Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 10 Here, the authors construct and validate an in vitro antibody library from a single, healthy donor. They demonstrate the functionality of the library using several therapeutically relevant targets and were able to obtain many different antibodies with the desired biophysical properties. Effective development of therapeutic antibodies demands high-quality molecules to meet strict manufacturing requirements and success in clinical applications. The first step in antibody discovery is the identification of potential candidate binding targets of interest, with two main methods used: hybridoma generation (or more recently single-cell cloning methods) from the immunization of normal or transgenic animals, and display methods based on naive or immune libraries. The source of diversity for libraries from which antibodies are selected has taken many formats: immune repertoires, generated from immunized animals or humans seropositive for a target; natural naive, derived from the B lymphocytes of nonimmunized donors14, yeast16, ribosome18, and mammalian display21, with phage and yeast display being the most commonly used. These methods couple phenotype to genotype: the gene encoding the displayed antibody is coupled to the antibody itself, allowing the cloning of antibody genes on the basis of the properties  of the encoded antibodies. Once selected, the affinity or specificity of an antibody can be further improved by applying the same display methods to derivative libraries.Specific binders are selected from libraries displayed using platforms such as phage/phagemid23. Diversity correlates with library functionality: the probability of discovering an antibody in the library with desired activity . In the case of antibody libraries, other desirable key features are “developability properties”: stability and resistance to aggregation in solution, thermodynamic stability, and low self-interaction, among others. Therefore, scientists strive to make antibody libraries as large as possible by performing numerous transformations or, less commonly, applying site-specific recombination approaches24. However, unless the underlying genetic diversity is sufficiently large, generating more transformants will not necessarily increase library functionality. This is less of a problem for synthetic or semi-synthetic libraries, in which the theoretical diversity far exceeds the number of transformants that can be routinely obtained. For example, the theoretical diversity of the HuCAL Gold library8 was estimated to be 1060, but the number of transformants, 1010, was the limiting factor. Although the assessment of genetic diversity is clearly an improvement over counting colonies, functional diversity is a more valuable measure, particularly in the case of synthetic libraries in which degenerate oligonucleotides may encode poorly folding, or polyreactive, antibodies, resulting in a larger chemical space that does not necessarily directly correlates with protein functionality.The key feature of any compound library, including antibody libraries, is diversity. In the case of antibody libraries, this is often measured by counting the number of clones obtained after library transformation. However, this is an inaccurate measure at best, plagued by uncertainties in colony counting and the underlying genetic diversity, which can often be significantly less than the number of transformants26. The question remains as to whether increasing the number of donors actually leads to improved antibody diversity and library functionality.For natural naive libraries, the general paradigm has been to increase the natural source of B-cell diversity by increasing the number of donors27. This may be a consequence of cloning or PCR inefficiencies, inherently restricted underlying diversity , or problems with harvesting the natural diversity, by failing to collect and reproduce the entirety of the diversity present in the starting material. It is also possible that the focus on increased donor numbers is misplaced. In a recent paper from DeWitt and colleagues28, using a combined flow cytometry/NGS analysis of B cells from healthy individuals, the authors inferred a ~109 diversity of circulating naive B cells in a donor at any given time, and proposed that naive B lymphocytes do not undergo  further cell division after VDJ recombination prior to encountering antigen. In fact, a library created from one of the greatest number of donors recorded to date (654)23, with a large number of transformants (3.1 × 1010) had one of the lowest VH diversities when assessed by NGS (105), indicating the importance of other factors in library generation beyond donor number. Here we tested the hypothesis that the final number of lymphocytes used to generate a library, and not the number of donors, determines library diversity, and hence library functionality. The main assumption is that harvesting a large number of B cells from a single individual may be as effective as increasing the number of donors. Because the capture and cloning of a diversity of 109 are well within antibody display state-of-the-art capabilities, we set out to determine whether, through the use of good library construction practices, we could produce a highly functional library from a single donor (single donor library or SDL), where the library is intended to maximize the diversity of V genes with favorable biophysical properties to isolate antibodies with therapeutic properties. For this reason, we used a restricted set of primers to bias amplification toward specific antibody families identified by Tiller and colleagues29 for their improved developability profiles including aggregation, solubility, and protease degradation. Further, several other families show increased representation in the naive repertoire which we tried to capture in this library.In recent papers examining library diversity with next-generation sequencing (NGS), estimated genetic diversity was far lower than anticipatedWe hypothesized that through the combined use of leukapheresis technologies, lymphocyte paramagnetic bead purification, careful gene-family focused PCR amplification, and NGS feedback, we could create a highly diverse and functional antibody library from a single donor.+ cells purified from a single individual  and variable heavy domains (IGHV).Here we describe the construction of a phage display library obtained from the population of CD19ual Fig. . Mononucely Fig. . The antely Fig. 31. The u34, ~40 IGKV35, and ~30 IGLV functional genes37, respectively. To maximize library functionality, we used derivatives of a subset of commonly used primers31 to avoid the amplification of germline scaffolds with undesired biophysical properties29 42. Likewise, the IGLV amplicons were generated by PCR using all the combinations of the 4 IGLV forward primers and 3 IGLV-J reverse primers and then mixed to represent the IGLV-J distribution in natural repertoires 40.More than 450 PCR reactions  we followed our previously established strategy3 , considered the most important component in conferring binding activity and specificity, as well as a more comprehensive merged CDR1 + 3 approaches to assess diversity.Because of the previously observed discrepancy between transformant count and actual diversity43, in which there are many low-abundance and a few highly-abundant clones, further complicating complete read coverage and analysis45. MiSeq has proved to be one of the more popular NGS platforms, able to provide a sequencing depth of up to 2.5 × 107 paired-end reads of 300 bp, sufficient to cover the entire IGHV, IGKV, or IGLV regions. However, this is still insufficient to cover the entire diversity of massive libraries, and as a consequence novel informatic approaches have been applied to precisely capture sequence diversity in massively undersampled sequence populations48. The best approach to overcome the undersampling problem is to generate more reads, for which we employed Illumina’s NovaSeq sequencing platform. With >100-fold deeper HCDR3 coverage over the MiSeq platform (~107), NovaSeq (~109) has the potential to provide an accurate measure, rather than an estimation, of the total diversity even though it comes with other limitations (read lengths of 150 bp severely limit V gene coverage whereby one loses CDR2 information).NGS has revolutionized the sequencing and analysis of antibody repertoires, revealing that immune repertoires adhere to heavily-tailed power distributions54 can be incorporated to reduce biological and technological sampling errors53, these approaches require consensus-building (~3–5 reads per identifier) and thus sequencing depth that greatly exceeds actual library size, making these methodologies most powerful for smaller libraries generated from selections . To overcome these hurdles, we employ a hierarchical clonotyping strategy for singletons similar to that previously described60 using both abundance filtering (N ≥ 2) and Hamming distances.Inherently, errors can be introduced throughout the library construction process, particularly during cDNA synthesis and PCR amplification of V genes. Despite deviating from the donor genetic material, these errors contribute to the diversity of a constructed library if they introduce functional amino acid changes. While unique molecular identifiers (UMIs) or molecular amplification fingerprinting (MAF)N ≥ 2). This approach retains singletons that would be lost through abundance filtering alone and would-be essential contributors in a massively undersampled population. After filtering for Phred score 61 and optimal V(D)J alignment E-value scores , to eliminate low-quality reads, we obtained a total of 1.82 × 109, 1.65 × 108, and 1.13 × 108 sequence reads for the IGHV, IGKV, and IGLV libraries, respectively, by NovaSeq analysis  are considered only if they are more than a given Hamming distance away from sequences with greater abundance  to 7.14 × 107 (Hamming = 0)  saturates at ~2.0 × 108 reads converging at ~7.0 × 105 sequences at Hamming 1–3 and ~1.5 × 106 for a Hamming of 0  Table  suggestsf 0 Fig. . Merged While the intended goal was primer-specific amplification of target scaffolds, due to the similarity of V genes and their corresponding primers, we also wanted to assess how well the primers amplified the V families and/or genes of interest. Of the 12 separate IGHV primers used , indicative that ≥80% of the population is constituted by 2 or more read counts for both IGKV and IGLV, respectively , with a lower and upper 10% quantile of 10 and 20, respectively. As expected, a larger proportion of extremely long HCDR3s ≥ 30 is seen within the IGHJ6 antibodies. Light chain CDR3s range from 3–21 to 3–23 for IGKV and IGLV, respectively. Median lengths for IGKV and IGLV are 9 (σ = 1.23) and 11 (σ = 1.45) with lower/upper quantile values of 8/10 and 9/12, respectively, reflecting previous trends of IGKV supporting longer CDR lengths. In contrast to IGHV/IGJHJ pairings, IGKV/IGKJ pairings do not show an increasing length trend across IGKJ families or IGKV families, though abnormal lengths (≥20) are apparent in IGKV1/IGKJ1, IGKV1/IGKJ3, IGKV1/IGKJ4, and IGKV3/IGKJ2.The SDL captures the full range of HCDR3 and LCDR3 lengths (3–42 amino acids) Fig. . The medσ = 1.31), and slightly greater than IGLV-HCDR3 at −0.82(±1.02). The overall net charge ranges from −6.0/6.0, −3.0/5.0, and −6.0/3.5 for IGHV, IGKV, and IGLV, respectively.We observed a broad and even HCDR3 net charge distribution above and below neutral charge using Lehninger’s pK scale at pH 7.0. Median HCDR3 net charge is ~0.00 (±1.39) similar to IGKV-CDR3 at ~0.00 (σ = 0.68) with upper/lower 10% quantile bounds of −1.37 and −0.56, respectively. This bimodal trend is not apparent in light chain CDR3 with Vκ Gravy index values are distributed around the median value of −1.31 (σ = 0.69) with upper/lower 10% quantile bounds of −2.11 and −0.43, respectively. Interestingly, Vλ families show index trends localized around the median of −0.18 (σ = 0.59) with upper/lower 10% quantile bounds of −0.92 and −0.18, respectively.We used the Gravy index score to understand the extent of hydrophobicity and hydrophilicity of given H/LCDR3 amino acid sequence. Sequences with index below 0 display more hydrophilic tendencies, while those above 0 exhibits more hydrophobic profiles. Across the entire spectrum of HCDR3 there is an apparent bimodal distribution with larger peak focalized to hydrophilic indices between 0 and −1.5 Fig. . A secon62. The final polyclonal populations displayed on yeast were tested for binding specificity against all four targets and no cross-reactivity toward the non-specific targets was observed for any of the selected populations  a tumor necrosis factor (OX40); (2) a T-cell inhibitor (B7-H4); (3) T-cell activation marker (CD40L); and (4) granulocyte-macrophage colony-stimulating factor (GM-CSF). The library was panned against the biotinylated antigens bound to magnetic streptavidin beads at saturation. After two rounds of phage selections, the resulting populations enriched for binding were transferred to a yeast-display system and two rounds of sorting were performed by FACS, as previously describedons Fig. . Additioons Fig. . For allSequence sampling with NGS  revealed a significant number of “HCDR3 clonotypes” (HCDR3 sequence clusters if Hamming ≤1 and abundance >1) and “HCDR3-Germline clonotypes” . On average 352 HCDR3, clonotypes and 852 HCDR3-germline clonotypes were identified per target, which differed by antigen . 51 of 77 (~66%) human-specific clones were successfully converted and showed binding activity, with a titer, after purification, ranging from 2.6 to over 600 µg/mL and affinities ranging from 71 nM to 156 pM Fig. . 42 of 51 was generated from two donors and was successfully used to select antibodies against a number of different targets, albeit with rather poor affinities. Since then, the general tendency has been to try and use as many donors as possible to maximize the library diversity63, based on the idea that the immune repertoire, in terms of germline IG loci, varies across human individuals and ethnicities, and this might influence and vary the quality of antibody responses, for instance during exposure to vaccination and disease. For this reason, there has been a tendency to generate libraries from more and more donors, with the idea that such libraries provide both quantitative (more antibodies) and qualitative  advantages.The first natural naive phage antibody library described27. The disadvantages of such clonal dominance may be offset by a greater percentage of natural IGHV/IGLV and IGHV/IGKV pairings in single-, as compared to multi-, donor libraries, claimed to be advantageous in immune libraries64. However, whether the numbers of natively paired antibodies present in a single donor are significant, or indeed relevant, to large naive antibody libraries, remains unclear.Here we show that a library carefully crafted from a single donor provides sufficient diversity to select hundreds of antibodies against different targets, notwithstanding the presence of a higher level of clonal dominance when compared to libraries created from multiple donors68, with the IGHV-D forming the central core of the binding site. The use of all three IGHV-D reading frames70, recombination between IGHV genes after rearrangement71, P-nucleotide72 or N-nucleotide74-mediated addition, and exonuclease mediated loss75, of nucleotides between IGHV/IGHV-D and IGHV-D/IFHV-J segments further increase diversity. Similar mechanisms apply to the IGKV and IGLV, although there is no D region, and less modification at the junctions. The combination of these diversification strategies with heavy and light chain assortment in an unbiased manner has been estimated to have the potential to generate over 1015 uniquely expressed B-cell receptors77, far exceeding the estimated ~1011 B cells in any individual78. Adequate sampling of B-cell diversity remains a problem due to the power-law distribution of sequenced populations (few high abundant clones and many low abundant clones), whereby some rearrangements occurring ≥1000 times more frequently than others79, dominate sequence analyses. This makes sufficient coverage unattainable, a problem aggravated by the short-read lengths of high capacity next-generation sequencing platforms (2 × 150bp with NovaSeq), which miss critical diversity information at the CDR2 region. Even though these issues make accurately measuring (rather than estimating) the true diversity of an antibody library challenging, the application of common standards to this problem should allow the determination of minimum diversity in a manner that allows libraries to be reliably compared and ensures that a given library retains sufficient diversity to select a wide array of putative leads for therapeutic development.Antibody recognition of a given epitope is mediated by the three complementarity determining regions (CDRs) of each of the heavy and light chains . IGHV diversity is generated by the sequential random assembly of ~50 IGHV, 23 IGHV-D, and 6 IGHV-J genes26, which gathered V genes from 809 different donors, unique reads obtained from 454 pyrosequencing resulted in the identification of only 7373 IGHV and 41,804 IGKV sequences, even though the total number of transformants was ~1010 CFU. In another publication23, also using 454 sequencing, the number of unique IGHV genes was estimated to be ~2 × 105, while the number of transformants was 105 fold higher. Finally, a publication27 combining three different NGS approaches  determined that a library3 of 7 × 107 transformants had an estimated HCDR3 diversity of only ~3 × 106. These publications exemplify why colony counting, the usual method used to estimate library diversity, while it may provide a reasonable assessment of combined IGHV/IGLV and IGHV/IGKV diversity, vastly overestimates IGHV diversity. The major problem of using NGS to estimate library diversity is significant undersampling. Even MiSeq (~107 paired-end reads) does not provide the necessary throughput to generate accurate estimates. In this respect, NovaSeq, as used here, with up to 3 × 109 paired-end reads may be able to approach the oversampling required for accurate diversity assessments.It is not clear how much of such natural diversity can actually be harvested in antibody libraries. In a recent study81; (2) hierarchical clustering linkage trees with a variety of mismatches81; (3) homology threshold clustering86; or (4) abundance filtering90. While it has been suggested that abundance filtering is optimal in many circumstances, Turchaninova and colleagues49 have pointed out that this type of filtering may be potentially deleterious, particularly in the analysis of naive B-cell repertoires (and for naive libraries as well): when individual clones are present at extremely low frequencies, and diversity exceeds the number of sequencing reads, abundance-based filtering will shrink the observed diversity of naive B cells by eliminating authentic clones sequenced only once. Nonetheless, due to inherent errors introduced through cDNA synthesis, PCR amplification, and Illumina sequencing, a robust clonotyping strategy remains a critical requirement when assessing CDR diversity.Another challenge in estimating naive library diversity is the definition of what constitutes a unique antibody. If antibodies differing by a single amino acid are considered to be different, diversity evaluations will be overestimated due to sequencing errors. Furthermore, functional differences between antibodies differing by single amino acids are usually minimal, unless the amino acid has markedly different biochemical properties and/or located in an area important in binding . One approach to overcome this problem is the use of clustering, which has been carried out in the past using a variety of methods, including (1) inference through a relationship with unmodified common ancestorsN ≥ 2). Our rationale for using amino acid Hamming distance is three-fold: (1) library diversity should be based on understanding clone functionality, not underlying nucleotide differences without binding consequences; (2) errors introduced during library preparation, especially at the cDNA and PCR amplification level are “real” sequences in the sense that introduced errors during library preparation become members of the final library, and (3) since most errors introduced during library Illumina sequencing are substitutions, not indels91 we reason that Hamming  would be more appropriate than other methods . With these factors under consideration, we show relative diversities at Hamming distances of 0–3  for CDR3 lengths of 10–30 amino acids, so providing upper and lower bounds for actual sequence diversity. Further, we also employed an abundance filtering cut-off for all unique CDR3 reads of two or more. Applying these methods to the single donor library described here, yields a measured HCDR3 diversity of 5.2 × 107 at a Hamming 0, which decreases to 2.2 × 107 at a Hamming of 3. These values increase to 7.1 × 107 (Hamming 0) and 2.7 × 107 (Hamming 3) when diversities are estimated using a non-linear regression model92. This leads to the conclusion that a significant proportion (>73%) of the true IGHV diversity of natural naive antibody libraries can actually be measured using NovaSeq, regardless of the Hamming distance used. Unfortunately, as NovaSeq is unable to cover HCDR2, it is not possible to determine IGHV germline gene identities. However, taking HCDR1 into account, increases these diversity figures to 1.5 × 108 (Hamming 0) and 7.02 × 107 (Hamming 3), reflecting the fact that identical HCDR3 sequence can be encoded by different IGHV and DH genes93. The case for the VL chains is more straightforward. As estimated and measured diversities coincide, saturation is reached, and the light chain diversity is measured at 2.2 × 105 to 4.7 × 105 for IGKV and 6.3 × 105 to 1.1 × 106 for IGLV for Hamming distances 3 to 0, respectively.To assess the diversity of the single donor library created here, we use abundance filtering coupled to amino acid Hamming distance analysis. We address Turchaninova’s singleton conundrum by including singletons within the analysis: however, “true singletons” are verified only if they fall outside the restrictive Hamming criteria relative to sequences that have undergone abundance filtering . If this new BCR has improved surface expression, further receptor editing is prevented by RAG downregulation96. Although it has not been shown for B cells, it is well known that antibody display levels on yeast, another eukaryotic organism, reflect the underlying thermostability and expression levels of the displayed component antibodies97, and directed evolution to improve antibody display on mammalian cells results in improved “developability”98 Similar mechanisms are also likely to be in play during B-cell development, with the immune system ensuring that antibodies have the potential to be expressed at high levels in plasma cells if required.One important advantage of using genes based on natural, rather than synthetic, antibodies is the “quality control” carried out by B cells during their development to ensure that antibodies are well expressed. B-cell viability is maintained by tonic signaling proportional to the level of surface-expressed B-cell receptor (BCR)50, but instead used individual 5′ primers with individual 3′ primers. Interestingly, although many of these primers were designed to be specific for particular V genes31, MiSeq analysis  of the PCR products obtained revealed that there was a significant degree of promiscuity in the V genes amplified. While some of this was expected, on the basis of sequence similarity , it is notable that all the primers, with the exception of IGHV2, also amplified other V genes. Two factors appear to drive this promiscuous amplification: similarity to the primer and the underlying abundance of a given gene within the cDNA source undergoing amplification. This is particularly striking with the IGHV6 primer, where approximately 50% of the product is IGHV4, a far more abundant gene family.In the library described here, we did not use multiplex PCR amplification strategies, which have been shown to bias PCR productsThe library functionality was also proven with successful selection campaigns where several highly specific leads for each of the targets were identified. Moreover, when a therapeutically relevant protein—CD73—was used as a target during a selection campaign a very high number of the selected antibodies, selected as scFvs, were successfully converted into full-length human IgGs retaining some remarkable affinity values. The importance of being able to convert a high number of selected scFv molecules into full-length immunoglobulins is essential because of the known therapeutic advantages of the IgG format: binding specificity, clinical safety profile, low intrinsic toxicity and immunogenicity, the inherent ability to recruit immune cell function to the site of antigen expression, and the prolonged serum half-life that reduces dosing interval required to elicit a therapeutic effect.In conclusion, we show here that it is possible to build a highly functional phage antibody library from a single donor, biased towards natural scaffolds with more favorable developability features. Furthermore, by using NovaSeq it is also possible to accurately measure the diversity of such a library. Future use of this approach, combined with common bioinformatic standards, should allow more accurate assessments of antibody library diversities in a manner that will allow different libraries to be compared.TG1 (Lucigen): [F′ traD36 proAB lacIqZ ΔM15] supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK– mK–).BS1365: BS591 F′ kan (BS591: recA1 endA1 gyrA96 thi-1 D lacU169 supE44 hsdR17) [lambda imm434 nin5 X1-cre].XL1Blue (Agilent): recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIq.Z∆M15 Tn10 (Tetr)].S. cerevisiae EBY100 (GAL1-AGA1::URA3 ura3–52 trp1 leu21his3200 pep4::HIS2 prb11.6R can1 GAL).3. Total RNA was prepared from 7 × 108 CD19+ cells purified from an entire single LeukoPak using the Straight From Leukopak CD19 (Miltenyi), with mRNA purified using the “Oligotex mRNA spin-column” kit (Qiagen). The mRNA was used to generate cDNA with Super Script IV (ThermoFisher) and primers specific to the constant region of IgM heavy chains and κ and λ light chains31.The SDL antibody phage library was created by assembling repertoires of light and heavy-chain variable regions into the scFv format and cloning into the pDAN5 display vector31, were used to generate 450 separate primary PCRs using the cDNA as template. The V genes were reamplified to add an overlapping region corresponding to the scFv linker as well to facilitate the restriction enzyme digestion for the cloning in the display vector.Twelve heavy and 10 light chain 5′ primers and 4 heavy and 8 light chain 3′ primers, based on previously published sequences3), and subsequently cloned in 24 separated sub-libraries for a total number of ~5.0 × 109 clones. We carried out 24 individual phage productions to generate the “primary” phage libraries. Those were combined in two phage pools, IGHV-IGKV and IGHV-IGKV, by mixing equal amounts of phage, and the two IGHV-IGKV and IGHV-IGKV mixes underwent lox-based-recombination3 to obtain the final tertiary phage library used in the selections.To maintain diversity, the 12 individual IGHV primary PCRs were PCR-assembled either with the pooled IGKV or IGLV PCR repertoires . We set a threshold whereby 90% of sequences had a minimum Phred100 quality score of ≥25. All clonotyping is performed on our in-house linux, R, and python-base software running using linear processing on a computing cluster composed of 256 nodes of 4× Quad Core AMD Opteron CPUs with 32GB RAM/node and 1TB disk/node. Sequences are annotated to germline V regions using the IgBlast command-line tool101, which uses the BLAST scoring algorithm102 to align query sequence to germline IGHV and IGKV/IGLV sequences from the international ImMunoGeneTics database® (IMGT®) database103. Appropriate V gene alignment calls were kept that retained an e-value less than 1 × 10−14 and a nucleotide percent identity match across the framework and CDR germline regions of ≥90% . After appropriate alignment amino acid sequences were subject to Hamming distance clonotyping strategy at 0–3 edit string distances for sequences of the equal length according to the following equation:dHD is Hamming distance of objects i and j with k index of given variable evaluating y from the total number of variables n. All data are presented at Hamming distance of 1, unless otherwise noted.All paired-end sequence reads derived from MiSeq were received from SeqMatic and merged using Paired-end fastq processing using PandaSeqUnsupervised hierarchical clustering performed using R hclust system, set to find relationships among the n observations, or 18 bp sequence dissimilarity is calculated using Ward’s minimum variance cluster agglomeration method. This particular approach minimizes total within-cluster variance, where each iteration step pair clusters with minimum between-cluster distances.pi is the proportion that clone i contributes to total sequence population and q takes on different values such as richness (i = 0), Shannon entropy (i = 1), or Simpson index (i = 2), with all estimates taken at q = 0, unless otherwise noted. Estimates for species accumulation curve are based on the non-linear negative exponential function.The diversity index was calculated according to the following general equation:104.Biophysical properties were calculated in R using subset of internal packages. Net charge is based on Lehninger’s pK scale at pH 7.0. Hydrophobicity is assessed using GRAVY index using the Kyte and Doolittle scale105. Briefly, the SDL phage library (1012 cfu) was used in 2 rounds of selection on recombinant human proteins using the automated Kingfisher magnetic bead system (ThermoFisher Scientific), applying 2 × 107 Streptavidin-conjugated magnetic beads (Dynabeads M-280) coated with the biotinylated proteins (100–400 nM), washed and incubated with the antibody phage library. After removal of non-binding phage, the remaining phage particles were recovered from the beads by acid elution and used to infect F’ pilus-carrying bacteria, and amplified by growth overnight at 30 °C. A second selection cycle was also performed; the first round of selections provided a 5 × 104 to 6 × 105 cfu while the outputs of the second round of selections provided 1.2 × 106–5 × 106 cfu. After the selection cycle was repeated, the pool of scFv clones was PCR amplified with specific primers introducing an overlapping region with the yeast-display vector pSpec yeast-display vector . The starting material for the PCR amplification consisted of 1 ng of plasmid DNA obtained from the second round of selections, consisting of more than 108 copies of the template, abundantly covering the diversity of the final round of phage selection.Antibody clones displayed as scFvs were isolated by combining phage- and yeast-display methodologies as previously described6 cells were screened , as described105. Up to 10,000 individual yeast cells with both positive antigen-binding signal and scFv display were sorted and propagated for further rounds of selection . Small scale expression (4 ml media) was performed for each clone and the expressed antibodies were protein G purified.Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary


Carriers of the APOE e4 allele are at higher risk of age-related cognitive decline and Alzheimer’s disease (AD). The underlying neural mechanisms are uncertain, but genotype differences in medial temporal lobe (MTL) functional activity and structure at mid-age might contribute. We tested 16 non-e4 and 16 e4 carriers (aged 45–55) on a subsequent memory task in conjunction with MRI to assess how hippocampal volume  and microstructure  differs by genotype and in relation to memory encoding. No previous study has investigated APOE effects on hippocampal microstructure using NODDI. Recall performance did not differ by genotype. A genotype by condition interaction in left parahippocampus indicated that in e4 carriers activity did not differentiate subsequently remembered from forgotten words. Hippocampal volumes and microstructure also did not differ by genotype but hippocampal volumes correlated positively with recognition performance in non-e4 carriers only. Similarly, greater hippocampal neurite orientation-dispersion was linked to better recall but only in non-e4s. Thus, we suggest that mid-age e4 carriers show a breakdown of normal MTL activation and structure-performance relationships. This could reflect an inability to utilise compensatory mechanisms, and contribute to higher risk of cognitive decline and AD in later life. APOE) gene has 3 variants ; the e4 allele has been subject to considerable research interest due to it being a well-established risk factor for late-onset Alzheimer’s disease (AD)1 with e4 carriers being approximately 3 times more likely to develop AD relative to e3 carriers2. The e4 allele also affects cognitive ageing trajectories in healthy individuals. Healthy older e4 carriers (from this point referred to as e4+) perform worse than non-e4 carriers (e4−) on measures of episodic memory, executive functioning and overall global cognitive ability3. Longitudinal studies in healthy populations point to more rapid age-related cognitive decline in e4+5. Small detrimental effects of e4 on cognition appear to be evident at mid age6 with these becoming more pronounced beyond the 6th decade7. However, midlife effects of APOE are not well studied, despite evidence that risk factor exposure during this life stage significantly influences AD risk in later life8. With accumulation of AD–related neuropathology beginning some decades before symptom onset9, midlife thus likely represents the optimal opportunity to intervene against cognitive decline and AD.In humans, the Apolipoprotein E , Dennis et al.16 reported significantly greater MTL activation to subsequently remembered items in e4+ aged 20–25, although recognition performance was matched. Likewise, we have shown increased activity in left MTL to subsequently remembered words in young adult e4+ with no differences in recognition performance17. Filippini et al.13 found that young adult e4+ (mean age 28) show a pattern of hippocampal over-recruitment in the recognition phase of a subsequent memory paradigm, in a task using non-verbal stimuli. MTL activity has also been reported in young adult e4+ during tasks not expected to engage that region, e.g. a Stroop task18 and a covert attention task19, and MTL coactivation differences have been shown in the resting state13. These findings have been interpreted as representing a compensatory mechanism whereby young adult e4+ recruit additional neural resources to maintain cognitive performance20.Brain structural differences have been detected in healthy e4 carriers. Imaging studies have tended to focus on the medial temporal lobe (MTL) since this structure is amongst the first to evidence AD-related pathologyet al.21 found that healthy mid age e4+  show reduced hippocampal activity during subsequent memory recognition. There were no differences in cognitive performance or MTL volumes, but performance on a verbal learning task correlated with MTL activation in e4− only. This suggests that the relationship between learning performance and MTL activity is disrupted in mid age e4+. Reduced MTL activity has also been reported in older (mean age 73) cognitively normal e4+ during encoding of visuospatial memory, in the absence of any performance differences. Again, MTL encoding activity correlated with performance in e4− only22. This is not attributable to a global decrease in e4+ neural activity, since follow-up work using the same task in a slightly younger age group (mean age 64) found that activity in MTL but not visual cortex was reduced in e4+23. These findings point to a specific downregulation of hippocampal activity in mid age and older e4+, and a breakdown of normal function-performance relationships.In contrast, at mid age there is evidence that hippocampal activation is instead reduced in e4+. Trivedi et al.24 found an age by genotype interaction in MTL activity during presentation of novel vs. familiar images: activity decreased with age in e4+ but increased in e4−, there were no performance or MTL volumetric differences. Likewise, in a sample aged 61–80, only e4+ showed a decrease in MTL activation with age during a working memory task, again no performance or brain volume differences were seen25. While MTL overactivity might characterise young adult e4+, these studies suggest that this effect is reversed perhaps as early as mid age. This could reflect early MTL vulnerability in this group, evident well before cognitive deficits become apparent. This would be consistent with evidence from functional and diffusion imaging studies suggesting accelerated neural ageing in e4+26, detectable at mid age27.Cross-sectional studies examining effects of age range add further evidence. Contrasting younger (20–35 yrs) and older (50–78) age groups, Filippini 28, recall of learnt word pairs29 and object/context encoding30. Choice of task could be a factor. Evidence suggests that, in older adults, activation levels during semantic memory tasks predict cognitive decline better than episodic memory tasks31. However recent work has shown that in older adults, hippocampal overactivation during episodic memory recall is associated with higher levels of tau in cerebrospinal fluid32. Since higher levels of tau in cerebrospinal fluid have been shown in older e4+33, this association could contribute to MTL overactivity in older e4+. Amyloid pathology could also be important since cerebral amyloid levels have been shown to be higher in healthy mid-age and older e4+, particularly in those with increased hippocampal activity35. It is worth noting that none of the aforementioned studies considered e4 dose effects due to insufficient numbers of homozygous e4 carriers being recruited, precluding stratification. Homozygous carriers of the e4 allele (where present) were included alongside those heterozygous for e4 to form an “e4 carrier” group. Most  studies excluded carriers of the e2 allele. Work by Cacciaglia and colleagues has examined e4 dose effects in a healthy mid-age sample (mean age 58), finding that while e4 dose did not affect cognitive performance, it did affect relationships between episodic memory performance and grey matter volumes (including in MTL)36, in a manner suggesting accelerated ageing in e4+; e4 dose was also associated with reduced volume in various brain regions including right hippocampus37.Conflicting results should be noted, however. Older e4+ have also been shown to overactivate MTL and other regions in response to picture encoding38.In this study, we sought to better characterize effects of APOE genotype status on MTL function and structure. We focused on ‘mid age’, recruiting participants within a narrow age range (45–55 years). This represents a critical life stage in terms of AD risk but is currently understudied. Mid age is likely the most informative age point in terms of detecting pathological changes that occur prior to detrimental effects on cognition, which can confound interpretations. This life stage also likely represents the optimal ‘window’ for monitoring and intervention in e4+39. Based on the literature we predicted reduced MTL activity in mid age e4+ and an a priori region of interest (ROI) approach was taken. The ROI mask encompassed hippocampus and parahippocampus. In e4+, we predicted reduced encoding-related activity within this ROI, indicating early MTL vulnerability. Based on previous findings, we did not expect e4+ to show memory performance deficits.Participants performed a subsequent memory task  during the acquisition phase) to assess functional activation to successfully encoded vs. subsequently forgotten items. The task used incidental encoding: in the acquisition phase participants were presented with a series of words and asked to make a classification response. In the subsequent recognition phase, all words presented at acquisition were presented again, interleaved with novel words: participants responded ‘old’ or ‘new’ to each. MTL activity to an item during incidental encoding predicts subsequent recognition success22. Relationships between white matter integrity and episodic memory also appear to be disrupted in healthy older e4+40. On this basis, we predicted that the normal MTL structure-function relationships observed in healthy individuals41 would be disrupted in e4+, with e4+ showing a breakdown in the relationship between hippocampal volume and memory performance measures.T1 structural images were also acquired to test for genotype differences in hippocampal volumes. Genotype effects on relationships between volumes and memory performance were also explored since previous studies point to disrupted relationships between MTL neural markers and memory performance in e4+42. Previous work has used DTI to investigate APOE effects on microstructure in healthy individuals but this is the first study to employ NODDI to address this question. Previous DTI results have been mixed. In young adult e4+, one study reported increased fractional anisotropy (FA), particularly in parahippocampal cingulum bundle43. Others have reported FA to be decreased44 or no different45. At mid-age, increased radial diffusivity (but no differences in FA) were found in e4 homozygotes46. In heathy older e4+, decreased FA has been reported in the left parahippocampal white matter47 and other white matter tracts48 although other studies have not replicated these49. The NODDI model offers several advantages over DTI. Unlike DTI, which assumes that each voxel contains a single tissue compartment with Gaussian diffusion, NODDI uses a three-compartment model that represents three distinct microstructural environments for each voxel – intracellular, extracellular and CSF. It differentiates two of the key contributing variables to FA by providing estimates of both neurite density and neurite orientation dispersion. Compared to DTI, the NODDI model better accounts for the confounding effects of free-water contamination, and has been shown to be superior in detecting clinically-relevant microstructural changes50 although some shortcomings should be noted. In particular, the technique assumes a fixed value for intrinsic diffusivity across the brain. If this assumption is violated it could lead to a bias in the extracted parameters52.A major strength of this study was the inclusion of neurite orientation dispersion and density imaging (NODDI). NODDI is an advanced diffusion MRI technique which is capable of detecting microstructural properties of the dentrites and axons in the brain53, but has not previously been used to explore APOE effects in healthy individuals. As we were focusing on effects in hippocampus/parahippocampus, we examined how neurite orientation dispersion (orientation dispersion index (ODI), the degree of dispersion of the fibre orientations) within MTL might differ by genotype. This was motivated by evidence that hippocampal ODI has a neuroprotective role, which we hypothesised might be lacking in e4+. In healthy individuals, higher ODI in bilateral hippocampus has been shown to protect against age-related differences in cognitive performance, this was the only region in which ODI exerted such an effect54. We hypothesised that differences in ODI could contribute to enhanced risk of poorer cognitive ageing and cognitive decline in e4+. ODI in bilateral MTL was therefore examined in terms of genotype effects and links to memory performance. We again predicted that relationships between memory performance and this structural measure would be disrupted in e4+. If so, this would add valuable new information regarding underlying mechanisms.NODDI has recently been used to show that e4+ with early onset AD have different patterns of white matter neurodegeneration56. The following inclusion criteria were employed: age 45–55, fluent English speaker, right handed, MRI-safe, no diagnosis of high blood pressure, and not currently being treated for a psychiatric condition. The genotyping procedure has been described previously17: APOE genotype was determined by buccal swab and Genotype analyses were performed by a third party  using fluorescence-based competitive allele-specific polymerase chain reaction (KASPar) targeting two APOE single-nucleotide polymorphisms (SNPs): rs429358 and rs7412. Protocols specified by the Human Tissue Act were followed throughout, participants consented to not being informed of their genotyping result, and volunteer call-back was performed by a third party so that the researcher remained blind. Invitation to take part in this study was based on a random sampling so genotype status could not be inferred from an invitation to take part. fMRI power calculation: We performed a power analysis using fMRIpower software (fMRIpower.org) developed by Mumford et al.57. This toolbox estimates the power to detect significant activation within specific ROIs, based on previous results. An appropriate group analysis  was entered into the toolbox, from data we have collected and published previously in mid-age e4+ and e4−, which used the same scanner and acquisition protocol, and a comparable design/analytical model27. The results from the power analysis were that testing 15 participants would provide at least 80% power to detect a significant difference between-groups with a corrected p-value of p < 0.05. Amongst those invited and subsequently consented to take part: 16 were e3/e3 carriers (these comprised the e4− group); 11 were e3/e4 and 5 were e4/e4 ; 1 participant was an e2/e4 and excluded from analyses based on evidence that the e2 allele might be protective58. Thus the final sample consisted of 2 groups: 16 e4− and 16 e4+. The Montreal Cognitive Assessment (MoCA59) was administered to ascertain the cognitive status of all participants. No participant scored below 26 on this measure.All participants volunteered under a written informed consent procedure. Ethical approval was granted by the Sussex University Schools of Psychology and Life Sciences Research Ethics Committee. All relevant guidelines and regulations were followed throughout. In the initial screening phase 165 healthy volunteers were recruited via advertisements at local clubs, universities and community centres, as part of a wider program of work investigating APOE behavioural effects in this age groupBetween the two groups, there were no significant differences in age, gender balance, NART IQ, MoCA score or years of education Table . Neverth17: it was constructed and run using MATLAB as a component of a one hour scanner session. The acquisition phase of the task was presented as a semantic categorisation task, and consisted of 80 words  presented sequentially. Each word was presented at a central point on-screen for 1 second. There was a variable ISI of 2.5 to 4.5 seconds. A mask (######) was presented between each stimulus. Participants were simply instructed to make a button press response to any word that described a profession, of which there were 8, quasi-randomly distributed throughout the set (ie. two profession words in each quarter). The acquisition phase duration was approximately 7 minutes. The surprise recognition phase began approximately 45 minutes after the acquisition phase. In the intervening period, participants completed the structural imaging and exited the MRI scanner. In the recognition phase (performed at a desktop PC), 140 words  were presented in random order. Each word was presented individually for 1 second followed by a mask. Participants were instructed to respond to each word using button 1 (‘old’) or 2 (‘new’) on the PC’s numeric keypad, to indicate whether they thought it was previously studied in the acquisition phase  and matched for lexico-semantic features of length , frequency, familiarity and imageability, according to Kucera-Francis norms, as this can impact recognition performance60.The task has been described elsewhereAll imaging data was acquired with a Siemens Avanto 1.5 T scanner  using a 32-channel head coil and gradient strength 44 mT/m.http://surfer.nmr.mgh.harvard.edu). The Freesurfer reconstruction pipeline normalizes, motion corrects, and excludes non-brain tissue via a hybrid watershed/surface deformation procedure before transforming the images into Talairach space and segmenting them62. Hippocampal volumes were calculated using the automated hippocampal and amygdala segmentation algorithm included with the development version of FreeSurfer 6.0 (dev-20190118), which uses a probabilistic atlas built with ultra-high resolution MRI data to segment the hippocampal substructures and nuclei of the amygdala. This offers an advantage over the hippocampal module released with FreeSurfer 6.0 since joint segmentation of hippocampus and amygdala ensures a more accurate estimate of hippocampal volume64. To account for differences in head size, we normalized the calculated volume of left and right hippocampus by dividing by the estimated total intracranial volume (from the Freesurfer segmentation) to give a volume ratio.A 3D T1-weighted MPRAGE sequence provided the anatomical images (5 minutes): parameters were TR = 1160 ms, TE = 4.44 ms, TI = 600 ms,FOV = 230 × 230 mm, acquisition matrix = 256 × 256, flip angle = 15°, with a resulting voxel size of 0.9 × 0.9 × 0.9 mm. Freesurfer 6.0 was used for cortical reconstruction and volumetric segmentation  were acquired. The total acquisition time was 15 minutes. The diffusion-weighted data were corrected for movement and eddy current distortion using a tool provided as part of FSL . Using the NODDI toolbox for MATLAB  a NODDI microstructural model was fitted to the data. This calculated neurite density index (NDI), ODI, and free water volume fraction (fiso) in native space. Using ANTS software (http://stnava.github.io/ANTs/)parameter maps for each of these were normalized into MNI space. First an optimal brain template was generated based on the mathematical average of all participants’ mean b0 images (from the NODDI acquisition) using a maximum of 30 × 90 × 20 interactions per registration using the greedy-syn transformation model and cross-correlation estimation to assess similarity. This process also provided the diffeomorphic transformation required to deform the native-space brain images to the study-specific template space. Next, the diffeomorphic transformation required to warp the study-specific template to standard MNI space was calculated and both transformations (native-group and group template-MNI) were combined so that each brain was warped to MNI space in a single step. Finally, binarized masks for left and right hippocampal grey matter  were applied to the ODI parameter maps using FSLmaths. ODI ranges from 0 (no dispersion of the fibre orientations) to 1 (fully dispersed).The following acquisition parameters were used for the diffusion-weighted imaging: TR = 8400 ms, TE = 99 ms, 60 slices, matrix size = 96 × 96, FOV = 240 × 240 mm65. After 2000 iterations, the distribution of bootstrap correlation differences was assessed to determine significance. This analysis was performed in Matlab using code adapted from https://github.com/GRousselet/blog/tree/master/comp2dcorr and is more robust than other approaches66. Volumetric differences were assessed separately for left and right hippocampal formations. NODDI data were averaged across left and right, for consistency with the ROI used by Nazeri et al.54.For all structural data, between-genotype differences were assessed using ANOVA in SPSS 25. Data met all assumptions for the use of these tests; gender was included as covariate in all analyses. To assess correlations with task performance, Spearman’s rank correlation coefficients were calculated. To test for differences in correlations between groups, a frequentist approach was taken using a percentile bootstrap method67: fMRI datasets were acquired using a T2* -weighted 2D-EPI sequence. To minimise signal artefacts originating from the sinuses, axial slices were tilted 30° from inter-commissural plane. Thirty-six 3 mm slices were acquired  acquisition, phase encoding direction = A/P) with a matrix size of 64 and an in-plane resolution of 3 mm × 3 mm . A total of 118 volumes were acquired (including 5 dummy volumes) yielding a total acquisition time of ~6.5 minutes. Field maps (phase and magnitude images) were also acquired for use in the unwarping stage of data preprocessing. Participants were positioned comfortably within the head coil; foam padding around the head minimized head movements. The experimental protocol was viewed on a screen via a mirror attached to the head coil. An MR compatible button box was used to collect responses: participants indicated a ‘sort’ detection using the first finger of their right hand.The fMRI acquisition and preprocessing protocol has been described previouslyhttp://www.fil.ion.ucl.ac.uk/spm/). Default settings were used throughout. After discarding the first 5 volumes, the remaining volumes were spatially realigned to correct for head motion, and unwarped. A mean image created from the realigned volumes was spatially normalized into standard stereotaxic space (at 3 × 3 × 3 mm3) using the Montreal Neurological Institute (MNI) template in SPM8. The derived spatial transformation was then applied to the realigned and unwarped volumes, which were finally spatially smoothed to facilitate group level statistics with a Gaussian kernel of 8-mm FWHM. fMRI data were analysed with the standard hierarchal model approach employed in SPM using methods described previously17. Design matrices were constructed for each participant, that modelled subsequently remembered , subsequently forgotten , and profession sort trials as separate regressors. Movement parameters were also entered. Processing of fMRI data was performed blind to group membership. Contrasts for subsequently remembered and forgotten trials were entered at the first level, and effects of condition (remembered/forgotten) and genotype (e4−/e4+) were analysed at the second level using a flexible factorial to test for the effects of condition and condition by genotype interaction, followed by a 2 sample t-test (with the 2 conditions averaged) to test for main effects of genotype. Gender was entered as a covariate.Images were pre-processed using SPM8 (RRID:SCR_007037) 69. For all comparisons, a voxel-wise FWE-corrected threshold of p < 0.05 was applied.For main task effects, correction was performed over the whole brain. For genotype effects, given our a priori hypothesis, a small volume correction was applied using a mask defined according to the automated anatomical labeling atlas (AAL) from the Wake Forest University PickAtlas toolbox (RRID:SCR_007378)Participants showed high levels of accuracy when identifying the 8 profession words  were observed between genotype groups Table  for leftet al.54. No significant genotype differences were observed  = 0.237, p = 0.630)  to subsequently remembered over forgotten trials Table .Table 5AThere was no main effect of genotype. Further, the directional contrasts e4+ > e4−, e4+ < e4−, over both conditions (remembered/forgotten) showed no effects at the whole brain level, or after small volume correction (SVC) with the bilateral hippocampus/parahippocampus ROI mask.Significant activity was observed in left hippocampus, after SVC for bilateral parahippocampus/hippocampus Table . The sigAPOE effects on MTL structure and function during subsequent memory acquisition. A consistent pattern of MTL disruption was identified in e4+. A blunted functional response and a breakdown in the relationship between MTL structure and memory performance was evident. Mid age represents a critical but understudied age point, and these findings advance knowledge of the possible mechanisms underlying enhanced risk of AD and cognitive decline in e4+.In this study we set out to comprehensively explore, in a group of mid age volunteers, 17, hypoactivation is reported with increasing age24. A major strength of the current study is the narrow 45–55 year age range: we focussed on the point in life where processes important for AD risk might be detectable , but before the onset of significant cognitive decline. Indeed, all volunteers had cognitively normal MoCA scores, and there were no recall performance differences between groups. In a previous study using a similar task, young adult e4+ (mean age 21) showed greater left MTL activity to subsequently recalled words, relative to e4− peers17. The current findings suggest that by mid age this difference is lost, with left MTL activity in e4+ no longer differentiating subsequently recalled from forgotten words. Some authors have suggested that e4+ MTL overactivity in youth might reflect compensation for early pathology20. Alternatively, overactivation might predispose e4+ to pathological changes due to a higher associated metabolic burden71. The decreased patterns of activity with age, demonstrated here and elsewhere24, favour the latter explanation. At mid age, compromised MTL function in e4+ might reflect increased risk for age-related cognitive decline. Trivedi et al. found that e4+ aged 43–65 showed significantly less activation in hippocampal regions to novel versus familiar words in the recognition phase of a subsequent memory task. Although (as here) there was no cognitive or MTL volumetric differences between the genotype groups, verbal encoding ability correlated with activity in left anterior hippocampus in e4−, but not e4+. This points to disrupted MTL functional relationships in mid age e4+.Previous studies suggest a pattern of MTL dysregulation in e4+ evident even in the absence of cognitive differences. In terms of functional response, we found a genotype by condition interaction in the left hippocampal formation, with activity in e4+ failing to differentiate subsequently remembered from forgotten items. There was no main effect of genotype, suggesting a specific disruption of memory-related MTL activity in e4+, rather than a more general pattern of over- or under- activity. Nevertheless, recall performance was equal between the genotype groups, suggesting that at this age point these disrupted MTL activity patterns do not significantly impact performance. These activation differences could signify a vulnerability marker and this finding accords with and adds to the literature showing MTL activation differences in e4+. While young adult e4+ often show MTL hyperactivation73 albeit not consistently. Evidence suggests that this linkage might become increasingly unreliable with age: Rajah et al.74 found that hippocampal volumes predicted context memory performance in young adults (mean age 24) though no such relationship was evident in an older group (mean age 68). In e4−, we found that left MTL volumes correlated with memory performance, whereas this relationship was completely absent in e4+. This absence suggests mid age e4+ show the characteristics of the older group reported by Rajah et al.74 and is consistent with evidence suggesting accelerated neuronal ageing in e4+27. It is possible that subtle MTL pathology in mid age e4+ might underlie these effects: early pathological changes not severe enough to undermine cognition or produce a group effect on structural volume, but able to disrupt volume-function relationships.Here, further evidence suggesting MTL disruption and pathological changes in e4+ was shown by the structural-performance correlation analyses. While hippocampal volumes and microstructure metrics were comparable between genotypes, interesting differences emerged when structural-performance correlations were contrasted between genotypes. Left hippocampal volume showed a significant positive correlation with recall performance in e4− but appeared to be absent in e4+, and these correlations were significantly different between genotype groups. Links between hippocampal volume and memory performance have been demonstrated in healthy adults, particularly at mid-age and youngerAPOE effects in healthy individuals. Previous work has relied on DTI techniques, but compared to DTI, NODDI is a better-specified model that provides additional microstructural measures. Here we were specifically interested in how ODI in hippocampal regions might be affected by e4 status. This was motivated by the findings of work by54 who investigated effects of age on grey matter neuritic organization and density, and relationship with cognitive performance. In healthy adults aged 21–84, a mediation analysis showed that higher hippocampal ODI was protective against cognitive aging. No other regions or diffusion measures showed this relationship. This could reflect extension and growth of the hippocampal dendritic tree as a compensatory response to partial deafferentation, and a marker of successful cognitive aging. Since e4+ are at higher risk of un-successful cognitive aging, we hypothesised that hippocampal ODI might be compromised in e4+, or fail to show a positive relationship with cognitive performance. Although no genotype differences were found, a disrupted relationship with performance was observed in e4+, analogous to the e4+ effects on volume-function relationships discussed above. Again, a positive relationship between ODI in hippocampal regions and recall performance was observed in e4− , but this relationship was absent in e4+. The difference in correlations was significant. Therefore in e4+ this neuroprotective mechanism, thought to be an important mediator of successful cognitive ageing, would appear to be non-functional. This is a novel and important finding. A failure to utilise greater dendritic branching to support memory performance highlights MTL vulnerability in e4+, and could contribute to the enhanced risk of age-related cognitive decline and AD. Further, the link between hippocampal ODI and cognitive performance in e4− adds valuable support to the findings of Nazeri et al.54 and justifies follow-up work based on these findings.Microstructure in the MTL was studied using NODDI. NODDI is an emerging technique and this is the first published study to use it to study The number of participants (N = 16 per genotype group) should be noted as an important limitation of the present study. Although we employed robust statistical tests, the findings  should be interpreted with caution pending replication. Unfortunately, small sample sizes are all too common in the APOE literature. Future work should aim to replicate these findings in an expanded sample and also investigate e4 gene dose effects Use of connectivity-based approaches would also be beneficial so as to explore how the MTL disruption identified here interacts with other brain regions and networks.In conclusion, we present a consistent set of findings in mid age e4+ that point to dysregulation of activity in the hippocampal formation, and disrupted relationships between structure and cognitive performance. The current work reinforces previous findings but add novel insights into possible mechanisms. A lack of hippocampal functional response in differentiating encoding success from failure was seen during memory acquisition in e4+. Hippocampal volumes correlated with memory performance in e4− but not e4+, suggesting that in e4+ this relationship might be disrupted by the presence of subtle early pathology. In terms of microstructure, hippocampal neurite orientation-dispersion correlated with better recall in e4− but again this relationship was absent in e4+. The inability to use this potential compensatory mechanism to support cognition could be an important contributor to enhanced risk of age-related decline and AD in e4+. This paper therefore provides novel insight into possible mechanisms linking APOE e4 to cognitive decline and AD.


Yaroshchuk; Scientific Reports 10.1038/srep45168 (2017).1. In this process the flow resulting from the osmotic pressure drop between fresh and saline water is used to drive a turbine. Unfortunately, at the current stage, in spite of its extreme simplicity and conceptual beauty, this process does not appear to be practically viable due to insufficient power efficiency2. This assessment could be radically changed by the recently theoretically predicted “Breakthrough” operation mode of PRO3. In this mode, the solute concentration at the interface between the porous support and the dense selective barrier layer of a non-perfect (‘leaky’) asymmetric membrane employed in PRO decreases with the increase of draw concentration, and, thus, the impeding effect of internal concentration polarization is eliminated, Fig. 4. In a still more recent study Wu and Field5, contested the physical feasibility of “Breakthrough mode” and casted doubt upon the suitability of SKK equations with constant coefficients to PRO. In this note we re-derive the SKK equations based on a very simple capillary friction model of membrane transport in the dense barrier layer and identify the problem with the constant coefficients’ assumption resulting in the occurrence of “Breakthrough mode”. Our derivation results in recovering the SKK equations in the dilute solution limit, albeit with hydraulic permeability dependent on the local solute concentration in the barrier layer . Taking into account this dependence, necessary for preserving the detailed force balance in the barrier layer, eliminates the existence of the “Breakthrough mode”.Osmosis—solution (solvent) flow through non-perfectly (perfectly) semipermeable membranes—is a fundamental classical phenomenon of major practical importance. One of its potentially useful technological applications is the Pressure Retarded Osmosis (PRO) employed for energy harvesting from salinity variationsIn Fig. 3 based on accurate analysis of the SKK model with constant coefficients for a particular narrow range of operation parameters , c(x), and w(x) are the solute and water number densities and P(x) is hydrostatic pressure; Following the common pattern of friction modelsSummation of  and 2),,2), takiHere, the identityEquation  implies , and referring to high dilution Defining the spatially constant water and solute molecular fluxes as mentclass2pt{minimΩw) Eqs.  assume tEquations , 7) may may7) mais solute diffusivity,For γΩw Eqs.  is reduc3), albeit with hydraulic permeability dependent on the solute concentration in the capillary (barrier layer) in accord with  ar ar13) arord with . So modiord with , as oppoIn the porous support the dimensionless solute flux readsIntegrating Eq. , we compIntegration of the dimensionless flux equation in the latter8, Fig. 5 and9, Fig. 5 therein). Substituting (18) into Eqs. :18\:18\\docunto Eqs. , 17) we we\documand, thus, the dimensionless velocity is subject to the following algebraic equationSince For high concentrations, the Eqs. , 21) re re\documIn Fig. The Modified Spiegler-Kedem-Katchalsky (MSKK) model is derived based on the capillary friction model. The detailed force balance in this model yields a solute concentration dependent hydraulic permeability and eliminates the breakthrough mode predicted by the classical local SKK equations with constant coefficients. The difference between the MSKK and the SKK models manifests itself only for extremely high solute concentration range for which the occurrence of breakthrough mode has been predicted.


PIEZO2 gene, who have major deficits in touch and proprioception, are nearly as good at sensing pressure as healthy control subjects. Together, these data support a role for Aβ afferents in pressure sensation and suggest the existence of an unknown molecular pathway for its detection.The sensation of pressure allows us to feel sustained compression and body strain. While our understanding of cutaneous touch has grown significantly in recent years, how deep tissue sensations are detected remains less clear. Here, we use quantitative sensory evaluations of patients with rare sensory disorders, as well as nerve blocks in typical individuals, to probe the neural and genetic mechanisms for detecting non-painful pressure. We show that the ability to perceive innocuous pressures is lost when myelinated fiber function is experimentally blocked in healthy volunteers and that two patients lacking Aβ fibers are strikingly unable to feel innocuous pressures at all. We find that seven individuals with inherited mutations in the mechanoreceptor  The mechanisms underlying deep pressure sensing are not fully understood. Here the authors demonstrate that while two individuals lacking Aβ fibers demonstrate impaired deep pressure sensing, seven individuals with PIEZO2 loss of function mutations display normal deep pressure responses. Sensory innervation of our muscles, joints and connective tissues provide a wealth of conscious and unconscious information about the state of our bodies when at rest or in motion2. Additionally, pressure sensation is a major component of our affective interactions, from the pleasure and comfort associated with hugs to the health benefits of stretching and massage therapy. While our mechanistic understanding of touch perception has grown significantly in recent years, how deep tissue sensations are detected and encoded remain less clear.Interoceptive signals from deep tissues are powerful modulators of our physiological state and serve as important indicators of potential tissue damage, inflammation or disease3. Generally speaking, thickly myelinated Aα and Aβ fibers are fast conducting and critically important for proprioception and touch, respectively, whereas the thinly myelinated Aδ and unmyelinated C fibers conduct more slowly and play prominent roles in thermosensation, chemosensation, and nociception4. There are notable exceptions to this generalization; for example, a prominent subtype of C fibers is activated by gentle stroking stimuli5 and a subset of Aβ fibers have been recently characterized that respond only to painful mechanical stimuli6.The somatosensory system detects both external and internal stimuli. Afferent projections of primary sensory neurons innervate sites throughout the skin and body where they are activated by a wide range of physical and chemical stimuli. The diverse nature of these stimuli is reflected by the heterogeneity of sensory neuron subtypes, their anatomical specializations, physiological properties, and the receptor molecules they express. For example, gentle cutaneous touch sensation is mediated by several types of mechanoreceptors, each having unique ending types, molecular profiles, and physiological properties2 or on the Aδ and C-type nociceptors that signal muscle pain8. Far less is known about non-painful pressure sensations from muscle, fascia, and deep tissues, despite the unique purposes they clearly serve. Intriguingly, some evidence exists suggesting that both myelinated and unmyelinated sensory afferents innervating muscles can respond to innocuous muscle pressures12. Nevertheless, the contributions of specific types of afferents to deep tissue sensations remain unknown.The majority of our understanding of peripheral somatosensory neurons comes from studying the afferents that target skin. What is known about non-cutaneous mechanosensation? Most research on interoception focuses on the vagal system. Interest in visceral sensation occurring via dorsal root ganglion neurons has largely centered on pain in relation to disorders such as Irritable Bowel Syndrome or Crohn’s Disease. Similarly, investigations of muscle sensation have mainly focused on either Aα-type proprioceptors that are required for tracking body position15. Third, we developed an assay to assess the roles of gentle touch and deep tissue sensation in an active behavioral task. For our studies, we worked with healthy adult volunteers and two cohorts of patients with rare conditions affecting their somatosensory primary afferent systems.Here, we investigated three aspects of pressure sensation in humans. First, we asked which sensory neuron fiber types mediate our ability to perceive deep pressure on the legs and hands. Second, we investigate whether this type of innocuous pressure stimulus engages the same molecular transduction pathway- ΡΙΕΖΟ2- as gentle touch sensation and proprioception18. These patients have been studied extensively  and have profound discriminative touch and proprioception deficits21. Despite these profound sensory deficits, both individuals perceive cutaneous temperature and pain and have affective responses to skin stroking, where unmyelinated afferents are known to be critical5. The phenotypes of the Aβ-deafferented individuals bear striking similarity to our second patient cohort, individuals with inherited bi-allelic loss-of-function mutations in the mechanically-gated ion channel ΡΙΕΖΟ2 (ΡΙΕΖΟ2 Deficiency Syndrome). ΡΙΕΖΟ2 is an essential receptor for mechanical stimuli in multiple species23 that is required for normal proprioception, vibration sensing and touch discrimination15. Individuals with ΡΙΕΖΟ2 Deficiency Syndrome also exhibit neonatal hypotonia, hip dysplasia, joint hypermobility and contractures, progressive scoliosis, delayed acquisition of motor milestones in the absence of muscle weakness, and deficits in interoceptive sensations such as from the mouth, stomach, and bladder25.Our first patient cohort consisted of two sensory neuronopathy individuals with selective loss of myelinated Aβ type sensory fibers after a rare virus or autoimmune induced neuronopathy syndromeOur findings demonstrate that, as for touch and proprioception, Aβ fibers are required for pressure sensation. However, unlike several other types of non-painful mechanosensation, pressure sensing does not require ΡΙΕΖΟ2 function. Together these findings offer insight into the neural and molecular pathways for pressure sensation in humans.28. We first confirmed that nerve blocks caused by blood pressure cuffs applied to the upper arm led to participants reliably losing cutaneous vibration sensation (known to be mediated by Aβ fibers) on their hands before heat detection by C fibers was affected . Aβ-deafferented patients were completely unable to discern differences of even 60 or 90 mmHg . We therefore developed an assay to assess the contribution of pressure detection in a task requiring multi-sensory feedback Fig. . Partici27, indicating the existence of afferents dedicated to sensation from deep tissues. For example, a study performing cutaneous anesthesia eliminated light touch sensation by von Frey filaments except for the single strongest von Frey fiber, which was described by participants as a sensation of “deep pressure”27. Consistent with this view, our study offers insights into pressure sensation by evaluating two groups of human patients with different rare mechanosensory deficits, in addition to healthy individuals undergoing a pressure block.How non-painful pressure is detected and perceived has remained unresolved. Previous studies in humans using anesthetic or pressure blocks have suggested that cutaneous sensation plays a minimal role32. Similarly, both Aβ-deafferented and ΡΙΕΖΟ2 Deficiency Syndrome patients are significantly impaired in sensing light touch and have difficulties actively maintaining light pressures with their fingertips. In contrast, we found significant differences when we probed deeper pressures; ΡΙΕΖΟ2 Deficiency Syndrome patients perceive deep pressure normally whereas Aβ-deafferented patients and healthy individuals with transient Aβ deactivation cannot. Thus, similar to touch, these findings support the idea that a specialized set of myelinated sensory afferents mediate innocuous pressure sensation whereas painful pressures are detected by thinly myelinated or unmyelinated fibers.The detection of light touch on the skin by the slowly adapting Merkel cell-neurite complex and the importance of ΡΙΕΖΟ2 in this type of response has been established35. However, we do not yet know how many functionally different sub-types exist. Studies on the different types of sensory neurons have largely focused on those projecting to skin36. For the sensation of deep tissue, it is clear that Aβ neurons innervating deep tissues go beyond the proprioceptors with endings in muscle spindles and tendon organs12. Recent advances in single cell sequencing37 offer the possibility of uncovering better genetic markers for these other Aβ subtypes, including those that might mediate innocuous deep tissue sensation.What are the fibers mediating pressure sensing? One possibility is that cutaneous myelinated mechanoreceptors with higher activation thresholds can signal pressure but without causing pain. This could be the case with the ΡΙΕΖΟ2-deficient individuals who retain the ability to detect von Frey filaments at the upper range of forces. Such an explanation is consistent with the existence of unknown molecular pathways for detecting higher forces in cutaneous sensory afferents. Additionally, our results favor that deep pressure sensation also arises from distinct and dedicated types of peripheral neurons. Somatosensory neurons are molecularly heterogenous, falling into at least 13 transcriptomic classes15. Studies in mice and humans demonstrate that high threshold mechanical stimuli, particularly acute mechanical pain, are detected by a different mechanism39. The ability of patients with ΡΙΕΖΟ2 Deficiency Syndrome to sense innocuous pressure exposes the existence of an additional type of mechanosensation that also does not require ΡΙΕΖΟ2. Recently, several genes have been proposed to function as mechanosensitive ion channels42 and await evaluation of their in vivo roles in pressure sensation.What is the molecular mechanism for pressure sensing? ΡΙΕΖΟ2 is critically required for touch discrimination, vibration sensing and proprioceptionThe current study is limited by the rarity of both Aβ-deafferentation and ΡΙΕΖΟ2 Deficiency Syndrome, leading by necessity to small sample sizes of patients and limited range of ages. It is possible that the pressure-sensing deficit observed in the Aβ-deafferented patients results in part from central changes due to many years without normal touch input. Yet, the preserved heat and pain thresholds in these patients - and the fact that healthy individuals lost deep pressure sensation after blockade of Aβ-fibers - suggests that these deficits relate more directly to their Aβ-deafferentation.44. While the cause of profound motor impairments in both the Aβ-deafferented and ΡΙΕΖΟ2 Deficiency Syndrome are certainly due to the loss of proprioception, it is notable that one major difference between these two syndromes is that people with ΡΙΕΖΟ2 Deficiency Syndrome retain the ability to sense pressure. Notably, the ΡΙΕΖΟ2 Deficiency Syndrome individuals are born without proprioception and develop alternative strategies throughout childhood to perform motor functions. It is tempting to speculate that, along with vision, deep tissue pressure sensation provides another key source of sensory input that these individuals use to partially compensate for their deficits.In our daily lives, we integrate information from our different sensory systems to perform basic tasks. Pressure sensation provides key information that, when absent, greatly affects quality of lifeThis study was approved by the NIH CNS Institutional Review Board. All participants provided written informed consent to participate and all relevant ethical guidelines for human subjects research were followed. Aβ-deafferented and ΡΙΕΖΟ2 Deficiency Syndrome patients were referred to our group by clinicians and collaborators for further study. Healthy control participants were selected based on age and sex from participants in a broad screening protocol at our institute (NCCIH). Potential participants were scheduled for a telephone screening interview during which the study procedures were described and eligibility criteria were reviewed. Participants underwent medical screening for unstable medical or psychiatric conditions and any abnormalities of the skin or nerves. All participants provided informed consent and were financially compensated for their time.Five healthy volunteers  completed the nerve block portion of the study. In addition to the placement of the nerve block, these volunteers underwent repeated tests of vibration, temperature, and deep pressure perception, both before the placement of the cuff and during the nerve block procedure . Following the application of the cuff, the participants placed their arm on a pillow and a visual barrier was positioned to obscure their view of the blocked arm and test stimuli. They were also given noise-isolating headphones playing white noise. These volunteers did not participate as controls in any other parts of the study.19), participated in the deep pressure discrimination, detection threshold, and sensory integration testing. Eight neurologically intact adults  participated as controls for the Aβ-deafferented group. Seven individuals with inherited loss-of-function mutations in the mechanically-gated ion channel ΡΙΕΖΟ2 (ΡΙΕΖΟ2 Deficiency Syndrome) also participated: three females  and four males . One of these participants had a medical contraindication (deep vein thrombosis) for leg compression tasks, therefore N = 6 completed this task. A total of 14 healthy volunteers  participated as controls for the ΡΙΕΖΟ2 Deficiency Syndrome group . All individuals with ΡΙΕΖΟ2 deficiency syndrome fit the conserved clinical presentation that includes congenital hypotonia, neonatal respiratory distress, and difficulty feeding. Hip dysplasia, joint contracture, and hypermobility were common early findings. Motor development was delayed with acquisition of independent ambulation in late childhood to adolescence in 5 of the 7 patients despite absence of muscle weakness. All patients developed childhood onset progressive scoliosis.Two patients with a rare sensory neuronopathy syndrome causing Aβ-deafferentation , who have been previously studied  was wrapped around the brachium of the participant’s left arm and rapidly inflated, using an electric pump, to a pressure ~100 mmHg above the participant’s systolic blood pressure. The participant then rested their arm, dorsal side down, on a pillow in front of them. Sensory testing included the measures delineated below. Participants were instructed that we were measuring their sensory perception at baseline, then placing a nerve block, then looking at how various sensations changed over time. The cuff was released after completion of testing procedures, or if C-fiber function was lost before completion of procedures, or upon request for any reason , or when one hour had elapsed.Each participant held their left arm above their head for ~1 min while a licensed healthcare practitioner exsanguinated the arm. Next, a blood pressure cuff . Vibration perception was operationalized as the percentage of onsets and offsets correctly identified. The vibration perception test was repeatedly administered until the release of the nerve block for any of the reasons listed above.Prior to and following the placement of the nerve block, the temperature at which the participants perceived warm stimuli was repeatedly assessed by a threshold task using a contact thermode . The thermode was placed against the left ventral forearm, approximately midway between the wrist and elbow, and set to 32 °C. Heat perception thresholds were assessed by increasing the temperature of the thermode at a uniform and gradual rate (1 °C/s) until the participant indicated their perception of heat by responding with a button press. The heat perception threshold test was repeatedly administered until the release of the nerve block for any of the reasons listed above.The ability of the participants to perceive deep pressure was assessed prior to the nerve block and again after participants showed complete  loss of vibration perception. Controlled, oscillating deep pressure was administered to each hand for ~20 s via a commercial hand massager device (Daiwa Felicity Electric Compression Hand Massagers). Each participant rated the intensity of the massage on each hand using a visual analog scale (VAS) with anchors of “no sensation” (coded as 0) to “highest possible intensity” (coded as 100).48 programming, causing pressure to reach the target peak and then to passively drop to the target baseline.A custom designed compression device, which allowed us to experimentally control the rate and amount of pressure, was used to inflate individual chambers of a commercial leg compression sleeve (Chattanooga Group PresSsion 8 Chamber Garments) fitted around the participant’s lower left calf See Fig. . The zonAβ-deafferented patients and matched healthy controls rated the intensity of two series of pressure stimuli, each containing eight trials. On each trial the sleeve oscillated five times between 10 and 30 mmHg (low series) and 15 and 65 mmHg (high series). Participants made a single rating of the perceived intensity at the end of each series on a VAS scale ranging from no sensation (coded as 0) to pain threshold  to pain tolerance (coded as 100). ΡΙΕΖΟ2 Deficiency Syndrome patients and matched healthy controls completed the same task except that each series contained six trials (rather than eight) and participants made ratings of perceived intensity twice per series  on the same visual analog scale described above. Median intensity ratings and interquartile intervals were calculated for data representation purposes.Using the same custom compression device, we used a two-alternative forced choice discrimination task to test all participants in their ability to discriminate between different intensities of pressure. On each trial, different levels of pressure were administered sequentially, and participants indicated whether the first or second stimulus was stronger. The first block of testing included 12 randomized trials with a difference of 30 mmHg between the two stimuli . If participants performed below 75% accuracy, task difficulty was decreased to a pressure difference of 60 mmHg (30 vs 90 mmHg) and then to a difference of 90 mmHg (30 vs 120 mmHg). Several ΡΙΕΖΟ2 Deficiency Syndrome participants received fewer trials due to time constraints and superior performance on a more difficult discrimination set (differences of 15 mmHg). For each participant the percentage of correct responses was calculated.Given the Aβ-deafferented patients’ inability to perceive pressure sensations up to 120 mmHg, we performed pressure perception threshold tasks using stimulation devices that allowed the application of high pressures to determine if there was a higher level of innocuous pressure the patients could perceive, as well as to ascertain pressure pain thresholds. These tasks were repeated on two body parts with different surface areas – the lower leg and the thumb – to test whether deficits were consistent. The pressure threshold task was not administered to the ΡΙΕΖΟ2 patients since they showed no deficit in pressure discrimination.Large Surface Area: A threshold task was used to determine what level of pressure first elicited percepts of pressure and pressure pain. The in-house device used during the discrimination task could not inflate to the pressure levels necessary for this task, so a clinical standard, manual blood pressure cuff was used instead. The blood pressure cuff  was wrapped around the upper calf of participants’ left and right legs and one cuff at a time was gradually inflated (~5 mmHg each second) using a manual hand pump. For trials of pressure detection, participants were instructed to verbally indicate when they perceived pressure around either leg. For trials of pain perception, they were instructed to report when they first perceived any kind of pain or “when the pressure started to hurt at all.” There were 10 trials of pressure detection and 2-4 of pressure pain perception for each participant, with an inter-stimulus interval of at least 10 s. Mean pressure levels and standard deviations were calculated for data representation purposes.2, with pressure increasing at a rate of ~0.5 kg/cm2 per second. Participants were instructed to indicate the moment that they felt any pressure or change in sensation (pressure threshold) or when they first perceived any kind of pain (pressure pain threshold). Mean pressure levels and standard deviations were calculated for data representation purposes.Small Surface Area: A pressure algometer (Wagner Instruments) with a blunted circular tip (1 cm diameter) was pressed manually into participants’ left thumbnail, with the thumb placed on a solid surface  were cupped over and touching the surface of a postal scale. The digital USB postal scale M-25  was modified so the internal load cell output was fed to PhidgetBridge  connected to a PC laptop computer through a Universal Serial Bus connector. Custom written Python software managed data acquisition from the load cell at 62.5 Hz. Scaled data were simultaneously saved and plotted in real time on the laptop screen. The laptop was placed on the table, with the scale in front, so that the screen was directly in the participant’s line of vision. Participants were instructed to apply as much force to the scale with their fingers as needed to keep a force readout line (in black) at the same level as a guide line (in red). At the start of the test, the red guide line appeared on the screen. Two different values of force setpoint (20 g and 150 g) were tested. 20 g spread over a ~1 cmt = 10 s the guide line would disappear and then re-appear at t = 20 s, and that their task meanwhile was to keep the applied force constant without visual feedback. Each trial lasted 30 s and trials were repeated three times per participant with force setpoints pseudo-randomized. Before data collection, all participants were allowed one test trial to familiarize themselves with the task. Recorded data were analyzed using Matlab48; data were smoothed over a 1 s window and trials were averaged. Performance was measured as the mean deviation from the target setpoint (MD (t)) at a given time point t) the last 5 s of the trial were averaged.The participants were also informed that at Further information on research design is available in the Reporting Summary


Hence, we consider mechanisms by which the evolution of marine eukaryotes, coupled with biogeochemical and ecological feedbacks, potentially between alternate stable states, could have caused changes in ocean carbon cycling and redox state, phosphorus cycling and atmospheric pO2. We argue that the late Tonian ocean ∼750 Ma was dominated by rapid microbial cycling of dissolved organic matter (DOM) with elevated nutrient (P) levels due to inefficient removal of organic matter to sediments. We suggest the abrupt onset of the eukaryotic algal biomarker record ∼660–640 Ma was linked to an escalation of protozoan predation, which created a ‘biological pump’ of sinking particulate organic matter (POM). The resultant transfer of organic carbon (Corg) and phosphorus to sediments was strengthened by subsequent eukaryotic innovations, including the advent of sessile benthic animals and mobile burrowing animals. Thus, each phase of eukaryote evolution tended to lower P levels and oxygenate the ocean on ∼104 year timescales, but by decreasing Corg/P burial ratios, tended to lower atmospheric pO2 and deoxygenate the ocean again on ∼106 year timescales. This can help explain the transient nature and ∼106 year duration of oceanic oxygenation events through the Cryogenian–Ediacaran–Cambrian.A ‘Neoproterozoic oxygenation event’ is widely invoked as a causal factor in animal evolution, and often attributed to abiotic causes such as post-glacial pulses of phosphorus weathering. However, recent evidence suggests a series of transient ocean oxygenation events ∼660–520 Ma, which do not fit the simple model of a monotonic rise in atmospheric oxygen (pO To first order, we do not expect evolutionary or ecologically driven changes in organic matter cycling within the ocean to have impacted atmospheric pO2, because the ocean P inventory adjusts to changes in P burial efficiency such that the P burial flux again matches the (unchanged) P input flux — and the corresponding oxygen source from Corg burial is left unchanged.The burial of photosynthetically derived elf seas  and reprCorg to total phosphorus buried in new sedimentary rocks, (Corg/P)burial, can change (thus affecting atmospheric pO2), because it is sensitive to the redox state of ocean bottom waters and hence to ocean nutrient levels and (ultimately) atmospheric pO2. Under anoxic and euxinic (sulfate-reducing) bottom-water conditions, phosphorus recycling to the water column is enhanced, increasing (Corg/P)burial  governed by the balance of supply from the atmosphere via high-latitude deep convection and  demand from inorganic reductant input at mid-ocean ridges 2] governn ridges ,43, consn ridges .Corg burial [Maintaining an oxygenated Proterozoic atmosphere (given tectonic reductant input) required greater than ∼25% of present-day g burial . A key pg burial  and of b2 [In this ‘DOM world’, balancing the P cycle would have demanded a larger ocean P inventory  to drive2 .Fossils indicate a diversification of heterotrophic eukaryotes in the late Tonian –48, inclThe absence of a eukaryotic algal biomarker record prior to the Cryogenian has been attributed to nutrient scarcity ,51. HoweInstead, we suggest two scenarios linking the biomarker record of eukaryotic algae  with an 2 demand over ∼104 years, thus tending to oxygenate deeper waters of shelf seas  that bridge the water-sediment interface accumulate polyphosphate from oxic waters and utilise it under anoxic conditions, triggering apatite precipitation [The combination of an oxygenated shelf water column overlying a sediment surface still covered by mats  produced a shelf-sea ecosystem structure unique to the Neoproterozoic, with a sharp sediment-surface redox gradient that would favour the formation of authigenic carbonate  and phospitation ,87. Neoppitation ,89 and spitation . Phosphopitation , then shpitation ,91.The first mobile trace-makers that scratched across mat surfaces appeared ∼565 Ma , but didCorg and O2 [Corg/P)burial, initially removed phosphorus and oxygenated the ocean (∼104 years) (2 and deoxygenated the ocean (∼107 years) restoring higher P levels [Bioturbating animals are well-known ‘ecosystem engineers’  that brig and O2 , suppresg and O2  and trapg and O2 , while ag and O2 . It is hg and O2 , and by 4 years)  and thenP levels . What is2 and ocean deoxygenation over the next ∼20 Myr [One view is that bioturbation increased in depth and intensity in the Cambrian ‘agronomic revolution’ in Stages 2–4 (∼530–510 Ma) ,102, cau ∼20 Myr . A count ∼20 Myr ,104. The ∼20 Myr .2 decline). Wider evidence shows an ocean oxygenation event ∼520 Ma (broadly coincident with the ‘Cambrian explosion’) followed by deoxygenation [These alternative hypotheses make distinct, testable predictions. Evidence that mid-depth waters of the Yangtze Platform oxygenated from Cambrian stages 2–4 has been used to question the early bioturbation model , but is genation ,107,108.The Cambrian evolution of large zooplankton would also have increased the efficiency of the biological pump , transfe2 or evolutionary innovations. We describe a series of eukaryotic innovations that created and strengthened the biological pump of POM from the ocean to the sediments, with major consequences for the phosphorus, carbon and oxygen cycles. We suggest that each phase of eukaryote evolution tended to lower P levels and oxygenate the ocean on ∼104 year timescales, but by decreasing Corg/P burial ratios tended to lower atmospheric pO2 and deoxygenate the ocean again on ∼106 year timescales. Coupled with tectonic drivers, and biogeochemical and ecological feedback, potentially between alternate stable states [6 year duration of oceanic oxygenation events through the Cryogenian–Ediacaran–Cambrian.The Neoproterozoic–Cambrian transition was not unidirectional or driven solely by either rising atmospheric pOe states , this coThe late Tonian ocean ∼750 Ma was dominated by rapid microbial cycling of DOM with elevated nutrient (P) levels due to inefficient organic matter removal to sediments.We hypothesise that abrupt onset of the eukaryotic algal biomarker record in the Cryogenian ∼660–640 Ma was linked to an escalation of protozoan predation (eukaryophagy).Corg and P to sediments.This ecological regime shift created a ‘biological pump’ of sinking POM, which transferred Corg and P transfer to sediments, contributing to the deposition of phosphorites.The Late Ediacaran advent of sessile benthic animals on top of microbial mats increased the efficiency of The Cambrian explosion of mobile burrowing animals broke the ‘mat seal’ on the upper sediments but by oxygenating them it enabled alternative means of efficient P retention.4 year timescales, but by decreasing Corg/P burial ratios, tended to lower atmospheric pO2 and deoxygenate the ocean again on ∼106 year timescales.Each phase of eukaryote evolution tended to lower P levels and oxygenate the ocean on ∼106 year duration of oceanic oxygenation events through the Cryogenian–Ediacaran–Cambrian periods.This can help explain the transient nature and ∼10