 is highly demanding, that for mammalian ZIP13 is not so since much less iron is needed to pump into the secretory compartments in mammalian cells.But iron is still needed in the mammalian ER/Golgi because some enzymes such as collagen crosslinking enzymes LH and PH4 need it as a cofactor. While it could be envisioned that the iron exporting function for Overall, evolutionarily speaking, ZIP13 may also work as an iron exporter in mammalian cells to provide iron to ER/Golgi organelle. But direct evidences are still needed to support this assertion.Our studies in the fly indicate that dZIP13 is an iron exporter  expression or zinc probe FluoZin-3 after ZIP13 overexpression, is detectable when 100 μM zinc is added to the media  were discovered by complementation of yeast zinc importer zrt1 zrt2 mutant  and Mn2+ (apparent Km of 18 ± 2 μM) can also be transported by ZIP14 while Zn2+ uptake by this protein could be inhibited by Cd2+, Mn2+, and Cu2+ , which is the ortholog of mouse Ke4 , found in cardiac muscle cells and elsewhere in the body, transports three Na+ ions into the cell in exchange for one Ca2+ ion transported out of the cell (Liao et al., Besides zinc transporters, many ion exchangers move different ions in opposite directions. NaDrosophila melanogaster has served as a good research platform to investigate the function of metal transporters and studies in this organism may advance our understanding of metal homeostasis and its relevance to human disease(Xiao et al., GX drafted the initial version of the manuscript; BZ drastically revised the manuscript. All authors made intellectual contributions, edited, and approved the manuscript for publication.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.


Epstein-Barr virus (EBV) infects greater than 90% of humans, is recognized as a significant comorbidity with HIV/AIDS, and is an etiologic agent for some human cancers. The critically endangered mountain gorilla population was suspected of infection with an EBV-like virus based on serology and infant histopathology similar to pulmonary reactive lymphoid hyperplasia (PRLH), a condition associated with EBV in HIV-infected children. To further examine the presence of EBV or an EBV-like virus in mountain gorillas, we conducted the first population-wide survey of oral samples for an EBV-like virus in a nonhuman great ape. We discovered that mountain gorillas are widely infected (n = 143/332) with a specific strain of lymphocryptovirus 1 (GbbLCV-1). Fifty-two percent of infant mountain gorillas were orally shedding GbbLCV-1, suggesting primary infection during this stage of life, similar to what is seen in humans in less developed countries. We then identified GbbLCV-1 in post-mortem infant lung tissues demonstrating histopathological lesions consistent with PRLH, suggesting primary infection with GbbLCV-1 is associated with PRLH in infants. Together, our findings demonstrate that mountain gorilla’s infection with GbbLCV-1 could provide valuable information for human disease in a natural great ape setting and have potential conservation implications in this critically endangered species. Lymphocryptovirus genus, is one of the most widespread and prevalent human pathogens, infecting over 90% of humans and persisting for their lifetimes1. In contrast with more developed countries, where people are primarily infected during adolescence, people living in less developed countries are usually first infected as infants3. In infants, primary infection is either asymptomatic or associated with non-specific signs of a viral infection4. The prevalence of histolopathologic changes associated with primary infections in infancy is largely unknown because tissue biopsies are rarely collected. However, EBV has been implicated in the development of a wide variety of benign and malignant diseases associated with congenital or acquired immune dysfunction and has become of increasing concern with the prevalence of HIV/AIDS globally. In young and adult humans with immune dysfunction, EBV has been associated with pulmonary reactive lymphoid hyperplasia, endemic Burkitt’s lymphoma, undifferentiated nasopharyngeal carcinoma, and extra-nodal B-cell lymphomas7.Epstein-Barr virus (EBV), a gamma herpesvirus in the 8. In captive primates, lymphocryptoviruses have been associated with B-cell nasal lymphoma in simian immunodeficiency virus (SIV)-infected rhesus macaques, non-Hodgkin lymphomas in SIV-infected rhesus and cynomolgus macaques, and T-cell lymphomas in SIV-infected pigtail and otherwise healthy Japanese macaques17. EBV-like lymphocryptoviruses are known to infect great apes: two types of lymphocryptoviruses similar to type 1 and type 2 EBV in humans have been detected in western lowland gorillas  and orangutans , and a lymphocryptovirus similar to type 1 EBV has been detected in chimpanzees (PtroLCV-1)19. Because it is difficult to conduct population-wide studies to detect viruses circulating in wild great apes, little is known about the epidemiology or associated pathologies of EBV-like infections in great apes, particularly those associated with primary infection in infancy.In nonhuman primates (hereafter referred to as primates), EBV-like lymphocryptoviruses have been associated with lymphoproliferative disorders and malignancies similar to those reported in immunodeficient humansGorilla beringei beringei) are at high risk for contracting human pathogens because approximately 60% of their population is habituated to the presence of humans21. Habituation involves gradually familiarizing selected gorilla groups to the close presence of humans to ensure gorillas are accessible for conservation-related management, research, and tourism22. While habituation is a highly effective conservation strategy, it can expose great apes to pathogens23. Herpesviruses are chronically shed in saliva  and can remain stable in the environment for up to one week providing opportunities for mountain gorillas to contact viruses that humans have shed in parks or captive enclosures26. The potential for close proximity contact in captivity has been demonstrated through the detection of human herpes simplex virus-1 (HSV-1), an alphaherpesvirus, in a confiscated eastern lowland gorilla (Gorilla beringei graueri) cared for by human caretakers using a similar management strategy to confiscated mountain gorillas27. Wild habituated mountain gorillas are also frequently documented to be in close proximity with humans28. While gammaherpesviruses are generally considered to be the most host specific of the herpesvirus subfamilies, a population level survey of potential cross-species herpesviral infections in great apes has not yet been performed. In experimental infections, EBV has the ability to infect multiple species including cotton-top tamarins, owl monkeys, common marmosets, rhesus macaques and cynomolgus monkeys33. In addition, a recent analysis of gammaherpesviral sequences from a wide range of mammals indicated that cross-species transmissions have historically occurred more frequently than previously estimated, with most attributable to bats and primates34.Mountain gorillas , consisting of lymphocytic interstitial pneumonia and/or follicular bronchiolitis7, a syndrome associated with primary EBV infection in immunosuppressed human infants and children37, has been seen histologically in lung tissues collected post-mortem from mountain gorilla infants.In previous studies, all wild mountain gorillas sampled and tested were serologically positive for antibodies to human EBV38. We implemented a novel chewed plant method to collect oral samples at a population-wide scale, for the first time in a great ape. We also conducted a retrospective study of infant necropsy cases over a 10-year period to examine mountain gorillas for naturally occurring asymptomatic primary EBV-like lesions. Our findings demonstrate that mountain gorillas are widely infected with a mountain gorilla-specific lymphocryptovirus with epidemiologic and pathologic similarities to EBV in humans.Here we investigated the epidemiology and origin of lymphocryptoviruses shed by wild human-habituated mountain gorillas in the two remaining mountain gorilla subpopulations, the Virunga gorillas residing in the Virunga Massif  and the Bwindi gorillas residing in the Bwindi Impenetrable Forest in Uganda. To date, investigation of great ape populations for orally shed viruses has been limited by the ability to non-invasively collect oral samples39. Using consensus polymerase chain reaction (PCR) we applied broadly reactive degenerative primers to characterize the herpesviral DNA present on chewed plants targeting the DNA polymerase (DPOL), terminase (TERM) and glycoprotein B (gB) herpesviral genes. We identified the first evidence of an EBV-like virus, a mountain gorilla-specific strain of lymphocryptovirus 1 (GbbLCV-1), in wild mountain gorillas as well as herpesviruses in the cytomegalovirus and rhadinovirus subfamilies. Amplified lymphocryptovirus sequences did not show significant differences between Bwindi and Virunga gorillas, with 98.9% nucleotide and 98.7% amino acid similarity across a 473 bp fragment of the gB gene ; 99.6% nucleotide and 100% amino acid similarity across a 230 bp fragment of the DPOL gene ; and 98.8% nucleotide and 97.9% amino acid similarity across a 424 bp fragment of the TERM gene . Analysis of the gB gene fragments showed 97.7–97.9% nucleotide and 99.4% amino acid similarity to GgorLCV-1 isolated from western lowland gorillas (AF534225) and 91.3–91.5% nucleotide and 96.2–97.5% amino acid similarity to human EBV (LN827799). Analysis of the DPOL fragment showed 98.9–99.3% nucleotide and 98.7–100.0% amino acid similarity to GgorLCV-1 (KU578068) and 89.7–90.2% nucleotide and 91.9–93.2% amino acid similarity to EBV (AB850649). TERM sequences for GgorLCV-1 were not available in the Genbank database. Sequences detected in all mountain gorillas were most similar to GgorLCV-1 and did not show a close similarity to GgorLCV-2 . Finding that GbbLCV-1 is most closely related to the western lowland gorilla lymphocryptovirus-1 is to be expected given the close phylogenetic relatedness of the host species.To study oral shedding of lymphocryptoviruses, discarded chewed plants were collected from 76.2% of the Bwindi and 47.6% of the Virunga human-habituated mountain gorillas (n = 294 gorillas from 26 family groups) Fig. . These t40. Prevalence of GbbLCV-1 was not significantly different among conservation regions, age, sex, or family group . We found oral shedding of GbbLCV-1 in 43.0% (143/332) of mountain gorillas, with 48.8% (83/170) of gorillas in the Virunga Massif and 37.0% (60/162) of gorillas in the Bwindi Impenetrable Forest orally shedding GbbLCV-1. Our estimates are likely lower than the true prevalence, given the rugged field conditions surrounding oral specimen collection from chewed plants and the likelihood of intermittent sheddingThe mountain gorilla-specific strain of LCV-1 was also detected in circulating white blood cells in 11 of 15 adult females, 1 of 2 adult males, 2 of 4 juvenile females, 0 of 1 juvenile males, and 1 of 1 infant females from whom blood samples were opportunistically collected. No significant differences were detected between age or sex and lymphocryptovirus presence in circulating white blood cells. While the availability of opportunistically collected blood samples was low, the high prevalence of GbbLCV-1 in gorilla oral secretions tested from the majority of the habituated population and frequent detection in gorilla peripheral white blood cell samples strongly suggest widespread infection with GbbLCV-1 in both mountain gorilla subpopulations.41. Fifty-two percent of infant mountain gorillas tested were orally shedding GbbLCV-1  indicating mothers are most likely the primary source of infection for their infants. Mountain gorilla mothers were frequently observed grooming their infants, which could expose them to saliva containing lymphocryptovirus. In addition, infants, while still primarily nursing, were frequently observed investigating and chewing on discarded chewed plants that their mothers had dropped. Samples chewed on by both mother and infant were not collected for diagnostic analyses.To determine under what circumstances mountain gorillas acquire GbbLCV-1 in the wild, we screened mountain gorillas from all age groups in which social structure was well known. Mountain gorillas from the 26 family groups sampled are followed daily and their reproductive states and social interactions are recorded. Thus, for the majority of mountain gorillas in these groups, their exact date of birth is known. The majority of mountain gorillas shedding GbbLCV-1 were infants (samples collected from infants aged 6 months to 3 years of age), suggesting primary infection during this stage of life, similar to what is seen in humans in less developed countries23. Evidence of immune dysregulation was present in the form of lymphadenopathy characterized by giant follicular hyperplasia in two of these cases and thymic atrophy in two.To study the role of lymphocryptoviruses in the development of PRLH in mountain gorillas, we examined archived tissue samples from infants demonstrating histologic evidence of PRHL at death. Pulmonary reactive lymphoid hyperplasia has been identified in 11 out of 62 mountain gorilla infants (17.7%) that died between 1988 and 2013. We examined available post-mortem tissues from eight infants that died between 2001 and 2011, which demonstrated histological signs of PRLH. Histological lesions included lymphocytic interstitial pneumonia exemplified by diffuse infiltration of the alveolar septa by lymphocytes , we tested direct oral swab samples from one confiscated mountain gorilla and five confiscated eastern lowland gorillas being reared in captivity in very close contact with human caregivers. We used the same pan-herpesvirus PCR assays, designed to detect herpesviruses in the alpha, beta and gamma subfamilies and the EBV specific PCR assay, for both wild and confiscated gorillas. The potential for cross species transmission of viruses by close proximity contact has been previously demonstrated with the detection of HSV-1 in one of the captive eastern lowland gorillas in this study35, our population-wide survey did not detect shedding of EBV in the wild or captivity during our study period and alternatively demonstrated that the lymphocryptovirus circulating in mountain gorillas is a mountain gorilla-specific strain of lymphocryptovirus 1, GbbLCV-1. Epstein-Barr virus serologic assays used previously were likely cross-reactive with GbbLCV-1. We also identified multiple epidemiologic and pathologic similarities between GbbLCV-1 and EBV including: primary infection during infancy, a likely mother-to-infant transmission pathway via saliva, latent infection in peripheral white blood cells, intermittent oral shedding and an association between primary infection and the presence of PRLH. Together these similar epidemiologic and pathologic findings suggest that GbbLCV-1 could be used to gain insight into human disease. While experimental manipulation of mountain gorillas would not be feasible or ethical in the wild, information can be gained from noninvasive health studies and pathological investigations of deceased individuals, providing valuable information for human disease in a natural great ape setting.Contrary to previous serologic evidence for ubiquitous EBV exposure in mountain gorillas41. Oral shedding of GbbLCV-1 was not detected in any infant under the age of 6 months. In less developed countries, human primary infection with EBV typically occurs during infancy through exposure to saliva by close contact with their mothers (in part due to the practice of pre-mastication of food for infants) or other family members and is either asymptomatic or results in nonspecific signs of a viral infection43. In developed countries, human primary infection with EBV typically occurs during adolescence when people begin to be exposed to saliva through contact with other adolescents (thus it’s common name as the “kissing disease”), and infection in this age class can cause mononucleosis44. In this study, we identified a correlation between mother and infant GbbLCV-1 oral shedding, which suggests mothers pass the virus to their infants through infected saliva. Infant mountain gorillas frequently explore and chew on discarded food items from their mothers and mother-infant grooming is common; both activities which present opportunities for exposure to GbbLCV-1 through saliva.Mountain gorilla infants had the highest prevalence of GbbLCV-1 oral shedding compared to other age groups. It is likely that infants are protected by maternal antibodies until approximately 6 months of age but become infected shortly thereafter when maternal antibodies wane, similar to what has been documented for lymphocryptoviruses in rhesus macaques and in humans in less developed countries45. Detecting GbbLCV-1 in both oral specimens and peripheral white blood cells in multiple age groups suggests a similar route of infection and persistence in mountain gorillas.While the highest prevalence of GbbLCV-1 oral shedding was in infants, lymphocryptovirus was detected in all age groups in both oral specimens as well as peripheral white blood cells. We know that after initial infection with EBV, the virus remains latent in peripherally infected B lymphocytesin situ in human case-control studies46. Although our sample size of mountain gorilla tissues viable for molecular diagnostics was small, identifying GbbLCV-1 in mountain gorilla PRLH lesions together with the knowledge that EBV is associated with PRLH in humans suggests a similar pathogenesis in mountain gorillas. None of the mountain gorilla infants in which PRLH lesions were identified showed signs of respiratory illness prior to their death, indicating that PRLH can occur without overt clinical signs.Our study localized GbbLCV-1 viral DNA to five infant lung tissues demonstrating lesions consistent with PRLH. A lymphocryptovirus could not be detected in any negative control mountain gorilla lung tissues. Epstein-Barr virus DNA has been associated with PRLH lesions 47 and other great ape-specific lymphocryptoviruses have been identified in wild chimpanzees, western lowland gorillas and orangutans, mountain gorillas present a more accessible model for lymphocryptovirus epidemiology and pathology in a natural free-ranging host. The majority of individuals in the mountain gorilla population are identified by name with researchers and park personnel monitoring them daily. More than four decades of data on life histories and behavioral interactions are recorded for these human habituated gorillas48. Until recently, population-wide studies of orally shed viruses in mountain gorillas were logistically impossible, limiting virus data to that which could be obtained from fecal samples or infrequently collected blood or oral swab samples. With the introduction of chewed plants as a way to collect great ape oral samples, mountain gorillas can now be frequently monitored for oral shedding of lymphocryptoviruses non-invasively with minimal behavioral disruption38.While primate models for human infection with EBV exist in captivity50. When infants die in the forest, every attempt is made to collect their bodies for necropsy. While only a small number of appropriately preserved tissue samples from these necropsies were available for our retrospective study due to inconsistencies in sample collection and variable length of formalin fixation, investigations into infant mortality are ongoing and additional PRLH cases are being identified and investigated.In addition, post-mortem infant mountain gorilla tissue samples are opportunistically available, permitting investigation of histopathologic lesions associated with primary lymphocryptovirus infection. Infant mountain gorilla mortality is high due to the practice of infanticide by adult males and other traumatic and infectious causese.g., Down syndrome, autoimmune diseases, and HIV/AIDS)7. Inferring from findings reported here in mountain gorillas, we hypothesize that pathology in humans is likely more common than has been reported to date and is more likely to be present in the absence of overt clinical signs. Our data warrant further investigation of the frequency of histopathologic lesions associated with primary infection with EBV in human infancy.Paired molecular and histopathologic findings in gorilla infants, in conjunction with the overall high prevalence of PRLH in mountain gorilla infant necropsy cases, indicates that histopathologic lesions associated with primary infection with a lymphocryptovirus is common in mountain gorilla infants. In contrast, little is known about the histopathologic lesions associated with primary infection with EBV in human infants and children for whom invasive lung biopsies are rarely performed. In children, in whom PRLH was diagnosed with an open lung biopsy, it is usually associated with congenital or acquired immune dysregulation , and the study was conducted in accordance with the relevant guidelines and regulations.38.Samples were collected from mountain gorillas in the Volcanoes National Park in Rwanda and the Mgahinga and Bwindi Impenetrable Forest National Parks in Uganda. Chewed plant samples (n = 383) discarded by wild human-habituated mountain gorillas were collected from 294 individual mountain gorillas between November 2012 and June 2013, as described previouslyWhole blood samples were opportunistically collected from human-habituated mountain gorillas between October 1997 and July 2008  while they were anesthetized for treatment of a presumed human-induced illness or injury. Tissue samples examined in this study were collected during necropsies of mountain gorillas that died in the Volcanoes National Park between 2006 and 2011 . Oral swab samples were collected and placed in viral transport media  from one orphan mountain gorilla and five eastern lowland gorillas in captivity in Rwanda while they were anesthetized for their annual routine health examinations between December 2009 and November 2010. All samples were frozen and stored at −80 °C. All samples were imported to the University of California Davis under CITES permit # 13US117181/9.38. DNA was extracted from whole blood samples using DNEasy® blood and tissue kits . Total nucleic acid was extracted from approximately 100 ug of frozen tissue. Tissue samples collected at necropsy were cut into small pieces with scissors disinfected with 10% sodium hypochlorite after handling each sample, to prevent DNA cross-contamination between tissues and between animals. Total nucleic acid was extracted using the NucliSENS® MiniMAG® system . DNA and total nucleic acid were stored at −80 °Celsius.Chewed plant samples were processed according to previously described techniques62. Primers used were designed to amplify all herpesviruses within the alpha, beta and gamma herpesvirinae subfamilies and were tested in our laboratory (unpublished data) as well as in previous studies to determine their ability to detect human herpesviruses, including EBV63. Purified sample DNA (2 ul) or products of first-round PCR reactions (1 ul) were used as templates in PCR reactions with 25 ul reaction mixture containing 1 μM of each PCR primer, 200 μM of each deoxynucleotide triphosphate, 0.75 units DNA Polymerase AmpliTaq Gold  and 5% DMSO . For the TERM assay, in first and second round PCR, the reactions were kept at 95 °C for 3 min and then cycled 55 times with 20 sec of denaturation at 95 °C, 30 sec of annealing at 48 °C and 30 sec of strand extension at 72 °C, followed by a final extension step at 72 °C for 10 min. For the DPOL assay the conditions were the same except that the annealing temperature was reduced to 46 °C. In order to confirm the absence of EBV, samples testing positive for a gorilla herpesvirus by degenerative herpes PCR assays were also screened using an EBV specific PCR assay using previously described primers for a fragment of the EBV DPOL gene64. PCR reactions were the same as described for degenerative assays with the exception of omitting DMSO. Reactions were kept at 95 °C for 3 min and then cycled 50 times with 20 sec of denaturation at 95 °C, 30 sec of annealing at 62 °C and 30 sec of strand extension at 72 °C, followed by a final extension step at 72 °C for 10 min. In addition, a representative subset of samples from gorillas from both the Virunga Massif and Bwindi Impenetrable Forest were analyzed by PCR to detect a fragment of the gB gene (509 bp) to generate larger sequences for phylogenetic analysis. Primers for the gB gene were described previously18 and PCR conditions were the same as described for the DPOL assay. Parallel reactions were run for all samples amplifying a fragment of the mammalian beta-actin gene to test sample quality. PCR products of appropriate size were cloned using TOPO® TA cloning kits , and sequencing was performed using Sanger sequencing at the University of California, Davis DNA sequencing laboratory. Sequences were compared to other published herpesviral sequences in the GenBank Database . Sequences were edited using Geneious version 7.0.6. Alignments were constructed using CLUSTAL W, executed through Geneious.DNA was analyzed by polymerase chain reaction (PCR) to detect herpesviral DNA utilizing degenerative primers amplifying a 450 bp region of the TERM gene and 225 bp region of the DPOL gene. Primers for the TERM and DPOL assays were described previouslyTissues collected at necropsy were fixed in 10% neutral buffered formalin. Fixation time was prolonged, up to more than a year in some cases, pending receipt of permits for importation into the United States. Fixed tissues were processed routinely for wax embedment and sectioning at 5 microns. Sections were stained with hematoxylin and eosin (H&E) and examined by light microscopy by a board certified veterinary pathologist (LJL).66. A total of 332 samples (out of 383) were included in statistical analyses (11 samples could not be properly assigned to an age group); samples that tested negative for herpesviruses but were also negative for mammalian beta-actin were excluded from statistical calculations because inadequate collection of quality sample material could not be ruled out for these samples. Period prevalence and 95% binomial exact confidence intervals (CI)67 for oral shedding of lymphocryptovirus were calculated across age class. Finite population correction factor was not used because sampling was conducted with replacement. Evaluation of the effects of conservation area, family group, age, and sex on gorilla lymphocryptovirus oral shedding was performed by both bivariate analysis using a 2-sided chi-square test and a mixed-effects logistic regression model69. Associations between lymphocryptovirus detected in whole blood and potential risk factors, such as age and sex, as well as associations between lymphocytic interstitial pneumonia status and lymphocryptovirus detected in lung and non-lung tissues were evaluated using a Fisher’s exact test70. Association between oral lymphocryptovirus shedding in infants and their mothers was evaluated by using a McNemar’s test71. All statistical analyses were performed using STATA version 13.172.Wild mountain gorillas were divided into age classes: infants were defined as gorillas less than three years of age, juveniles as three to five years, subadults as six to seven years and adults as eight years and olderThe datasets generated during the current study are available from the corresponding author on reasonable request with permission from the Rwanda Development Board.Supplementary Figure 1


SPOCK1 gene showing association with mathematics ability . The SPOCK1 gene is located on chromosome 5q31.2 and encodes a highly conserved glycoprotein testican-1 which was associated with tumor progression and prognosis as well as neurogenesis. This is the first study to report genome-wide significant association of individual SNPs with mathematics ability in general populations. Our preliminary results further supported the role of SPOCK1 during neurodevelopment. The genetic complexities underlying mathematics ability might contribute to explain the basis of human cognition and intelligence at genetic level.Mathematics ability is a complex cognitive trait with polygenic heritability. Genome-wide association study (GWAS) has been an effective approach to investigate genetic components underlying mathematic ability. Although previous studies reported several candidate genetic variants, none of them exceeded genome-wide significant threshold in general populations. Herein, we performed GWAS in Chinese elementary school students to identify potential genetic variants associated with mathematics ability. The discovery stage included 494 and 504 individuals from two independent cohorts respectively. The replication stage included another cohort of 599 individuals. In total, 28 of 81 candidate SNPs that met validation criteria were further replicated. Combined meta-analysis of three cohorts identified four SNPs  of  Mathematics serves as a fundamental instrument in modern society as it plays an important role in many fields including science, engineering, and economics. It also used as a key index of human intelligence. Exceptional mathematics ability was frequently observed among genius from many domains. Meanwhile, dyscalculia, characterized by impaired number processing skills, is a specific developmental disorder of mathematics ability that affects approximately 3 to 6% of childrenRecent years, genome-wide association study (GWAS) has been widely applied to investigate genetic components underlying complex traitsMYO18B and is associated with intraparietal sulcus morphologyTo date, none of studies has identified genome-wide significant association with mathematics ability in general populations due to small effects of common variants. However, research with specific populations might increase statistical power to detect significant association as it was reported that prevalence of mathematic disability was higher among children with neurodevelopmental disorders such as reading disability, attention-deficit/hyperactivity disorder (ADHD) and autism7Previous GWAS of mathematic ability is mainly performed in Western populations of which genetic backgrounds are substantially different to Chinese populations. In the present study, we performed GWAS of mathematic ability in the Han Chinese elementary school students through QTL-based approach. In total, we identified four SNPs exceeding genome-wide significant threshold which is the first time to report genome-wide significant association of individual SNPs with mathematics ability in general populations. Our results provide novel evidence to explain genetic complexities underlying mathematic ability and the basis of human intelligence at the genetic level.−5 were identified . Each minor allele of these four SNPs was associated with decrease of math score ranges from 2.33 to 2.43 points approximately. To our knowledge, it is the first time to report association of a single SNP with mathematic ability at genome-wide significant level in general populations.Despite substantial heritability underlying mathematics ability, contribution of SNPs to this complex cognitive trait remained inconclusive. Previous GWAS of mathematics ability in general populations proposed candidate SNPs spanning chromosome 2, 3, 4, 5, 6, 7, 11, 12, 13, 20 and 21451112SPOCK1 and in highly linkage disequilibrium. The most significant SNP rs1012694 is located between exon 3 and 4 of SPOCK1. This gene is located on chromosome 5q31.2 and encodes sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 1. Testican-1 is a highly conserved glycoprotein that involved in regulating proliferation, cell-cycle progression, apoptosis, adhesion, and cell-matrix interactionSPOCK1 and its gene product testican-1 have been associated with tumor progression and prognosis of different cancer types. Expression of SPOCK1 at mRNA and protein level was upregulated by the transcription factor CHD1L which could directly bound to promoter region of SPOCK1SPOCK1 resulted in activation ofPI3K/AKT signaling which could block apoptosis and promote proliferation, invasiveness and metastasis of cancer cells16SPOCK1 was implicated in epithelial-to-mesenchymal transition (EMT) which promoted migration and invasion in lung cancer and esophageal squamous cell carcinoma and conferred acquired drug resistance in gastric cancer1819SPOCK1 has been considered as a novel prognostic and therapeutic target for various cancer types.These SNPs are intron variants of SPOCK1 in cancers has been relatively well understood, its contribution to neurological and cognitive development remains elusive. Recently, novel de-novo SPOCK1 mutation was reported in a female proband with developmental delay, microcephaly and agenesis of corpus callosumSPOCK1 located within 5q31 was suggested to be a candidate gene of observed developmental abnormalities. The identified de-novo mutation of SPOCK1 might be protein-damaging which could potentially lead to developmental delay and microcephaly. Therefore, SPOCK1 might play a critical role during neurogenesis. Indeed, testican-1, encoded by SPOCK1, was shown to inhibit attachment of Neuro-2a cells and their ability to form neurite extensionsAlthough the role of Some potential limitations of the present study should be noted. The effect sizes might have been slightly overestimated due to lack of the adjustment for risk factors such as family socioeconomic status. Using nonverbal intelligence as an exclusion criteria might result in biased distribution of children’s math scores as it was assumed that children with lower nonverbal intelligence seems more likely to have lower math scores as well. In addition, sample size of our study were relatively small compared with genome-wide studies of chronic diseases such cancer or diabetes. However, the strengths of our study include its stringent quality control procedures and all participants genotyped by using the Affymetrix Axiom Genome-Wide CHB1 and CHB2 arrays which contain over one million SNPs specifically designed for Chinese population.SPOCK1 that showed genome-wide significant association with mathematic ability in Chinese children. Mathematic ability is a complex trait that involved polygenic and environmental factors. SPOCK1 and its gene product tesican-1 showed potential functional relevance to neurodevelopment. Our study has identified a susceptibility gene, SPOCK1, which provides novel genetic insights into development of mathematics ability and the basis of human intelligence.In conclusion, we reported four genetic variants of th percentile were excluded from this study. In total, 1622 participants  were eligible for subsequent genotyping and association analysis. Mathematic ability was measured by children’s academic performances of mathematics according to their mid-term and final exam of each semester. The examination papers were designed by education authorities of Shandong Province for each grade respectively according to the curriculum. Therefore, different tests will be applied to children in different grades but children in same grade will take exactly the same test. The tests aimed to evaluate children’s academic performances of mathematics from three perspectives including “understanding numbers”, “computing and knowledge” and “non-numerical processes”. The teacher’s rating process was double-blinded as student’s answer sheet will be randomly and anonymously distributed to different teachers. Answers of all questions are clear and definite. There was no arbitrariness in scoring as teachers from different schools received unified training to ensure that their rating criteria for each student are standardized and objective. The mean score of mid-term and final exam within in same semester that were usually conducted within a 3-month interval was calculated for the analysis. This study was approved by the ethical committee of Tsinghua University School of Medicine. The methods were carried out in accordance with the relevant guidelines. Informed consent was obtained from all subjects.We recruited 2,425 grade two to grade six primary students aged 7 to 13 from three counties, Liangshan, Dongming and Cao, in Shandong Province in China. In the first step, Raven’s Progressive Matrices test for nonverbal intelligence was administered to these eligible children individually, whose nonverbal intelligence scores lower than the 25−5). After quality control procedures had been performed, 494 children with 1182517 SNPs from Liangshan and 504 children with 1108437 SNPSs from Dongming were included in the final analysis.DNA was extracted from blood samples and SNP genotyping was performed with the Affymetrix Axiom Genome-Wide CHB1 and CHB2 arrays  by CapitalBio Technology . Quality control was performed followed by standard quality control metricsTM DNA self-collection kit according to the manufacturer’s instructions . DNA concentration was determined by Nano Drop 1000 . Specific assays were designed using the MassARRAY Assay Design software package (v3.1). Mass determination was carried out with the MALDI-TOF mass spectrometer and Mass ARRAY Type 4.0 software was used for data acquisition.Replication samples were typed at CapitalBio Technology  with Sequenom MassARRAY platform  according to the manufacturer’s protocol. Briefly, genomic DNA was extracted from saliva of each individual through Oragene−5 were selected for replication in Cao population. Finally, a meta-analysis was conducted to combine results from the three populations. A fixed-effect model with inverse variance weighting was used when there was no indication of heterogeneity (P for Cochran’s Q statistic >0.05); otherwise, a random-effect model for the corresponding SNPs was adopted. A Manhattan plot of −log10P was generated using the ggplot2 packageAfter quality control, association analyses and meta analyses were performed using PLINK1.9http://biostats.usc.edu/Quanto.html). Under the additive model, it had 80% power at the p < 0.05 level to detect association with an allelic variant of 20% frequency accounting for 1.58% and 1.55% of the variance in math score in Liangshan and Dongming populations respectively.Power calculations were performed using Quanto version 1.2.4 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.