AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three –omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery . We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two –omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes.  The molecular mechanisms underlying OA remain incompletely understood and there is no curative therapy for OA; disease progression culminates in joint replacement surgery. OA is a complex disease, with both heritable and environmental factors contributing to susceptibility3.Osteoarthritis (OA) affects in excess of 40% of individuals over the age of 70 yearsCartilage degeneration is one of the key features of OA. Cartilage tissue is readily accessible at joint replacement surgery, providing an opportunity to characterize the molecular processes underpinning disease development in the right tissue, both to improve our fundamental understanding of disease biology and to identify novel therapeutic opportunities. In recent years, –omics studies in OA have expanded our understanding of disease pathogenesis, reviewed in refs For full details of methods see Supplementary Methods.9: one sample with high OARSI grade signifying high-grade degeneration (“degraded sample”), and one sample with low OARSI grade signifying healthy tissue or low-grade degeneration (“intact sample”) . Cartilage was separated from bone and chondrocytes were extracted from each sample. DNA, RNA, and protein were extracted from the isolated chondrocytes of each sample. All patients provided full written informed consent prior to participation. We obtained two cartilage samples from each patient. Knee samples were scored using the OARSI cartilage classification systemLC-MS analysis was performed on the Dionex Ultimate 3000 UHPLC system coupled with the high-resolution LTQ Orbitrap Velos mass spectrometer. Proteins were defined as differentially abundant if the absolute median abundance ratio between degraded and intact samples was ≥ 0.75, the absolute median abundance ratio was greater than the standard deviation of abundance ratios, and the protein was detected in paired samples from at least 5 of 11 patients post-QC.10, and reads aligning to each gene in each sample were counted using the HTSeq package11. Absolute transcript abundance was calculated using the FPKM (fragments per kilobase of transcript per million fragments mapped) measure across 15,418 genes post-QC. We used edgeR v3.012 to identify differentially expressed genes, applying a generalized linear model for tissue status (degraded or intact) with individual ID as covariate, and defined significance at 5% false-discovery rate (FDR).Multiplexed libraries were sequenced on the Illumina HiSeq 2000 (75 bp paired-end read length) and individual indexed library BAM files were produced. Reads that passed quality control (QC) were re-aligned using tophat213; the probe beta values were quantile-normalized using the ‘dasen’ method from the wateRmelon package14. Differential methylation on 425,694 probes post-QC was tested using the CpGassoc package15, applying a linear model for tissue status at each probe, with individual ID as covariate.Methylation was assayed using the Illumina 450k BeadChip. Data were parsed and QCed using ChAMP16, differentially methylated regions (DMRs) were defined as regions of at least 3 probes with FDR ≤ 5% (DMPs) and no more than 3 non-significant probes, with no more than 1 kb between adjacent probes. We used bedtools17 to identify genes overlapping each DMR, extending gene boundaries to include likely promoter regions.Following previous analysesFor a promoter-level analysis, we used the probe annotations from ChAMP to assign promoter probes to each gene. After computing the mean beta value in the promoter of each gene in each sample, we used a paired t-test to identify genes with differential promoter-region methylation between degraded and intact samples. Significance was set at 5% FDR.Gene expression and DNA methylation in both replication cohorts were measured using the same procedure as for the discovery data. We considered the expression of 14,762 genes that passed QC across all datasets; this included 332 of 349 genes with FDR ≤ 5% in the knee discovery data.We considered 416,437 probes that passed QC across all datasets; this included 9,723 of 9,867 probes with FDR ≤ 5% in the knee discovery data (“DMPs”).18. This dataset comprised a comparison of affected and macroscopically preserved OA cartilage from 33 patients undergoing total joint replacement surgery , measured using the Illumina HumanHT-12 v3 microarrays.We carried out an additional replication analysis for gene expression changes using an available published dataset from the ongoing Research Arthritis and Articular Cartilage (RAAK) study19, Reactome20, and, separately, Gene Ontology (GO) annotations21. Genes annotated to the same term were treated as a “pathway”. Enrichment was assessed using a 1-sided hypergeometric test, with significance at 5% FDR. Empirical p-values for the enrichments were obtained from randomisations.We tested whether particular biological gene sets were enriched among significant genes from each of the RNA-seq, methylation, and proteomics datasets. We used KEGGp-values) was higher than expected by chance, obtaining empirical p-values from 100,000 randomisations.For each gene set, we asked whether the association across the three –omics datasets . Tissue samples were collected under National Research Ethics approval reference 15/SC/0132, South Yorkshire and North Derbyshire Musculoskeletal Biobank, University of Sheffield; and under National Research Ethics approval reference 11/EE/0011, Cambridge Biomedical Research Centre Human Research Tissue Bank, Cambridge University Hospitals, UK. All participants provided written informed consent. All methods were carried out in accordance with the relevant guidelines and regulations.https://www.ebi.ac.uk/ega/studies/EGAS00001001203. All discovery methylation data are available in the EGA repository, https://www.ebi.ac.uk/ega/studies/EGAS00001001213. The discovery proteomics data has been deposited in the PRIDE archive under study ID PXD002014 [publication in process].All genome-wide summary statistics generated in the discovery component of this study are available as Supplementary Tables. All discovery RNA sequencing data are available in the EGA repository, We compared the degraded and intact tissue across patient-matched samples.We identified 209 proteins with evidence of differential abundance  inhibitor that has been reported to be lost from chondrocytes of the superficial zone and shifted to the middle zone in OA cartilage in a targeted study22.We identified 349 differentially expressed genes: 296 genes showed higher and 53 lower expression levels in the degraded samples  , while the DMR associated with CLEC3B showed evidence of increased methylation. Using IHC we independently confirmed the presence of AQP1, COL1A1 and CLEC3B in articular cartilage chondrocytes  have not previously been implicated in OA ; all five are annotated as secreted proteins in the Human Protein Atlas23 (see Supplementary Note).Among the 209 proteins with evidence of differential abundance in the proteomics data, 31 were also differentially expressed between degraded and intact samples at the RNA level . This correlation became substantially higher when we only considered the 31 genes that were differentially expressed in both datasets .Using all samples irrespective of tissue status, we found a significant correlation between gene expression levels and protein abundance  and excellent replication rates for differentially expressed genes and probes with differential methylation , we again found good agreement of the log-fold-changes for the differentially expressed genes from the discovery data .We additionally pursued replication in a further independent, published microarray gene expression dataset from the RAAK studyWe identified biological processes with consistent evidence of involvement in OA progression at multiple levels Fig. –S10. A s24. Indeed, histological examination of the samples we investigated showed greater blood vessel ingrowth in tissues with more advanced OA . We also found enrichment of genes involved in the regulation of angiogenesis at multiple levels, implicating pathways involved in blood vessel and nervous system formation . The growth of blood vessels and nerves are closely linked processes that share regulatory mechanisms, including the ERK cascade and heparin-binding proteins25 for the 49 genes differentially regulated across at least two –omics levels. We identified ten agents with investigational or established actions on nine of the dysregulated proteins, which already have Food and Drug Administration Marketing Authorization for use in humans (Table 1), an agonist of osteocalcin (BGLAP gene). Periostin (POSTN gene), a protein with elevated expression in OA in this study and others28, is a vitamin K-dependent protein that induces cartilage degeneration29. A recent study has associated sub-clinical vitamin K deficiency with knee OA incidence30, adding evidence that this compound could be a disease-modifying agent in OA.To identify existing drugs that could be repurposed for OA, we searched Drugbankns Table . Among t31. Previous studies of osteoarthritis have investigated methylation33, transcription37, or protein expression39 separately, or a combination of up to two of these –omics assays41, some with the addition of genetic data42. By contrast, this study provides a systematic analysis of biological changes involved in OA across genome-wide methylation, gene and protein expression levels.Characterisation of the molecular landscape of OA is now increasingly feasible and catalyzed by technological advances in functional genomics, and the accessibility of the relevant tissue at joint replacement surgery. Indeed, the field of functional genomics in OA is now buddingAQP1, COL1A1, and CLEC3B in disease progression.We identify 49 genes with evidence of changes between intact and degraded cartilage on multiple molecular levels, including some novel molecular players in OA. We provide robustly-replicating evidence with convergence of all three –omics levels of the involvement of AQP1 encodes aquaporin-1, a protein that facilitates water transport across biological membranes. Chondrocyte swelling and increased cartilage hydration has been suggested as an important mechanism in OA43. AQP1 has been found to be over-expressed in the meniscus of a rat model of knee OA44 and in degraded compared to intact articular cartilage in human patients with knee OA45. CLEC3B encodes tetranectin, which binds human tissue plasminogen activator (tPA)46. CLEC3B has previously been found to be up-regulated in OA47. COL1A1 is one of several collagens that were differentially abundant at both the RNA and protein levels  is a broad specificity aminopeptidase that has previously been detected in the synovial fluid of OA patients55 and therefore has potential as a novel OA biomarker. CHRDL2 is a bone morphogenetic protein (BMP) inhibitor that has been reported to be lost from chondrocytes of the superficial zone and shifted to the middle zone in OA cartilage in a targeted study22. WNT5B, a ligand for frizzled receptors in the WNT signaling pathway, has been found to be differentially transcribed in osteoarthritic bone, consistent with current understanding that OA is a disease involving both cartilage and bone56.7. These results corroborate that increased ECM turnover is a crucial component in OA pathogenesis. We found suggestive evidence of a link between some of these pathways and genetic OA risk loci (see Supplementary Note, Supplementary Table 58. This is not surprising, as all of the patients studied here had a diagnosis of OA and clinically advanced disease in at least one location within the joint. Inflammatory mediators are soluble factors present throughout the joint, to which both the healthy and the diseased chondrocyte populations are potentially exposed, and for which the regulatory molecules may differ compared to the articular cartilage tissue studied here.We identify extracellular matrix organization, collagen catabolism and angiogenesis as biological pathways that have significant association with disease progression, in agreement with the consensus findings of previous methylation and gene expression studies of OA59 or by pooling intact and degraded cartilage16, as neither of these studies examined differences between intact and degraded cartilage, and whether such differences are joint-independent.Based on our discovery data, we estimate that ~95% of significantly different genes identified in this study are true positives (Supplementary Fig. Notably, this study is a proof-of-concept for integrative deep molecular phenotyping across methylation, gene expression, and protein abundance. As such, it was not powered to provide an exhaustive list of molecular targets and pathways. Indeed, we estimate that only ~10% of the true differentially expressed genes are statistically significant in this study (Supplementary Fig. In summary, the integrative functional genomics approach undertaken here has identified biological changes in disease-relevant tissues, highlighting three genes and several pathways that are involved on all three levels examined. Moreover, the approach identified nine genes with changes on multiple molecular levels that are already targeted by drugs approved for human use, highlighting the potential of discovering targets for intervention. These drugs have established safety profiles and pharmacokinetic data for use in humans, which would shorten the investigative pipeline to clinical use in OA. These agents cover a broad range of mechanisms of action and represent novel investigational targets for ‘first in disease’ studies of OA progression. Further studies will be necessary to comprehensively characterize the molecular signatures of OA.Supplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9Supplementary Table 10Supplementary Table 11Supplementary Table 12Supplementary Table 14


Planctomycetes and the metabolic potential was consistent with detrital marine snow degradation. By contrast, in seep samples with methane as the principal hydrocarbon, methane oxidation by abundant members of ANME-1 was likely the predominant process. Seep samples characterized by fluids containing both methane and complex hydrocarbons, were characterized by abundant Chloroflexi (Anaerolinaceae) and deltaproteobacterial lineages and exhibited potential for complex hydrocarbon degradation. These different metabolic capacities suggested that microorganisms in cold seeps can potentially rely on other processes beyond methane oxidation and that the hydrocarbon composition of the seep fluids may be a critical factor structuring the seafloor microbial community composition and function.Oil and gas percolate profusely through the sediments of the Gulf of Mexico, leading to numerous seeps at the seafloor, where complex microbial, and sometimes animal communities flourish. Sediments from three areas (two cold seeps with contrasting hydrocarbon composition and a site outside any area of active seepage) of the Gulf of Mexico were investigated and compared. Consistent with the existence of a seep microbiome, a distinct microbial community was observed in seep areas compared to sediment from outside areas of active seepage. The microbial community from sediments without any influence from hydrocarbon seepage was characterized by  These seeps sustain conspicuous microbial communities identifiable on the seafloor by the presence of white or colored microbial mats, aggregates of vesicomyid clams and/or tubeworms depending on the fluid flow rate and the hydrogen sulfide concentrations14. Numerous studies have focused on the microbial communities present in these environments and a large diversity of microorganisms dependent on methane and sulfur cycling has been observed15.Gulf of Mexico sediments harbor numerous shallow sources of methane and other hydrocarbons. These include methane seeps15. Although the mechanism of anaerobic methane oxidation (AOM) has been investigated since 200016, and seems to involve a reverse methanogenesis pathway19, the exact process remains unclear and different variants may occur depending on the species involved20. Indeed, different groups of ANME 23, closely affiliated to the Methanosarcinales and Methanomicrobiales, have been identified. In addition, sulfate-reducing Deltaproteobacteria  have been identified in syntrophic methane-oxidizing consortia in these and other environments28. However ANME-1 and ANME-3 have been observed as monospecific aggregates questioning whether the association between ANME archaea and sulfate-reducing deltaproteobacteria is obligate29. AOM is widely considered to be the main process occurring in cold seep sediments due to the ubiquity of ANME sequences in 16S rRNA gene surveys and the microscopic detection of the striking aggregates of ANME and SRB30. Consequently, the intriguing microbial consortia involved in AOM have been the focus of the majority of the microbial investigations of marine seep sediments.Particular attention has been devoted to consortia of archaeal anaerobic methanotrophs (ANME) and sulfate-reducing bacteria (SRB) that couple the anaerobic oxidation of methane to sulfate reduction14. The relative proportion of non-ANME/SRB lineages has been shown to increase with the distance from the seep source and the proximity of faunal assemblages14, suggesting the existence of ecological niches other than AOM in these seep sediments. Furthermore, the presence of oil and higher hydrocarbons in the percolating fluid is also suspected to play an important role in shaping the microbial communities present, by selecting for non-methane-hydrocarbon degraders, methanogens and acetogens11. Indeed, activity measurements in Gulf of Mexico seeps suggest that significant rates of sulfate reduction are supported by non-methane hydrocarbon degradation and that an uncultured group of Deltaproteobacteria may be responsible for degrading higher molecular weight hydrocarbons11. This has been confirmed by the detection of genes involved in anaerobic degradation of alkanes in sulfate-reducing bacteria31, successful cultivation of hydrocarbon degrading sulfate reducers from seep sediments33, and stable isotope probing experiments34. Furthermore, single cell genomics and assembly of genomic bins from large metagenomic datasets, has highlighted the metabolic potential of some of the microbial “dark matter” from marine sediments. These approaches have revealed the capacity for organic matter degradation in Marine Benthic Group D archaea, Bathyarchaeota, Atribacteria and Dehaloccocoidia, and potential for methanogenesis in Bathyarchaeota39. However genome-centric metagenomics only provide single pieces of the puzzle and the overall picture of microbial community function in marine deep-sea ecosystems remains fragmented.Other members of the microbial communities in these environments have been somewhat overlooked even though numerous uncultured archaeal and bacterial lineages co-occur with the ANME/SRB consortia40. Two cold seep sites with different hydrocarbon compositions, potentially reflecting methane and thermogenic gas hydrates and samples from outside any area of active seepage were compared. Common and unique metabolic features of the microbial communities were identified and complemented with geochemical data to provide new insights into the different microbial processes in marine hydrocarbon seep environments.To better understand the community structure and different functions present in marine seep ecosystems, a comparative multigenic and metagenome sequencing approach was used on samples from three area of the Mississippi Canyon in the Gulf of Mexico. The Mississippi Canyon harbors numerous sea floor mounds with shallow methane and thermogenic gas hydrates40. Methane was the only hydrocarbon detected in sediments from Site 2 (PC9 and PC10).Push core sediment samples were collected in October 2015 at three different locations in the Mississippi Canyon of the Gulf of Mexico. Site 1 (PC5 and PC6) and Site 2 (PC9 and PC10), located 70 meters appart, presented unambiguous geochemical evidence of seepage and white mat-like traces at the sediment surface. Two push cores (PC11 and PC12) were also taken 200 meters away, at locations remote from any area of visible active seepage as ‘background’ reference samples. Different pore water compositions were observed in these sediment cores , total 16S rRNA gene abundance was up to 10 times higher than in the non-seep sediments with an average of 9.7 ± 2.1 × 108 16S rRNA genes.g−1 in the 3–4 cmbsf sediment layer of PC5 and throughout PC6. Microbial abundance increased in the bottom (10–12 cmbsf) sediment layer of PC5 which contained 3.13 × 109 16S rRNA genes.g−1. In both PC5 and PC6 sediment cores, bacteria were predominant in the 3–4 cmbsf sediment layer  whereas a similar amount of Bacteria and Archaea was detected in the 10–12 cmbsf sediments layers. In Site 2 sediments (PC9 and PC10), microbial abundances were heterogeneous probably due to extremely localized seepage. In PC9 sediment core, 3.6 × 108 16S rRNA genes.g−1 were quantified and bacterial genes represented 80% of the total 16S rRNA gene abundance. By contrast, more than 1.3 × 109 16S rRNA genes.g−1 were detected throughout the PC10 sediment core with the archaeal genes representing 52% and 57% of the prokaryote total 16S rRNA genes at 3–4 cmbsf and 10–12 cmbsf respectively.Microbial abundances, estimated using quantitative PCR, were different in the Gulf of Mexico sediments from seep and non-seep sites Table . In the 4 reads per sample. Both archaeal and bacterial sequence analyses indicated a similar clustering of the samples , with the exception of the PC9 sediment core that exhibited a geochemical and microbial abundance profile similar to sediments from outside the seep area  and explained up to 19% of the dissimilarity between the seep samples and those from outside the seep areas. The archaeal community of the seep samples was characterized by dominance of members of Marine Benthic Group D  and the presence of ANME-1 at 10–12 cmbsf  , and the SEEP SRB1b affiliated to the Desulfosarcina/Desulfococcus group (Desulfobacteraceae)  related sequences were also identified only in the seep sediment samples. JS1 sequences represented a large proportion of the reads at 10–12 cmbsf . Additionally, differences between seep samples (PC10 vs PC5 and PC6) were observed. A larger proportion of Chloroflexi  was detected in Site 1 (PC5 and PC6) whereas larger proportions of SEEP SRB1b and SEEP SRB2 related sequences  were observed in PC10  Fig. . Bacteriae) Fig. . CandidaC10 Fig. .Figure 1mcrA) and dissimilatory sulfite reductase (dsrAB) genes  were detected in PC10 sediment samples whereas a larger proportions of reads from the Syntrophobacteraceae (28 ± 6% of the dsrAB sequences) were identified in samples from outside seep areas -related sequences (13 ± 9% of the dsrAB sequences) were also detected at higher relative abundance in seep samples , which lack any known cultured representative . Despite numerous attempts using MetabatArchaea , archaeal diversity obtained by shotgun metagenomics presented a different picture, notably for the PC10 sample . In the metagenome data ANME-1 16S rRNA genes represented up to 80% and 14% of the archaeal 16S rRNA reads in PC10 and PC5 sediments respectively  affiliated with ely Fig. . This disis Fig. , represenarGH, hao), chlorinated compound degradation , breakdown of sulfonated heteropolysaccharides  and degradation of other polysaccharides and sugars . Based on the taxonomic signature of these genes, nitrogen cycle genes (25% of narG reads and 95% of hao reads) were mainly affiliated with marine Planctomycetes  , whereas the genetic potential for the initial breakdown of sulfonated compounds and polysaccharides were identified in members of the Planctomycetes (31%), Bacteroidetes (31%) and Firmicutes (13%)  and anaerobic methane oxidation pathways  were detected in the sediment core from the methane-dominated site 2 seep sample PC10 , ANME-1 (30%) and Desulfobacteraceae (24%), whereas genes involved in AOM were mainly affiliated with the ANME-1 lineage (47%)  as well as genes from the sulfate reduction pathway  were over represented in sediment samples from seep site 1 (PC5) which contained more complex hydrocarbons  and Firmicutes (17%). Taxonomic affiliations of hydrocarbon degradation genes indicated potential for toluene degradation (bssA) in Deltaproteobacteria (Desulfobacteraceae (45%) and Syntrophobacteraceae (47%)), cyclohexane degradation in Actinobacteria (23%), Firmicutes (34%), Alpha- (14%) and Deltaproteobacteria (Desulfobacteraceae 22%) and polycyclic aromatic hydrocarbons degradation (bnsE) in Deltaproteobacteria (Desulfobacteraceae 27%) and Chloroflexi (72%)  and the accumulation, burial and maturation of this organic matter over geological timescales has generated widespread natural hydrocarbon seep environments such as cold seeps, gas hydrates, brines and asphalt volcanoes44. Geochemical analysis of the sampled seep areas indicated that the seep fluids were distinct with respect to their composition. In Site 1 (PC5 and PC6) methane and more complex hydrocarbons  were detected in the sediments, as previously observed in thermogenic gas hydrates and seep of the Mississippi Canyon40 whereas methane was the only hydrocarbon identified in Site 2 sediments, which is consistent with methane hydrates and seep also observed in the Canyon40. Therefore, in this study, microbial community composition and metabolic potential among three contrasting environments in the Gulf of Mexico were compared: a methane seep (PC9 and PC10), a hydrocarbon seep (PC5 and PC6) and sediments from outside the seep areas (PC11 and PC12). Potential for CO2 fixation via the reverse tricarboxylic acid cycle and the Wood Ljungdahl pathway as well as the potential for fermentation (formate and acetate) were highly represented in all samples  indicated a specific community composition with an overrepresentation of the Planctomycetes, Marine Group I and Marine Benthic Group E archaea as previously observed in seafloor sediments from the Guaymas basin, with minimal influence of hydrocarbon seepage22. Comparative analysis of the metagenomes indicated an overrepresentation of several metabolic genes, notably affiliated to the Planctomycetes and Bacteroidetes, involved in the degradation of various glycans as well as chlorinated and sulfonated compounds, as previously detected by fosmids sequencing45. In marine environments, chlorinated compounds such as chloromethane and other chloroalkanes are derived from phytoplankton and algae , while sulfonated compounds have been found as a major constituent of humic acids and algae (agar and fucans)46. Likewise glycans are abundant polysaccharides in marine invertebrate tissues and algae45. The communities in the non-seep site were therefore likely adapted to grow on detrital organic matter delivered to the sediment as marine snow and decaying organic matter particles. As has been previously observed22 it was not possible to extract RNA of sufficient quality or quantity for subsequent analysis, from the non-seep sediments (data not shown). This observation, as well as the frequent detection of Marine Group I archaea and Planctomycetes in the water column47 and/or attached to macroscopic detrital aggregates48, suggest that these microbes may predominantly have a pelagic lifestyle, degrading the heteropolysaccharides of the marine snow, and primarily represent organisms deposited in the sediment with water column-derived organic matter and have limited activity in situ in these marine sediments. Nitrate reduction potential, inferred from narG genes was detected in members of Planctomycetes, candidate division OP3, Euryarchaeota and Proteobacteria. However, metabolic activity of these bacteria and archaea might be limited in marine sediments by the low availability of nitrate  and nitrite. Sulfate-reducers were detected by targeted amplifications (16S rRNA and dsrAB genes) but were represented by a minority of reads in shotgun metagenome data, and sulfate was only depleted in the upper sediment layer. If sulfate reduction is not balanced by the considerable potential for sulfate generation from sulfonated organic compounds (a high proportion of sulfatase genes were identified in the metagenome data) or sulfide oxidation by Alpha- and Gammaproteobacteria detected in the sediments, these results suggest that the activity of the sulfate reducers detected in these sediments, is limited. Thus it is likely that the ecosystem in non-seep sediments is driven largely by utilization of water column-derived organic carbon, and that this is insufficient to support complete reduction of seawater derived sulfate. The corollary of this is that sulfate-reduction is not supported by upwelling sources of organic carbon, consistent with the lack of evidence of hydrocarbon seepage in these sediments. The detection of sulfate at seawater levels at 10–12 cmbsf in these sediments suggests that the surface sediment sulfate-reducing communities may to some extend be fed by seawater advection into the sediments but that even then the organic matter delivered to the sediments from the water column, is insufficient to support complete sulfate reduction.Although non-seep areas represent the vast majority of all marine sediments, they remain relatively poorly investigated compared to seep sediments. Methane concentration was extremely low (<0.05 µM) in the non-seep sediments analyzed. Moreover, no 49. Previous global analysis of 16S rRNA genes recovered from seafloor ecosystems identified a specific microbiome in cold seeps environments30, however distinctions between methane and oil seep microbial community and metabolic profiles have received much less attention11. Multigenic  and metagenome sequencing highlighted a qualitatively similar microbial community composition between the methane and oil seeps. Archaeal  and bacterial  lineages were present as had previously been noted in Gulf of Mexico cold seep sediments11, supporting the concept of a characteristic seep microbiome30. However, shotgun metagenomic sequencing highlighted different relative proportions of these lineages depending on the nature of the seep fluids. A large proportion of ANME-1  was detected in methane seep sediments, as previously observed11. In line with this observation, the complete anaerobic methane oxidation pathway (reverse methanogenesis without the mer gene), previously identified in ANME-150, was overrepresented in metagenomes from these sediments  and SEEP SRB2 , consistent with findings in other anoxic cold seep sediments30. However, these SRB are not known as direct syntrophic partners for the anaerobic oxidation of methane by ANME-1 in cold sediments. Furthermore, the representation of ANME-1 16S rRNA genes in the metagenome dataset (14 and 80% in PC5 and PC10 respectively) was disproportionate with any potential syntrophic bacteria (0.8% of 16S rRNA reads affiliated to SEEP SRB1a or Desulfobulbus). Therefore these results could suggest that ANME-1 in our methane seep sediments are likely to oxidize methane by the reverse methanogenesis pathway in a bacterial syntrophy-independent process, as previously proposed51. The particular abundance of ANME-1 in the methane rich seeps would suggest that anaerobic methane oxidation was the predominant microbial process in these seeps.Cold seeps are defined by the upward advection of methane and other hydrocarbons from the subsurface seabed to the seafloorDesulfobulbaceae, Syntrophobacteraceae, Desulfatiglans and Chloroflexi were identified in the hydrocarbon seep sediments, where residual oil and aromatic hydrocarbons were observed. Gene markers for anaerobic degradation of aromatic hydrocarbons  were overrepresented in these sediments, suggesting that in the presence of other hydrocarbons along with methane, the metabolic potential of the microbial community may be based on breakdown of more complex hydrocarbons rather than on methane oxidation. The hydrocarbon degradation genes were mainly affiliated to members of the Desulfobacteraceae and Syntrophobacteraceae, as previously observed by a masD/assA/bssA amplicon survey of cold seep sediments31. 16S rRNA genes of from Desufatiglans, which are related to cultivated species known to degrade alkyl-substitued and unsubsituted aromatic hydrocarbons32, and other sub-groups of the Desulfobacteraceae (SEEP SRB1d) were overrepresented in sediment samples affected by seepage of hydrocarbons other than methane. It is therefore likely that these uncultured Deltaproteobacteria may degrade aromatic hydrocarbons, as previously suggested34 and contribute to the UCM, characteristic of biodegraded oil, detected in these sediments. An overrepresentation of sulfate reduction genes  from Desulfobacteraceae, and pore water sulfate depletion were observed in these samples, as in other oily seeps52. Therefore, energy for hydrocarbon degradation in marine seepages is likely provided by sulfate reduction, supporting previous activity measurements in Gulf of Mexico seep sediments that suggested sulfate reduction activity was fuelled by non-methane hydrocarbons52. An overrepresentation of sulfide oxidation genes (Sox), from known mat-forming Gamma- and Epsilonproteobacteria (Beggiatoa and Sulfurovum spp.) was also apparent in the metagenome data from these sediments -affiliated genes for aromatic hydrocarbon degradation and sulfate reduction were also detected in the metagenomic dataset, though they represented a minority in 16S rRNA and dsrAB gene surveys. Members of the Firmicutes phylum such as Clostridia and Desulfotomaculum are known oil degraders33, therefore they may also contribute to the degradation of hydrocarbon in these seep sediments. Furthermore, the potential for polycyclic aromatic hydrocarbon  degradation (bnsE) was also identified in members of the Chloroflexi phylum. Chloroflexi have been frequently detected in marine sediments53 and have been implicated in a number of metabolic processes  depending on the particular Chloroflexi lineage. BLAST analysis of full length 16S rRNA sequences reconstructed from metagenomic data (data not shown) indicated that Chloroflexi sequences were affiliated to Anaerolinaceae and closely related to Chloroflexi sequences previously amplified from mud volcanoes56. Anaerolinaceae have been identified as abundant components of anaerobic hydrocarbon-degrading enrichments59. Together, this suggests that considerable metabolic variability occurs within the Chloroflexi phylum and some cold seep-associated Anaerolinaceae lineages may be hitherto unknown aromatic hydrocarbon degraders. Consistent with the UCM detected by GC analysis, the metabolic potential of the resident microbial community appears to be adapted to the composition of the seepage with different catabolic pathways for the degradation of aromatic hydrocarbons being present.By contrast, a larger proportion of Due to the prevalence of seep environments, Gulf of Mexico sediments represent a unique opportunity to observe the influence of seep hydrocarbon composition on microbial community structure and function. Although, the microbial community analysis presented here supports the concept of a characteristic seep microbiome, our sampling strategy and comparative metagenomic approaches indicated different microbial communities and metabolic traits depending on the composition of the seep fluids. Therefore, even if these metabolic capacities remain to be confirmed by larger scale investigation, activity measurements or transcriptomic analyses, they indicate that seafloor ecosystems can be sustained by different microbial activities depending on percolating fluid composition.Sediments push core samples were collected in October 2015 at three different locations in the Mississippi Canyon of the Gulf of Mexico using an ROV. Duplicate push cores were taken approximatively 20 cm apart at each sampling site. Site 1 (PC5 and 6) at 1055 meters water depth, was covered by a thin microbial mat and had unambiguous geochemical evidence of seepage. Site 2 (PC9 and 10), located approximatively 70 meters away, at 1129 meters water depth had scattered white mat-like traces at the sediment surface. Site 2 was identified by seafloor acoustic reflectivity but no fluid emission were observed on the seafloor. Finally two sediment push-cores (PC11 and 12) were taken 200 meters away at locations remote from any visible active seepage area (1128 meters water depth) as ‘background’ reference samples.60 and re-suspended in 200 µl of DNA-, RNA-, RNase- free water. Extracted nucleic acids were purified using a Wizard DNA clean-up kit  and were stored at −20 °C until further analysis. During nucleic acid extraction, visible oil was present in the 11–12 cmbsf sample from PC5 and PC6.Sediment cores were recovered to the research vessel and immediately sectioned aseptically. Sediment layers from 1–3, 5–10 and 12–15 centimeters below the seafloor (cmbsf) were subsampled for oil and gas analysis whereas samples from 3–4 and 10–12 cmbsf were subsampled for microbiology and porewater analysis. Samples for microbiology were immediately stored in 50 ml sterile tubes at −80 °C until nucleic acid extractions could be performed. Nucleic acids (DNA and RNA) were extracted from 5 grams of sediments as previously describedMethane concentration and hydrocarbon composition in sediment samples were determined by gas chromatography and GC MSxMS . After nucleic acid extraction, pore water was collected from all the samples following centrifugation. Anions were quantified using a Dionex ICS-2000 ion chromatograph with suppressed conductivity detection. Anions were separated on an AS11 column using a KOH gradient.Bacteria and Archaea in the Gulf of Mexico sediments was estimated using quantitative (q)PCR targeting 16S rRNA genes with primers Bact1369f/Bact1492r and Arc787f/Arc1059r respectively (Supplementary Table Desulfobulbus propionicus (ATCC 33891) and Methanococcoides methylutens (ATCC 33938). The R2 of standard curves obtained by qPCR were above 0.99 and PCR efficiencies above 94%. qPCR results were expressed in terms of 16S rRNA gene copies per gram of sediments.Abundance of 14. Additionally, sulfate reducers and methanogens were investigated by amplification and sequencing of the dissimilatory (bi)sulfite reductase (dsrAB) gene using primers DSR1728/DSR4R (380 bp product) and the methyl coenzyme M reductase (mcrA) gene using primers MLf/MLr (550 bp product)  and extension for 30 s at 72 °C followed by a final extension step at 72 °C for 5 min. Replicate amplicons were pooled and purified from agarose gels using a Qiagen MinElute Gel purification kit . PCR products were indexed using a Nextera XT kit  according to manufacturer’s recommendations. Indexed amplicons were quantified using a Qubit dsDNA HS Assay Kit  and diluted to give an equimolar mix of products at a final concentration of 4 nM for Miseq library preparation. DNA libraries were diluted to 4 pM then pair-end Illumina MiSeq sequencing was performed using an Illumina Miseq v3 kit , as recommended by the manufacturer. This generated 2 × 300 bp pair-end sequences. Datasets were split into reads from individual indexed amplicons in silico using Miseq reporter software. Reads were assembled into single pair-end sequences which were curated using Qiime61. OTU picking was carried out using the de novo OTU picking option. Sequences with low quality scores or flagged as chimeras using UChime were removed. Alignment and determination of the taxonomic affiliation of the reads were carried out using BLAST against Silva release 11962, dsrAB63 and mcrA64 sequences databases as references. Raw sequences were deposited in the NCBI public database under Bioproject PRJNA385797.Microbial community composition of the Gulf of Mexico sediments was determined by high throughput 16S rRNA gene sequencing. The V4-V5 region of bacterial 16S rRNA genes (420 bp product) was amplified using primers S-D-Bact-0516-a-S-18/S-D-Bact-0907-a-A-20 whereas the V1-V2-V3 region of archaeal 16S rRNA genes (530 bp product) was amplified using primers S-D-Arch-0008-b-S-18/S-D-Arch-0519-a-A-19 , using default settings. Assembly was performed from paired-end joined reads and unpaired reads passing quality filtering using MEGAHIT66, using default settings. After assembly, all reads which passed quality filtering where mapped back to the assembled contigs to detect reads which were not included in the assembly, using BBMap67, with default settings. Reads which were not mapped to the assembly were concatenated with assembled contigs into a single fasta file for upload to the IMG/M analysis pipeline68 for gene calling and functional annotation. For each sample, coverage and mapping data file (BAM file) was also uploaded to IMG/M pipeline to preserve relative abundance of the genes. Metagenomes were normalized by rarefaction for sample comparison. Metagenome data are available in IMG/M under the following accession numbers: 3300008340, 3300008410, 3300008416, 3300008417, 3300008465 and 3300008468 . Reconstruction of full length 16S rRNA genes was done using EMIRGE70.Metagenomes for 6 sediment samples were constructed from 1 ng of metagenomic DNA using a Nextera XT Library Kit  according to manufacturer’s recommendations. Tagmentation and indexing were checked using a High Sensitivity DNA chip on an Agilent Bioanalyzer 2100 . Metagenomes were normalized and diluted to 4 nM based on the average DNA fragment size and concentration determined from the Bioanalyzer analysis. Two metagenomes were pooled in equimolar quantities for each sequencing run. Metagenome libraries were diluted to 14.3 pM and sequenced using an Illumina Miseq V3 kit . Barcode and adapter sequences were removed from the metagenome data on-instrument using Illumina’s MiSeq Reporter software and the sequence data were exported as fastq files. Datasets were quality filtered using Trimmomatic71 according to the guide for statistical analysis in microbial ecology72. For amplicon dataset, distance matrices between samples were determined on 97% operational taxonomic units using the Bray-Curtis dissimilarity index. Differences in community composition and metabolic potential (Number of identified Kegg orthologies) were tested using One-way NPMANOVA and SIMPER tests based on Bray-Curtis dissimilarity measures.Statistical analyses  were performed using PAST softwareSupplementary Material


BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. This study explored the utility of nanopore sequencing to examine whole BRCA1 mRNA transcripts and to provide accurate categorisation of in-frame and out-of-frame splicing events.Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 transcripts from a previously studied control lymphoblastoid cell line were assessed using MinION nanopore sequencing of long-range reverse transcriptase-PCR amplicons.The exon structure of BRCA1 isoforms, including 18 novel isoforms which showed skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as Δ and Δ21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Fourteen novel isoforms were formed entirely from a combination of previously identified alternative splice junctions, suggesting that the total number of BRCA1 isoforms might be lower than the number of splicing events reported previously.Our study identified and characterised 32 complete BRCA1 transcript structure that has not been described previously. This finding has key implications for predicting the translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full-length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.Our results highlight complexity in The online version of this article (doi:10.1186/s13058-017-0919-1) contains supplementary material, which is available to authorized users. BRCA1 is typically performed for individuals from suspected high-risk breast (and ovarian) cancer families to identify the genetic cause for their disease. However, an important practical issue associated with genetic testing is the identification of rare sequence variants with unknown clinical significance. Interpreting the clinical meaning of unclassified variants is a key challenge facing the future of genomic-based health initiatives .fasta > [alignmentFile].gff3 as gmap -g BRCARD1_geneseq.fasta -f 2 -n 0 -t 16 all.fasta > all.gff3. The output file was manipulated using an in-house R script to select for reads spanning the full length of the gene by ensuring it contained sequences from the first and last exons. The full-length reads were then grouped into isoforms based on their composition of exons and introns.The Oxford Nanopore MinION Genomic DNA Sequencing Kit (R9 flow cell chemistry) was used to prepare the DNA libraries according to the manufacturer’s instructions. Briefly, PCR products were purified and then quantified using the Qubit® Fluorometer (ThermoFisher Scientific) followed by end repair and dA tailing using the NEBNext Ultratm End Repair/dA-Tailing Module (New England BioLabs Inc.). The DNA library entailing adaptor ligation and purification of double-stranded DNA with hairpin adaptor was prepared using the Nanopore Sequencing Kit SQK-NSK007 (R9 version). The MinKNOW program was used for running MinION for 48 hours. Additional sample mix was applied to the flow cell when the number of pores being used was less than 20, until the entire sample was used. The raw electrical signal was uploaded to Metrichor (version 1.107), using the 2D Basecalling RNN for SQK-NSK007 which returned basecall data in MinION fast5 file format. The Poretools package was used to extract fasta files for high-quality 2D reads . SequencBRCA1 splicing changes in the clinical setting for variant evaluation [BRCA1 transcripts, we carried out long-range RT-PCR for BRCA1 transcripts using primers targeting the 3′ end of exon 1 and the 5′ end of exon 24 to generate a 5.8-kb amplicon  were observed in 28/47 PCR assays. A preliminary assessment of fragments from 10 PCR assays by Sanger sequencing confirmed BRCA1 identity  have been widely used as a cell model for analysing aluation . For thialuation . To obtaQ-value of 9 were obtained, of which 95% aligned to the target sequence. Reads failing the 2D filter were possibly due to shearing of DNA by pipetting or incomplete ligation of hairpin adaptors, thus resulting in shorter reads lacking a complementary strand signal. Our library contained pooled PCR products obtained from cDNA generated from BRCA1 and BARD1 (part of a separate study) transcripts. Nanopore sequencing resulted in 10.7% of passed 2D reads aligning to BRCA1 and a higher proportion (84.4%) of passed 2D reads aligned to a shorter (≤2 kb) BARD1 cDNA sequence. A summary of the mapping and filtering process is shown in Additional file BRCA1), a total of 177 reads were identified that revealed the complete structure of different BRCA1 isoforms.A total of 117,504 reads were obtained from MinION sequencing run from two DNA libraries  derived from pooled RT-PCR products on a R9 flow cell over a period of 48 hours. Approximately 21% of 2D reads containing both template and complement strand with a BRCA1 isoforms   [n ≤ 3) of reads, and this would be particularly important for potential splice shift events not identified previously.Together, these results suggest complexity in transcript structure that has not been described previously for N (>10%) , a higheBRCA1 transcripts lead to abnormal and potentially deleterious proteins requires knowledge about the structure of the coding isoforms. Sixteen of the 20 novel isoforms lacked sequences coding for the RING and/or BCRT domains, which have been previously shown to harbour amino acid residues of clinical importance [Determining whether portance , althougWe detected the predominant splicing events, Δ11q and Δ9,10, which individually would be predicted to generate modified in-frame transcripts that are not considered deleterious based on protein coding. However, our study has shown these splicing events can co-occur with skipping and insertion events causing out-of-frame coding in these isoforms  have been found previously [BRCA1 and has potential implications for interpreting the biological and clinical significance of spliceogenic variants.Laboratory assays assessing the effect of DNA sequence variants on evidence , 6. Howenscripts . For theeviously ; howeverBRCA1 mRNA splicing events occur concurrently, our non-quantitative study was unable to establish the likelihood of such events being expressed in the same transcript. If future studies show that many of the detected BRCA1 exon skipping events exclusively co-occur, then the total number of isoforms might be lower than the number of splicing events reported previously by Colombo et al. [BRCA1. Further studies will therefore be required to measure the cellular levels of sequenced isoforms, and to investigate the possibility of further transcript complexity due to splice site shifts involving a small number of nucleotides. Such studies will require improved data analysis tools to take full advantage of the sequencing information generated, although we note that this field continues to advance as evidenced in a recent report by Hu et al. [While our work highlights that many o et al. . Such a u et al. .BRCA1 transcripts. Together, our results highlight the potential of this technology to overcome limitations of traditional PCR-based techniques.The ENIGMA Splicing Working Group previously led a multicentre study which highlighted methodological issues that confounded the interpretation of splicing results . A majorBRCA1 transcript structure that has not been described by previously reported studies. Assessment of whole BRCA1 transcripts is now possible and has key implications for predicting translation frame of splicing transcripts, which is important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full-length BRCA1 transcript levels, to detect additional novel isoforms involving small nucleotide shifts and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants. Furthermore, the application of MinION or similar platforms may be extended to other disease-associated genes to establish whether they display similar complex splicing patterns to BRCA1.Our study highlights complexity in


Growth factor profiles could be influenced by the utilization of exogenous insulin. The data presented shows the relationship between pre-existing use of injectable insulin in women diagnosed with breast cancer and type 2 diabetes mellitus, the growth factor profiles at the time of breast cancer diagnosis, and subsequent cancer outcomes. A Pearson correlation analysis evaluating the relationship between growth factors stratified by of insulin use and controls is also provided. Specifications TableValue of the data•This dataset represents the observed relationship between injectable insulin use, circulating growth factors at breast cancer diagnosis and outcomes.•Reported data has the potential to guide future research evaluating insulin-induced growth factor modulation in breast cancer.•Our observations may assist future studies in evaluating the relationship between insulin safety and effectiveness and growth factors production in cancer.1Reported data represents the observed association between use of injectable insulin preceding breast cancer and the growth factor profiles at the time of cancer diagnosis in women with diabetes mellitus . Data in2Evaluation of growth factor profile association with injectable insulin use and BC outcomes was carried out under two protocols approved by both Roswell Park Cancer Institute (EDR154409 and NHR009010) and the State University of New York at Buffalo (PHP0840409E). Demographic and clinical patient information was linked with cancer outcomes and growth factor profiles of corresponding plasma specimen harvested at BC diagnosis and banked in the Roswell Park Cancer Institute Data Bank and Bio-Repository.2.1All incident breast cancer cases diagnosed at Roswell Park Cancer Institute (01/01/2003−12/31/2009) were considered for inclusion (n=2194). Medical and pharmacotherapy history were used to determine the baseline presence of diabetes.2.2All adult women with pre-existing diabetes at breast cancer diagnosis having available banked treatment-naïve plasma specimens  in the Institute׳s Data Bank and Bio-Repository were included.Subjects were excluded if they had prior cancer history or unclear date of diagnosis, incomplete clinical records, type 1 or unclear diabetes status. For a specific breakdown of excluded subjects, please see the original research article by Wintrob et al. A total of 97 female subjects with breast cancer and baseline diabetes mellitus were eligible for inclusion in this analysis.2.3Each of the 97 adult female subjects with breast cancer and diabetes mellitus (defined as “cases”) was matched with two other female subjects diagnosed with breast cancer, but without baseline diabetes mellitus (defined as “controls”). The following matching criteria were used: age at diagnosis, body mass index category, ethnicity, menopausal status and tumor stage (as per the American Joint Committee on Cancer). Some matching limitations applied 2.4Clinical and treatment history was documented as previously described 2.5All the plasma specimens retrieved from long-term storage were individually aliquoted in color coded vials labeled with unique, subject specific barcodes. Overall duration of freezing time was accounted for all matched controls ensuring that the case and matched control specimens had similar overall storage conditions. Only two instances of freeze-thaw were allowed between biobank retrieval and biomarker analyses: aliquoting procedure step and actual assay.2.6A total of 6 biomarkers  were quantified according to the manufacturer protocol. The following Luminex® biomarker panels were utilized in this study: TGFB-64K (tumor growth factor-β), HCYTOMAG-60K (platelet-derived growth factor BB), and HAGP1MAG-12K  produced by Millipore Corporation, Billerica, MA. C-peptide determinations were done according to the manufacturer protocol as previously reported 2.7Biomarker cut-point optimization was performed for each analyzed biomarker. Biomarker levels constituted the continuous independent variable that was subdivided into two groups that optimized the log rank test among all possible cut-point selections yielding a minimum of 10 patients in any resulting group. Quartiles were also constructed. The resultant biomarker categories were then tested for association with type 2 diabetes mellitus therapy and controls by Fisher׳s exact test. The continuous biomarker levels were also tested for association with diabetes therapy and controls across groups by the Kruskal–Wallis test and pairwise by the Wilcoxon rank sum. Multivariate adjustments were performed accounting for age, tumor stage, body mass index, estrogen receptor status, and cumulative comorbidity. The biomarker analysis was performed using R Version 2.15.3. Please see the original article for an illustration of the analysis workflow Correlations between biomarkers stratified by type 2 diabetes mellitus pharmacotherapy and controls were assessed by the Pearson method. Correlation models were constructed both with and without adjustment for age, body mass index, and the combined comorbidity index. Correlation analyses were performed using SAS Version 9.4.Wadsworth Foundation Peter Rowley Breast Cancer Grant awarded to A.C.C. .This research was funded by the following grant awards:


Basal ganglia injury at term remains a major cause of disability, such as cerebral palsy. In this study we tested the hypotheses that blockade of astrocytic connexin hemichannels with a mimetic peptide would improve survival of striatal phenotypic neurons after global cerebral ischaemia in term-equivalent fetal sheep, and that neuronal survival would be associated with electrophysiological recovery. Fetal sheep (0.85 gestation) were randomly assigned to receive a short or long (1 or 25 h) intracerebroventricular infusion of a mimetic peptide or vehicle, starting 90 minutes after 30 minutes of cerebral ischaemia. Sheep were killed 7 days after ischaemia. Cerebral ischaemia was associated with reduced numbers of calbindin-28k, calretinin, parvalbumin and GAD positive striatal neurons  but not ChAT or nNOS positive neurons. Short infusion of peptide (n = 6) did not significantly improve survival of any striatal phenotype. Long infusion of peptide (n = 6) was associated with increased survival of calbindin-28k, calretinin, parvalbumin and GAD positive neurons (P < 0.05 vs. ischaemia + vehicle). Neurophysiological recovery was associated with improved survival of calbindin-28k, calretinin and parvalbumin positive striatal neurons . In conclusion, connexin hemichannel blockade after cerebral ischaemia in term-equivalent fetal sheep improves survival of striatal GABA-ergic neurons. Basal ganglia injury occurs in approximately 25% of cases of encephalopathy at term and is strongly associated with poor neurophysiological outcomes, such as increased severity of neonatal seizures3, and discontinuity of electroencephalographic activity5. Although therapeutic hypothermia reduced the incidence and severity of injury and improved survival without disability after moderate to severe encephalopathy6, nearly half of treated infants still die or survive with disability. Therefore there is a crucial need to identify other therapies to reduce the incidence and severity of hypoxic ischaemic encephalopathy (HIE) at term.Basal ganglia injury after hypoxia ischaemia (HI) at term remains a major contributor to poor neurodevelopmental outcomes such as cerebral palsy, learning disability and epilepsy9. Recent work has shown that opening of astrocytic connexin hemichannels (Connexin 43 (Cx43)), the constituents of gap junctions, during ischaemia is implicated in the pathogenesis of HIE12. Post-insult blockade of Cx43 hemichannels using a mimetic peptide (Peptide 513), targeting the extracellular loop of Cx43 was associated with improved recovery of EEG power, reduced electrographic seizures and improved survival of oligodendrocytes after global cerebral ischaemia, but only an intermediate improvement in survival of cortical neurons11. It remains unclear whether the apparent neuroprotective effect of Cx43 hemichannel blockade is selective for particular cell types. To examine this further, we tested the effect of Cx43 hemichannel blockade on the striatum, a major nucleus within the basal ganglia that is highly susceptible to cerebral ischaemia and where neuronal phenotypes have been well-defined14.Consistent with clinical HIE, global cerebral ischaemia in term-equivalent fetal sheep, induced by complete bilateral occlusion of the carotid arteries, is associated with profound loss of EEG activity, electrographic seizures and basal ganglia injury16.The aim of this study was to test the hypothesis that blockade of Cx43 hemichannels with a mimetic peptide would selectively improve survival of striatal phenotypic neurons after global cerebral ischaemia in term-equivalent fetal sheep at 0.85 of gestation. Further, we tested the secondary hypothesis that improved survival of GABA-ergic striatal neurons would be associated with reduced seizure burden, faster rate of return to sleep state cycling and recovery of EEG activity. At this gestational age, brain maturation is equivalent to the human infant at term10. During ischaemia, there was rapid suppression of EEG power that did not differ between groups  and parvalbumin positive striatal neurons . The day of return of sleep state cycling was inversely related to improved survival of calbindin-28k  and parvalbumin positive striatal neurons . Recovery of EEG power after 7 days was positively associated with improved survival of calbindin-28k . There was no significant correlation between recovery of EEG power and survival of calretinin, nNOS or ChAT positive striatal neurons.Electrographic seizure burden was inversely related to improved survival of calbindin-28k 23, and excessive cell swelling and rupture25. We have previously shown that Cx43 hemichannel blockade started 90 minutes after cerebral ischaemia or asphyxia and continued for at least 25 hours markedly reduced status epilepticus, improved recovery of brain activity and reduced oligodendrocyte loss12. While there are multiple connexin hemichannel isoforms, there is strong evidence that Cx43 is the predominant astrocytic hemichannel isoform involved in the pathogenesis of neuronal injury and is one of the key therapeutic targets for research into neuroprotection27. Additionally, it has recently been reported that extracellular loop homology is distinct between Cx43 (Peptide 5) and other connexin isoforms at amino-acid position 5 (leucine)28. This is a highly sensitive position where a single amino-acid substitution causes complete loss of function28. This suggests that the Cx43 mimetic peptide is isoform specific and is unlikely to be effective against other connexin isoforms found on neuronal, vascular or glial cells.Basal ganglia injury at term is strongly associated with poor neurodevelopmental outcomes that include learning disability, epilepsy and cerebral palsy30. Collectively, these data suggest that the GABA-ergic striatal neurons, particularly those expressing the intracellular calcium binding proteins: calbindin-28k, calretinin and parvalbumin, are highly susceptible to ischaemic injury in the term-equivalent fetus.In the present study, cerebral ischaemia was associated with reduced survival of cabindin-28k, calretinin, parvalbumin and GAD positive neurons after 7 days of recovery. However, there was no effect of ischaemia on nNOS or ChAT positive neurons after 7 days. These findings are broadly consistent with observations after shorter periods of recovery from asphyxia or ischaemia in preterm and term-equivalent fetal sheep32 and developing brain33. Furthermore, striatal mitochondria show greater sensitivity to disturbances in calcium homeostasis compared to mitochondria from cortical and hippocampal neurons34. Collectively, these data suggest that developing striatal GABA-ergic neurons are susceptible to increased extracellular glutamate after HI, possibly due to greater susceptibility to disturbances in calcium homeostasis and subsequent mitochondrial dysfunction.The vulnerability of GABA-ergic neurons that express calcium binding proteins may reflect greater dependency of these neurons on their calcium buffering system for homeostasis, such that impaired calcium buffering capacity during and after HI may underlie their susceptibility to injury. Furthermore, we did not observe significant neuronal injury in striatal neurons that lacked calcium binding proteins, such as those expressing nNOS and ChAT. These data further support the idea that neurons expressing calcium binding proteins show greater vulnerability to ischaemic injury. Critically, excessive intracellular calcium accumulation has been implicated in the pathogenesis of mitochondrial dysfunction and neuronal apoptosis and necrosis in the adult36. In peritraumatic areas, extracellular ATP can activate neuronal and glial purinergic receptors and induce cellular injury by promoting extracellular calcium influx through the receptor channel37, as well as augmenting the pro-inflammatory function of activated microglia39. Furthermore, activation of astrocytic purinergic (P2Y) receptors by extracellular ATP has been shown to increase intracellular calcium accumulation, which enhances connexin hemichannel opening and leads to ‘ATP-induced ATP release’41, increasing the potential for ATP-mediated neurotoxicity. Taken with the finding discussed next that prolonged connexin hemichannel blockade after ischaemia was selectively protective, these data strongly support a key role for ATP in the pathogenesis of basal ganglia injury in the developing brain.Alternatively, GABA-ergic neurons may be particularly susceptible to ATP released into the extracellular space from open astrocytic connexin hemichannels after HI. Glia and striatal GABA-ergic neurons express purinergic receptors43. Thus, these data demonstrate that prolonged connexin hemichannel blockade markedly improved the overall survival of striatal neurons. The improved survival of striatal GABA-ergic neurons observed in this study was similar to previous reports that have shown improved striatal GABA-ergic neuronal survival after inhibition of neuronal nitric oxide synthase14 and therapeutic hypothermia29 after cerebral HI. Conversely, supplementation with low dose recombinant insulin-like growth factor-1 improved survival of GAD-positive striatal neurons but did not improve survival of parvalbumin-positive cells9. Based on the likely cellular mechanisms responsible for neuroprotection in these studies and ours, we can reasonably suggest that post-insult production of reactive nitrogen species, reduced trophic support, excessive purinergic receptor activation, secondary inflammation and intracellular accumulation of excitatory amino acids and calcium are potentially key pathophysiological mechanisms involved in basal ganglia injury and should be the subject of further investigation.The short infusion of the mimetic peptide was not associated with improved survival of striatal neurons. In contrast, a long infusion for 25 hours was associated with improved survival of calbindin-28k, calretinin, parvalbumin and GAD positive GABA-ergic neurons. Critically, GABA-ergic neurons, particularly those expressing calbindin-28k, calretinin and parvalbumin calcium binding proteins make up the majority of the striatal neural population44. Resistance of striatal cholinergic interneurons to injury may be partially mediated by their increased expression of the free radical scavenger superoxide dismutase46. Furthermore, striatal GABA-ergic interneurons containing nNOS have previously been shown to be resistant to hypoxic ischaemic injury48 and neurodegeneration in Huntington’s and Alzheimer’s disease patients50. While the exact mechanisms of their resistance to injury is unclear, it may include a nitric oxide mediated reduction of N-methyl-D-aspartate-mediated influx of calcium into the intracellular space and/or increased regional blood flow mediated by local nitric oxide release47.Immunoreactive ChAT and nNOS interneurons were relatively resistant to ischaemic injury in the present study. In adult rats, cholinergic interneurons were more tolerant to glutamate receptor mediated excitotoxicity than spiny projection neurons, possibly due to their lack of the glutamate receptor sub-units: GluR1, GluR2/3 and GluR453, reflecting reduced neural injury. These data suggest that improved survival of striatal GABA-ergic neurons, in addition to partial preservation of cortical neurons, as previously reported in Davidson et al.10, contribute to improved recovery of EEG activity after HI.We observed improved recovery of EEG power in fetuses treated with the long infusion of peptide. In line with this, a positive relationship between improved recovery of EEG power at 7 days and survival of striatal calbindin-28k and parvalbumin positive neurons was observed. This is consistent with the clinical finding in term infants that improved recovery of EEG power is strongly associated with better neurodevelopmental outcome55, and are strongly associated with poor neurodevelopmental outcomes58. Compared to adults, the neonatal brain is susceptible to electrographic seizures, possibly related to peak synaptic and dendritic spine density and/or relative abundance of glutamatergic (pro-excitatory) neurons59. During seizures, blood flow to the basal ganglia is increased compared to the cortex, cerebellum or brainstem, suggesting increased local electrical activity and metabolic demand60. Furthermore, in term infants, seizures were associated with an increase in lactate:choline within the basal ganglia, suggesting that seizures may independently increase local metabolic stress57. In this study peptide infusion was associated with a reduction in electrographic seizures, with the greatest effect in the long infusion group. We observed an inverse relationship between electrographic seizure burden and numbers of surviving calbindin-28k, calretinin and parvalbumin positive striatal neurons; parvalbumin positive neurons showed the strongest correlation with reduced seizure burden. This raises the possibility that loss of GABA-ergic interneurons within the basal ganglia, particularly those expressing the parvalbumin protein, facilitates disinhibition and thereby promotes seizure propagation. Supporting this, parvalbumin deficient mice are significantly more susceptible to pentylenetetrazole-induced seizures than controls61. Alternatively, it may reflect a differential susceptibility of GABA-ergic neurons to seizures, such that neurons expressing the parvalbumin protein are more susceptible to seizure-induced metabolic stress.Seizures are common after HI62. Furthermore, striatal medium spiny neurons generate distinct firing patterns during the sleep-state cycle, mainly characterised by brisk rhythmic neuronal firing during non-rapid eye movement sleep due to fluctuations between hyperpolarised quiescence and a depolarised state63. These data suggest that basal ganglia neurons, particularly medium spiny neurons, contribute to the neural circuitry that controls sleep state. Consistent with this, improved survival of striatal GABA-ergic neurons, particularly the calbindin-28k medium spiny neurons, was highly associated with faster return of sleep state cycling.Striatal lesions have been associated with destabilisation of sleep state architecture and impaired electrocortical arousal10. However, in the present study, short infusion of peptide was not associated with improved survival of any striatal neuronal phenotype. These data demonstrate that the relationship between neuronal loss and brain activity is complex and not purely dependent on improved cell survival. Speculatively, improved functionality of surviving neurons and glia may have contributed to the intermediate improvement in neurophysiological recovery after the short infusion.Intriguingly, peptide infusion was associated with a graded improvement in neurophysiological recovery, such that short infusion was associated with an intermediate reduction in electrographic seizure burden and improvement in the rate of return to sleep state cycling. We have previously shown that short infusion of Peptide 5 was associated with improved recovery of EEG power after cerebral ischaemia56. However, using a prolonged recovery time precluded us from examining specific cellular mechanisms that mediated neuroprotection in this study. This is one of the limitations of performing large animal translational studies which are expensive relative to rodent studies64. Thus, identifying specific cellular mechanisms would require a separate series of experiments, which will be the subject of future investigations in our laboratory. Similarly, it is not possible to be certain whether the mimetic peptide acts directly on striatal Cx43 or potentially via paraventricular actions. Nevertheless, we have previously shown that propidium iodide uptake is reduced by i.c.v. peptide infusion in the parasagittal white matter and in grey matter10. Given that Peptide5 is a small peptide that is highly specific to the extracellular loop of Cx4328, this supports a local effect.Some limitations of the present study should be considered. It is important to reflect that correlation is of course not causation. In this study we used correlative analysis to better understand the relationship between EEG function and survival of phenotypically distinct striatal neurons and thus to highlight hypotheses that can be studied further. Further, the present study used a relatively prolonged recovery period (7 days) to allow us to test the efficacy of Cx43 hemichannel blockade on the evolution of the secondary phase of neuronal injury, which occurs for several days after the insultIn conclusion, prolonged infusion of a mimetic peptide targeting Cx43 hemichannels was associated with improved survival of striatal GABA-ergic neurons, reduced electrographic seizure burden, faster return to sleep state cycling and improved recovery of EEG power 7 days after HI. These data suggest that connexin hemichannel blockade has potential to reduce the severity of basal ganglia injury in term infants with HIE.2 . During surgery, ewes received an i.v. infusion of isotonic saline (250 mL/h) to maintain fluid balance. Depth of anaesthesia, maternal heart rate and respiration were continuously monitored by trained anaesthetic staff.All procedures were approved by the Animal Ethics Committee of The University of Auckland following the New Zealand Animal Welfare Act, and the Code of Ethical Conduct for animals in research established by the Ministry of Primary Industries, Government of New Zealand. Romney Suffolk fetal sheep underwent aseptic surgery between 118 and 124 days gestation (term = 147 days). Food but not water was withdrawn 18 h before surgery. Ewes were given long acting oxytetracycline  intramuscularly 30 minutes before the start of surgery. Anaesthesia was induced by intravenous injection of propofol  and maintained using 2–3% isoflurane in O10. In brief, following a maternal midline abdominal incision, the fetus was exposed, and polyvinyl catheters were inserted in the right and left brachial arteries and amniotic cavity. The vertebral-occipital anastomoses were ligated and a pair of inflatable carotid artery occluder cuffs was placed around both carotid arteries66. Two pairs of electroencephalograph (EEG) electrodes  were placed through burr holes onto the dura over the parasagittal parietal cortex  and secured with cyanoacrylate glue. A reference electrode was sewn over the occiput. An intracerebroventricular (ICV) catheter was placed into the left lateral ventricle . Accurate placement of the ICV catheter was shown during the surgical procedure by confirming that cerebrospinal fluid could flow freely out of and into the lateral ventricle. Further, we have previously shown that this was associated with accurate placement of the ICV catheter (~4 days after surgery) in a pilot study, whereby fluorescent peptide was infused into the left lateral ventricle from 90 min until 6 hours after ischaemia. Inspection of freshly cut coronal sections revealed a high concentration of peptide surrounding the left lateral ventricles with widespread diffusion into subcortical and cortical areas10.Instrumentation of fetuses has been previously describedad libitum in a temperature-controlled room  with a 12 h light dark cycle. Ewes received intravenous antibiotics daily for 4 days . Fetal catheters were maintained patent by continuous infusion with heparinised saline (20 IU/mL), and the maternal catheter was maintained patent by daily flushing.Antibiotics  were administered into the amniotic sac before closing the uterus. A maternal long saphenous vein was implanted to provide access for postoperative care. Sheep were housed in separate metabolic cages with access to water and food 67 and was normalised by log transformation .Continuous fetal EEG recordings began 1 d before and continued until 7 d after bilateral carotid artery occlusion. The analogue fetal EEG signal was low pass filtered with a cut off frequency set with the −3 dB point at 30 Hz, and digitised at a sampling rate of 512 Hz. EEG power was derived from the power spectrum signal between 0.5 and 20 Hz28) as either a short infusion; 50 µmol/kg, dissolved in 1 mL of artificial cerebrospinal fluid and infused over 1 h (n = 6), or a long infusion; 50 µmol/kg over 1 h then 50 µmol/kg over 24 h (n = 6) at a rate of 1 mL over the first hour then 1 mL over 24 h. Controls received either sham ischaemia (n = 6) or cerebral ischaemia (n = 6) followed by an equivalent ICV infusion of vehicle . The ICV infusion was delivered using an external infusion pump . For all groups, the dead-space in the ICV cannula was cleared by preinfusing 0.7 ml of artificial CSF at 1 ml/h, before the start of the peptide or vehicle infusion. Randomisation was stratified by cohort to control for the time of year and breeding season to avoid any potential confounding related to intra-or inter-seasonal differences in fetal growth due to changes in flock management, nutrition, climate or paternal genotype68. Ewes and fetuses were killed 7 days after ischaemia with an overdose of sodium pentobarbitone .Experiments began at 128 ± 1 (mean ± SD) days gestation. Global cerebral ischaemia was induced by reversible inflation of the carotid artery occluder cuffs with saline for 30 minutes. Successful occlusion was confirmed by rapid sustained suppression of EEG activity. Ninety minutes after the end of ischaemia, fetuses were randomly assigned to receive an ICV infusion of a mimetic peptide that blocks the extracellular loop of Cx43  using a microtome . Brain regions of the forebrain used for analysis included sections taken at the level of the mid striatum, 26 mm anterior to stereotaxic zero according to the fetal sheep stereological atlas69. Slides were dewaxed in xylene and rehydrated in decreasing concentrations of ethanol and then washed in 0.1 mol/L phosphate buffered saline (PBS). Antigen retrieval was performed in citrate buffer using the pressure cooker technique in an antigen retrieval system . Endogenous peroxidase quenching was performed by incubation in 0.1% H2O2 in methanol. Non-specific antigens were blocked using 3% normal goat serum. The sections were labelled with 1:200 rabbit anti-Calbindin 28k , 1:500 rabbit anti-neuronal nitric oxide synthase , 1:200 rabbit anti-glutamic acid decarboxylase , 1:200 rabbit anti-Calretinin , 1:200 rabbit anti-parvalbumin (medium to large GABA-ergic aspiny interneurons Swant®) and 1:200 mouse anti-cholinergic acetyltransferase  overnight at 4 °C. Sections were incubated in biotin conjugated IgG  for 3 h at room temperature. Sections were incubated in ExtrAvidin®-Peroxidase  for 2 h at room temperature and allowed to react with 3,3′-diaminobenzidine tetrahydrochloride (Sigma Aldrich). The reaction was stopped by washing in PBS before sections were dehydrated and mounted.At post mortem, the fetal brains were fixed 70. When the investigator (RG) was unsure whether a neuron was morphologically normal, 40x magnification was used to confirm whether the cell should be excluded from the count. Average scores from both hemispheres from two sections were calculated for each subject.Estimates of striatal neuron phenotype density were quantified by light microscopy using Stereoinvestigator software (Microbrightfield Bioscience (MBF), Williston, VT, USA). The striatum, including the caudate nucleus and putamen, was outlined at 2x magnification Fig.  and neur71. Sleep state cycling was defined as a repetitive alternating pattern of high and low voltage EEG activity with each phase lasting approximately 20 minutes. All neurophysiological assessments and neuronal counts were performed by an assessor who was blinded to the treatment groups.Off-line physiological data analysis was performed using LabVIEW based customised programs . Seizures were identified visually and defined as sudden repetitive and evolving waveforms in the EEG signal lasting more than 10 seconds and of an amplitude greater than 20 μVStatistical analysis was undertaken using SPSS  and GraphPad software . Between groups comparison was performed using a one-way ANOVA with Fisher’s LSD test for multiple comparisons. Linear and non-linear regressions were used as appropriate to compare the relationship between EEG recovery  and survival of striatal phenotypic neurons. Statistical significance was accepted when P < 0.05. Data are means ± SD.


Our results illustrate how once unusual, but increasingly frequent Arctic weather events affect some cold-adapted mammals, and suggest that an understanding of species responses to a changing Arctic can be enhanced by coalescing groundwork, rare events, and insights from local people.Despite the growth in knowledge about the effects of a warming Arctic on its cold-adapted species, the mechanisms by which these changes affect animal populations remain poorly understood. Increasing temperatures, declining sea ice and altered wind and precipitation patterns all may affect the fitness and abundance of species through multiple direct and indirect pathways. Here we demonstrate previously unknown effects of rain-on-snow (ROS) events, winter precipitation, and ice tidal surges on the Arctic’s largest land mammal. Using novel field data across seven years and three Alaskan and Russian sites, we show arrested skeletal growth in juvenile muskoxen resulting from unusually dry winter conditions and gestational ROS events, with the inhibitory effects on growth from ROS events lasting up to three years post-partum. Further, we describe the simultaneous entombment of 52 muskoxen in ice during a Chukchi Sea winter tsunami ( Ovibus moschatus) died1. Nearly 150 years earlier, Charles Darwin noted the primacy of abiotic conditions in determining the fitness of species at the top of the world by observing: “Only in extreme conditions in arctic regions or on the borders of an utter desert will competition cease and the struggle for life become almost exclusively with the elements”2.In August 2015, President Barack Obama visited the small town of Kotzebue in Arctic Alaska to garner an increased understanding of the immediacy and potential consequences of warming global temperatures. On a day in February that year, the low temperature in Kotzebue failed to drop below 0 °C for the first time; the snow melting as it rained. Fifteen hundred kilometers northeast and thirteen years before, a major winter rain-on-snow (ROS) event encased the ground in ice preventing the Arctic’s largest land mammal from accessing food; an estimated 20,000 muskoxen  owing to the catastrophic effects of such events on wildlife11. In the arid regions of Beringia, extreme ROS events are rare, yet the frequency of minor events (<10 mm) is increasing12. While not likely to be as catastrophic to wildlife as extreme ROS, smaller events may still lead to partial or complete ground icing11 and less access to food, with potentially negative effects on fitness.The Arctic is a realm of extremes, and Arctic species have adapted to maximize short growing seasons and endure long winters that may include temperatures below −40 °C, driving winds, and deep snow. A changing climate now brings new challenges for arctic species; warming temperatures are altering air velocity and the extent and duration of sea ice, all of which conflate to modify ocean currents and the timing and intensity of precipitation13. For example, warmer winter temperatures coupled with precipitation can have indirect negative effects on herbivores by increasing the probability of ROS ground-icing events which limit access to food resources14. Yet warmer winter temperatures may also have a positive effect on the energy balance of an individual by ameliorating thermoregulatory costs. Likewise winter precipitation falling as rain can reduce snowpack, thereby reducing the metabolic costs of foraging in deep snow16. However snowpack also insulates ground vegetation18, and thus reduced snowpack could negatively affect plant productivity and resource availability in the following growing season.Assessing the effect of weather variance associated with warmer temperatures on herbivorous Arctic species is further complicated by the myriad direct and indirect pathways by which weather can affect the energy balance of herbivores19 - to model head size as a function of extreme cold, mean winter precipitation, number of ROS events and growing season plant productivity  from 1-, 2-, and 3-year old individuals across six years at two sites in Arctic Alaska  Fig. . ResultsThe relationship between mean head size and precipitation for 1- and 2-yr olds was driven by the extremes in our dataset Fig. . On aver29 although the extent to which Wrangel’s ROS, temperature and summer NDVI are conflated is unclear.In the East Asian sector of Beringia on Wrangel Island, minor ROS events were more than 2× more frequent, temperatures colder and growing seasons shorter than at Alaskan sites Fig. . Correspivu or ivuniq in Iñupiaq, the language of indigenous northern-coastal Alaskans, these wind-driven polar ice-override surges pile ice to 4 m high, and distribute marine subsidies inland to 20 kms30.In contrast to the subtle interactive effects detailed above, a one-time extreme icing event caused by a tidal surge produced a more certain effect – an extraordinary mass mortality event. On February 14, 2011, 55 muskoxen clustered in a lagoon at the northern coast of Bering Land Bridge peninsula Figs . A few d33. However further longitudinal studies will be required to determine whether the negative effects of dry winter conditions and minor ROS events on juvenile growth ultimately affect the survival or reproduction of muskoxen, or whether compensatory growth occurs at later ages35.Our findings on the inhibitory effects of exceedingly dry winter conditions and minor ROS events on the juvenile growth of muskoxen, and of mass ice–induced mortality due to a wind-driven tidal surge, offer potent new insights into the effect of unusual weather variance on the biology of cold-adapted species. Smaller-size in juveniles and sub-adults is typically associated with lower fitness due to delayed puberty and increased mortality20, or both. However extremely dry conditions and lack of access to water in winter could become serious for some cold-adapted mammals under climate change. For instance, when snow patches (the only available winter water source) are scarce above 4500 m on the Tibetan Plateau, endangered wild yaks (Bos mutus) are less able to support lactation during winter36.The exceedingly dry winter in our dataset was part of a longer precipitation anomaly at Cape Thompson that began in April 2007 Fig. , with toWhy does the skeletal growth of 1- and 2-yr old muskoxen appear to be most negatively affected by exceedingly dry conditions, while the growth of 3-yr olds is most affected by ROS events during gestation? One potential explanation for this apparent discrepancy is the interdependence of ROS events and precipitation. In particular, the driest winter had no ROS events (0 events), meaning that for 1- and 2- yr olds the smallest head sizes were associated with the fewest ROS events. Our initial models suggested a weak negative association between gestational ROS events and head size for 1- and 2-yr old muskoxen, but these pathways were not retained during model selection. For these cohorts, it appears that negative effects associated with small increases in the number of ROS events were superseded by the positive effects of increasing precipitation.In contrast, some of the smallest 3-yr olds in our dataset experienced both the driest winter as juveniles, and the most ROS events while still in utero (8 events). Our models identified ROS to be the stronger predictor of head size in 3-yr olds, but it is possible that both dry conditions and ROS events contributed to the slower growth of these individuals. A longer or more spatially extensive dataset - capturing greater variability in precipitation and ROS events -would allow for a more comprehensive evaluation of the relative strengths of these environmental conditions on each age class, and for the exploration of possible non-linear relationships between variables.38, there is far more uncertainty than certainty about pattern and effect for a broader realm of species and taxa. Yet one-time events resulting from severe storms and changing ice conditions have been shown to affect species beyond the one-time iced entombment of 52 muskoxen , 170 belugas (Delphinapterus leucas), and sea otters (Enhydra lutris) in southern Beringia, and also of a convergence of nine polar bears at a 20 × 6 m opening in sea ice, killing an estimated 100 belugas in the eastern Arctic40  from Nunivak were used in all subsequent translocation throughout Alaska and from Nunivak directly to Wrangel Island (WI) in the Chukotka region of Russia. As a result, all animals at our study sites stem from the same set of founders47, for juvenile cohorts born from 2005–2012 Specifically, we used non-invasive ground photogrammetry to assess linear head dimensions of these readily distinguishable juvenile-aged cohorts48. Head size photo images were gathered in March and April (from 2008 through 2013) by on-the-ground approaches to muskoxen along the Alaskan Chukchi realm in and adjacent to National Park Service units involving Bering Land Bridge to Cape Thompson including Cape Krusenstern. We repeated these same methods on WI in March and April, 2014.We approximated skeletal growth of muskoxen by using head size dimensionality of 1-, 2-, and 3-yr olds, the ages of most rapid growth19 with covariate data collected from weather stations during the winter, and MODIS vegetation indices during growing seasons, to disentangle potential direct and indirect effects of weather variance on muskoxen and their food resources. Our general conceptual model of hypothesized relationships between covariates of interest (Table 49) during the previous growing season and during an individual’s first growing season  simulations to fit path models, as well as parameter estimates for best fit models are in the Supplemental Information and Table To assess spatial variation in the skeletal growth of muskoxen, we estimated mean age-graded head size of juveniles born from 2005–2012 in the two Alaskan populations and subsequently asked whether similar metrics in the Wrangel Island . Data used to calculate ROS events on Wrangel Island were from a digital hard-copy offered by the continuously-manned weather station of the Federal Hydro-meteorological Service of Russia in Ushakovskoe  whose weather records date to 1926.We compiled winter weather data for our Alaskan sites (Cape Thompson and Bering Land Bridge) from the closest NOAA-maintained weather stations with data coverage from 2004 to 2013. These were: Deering  for the Bering Land Bridge site, and Kivalina Airport  for the region from Cape Thompson to Krusenstern. Alaskan weather data were accessed from NOAA’s National Center for Environmental Information  >0 °C and precipitation occurred, and conditions appropriate for snow to already be on the ground (e. g. previous precipitation had occurred when T < 0 °C). While ground icing can form from repeated melt and freeze events, even when temperatures remain <0 °C, we focus here on conditions that are most likely to result in ground icing.Detection of Arctic ROS events is difficult because weather stations are few, monitoring inconsistent, and ROS relatively rare54. Our NDVI data covered the ten year period from 2004–2013, were for our two study sites, and obtained from the University of Alaska Geographic Network of Alaska (GINA) , which receives and publishes MODIS NDVI (eMODIS) datasets from the USGS as 7-day 250 meter resolution composite rasters spanning 42 weeks of each year for the entire state of Alaska . Various annual metrics are then derived for these datasets by GINA to provide several seasonal values of NDVI for each 250 meter pixel, which are then embedded into individual 12-band, 32-bit floating point GeoTiff rasters49. Of these metrics, time-integrated NDVI, measured between season onset and season end, were used for analyses.Time integrated NDVI (iNDVI) sums NDVI values of a pixel over an entire growing season, thereby combining information on both peak productivity and growing season length55.Our two study areas, Cape Thompson and Bering Land Bridge, were delineated using Google Earth by manually constructing two polygon files. These were then imported into ArcGIS and converted to feature classes. They were then projected to Alaska Albers Equal Area (NAD83)(meters) for further analyses with the NDVI data, which also natively uses this projection system. All geo-processing and spatial analyses were completed using ESRI’s ArcGIS Desktop version 10.2 software6 to prevent any truncation of significant digits in the original data prior to analysis. Because we did not have information about individual foraging locations for muskoxen during growing seasons, we assigned each site a yearly mean iNDVI value by averaging the iNDVI values for all pixels at each site in each year.For each data year, NDVI metric data were exported locally from GINA’s Web Coverage Service (WCS) and clipped to the two study area extents. The time-integrated NDVI metric band was then extracted from each raster along with an additional band indicating pixel value quality (good or bad) using the Composite Band tool in the ArcGIS Spatial Analyst extension. Resulting rasters were then filtered using only good pixel values for each year and study area. Actual time-integrated NDVI values were then inflated by a factor 10https://tidesandcurrents.noaa.gov/noaatidepredictions/NOAATidesFacade.jsp?Stationid=9491094) for the Red Dog Dock Station (Chart # 16005) which has operated since 2003. The highest observed water level (2.27 m on 2/26/2011) is appreciably greater than mean high water (0.24 m), mean tide level (0.14 m), and mean sea level (0.14 m) across this period.To assess storm surges, we coalesced data from NOAA measures of tides and currents (Our work has been performed in accordance with all relevant guidelines and regulations of an animal care institutional committee at Colorado State University and is approved under CSU Protocol ID: 15–6330 A.Supplementary MaterialsSupplementary File S1Supplementary File S2Supplementary File S3Supplementary File S4