Genome-wide association studies (GWAS) have successfully identified genetic susceptible variants for complex diseases. However, the underlying mechanism of such association remains largely unknown. Most disease-associated genetic variants have been shown to reside in noncoding regions, leading to the hypothesis that regulation of gene expression may be the primary biological mechanism. Current methods to characterize gene expression mediating the effect of genetic variant on diseases, often analyzed one gene at a time and ignored the network structure. The impact of genetic variant can propagate to other genes along the links in the network, then to the final disease. There could be multiple pathways from the genetic variant to the final disease, with each having the chain structure since the first node is one specific SNP (Single Nucleotide Polymorphism) variant and the end is disease outcome. One key but inadequately addressed question is how to measure the between-node connection strength and rank the effects of such chain-type pathways, which can provide statistical evidence to give the priority of some pathways for potential drug development in a cost-effective manner.We first introduce the maximal correlation coefficient (MCC) to represent the between-node connection, and then integrate MCC with K shortest paths algorithm to rank and identify the potential pathways from genetic variant to disease. The pathway importance score (PIS) was further provided to quantify the importance of each pathway. We termed this method as “MCC-SP”. Various simulations are conducted to illustrate MCC is a better measurement of the between-node connection strength than other quantities including Pearson correlation, Spearman correlation, distance correlation, mutual information, and maximal information coefficient. Finally, we applied MCC-SP to analyze one real dataset from the Religious Orders Study and the Memory and Aging Project, and successfully detected 2 typical pathways from APOE genotype to Alzheimer’s disease (AD) through gene expression enriched in Alzheimer’s disease pathway.https://github.com/zhuyuchen95/ADnet).MCC-SP has powerful and robust performance in identifying the pathway(s) from the genetic variant to the disease. The source code of MCC-SP is freely available at GitHub ( APOE gene has been well identified to be associated with Alzheimer’s disease from large scale GWAS . When r = 0, there is no linear relationship between the two variables.The Pearson correlation coefficient is used as an indicator to measure the strength of linear correlation between two random variables X and Y.X and Y, which is essentially a linear correlation of the ranks of X and Y. It is free of the distribution of variables and is defined as follows:ri (si) represents the rank of xi (yi) in sample X (Y), and the range of ρ is . When ρ = 0, there is no monotonic relationship between the two variables. When ρ > 0, the relationship between the two variables increases monotonically; when ρ < 0, it decreases monotonically.The Spearman correlation coefficient indicates the degree of monotonic correlation between two variables R is different from the previous correlations based on the covariance matrix and variance matrix. It measures the correlation between variables by calculating the Euclidean distance of the sample itself. R is non-negative and can be used to measure the correlation between X and Y with any dimension. R = 0 indicates that X and Y are independent.The distance-related X, Y) = { : k = 1, …, n}, and define the following quantitiesSuppose the sample data is  is defined bySimilarly, Rn is defined as:The empirical distance correlation coefficient p is the joint probability density function of , and p(x), p(y) are the corresponding marginal density functions of , respectively. I can be viewed as the expected value of Mutual Information is a useful measure of information in information theory. It can be seen as the amount of information about a random variable contained in a random variable, or a random variable due to the knowledge of another random variable. Mutual Information does not need to make any assumption about the nature of the relationship between variable characteristics and is defined asH(Y) is the entropy of Y, referring to the uncertainty of Y, and H(Y| X) is the uncertainty of Y given X. Thus, I can be interpreted as a quantity introduced by X to reduce the uncertainty of Y. Therefore, the closer the relationship between X and Y, the greater the I. I = 0 when two variables are independent.The value of mutual information can also be expressed as Kullback–Leibler divergence ,x1, x2, …, xn, are taken from the continuous distribution p(x). A kernel density estimate is defined ash is the bandwidth and K(∙) is the kernel function, K(x) > 0, ∫K(x)dx = 1. At present study, we chose the commonly used Gauss kernel function.The calculation of MI requires the estimation of the density functions, we adopt the kernel density estimation method. For the one-dimension marginal density, we assume that the data X, Y be a bivariate sample drawn from a common distribution described by the density function. The bivariate kernel density estimation can be defined to bex, y)TZi = T, i = 1, 2, …, n.For the two-dimension joint density, assuming that the data H is the bandwidth (or smoothing) 2 × 2 matrix which is symmetric and positive definite; K(∙) is the bivariate kernel function which is a symmetric multivariate density and Here X, Y) the largest possible mutual information achievable by any X -by- Y grid applied to the data . We set the proportion of nonlinear relationship to be 30% (5 edges having cosine and 2 edges having quadratic relationship), 40% (6 edges having cosine and 3 edges having quadratic relationship), 50% (8 edges having cosine and 4 edges having quadratic relationship) and 60%  all-right: it is the most stringent and means that the top 4 pathways can be precisely allocated with the same order as their effects from large to small; the more times to find the top 4 pathways, the better the method; and 2) range-right: it means that the top 4 pathways with some effects are ranked in top 4, while the order can be allowed to be chaotic. For instance, if the top 4 paths are sorted as APOE genotype to AD, with the network constructed from KEGG-based Alzheimer’s disease pathway , and more recently, it has been used to generate genome-wide genotyping data generated on a Affymetrix 6.0 platform and imputed to 2.2 million single nucleotide polymorphisms (SNPs) with HapMAP [APOE genotype is defined as a 0–1 variable. Following previous studies [combat” package.DNA has been used to characterize apolipoprotein E allele status . It is necessary to explore the pathway mechanism behind this association. We mapped all gene expression from ROSMAP to the KEGG Alzheimer’s disease pathway to determine the candidate gene expression. The APOE genotype were linked to the three genes , which located on the left of the Alzheimer’s disease pathway. Then, the other downstream genes are connected following the Alzheimer’s disease pathway structure, and finally connected to AD. There is a total of 24 genes and 26 nodes in the network. The starting node of the whole network is the APOE genotype and the terminating node is AD  from ROSMAP, with 163 from MAP and 201 from ROS. The age of death was between 67 and 90 years. Among these samples, 192 samples had AD and 172 were normal controls. We first performed a simple logistic regression between Additional file 1: Table S1. The rank of the total 33 pathways for 6 integration methods. Figure S1. The proportion correctly pinpointing top 4 pathways under sample size 500 and nonlinear proportion 60%. Figure S2. The proportion correctly pinpointing top 4 pathways under sample size 500 and nonlinear proportion 30%. Figure S3. The proportion correctly pinpointing top 4 pathways under sample size 500 and nonlinear pattern being φ(xi) = sin(2xi) + ε. Figure S4. The proportion correctly pinpointing top 4 pathways under sample size 500 and nonlinear pattern being exponential and reciprocal. Figure S5. The proportion correctly pinpointing top 4 pathways under sample size 500 and nonlinear proportion 40% (including NPR). Figure S6. The proportion correctly pinpointing top 4 pathways under sample size 300 and nonlinear proportion 30%. Figure S7. The proportion correctly pinpointing top 4 pathways under sample size 300 and nonlinear proportion 40%. Figure S8. The proportion correctly pinpointing top 4 pathways under sample size 300 and nonlinear proportion 50%. Figure S9. The proportion correctly pinpointing top 4 pathways under sample size 300 and nonlinear proportion 60%. Figure S10. The proportion correctly pinpointing top 4 pathways under sample size 300 and nonlinear pattern being φ(xi) = sin(2xi) + ε. Figure S11. The proportion correctly pinpointing top 4 pathways under sample size 100 and nonlinear proportion 30%. Figure S12. The proportion correctly pinpointing top 4 pathways under sample size 100 and nonlinear proportion 40%. Figure S13. The proportion correctly pinpointing top 4 pathways under sample size 100 and nonlinear proportion 50%. Figure S14. The proportion correctly pinpointing top 4 pathways under sample size 100 and nonlinear proportion 60%. Figure S15. The proportion correctly pinpointing top 4 pathways under sample size 100 and nonlinear pattern being (xi) = sin(2xi) + ε . Figure S16. The network when there are 30% nonlinear between-node connection in the 4 effective pathways. Figure S17. The network when there are 40% nonlinear between-node connection in the 4 effective pathways. Figure S18. The network when there are 50% nonlinear between-node connection in the 4 effective pathways. Figure S19. The network when there are 60% nonlinear between-node connection in the 4 effective pathways. Figure S20. The Alzheimer’s disease pathway downloaded from KEGG.


We conclude that BP1–BP2 deletion moderately increases the risk of the generally milder, but commoner, CVM phenotypes seen in this unselected population, in addition to its previously demonstrated association in case/control studies ascertained for CVM.Deletion of a non-imprinted 500kb genomic region at chromosome 15q11.2, between breakpoints 1 and 2 of the Prader–Willi/Angelman locus (BP1–BP2 deletion), has been associated in previous studies with phenotypes including congenital cardiovascular malformations (CVM). Previous studies investigating association between BP1–BP2 deletion and CVM have tended to recruit cases with rarer and more severe CVM phenotypes; the impact of CVM on relatively unselected population cohorts, anticipated to contain chiefly less severe but commoner CHD phenotypes, is relatively unexplored. More precisely defining the impact of BP1–BP2 deletion on CVM risk could be useful to guide genetic counselling, since the deletion is frequently identified in the neurodevelopmental clinic. Using the UK Biobank (UKB) cohort of ~500,000 individuals, we identified individuals with CVM and investigated the association with deletions at the BP1–BP2 locus. In addition, we assessed the association of BP1–BP2 deletions with neuropsychiatric diagnoses, cognitive function and academic achievement. Cases of CVM had an increased prevalence of the deletion compared with controls (0.64%; OR = 1.73 [95% CI 1.08–2.75];  NIPA1, NIPA2, CYFIP1 and TUBGCP5) within a 500-kb region  on the long arm of chromosome 15 and encompass 4 genes (p = 0.02) [Genomic deletions are known to be a significant cause of congenital malformations, among which congenital cardiovascular malformations (CVM) have the highest population prevalence of around 9 per 1000 live births –24. We p = 0.02) . Other s = 0.02) , 27 and  = 0.02) , 29. HowThe UK Biobank (UKB), comprising ~500,000 individuals with genotype data, hospital admission records, baseline cognitive function testing, and a variety of other collected information, provides a large-scale resource for investigating complex genotype–phenotype associations. Here, we determine the prevalence of BP1–BP2 deletions within the UKB cohort and explore its association with CVM; and with neurodevelopmental diagnoses and cognitive function.n = 488,366), were used to generate CNV calls using the PennCNV software [n = 301). Samples containing an excess of CNVs (>40) (n = 1401) were also removed at this stage. This threshold intentionally retained samples with a relatively high number of CNVs as global CNV burden has previously been found to contribute to the risk of sporadic CVM [B-allele frequency and log2 ratio transformed intensity value files, generated and made available by UKB using the Applied Biosystems UK BiLEVE Axiom and UKB Axiom arrays  between the outermost array probes covering this region .15q11.2 deletions were identified where a heterozygous deletion spanned at least 95% of the four genes involved  data available in the UKB contain information on hospital admissions for the cohort. It includes primary and secondary diagnoses in the form of ICD9 and ICD10 codes, as well as details of operations and procedures as recorded through OPCS-4 codes. Using the classification schema shown in Fig. In addition to codes covering ‘congenital malformation of the circulatory system’, we used additional codes to identify, amongst others, certain defects of congenital origin presenting in later life /Advanced Subsidiary levels (AS levels) or equivalent’ and each of these categories compared with the baseline group by fitting a multinomial log-linear model, accounting for age and sex. Fecundity was assessed in males and females separately using Poisson regression fitted to the number of children fathered and number of live births per person, respectively. Outliers were removed if greater than five times the standard deviation from the mean. This excluded samples with >8 children fathered in males and >7 live births in females. An interaction test was performed to confirm the differences between male and female subgroups.The frequency of BP1–BP2 deletion in cases of CVM, cases of neuropsychiatric disorder, and controls was compared using chi-squared tests. Associations between the deletion and quantitative measures of cognitive function were assessed using different statistical models dependent upon the distribution of the data, and adjusting for age and sex. Linear regression was used for reaction time . Poisson regression was fitted for fluid intelligence score. For the pairs matching test where values are heavily skewed towards zero, values were converted to a binary variable (0(0), 1(>0)) and logistic regression fitted on the binary outcome. Assessment of the numeric memory test was again assessed with logistic regression by converting the values into a binary variable (0(≤6), 1(>6)). The association between the deletion and the highest qualification achieved was assessed by combining ‘O levels/General Certificate of Secondary Education or equivalent’, ‘Certificate of Secondary Education or equivalent’, ‘National Vocational Qualification or Higher National Diploma or Higher National Certificate or equivalent’, ‘Other professional qualifications’, ‘None of the above’ and ‘Prefer not to answer’ into a single category and treating this as a baseline group. Further details on UK specific qualifications are available at In total, 2792 participants with genetic data available were identified as having non-syndromic CVM and 472,378 were classified as controls (see “Methods”). Table p = 0.03). In keeping with previous observations, no participant was homozygous for the BP1–BP2 deletion.In total, 1832 BP1–BP2 deletions were identified in UKB participants with genotyping data available. The CVM and control groups accounted for a total of 1787 of the samples with BP1–BP2 deletion, with 45 deletions present in participants who were excluded according to the criteria outlined in “Methods”. Of the 2792 non-syndromic CVM samples, 18 carried the BP1–BP2 deletion resulting in an estimated prevalence of 0.64% Table . The speTwo of the eighteen CVM case samples carrying the BP1–BP2 deletion also had neuropsychiatric diagnoses. Those individuals had cardiovascular diagnoses of ASD and aortic valve disease/replacement; and neuropsychiatric diagnoses of bipolar disorder and scholastic developmental disorder, respectively.p = 0.004)  Table . The speAfter removing participants with relevant neuropsychiatric disorders, 483,330 samples remained. Of these, 1808 carried the BP1–BP2 deletion. Figure p = 5.60E−22). Similarly, a lower proportion of participants with BP1–BP2 deletion had attained A/AS levels as their highest educational qualification than participants without the deletion .Carriers of the BP1–BP2 deletion were compared with non-carriers in terms of the highest academic qualifications that they had achieved. Table p = 0.00175). This difference was not evident in females . A test for interaction confirmed the differences between the male and female subgroups (p-interaction = 5.46E−04).Fecundity was assessed in those individuals with and without the BP1–BP2 deletion. Self-reported number of children fathered  and number of live births  in each cohort are shown in Fig. p = 0.03) and, in broad agreement with recent findings from others [p = 0.0043), measures of cognitive function, academic achievement and fecundity.The 15q11.2 (BP1–BP2) deletion (sometimes referred to as the Burnside-Butler syndrome susceptibility locus) has previously been associated with phenotypes including developmental delay, autism, schizophrenia and CVM; the great majority of the evidence regarding the deletion thus far originates from cohorts specifically selected for one or other of those phenotypes. We estimated the prevalence of the deletion and its associations with CVM and other diagnoses in the UKB cohort. We confirmed the association of the deletion with CVM   Fig. , supportTUBGCP5, CYFIP1, NIPA1 and NIPA2, are located in the deleted region. The effects of hemizygosity at each individual gene in the region have not been systematically described in mouse models, nor has any mouse model of the entire deletion been reported as yet. Therefore, the mechanisms whereby the deletion causes its associated phenotypes, and the interaction between hemizygosity for the region and other genetic and environmental factors to result in the wide variety of phenotypes described, remain to be elucidated.The clinical and genetic aspects of BP1–BP2 deletion have been recently reviewed [Regarding the classification of the deletion for the purposes of clinical genetics and genetic counselling, we tend to agree with Jønch et al. that the deletion should be classified as “pathogenic, of mild effect size” . The effSupplementary Table LegendsSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5


Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park  and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla  SIP experiments have been limited by their need for isotope-labeled molecules that can be prohibitively expensive or unavailable because of compositional complexity. An alternative to SIP targeted at a specific assimilatory pathway is to label all anabolically active cells in a sample with deuterated water . HPG is a structural analog of L-methionine and is incorporated into newly synthesized proteins due to the substrate promiscuity of methionyl-tRNA synthetase [Bioorthogonal non-canonical amino acid tagging combined with fluorescence-activated cell sorting (BONCAT-FACS) is a recently developed Next-Generation Physiology approach  that cannthetase . HPG-connthetase , 6, 11. nthetase . BONCAT-nthetase  and deepnthetase , suggest0 sample was taken from the homogenous mixture and frozen in 10% glycerol-TE [L-homopropargylglycine  was added to the remaining bulk slurry to yield a final concentration of 50 µM. Afterwards, 6 mL of the HPG-containing slurry was transferred to all incubation vials that had already been amended with a substrate (Supplementary Table 2). Two sets of triplicate incubations tested headspace composition on microbial activity, 2% O2 (microoxic), and 100% N2 (anoxic) were prepared by flushing the headspace with 0.2 µm filtered N2 gas for 3 min. The vials for 2% O2 incubations had ~10% of the N2 headspace volume removed and replaced with 0.2 µm filtered ambient lab air. All incubations were performed in triplicate and were incubated at 74 °C in the dark for 48 h without shaking. This incubation time was chosen after preliminary experiments had been performed with 6, 24, and 48 h incubation times. An incubation time of 48 h was selected to increase cell labeling rates to allow for the reliable sorting of BONCAT-positive cells. At the completion of the incubation, vials were uncapped, and the content of each vial was immediately transferred to a 15 mL conical tube with 10% glycerol-TE [All work in YNP was conducted under US National Park Service permit no. YELL-SCI-8010 (2017–2019). A sediment slurry sample  was collected using a stainless-steel measuring cup fixed to the end of a 12-foot telescoping pole from a hot spring within the Five Sisters hot spring group in YNP  , 100 µM copper sulfate pentahydrate (Sigma Aldrich), 500 µM THPTA (Click Chemistry Tools), and 4 µM Cy3 picolyl-azide dye (Click Chemistry Tools) in 1× PBS. The reaction mixtures were vortexed briefly to mix and then rotated in the dark at room temperature for 1 h. After this incubation step, all samples were washed three times by a series of centrifugation steps at 14,000 × g for 5 min and resuspended in 1 mL of 1× PBS. Following the final wash, cells were resuspended in 500 µL of 1× PBS and stored at 4 °C in the dark before being sorted on a fluorescence-activated cell sorter (FACS) the subsequent day. One replicate of each incubation condition was extracted per day and all samples were checked for successful completion of the click reaction by epifluorescence microscopy (Leica DM4B microscope with DAPI and Cy3 filter sets).For cell extractions, samples preserved in glycerol-TE were thawed at 4 °C, vortexed briefly at maximum speed, and 1 mL of slurry were transferred to a 15 mL conical tube containing 4 mL of 1× phosphate buffered saline (PBS) with Tween 20 (Promega) at a final concentration of 0.01%. The conical tubes were vortexed for 5 min at maximum speed to detach cells from particles before being centrifuged for 5 min at 500 × prepared  and 200 g for 5 min and the supernatants were discarded by careful aspiration before the cell pellets were resuspended in 20 µL of nuclease-free water. Epifluorescence microscopy was used to check the purity of sorted cells, confirming the presence of only fluorescent cells in the “BONCAT gate” (not shown). Cells were stored at 4 °C until all samples were sorted. SYBR Green I nucleic acid stain (Invitrogen) was tested as a counterstain to fluorescently identify all events containing biomass prior to gating on a BONCAT fluorescent signal. However, after testing several different combinations of SYBR Green I with clickable dyes  it was determined that too much bleed-over across fluorescence channels would prohibit the reliable sorting of SYBR and BONCAT-positive events with the current laser and detector setup.On the day following cell extraction and click staining, cells were sorted based on Cy3 fluorescence intensity using a Sony SH800S FACS. Prior to sorting, a 50 µL aliquot of each sample (“Presort” sample) was transferred to a new tube and stored at 4 °C for later processing. For sorting, a first gate was drawn based on forward scatter area (FSC-A) and back scatter area (BSC-A) to exclude any large particles that remained after filtration . Amplification of bacterial and archaeal 16S rRNA genes was performed by PCR using the Earth Microbiome protocol [Escherichia coli (E. coli) DNA were included. After confirming correct band length, each PCR reaction was again purified with Ampure XP beads to remove excess primers. Triplicate Quant-iT Picogreen dsDNA Assay (Invitrogen) reactions were performed for all samples following manufacturer’s guidelines and quantified with a Biotek Synergy H1 Hybrid microplate reader. For each sample, excluding E. coli positive controls, 10 ng of barcoded and cleaned DNA per sample was pooled. Additional samples that were amplified and included in the sample pool were reagent extraction blanks and sheath fluid aliquots from the sorter sheath fluid tank. Half of the pooled volume was concentrated with the QIAquick PCR purification spin column kit (Qiagen) following manufacturer’s guidelines. Sequencing was performed by Laragen Inc.  using Illumina 2 × 250 paired end read MiSeq sequencing. A total of 12,071,436 raw reads were generated from 209 samples.For DNA extraction, three cycles of freeze–thaw were used to lyse cells. Cells stored in 20 µL nuclease-free water at 4 °C were transferred into 96-well microtiter plates that were sealed with sterile adhesive foil sheets. Freezing was performed by placing the plate into a −80 °C freezer for 20 min. Thawing was done at 99 °C for 10 min in a thermal cycler (Eppendorf Mastercycler nexus GSX1). Prior to each subsequent freezing step, the plates were pulse centrifuged at 500 × protocol  with updprotocol  , Desulfobacterium autotrophicum (DSMZ 3382), Rhodopirellula baltica (DSMZ 10527), and Aliivibrio fischeri (DSMZ 507). The environmental sample tested was sediment-water slurry from hot spring FS5. DNA was quantified by Qubit high sensitivity dsDNA assay (Invitrogen) on varying amounts of cell numbers (achieved by consecutive 1:10 dilutions) for each test sample. The lowest quantity of cells required for detection with this Qubit assay was determined to be 100,000 cells for R. baltica while all other cultures needed a minimum of 250,000 cells. DNA was extracted from FS5 with the FAST DNA spin kit for soil  and 16S rRNA genes were amplified and sequenced as described above. The community composition of the environmental sample based on 16S rRNA gene sequence analysis was comparable to the sequence composition of the “presort” samples that were lysed with repeated freeze–thaw cycles. Sequence composition from the DNA extraction kit samples were similar to the composition of samples extracted with freeze–thaw cycles.Validation of successful DNA extraction through consecutive freeze–thaw cycles was confirmed with four pure culture isolates and one environmental sample. The following cultures were purchased from the German Collection of Microorganisms (DSMZ) and grown in the recommended media: E. coli was grown overnight in an Erlenmeyer flask in M9 minimal media with 0.04% glucose. The culture was then split into equal volumes and received new M9 media as before but containing different substrate amendments. Five conditions were tested in triplicate with the following addition to the base medium (0.04% glucose) and a final concentration of 50 µM HPG; 0.36% sucrose, 0.36% sorbitol, 0.36% glucose, 0.36% glucose and 10 µg/mL chloramphenicol, or no substrate addition. A sixth condition was tested without additional substrate or HPG. After 2 h of incubation, E. coli cells were fixed with 2% paraformaldehyde for 1 h at room temperature, washed three times with 1× PBS, and click stained with Cy3 picolyl-azide dye. Click stained cells were visualized with an epifluorescence microscope and imaged with Leica software. Images were analyzed using daime software package [ package  for reladecontam (version 1.1.2) [decontam identified 438 contaminating ASVs which represented 399,454 reads . Samples represented by at least 5000 sequence reads were used for further analysis. ASVs were collapsed at the genus level, relative sequence abundance was calculated for each sample, and only taxa above 0.01% across all samples were analyzed. From here on, relative abundance is referring to the relative abundance of sequencing reads. Outlying replicates were determined using the DESeq2 package [decontam. Microbial community analysis was performed in R [lme4 package [p values adjusted for multiple comparisons [DESeq2. All sequence data associated with this study have been deposited in Genbank under BioProject number PRJNA601738. All files generated during data processing are available in the Sequence reads were processed using QIIME 2 version 2018.6)  package 18.6  pac package  by clustmed in R  using PEmed in R  with Bra package  with subparisons . Log2-foE. coli, which demonstrated that translational activity as measured by BONCAT can only be detected when cultures were grown with glucose or sorbitol as the sole carbon and energy source but not with sucrose , with the next most abundant community members belonging to bacterial candidate phylum Fervidibacteria (9.57%) and phylum Deinococcus-Thermus (7.98%)  hot springs over a wide pH range (2.9–9.3) [Aigarchaeota remain recalcitrant to cultivation and no experimental data on their functional activity are currently available. Reconstructed genomes suggest the capacity for aerobic respiration or anaerobic respiration with nitrate as an electron acceptor [Aigarchaeota metagenome bin [For this benchmark study, we selected a high temperature (74 °C), alkaline pH 8.2) hot spring  in the Lower Geyser Basin of YNP Fig. . Hot spr hot spri2.9–9.3) . Despiteacceptor . In addinome bin . One of 2), microoxic (2% O2), and anoxic (100% N2) headspace conditions. Laboratory incubations were established with a slurry containing FS5 water and sediment to which HPG and substrates were added. After 48 h of incubation, biomass was resuspended in glycerol Tris-EDTA buffer [We applied BONCAT-FACS combined with 16S rRNA gene sequencing to identify the active microbial community members in hot spring material when incubated in the presence of 23 different substrates or growth conditions   Fig. . This reFervidibacteria were observed in higher proportion in most presort populations as compared to the original T0 bulk sample. This could be attributed to either favorable growth of this yet uncultured lineage or be a result of preferential cell extraction or cell lysis during freeze–thaw cycles as compared to bulk sample DNA extraction. Alternatively, their increase in relative abundance could have resulted from sample cooling (72 to 55 °C) during transit from the field to the laboratory (4 h).Although the overall composition of the active communities did not vary significantly in response to incubation conditions, changes within the richness and evenness of the communities were detected. To further describe the community composition, the Shannon’s diversity index of each incubation was calculated Fig. . For eacp = 0.00349 and p = 0.0334, respectively). Cellobiose is a disaccharide of repeating glucose monomers that compose cellulose polymers. Cellobiose can be cleaved by β-1,4 hydrolases into glucose monomers which serve as initial substrate for most central metabolic pathways. The Shannon’s diversity index of cells active with cellobiose amendment was the highest we observed alluding to wide use of cellobiose increasing the evenness of active microbes in these incubations (p = 0.00349).The Shannon’s diversity indices of the active, sorted fractions were more varied when standardized to the HPG-only control than the presort fractions. This indicates that the activity response among different taxa varies between treatments. For example, incubations with cellobiose or under anoxic conditions led to statistically significantly different diversity indices on average than the HPG-only control incubations . Headspace exchange from aerobic  to anoxic conditions (100% N2) in the incubations caused the active community to have a significant increase in Shannon’s diversity (p = 0.0334) compared to the HPG-only control. This is consistent with the idea that facultative anaerobes persist in the hot spring and their activity varies with oxygen availability.The perimeter of FS5 has a photosynthetic zone  when compared to the HPG-only sorted fraction (p < 0.10). This suggests that cellobiose has an impact on the activity of several taxa despite the hot spring lacking noticeable large carbon inputs or organic matter that would be degraded regularly in situ. Uncultured phylum BP4 had an LFC of 3.62 in response to cellobiose amendment . These results demonstrate the importance to functionally test genomic predictions under environmentally relevant conditions.The relative abundance of six taxa increased and two decreased statistically significantly in the active, sorted community in response to cellobiose-amendment based on logradation ; furtherThermus increased , whose taxonomic affiliation could not be resolved beyond phylum level, decreased  and under anoxic conditions (LFC = 8.12)  geothermal environments, including hot springs throughout YNP [Sequences related to the genus 12) Fig. . Pyrobacpiration –37. Thishout YNP  and likeThermocrinis and uncultured phylum Gal15 increased in relative abundance in both the cellobiose  and anoxic  incubations’ sorted fractions alluding to possible favorable cultivation conditions for these organisms. Other organisms were also detected with large LFCs, though they were not statistically significant when averaged across all three biological replicates. For some samples high variability was observed, emphasizing the need for replication in order to draw biologically meaningful conclusions.Cellobiose and anoxic incubations were the only treatments to inform us of community-wide substrate and headspace selectivity with statistical significance. However, organisms belonging to Aigarchaeota represented a smaller proportion of presort reads (31.4%) after the 2-day incubation than in the original sample (41.9%) . In contrast, the BONCAT-labeled, active fraction of cells were dominated by the uncultured candidate phylum Fervidibacteria [Fervidibacteria also comprised 48.0% of the total reads across all presorted, bulk communities and the active, sorted cell fraction  and anoxic (21.7%) treatments despite not being statistically significant (p = 1) (HPG-only = 13.3%). The ability to analyze microbial activity under varying headspace compositions demonstrates that BONCAT-FACS is an approach with great potential for studying cellular responses involving non-assimilatory pathways.An uncultured member of the bacteria . Fervididictions  informedc growth  and our Fervidibacteria populations in presort samples, possibly attributing to higher proportions in sorted, active samples. Alternatively, sampling too early might not provide enough opportunity for organisms to respond to the conditions provided, attributing the BONCAT signal to activity independent of sample treatment, or a general lack of signal. This is a problem with any methodology surveying activity in response to substrate amendment including BONCAT and heavy water SIP [Preliminary experiments determined that 48 h was to be the optimal time for incubation of this sample type to allow for enough HPG incorporation to be detected efficiently on our cell sorter (data not shown). However, this incubation time showed high baseline activity in the HPG-only control making activity variation due to substrate amendment difficult to determine. Extended incubation times with HPG have been shown to not be detrimental to the survival or activity of microorganisms in other sample types –7, 9, 11ater SIP .In this study, the microbial community activity response to substrate amendments and varying oxygen conditions in the headspace was compared to HPG-only incubations that represented close to in situ conditions. This allowed us to determine substrate or oxygen preference of microorganisms without the need for prior cultivation or genomic analysis. The overall composition of the active microbial community did not vary significantly from the HPG-only control (according to Bray–Curtis dissimilarity), but both the Shannon’s diversity indices and the relative abundances of specific microbial populations (LFC) in the active communities were affected by some treatments.Our benchmark study demonstrates that BONCAT-FACS can be used to identify which taxa in a microbial community are anabolically active and experimentally test genomic predictions about their metabolism in a high-throughput manner and at single cell resolution. Most importantly, BONCAT-FACS is not constrained by the limits of cultivation in order to identify conditions beneficial to the activity of cultured or uncultured archaea and bacteria and can be conducted under as close to in situ conditions as experimentally possible. This information could, in the future, be used for targeted cultivation attempts by informing the design of specific enrichment media or be combined with metagenomic sequencing to provide direct links between cellular function and genetic make-up. With the use of a small set of inexpensive chemicals to label active cells and widely available FACS infrastructure to sort fluorescently labeled cells, BONCAT-FACS can be easily adapted by other labs. Thus, we believe that BONCAT-FACS has strong potential to further progress our understanding of microbial ecophysiology in any natural or human-made system and become a valuable complement to SIP-Raman studies.SI Figure and table legendsSI Figure 1SI Figure 2SI Table 1SI Table 2SI Table 3SI Bioinformatics files


White matter hyperintensities (WMH) occur in association with dementia but the aetiology is unclear. Here we test the hypothesis that there is a combination of impaired elimination of interstitial fluid from the white matter together with a degree of hypoxia in WMH. One of the mechanisms for the elimination of amyloid-β (Aβ) from the brain is along the basement membranes in the walls of capillaries and arteries . We compared the dynamics of IPAD in the grey matter of the hippocampus and in the white matter of the corpus callosum in 10 week old C57/B16 mice by injecting soluble Aβ as a tracer. The dynamics of IPAD in the white matter were significantly slower compared with the grey matter and this was associated with a lower density of capillaries in the white matter. Exposing cultures of smooth muscle cells to hypercapnia as a model of cerebral hypoperfusion resulted in a reduction in fibronectin and an increase in laminin in the extracellular matrix. Similar changes were detected in the white matter in human WMH suggesting that hypercapnia/hypoxia may play a role in WMH. Employing therapies to enhance both IPAD and blood flow in the white matter may reduce WMH in patients with dementia. White Matter Hyperintensities (WMH) on magnetic resonance imaging (MRI) occur in the cerebral hemispheres mainly in elderly patients and are particularly associated with dementia . HyperinThe brain has no conventional lymphatic vessels. Early physiological studies using radio-iodinated tracers have shown that fluid, soluble metabolites and tracers drain along the walls of cerebral arteries to cervical lymph nodes and only an estimated 10–15% leaks into the CSF . DetaileIt appears, therefore, that two of the major functions of arteries, arterioles and capillaries in the brain are (a) maintaining blood flow with the supply of nutrients and immune cells to brain tissues and (b) drainage of interstitial fluid and soluble metabolites from the brain to cervical lymph nodes by the IPAD pathways to maintain homeostasis in the brain , 14. ChaThe process of IPAD ceases upon cardiac arrest , therefoCerebrovascular BM consist of a fine extracellular matrix (ECM) of glycoproteins and proteoglycans that ensheath the abluminal side of endothelia separating the endothelia from pericytes or smooth muscle cells and pericytes or smooth muscle cells from astrocytes, encircling the different cell types . Each ceIn this study we aim to firstly ascertain if ISF and solutes are eliminated from the white matter by IPAD. We use young adult wild-type mice to compare the drainage of amyloid-β (1–40) from hippocampal grey matter and the white matter tracts of the corpus callosum. We then use human post-mortem brains with WMH to compare the levels of extracellular matrix proteins fibronectin and laminin with those of age-matched controls. Finally, we apply hypercapnia to model hypoxic hypoperfusion and analyse the production of fibronectin and laminin by cultured human brain vascular smooth muscle cells (HBVSMC). Involvement of failure of IPAD in the aetiology of WMH has previously been deduced largely from circumstantial evidence and involvement by association. Direct evidence is difficult to obtain, especially from post-mortem human brain tissue. In this study we seek more direct evidence for the involvement of failure of IPAD in the aetiology of WMH. We show evidence of hypoxia in the white matter in WMH and we also demonstrate, experimentally, a reduced capacity of IPAD in the white matter compared with grey matter.Hypothesis 1: The dynamics of IPAD in the cerebral white matter differ from IPAD in the grey matter of the hippocampus. The hypothesis is tested by a) comparing the density of capillaries in grey and white matter and b) injecting soluble Aβ as a tracer independently into the grey matter of the hippocampus and into the white matter of the corpus callosum of mice and comparing the dynamics of drainage of tracer along IPAD from each of these regions of the brain. Hypothesis 2: Changes induced by hypercapnia as a model of hypoxia in the extracellular matrix of vascular smooth muscle cells are similarly expressed in human white matter exhibiting WMH. In order to test Hypothesis 2 we selected two proteins in the BMs of smooth muscle cells, a) laminin and b) fibronectin and established the effects of hypercapnia as a model of hypoxiaon cultures of vascular smooth muscle cells. We then compared the changes observed in culture with the changes in extracellular matrix in WMH.We test two hypotheses: n = 10) were anaesthetised with Isoflurane mixed with concentrated O2 (1.7 L min-1) . Isoflurane was used rather than injectable anaesthetics based on our previous study which showed that isoflurane is better at maintaining a more physiologically normal heart rate and oxygen saturation level [All procedures were carried out in accordance with animal care guidelines stipulated by the United Kingdom Animals (Scientific Procedures) Act 1986, Home Office licence P12102B2A. 10-week-old C57/BL6 wild-type mice  - Anterior-Posterior − 2 mm; Medial-Lateral 0.5 mm; Dorsal-Ventral - 1.3 mm, n = 5)  and viewed using a Zeiss Axioskop 2 fitted with a rhodamine filter to identify the section containing the site of injection. In our previous studies we showed that, in mice, the drainage of Aβ40 occurs predominantly in a posterior direction and can be visualised in the walls of blood vessels as close as 200 μm to the injection site [Anaesthetised mice were placed in a KOPH instruments stereotaxic frame (Model 900) and the head secured with jaw bars. A midline incision was performed and a Tech2000 Micromotor drill  with 0.7 mm burr was used to create a burr hole in the skull above the injection site  for 1 h at room temperature. Sections were then incubated in rabbit anti-collagen IV 1/400 in 0.01 M PBSt  and anti-smooth muscle actin (SMA) FITC conjugated 1/200 in 0.01 M PBSt  overnight in a moist chamber at 4 °C. Sections were then washed 3 × 10 mins in 0.01 M PBS and incubated in conjugated secondary antibody goat antirabbit Alexa Fluor 633 0.01 M PBSt  for 1 h at room temperature. Sections were further incubated in 1% Sudan Black for 5 min to remove auto fluorescence before being mounted in Mowiol Citifluor and imaged using confocal microscopy.2) and multiplying this value by 500,000 to be expressed as number of vessels per 0.5mm2. This was also performed for the number of capillaries, arterioles or venules with Aβ40 in their vessel walls. Vessels were identified based on lumen diameter and immunoreactivity to SMA  [P < 0.05.For each section, tile scans of the left hippocampus or corpus callosum were captured using a Leica SP8 confocal microscope fitted with × 20 objective set at an optical zoom of 1. Laser power and detection windows were kept consistent for all scans. Sequential imaging was used to prevent cross excitation of fluorophores. Quantification of IPAD of Aβ40 HiLyte Fluor 555 injected into the left hippocampus or corpus callosum was performed using a max projection of each tile scan uploaded into Adobe Photoshop CS6. We chose to use maximum projections of each tile scan as they provide a more precise and accurate method to assess IPAD, particularly when assessing vessel density. IPAD was assessed by using Adobe Photoshop CS6 to manually measure vessel density of capillaries, arterioles and venules and counting the number of vessels containing Aβ40 HiLyte Fluor 555 in their vessel walls. Vessel density was calculated by dividing the total number of capillaries, arterioles or venules by the overall surface area (in μmvenules) , 10. Thevenules)  was outlHuman autopsy CNS tissue was obtained from the MRC Cognitive Function and Ageing Study (CFAS) that had a post-mortem MRI confirming white matter hyperintensities , 28. ConTwo tissue sections of 10 μm thickness of the white matter from each case  were deparaffinised and then rehydrated through a graded series of alcohols. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 min at room temperature. Heat mediated antigen retrieval was then performed by microwaving in 0.01 M citrate buffer (pH 6). After blocking in 15% goat serum  for 1 h at room temperature, sections were incubated in rabbit polyclonal anti-laminin antibody 1/50  or rabbit polyclonal anti-fibronectin antibody 1/400  in 0.01 M PBS with 0.1% triton in a moist chamber at 4 °C for 48 h. Sections were then washed 3 × 10 mins in 0.01 M PBS and incubated in goat anti-rabbit IgG 1:200 (Vector Laboratories) in 0.01 M PBS with 0.1% triton for 1 h at room temperature. Sections were further incubated with the alkaline phosphatase substrate  for 1 h at room temperature. Anti-laminin or anti-fibronectin immunoreactivity was visualised by incubating tissue sections in glucose oxidase diaminobenzidine nickel (DAB) solution for 5 mins . Sections were then washed, dehydrated, cleared in xylene and coverslipped with DPX (Thermo Scientific).P < 0.05.Tissue sections were examined using an Olympus dot slide microscope. Randomly selected regions (1.7 mm × 2.2 mm) of white matter (1 per slide) were captured from each section and imported into Image J software   to calcu2/95% air) at 37 °C and the medium was refreshed every 2–3 days according to the manufacturer’s instructions. To confirm that the cells are indeed smooth muscle cells, we analysed the presence of smooth muscle actin through immunofluorescence with anti-alpha smooth muscle actin 1/200 in 0.01 M PBSt .Human brain vascular smooth muscle cells (HBVSMC) were obtained from Sciencell (sc-1100). Cells were grown in the smooth muscle cell medium purchased from Sciencell (sc-1101) supplemented with smooth muscle cell growth supplement (sc-1152), 100 U/mL penicillin, 100 μg/mL streptomycin (sc-0503) and 2% foetal bovine serum. Cells were maintained in a humidified atmosphere . Cells were seeded into two 96 well plates with poly-L-lysine coated wells at a density of 104 cells per well. Plates were incubated for 72 h in either normoxic conditions  or conditions of increased CO2  and maintained at 37 °C. 20 μL of MTS reagent was added to the cells and the plates were wrapped in foil and incubated for a further 2 h for the reagent to develop. Blank wells containing no cells were also included and their absorbance value was subtracted from the test wells. Absorbance was measured at 490 nm with a Fluorostar optima plate reader and the results were exported to Microsoft Excel.We first assessed for cell proliferation and metabolic activity of HBVSMC in either a normoxic environment or conditions of increased CO2. HBVSMC were plated onto poly-L-lysine coated coverslips in a 24-well plate at a density of 0.5 × 105 cells per coverslip with 1 mL of smooth muscle medium. Cells were then exposed to either normoxic (5% CO2/95% air) or conditions of increased CO2  for 72 h and fixed with 4% paraformaldehyde for 10 min before immunofluorescent staining with either with anti-laminin  or anti-fibronectin  antibodies, diluted to 1:200 and 1:400 in 0.01 M PBS respectively. Cells were examined by using a Leica SP8 confocal microscope and three non-overlapping z-stacks were captured per stained coverslip (each an area of 0.15 mm2). The fluorescence intensity of laminin and fibronectin staining was calculated using the RGB measure tool in ImageJ. The results from individual images were averaged to give a mean value per coverslip and are based upon data gathered over three independent experimental runs (n = 3). Statistical analysis was performed using SPSS Statistics version 26.0 (IBM) and a Mann-Whitney U test with significant set at P ≤ 0.05.We next assessed for expression of laminin or fibronectin from HBVSMC in normoxic and conditions of increased COTo determine the pattern of intramural Peri-Arterial Drainage (IPAD) in the white matter, the pattern of drainage of fluorescent Aβ40 injected into the corpus callosum was compared with the drainage of Aβ40 injected into the hippocampus of young adult (10-week-old) C57/Bl6 mice. Immunohistochemistry and confocal microscopy was used to assess differences / similarities in vascular density and IPAD between the hippocampus and corpus callosum.Qualitative assessment revealed the pattern of IPAD normally observed in the hippocampus , charact2, p < 0.05). Conversely, the density of capillaries with Aβ40 in their vessel walls was significantly higher in the corpus callosum versus hippocampus   but we observed no significant differences in density of arterioles  or venules   and a significant decrease in fibronectin expression  in the white matter of subjects with WMH compared to aged-matched controls  .. Fibronectin was decreased but not significantly (p = 0.827)  and there is a complex relationship between fibronectin and MMP2 in humans that varies according to the changes in the time from the hypoxic event , 44. OurIt has been demonstrated in experimental work in rodents that laminin increases after hypoxia , 47, butIt has been known for several decades that the majority of the interstitial fluid in the white matter drains along the white matter tracts , 52. In Many of the fine details of fluid dynamics withn the brain tissue and IPAD await explanation but what seems certain is the involvement of arteries and capillaries not only in the delivery of blood but also in the drainage of fluid and soluble matabolites from the brain. It may be possible therefore to combine therpeutic approaches to resolving ischaemia/hypoxia and failure of IPAD in WMH.


The flow procedure was optimized and validated, paying special attention to its selectivity and sensitivity. The LOD was 31 nmol·L−1, while the within-day and day-to-day precisions were better than 1.0% and 5.0%, respectively (n = 6). Random urine samples from adult volunteers (n = 7) were successfully analyzed without matrix effect (<1%). Endogenous HIS content ranged between 116 and 1527 μmol·L−1 with percentage recoveries in the range of 87.6%–95.4%.In the present study, the determination of histidine (HIS) by an on-line flow method based on the concept of zone fluidics is reported. HIS reacts fast with  The order of mixing proved to have negligible impact on the sensitivity and the aspiration order of OPA/Buffer/Sample was adopted for all subsequent experiments.In a second series of preliminary studies, it was confirmed that the reaction can proceed under zone fluidic conditions and that was promising for further development and optimization. Initial ZF experiments were performed under the following starting variables: [OPA] = 10 mmol·L−1), and the volumes of the sample (50–125 μL) and the OPA/Buffer (25–75 μL).The development of the ZF method involved experimental studies of the various chemical and instrumental parameters that are expected to affect the performance of the procedure, such as: the pH of the reaction (6.0–9.0), the reaction time (0–120 s), the reaction temperature (25–40 °C), the amount concentration of OPA (5–15 mmol·LAs can be seen in −1. The reaction mixture is “trapped” in the reaction coil (RC) and the flow is stopped for increasing time intervals. As can be seen in The effect of the reaction time was investigated under stopped-flow conditions. In brief, following aspiration of the zones in the holding coil (HC) of the ZF setup, the flow was reversed for 30 s at 0.6 mL·min−1 HIS) and the signals practically doubled in this range. A sample injection volume of 100 μL was selected in terms of satisfactory sensitivity, sampling rate, and sample consumption. On the other hand, the volumes of the OPA reagent and the buffer had a rather moderate effect on the method. In both cases, 50 μL were selected taking into account the sensitivity and the consumption of the reagents.The volumes of the samples and reagents zones are an important variable that affects both the mixing efficiency and the dispersion/sensitivity. The sample injection volume vs. the fluorescence intensity had an almost linear profile in the range of 50–125 μL (at 2 μmol·L−1. The criteria were the sufficient excess of the reagent and its effect on the kinetics of the reaction (sensitivity). The experimental results confirmed that the method was unaffected at OPA amount concentrations >10 mmol·L−1, where the signals reached a plateau. The latter value was therefore selected for subsequent validation experiments.Finally, the amount concentration of the reagent was investigated in the range of 5–15 mmol·LAn overview of the studied instrumental and chemical variables, the investigated range and the selected values is included in The proposed method has been validated for linearity, limits of detection (LOD) and quantification (LOQ), precision, selectivity, matrix effect, and accuracy.−1 (20–310 μg·L−1) HIS. The regression equation was obtained in a “cumulative” way by incorporating the results from more than 90 standard solutions analyzed in different working days (n = 8). In this way, the calibration curve is more representative including potential day-to-day variations. The following regression Equation (1) was obtained:F is the fluorescence intensity as measured by the detector. Linearity was further validated using the back-calculated concentrations . The percent residuals were distributed randomly around the “zero axis” and ranged between −13.2% and +9.5%. It should be noted that the highest positive and negative values correspond to residuals at the lowest point of the calibration curve close to the LOQ of the method.The method proved to offer satisfactory linearity in the range of 125–2000 nmol·Lb is the standard deviation of the intercept and s is the slope of the respective regression lines. The calculated LOD/LOQ for the analyte was 31 and 93 nmol·L−1, respectively, corresponding to 4.8 and 14.4 μg·L−1 HIS.The LOD and LOQ was estimated based on the following Equations (2):−1 level by repetitive injections (n = 8). The relative standard deviation (RSD) was 0.5 % . As can be seen in The day-to-day precision was evaluated by independent calibration curves obtained at different working days ((i)against amino-acids and biogenic amines that can potentially react with OPA;(ii)using an artificial urine matrix spiked with the analyte;(iii)using a pooled human urine sample also spiked with the analyte.The selectivity/matrix effect of the proposed method was evaluated towards three axes:−1 (10-fold excess compared to HIS) with the exception of Histamine and Glutathione that were examined at equimolar concentrations to HIS (1.0 μmol·L−1). Based on the experimental results the selectivity factor (S.F.) for each potential interfering compound (INT) was calculated according to the following Equation (3):As can be seen in (i)Alanine, Lysine, Threonine, Serine, and Tyrosine do not seem to react with OPA at the experimental conditions of the ZF method since the obtained signals were not different compared to the blank values.(ii)Glycine, Cysteine, and Glutamate react with OPA, but the selectivity factors are high, ranging between 32–148.(iii)Histamine and Glutathione seem to cause the most serious interference at equimolar levels to HIS. Both compounds are known to react with the tagging reagent in the absence of nucleophilic compounds, yielding fluorescent derivatives ,24. HoweThe results can be categorized as follows:To evaluate the matrix effect, a widely accepted artificial urine sample has been prepared as described in the experimental section. As can be seen in n = 8). In brief, 500 μL of each urine sub-sample were transferred in a centrifuge tube and mixed. An equal volume of ice-cold acetonitrile (4 mL) was added to precipitate proteins followed by centrifugation. The obtained solution was diluted 250-fold and spiked with HIS in the range of 250–1500 nmol·L−1. The experimental regression Equation (4) was:Further evaluation of the matrix effect was carried out using a real pooled urine sample (−1) preparing two independent series of samples in the pooled urine matrix. Quantification was carried out using the cumulative aqueous calibration curve described in The accuracy of the method was validated at three concentration levels  obtained in an extensive study (>800 samples) that has been reported in the literature [Seven random urine samples from male and female volunteers were analyzed according to the optimized and validated method described above. Samples were preliminarily screened to determine the necessary dilution factor in order to fall within the range of the calibration curve. In all cases, the dilution factors were either 500 or 1000 being consistent with the validation of the method in terms of potential matrix effects. The experimental findings are tabulated in terature .The ZF setup was consisted of the following parts: a Minipuls3 peristaltic pump ; a low-pressure micro-electrically actuated 10-port valve ; a RF-551 flow-through spectrofluorimetric detector operated at high sensitivity ; PTFE tubing was used for the connections of the flow configuration (0.5 or 0.7 mm i.d.); and Tygon tubing was used in the peristaltic pump. An HPLC column heater  was employed for the temperature control (60 ± 0.5 °C) of the reaction coil (100 cm/0.5 mm i.d.); the latter was tightly wrapped around the stainless-steel body of an old HPLC column (4.6 mm i.d.).® software . Off-line spectra were recorded using a RF-5301PC batch spectrofluorophotometer .Control of the ZF system was performed through a LabVIEW  based program developed in house; while data acquisition (peak heights) was carried out through the Clarityo-phthalaldehyde , KH2PO4 , NaOH (Merck) and HCl (Sigma) were all of analytical grade. Doubly de-ionized water was produced by a Milli-Q system .Histidine  was purchased by Sigma ; −1 level in water. Working solutions were prepared by serial dilutions in water. The derivatizing reagent (OPA) was prepared at an amount concentration of 10 mmol·L−1 by firstly dissolving in 0.5 mL methanol and subsequently adding 9.5 mL water [−1) was also prepared daily and regulated to the desired pH value (pH = 7.5) by drop-wise addition of NaOH (1 mol·L−1).The standard stock solution of the analyte was prepared daily at the 1000 μmol·LmL water . This so−1), citric acid (2.0 mmol·L−1), sodium bicarbonate (25 mmol·L−1), urea (170 mmol·L−1), calcium chloride (2.5 mmol·L−1), sodium chloride (90 mmol·L−1), magnesium sulfate (2.0 mmol·L−1), sodium sulfate (10 mmol·L−1), potassium dihydrogen phosphate (7.0 mmol·L−1), di-potassium hydrogen phosphate (7.0 mmol·L−1), and ammonium chloride (25 mmol·L−1). The pH of the solution was adjusted to 6.0 by addition of 1.0 mol·L−1 HCl.Synthetic urine (200 mL in water) was prepared according to the literature  and was All other amino-acids and biogenic amines employed in the selectivity studies were of analytical grade and were supplied by Sigma. All solutions were prepared in water at the levels mentioned in the respective section.−1), buffer  and sample/standards (100 μL) were sequentially aspirated in the holding coil (HC).The experimental conditions of the optimized ZF sequence for the determination of HIS can found in −1 and the reaction was allowed to develop for 60 s under stopped-flow conditions. Downstream fluorimetric detection was carried out at λex/λem = 360/440 nm. The sampling throughput was 16 h−1.Upon flow reversal, the stacked reaction zones were propelled towards the thermostated reaction coil  at a flow rate of 0.6 mL minRandom urine samples were kindly donated voluntarily  by male and female members of the laboratory. Samples where either processed immediately or stored at −20 °C until analysis .(i)protein precipitation with addition of ice-cold acetonitrile (1 + 1);(ii)centrifugation ;(iii)500–1000-fold dilution depending on the levels of HIS in the real samples;(iv)Analysis by the ZF method.Due to the selectivity and sensitivity of the developed method, sample preparation included the following simple and rapid steps:It utilizes readily available reagents and due to the zone fluidics-based concept the consumption and generation of wastes is minimal compared to continuous flow techniques such as HPLC and Flow Injection Analysis.The method is based on direct reaction and is advantageous compared to indirect methods based on inhibitory effects.The high sensitivity of the method down to the nano-molar level enables the direct analysis of Histidine in human urine with minimum sample preparation.The unique mechanism of the derivatization reaction excluded interference from most amino-acids and biogenic amines offering a highly selective platform for the determination of the analyte in the complicated samples without matrix effects.−1.Application in random urine samples was successful at a reasonable sampling frequency of 16 hThe developed on-line fluorimetric method for the determination of Histidine offers some interesting features:


Castor) are prolific ecosystem engineers and dramatically alter the landscape through tree harvesting and dam building. Little is known, however, about the evolutionary drivers of their woodcutting behaviour. Here we investigate if early woodcutting behaviour in Castoridae was driven by nutritional needs. We measured stable carbon and nitrogen isotopes (δ13C and δ15N) of coeval subfossil plants and beaver collagen (Dipoides sp.) from the Early Pliocene, High Arctic Beaver Pond fossil locality (Ellesmere Island), in order to reconstruct Dipoides sp. diet. Isotopic evidence indicates a diet of woody plants and freshwater macrophytes, supporting the hypothesis that this extinct semiaquatic beaver engaged in woodcutting behaviour for feeding purposes. In a phylogenetic context, the isotopic evidence implies that woodcutting and consumption of woody plants can be traced back to a small-bodied, semiaquatic Miocene castorid, suggesting that beavers have been consuming woody plants for over 20 million years. We propose that the behavioural complex  preceded and facilitated the evolution of dam building. Dam building and food caching behaviours appear to be specializations for cold winter survival and may have evolved in response to late Neogene northern cooling.Modern beavers ( Castor harvests trees and shrubs for sustenance (particularly during the winter1), but also for the purpose of lodge and dam building. These behaviours have a profound effect on regional hydrology, nutrient flow across the landscape, and local biodiversity, thus making them exemplary “ecosystem engineers”4. Modern beavers also are known to feed on trees. This food source is particularly important for northern populations that survive freezing winters, subsisting on their underwater caches of leafy branches6.Beavers today are renowned for their woodcutting behaviour and construction abilities. The extant genus 9. The only definitive evidence of woodcutting in extinct beavers is from the Pliocene-aged High Arctic Beaver Pond fossil site, which preserves evidence of beaver-cut wood and a possible dam-core associated with the extinct beaver genus, Dipoides (species not yet described)11. Examination of Dipoides sp. incisors and cut marks on wood from the Beaver Pond site suggest that Dipoides sp. woodcutting performance was poorer than that of modern Castor11. The appearance of woodcutting in Dipoides sp., a distant relative of Castor, implies that this behaviour originated 20 to 24 Ma ago, in a group of semiaquatic beavers that includes both extant species (Castor canadensis and Castor fiber), the small Holarctic genus Dipoides, and the North American Ice Age giant beaver Castoroides12. And yet recent research shows that the diet of Ice Age Castoroides, a close relative of Dipoides, was dominated by submerged plants, not trees and shrubs13. This finding, alongside a lack of definitive evidence of lodges or dams at the Beaver Pond site highlights the question: Does the cut wood at the Beaver Pond site represent a means of gathering food?Castoridae is a diverse family of herbivorous Holarctic rodents, originating during the late EoceneDipoides sp. subfossil remains. The specimens originate from a peat deposit at the Beaver Pond fossil site, located on Ellesmere Island , situated within the Canadian Arctic Archipelago   plant macrofossils and (ii) bone collagen from High Arctic  W) Fig. . We reco14. The thickest peat deposit within this sequence, which yielded material for this study, sits 380 m above present day sea-level overlooking Strathcona Fiord. Most recent terrestrial cosmogenic nuclide burial dating of sands above this peat layer have yielded a date of 3.9 + 1.5/− 0.5 Ma15, placing the minimum age of peat formation during the late Early Pliocene (late Zanclean). This peat unit is characterized by an abundance of beaver-cut sticks11 and has yielded a wide array of beautifully preserved plant macrofossils, invertebrates, and vertebrate fossil remains . It is hypothesized that northwest passages did not exist during the Pliocene, as they had yet to be incised by fluvial and glacial erosion26. This unbroken coastal plain altered ocean circulation patterns in the High Arctic, and along with the Bering Isthmus that connected North American and Eurasia until 5.5 Ma ago27 would have enabled the migration of terrestrial species across the Neogene High Arctic10.Ellesmere Island today is a polar desert, with sparse flora and very little precipitation28 and high latitude regions experienced amplified warming. Pliocene Arctic mean annual temperature was near freezing, which is ~ 15–22 °C warmer than present day, and tree line was ~ 2000 km further north33. Summer temperatures at the Beaver Pond site reached 20 °C, while winter temperatures were more moderate than present day, with a low of ~ – 12 °C24. Despite the relatively mild conditions, the Beaver Pond site still experienced total darkness and subzero temperatures during the winter months.Late Early Pliocene mean global temperature was 2–3 °C above modern36. The Beaver Pond macrofossil assemblage indicates a larch forest, although birch, alder, spruce, pine, cedar, and cold-adapted woody shrubs were also present (see Matthews and Fyles17 for a complete list of identified flora). The Beaver Pond site supported higher faunal biodiversity than any modern near-tree line communities.There is no modern analog for the ecological community found at the Beaver Pond site, although the flora of present-day Labrador (Canada) is considered to be similarDipoides sp., see below), three-toed horse, bear, badger, “deerlet”, water fowl, fish and a rabbit relative22. Pliocene-age sites with similar fauna and flora community composition are very rare—Idaho in mid-continent North America, and the high altitude Yushe Basin, in northeastern China are the only known sites with similar  faunal assemblages38.The remains of a complex faunal community were discovered at the Beaver Pond site, including beaver  the ecological impact of Dipoides sp. on the Pliocene landscape, (ii) Dipoides sp. potential for winter survival strategies such as underwater food caching, and (iii) the evolutionary context of tree-exploitation within the Castoridae family.Here we use stable isotope data to understand δ13C) and nitrogen (δ15N) isotope compositions of an animal’s bodily tissues correlate closely with that of its diet, when adjusted for 13C- and 15N-enrichment that occurs during collagen formation and with each successive trophic level44. Well-preserved bone collagen is therefore a useful integrator of an animal’s diet. In addition, sufficient context is required to accurately describe the nutrient flow between subsequent trophic levels of an ecosystem. In particular, the diet of an organism must be interpreted within the context of an appropriate dietary baseline. This baseline is composed of isotopically defined food or “menu-items” available to the organism.The stable carbon (δ13C and δ15N of the dietary baseline for herbivores. The δ13C and δ15N of primary producers depend on physiology (i.e. which photosynthetic pathway the plant employs) and the isotopic composition of bioavailable sources of C and N (i.e. atmospheric CO2). Casey and Post45 provide a thorough review of how primary producer δ13C and δ15N vary with physiology and local terrestrial and aquatic environmental conditions. A particular challenge in many forested-wetland environments, however, is that the carbon and nitrogen isotope range of terrestrial and freshwater plants overlap. There are, however, sufficient differences between the δ13C and δ15N of terrestrial vegetation utilizing the C3-photosynthetic pathway and vascular freshwater plants (macrophytes) for them to serve as useful endmembers of herbivore diet in such environments (see Methodology).The isotopic composition of primary producers at the base of the food chain control the 47. Hence, reconstructing the diet of herbivores that lived thousands or millions of years ago can be problematic when using isotopic data, as is very rare to find sufficiently preserved coeval plant material and faunal remains from the same geologic locality. Typically, isotopic data for modern plants are all that are available in palaeodiet studies. Fortunately, much of this concern is alleviated at the Beaver Pond site, given the excellent organic preservation of coeval plant and animal tissues.Another challenge is that the isotopic composition of regional and global C and N baselines  can change over timeDipoides sp. (n = 5) bone collagen stable isotope results (δ13Ccol and δ15Ncol) are presented in Table Dipoides sp. δ13Ccol ranges from − 20.8 to − 19.1‰, with a mean of − 20.3‰, and δ15Ncol ranges from + 3.2 to + 5.8‰, with a mean of + 4.7‰ .Atomic C:N ratio and carbon and nitrogen contents (wt%) were used to assess collagen preservation for al Table . All speScorpidium scorpioides, Larix, Betula, Stuckenia filiformis, Scheuchzeria sp., Cornus sericea, Menyanthes trifoliata) (Table Scorpidium scorpioides (hooked scorpion moss). Larix (larch—a deciduous conifer) was the only conifer species identified from this peat sample  and from three species of Carex  were also recognized, but in insufficient quantities for stable isotope analysis.By volume, the Beaver Pond peat sample examined in this study consisted of 85% bryophytes, 10% wood and twigs, and 5% macrofossils. Eleven genera were identified, representing a diverse assemblage of terrestrial and freshwater plants Table . Seven ta) Table . The domδ13C, δ15N, C (wt%), N (wt%), C/N (wt%), and atomic C:N ratios are all within the range expected for terrestrial and freshwater plants  values obtained in the present study are within or close to the mean carbon content for modern plants (between ~ 40 and 47%) . Plant macrofossil N (wt%) values are lower than the mean nitrogen contents of modern plants (between ~ 1 and 3%) but are not outside the reported range for modern plants.The chemical and elemental compositions of plants vary widely by species, life history stage (i.e. senescence), and environment conditions. For these reasons, atomic C:N ratio and carbon and nitrogen content are not considered to be infallible indicators of organic preservation in subfossil plantsDipoides sp. δ13Ccol and δ15Ncol within the context of the isotopic dietary baseline composed of coeval terrestrial and freshwater vegetation from the High Arctic Beaver Pond fossil site. The faunal and plant macrofossil isotope data were incorporated into a Bayesian mixing model to determine the relative input of terrestrial versus freshwater plants to Dipoides sp. diet  to estimate the relative input of different sources to a product or mixture. In ecology, mixing models use the stable isotope compositions of different food sources to infer their relative contributions to the composition of overall diet, and assess the probability that the inferred proportions are correct. There are systematic differences in the isotopic composition between a consumer’s collagen and its diet, both for carbon and for nitrogen. Hence, a correction factor must be applied to render data for consumers and possible diet items directly comparable.Dipoides sp. diet. The functional types were statistically defined and their relative contribution to diet was assessed using scripts from SIAR V4 in R Studio 3.1.2 . Based on existing literature, respective bone collagen-to-diet offsets of + 4.2‰ and + 3.0‰ were subtracted from Dipoides sp. δ13Ccol and δ15Ncol when incorporated into the mixing model53.Plant macrofossils were divided into three sources, or functional types: terrestrial woody plants, vascular freshwater macrophytes, and bryophytes (mosses). These groupings were created to assess how terrestrial and freshwater resources contributed separately to Dipoides sp. consumed both woody plants and freshwater macrophytes in approximately equal proportions  or environmental conditions in the water column (i.e. boundary-layer effect)56. In addition, tree bark is more enriched in 13C than tree foliage57 and may have been a key resource for Dipoides sp.The distribution of zed Fig. . This isDipoides sp. diet. It is likely that Dipoides sp. also used shrubs and trees as a source of construction material11, but more evidence is needed to confirm this. Similar to extant Castor, Dipoides sp. may have also demonstrated regional differences in diet, where northern and southern populations utilized different resources according to their availability.The results of the dietary mixing model support the interpretation that woody plants were an important contributor to 58. Beaver Pond plant macrofossil N (wt%) and C/N are highly variable (Table Plant macrofossil nitrogen content (N wt%) and C/N are indicators of forage quality and may be used to interpret the relative nutrition of dietary inputs. Plants with high N (wt%) contain more protein and energy—likewise, low N (wt%) correlates with low plant digestibility, high fiber and high lignin compound contentle Table , Fig. 5.60.In extremely seasonal environments such as the High Arctic, herbivores must use plant resources in a highly efficient manner. Herbivores must consume the highest quality forage possible during the brief growing season to maximize nutrient and energy gain. High quality forage typically includes young leaves with high nitrogen content, minimal structural (fibrous) tissues, and low defense compound contentDipoides sp. at the Beaver Pond site. It should be noted that forage quality is not the only factor that governs herbivore feeding behaviour. Animals may preferentially target plants with higher biomass to minimize energy expenditure traveling between forage sites or select plants that grow in locations that minimize the risk of predation.Within the Beaver Pond macrofossil assemblage, pod grass (an emergent macrophyte) and birch have the highest nitrogen content and lowest C/N Fig. . A large58. As there is no time-constraint on macrofossil deposition at the Beaver Pond site, variation in C/N may also be due to differences in plant phenological stage at time of incorporation into the peat layer. The incorporation of senescent plants into the peat deposit at the end of each growing season may in part account for the lower than expected macrofossil N (wt%) values reported from this site.The C/N of high Arctic shrubs decreases over the course of the growing seasonThe Beaver Pond macrofossil assemblage contains a diverse range of terrestrial and freshwater plant species. The identified plant species in this study concur with previous interpretations that this was an open-forest landscape interspersed with shallow wetlands. Larch trees and cool-climate woody shrubs dominated the forest community. The wetlands supported both vascular macrophytes and dense assemblages of bryophytes.δ13C are all within the range expected for primary producers utilizing the C3 photosynthetic pathway and accessing either ambient or dissolved atmospheric CO2 as their dominant carbon source. The δ15N of the macrofossils are also within the expected range for a riparian ecosystem in a cool climate biome.The macrofossil Dipoides sp. most likely consumed leafy tree branches and woody tissues (cambium), it is worth noting that plant seeds and cone bracts were analyzed in this study due to ease of macrofossil taxonomic identification. Leaf δ13C is typically lower than that of other plant parts61, although there is no clear pattern in intra-plant variation of δ15N.While Scorpidium (hooked scorpion moss), have very low δ13C for a primary producer (− 36.6 to − 34.6‰). This pattern is consistent with modern mosses collected from freshwater habitats in Subarctic and Arctic regions64.Samples of the dominant Beaver Pond site bryophyte, δ13C rather than species-specific physiological differences. Peat mosses can grow partially or fully submerged in water. Given that mid-Pliocene atmospheric CO2 concentration levels were similar to modern (~ 400 ppm)66, moss exposed to the atmosphere would preferentially have used the abundant 12CO2, resulting in low δ13C. Alternatively, low δ13C in peat mosses can also indicate an underwater growing environment rich in 13C-depleted respired CO2 from surrounding plants62.Environmental conditions dictate moss δ15N is relatively high for a photosynthetic organism (mean =  + 4.8‰). This is indicative of either the presence of 15N-enriched sources of bioavailable N , or increased nutrient availability61. Unlike vascular plants, mosses do not uptake compounds through their roots. Rather, they obtain nutrients from wet or dry deposition through their leaves68. Today, beaver ponds are considered to be N sinks, with elevated rates of bacterially mediated denitrification69. These bacterial processes result in 15N-enriched products that are readily dissolved and used by plants (including moss) living in an aqueous environment. Decomposition processes also increase plant δ15N over time47 and remineralized organic debris decomposing in wetlands may be particularly 15N-enriched.Moss macrofossil δ13C and high δ15N), which suggests that hooked scorpion moss contributed substantially to peat biomass accumulation at the Beaver Pond site.Beaver Pond bulk peat samples and moss macrofossils show a similar isotopic pattern . It is reasonable to assume, however, that the very small sample size in this study hides the potential extent of the carbon isotope variability of macrophytes at the site.Beaver Pond macrophyte 13C as the dissolved CO2 pool (the dominant carbon source) becomes increasingly limited54.Pod grass and bogbean are classified as emergent macrophytes , while pondweed grows entirely submerged. Submerged macrophytes become more enriched in δ13C is relatively low (− 26.5‰) for a submerged macrophyte. This indicates that it grew in an aquatic environment with adequate dissolved CO2. This is in keeping with the interpretation that the Beaver Pond was a fen  during the Pliocene. A low δ13C may also indicate high influx of terrestrial organic biomass or mosses (with low δ13C) into the water that subsequently remineralized and contributed to the dissolved inorganic carbon pool.The Beaver Pond site pondweed δ15N. Beaver Pond macrophyte δ15N (range =  + 0.2 to + 2.7‰) indicate interspecific access and use of a variety of different sources of bioavailable N within the water column and substrate. The most likely N sources are microbial-fixed atmospheric N2 (which ranges from –2 to + 2‰), the products of nitrification/denitrification processes (15N-enriched NH4+ or NOx), and remineralized 15N-enriched organic material 73.Environmental conditions strongly influence aquatic plant Larix groenlandii) is the most common vascular plant species in this macrofossil assemblage.Larch (the extinct species δ13C of (i) larch shoots/buds (which bear the needles) and cone bracts, and (ii) larch seeds. Larch shoots and cones  are more depleted of 13C than larch seeds . This could be indicative of seasonal physiological or environmental conditions experienced by larch trees at the Beaver Pond site. The cones and shoots of extant larch trees begin growing in the early spring and have lower δ13C, whereas their seeds (higher δ13C) do not develop and mature until mid to late summer74.There is an offset of ~ 2‰ between the 75. This restricts not only the rate of water leaving the needle/tree, but also that of atmospheric CO2 entering it. Stomatal closure reduces CO2 entry and results in less discrimination against 13CO2. High levels of solar irradiance in the summer increase the rate of CO2 assimilation. Plants growing at very high latitudes experience 24-h of daylight during the summer. This creates a greater demand for CO2 to maintain photosynthesis and less discrimination against 13CO2. Both aridity and increased light levels could contribute to why Beaver Pond larch tissues grown late in the summer are more 13C-enriched than those grown in the early spring/the previous fall.A number of physiological and environmental conditions could be responsible for this offset between needle/-bearing structures and seeds. Atmospheric vapor pressure deficit (aridity) induces stomatal closure in vascular plantsδ13C of photosynthetic conditions from the previous growing season77. In addition, differences in the macromolecular  composition of larch buds/needles versus seeds could account for their offset in δ13C .Alternatively, trees can use water and carbon (in the form of sugars) stored during the previous year to promote new growth during the early spring when leaves are absent and light levels are low. Tissues that develop early in the growing season (i.e. needle-bearing buds and shoots) can therefore reflect the δ15N (mean =  + 2.7‰) indicate that these conifer trees had access to N sources other than “light” fixed atmospheric N2. Given the proximity of wetlands, the root systems of larch trees may have had access to 15N-enriched dissolved nitrates in the surrounding water-logged soils. Increasing foliar N concentration due to atmospheric N deposition also drives up plant δ15N79.Larch δ15N, where rainier ecosystems tend to produce more 15N-depleted plants80.Aridity may also have influenced terrestrial plants growing at the Beaver Pond site. Higher rainfall is inversely correlated with δ13C, there is an offset in δ15N (and N wt%) between larch needle-bearing structures  and larch seeds . This could indicate differences in the macromolecular composition of these different tissue types .Similar to Dipoides sp. diet differs from that of other members of Castoridae that lived in North America during the late Cenozoic. Pliocene High Arctic Dipoides sp. (n = 5), modern subarctic Castor canadensis (n = 4) (Table Castoroides ohioensis (n = 11)  Table , and latnsis n = 1 (Table δ13Ccol of modern C. canadensis (collected in 2013 and 2014) and Castoroides (late Pleistocene in age), respectively. These corrections were based on the average δ13C of atmospheric CO2 (δ13CCO2) calculated from Pliocene dual-benthic and planktonic foraminifera proxy records, spanning from ~ 4.1 to 3.8 Ma (average δ13CCO2 = − 6.55‰)76. These foraminifera proxy records are approximately contemporary with the Beaver Pond site. Average δ13CCO2 for 2014 (− 8.57‰) was compiled from the Scripps CO2 monitoring program. Average δ13CCO2 for the late Pleistocene (− 6.45‰) was compiled using ice core data from Schmitt et al.82.Suess effect corrections of + 2.02‰ and − 0.1‰ were applied to the δ13C of contemporary atmospheric CO2. Therefore, changes in δ13CCO2 help explain differences in δ13C between Dipoides sp. and modern C. canadensis. Additional factors, however, are important in explaining the wide range of δ13C and large enrichment in 13C measured for Castoroides.Plants growing during these three different time periods  would reflect the Dipoides sp. diet composition differs from that of Castoroides (the Pleistocene giant beaver)  indicate a diet composed predominantly of aquatic (particularly submerged) macrophytes and minimal woody plant material (Table 13.r) Figs.  and 7. CCastoroides, both Dipoides sp. and C. canadensis have a relatively small range of δ13Ccol and δ15Ncol .δ15N baseline of flora in terrestrial high latitude environments during the Pliocene would be a valuable avenue of future research.Changes in the isotopic composition of the C and N baseline between the Pliocene and the present could also account for the isotopic offset between beaver species. Further investigation of possible changes in the Dipoides sp. ecology. These data also contribute to our understanding of the evolution of behavioural transitions within Castoridae. In particular, how Castor’s distinctive complex of behavioural traits  may have evolved. A new hypothesis of behavioural evolution in castorids based on evidence from the fossil record  and skeletal-dental morphology is mapped onto a simplified phylogenetic tree in Fig. 887.Evidence from the Beaver Pond site has implications for our understanding of 90. The latter includes Castor and Dipoides. Members of the fossorial clade (~ 7 genera) possess striking specializations such large digging claws, extremely reduced tails, and broad, procumbent incisors for digging. In some cases, specimens have been found within fossil burrows . The semiaquatic group comprises two subfamilies, Castorinae , and Castoroidinae . The oldest definitive Castorinae in the fossil record is Steneofiber eseri from the early Miocene . S. eseri shows evidence of living in family groups and swimming specializations91. This, in combination with aDNA evidence12, suggests Castorinae and Castoroidinae are derived from a semiaquatic ancestor in the early Miocene.Castoridae is a group of herbivorous rodents comprising roughly two dozen genera. Most fossil castorids fall within two major groups: a clade of fossorial specialists  and a semiaquatic cladeCastor, though not morphologically highly specialized for the task, digs bank burrows and creates extensive canal systems92. In addition, the extinct semiaquatic beaver Steneofiber eseri was found within a burrow91. Considering the phylogenetic distribution of burrowing behaviour within the Castorid tree , Myocastor (nutria), and Ondatra (muskrat)92. It is also possible that Dipoides constructed lodges. Extant Castor and Ondatra are known to construct burrows and lodges, depending on the characteristics of the habitat. Bank burrows are associated with stream environments, whereas lodges are better suited to calmer waters92. Unlike Castor, extant Ondatra construct their push-up lodges using cattails and other fibrous vegetation rather than wood. The abundance of cut wood at the Beaver Pond site11 suggests that Dipoides sp. had the option to incorporate wood into their nesting structures, and possibly built lodges.If burrowing behaviour in semiaquatic castorids is the primitive condition, it is likely Castor and Dipoides in Fig. Castor canadensis does not hibernate, but builds and sink rafts of branches and foliage to use as a source of fresh food during the winter months93. Dipoides sp. may have also engaged in this behaviour and used underwater caches of branches as a primary food source to survive the consecutive months of darkness during the high latitude winter when plants become dormant. The use of woody plants in this way may have been key to allowing beavers to disperse between North American and Eurasia, which required crossing the Bering Isthmus94, a high latitude landmass. Curiously, given that a diet rich in woody plants appears to be the primitive condition of semiaquatic castorids, the absence of woody plant consumption seen in the Pleistocene giant beaver Castoroides13 must be interpreted here as an evolutionary loss and potentially a leading factor in their extinction  and Steneofiber eseri (Castorinae) were small , suggesting that the common ancestor of the semiaquatic lineage was also small bodied. Although the common ancestor of the semiaquatic beaver lineage is inferred to have consumed woody plants (this study), and may have used branches in creating food piles and wood for lodge construction, it would have been too small to have had the capacity to build and maintain dams. As such, if Dipoides sp. did exhibit dam building behaviour, it would be the result of parallel evolution within the Castoroidinae and Castorinae lineages.It seems unlikely that the common ancestor of all semiaquatic beavers was a dam-builder. Extant Dipoides sp. palaeodiet from bone collagen δ13C and δ15N within the context of an isotopic dietary baseline composed of coeval ~ 4 Ma old terrestrial and freshwater plant macrofossil remains. The Beaver Pond site provides a very rare opportunity for such a palaeodiet reconstruction using coeval herbivore and plant remains. A Bayesian mixing model indicates that Dipoides sp. diet was composed of approximately equal proportions of woody plant material and freshwater macrophytes, with slightly more emphasis on macrophyte consumption. Dipoides sp. dietary preferences lie somewhere in between those of other North American late Cenozoic semiaquatic beavers .Here, we reconstruct Pliocene High Arctic Dipoides sp. at the Beaver Pond fossil site was the result of harvesting for consumption, possibly as part of an underwater winter food cache. The results also suggest that the early Miocene ancestor of the semiaquatic beaver lineage engaged in woodcutting and consumed woody plants as part of its diet. Swimming, woodcutting, and a diet of woody plants could have set the stage for the evolution of dam building behaviours—advantageous traits that may have been selected for by the cooling climate of the late Neogene, and which have resulted in Castor’s modern role as a keystone species and ecosystem engineer.The consumption of woody plant material suggests that a proportion of the assemblage of the wood cut by Dipoides sp. skeletal material and the plant macrofossils used in this study originated from the Beaver Pond fossil site, Unit III, as defined by Mitchell et al.21. Unit III is a peat layer that yielded the majority of the beaver-cut sticks and vertebrate faunal remains discovered at the site. It is interpreted to have been a rich fen connected to open water, within a larch-dominated forest ecosystem21.The Plant macrofossils were isolated from bulk samples of Unit III peat and identified to taxon. Macrofossils were extracted from the peat using a combination of water-flotation and wet-sieving. Organic material greater than 0.425 mm was retained for further cleaning. Adhered sediment and moss were removed from the macrofossils using surgical forceps and repeated ultrasonic water baths. Cleaned macrofossils were dried at 26 °C for 24 h and identified to taxon using a binocular microscope.Dipoides sp. bone collagen was extracted and its δ13C and δ15N measured at the Alaska Stable Isotope Facility (UAF). Collagen extraction was performed using a modified Longin98 method of gelatinization. Organic contaminants were removed using XAD-2 resin100 and collagen purification was performed according to methods developed by Matheus101. Non-soluble collagenous portions were rinsed to neutral pH, but not subjected to base treatment. Collagen was gelatinized in weak HCl (pH 3) under N2 gas at 105° C until dissolved (2 to 6 h). The solution was centrifuged and filtered with a 0.45 µm syringe-type PTFE filter and the supernatant containing dissolved collagen was lyophilized and weighed to determine the percent collagen yield. Lyophilized collagen was then hydrolyzed in 6 N HCL under N2 gas for 4 h at 120 °C. The hydrolyzates were passed by gravity flow through 2 cc of compacted Serva XAD-2 HPLC resin in syringe columns to extract humates and other long-chain organic contaminants that can adhere to fossil collagen. The hydrolyzates were passed through a 0.45 µm PTFE filter placed at the distal end of each syringe column and were dried by rotary evaporation. Stable carbon and nitrogen isotope analysis of the hydrolyzed collagen was performed using a GC-Isolink gas chromatography combustion system coupled to a Thermo Scientific Delta V Plus isotope ratio mass spectrometer operated in continuous flow mode, using helium as the carrier gas.δ13C and nitrogen content ; values of δ15N were determined in the second session, using individually tailored weights based on each sample’s N wt%.Plant macrofossils were powdered using a ball-bearing mill and weighed into tin capsules (0.38 ± 0.02 mg). Stable carbon and nitrogen isotope analysis of macrofossil remains was conducted at the LSIS-AFAR facility at the University of Western Ontario . Samples were analyzed in continuous flow mode using a Costech elemental analyzer (ECS 4010), coupled to a Thermo Scientific ConFlo IV and Delta V Plus isotope ratio mass spectrometer in continuous flow mode, using helium as the carrier gas. One method duplicate  and one analytical duplicate  were included for every ten samples. The carbon and nitrogen isotope measurements of the plant macrofossils were completed in separate analytical sessions. The first session was used to determine δ-notation in per mil (‰) relative to international standards. Collagen δ13C and δ15N were calibrated to VPDB and AIR, respectively. Analytical accuracy and precision were 0.0‰ for δ13C measurements, and 0.2‰ for δ15N measurements.All isotopic results are reported in δ13C and δ15N were calibrated to VPDB and AIR, respectively using USGS40  and USGS41a . Additional reference materials IAEA-CH-6  and NIST-1547 (Peach Leaves)  were used to evaluate instrument precision and accuracy for δ13C and δ15N, respectively. A keratin powder  was also included to monitor instrument drift. Combined instrument and analytical errors were ± 0.1‰ for δ13C, and ± 0.2‰ for δ15N.Plant macrofossil Dipoides sp. and plant macrofossil isotopic results were incorporated into a statistically-based Bayesian mixing model (SIAR V4). This approach provides a statistically robust means of evaluating the relative dietary contributions of woody plants and aquatic primary producers to Dipoides sp. Diet102.The