Understanding deltaic resilience in the face of Holocene climate change and human impacts is an important challenge for the earth sciences in characterizing the full range of present and future wetland responses to global warming. Here, we report an 8000-year mass balance record from the Nile Delta to reconstruct when and how this sedimentary basin has responded to past hydrological shifts. In a global Holocene context, the long-term decrease in Nile Delta accretion rates is consistent with insolation-driven changes in the ‘monsoon pacemaker’, attested throughout the mid-latitude tropics. Following the early to mid-Holocene growth of the Nile’s deltaic plain, sediment losses and pronounced erosion are first recorded after ~4000 years ago, the corollaries of falling sediment supply and an intensification of anthropogenic impacts from the Pharaonic period onwards. Against the backcloth of the Saharan ‘depeopling’, reduced river flow underpinned by a weakening of monsoonal precipitation appears to have been particularly conducive to the expansion of human activities on the delta by exposing productive floodplain lands for occupation and irrigation agriculture. The reconstruction suggests that the Nile Delta has a particularly long history of vulnerability to extreme events (e.g. floods and storms) and sea-level rise, although the present sediment-starved system does not have a direct Holocene analogue. This study highlights the importance of the world’s deltas as sensitive archives to investigate Holocene geosystem responses to climate change, risks and hazards, and societal interaction. A key challenge concerning continental rivers is to better understand past, present and future river fluxes in the face of climate shifts, land-use alterations, river catchment modifications and their impact upon base-level geosystems -3. Withiet al.[The recent worldwide degradation of deltaic wetlands is often highlighted as an expression of global warming and human impacts . For theet al.. found tet al.. By contet al.-28.The Nile is the world’s longest river (>6500 km) and shaped the development of numerous complex societies, providing a reliable source of water for farming and linking populations between sub-Saharan Africa and the Mediterranean ,30. Its To assess the Nile Delta’s state of health over centennial to millennial timescales, we generated a well-resolved 8000-year sediment accretion record using more >100 cores studded across the present deltaic fringe , a series of centennial-scale fluctuations evoke shorter-scale, non-precessional forcing agents as pacemakers of deltaic change. These include, for instance, relative sea-level changes and ENSO variability [Around 5000 cal. BP, the accretion record presents decreases in sediment pulsing to the delta driven by a southerly migration of moisture-bearing monsoon rains (mean summer maximum ~15° N), which impacted upon both the watershed’s geosystems and the geography of human occupation along the Nile corridor and its bordering regions . The endiability . Our reciability . BecauseDeltaic archives bear the signatures of climate-induced modifications in delta-front geomorphology around this time . A stronThe onset of the mass balance losses coincides with an intensification of human occupation of the Nile Delta, when failing rains in the ‘Green’ Sahara focused populations along the Nile corridor ,58,59. TThis Nile Delta sediment accretion record reveals new insights into the delta’s long-term vulnerability to climate change and human impacts. We have documented a >70% decrease in the Nile Delta’s accretion status between the early and late Holocene, consistent with a gradual decrease in climate-driven sediment supply. The precise spatial expression of these sediment losses invites closer scrutiny . At the regional scale, the Nile Delta experienced a geomorphological restructuring from a river-dominated cuspate delta to a wave-dominated arcuate delta following the end of the ‘African Humid Period’. A corollary of the millennial-scale ebb in Nile flow and human channelling of the delta was a gradual reduction in the number of fluvial distributaries from up to nine around 5000 years ago to just two at present. Furthermore, we suggest that reduced river flow linked to a weakening of the Ethiopian Monsoon was particularly conducive to the expansion of human exploitation of the delta by exposing productive floodplain lands and producing a sharper contrast between seasonally-driven Nile minima and maxima. In terms of risks and vulnerabilities, the impact of sediment deficits would have been particularly acute along coastal margins and in areas of young Holocene strata, where subsidence rates in excess of 5 mm/yr are presently attested . AlthougFile S1Figure S1. Rank diagram of the 318 radiocarbon dates and calibrations included in the statistical analyses. The 2 sigma error bars of calibrations are denoted. Figure S2. Example of the calculation of sedimentation rates from a Maryut lagoon core. Table S1. Statistical correlation of Nile Delta accretion rates versus other archaeological and palaeoclimate proxies from the Nile valley and neighbouring regions.(DOC)Click here for additional data file.


Transport of PGF2α to the ovary results in release of OT by luteal cells in a positive feedback loop leading to luteal regression. This signaling cascade can be rapidly blocked by exposing endometrial cells to physiologic levels of P. This mini review will focus on the mechanisms by which P may act to block OXTR signaling and the luteolytic cascade in the ruminant endometrium, with special focus on both non-genomic signaling pathways and non-receptor actions of P at the level of the plasma membrane. While this review focuses on ruminant species, non-classical blockage of OXTR signaling may be important for fertility in women.Expression of the oxytocin receptor (OXTR) in the endometrium of ruminant species is regulated by the ovarian steroids progesterone (P) and estradiol (E). Near the end of the estrous cycle, long-term exposure of endometrial epithelial cells to P results in loss of genomic P receptors (PGRs), leading to an increase in E receptors (ERs). Genomic regulation of the OXTR is mediated via suppression of ER signaling by P. Upon OT binding at the plasma membrane of endometrial cells, a signaling cascade is generated stimulating release of prostaglandin F In mammals, signaling by the neuropeptide oxytocin (OT) via receptors (OXTRs) in the uterus results in initiation of parturition [as reviewed by Kamel ] and con2; OT is secreted primarily from the posterior pituitary, but also by the corpus luteum (CL) of several mammalian species such as swine, ruminants, and primates . HoweveIncreased expression of OXTR near parturition is noted in all mammalian species, including primates and ruminants; OXTR signaling is critical for initiation of myometrial contractions in ruminants  concentrations were all able to interfere with OT-stimulated PGF2α secretion and intracellular calcium release . The observed inhibition by P of Oxtr function is at the level of the plasma membrane: P conjugated to bovine serum albumin, which prevents P from crossing through the plasma membrane easily, also inhibited membrane binding of OT. Specific and high-affinity binding sites for P were reported only in transfected CHO cells expressing the Oxtr. Moreover, high-affinity binding of P to Oxtr-expressing membranes was regulated by the state of the G-protein coupling to the receptor, based on additional experiments performed with the G-protein uncoupling reagent GTPγ S. These observations indicate that the interaction of P with the rodent Oxtr is possibly a direct steroid-receptor interaction, either by altering the conformation of the receptor or directly blocking the binding site for OT on the OXTR . E, cortisol, testosterone, and arginine vasopressin failed to compete for the P binding site, however, P, R5020 (synthetic progestin), RU 486, and OT all bound to the same site on plasma membranes. A radioreceptor exchange assay demonstrated that pre-exposure of membranes to P significantly increased the number of P binding sites. Inhibition of OT binding to endometrial membranes by P was dose-dependent at a low (nM) steroid concentration , P might be acting to exclude cholesterol from the plasma membrane, thus altering membrane fluidity in these SLCs . The effect these mPRs might exert on OXTR signaling in uterine tissues remains to be investigated.Other novel, non-genomic membrane PRs (mPRs) have been identified in the ovine uterus which may act to block local OXTR signaling (Ashley et al., The genomic PGR also rapidly activates the MAPK pathway upon stimulation in breast cancer cells via a SRC kinase-like SH3 domain (Faivre et al., 2α from endometrial tissue by P, which could lead to delayed luteolytic events. The best-characterized tissue to date of regulation of OXTR expression and OT signaling is the ruminant uterus, however, rapid inhibition of OT action by P remains to be investigated in the ruminant uterus during pregnancy. Inhibition of OXTR signaling may be due to non-receptor mediated events related to steroid overloading of plasma membranes, or via specific binding of P to one of several membrane-associated P receptors (Figure situ, further studies are needed to elucidate any involvement of membrane P receptors in blockage of OXTR signaling. The role of an ovarian OXTR in regulation of primate luteal tissue remains unclear, but P may act to block local OXTR signaling. Given recent studies identifying functional OXTR in primate luteal cells and P inhibition of OT-signaling in bovine SLCs (Davis et al., There is evidence that P directly antagonizes OXTR signaling via a non-genomic mechanism, however, there are only a few studies, some with conflicting results (summarized in Table s Figure . NeverthThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria. As a result, the absorption of bioactive and water soluble ferrous ion (Fe2+) became a challenge for all forms of life and left a great impact on evolution All aerobic living organisms face a dilemma when confronted with the need to assimilate the essential element iron. Indeed, iron is the second most abundant metal on earth, yet the primary form found in the environment is the water insoluble and metabolically inactive ferric ion (Fe3++e− = Fe2+ (ferric reductase) and O2+ e− = O2− (NADPH oxidase).Ferric reductases (FRE) and NADPH oxidases (NOX) are homologs NOXs transfer electrons to oxygen to produce short-lived superoxide which is the primary reactive oxygen species (ROS), which is then transformed into various other ROS, such as hydrogen peroxide, hypochlorite or ozone Detailed bioinformatics analyses highlighted both the gene phylogeny and the structure of family members In the present study, we characterized the evolutionary history of the FRD superfamily. By adding homologs of species from deep-branching nodes of the species tree, we showed that families and subfamilies emerged earlier than had been previously thought. The gene phylogeny is discussed in the context of structural and functional features. Findings to be highlighted include 1) conserved residues predicted to be crucial for NADPH-oxidases; 2) the probable lateral inheritance of the metazoan STEAP family from bacteria and the synchronous loss of the ancient ferric reductases in this clade; 3) the emergence of the NOX family from EF-hand containing superfamily members; 4) the origin of the FRD superfamily from a bacterial homolog that consists solely of a ferric reductase domain.Eukaryotic homologs of the FRD superfamily were obtained from UniProtKB (release 2011_07) Sequences were aligned using MAFFT (version 6) http://www.phylosoft.org/archaeopteryx).For the analysis of the superfamily, the sequence redundancy of the predicted ferric reductase domains was reduced to 90% using Jalview. Truncated and dubious sequences were removed and sequences were realigned with MAFFT, using settings optimized for data with one conserved domain and long gaps (L-INS-i) and the scoring matrix JTT200. No other default parameter values were changed. The alignment was edited again with Jalview. This time, the data model was selected in a less stringent way to keep a maximum number of positions for the analysis. Sequence redundancies above 82% were removed from the data model. An ML tree was constructed with PhyML using the same evolutionary model as described above. Phylogenetic trees were inspected and drawn using Archaeopteryx , NOX-EF group (EF-hand(s) containing members of the NOX group), ppFRE group (protist and plant homologs of the FRE group), and fuFRE  families, as well as the bacterial short and long forms and the STEAP family were obtained from the UniProt server and previous datasets. Sequences of each family were aligned using MAFFT. The alignments were then inspected manually and sequences that possessed atypical insertions within the transmembrane domains were removed from the MSA. The monophyletic group of YedZ-like proteins was removed from the MSA of bacterial short forms; in the following the group was treated as a group on its own. Well-aligned regions – including the predicted transmembrane domains (TM3–TM5) – were extracted from the alignments. Finally, sequence conservation logos were constructed from the eight MSAs using the WebLogo server (weblogo.threeplusone.com). The alignments used for this purpose included 443 , 107 , 75 (STEAP), 22 , 123 (fuFRE), 54 (ppFRE), 66 (NOX-EF), and 54 (NOX) sequences, respectively.For the identification of conserved sites within transmembrane domains and conserved adjacent regions, representative homologs of the NOX family , mafft.cbrc.jp/alignment/server (MAFFT), guidance.tau.ac.il (GUIDANCE), darwin.uvigo.es/software/prottest.html (ProtTest), www.phylogeny.frwww.cbs.dtu.dk/services , phobius.sbc.su.se (Phobius), pfam.sanger.ac.uk (Pfam), www.ebi.ac.uk/interpro (InterPro), weblogo.berkeley.edu (WebLogo) and local computers.The analyses were performed on the Vital-IT High Performance Computing Center , Diplomonadida (3), Euglenozoa (2), Microsporidia (4), Parabasalia (1), stramenopiles (1), Platyhelminthes (2) and Arthropoda (2). For bacteria, FRD homologs are annotated in only 37% of all complete proteomes.At the highest level of structural organization, members of the FRD superfamily are characterized by a common transmembrane ferric reductase domain (Pfam: PF01794) with structural similarity to cytochrome b of the mitochondrial bc(1) complex. Our analysis initially focused on eukaryotic homologs, whose superfamily members also possess a dehydrogenase module consisting of an FAD-binding domain (Pfam: PF08022) and an NADPH-binding domain (Pfam: PF08030), both oriented towards the cytoplasm. Conserved blocks of these homologous regions account for the data model used in the phylogenetic analyses of the eukaryotic genes; the phylogeny of the FRD superfamily is inferred based on the homologous regions of the ferric reductase domain . The manNematostella vectensis currently represents the most basal metazoan, and its proteome contains three gene copies, i.e. two ortholog to the NOX1-3 subfamily and one ortholog to the NOX4 subfamily. There is even some evidence that the divergence of these two subfamilies took place prior to the emergence of animal – NOX homologs of the choanoflagellates Monosiga brevicollis and Salpingoeca sp. (strain ATCC 50818) cluster steadily with the NOX1-3 clade. Furthermore, the NOX family includes the independently duplicated genes from fungi, Amoebozoa and Naegleria gruberi. The presence of these co-orthologs reveals that a gene of the NOX family was probably already present in very early eukaryotes.The phylogenetic tree of the FRD superfamily suggests three major protein groups. Clade 1 includes homologs of the ROS-generating protein families (NOX group) as well as homologs of ferric reductases (FRE) predominantly from plants and protists (ppFRE). This group is of special interest in this study, because – based on functionally characterized family members – we presume that the functional shift from a metalloreductase to a ROS-generating protein took place within this branch in an ancestral gene of very early eukaryotes. The phylogeny of the ROS-generating NADPH oxidases  was analyzed previously What is more challenging is the interpretation of the evolution of the EF-hand containing protein group . It has been suggested that the EF-hand domain was acquired only once At the base of clade 1, metalloreductases predominantly from protists and plants (ppFRE) diverge . Minor cClade 2 includes solely fungal metalloreductases (fuFRE), and the topological placement of this branch outside other eukaryotic metalloreductases is notable . Our anaSo far, no metazoan eFRE orthologs have been identified. Indeed, animals possess enzymes with the conserved ferric reductase domain and a ferric reductase activity, but this family (STEAP) is probably xenolog to eFRE .Acaryochloris marina (strain MBIC 11017) (UniProtKB: B0CEP3), which could have been obtained from an ancestral gene of the NOX5 family . This clade is composed of bacterial ferric reductases (bFRE) including the bacterial YedZ family and the eukaryotic STEAP family. Bacterial members can be roughly classified into short and long forms; the short forms consist basically of the ferric reductase domain, and the long forms of all three domains conserved in the eukaryotic families . Accordi5 family . UniProt5 family .The basic structural component of this superfamily is the transmembrane ferric reductase domain that is present in all the analyzed superfamily members. This domain includes four canonical conserved heme-coordinating histidines in transmembrane domain 3 (TM3) and transmembrane domain 5 (TM5). Their positions in the human NOX2 sequence are 101 and 115 (TM3), and 209 and 222 (TM5). The heme close to the cytoplasm is expected to bind to His-101 and His-209, while the heme close to the cell surface is expected to bind to His-115 and His-222. Most of the bFRD clade homologs consist solely of this domain, but seven distinct modules have been identified, which contribute to the extension of this simple ‘bacterial structural core’ . InteresIn the following sections, the different domain architectures are discussed along with examples of well-studied proteins . An overProteobacteria, which are involved in redox regulation and transmembrane electron transfer E.coli homolog of this family has been shown to form an oxidoreductase complex with the soluble catalytic molybdoenzyme YedY Typical representatives of the ‘one-domain’ homologs from bacteria are members of the YedZ family found in Most structural modifications are found in orthologs of closely related species, thus indicating that variations of the basic core are generally not persistent in bacteria, a fact which is typical for the evolution of prokaryotic proteins. Hence, the enduring bacterial ‘long form’ might be more efficient than functional equivalents or could have a unique function.Saccharomyces cerevisiae homologs are among the best-studied examples of this group. As they are important components of the high affinity uptake system for iron and copper ions, FRE1 and FRE2 are involved in both ferric and cupric ion reduction. There is evidence that FRE3-6 homologs are specific iron reductases, whereas the FRE7 homolog is a specific copper reductase Most, if not all, fungal metalloreductases (fuFRE) contain an additional predicted transmembrane domain that precedes the three conserved domains of the eukaryotic structural core. The nine Viridiplantae and stramenopiles. A well-studied example is the tissue-specific expression of the eight paralogs of the ferric chelate reductase (FRO) family from Arabidopsis thalianaIn some ways similar to the fungal metalloreductases, the majority of protist and plant homologs (ppFRE) in this group bear predicted transmembrane regions in addition to those of the ferric reductase domain. Two or six transmembrane domains are found in the N-terminal of these homologs, but additional transmembrane helices are also predicted close to the C-terminus, within the NADPH-binding domain in clade members from Heterolobosea N. gruberi and homologs from the stramenopiles Albugo laibachii (UniProtKB: F0X089) and Phytophthora infestans (UniProtKB: D0MUZ3) – contain one or two extracellular DOMON domains (Pfam: PF03351). The DOMON domain was predicted to possess a hydrophobic pocket that binds heme and sugar Besides additional transmembrane domains, a single clade of the eFRE group has acquired a new structural module. Members of a subfamily – including seven paralogs from the Note that no FRE-EF forms were observed in bacteria and eukaryotes, suggesting that the regulatory EF hand domains were acquired by superoxide-producing NOX enzymes.Rhodophyta, stramenopiles and Viridiplantae contain four predicted additional transmembrane helices between the FAD-binding and NADPH-binding domains. One study revealed a potential ROS-generating NADPH oxidase activity for the homolog of the red algae Chondrus crispus, At the base of the NOX group, homologs from 2+ to EF-hands can cause conformational changes linked to regulatory functions, as for instance in calmodulin 2+, representative family members were inspected manually . All the studied homologs possessed at least two EF-hands, of which at least one was canonical and hence assumed to be Ca2+-binding. Fungal members of the paraphyletic group of NOX-EF have a single Ca2+-binding EF-hand motif. They seem to exhibit ROS-generating NADPH oxidase activity and have been suggested to be regulated by Ca2+2+-regulated ROS-generation The functional shift from metalloreductases to ROS-generating NADPH oxidases probably occurred early on in the NOX group and, to some extent, seems to be linked to the occurrence of EF-hands in the N-terminus. The binding of CaD. discoideum. Preliminary studies using a NOXC knockout strain showed reduced levels of stimulated ROS generation in vegetative and starved cells . We therefore assume, that the second conserved residue, Thr-178, is equally important for NADPH oxidase activity, but a gating function seems more likely than oxygen-binding. According to an analysis of the functional roles of catalytic residues A comparison of the eFRE metal reductases with expected NADPH oxidases indicated two positions that are conserved in members of the latter group: a histidine in addition to the two heme-binding histidines in transmembrane domain 3 (TM3), and a conserved threonine in transmembrane domain 4 (TM4) . In accostramenopiles and rhodophytes – but not the genes of the chlorophytes analyzed here – are likely to possess a ROS-generating activity that is not regulated by Ca2+. All other homologs of the NOX-EF group possess two or more EF-hands. In addition, two other structural components are found in members of this group: a domain of unknown function that encompasses the N-terminus of the NADPH oxidase  and the heme-containing peroxidase-like domain (Pfam: PF03098). These two additional domains are the hallmark of two separate NOX families. The cytoplasmic NADPH-oxidase domain is attached to the N-terminus of the EF-hands in the RBOH family of land plants. Interestingly, closely related homologs from stramenopiles (RBOH-like) seem to possess EF-hands, but show no predicted domains at the N-terminus. Members of the DUOX family comprise up to three predicted EF-hand motifs, and a further transmembrane domain links to an extracellular peroxidase-like domain. The predicted signal peptide might be required to translocate the peroxidase-like domain to the extracellular space. Members from both families have been biochemically characterized as ROS-generating and Ca2+-regulated enzymes, strongly suggesting a conserved regulation pattern within the NOX-EF group  and Naegleria. The loss of the EF hand-containing N-termini occurred concomitantly with the emergence of separate cytosolic and membrane bound regulatory subunits. An advantage of multi-domain proteins is the colinear expression of functional units that are essential for a specific cellular task, bypassing the need for coordinated expression of separate gene products. However, a more fine-grained regulation is possible when multiple differentially expressed and regulated subunits assemble into a functional complex.Analyses of the extremely divergent domain architectures suggest that two distinct series of events appear to have led to an increased complexity of regulation of ferric reductase domain enzymes. First, as detailed in previous sections, during the early evolution of the superfamily, NOX enzymes acquired longer N-termini with various regulatory and functional domains. Second, the N-terminus was lost subsequent to the gene duplication event that gave rise to the emergence of the NOX1-4 family, which includes the meatozoan NOX1-4 homologs, and the independently duplicated co-orthologs of fungi , Developmental steps to a higher regulation complexity from NOX4, to NOX3, NOX1 and NOX2 can be traced in the NOX family. Of all these, the best studied NOX ortholog is NOX2 and its regulatory network has shown to be particularly complex. Indeed, its fine regulation requires an interaction both with the membrane component p22phox and the cytosolic components p47phox, p67phox, and p40phox. Finally, it also associates with a Rac GTPase that is responsible for the activation of the protein complex Heterolobosea N. gruberi, for instance, contain an SH3 domain (Pfam: PF00018) within their C-terminal NADPH binding domain. Usually, the SH3 domain mediates protein complex assembly via binding to proline rich regions (PRR) of binding partners N. gruberi NOX homologs suggests that some unknown PRR-containing protein partner is involved in their regulation Dictyostelium discoideum carries a functionally analogous WW domain for protein-protein interactions (Pfam: PF00397) Pezizomycotina, including Aspergillus nidulans, Neurospora crassa and Epichloe festucaeD. discoideum NOXA and NOXB, Rac1A, -B and -C show the greatest homology with Rac2 in human neutrophils. In addition, all of the Rac2 residues at the interface with p67phox are conserved, suggesting that a similar activation process might also have occurred in D. discoideumPrecursors of the domains that interact with these essential regulatory subunits can already be found in the NOX homologs of unicellular organisms. The two orthologs of Domain architectures evolve at different modes in prokaryotes and eukaryotes and our results are characteristic for the two superkingdoms. Prokaryotic proteins generally possess a less complex domain composition than eukaryotes, but undergo fusion and fission events more frequently. Hence, proteins with a more complex domain organization are found in small clades . In contAt the sequence level, divergent evolution can be observed at functional sites, which are assumed to reflect changes in substrate selectivity and possibly the reaction mechanism. The functional shift from metal reductase to NADPH oxidase activity probably occurred early on in evolution at the base of the NOX clade. Four amino acids have been identified to distinguish metalloreductases from ROS-generating NADPH oxidases. The replacement of the first canonical heme-spanning histidine by a highly conserved arginine in members of the prokaryotic YedZ family and the eukaryotic STEAP family may indicate a change in the reaction mechanism.The ferric reductase domain may well have emerged from cytochrome b. The phylogeny of the FAD-binding domain suggests the fusion of a ferric reductase domain to a dehydrogenase module . Based oThis study reveals that the ferric reductase domain superfamily probably originated from a short bacterial homolog that consisted solely of a ferric reductase domain. During evolution these ‘one-domain’ homologs were extended by different modules that either participate in redox systems or are regulatory components. The C-terminal fusion of an FAD- and NADPH-binding domain probably resulted in the core of the long homologs, which are mostly found in eukaryotes. Phylogenetic analysis reveals that the highly diverse number of gene copies per species is derived by extensive lineage-specific gene gain and gene loss throughout evolution. The functional shift from metal reductase to NADPH oxidase activity probably occurred early in evolution at the base of the NOX clade. The generation of potentially toxic ROS is accompanied by increased complexity of regulatory systems. One such regulatory component is p22phox, which appears with the emergence of the NOX family; its origin is still unknown.Four amino acids have been identified to distinguish metalloreductases from ROS-generating NADPH oxidases. We believe that it might be possible to detect further sites for the construction of even more specific function predictors based on the comparison of clade-specific conservation signatures. Finally, we plan to use conservation signatures for the revision of unresolved nodes. Preliminary results suggest taking the option of classifying the superfamily into three main groups, namely NOX, FRE and YedZ/STEAP. These and other related issues will be the subject of future interesting studies.File S1Sequence data and domain predictions.(XLSX)Click here for additional data file.File S2Maximum likelihood phylogeny of eukaryotic gene families of the FRE group and the NOX group. The phylogenetic tree is a detailed presentation of (TIF)Click here for additional data file.File S3Exploration of the tree space. Phylogenies of the FRD superfamily from multiple analyses. Gene families are colored in the phylogenetic tree and family names are given. Branch support values of major internal nodes are indicated.(PDF)Click here for additional data file.File S4Analysis of bacterial FRD superfamily members.(PDF)Click here for additional data file.File S5Evolution of the FRD superfamily from the view of its core domains.(PDF)Click here for additional data file.


A key goal of anemia management in dialysis patients is to maintain patients' hemoglobin (Hb) levels consistently within a target range. Our aim in this study was to assess the association of facility-level practice patterns representing Hb measurement and erythropoiesis-stimulating agent (ESA) dose adjustment frequencies with facility-level Hb variation.This was a retrospective observational database analysis of patients in dialysis facilities affiliated with large dialysis organizations as of July 01, 2006, covering a follow-up period from July 01, 2006 to June 30, 2009. A total of 2,763 facilities representing 436,442 unique patients were included. The predictors evaluated were facility-level Hb measurement and ESA dose adjustment frequencies, and the outcome measured was facility-level Hb variation.First to 99th percentile ranges for facility-level Hb measurement and ESA dose adjustment frequencies were approximately once per month to once per week and approximately once per 3 months to once per 3 weeks, respectively. Facility-level Hb measurement and ESA dose adjustment frequencies were inversely associated with Hb variation. Modeling results suggested that a more frequent Hb measurement (once per week rather than once per month) was associated with approximately 7% to 9% and 6% to 8% gains in the proportion of patients with Hb levels within a ±1 and ±2 g/dL range around the mean, respectively. Similarly, more frequent ESA dose adjustment (once per 2 weeks rather than once per 3 months) was associated with approximately 6% to 9% and 5% to 7% gains in the proportion of patients in these respective Hb ranges.Frequent Hb measurements and timely ESA dose adjustments in dialysis patients are associated with lower facility-level Hb variation and an increase in proportion of patients within ±1 and ±2 g/dL ranges around the facility-level Hb mean. A central aim of anemia management in dialysis patients is to maintain patients' hemoglobin (Hb) levels consistently within a target range. Anemia management guidelines and protocols thus require appropriate adjustment of erythropoiesis-stimulating agent (ESA) doses in response to Hb deviations relative to the target range. There are varied opinions among clinicians, however, regarding practice patterns that help achieve this objective -7.The current US Food and Drug Administration-approved ESA label recommends an Hb target range of 10 to 12 g/dL when treating dialysis patients ,9, whichRecently, the Centers for Medicare and Medicaid Services proposed a rule for the End-Stage Renal Disease Prospective Payment System that includes a payment penalty for dialysis facilities that maintain more than a specified proportion of patients in Hb <10 or >12 g/dL ranges . It is tA retrospective observational database was constructed from electronic patient-level data contributed by large dialysis organizations (LDOs) in the United States. An LDO was defined as a chain of >500 affiliated dialysis centers. All data were de-identified in accordance with the Health Insurance Portability and Accountability Act Privacy Rule for limited use datasets; thus no additional institutional review board approval was sought for this study. This database has been described in detail in a previous publication . Data frThe dialysis facility was regarded as the unit of analysis. For each dialysis facility, the following sets of variables were calculated for each month during the study. They were (1) Hb variation, defined as the SD of all Hb measurements across all patients; (2) the number of patient-days, defined as the summation across all patients of the following measure: date of last patient-level dialysis session - date of first patient-level dialysis session + (7/3), with 7/3 applied to adjust for the follow-up truncation at the last observation in a month, assuming the average sequence of dialysis sessions was 3 times per week (TIW) for HD patients; (3) Hb measurement frequency, defined as the number of Hb measurements across all patients divided by the number of patient-days/30; and (4) ESA dose adjustment frequency, defined as the number of ESA dose adjustments across all patients divided by the number of patient-days/30.Only persistent dose changes were regarded as true clinical dose adjustments . Dose changes were considered to be persistent if they lasted for at least 3 TIW sessions for non-zero doses and at least 9 TIW sessions for zero doses. The longer time requirement for zero-dose adjustments helped reduce potential misclassification of intentional clinical adjustments to zero doses with hospitalization events. We validated the methodology with data from patients for whom hospitalization information was available (approximately 50% of the study cohort). The validation step indicated that this methodology would result in a very low proportion (approximately 0.3%) of dose adjustments that could be potentially misclassified. We believe that this level of error is unlikely to cause a meaningful impact on the results. The algorithm adopted for identifying dose changes considered the entire patient-level longitudinal data before further categorization by calendar months. The calendar month boundaries thus did not interfere with the definition of a dose change.We summarized the aggregate variation in facility-level Hb variation, Hb measurement frequency, and ESA dose adjustment frequency by pooling these measurements across all facility-month combinations and calculating basic descriptive statistics. A continuous distribution curve for these measures was created by calculating the percentage of facility-months within specific intervals. By adopting a method similar to one in a previous publication , we prodWe studied associations between facility-level Hb variation and Hb measurement frequency and between facility-level Hb variation and ESA dose adjustment frequency by using a mixed-modeling regression approach that partitioned cross-sectional and longitudinal information in the data -26. ThisWe considered 2 separate models for studying the associations between facility-level Hb variation and Hb measurement frequency and between facility-level Hb variation and ESA dose adjustment frequency due to the relatively high collinearity between Hb measurement and ESA dose adjustment frequencies. An additional reason for considering 2 separate models was that a model which simultaneously considered both these factors could introduce a conceptual possibility that the benefit from these factors in terms of their implications on Hb variation could be regarded as additive and can accrue independently. Though the benefit from Hb measurement frequency can be considered to accrue independently, the same cannot be said about the ESA dose adjustment frequency since any practical anemia management protocol would consider Hb measurement frequency to exceed the ESA dose adjustment frequency. The fixed- and random-effects part of these mixed models included an identical set of parameters representing an intercept term, a linear cross-sectional slope parameter, and a linear longitudinal slope parameter. The number of patient-days was introduced as a weight in the preceding models. The mixed models were implemented using Proc MIXED in SAS . All analyses were performed in SAS, version 9.1.3.We consider the model relating facility-level Hb variation and Hb measurement frequency as not being influenced by an endogeneity problem due to omission of the number of Hb measurements from the model. This is because we defined Hb variation in terms of SD. The expectation of Hb variation is therefore independent of sampling more or less Hb observations, which would influence the precision of estimating the SD. We would highlight that we have not defined Hb variation as the precision of SD, but SD itself, which is a summary measure of the width of the facility-level Hb distribution curve.Please see the Technical Appendix  were available for all 36 months of follow-up, with the remaining facilities (1%) lost to follow-up owing to closure. The total number of unique patients and facility-month combinations represented in the analysis dataset during the 36-month study period were 436,442 and 98,717, respectively.Figure P < 0.001).Figures Figure P < 0.001). The estimates for cross-sectional and longitudinal parameters for both models were negative, which indicated a consistent negative association between facility-level Hb variation and Hb measurement frequency and between facility-level Hb variation and ESA dose adjustment frequency. The magnitude of associations as informed by the cross-sectional and longitudinal parameters was not markedly different for both of these models. Based on the mean values of Hb measurement and ESA dose adjustment frequencies, the difference between cross-sectional and longitudinal parameter estimates translated to a relatively low facility-level Hb variation difference of approximately 0.03 g/dL to 0.05 g/dL as measured by the standard deviation. Formal testing, however, indicated that the cross-sectional and longitudinal parameters were statistically different from one another with a P-value for the difference <0.001 for both models.Table Figures We can suggest plausible mechanisms that may be responsible for driving the observed variation in facility-level anemia management practice patterns and their association with the facility-level Hb variation reported in this study. The relatively wide variations observed in facility-level Hb measurement and ESA dose adjustment frequencies suggest that physicians exercise a significant level of control over these practice patterns in their individual units. Thus, facilities with a higher rate of Hb monitoring may represent physicians who value an assessment of both point-in-time Hb measurement as well as the trend. Assessment of an Hb trend can be helpful; if the trajectory of Hb levels is suggestive of a systematic decline despite stable ESA dosing levels , protocols that encourage timely upward adjustment of ESA doses will be associated with better Hb control. Similarly, if the reverse situation occurs, ESA dosing protocols that encourage a timely reduction or temporary discontinuation of ESAs will result in significantly better control in managing unintended excursions towards the higher Hb ranges. If ESA doses are temporarily withheld to manage high Hb level excursions, the practice of frequent Hb monitoring also allows for a timely re-initiation of treatment before Hb levels decrease too much.When the range of facility-level Hb variation from modeled scenarios was compared with the range represented in the 99th to 1st percentile behavior, it indicated that practice patterns considered in the current study represented only 20% to 30% of the potential for reducing the facility-level Hb variation. This is to be expected because there are a number of other unrelated factors and practice patterns that could influence the facility-level Hb variation. For example, one such practice pattern is the detailed nature of ESA dose adjustments as a function of specific Hb levels. Similarly, iron administration and iron indices can also influence response to erythropoietin: intravenous iron supplementation is independently associated with the risk of exceeding targeted Hb levels  and chanThe bimodal distribution of facility-level Hb measurement frequency reflects the fact that the most popular protocols concerning Hb measurement frequencies were either once per week or once per 2 weeks, with once per 2 weeks being the more prevalent protocol. The increase in mean facility-level Hb measurement frequency seen in Figure Our method of simultaneously modeling cross-sectional and longitudinal associations in the data is similar to that described elsewhere -26. DetaThere are a number of important limitations of our study that warrant careful consideration. It is possible that the set of studied facility-level practice patterns did not exert a direct causal impact on Hb variation, but were surrogates for diligent anemia management that could have resulted in a simultaneously elevated likelihood of the implementation of the described practice patterns as well as a reduction in Hb variation owing to other unrelated factors. These unrelated factors, for example, could represent independent facility-level practice patterns implemented to address the need for managing Hb levels within a narrower target range over time. An important limitation of the analysis dataset was the unavailability of facility-level demographic characteristics or comorbidity information. However, we have provided a summary description of how the adopted modeling framework could have moderated this limitation. The modeling framework was able to effectively utilize the main strengths of the database, which included the availability of consistent longitudinal data at a patient-level and the ability to map patients to individual dialysis facilities. Other important limitations of the study include the fact that only HD patients within the LDO segment receiving intravenous ESA were represented, which limits the extrapolation of results to other patient and dialysis provider segments. Additionally, patients included in the analysis received only intravenous epoetin alfa and, consequently, these results should be considered valid only for this drug and route of administration. In addition, impact on facility-level Hb variation due to potential changes in patient-level iron markers and intravenous iron administration were not captured in the analysis due to unavailability of this data. We have implicitly assumed that the longitudinal changes in the Hb distribution curve at the facility-level during the study time period were primarily influenced by changes in practice patterns concerning Hb measurement and ESA dose adjustment administration frequencies.This study offers useful hypotheses regarding the nature of anemia management practice patterns in dialysis patients that could result in lower facility-level Hb variation and an increase in the proportion of patients within ±1 and ±2 g/dL ranges around the facility-level Hb mean. In particular, this study suggests that the practice patterns of frequent Hb measurements and timely adjustments of ESA doses could be helpful in attaining this objective.Drs. Khan and Ashfaq and Mr. Kothawala are employees of Amgen Inc. Dr. Krishnan is a former employee of Amgen Inc. and has also served on advisory boards and speaker bureaus for Amgen Inc.IK and AK analyzed the data in this study, and all authors interpreted the data. All authors contributed substantially to the writing of this manuscript and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/12/22/prepubTechnical Appendix. This appendix contains additional description regarding the model formulation, covariance structure, parameter estimation, and confidence intervals. The appendix also contains residual diagnostics for assessing model adequacy and results from an unweighted analysis.Click here for file


Steroid-induced osteonecrosis of the femoral condyle is a relatively uncommon condition and is often difficult to select appropriate treatment especially in young patients. Three young men  presented with severe pain and dysfunction of the knee diagnosed as steroid-induced osteonecrosis of the femoral condyle by magnetic resonance imaging (MRIs). Full-thickness cartilage defects sized 20 × 10, 15 × 10, and 30 × 20 mm respectively were classified as International Cartilage Repair Society Grade IV lesions and treated with osteochondral autograft transplantation. They were treated successfully with osteochondral autograft transplantation certificated by post-operative MRI and second look arthroscopy. Steroid-induced osteonecrosis of the femoral condyle is a relatively uncommon condition and its clinical course and established treatment remain controversial, mainly because of the limited number of cases ,2. SteroWe encountered three young patients with steroid-induced osteonecrosis of the femoral condyle and performed osteochondral autograft transplantations. In this report, we outline the cases and give an overview of their treatment.A 25-year-old man suffering from severe right knee pain presented at our institution. He had received steroid-pulse therapy several times for nephrotic syndrome and was subsequently diagnosed with steroid-induced osteonecrosis of the lateral femoral condyle. The preoperative X-ray and MRI showed an osteochondral defect of the lateral femoral condyle Figure A-C. ArthAn 18-year-old man suffering from severe left knee pain had received a renal transplant because of focal segmental glomerulosclerosis and was subsequently diagnosed with steroid-induced osteonecrosis of the medial femoral condyle. The preoperative X-ray and MRI showed an osteochondral defect of the medial femoral condyle Figure A-C. PreoA 24-year-old man suffering from severe right knee pain had received a renal transplant because of focal segmental glomerulosclerosis and was subsequently diagnosed with steroid-induced osteonecrosis of the medial femoral condyle. The preoperative X-ray and MRI showed an osteochondral defect of the medial femoral condyle Figure A-C. FollAs steroid-induced osteonecrosis of the femoral condyle is a relatively rare disease, literature regarding its treatment and histology is sparse ,2, and t2 treated by osteochondral graft transplantation with a satisfactory result [2 defect undergoing osteochondral autograft for steroid-induced osteonecrosis of the femoral condyle [2 (range 1.5 to 5.0 cm2) and all patients received satisfactory results. One of the factors for the success of autogenous osteochondral mosaicplasty is the size of osteochondral articular cartilage defect. Hangody et al. reported that the defect size between 1 and 4 cm2 is the promising factor for the success of the procedure. Lane et al. suggested that because of the difficulty of matching the topography of recipient and donor joint surfaces, the amount of tissue that can be successfully transferred in most surgeons’ hands is limited to less than 2 cm2. Since autogenous osteochondral graft transplantation is a surgical procedure with free bone graft transplantation, the engraftment of the transplanted graft is an important issue, however no detailed analysis of this issue has been reported in cases of surgically treated steroid-induced osteonecrosis of the femoral condyle. Our study has shown that the transplanted grafts remained viable up to two years after surgeries based on MRIs examination and arthroscopic evaluation. However, the lesion in case 2 was partially associated with fibrous tissue formation according to a second-look arthroscopic evaluation despite a good clinical outcome. In this patient we were unable to completely perform osteochondral autograft transplantation and only performed microfracture at the deep posterior part of the lesion because of the location of the lesion. In case 3, similarly, the posterior aspect of the lesion was difficult to reach for grafting even with the knee fully flexed. A previous study suggested that osteochondral graft stability plays an important role in preserving the histologic properties of the cartilage [Recently, use of an autogenous osteochondral graft has gained in clinical popularity because of its technical feasibility and cost effectiveness. Animal and clinical studies have shown that osteochondral plugs maintain hyaline cartilage coverage over the subchondral bone . Howevery result . In cont condyle . In our artilage . As a reOsteochondral autograft transplantation for focal full thickness articular cartilage defects induced by steroids achieved excellent clinical results in three knees. Even though the lesion of osteonecrosis extended to the non-weight-bearing deep posterior site where it was difficult to perform the perpendicular graft transplantation and we only did the bone marrow stimulation with microfracture technique, the lesion including the non-weight-bearing area was successfully healed with smooth articular surface and fibrous tissue, leading to excellent clinical outcomes in the short-term follow up. We believe the osteochondral graft has the potential to prevent or delay the development of degenerative changes of the knee and is a good treatment method for focal steroid-induced osteonecrosis of the femoral condyle.Informed consent was obtained from the patient for publication of this case report and any accompanying image.None of the authors received financial support for this study.YH, KN, MK and RK carried out the surgical treatment and NF, TM, SK, TM, KI and RK discussed the results and commented on the manuscript. All authors have read and proved the final manuscript.


The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents.in vitro.In this study, the tumoursphere assay was validated in MCF-7 cells and used to screen novel marine algal compounds for potential anti-cancer stem cell (CSC) activity high/CD24low marker-bearing cells and Oct4 expression compared to those in the bulk population grown in regular adherent conditions. Using this assay, a set of algal metabolites was screened for the ability to inhibit mammosphere development as a measure of potential anti-CSC activity. We report that the polyhalogenated monoterpene stereoisomers RU017 and RU018 isolated from the red alga Plocamium cornutum, both of which displayed no cytotoxicity against either adherent MCF-7 breast cancer or MCF-12A non-transformed breast epithelial cells, were able to prevent MCF-7 mammosphere formation in vitro. On the other hand, neither the brown algal carotenoid fucoxanthin nor the chemotherapeutic paclitaxel, both of which were toxic to adherent MCF-7 and MCF-12A cells, were able to inhibit mammosphere formation. In fact, pre-treatment with paclitaxel appeared to enhance mammosphere formation and development, a finding which is consistent with the reported resistance of CSCs to traditional chemotherapeutic agents.MCF-7 breast cancer cells were observed to generate tumourspheres or mammospheres after 3-5 days growth in anchorage-independent conditions and an apparent enrichment in potential CSCs was observed by an increase in the proportion of CD44Due to the proposed clinical significance of CSC in terms of tumour initiation and metastasis, the identification of agents able to inhibit this subpopulation has clinical significance. The cancer stem cell theory challenges the traditional monoclonal models of cancer development and has revolutionized the way we think about the origin and progression of cancers ,2. This +CD24- marker-bearing subpopulation could regenerate a tumour from as little as 100 cells, whereas tens of thousands of cells from the bulk population failed to do so  and isolated mammospheres in this study showed an increase in cells expressing the CD44+/CD24- breast cancer stem cell (BCSC) phenotype, the ability of RU017 and RU018 to inhibit mammosphere formation suggested that these compounds have putative anti-CSC activity and may inhibit a signal transduction pathway which is essential in mammosphere formation. Compounds RU017 and RU018 were not, however, able to remove existing mammospheres when added on Day 4. This could be attributed either to the inability of the compounds to penetrate the mammospheres or could indicate that they were only inhibitory during the early stages of mammosphere formation. This was supported by the fact that pre-treatment of MCF-7 cells with either RU017 or RU018 prior to seeding into anchorage-independent conditions had no effect on the ability of the cells to form mammospheres under these conditions. A possible explanation for this is that the effect of the compounds on the MCF-7 cells may be specific to their initial propagation under anchorage-independent serum-free growth conditions. This is supported by the fact that dissociated untreated mammospheres seeded into regular anchorage-dependent conditions and then treated with the algal compounds experience only a minor decrease in viability as opposed to the large decrease in viability seen when cells are treated with these compounds upon seeding into anchorage-independent conditions.In the case of the halogenated monoterpene stereoisomers RU017 and RU018, both of which were shown to have no effect on the survival and proliferation of either breast cancer cells or non-transformed breast epithelial cells To further assess the efficacy of the algal compounds, RU017 and RU018, their effect on sphere forming efficiency was assessed for second and third generation mammospheres. In both cases, although the compounds were unable to prevent second or third generation mammosphere formation as was the case for primary mammospheres, they did result in a dose-dependent decrease in sphere forming efficiency. This reduced ability of the algal compounds to inhibit mammosphere formation in subsequent generations may be due to accumulated changes in growth characteristics of the cells as a consequence of prolonged culturing under anchorage-independent serum-free conditions, resulting in a more aggressive phenotype. This could also account for the increase in sphere forming efficiency in the vehicle control for the third generation as compared to that of the second generation. Interestingly, RU018 appeared more effective than RU017 in inhibiting second and third generation mammosphere formation, a finding which was consistent with the trend observed in terms of first generation mammosphere cell viability. This data implies that the stereochemistry of the monoterpene compounds plays a role in their mechanism of action against MCF-7 mammospheres.The identification of CSCs within breast tumours has resulted in a major shift in focus in terms of the development of novel therapies to treat this disease . It is nPlocamium corallorhiza and Plocamium cornutum at room temperature for 30 minutes. A primary antibody solution containing anti-CD44 conjugated to APC in 0.1% (w/v) BSA/TBS was added to the cells and incubated overnight at 4&z.ousco;C. Cells were washed twice with agitation in blocking solution, after which the cells were washed in distilled water containing 1 μg/ml of DNA binding dye Hoechst 33342. The coverslips were mounted onto glass slides using Dako mounting media (Dako). Differential contrast (DIC) and fluorescent images were taken using a Zeiss LSM510 meta confocal microscope.MCF-7 cells were seeded at a density of 4000 cells per well in anchorage independent mammosphere conditions and mammospheres allowed to develop over 8 days. In addition MCF-7 cells were cultured in anchorage-dependent regular culture conditions and lifted using 0.25% (v/v) trypsin. Whole cell lysates were prepared from adherent and mammosphere cultures. Briefly, cells were collected by centrifugation at 2000 rpm for 3 minutes and resuspended in 100 μM SDS sample buffer  SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue) before boiling for 5 minutes. Thereafter, 30 ug of protein from each lysate was loaded onto a 12% SDS polyacrylamide gels, separated by electrophoresis and transferred onto a nitrocellulose membrane, before probing with mouse anti-Oct3/4 (1 in 2000) at 4&z.ousco;C overnight. The HRP-conjugated anti-mouse secondary antibody was detected using the ECL Advanced Western Blotting Kit  and visualized using the Molecular Imager ChemiDoc XRS System (BioRad). Lysates were probed with rabbit anti-histone H3 antibody (1 in 2500) as a loading control.Plocamium cornutum were gathered off the south-east coast of South Africa and the halogenated monoterpenes extracted as previously described [Specimens of the red alga escribed .Cell viability after treatment with the compounds was evaluated by means of the MTT assay in the case of adherent cells and WST-1 assay in the case of mammospheres, according to manufacturer’s instructions. For both assays, percentage viability after treatment with compounds was calculated relative to the DMSO vehicle control (taken as 100% viability) when equal numbers of cells are seeded. For the MTT Cell Proliferation Kit 1, MDA-MB-231, MCF-7 or MCF-12A cells were seeded at a density of 6000 cells/well in 96-well plates and assessed for viability as previously described . In the APC: Allophycocyanin; BCSC: Breast cancer stem cell; BSA: Bovine serum albumin; CSC: Cancer stem cell; DMEM: Dulbecco’s Modified Eagle Medium; DMSO: Dimethyl sulphoxide; EGF: Epidermal growth factor; FCS: Fetal calf serum; FGF: Fibroblast growth factor; FITC: Fluorescein isothiocyanate; FXN: Fucoxanthin; PBS: Phosphate-buffered saline; PSA: Penicillin-streptomycin-amphotericin; Ptx: Paclitaxel; SFE: Sphere forming efficiency; TBS: Tris-buffered salineThe authors declare that they have no competing interests.ALE, JdlM and GLB were involved in the conception and design of the study. JdlM and ALE drafted the manuscript. JdlM carried out the MCF-7 mammosphere assays and screening of marine compounds. JNS carried out the flow cytometry and MGS the Oct4 Western analysis. MTC and DRB were responsible for the purification of the compounds from marine algae. All of the authors read and approved the manuscript.


We applied this approach to a peripheral whole blood expression study of kidney allograft rejection. The patterns of differential composition uncovered are consistent with previous studies carried out using flow cytometry and provide a relevant biological context when interpreting cell type-specific differential expression results. We identified cell type-specific differential expression in a variety of leukocyte sub-populations at the time of rejection. The tissue-specificity of these differentially expressed probe-set lists is consistent with the originating tissue and their functional enrichment consistent with allograft rejection. Finally, we demonstrate that the strategy described here can be used to derive useful hypotheses by validating a cell type-specific ratio in an independent cohort using the nanoString nCounter assay.Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. Various populations of lymphocytes are principal mediators of this process, infiltrating graft tissues and driving cell-mediated cytotoxicity. Understanding the lymphocyte-specific biology associated with rejection is therefore critical. Measuring genome-wide changes in transcript abundance in peripheral whole blood cells can deliver a comprehensive view of the status of the immune system. The heterogeneous nature of the tissue significantly affects the sensitivity and interpretability of traditional analyses, however. Experimental separation of cell types is an obvious solution, but is often impractical and, more worrying, may affect expression, leading to spurious results. Statistical deconvolution of the cell type-specific signal is an attractive alternative, but existing approaches still present some challenges, particularly in a clinical research setting. Obtaining time-matched sample composition to biologically interesting, phenotypically homogeneous cell sub-populations is costly and adds significant complexity to study design. We used a two-stage,  Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. It is caused by an immune response, with both innate and adaptive components, mounted by the host against alloantigen in the donor tissue. Various lymphocyte sub-populations are known to be principal mediators of this immune response, infiltrating graft tissues and driving cell-mediated cytotoxicity e.g.; eosinophils, Tregs), whose signal might otherwise be drowned out by the more abundant cell type compartments . An ability to study both of these systems and their interplay all within the same sample would be very useful.Measuring genome-wide changes in transcript abundance in circulating blood cells  can deliver a comprehensive view of the status of the immune system and has been useful in studying the pathobiology of many diseases, including kidney allograft rejection Experimental separation of the component cell types of complex tissue samples is an obvious solution. Following isolation and quantification, one could perform between-group differential expression analysis for each of the cell types in a tissue to assess cell type-specific gene expression changes in the experimental context. However, experimental methods for isolating cell type subsets from complex tissues, such as fluorescence-activated cell sorting (FACS) or enrichment columns, are expensive and the need to process additional gene expression assays for each cell type of interest further exacerbates the problem. Ignoring the additional costs incurred, such isolation methods rely on the availability of unique cell-surface markers and appropriate antibodies that may or may not exist for all cell types of interest et al.S. Cerevisiae. Other groups subsequently demonstrated that microarray expression deconvolution can reasonably quantify the constituents of peripheral whole blood et al. demonstrated that cell-specific gene expression may be inferred from peripheral whole blood expression data using total leukocyte differentials  The cell-composition-dependent signal and cell-function-dependent signal in complex tissue gene expression data may, however, be statistically deconvolved. Lu For simplicity, inferring cell type-specific expression from the peripheral whole blood sample expression data can be referred to as the forward case of deconvolution, while inferring composition from the peripheral whole blood sample expression data can be referred to as the reverse case of deconvolution.in situ. Finally, we have previously hypothesized that the sensitivity of forward deconvolution may be poor when phenotypically heterogeneous compartments are used in the model  These approaches are complementary. While total leukocyte differentials have been proposed as a readily available source of composition information to enable forward deconvolution approaches in a clinical setting, they offer insufficient granularity, most obviously within the lymphocyte (lymphoid) compartment. Flow cytometry can provide much higher granularity, but at higher cost and increased complexity. The use of reverse deconvolution addresses these issues directly by providing more granular composition data without incurring any additional costs or requiring additional data collection. More generally, the use of whole genome expression data to infer cell type composition could theoretically allow for quantification and study of otherwise non-trivially isolatable cell types in silico deconvolution strategy and its application to the study of the lymphocyte compartment of peripheral whole blood during acute kidney allograft rejection to highlight its utility. First, we detail the construction of a suitable basis matrix: an estimate of the cell type-specific expression profiles of the various components of peripheral whole blood that allows us to infer the fractions of these components in each sample from the observed expression in peripheral whole blood (reverse deconvolution), and we establish its performance in three separate cohorts of transplant recipients. Next, we demonstrate that including lymphocyte sub-populations may yield additional discovery when performing cell type-specific differential expression analysis in peripheral whole blood and provide a more relevant biological context for this discovery. We establish the plausibility of the cell type-specific probe-sets identified by this approach. Finally, we apply this two-stage, in silico deconvolution approach to a timecourse study of acute kidney allograft rejection, including samples and time points for which no independent composition data was available, to highlight its utility when attempting to derive value from existing clinical samples.We describe below the implementation of a two-stage, This prospective observational study was conducted at 4 renal transplant centres  between January 2005 and September 2009, and expanding to include The Ottawa Hospital  and Princess Alexandra Hospital  between September 2009 and May 2012) and was approved by the UBC Providence Health Care Research Ethics Board , UBC Clinical Research Ethics Board , Ottawa Hospital Research Ethics Board , and Metro South Health District Human Research Ethics Committee , respectively. All eligible patients undergoing a kidney transplant were invited to participate in the study. Recipients eligible for the study were >18 years of age and able to provide informed consent. Recipients who were under 18 years of age, received multiple, different solid organ transplants, HIV positive, or received organs from donors who tested positive for HIV were excluded. All study participants provided informed written consent.Recruited patients received a standardized treatment protocol including basiliximab 20 mg i.v. on days 0 and 4, methylprednisolone 125 mg i.v. on the day of transplantation tapering to zero by day 3 post-transplant, tacrolimus 0.075 mg/kg b.i.d. and mycophenolate 1000 mg b.i.d. Tacrolimus concentrations were measured by tandem mass spectrometry, and the dose was adjusted to achieve 12-hour trough levels of 8–12 ng/mL in month 1, 6–9 ng/ml in month 2, and 4–8 ng/ml thereafter. Allograft rejection was diagnosed by normal clinical and laboratory parameters, confirmed by biopsy, and graded according to the Banff 97 working classification of renal allograft pathology We employed a case-control design Table S1. Peripheral whole blood gene expression was assayed on Affymetrix U133 Plus 2.0 microarray pre-transplant (baseline), at the time of rejection (rejection) and at the first available time point at least 7 days following rejection (post-rejection). Total leukocyte differentials time-matched to the RNA extraction blood draw (within 24 hours) were available for 41 subjects , only at the rejection time point.This selection process yielded 48 suitable subjects  from a primary cohort , described in previous work by our group A secondary cohort of 44 subjects  was assembled by applying the same selection process to kidney transplant recipients enrolled between September 2009 and May 2012 across all four sites. Peripheral whole blood gene expression was assayed on the nCounter GX Human Immunology Assay  at the time of rejection only. Total leukocyte differentials time-matched to the RNA extraction blood draw were not available for these subjects. This secondary cohort was used to test a cell type-specific hypothesis formulated at the rejection time point in the primary cohort.training set, is an aggregate of two previously described cohorts of heart (n = 26) test set to assess the performance of our deconvolution model. The various datasets used in this analysis are tabulated in The statistical model used to infer the cellular composition of peripheral whole blood samples was constructed, and preliminary validation on it performed, using two aggregate datasets of the expression profiles of leukocyte sub-populations isolated from peripheral whole blood, obtained from the Gene Expression Omnibus  website. Two additional groups of patients were used to train and validate the statistical model used to infer the composition of peripheral whole blood samples. The first group, our Blood samples for all subjects and time points were collected in PAXgene tubes and stored at −80°C until analysis. Total RNA was extracted using PAXgene Blood RNA Kits , and integrity and concentration determined using an Agilent 2100 BioAnalyzer . Affymetrix Human Genome U133 Plus 2.0  microarrays were processed at the Microarray Core Laboratory at Children's Hospital, Los Angeles in order to assess whole genome expression. The microarrays were checked for quality using the “affy” (version 1.16.0) and “affyPLM” (version 1.14.0) libraries, part of the BioConductor project, as well as “mdqc”  All statistical analyses were performed using the R Statistical Programming Language ijX for sample i = 1, 2, …, n and genes j = 1, 2, …, p measured cell-type proportions ikw for samples i = 1, 2, …, n and cell types k = 1, 2, …, K, cell type-specific contribution to the observed gene expression kjh for cell-types k and gene j, and a random error term ije yields the following equation:Peripheral whole blood expression was modeled as follows: assume observed expression values X, W, H be matrices with entries ijX (sample observed expression), ikw (sample composition), and kjh (sample cell type-specific contribution to the observed expression) respectively. Having measured X, the convolved peripheral whole blood expression, we wish to study W and H in isolation; that is, we wish to deconvolve X.Let W, the sample composition, is known, then H, the cell type-specific contribution to the observed expression, can be inferred by fitting the above model by regression of each column of X on W, to yield the coefficients in the corresponding column of H, as in csSAM kjh as the average gene expression for cell-type k in the group of samples. The coefficients can then be compared across groups to assess cell type-specific differential expression. Statistical testing employs a permutation scheme to estimate false discovery rate cut-offs. We refer to this as the forward case of deconvolution.If W can be inferred by fitting the above model by regression of each column of X on H, to yield the coefficients in the corresponding column of W. In this case the cell type-specific contribution to the observed expression, H, can be estimated from the expression profiles of isolated cell populations, at least for a minimal subset of genes that exhibit cell type-specific expression. We can then use this estimate of H, termed the basis matrix, to deconvolve. The approach was first demonstrated by Lu et al., studying cell-cycle regulation of the yeast S. CerevisiaeSimilarly, H, for a subset of cell type-specific genes. In the case of peripheral whole blood, this estimate can be obtained from a collection of expression profiles of leukocyte populations isolated from blood. We identified a suitable dataset (GSE28490) on our target platform (Affymetrix U133 plus 2.0) via the Gene Expression Omnibus The basis matrix for deconvolution is an estimate of the cell type-specific contribution to the observed expression, 2-transformed expression obtained from GSE28490 and fit a multinomial elastic net model 2 fold change between any two cell types by using “limma” training set by reverse deconvolution. We employed a quadratic programming approach (via the “limSolve” library), as in training set. Predicted lymphocytes proportions are the sum of the predicted proportions for B cells, CD4+, CD8+ T cells and NK cells. The final basis matrix was that constructed from the largest alpha parameter value  that minimized the root mean squared error (RMSE) in lymphocytes. Performance of the selected matrix was then validated in an independent test set.Identifying a minimal subset of cell type-specific genes from this collection of isolated leukocyte expression profiles can be framed as a multinomial classification problem with feature selection. Feature selection is necessary because we expect most genes to be non-informative with respect to discriminating between cell types Traditional, two-class differential expression analysis in peripheral whole blood was carried out using Significance Analysis of Microarrays  We lacked suitable biological starting material to carry out direct experimental validation of the putative cell type-specific differentially expressed probe-sets identified by csSAM (FDR ≤0.30) in this analysis. In order to establish their plausibility, we assessed both their tissue-specificity and functional enrichment in the context of acute allograft rejection.th percentile of the enrichment data across all tissues was used as a threshold. For each tissue, the corresponding gene set was composed of probe-sets with enrichment greater than the threshold value. The tissue-specific enrichment of the candidate cell type-specific gene lists was assessed via a hypergeometric test of their overlap.The tissue-specificity of all cell type-specific probe-set lists was evaluated in three ways. First, we visualized the median tissue-specific enrichment score  are reported. Prediction accuracy was very good in lymphocytes , but generally poor in neutrophils  and monocytes  and eosinophils , which are included, but not plotted, in all subsequent analyses.For the selected basis matrix, predicted vs. measured proportions , were plotted for neutrophils, lymphocytes and monocytes  for both our Having established prediction performance, we next applied reverse deconvolution with the selected basis matrix to a cohort of 48 kidney transplant recipients  at the time of a treatable acute rejection episode. The cell type composition of each peripheral whole blood sample was inferred by reverse deconvolution of the mixed expression data to all seven cell types present in the basis matrix . Repeating the experiment, but substituting in the predicted and summed lymphocyte proportions yielded similar results (not shown). Using the predicted composition data resulted in the identification of 456 probe-sets down-regulated in neutrophils, five probe-sets up- and 445 probe-sets down-regulated in B cells, 221 probe-sets up-regulated in CD4+ T cells and 221 probe-sets up-regulated in NK cells.Next, cell type-specific differential expression analysis csSAM was carret al. dataset et al. dataset (see Methods). The adjusted p-values for these tests  using predicted cell type proportions at the time of a treatable acute rejection episode, we assessed their tissue specificity across a broad range of tissue typesFinally, the csSAM output was used to create a cell type-specific ranking statistic analogous to the Signal to Noise Ratio employed by GSEA, as described previously by our group.Peripheral whole blood samples suitable for flow cytometry were never collected for the subjects used in the current study. As a result, experimental validation of the intermediate lymphocyte composition predictions was impossible. Instead, a lymphocyte-specific hypothesis was formulated based on the two-stage deconvolution results (see Methods) and tested in samples from an independent cohort, using a different gene expression assay. A similar approach was recently used to identify a patient's risk of active tuberculosis infection from peripheral whole blood expression data Finally, we wished to demonstrate the power of this approach in enriching existing clinical datasets, by allowing the exploration of cell specific biology without the need for previously collected, time-matched composition information. To this end, we applied deconvolution to study the peripheral whole blood cell type composition (reverse deconvolution) and cell type-specific biology (forward deconvolution) of kidney rejection. For the 48 subjects  from The composition of the peripheral whole blood samples was inferred from mixed expression data Next, cell type-specific differential expression analysis was performed using the inferred sample composition obtained above for all 48 subjects  at the time of a treatable acute rejection episode, as well as at baseline  and after rejection had resolved  when expression data was available. The number of probe-sets called differentially expressed at various FDR cutoffs is plotted for the one-tailed up and one-tailed down hypotheses . We therefore elected to construct our own U133 plus 2.0-based basis matrix, in order to study the cell types we deemed interesting in this context. A suitable collection of the isolated expression profiles of various leukocyte sub-populations was identified on the GEO website and an optimal basis matrix constructed by fitting a multinomial elastic net model to the isolated expression profiles of seven leukocyte sub-populations. This was performed in the RMA normalized, log2-transformed data . The size of the resultant basis matrix was tuned to maximize prediction performance of reverse deconvolution for lymphocytes in a training set of 50 heart and pediatric kidney allograft recipients for which total leukocyte differentials time-matched to the RNA blood draw were available. We observed no bias in prediction performance between subjects either undergoing acute allograft rejection or not (data not shown). The selected basis matrix was then used to predict the blood leukocyte composition of samples from 48 kidney allograft recipients using reverse deconvolution. We confirmed our ability to predict lymphocyte proportions in a subset of these 48 subjects for which total leukocyte differentials time-matched to the RNA blood draw were available. These 41 subjects acted as an independent test set to assess the prediction performance of the selected basis matrix.We first evaluated the previously published Abbas Prediction performance in lymphocytes was comparable to current state of the art total error rate for lymphocyte measurement by total leukocyte differentials . The elastic net alpha parameter was tuned from 1 to 0 and RMSE of the predicted lymphocyte proportion was computed as the number of genes included in the basis matrix increased. We observed that, for most values of alpha , the RMSE was ≤10%. Predicted neutrophil proportions were similarly robust (not shown). An initial concern was that basis matrix genes might be under differential regulation in the perturbed state of interest. The similar (and robust to gene membership) prediction performance in both AR and NR subjects in both our training and test cohort suggests that the feature selection strategy we adopted resulted in an unbiased basis matrix.Varying the composition of the basis matrix did not negatively affect prediction accuracy in lymphocytes  B, CD4+ T and NK cell proportions in AR subjects could be the result of increased infiltration of these cell types into the allograft, which would be consistent with our current understanding of solid organ rejection, in which NK cells act as facilitators of solid organ rejection, amplifying early graft inflammation and supporting the activity of alloreactive T cells Having selected an optimally performing basis matrix, we wished to demonstrate the utility of this approach in studying a complex biological process. We studied the proportions of various lymphocyte sub-populations during kidney allograft rejection in the inferred composition data. Our results were consistent with a previously published flow cytometry analysis of the peripheral blood of kidney transplant recipients by Sagoo The inferred composition estimates were then used as input to csSAM. The inclusion of phenotypically homogeneous and biologically relevant lymphocyte sub-populations provides more useful context when interpreting cell type-specific differential expression results. More importantly, deconvolving to more phenotypically homogeneous components should improve the sensitivity of the approach .e.g., total leukocyte differentials).Validating the results of statistically powered cell type-specific differential expression analysis is generally challenging because experimental separation protocols are known to result in modified gene expression Figure S5). Lists that overlap significantly between cell types with opposite directionality , serve to highlight the issue of convolution in peripheral whole expression data. Tissue specific enrichment results, using both the Benita et al. and the MSigDB C7 collection of immunologic gene sets, were consistent with the cell type origin. Hypergeometric test of the overlaps between tissue specific gene sets and the cell type-specific probe-set lists in the Benita et al. data suggest that both the neutrophil and NK cell lists were very cell type-specific, while the B and CD4+ T cell lists were more broadly representative of mature, circulating B or T lymphocytes. While no C7 gene sets were significantly enriched in the B cell list, the CD4+ T cell list was highly specific for CD4+ T cell states and perturbations. Tissue-specific enrichment results thus suggest that our two-stage, in silico deconvolution strategy is yielding truly cell type-specific probe-sets.To evaluate the plausibility of the derived cell type-specific probe-set lists, we turned to tissue and functional enrichment strategies, as well as the literature. We first observed that the cell type-specific probe-set lists are distinct, though some lists exhibit significant overlap  had skewed cytokine response, with a higher propensity for TGF-β production than B cells from AR subjects. TGF-β was found to be down-regulated in B cell in AR subjects and TGF-β signaling was one of the most negatively enriched gene sets in these subjects, though it did not reach statistical significance.Furthermore, functional enrichment results using the MSigDB C2 (REACTOME) collection of gene sets were consistent with our current understanding of solid organ rejection, in which NK cells act as facilitators, amplifying early graft inflammation and supporting the activity of alloreactive T cells Having established plausibility, we attempted to validate some of these cell type-specific results in an independent patient cohort using the nCounter GX Human Immunology Assay. A lymphocyte-specific ratio for each patient was computed by taking the mean expression of probe-sets up-regulated in CD4+ T and NK cells in AR subjects at the time of rejection and dividing it by the mean expression of probe-sets down-regulated in B cells in AR subjects at the time of rejection, for probe-sets that could be mapped to the nCounter assay. The ratio was constructed so as to maximize the difference between AR and NR subjects. It was significantly different between AR and NR subjects when computed from the peripheral whole blood microarray data , demonstrating it could overcome the convolution issue in peripheral whole blood expression data, and this result was replicated on the nCounter platform, in independent samples. While it is likely that the cell type-specific lists generated by this two-stage deconvolution approach include false positives, this replication result demonstrates that it can be a fruitful strategy, particularly if the goal is hypothesis generation.in silico deconvolution strategy enables using existing clinical samples.Finally, we wished to demonstrate the utility of this approach by applying it to a timecourse study of acute kidney allograft rejection. Such a study would have been extremely challenging to implement had we been reliant on the availability of time-matching of the RNA blood draw (PAXgene tube) to total leukocyte differentials (EDTA whole blood tube) to assess sample composition. It is presented below as an illustrative example of the kind of retrospective analysis that a two-stage, The pre-transplant time point (baseline) is included as a control. We expect both groups to be similar at this time. The identification of approximately 50 differentially expressed probe-sets in the peripheral whole blood analysis, including eight at FDR ≤10% and three probe-sets at FDR ≤5%, serves to reinforce that peripheral whole blood expression data can be convolved by differences in composition across subjects. Conversely, no statistically significant, cell type-specific, differential expression is identified between AR and NR subjects at this pre-transplant time point. The composition of peripheral whole blood from AR and NR subjects is also comparable pre-transplant, with the exception of NK cell proportions, which are significantly lower in AR subjects. Though this may simply be an artefact of inaccurate prediction, NK cells have been previously reported to facilitate the induction of tolerance Post-rejection, the cell type-specific signal seen at the time of rejection resolves in neutrophils and NK cells. It is interesting that these cell types, both involved with the early, innate immune response Figure S2), confirming that our selected basis matrix can accurately predict the proportions of pure leukocyte populations isolated from peripheral whole blood. However, absent more granular independent measurement of composition we are unable to assess the prediction accuracy at the lymphocyte sub-population level  in mixtures. This is an important limitation of the current work, which we hope to address in future studies . Unfortunately, the required biological materials were not available for the subjects used in this study. Sample composition was not, however, the primary focus of the current work. Rather we wished to study cell type-specific differential expression in lymphocyte sub-populations before, during and after acute rejection of a renal allograft. Prediction error in the composition estimates used as input for this analysis, though undesirable, is carried forward into the cell type-specific differential expression results, potentially leading to an inflated rate of false positives. We also note that the csSAM procedure does not account for errors in the composition estimates used as input when estimating FDR cutoffs. Ultimately, any specific hypotheses based on the cell type-specific differentially expressed gene lists would need to be validated in independent samples and using a different technology to assay gene expression.The current study has two main limitations. First, while neutrophil and monocyte prediction performance can be directly validated using total leukocyte differential data, predictions for the various lymphocyte sub populations cannot. The validity of applying reverse deconvolution using the selected basis matrix to peripheral whole blood was assessed by experimenting with expression profiles obtained from leukocyte sub populations isolated from peripheral whole blood . These profiles were appropriately deconvolved  under different experimental conditions and assessing expression in these isolated components, as well a reconstituted, equal volumes mixture of these components. The cell type-specific expression can then be obtained by deconvolution of the reconstituted mixture expression data using the measured proportions of the components in the original sample and compared to that observed in the isolated components. This is analogous to the in silico proof of concept originally presented by Shen-Orr et al. Second, validation of the cell type-specific differentially expressed probe-sets identified by csSAM is challenging in practice because it is likely that experimental separation affects expression in silico deconvolution approach described here has allowed us to deconvolve the lymphocyte compartment of peripheral whole blood and study both the cell type composition of, and cell type-specific expression in, existing clinical samples. This did not require the collection of time-matched sample composition information. The inferred composition data provided a more informative context for interpretation of the cell type-specific differential expression results. The predicted cell type proportion and cell type-specific differential expression results at the time of rejection were consistent with the experimental context, consistent with previously published work, and suitable for exploratory analysis and hypothesis generation. Finally, we demonstrated the power of this approach in allowing us to gain additional insight from existing clinical samples for which only peripheral whole blood expression data is available. While we focused on acute allograft rejection in kidney transplantation, this two-stage, in silico deconvolution approach should allow for similar studies to be carried out in any peripheral whole blood expression datasets on the Affymetrix U133 Plus 2.0 platform, allowing for re-examination of more than 80000 arrays, comprising nearly 3000 experiments on GEO alone. The approach should be broadly applicable to the study of any complex tissue for which isolated component expression profiles exist on the same technology platform, including next generation sequencing, and may prove a fruitful strategy in many contexts.The two-stage, Figure S1Robustness of the basis matrix. The condition number (kappa) of the basis matrix and root mean squared error (RMSE) of the predicted lymphocyte proportion in AR and NR subjects is plotted as alpha, the elastic net tuning parameter, is adjusted between 1 and 0. Corresponding number of genes included in the basis matrix thus constructed is indicated.(TIFF)Click here for additional data file.Figure S2Performance of expression deconvolution on purified leukocytes supports using it on peripheral whole blood. Reverse deconvolution of an independent test set of leukocytes isolated from peripheral whole blood (GSE28491) demonstrates that various cell types are accurately deconvolved. Plotted data is the predicted proportion of that cell type in the whole sample produced by reverse deconvolution of each of seven purified cell type expression profiles. Data points are from independent subject samples.(TIFF)Click here for additional data file.Figure S3Reverse deconvolution is more accurate when data is quantile normalized. The performance of reverse deconvolution using the optimal basis matrix is assessed by visualizing measured and predicted cell type proportions for neutrophils, lymphocytes and monocytes in the training set , either quantile normalized (A) or not (B). Predicted lymphocyte proportions are the sum of the predicted proportions for B cells, CD4+, CD8+ T cells and NK cells. Measured and predicted proportions are plotted and the adjusted coefficient of determination (adj. R2) and root mean squared error (RMSE) reported.(TIFF)Click here for additional data file.Figure S42-transformed.Reverse deconvolution is more accurate when data is log The performance of reverse deconvolution using the optimal basis matrix is assessed by visualizing measured and predicted cell type proportions for neutrophils, lymphocytes and monocytes in both the training  and test  sets, either log2-transformed (top) or not (bottom). Predicted lymphocyte proportions are the sum of the predicted proportions for B cells, CD4+, CD8+ T cells and NK cells. Measured and predicted proportions are plotted and the adjusted coefficient of determination (adj. R2) and root mean squared error (RMSE) reported.(TIFF)Click here for additional data file.Figure S5Overlap between the various cell type-specific differentially expressed probe-set lists at the time of rejection. A Venn diagram showing the overlap between the various cell type-specific differentially expressed probe-set lists obtained in (TIFF)Click here for additional data file.Table S1Subject Demographics.(XLS)Click here for additional data file.


For the development of biosimilar monoclonal antibodies (mAb) or related substances containing the IgG Fc part it is mandatory to fully compare immunological properties between originator and biosimilar in a "comparability exercise" . The mosRetroviral vectors were used to generate cell lines with stable genetic modification. Vector particles were generated by transient transfection of 293T cells with plasmids encoding gag, pol/env and an expression plasmid containing the packaging region and the sequences of promotor and the transgenes, i.e. selection marker and gene of interest. Multiple gene expression was achieved either by using a bicistronic design enabling transcription from two promotor sequences, or by using an internal ribosomal entry site. Transduction of cells in log phase was followed by a selection of transduced cells and clonal selection by limiting dilution. Cell clones were expanded for primary and secondary cell banks and further characterised with regard to transgene expression and functional characteristics. We developed a human transgenic NK-cell line  with stable expression of Fc gamma-receptor IIIA  and stable functional characteristics. Target cell lines were generated similarly using different expression plasmid constructs.ADCC assays were developed by using design of experiments (DoE) to determine experimental factors of importance for assay suitability. To show assay suitability goodness of fit, the amplitude of sigmoid curve, slope and parallelism was determined for each sample compared to a standard. Hypo- and hyperpotent samples  of Rituximab, Trastuzumab, Adalimumab and Infliximab were analysed to determine accuracy and linearity of each method. Optimisation of each assay requires determining the relative importance of factors including E:T ratio, incubation time, target cell density and pre-assay schedules for target and effector cells. Analysis of critical factor interaction was performed using Minitab software. A list of established ADCC assays is shown in Table CD16 expression was analyzed and quantified by flow cytometry. Cells were stained using anti-CD16 PE-conjugated antibodies. PE-fluorescence was correlated to number of PE-molecules per cell using BD Quantibrite beads. Primary NK-cells were isolated using Dynal beads (purity > 95%) from 3 healthy donors and used immediately after isolation.In order to prove genetic stability of the transgenic NK cell line CD16 expression was analysed by flow cytometry for up to 22 passages. More than 95% of cells were CD16 positive, viability of cells was >90%. CD16 expression level was stable (19.000 - 28.000 CD16 molecules/cell).Functional stability of the effector cell line was shown for more than 30 passages. This was shown by a stable EC50 value obtained for a reference antibody in the Trastuzumab ADCC assay.The effector cell line was compared with primary NK-cells (purity > 95%) from 3 donors in a Trastuzumab ADCC assay. The data show high donor variability, mostly incomplete dose-response curves and a killing activity with a low dynamic range (baseline to top ratio: 3). For primary NK-cells the amplitude of the dose-response curve is dependent on both donor variability and the type of target cell. Using the effector cell line this is dependent on the target cell only. Assay variability was strongly reduced and sample throughput could strongly be increased by using the effector cell line in comparison to primary NK-cells. Optimization of each assay by DoE required determining the relative importance of various factors including effector to target cell ratio, incubation time, target cell density and pre-assay culture schedules for target and effector cells. Accuracy of these ADCC assays could be shown in between a range of 50% to 200% potency. Linearity was shown by a high coefficient of determination (>0.97) and other statistical methods. Inter-assay precision of all ADCC assays was <20%.ADCC assays for Infliximab and Adalimumab require a membraneTNFalpha expressing target cell line Table . In thisAccuracy and linearity of the Infliximab ADCC assay was analysed by measuring items containing varying theoretical antibody concentrations to simulate hypo-potent and hyper-potent samples. Linearity was shown by a high coefficient of determination or by testing if the 2nd order polynomial model is non-significant . For precision analysis the relative potency of a sample was repeatedly analyzed on 4 days with 3 assays per day.Altogether these data show the feasibility of providing suitable tools for validation and routine testing of various mAbs in ADCC potency assays scalable to the analytical needs of biosimilar testing.