Although high-throughput marker gene studies provide valuable insight into the diversity and relative abundance of taxa in microbial communities, they do not provide direct measures of their functional capacity. Recently, scientists have shown a general desire to predict functional profiles of microbial communities based on phylogenetic identification inferred from marker genes, and recent tools have been developed to link the two. However, to date, no large-scale examination has quantified the correlation between the marker gene based taxonomic identity and protein coding gene conservation. Here we utilize 4872 representative prokaryotic genomes from NCBI to investigate the relationship between marker gene identity and shared protein coding gene content., or single copy orthologs through a multi-locus sequence analysis. An important aspect related to this observation is the intra-organism variation of 16S copies from a single genome. Although the majority of 16S copies were found to have high sequence similarity (> 99%), several genomes contained copies that were highly diverged (< 97% identity).Even at 99–100% marker gene identity, genomes share on average less than 75% of their protein coding gene content. This occurs regardless of the marker gene(s) used: V4 region of the 16S rRNA, complete 16S rRNAThis is the largest comparison between marker gene similarity and shared protein coding gene content to date. The study highlights the limitations of inferring a microbial community’s functions based on marker gene phylogeny. The data presented expands upon the results of previous studies that examined one or few bacterial species and supports the hypothesis that 16S rRNA and other marker genes cannot be directly used to fully predict the functional potential of a bacterial community.The online version of this article (10.1186/s12864-019-5641-1) contains supplementary material, which is available to authorized users. Tools such as PICRUSt [Characterizing the diversity, abundance, and functional capacity of microbial communities has remained an important but difficult task for scientists. Current next-generation sequencing studies typically employ either full metagenome analysis, in which the entire genomic content of a community is sequenced, or marker gene analysis  where individual genes, most often 16S rRNA, are targeted using amplification with conserved primers. While these amplicon-based studies provide valuable insight into the diversity and relative abundance of taxa within communities, they provide no direct insight into the function or genomic content of a community. Recently, there has been a surge in the desire to predict functional capacity based on taxonomic assignment from these amplicon studies. In fact, phylogeny has been used to infer the molecular functions of microbes in the past and in recent papers . Tools s PICRUSt  and Viko PICRUSt  have bee PICRUSt , and it  PICRUSt . While aEscherichia coli (two pathogenic and one non-pathogenic) share less than 40% of their gene products, even though their 16S sequences are identical [Roseobacter spp. and Microbacterium spp. [There are multiple lines of evidence that 16S rRNA is not an ideal marker for characterizing functional content –12. One dentical . Recent ium spp. , 15. ThuE. coli and Vibrio spp., are more prone to and efficient in laterally transferring DNA, this results in a large pool of genes that are unshared between phylogenetically related organisms [This study aims to survey and quantify the variability of the 16S rRNA gene and select conserved single-copy ortholog genes (housekeeping genes) to examine its relationship with shared gene content. For this relationship, we hypothesized that the correlation would follow two general rates of change. (1) There is an unpredictable proportion of the genome typically acquired by horizontal gene transfer  , 18. Furrganisms , 20. On rganisms .To test these hypotheses, we conducted a comparative genomic study using 4872 well-annotated prokaryotic reference genomes from the publicly available RefSeq database on NCBI Fig. . UtiliziA total of 4872 complete representative prokaryotic genomes are available through the NCBI ftp portal, spanning 28 of the 29 accepted bacterial phyla  and bothn = 2485) to twenty , with an average of 2.3 copies per sample (σ = 2.1). The majority (99.1%) of the 16S rRNA copies with each genome examined have high sequence similarity (> 97%), however, a total of 38 genomes were found to have 16S rRNA copies that are less than 97% identical , all genomes with less than 97% intra-genomic 16S copy identity have less than nine copies of the gene.For genomes within this dataset, 16S rRNA copy number ranged from one n = 485 to twThrough sequence alignment validation of the low percent identity copies we found that 15 of the genomes contain one or more 16S rRNA copies with long stretches of unidentified nucleotides (represented by N’s sequence) or had large gaps within the alignment. For example, the genome GCF_000332335 has five copies of the 16S rRNA gene, four of which were nearly identical while one sequence was highly diverged and contained several stretches of unidentified nucleotides. In such cases, these highly diverged copies were not included in the analyses comparing 16S copy number and sequence variation.To explore the relationship between gene identity and shared gene content, we first clustered all marker gene sequences at intervals between 95 and 100%. This was completed separately for each of the three datasets . For the complete 16S rRNA and the V4 16S rRNA datasets, a large proportion of the 16S rRNA copies from the genomes clustered with 16S rRNA copies from different genomes at or above 95% sequence similarity (71 and 80% respectively), thus retaining a large number of comparisons for this analysis. Because only 8% of the MLSA dataset concatenated sequences clustered with sequences from other genomes at 95% or greater, we included further comparisons at 93.0–93.9% and 94.0–94.9%. At this range 520 marker gene sequences (13%) clustered into groups with two or more unique genomes.A representative graph depicting the sequence clustering of the complete 16S rRNA dataset for each percent identity group can be seen in Fig. As previously shown, many of the genomes in this dataset have low sequence similarity across their 16S rRNA copies. To investigate how this affected sequence clustering we tracked the 16S rRNA copies for each genome across the clustering groups. Figure Determining proper phylogenetic identification is often an important step in metabarcoding analyses, we therefore examined the taxonomic relationships between genomes at various marker gene sequence identity thresholds. As shown in Fig. After clustering marker gene sequences into sequence similarity intervals, pairwise comparisons of protein coding gene content were completed for each genome using Orthofinder. Percent shared genes is defined here as the ratio between the number of genes matched among two genomes and the total number of genes present in both. Figure p < 0.05). The group termed “Other” is comprised of highly similar 16S sequences that span different bacterial classes. As expected, these comparisons contain a significantly lower number of shared genes . Other classes of bacteria, like Bacilli, Clostridia, and Alphaproteobacteria contain similar amounts of shared genes when compared to the overall dataset.To expand upon these findings, we wanted to determine if there are certain groups of bacterial lineages where the relationship between marker gene identity and shared genome composition is higher or lower than the combined dataset Fig. . We examTo further validate these findings and test if the unshared genes may have been misannotated or if their functionality was lost due to rapid evolution, we subjected unshared genes to a tBLASTn search against complete genomes in the clustering group rather than their associated protein FASTA file. We found that most of these genes  are not found in the closely related genome at > 70% identity and only 1.8% on average are found at greater than 95% identity   of the matched and unmatched proteins identified from Orthofinder. We chose to focus on genomes whose V4 16S rRNA clustered at 99% sequence similarity or greater. This dataset consists of 6,324,117 protein accessions and 3515 total genome-genome comparisons. Of these accessions, 3,791,226 are found in the UniprotKB databases with a total of 2,803,829 containing gene ontology metadata. Results show 1794 GO terms significantly more likely to be shared, and 1119 GO terms more likely to be novel (unshared)  for each of the three broad categories of ontology: biological process, molecular function, and cellular component. ‘Count-shared’ and ‘Count-unshared’ refer to the number of times that particular GO term was found to be shared or unshared in the genome wide protein-coding content comparisons with Orthofinder. For an expanded summary of significant GO terms, see Table S5 in Additional file The current study quantifies the functional evolution of microbial genomes by describing the relationship between marker gene identity and shared protein-coding gene content. Results show that prokaryotic genomes exhibit a dynamic rate of evolutionary change. Although most of the genome mimics a rate of change following marker gene divergence, on average, 22–28% of the genome is independent of phylogenetic identity Fig. . This dyStaphylococcus aureus [We analyzed the complete 16S rRNA, the variable portion of the 16S rRNA, and various single-copy orthologs through an MLSA-like approach. We expected that the greater resolution by the complete 16S dataset and MLSA approach would significantly dissect the observed rapid change in gene content, but it was only marginally improved. Although marginal, these results support the use of an MLSA approach to improve the resolving power between shared protein-coding gene content and percent identity. This style of analysis has been routinely used in genotyping pathogens, such as methicillin-resistant s aureus  or diffes aureus , 31.Evidence shown in both Fig. Figure The field is moving away from using OTU sequence clustering for amplicon analyses and has begun to use exact sequence variants instead  . The res. From the thousands of pairwise comparisons, we found that these functions are more likely to be found in unshared gene datasets. Within the shared datasets we observed GO terms such as ‘DNA repair’, ‘DNA binding, and ‘integral component of the plasma membrane’. These are essential components that are necessary for a microbe to function, regardless of environment.The purpose of the GO enrichment analysis was to determine if the functions that change rapidly are unique and predictable. Additionally, we wanted to identify what functions are lost when a close correlation between marker gene identity and overall functional capacity is assumed. We found that although most gene ontology terms are shared across the genome dataset, many important and unique functions are significantly more prevalent in the novel/unshared gene sets Table . Key funThese findings related to the novel/unshared PCG functions are expected and similar to the observation of a core and accessory genome within microbes and supports the pan-genome concept, which is the collection of shared genomic resources that varies across environments . When scThe RefSeq representative prokaryotic genome database contains a large and diverse representation of major bacterial taxa for a comprehensive microbial dataset. All included genomes underwent a consistent annotation pipeline and nearly all protein-coding genes are linked to RefSeq GenBank files, so annotations and gene functions can be determined programmatically in an efficient manner. However, because many of the genomes available are biased towards biomedically and clinically relevant taxa, we anticipate some level of bias in the functional content of these organisms.Proteobacteria, which comprises of 46% of the genomes sequenced [Authors of programs aimed at inferring functional content from amplicon data are quick to acknowledge the limitations that are expanded upon here. PICRUSt does provide a QC metric, Nearest Sequenced Taxon Index (NSTI), which can help elucidate the limitation of database coverage and aid in interpretation of data. However, databases such as GenBank are severely biased towards easily culturable bacteria, like equenced , leavingequenced . Thus, pThe central hypotheses in this study address the relationship between marker gene identity and protein coding gene content. We observed with overwhelming evidence that even phylogenetically identical organisms do not share substantial proportions of their gene products, highlighting the gap between marker gene identity and protein-coding gene content. Specifically, we found that 22–28% of an organism’s functional capacity cannot be determined from marker gene(s) alone, even with MLSA. This is true even when analyzing 100% identical sequences, demonstrating the limitations of amplicon-based studies and their ability to characterize the functional capacity of microbial communities. Future studies using additional marker genes or other variable portions of the 16S gene, along with environmental datasets, would build on the results presented here and further elucidate the dynamics of microbial evolution.Prokaryotic genome and assembly accessions were identified from the NCBI representative genome report file. Corresponding genome/assembly FASTA, general feature format (GFF), amino acid FASTA (FAA), and GenBank feature format (GBFF) files were then downloaded via the NCBI ftp server  forward primer and 926r (CCGYCAATTYMTTTRAGTTT) reverse primer.We constructed a third and final dataset consisting of concatenated single-copy orthologs to test a multi-locus sequence analysis (MLSA) like approach. Following the example of previous studies –45, singFor each of the three datasets , we used UCLUST software v1.2.22q  to clustTo investigate how the 16S rRNA gene copies from a single genome fell out into clustering groups, we examined the number of unique clustering groups per genome and identified any genomes whose rRNA copies were found in different clustering groups. In addition, we identified the number of unique genomes represented in each clustering group and their taxonomic assignments.Next, we wanted to calculate the shared gene content between all genomes represented within the marker gene clustering groups using the program OrthoFinder v0.4, with default settings . For valThe shared gene content comparisons were then linked back to the marker gene clustering groups obtained from UCLUST. Each comparison is only included in the highest percent identity group, effectively dividing the data into comparisons from 95.0–95.99, 96.0–96.99, 97.0–97.99, 98.0–98.99, 99.0–99.99, and 100% marker gene identity. Using R v2,14.2, we created box plots depicting shared content in relation to percent marker gene identity for each of the three datasets.To determine if different lineages exhibit a higher or lower relationship between shared gene content and marker gene percent identity compared to the complete dataset, we split the V4 16S dataset into each of the major bacterial classes and completed the marker gene clustering and percent shared gene calculations outlined above. A Kruskal-Wallis test followed by a Dunn test for multiple comparisons with a Bonferroni correction was then completed to determine if the mean percent shared genes for each lineage was significantly different than the complete dataset.To better understand the differences in shared and novel functions of closely related genomes, we examined all protein-coding genes from genomes whose V4 16S clustered together at 99% identity. First, the count of each protein accession within a matched or unmatched Orthofinder output file was determined across all comparisons. We linked accessions to gene ontology (GO) using the UniprotKB Swiss-Prot and TrEMBL databases . Protein accessions and their respectively mapped GO terms were imported into the topGO software v3.8 . For eacAll routine statistical analyses were performed in either Python v3.4 or R v2.14.2 with plottrix package .Additional file 1:Table S1. The table provides the genome accessions and taxonomy for all reference sequences used in this study. (XLSX 169 kb)Additional file 2:Table S2. Summary of 16S rRNA copy statistics for each bacterial genome. Included is the number of 16S rRNA copies, the minimum and maximum length among copies, the average length of copies, and the minimum and maximum percent identity among copies. (XLSX 93 kb)Additional file 3:Table S3. A list showing the ratio of shared genes for each bacterial genome comparison. Data is included for each of the three datasets; Full-length 16S rRNA, V4 16S rRNA, and MLSA. (XLSX 1048 kb)Additional file 4:Table S4. Summary of BLAST results comparing the unshared gene content of bacterial genomes whose V4 16s rRNA clustered at 99% identity. (XLSX 23 kb)Additional file 5:Table S5. The table provides details of all significantly shared and unshared GO terms for bacterial genomes whose V4 16S rRNA sequences clustered at greater than 99% identity. (XLSX 158 kb)


The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity.We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells.hi (n = 3), CEAlo (n = 1) and CEAmixed PDOs (n = 4), that stably maintained populations of CEAhi and CEAlo cells, which has not previously been described in CRC cell lines. CEAhi PDOs were sensitive whereas CEAlo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEAlo cells maintain cancer cell growth. Culture of FACS-sorted CEAhi and CEAlo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/β-catenin pathway activity in CEAlo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/β-catenin pathway.PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEABased on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/β-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic.The online version of this article (10.1186/s40425-019-0575-3) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is the third most common cause of cancer related mortality worldwide . Median Checkpoint-inhibiting immunotherapies recently led to substantial increase in survival in some tumor types. These include hypermutated microsatellite instable (MSI) CRCs , 6 but cThe T cell bispecific antibody cibisatamab (CEA-TCB) is a novel immunotherapy that redirects T cells independently of their T cell receptor specificity to tumor cells expressing the carcinoembryonic antigen (CEA) glycoprotein at the cell surface . A majorCibisatamab has a single binding site for the CD3 epsilon chain on T cells and two CEA binding sites which tune the binding avidity to cancer cells with moderate to high CEA cell surface expression . This avAPC, TP53, KRAS and BRAF.Molecular mechanisms of cibisatamab activity have been investigated in CRC cell lines in vitro using killing assays with peripheral blood mononuclear cells . This idRecently developed protocols allow the expansion and long term propagation of cancer cells as so called patient derived organoids (PDOs) from CRC biopsies . PDOs haIdentification of resistance mechanisms for novel immunotherapies in patient-relevant preclinical models should define candidate biomarkers for the identification of sensitive patient subgroups or of combination therapy strategies that enhance efficacy by co-targeting of resistance pathways. This could maximize trial success rate and prevent drug attrition, which remains common for new cancer drugs in the absence of biomarkers. The aim of this study was to investigate mechanisms of resistance to cibisatamab immunotherapy beyond those identified in cell lines by using PDO models from treatment refractory metastatic CRCs. To this end, we established seven PDOs from chemotherapy resistant metastatic CRCs and one from a treatment naïve primary CRC and developed an in vitro co-culture assay with CD8 T cells to assess cibisatamab efficacy.Imaging-guided core biopsies from metastatic colorectal cancers that had been treated with at least two prior lines of chemotherapy were obtained from the Prospect C and Prospect R trials . One endoscopic biopsy from a treatment naïve primary colorectal cancer was obtained from the FOrMAT trial . Trials were run at the Royal Marsden Hospital and all patients provided written informed consent before trial inclusion. Anonymized buffy coats from healthy donors were obtained from the local blood bank  or through the Improving Outcomes in Cancer biobanking protocol at the Barts Cancer Institute  from individuals providing written informed consent. DLD-1 and MKN-45 cell lines were obtained from the American Type Culture Collection and were maintained in RPMI 1640 medium supplemented with 10% FBS, 1X Glutamax and 100units/ml penicillin/streptomicin (Thermo Fisher).PDO cultures from CRC-01, CRC-02 and CRC-06 were established directly from core biopsies by rough chopping followed by embedding in growth factor reduced Matrigel (Corning). Very small biopsy fragments were available from CRC-03, CRC-04, CRC-05, CRC-07 and CRC-08 and these were first grafted subcutaneously or under the kidney capsule of female CD1 nude mice by the Tumour Profiling Unit at the Institute of Cancer Research (Home office licence number PD498FF8D). Mice were culled once tumors had grown and tumors were removed and dissociated in a gentleMAX Octo dissociator using the Human Tumour Dissociation Kit (Miltenyi Biotec). Mouse cells were magnetically removed using the Mouse Cell Depletion Kit (Miltenyi Biotec), and purified human tumour cells were embedded into growth factor reduced Matrigel. PDOs were expanded in Matrigel as described  using Adhttp://n2t.net/addgene:12260; RRID:Addgene_12,260) and 0.75 μg of pMD2.G envelope plasmid  using TransIT-293 transfection reagent (Mirus). The cells were media changed the following day, virus harvested after 24 h and passed through a 0.45uM filter before use. For lentiviral transduction PDOs were harvested from the cultures in Matrigel and dissociated to single cells using TrypLE Express (Thermo Fisher), and pelleted. The pellets were resuspended in media with the addition of virus and 1nM polybrene (Sigma Aldrich) and centrifuged at 300g for 1 h. The samples were resuspended and plated in culture for between 6 h and overnight, before replacing the media. Following recovery and expansion, eGFP positive cells were sorted by flow cytometry and further expanded before use.The nuclei of PDOs were labelled by introducing an eGFP tagged histone 2B construct (pLKO.1-LV-H2B-GFP)  to enabl5 cells were stained with 20nM of the human anti-human CEA antibody CH1A1A (Roche) and 25μg/ml of the R-Phycoerythrin conjugated secondary antibody AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (Stratech). DRAQ7 (Biostatus) staining was included for dead cell exclusion. CEA expression was analysed on a Sony SH800 flow cytometer. Gate boundaries were set at the trough between high and low CEA populations in PDOs with mixed CEA expression and identical gates were used across all samples. The percentage of CEAhi and CEAlo populations and mean fluorescence intensities (MFI) were calculated for each PDO.Cell lines were harvested using enzyme-free Cell Dissociation Buffer (Thermo Fisher) and PDOs with TrypLE Express (Gibco). 2 × 10www.proteinatlas.org) with the rabbit polyclonal antibody HPA019758 (Sigma Aldrich) which has been validated against RNA expression data, immunofluorescence staining with an independent antibody and by Western blot were downloaded and assessed for expression heterogeneity by two pathologists .Microscopic images of 11 CRC samples stained by the Human Protein Atlas  were isolated from buffy coats with Ficoll-Paque according to the manufacturer’s protocol . CD8 T cells were isolated from PBMCs with Human CD8 Dynabeads FlowComp (Thermo Fisher). The purity of CD8 T cells was assessed by flow cytometry  and only populations with at least 90% CD8 positive cells were used for expansion with the CD3/CD28 Dynabeads Human T-Activator kit (Thermo Fisher) in RPMI 1640 supplemented with 10% FBS (Biosera), 1X Glutamax, 100units penicillin/streptomycin and 30 U/mL IL-2 (Sigma Aldrich) following the manufacturer’s protocol.PDOs were harvested with TrypLE Express and neutralised with DMEM/F12 Ham medium (Sigma Aldrich) with 10% FBS. Cells were filtered through a 70um filter, counted and re-suspended in phenol-red free RPMI medium (Thermo Fisher) supplemented with 10% FBS (Biosera), 1XGlutamax and 100units penicillin-streptomycin. On day 0, 5000 tumor cells per well of a 96 well-plate  were plated. CD8 T cells were added on day 1 at the indicated effector to target (E:T) ratios with 20nM of cibisatamab or 20nM of the untargeted negative control antibody DP47-TCB (both provided by Roche). Tumor cells without CD8 T cells and without antibody were also included as controls. All conditions were plated in triplicates and at least 3 different healthy donors were tested on each of the 8 PDOs.The GFP confluence was quantified every 48h–72h over a 10-day period using the GFP confluence application on the Celigo Imaging Cytometer (Nexcelom Bioscience). GFP confluence analysis was able to track the growth of GFP positive PDO cells over multiple timepoints without erroneously counting the T cells in the co-culture. Confluence analysis was furthermore superior to the counting of cell nuclei which generated inaccurate results in areas of high cancer cell density such as the PDO centre. The main advantage of confluence analysis over measuring spheroid diameters is the ability to track even the the growth of PDOs showing highly variable shapes. Growth curves were generated with CD8 T cells from three different healthy blood donors. The percentage growth reduction was calculated from readings taken between days 7 to 9, before PDOs showed growth retardation, likely due to exhaustion of the growth media. In order to calculate the percentage of growth reduction, confluence at day 1 was subtracted and the confluence in wells treated with the DP47-TCB control antibody at the endpoint was set to 100%.6 cells were stained with DAPI for dead cell exclusion, 20nM of the CH1A1A antibody (Roche) and 25μg/ml of the R-Phycoerythrin conjugated secondary antibody (Stratech) and sorted in CEAhi and CEAlo cells in an ARIAIII cytometer (BD Biosciences) in the FACS core facility at the Institute of Cancer Research. Cells were collected in cold DMEM supplemented with 10% FBS and pellets were frozen at -80°C after centrifugation for RNA-sequencing. CEAhi and CEAlo cells sorted from CRC-06 and CRC-08 were also grown for 1 month in DMEM/F12 Ham medium with 20% FBS, penicillin/streptomycin and 2% Matrigel.PDOs cells were harvested with TrypLE Express, filtered through a 70um filter to obtain a single cell solution and 2 × 10hi and CEAlo populations from CRC-03, CRC-06 and CRC-08 using RNAeasy Plus (Qiagen) and quantified with Qubit RNA High Sensitivity assay (Thermo Fisher). RNA sequencing libraries were prepared using the QuantSeq 3′-mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) using the manufacturer’s protocol. A minimum of 5 ng input RNA was used from each sample and qPCR was performed on all samples before PCR amplification of the library to determine optimal cycle number following the manufacturer’s recommendations. Final libraries were quantified in duplicate using Qubit-HS and Bioanalyzer High Sensitivity (Agilent) before pooling. Libraries were sequenced with 100 single read cycles on an Illumina HiSeq2500 in rapid mode. Alignment and gene counting were performed on the Lexogen QuantSeq data analysis pipeline on the Bluebee cloud. Annotation data on the GRCh38.p12 genome assembly was exported from Ensembl with Biomart. The ID’s of protein coding genes were identified using the gtf file filtered on the ‘gene_biotype’ field. The Quantseq raw count data were then filtered for this gene set and the Ensembl ID’s converted to gene symbol using the Homo.sapiens Bioconductor package. Lowly expressed genes containing fewer than 10 counts were removed from the dataset. The count data were then normalised using the estimateSizeFactors and counts functions from the DESeq2 Bioconductor package [Total RNA was extracted from frozen pellets of flow sorted of CEA package .5 PDO cells/well were seeded in 12 well plates and allowed to attach overnight. Media were changed and cells were treated with DMSO control or with 10uM tankyrase inhibitor  [10of Lord)  or 10uM p values are two tailed. Gene set enrichment analysis was performed with the GSEA software V3.0 using 5000 gene set permutations and the Hallmarks V6.2 gene set collection [Pearson correlation analysis and the paired t-tests were performed with the GraphPad Prism software. All llection .CRC PDOs were established as 3D cultures in Matrigel a) directly from core biopsies from three chemotherapy resistant metastatic CRCs , b) from small core biopsies of four chemotherapy resistant metastatic CRCs which were first expanded as xenografts in immunodeficient mice  and c) from an endoscopic biopsy taken from a treatment naive primary CRC (CRC-08). Each PDO was continuously grown for at least 2 months in Matrigel to test for long term viability. They were labelled with a lentivirus encoding a histone tagged nuclear enhanced green fluorescent protein (eGFP) and were subsequently transferred into culture conditions with 2% Matrigel dissolved in growth medium. Matrigel does not form a solid culture matrix at this dilution and allows PDOs to attach to the bottom of the plastic plate. These culture conditions facilitate interaction with T cells and allow monitoring of PDO growth with wide field fluorescence light microscopy.PDOs were dissociated into a single cell suspension and CEA cell surface expression was analysed by FACS using the CH1A1A antibody which has identical CEA antigen binding sites to the cibisatamab bispecific antibody  and moderately lower in two others . A proportion of the CEAlo cells in each of these four PDOs showed CEA expression levels which were as low as in the DLD-1 cell line, demonstrating heterogeneity across a broad range of CEA expression levels.The most striking CEA expression heterogeneity was detected in 4 PDOs  which each contained large subpopulations of CEAThe heterogeneous CEA expression profiles of these PDOs is reminiscent of the CEA expression heterogeneity which has been described in CRC samples from patients . We furthi PDO CRC-01 and showed no activity in the presence of the untargeted TCB antibody (DP47-TCB) which was used as a negative control  by magnetic bead sorting and expanded in vitro with IL2 and CD3/CD28 beads for 7–14 days. GFP-tagged CRC PDO cells were then seeded in 96 well plates, T cells were added the following day . All three PDOs in which GSEA analysis had shown different WNT/β-catenin signatures activity in FACS sorted CEAhi and CEAlo subpopulations harbored APC mutations. This demonstrated that the WNT pathway remains regulated in these PDOs despite the presence of APC mutations, a phenomenon previously observed in CRC model systems and patient tumors [lo and CEAhi cells in these eight PDOs.Our aim was to identify regulatory and potentially actionable pathways that control CEA expression and we hence focused subsequent analyses on the WNT/β-catenin signalling pathway. This pathway is genetically activated in the majority of CRCs, most frequently through mutations and loss of heterozygosity of the 1 itself , 22. Sevene Fig. d. Two int tumors –25. Morelo compared to CEAhi cells  the porcupine inhibitor LGK-974 which prevents WNT ligand secretion and hence autocrine and paracrine WNT-receptor activation and b) the tankyrase inhibitor compound 21 which inhibits the downstream WNT/β-catenin pathway by stabilizing the β-catenin destruction complex , 30. BotDOs Fig. f. The taThis analysis of cibisatamab immunotherapy in a co-culture model of allogeneic T cells and PDOs, established mainly from metastatic chemotherapy resistant CRCs, confirmed high cell surface CEA expression as a key determinant of treatment sensitivity in vitro. Importantly, in contrast to cell lines, we found that CEA expression is frequently heterogeneous within PDO populations, resulting in bimodal CEA expression patterns in its most extreme form. We furthermore demonstrated high phenotypic plasticity that enables CRC cells to switch between high and low CEA expression. These results are likely highly relevant for CEA targeting immunotherapies as all 4/8 PDOs with mixed CEA expression profiles were at least partially resistant to cibisatamab treatment in vitro.hi cells from the predominantly CEAlo PDO CRC-06 more rapidly converted back to the CEAlo state than those from the CEAmixed PDO CRC-08 furthermore suggest that differences in the transition rate from high to low CEA and vice versa may determine the abundance of CEAhi and CEAlo cells at the long term equilibrium. Moreover, gene expression profiles of CEAhi and CEAlo cells from individual PDOs demonstrated that cell surface expression of CEA is regulated at the gene expression level and negatively correlates with WNT/β-catenin pathway activity. CEA expression is tightly regulated in healthy colonic crypts where expression is absent at the crypt bottom that harbors intestinal stem cells and gradually increases in epithelial cells as they become more differentiated towards the top of the crypt. The activity of WNT/β-catenin signalling physiologically decreases along the same axis, from the highest levels at the crypt bottom to low signalling activity at the top of the crypt. Together with our results that demonstrate a negative correlation between CEA expression and a WNT/β-catenin activity signature in PDOs, this suggests that the regulation of CEA expression heterogeneity in PDOs and in healthy colon epithelial cells is similar.The observation that FACS sorted CEAAPC protein truncation by distinct mutations, which is likely to lead to different levels of β-catenin signalling pathway activation [We found no correlation between the degree of tivation , with CEtivation –25. Similo cells highlights further similarities to intestinal stem cells at the crypt bottom which are more dependent on oxidative phosphorylation than their more differentiated progeny towards the top of the crypt [lo cells in PDOs and the crypt bottom where stem cells reside, specific genes which have frequently been associated with stemness in CRC were not upregulated in the CEOlo cells from our PDOs. This may indicate a dissociation of the mechanisms that regulate differentiation processes including CEA expression and stemness in PDOs from advanced and drug resistant tumours but this requires further investigation.The upregulation of a signature of oxidative phosphorylation in CEAhe crypt . DespiteThe critical role of CEA expression heterogeneity and plasticity as determinants of treatment resistance in our in vitro models may be relevant for the characterization of predictive biomarkers for CEA-targeting immunotherapies in CRC where heterogeneous CEA expression has been described. Moreover, finding strategies to increase CEA expression may increase the clinical responses of cibisatamab.hi cells were highly sensitive to cibisatamab treatment in vitro but the low level of expression plasticity we observed may nevertheless enable gradual loss of the target antigen during prolonged treatment. The plasticity of CEA expression which re-establishes a CEAhi population after FACS sorting of CEAlo cells suggests that re-treatment with cibisatamab after a prolonged treatment break may lead to further responses. Phenotypic plasticity may furthermore be amenable to combination therapies which either increase CEA expression in the CEAlo population or which co-target vulnerabilities of the CEAlo subpopulations while also administering cibisatamab. Our results provided proof of principle that CEA expression can be pharmacologically perturbed and indicate WNT pathway inhibitors, which are in clinical development, as a potential strategy to boost CEA expression and increase therapeutic benefit from bispecific CEA-targeting antibodies. However, the WNT pathway has an essential role in the development and the homeostasis of many normal tissues in the human body. As a consequence, targeting of the WNT pathway has been challenging in preclinical models and clinical trials, particularly due to gastrointestinal toxicities [lo cells may be susceptible to inhibition of oxidative phosphorylation as an alternative strategy to address this subpopulation. Several CEA targeting immunotherapies beyond bispecific T cell redirecting antibodies are currently in preclinical or clinical development, including CEA vaccines and CEA targeting CAR T cells or TCR engineered T cells [PDOs harboring predominantly CEAxicities . Howeverxicities . Our res T cells . CEA exp. This addresses a major need in translational immunotherapy research, particularly as studies in immunocompetent animal models are often slow and expensive and sometimes even impossible as animal models are not available for all tumor types, or are incompatible with human-specific reagents, including many monoclonal antibodies. Allogeneic systems can be hampered by alloreactivity where donor T cells recognize MHC molecules on target cells as non-self. This can lead to high background killing activity that may impair assay sensitivity and specificity compared to autologous systems. Cancer cells alone as well as in co-culture with T cells but without antibody were included as controls in our assays. This allowed us to identify and exclude the small number of experiments in which alloreactivity impaired cancer cell growth. Using allogeneic T cells in PDO co-culture models hence represent a valid alternative strategy when autologous T cells are unavailable. These PDO T cell co-culture models can accelerate the discovery of immune-oncology candidate resistance mechanisms, biomarkers of efficacy and define rational combination therapy strategies for clinical testing. The ability of PDOs to represent intra-tumor heterogeneity to some degree, and the opportunity to establish them from tumors which match the stage and the treatment history of patients in which novel drugs are tested, are decisive advantages for pre-clinical discovery.Finally, this study shows the potential of PDO and allogeneic T cell co-cultures as novel tools to dissect mechanisms of immunotherapy resistance in vitroCEA expression heterogeneity is common in CRC PDOs from therapy resistant metastatic CRCs and this confers cibisatamab resistance in vitro. Whether CEA expression heterogeneity is associated with cibisatamab resistance should be investigated in clinical trial samples. CEA expression can be pharmacologically enhanced through WNT/β-catenin pathway inhibition, suggesting opportunities for combination therapies that should increase cibisatamab efficacy in the clinic.Additional file 1:Table S1. Genome-wide RNA expression analysis of FACS sorted CEAhi and CEAlo PDO cells. (XLSX 1049 kb)


Expert psychiatrists conducting work disability evaluations often disagree on work capacity (WC) when assessing the same patient. More structured and standardised evaluations focusing on function could improve agreement. The RELY studies aimed to establish the inter-rater reproducibility (reliability and agreement) of ‘functional evaluations’ in patients with mental disorders applying for disability benefits and to compare the effect of limited versus intensive expert training on reproducibility.alternative.work), 100–0%. Secondary outcomes were WC in last job (WClast.job), 100–0%; patients’ perceived fairness of the evaluation, 10–0, higher is better; usefulness to psychiatrists.We performed two multi-centre reproducibility studies on standardised functional WC evaluation (RELY 1 and 2). Trained psychiatrists interviewed 30 and 40 patients respectively and determined WC using the Instrument for Functional Assessment in Psychiatry (IFAP). Three psychiatrists per patient estimated WC from videotaped evaluations. We analysed reliability (intraclass correlation coefficients [ICC]) and agreement (‘standard error of measurement’ [SEM] and proportions of comparisons within prespecified limits) between expert evaluations of WC. Our primary outcome was WC in alternative work  and RELY 2 . Agreement was low in both studies, the ‘standard error of measurement’ for WCalternative.work was 24.6 percentage points (20.9–28.4) and 19.4 (16.9–22.0) respectively. Using a ‘maximum acceptable difference’ of 25 percentage points WCalternative.work between two experts, 61.6% of comparisons in RELY 1, and 73.6% of comparisons in RELY 2 fell within these limits. Post-hoc secondary analysis for RELY 2 versus RELY 1 showed a significant change in SEMalternative.work , and in the proportions on the differences ≤ 25 percentage points WCalternative.work between two experts (p = 0.008). Patients perceived the functional evaluation as fair , psychiatrists as useful.Inter-rater reliability for WCEvidence from non-randomised studies suggests that intensive training in functional evaluation may increase agreement on WC between experts, but fell short to reach stakeholders’ expectations. It did not alter reliability. Isolated efforts in training psychiatrists may not suffice to reach the expected level of agreement. A societal discussion about achievable goals and readiness to consider procedural changes in WC evaluations may deserve considerations.The online version of this article (10.1186/s12888-019-2171-y) contains supplementary material, which is available to authorized users. Western countries have social security systems in place that provide wage replacement benefits to individuals whose reduced health restricts or prevents them from working . Over thMany treating psychiatrists , 4 are eOur research on the reproducibility of WC evaluations evolved from widespread dissatisfaction with medical evaluations in Switzerland, where two nationwide surveys highlighted serious concerns regarding psychiatric evaluations of WC , 6. RespReproducibility is an umbrella term that encompasses two related concepts , 13. FirGood reproducibility, which encompasses both reliability and agreement, is a prerequisite for implementing a procedure in routine practice. We explored the effect of standardised training in functional evaluation for psychiatrists assessing the WC of patients reporting disability due to mental illness. We focused on patients with mental disorders, as this population is perceived as being particularly vulnerable to subjectivity regarding the evaluation of work disability , 14, 15.Two major administrative governmental changesWe performed two multi-centre reproducibility studies, RELY 1 and 2, using a partially crossed design in which four expert psychiatrists  independently rated the WC of actual patients claiming disability benefits . Psychiatrist recruitment took place in five assessment centres. Eligible patients had submitted an application for disability benefits from the Zurich office of the National Disability Insurer or from Suva, were fluent in German, and were attending an independent psychiatric evaluation for the first time. In line with routine procedures of the commissioning organisationIn RELY 2, all but one RELY 1-experts were recruited as interviewers. The recruitment of new video raters was carried out through Swiss Insurance Medicine, the professional society of insurance medicine. Patient recruitment for RELY 2 followed the procedures of RELY 1 and took place between July 2015 and April 2016. To compensate for the time loss in RELY 1 (see below), RELY 2 re-used 15 videos from RELY 1 that scored highest for functional interviewing criteria .Our functional evaluation approach incorporated three tools to systematically collect and document information for judging the patients’ work disability: (1) a semi-structured interview about their work and self-perceived work limitations; (2) concise descriptions of exemplary reference jobs for alternative work, and (3) a three-part instrument for documenting work-related limitations  used by the insurers to calculate the patients’ benefits. Secondary outcomes were WC for patients’ last job (IFAP 3a), experts’ certainty in their own judgements of WC , patients’ perceived fairness of the evaluation   with imp+ 0.15 from the observed ICC, assuming a true ICC value of 0.6  and 0.47 [fair]), i.e., experts were not enabled to better distinguish between individuals with higher and those with low remaining WC.While 24% of variance in WC for ‘last job’ in RELY 1 was attributable to the psychiatrists, more intensive standardisation reduced this variation to 7% in RELY 2. The reliability of the expert judgement on the patients’ WC did not change in any of the four situations , indicating that our training programme in functional evaluation did not improve the reliability of expert judgements (ICCp = 0.008). The comparison of SEMalternative.work showed a significant change by − 5.2 percentage points  in RELY 1 and 9.4 (95%CI 9.1–9.7) in RELY 2 for ‘Overall perception of fairness’  of expert psychiatrists assessing WC in patients with mental disorders found that more intensive training in functional evaluation of WC reduced variance but did not change psychiatrists’ low ability to discriminate patients with different degrees of WC for alternative work from each other. Post-hoc comparisons of RELY 1 and 2 indicated that intensive training achieved higher agreement between experts for WCStrengths of our studies include the use of real-life disability evaluations with their heterogeneous mix of typical patients, a broad spectrum of experts, and calibration of experts and description of work demands as reference. Despite clear differences in concepts , 13, 25,alternative.work. There, experts considered fewer patients as fully able or fully unable to work in alternative work compared to RELY 1, and consequently, almost all patients were attributed some remaining WC. The reduction of patient variance in RELY 2alternative.work indicates that patients were perceived as more homogeneous than those in RELY 1. However, the equal reduction of variance across all variance components resulted in unaltered low discrimination of remaining WC across patients  fibromyalgia will not be able to work, while others feel very differently.Prior evidence to inform our study design was very limited , 8: We lThe original research plan proposed a reliability study on functional evaluation and WC judgements (RELY 1), followed by a randomised comparison with current practice . Since gExternal factors interfered with the planned randomised comparison. It remains untested whether patients and lawyers would have indeed consented to a chance -rather than a ‘preferred option’- allocation to either type of disability evaluation. In case of objections, a non-randomised comparison might have been the best alternative to test the effectiveness of training on reproducibility. Since the RELY studies lacked randomisation , findings need to be interpreted with caution.Some might argue that our study examines videos of patients rather than actual patients. However, our design purposefully mimics real-life disability assessments  disciplines which adds challenges that were beyond our study.The complexity of WC evaluation brings about many more sources of variation than we could address in our study Table  5, 30]., 30.5, 3Systematic research on direct evaluation of WC is sparse . Our recWas training in functional evaluation sufficient to tackle the tasks as medical expert? A critical revision would include a review of training material, training intensity, and duration, and documention of success in expert calibration. This requirement is analogous to medical training where trainees acquire skills, e.g. in ultrasound imaging, by performing hundreds of scans under supervision in order to distinguish normal images from pathologies and discriminate similar but different pathologies.Current expert-based WC evaluations contain many discretionary judgements that contribute to low reproducibility. Standardising the process as done in RELY 2 appears to have some but not sufficient impact. Experts have called for more tools to complement their functional judgements , such asMore far-reaching approaches would restrict the physicians’ role to their professional core competences: reporting the impact of impaired health on the patients’ functional capacities. Work capacity is legally defined as expected earning capacity in suitable work . Most phWhat level of variation is acceptable for WC evaluation Figs. , Table 1Acceptable level of variation in WC evaluation is a social policy issue that requires a societal discussion, adressing the balance between the principles of fairness (‘similar treatment for similar cases’) versus the principle of treating each case individually – implying discretionary expert judgements and highly variable WC ratings across cases. Our stakeholder survey demonstrates a strong preference for fair and equal evaluations.What level of agreement would be required to reach these objectives? Widely accepted guidance for evaluating psychological tests  require Unexplained variance remained a major concern in the RELY studies. Research needs to identify additional potentially modifiable sources of variance , such aAll aspects of WC evaluations are seriously under-researched which challenges the planning of studies. We need data about potential effect sizes, sources and extent of variations, impact of expert calibration on reproducibility, criteria to decide on outcome measure, data to feed power calculations. Training experts alone may not result in acceptable reproducibility. Nevertheless, a better understanding of the cognitive approaches how medical experts come up with WC ratings may inform training. Furthermore, teaching ‘functional evaluation’, a novel technique, needs iterative refinement integrating experience from practice into training curricula, including material, intensity, duration, teaching techniques, and evaluation of learning.No study in our systematic review  providedEvidence from non-randomised studies suggests that intensive training in functional evaluation may increase agreement on WC between experts, but fell short to reach stakeholders’ expectations. It did not alter reliability. Isolated efforts in training psychiatrists may not suffice to tackle the complexity of the task to reach the expected level of agreement. Adding additional components in the procedures of WC evaluations may deserve considerations.Additional file 1:. Comparison of the design for the RELY study as planned with the actual conduct of the studies as RELY 1 and RELY 2. *SIM = Swiss Insurance Medicine, professional society of medical experts (DOCX 15 kb)Planned design versus actual conduct of the studiesAdditional file 2:. Both RELY studies recruited psychiatrists from the same population (practicing experts being SIM members). Training differed in training intensity and duration to implementation. Patients were recruited from the same population through the National Disability Insurer and Suva. In RELY 2, we re-used 15 interviews from RELY 1. The 25 new RELY 2-interviews were conducted by 11 RELY 1-interviewers who were re-trained for rating. Both studies used the same implementation procedure. (PNG 45 kb)Design of the RELY studiesAdditional file 3:Questionnaire on Perceived Fairness. Patients’ perception of the fairness of the work disability evaluation. The questionnaire had 29 items on a scale from 1 to 5 (higher scores indicate stronger affirmation) and a single item on overall perception of fairness on a scale from 10 to 0. The table shows five typical items. (DOCX 18 kb)Additional file 4:. * n = 1 missing due to violation of rating rules (JPG 67 kb)Patient flow in RELY 1Additional file 5:. *: n = 5 missing due to violation of rating rules (JPG 74 kb)Patient flow in RELY 2


Objective: Recent advances in non-invasive prenatal diagnosis (NIPD) through cell free fetal DNA (cffDNA) has highlighted cffDNA purification as a critical initial step. Herein, we aimed to compare the efficiency of one proposed protocol with two commercial kits for isolation of cffDNA. Materials and methods: cffDNA was isolated from whole blood of 50 normal pregnancies using one proposed manual protocol compared with QIAamp DNA Blood Mini and Bioneer Kits. Methylated DNA immunoprecipitation real time polymerase chain reaction  was performed to quantify three fetal specific sequences.Results: Maximum cffDNA quantity was obtained by suggested protocol (248.79 ± 14.07 ng/µl) and the best quality was achieved by Bioneer Kit  (p < 0.001). Enrichment of fetal specific sequences was significantly higher when proposed protocol was used to isolate cffDNA (p = 0.01).Conclusion: Inhibitory effect of NaI on nucleases and double digestion of DNA associated proteins may be the main reasons behind the superiority of suggested protocol. Significantly higher amplification of fetal specific sequences in suggested protocol would be a strong evidence on recovery of small fetal fragments as demonstrated with its maximum total DNA quantity and amplification in different PCR reactions. Introduction of various non-invasive prenatal diagnosis (NIPD) tests heralds for promises against conventional prenatal diagnosis associated with minimal, but finite risk of various fetal and maternal complications. NIPD have been extended to be used in determining fetal RhD statue  as well Maternal blood is the main non-invasive source of fetal cells and cell free fetal DNAs (cffDNA) for different NIPD tests. Disruption of placental barriers during apoptosis process opens the door for fetal cells and DNAs to gain entry into the maternal circulation. It was shown that the quantity of fetal cells or cffDNA is a valuable and early biomarker of fetal abnormalities and second or third trimester placental complications such as pre-eclampsia . Fetal cells have longer half-life and remain in maternal circulation even until the next pregnancy. In contrast, cffDNA disappears from the maternal body within 2 hours postpartum, renders them a more appropriate and sensitive choice for non-invasive genetic diagnosis . HoweverHerein, we compared one proposed manual and two kit based methods of DNA isolation with each other to define which method is preferred for performing NIPD. To achieve this aim, cffDNA was isolated from whole blood of pregnant women with all of three protocols.Sample preparation: EDTA containing falcon tubes was used to collect 5 ml peripheral whole blood from 50 pregnant women whom were defined as high risk by doing first and second trimester screening tests and were referred for doing either chorionic villous sampling (CVS) or amniocentesis. Enrolled women were among 10-16 weeks of pregnancy (aged 18-40) and filled the informed consent form according to the code of Tehran University of Medical Sciences ethical committee (92-02-30-21577) for experiments involving human uniform requirements for manuscripts. All the included samples had a confirmatory karyotype analysis report. Twelve samples which have been drawn after amniocentesis and CVS or after 16 weeks of pregnancy were excluded from further assessments. All the whole blood samples were stored in -80˚C within 2 hours after venue puncture till the DNA isolation process being initiated.AccuPrepTM Genomic DNA Extraction Kit (Bioneer) were followed to extract total DNA from 1 milliliter of maternal whole blood. The quality and quantity of DNA was determined using NanoDrop ND-1000 spectrophotometer . Five micro liters of achieving DNA from all methods was amplified through standard PCR to confirm its quality again. Of note, to have minor changes on DNA isolation efficiency using various protocols, all of used protocols were performed tested in the similar situations and sites and by using the same tools and stock materials.The manual method of DNA isolation in comparison with QIAamp DNA Blood Mini Kit (Qiagen) and DNA isolation protocols: Proposed method: The proposed method was based on protein digestion in two sequential steps. Every milliliter blood samples was dispensed in 3 ml of lysis buffer containing 6 M NaI (Sigma), 1% SDS, 10 mM EDTA, 20 mM Tris-HCL and proteinase K  and the mixture was incubated at 65 ˚C for 20 minutes in water bath. DNA was dehydrated in 70% ethanol and dissolved in ddH2O. Remained nucleoproteins were digested through incubating DNA molecules in 6 ml digestion buffer  including 20 mg/ml proteinase K for 20 minutes. DNA, was eventually eluted in ddH2O following two rounds of washing with 250 µl 70% ethanol.AccuPrepTM Genomic DNA Extraction Kit according to their manufacturer’s instructions and then was dissolved in ddH2O. Due to required less starting material (< 1ml) for both kit based protocols, we had to isolate DNA from one sample in multiple reactions according to instructions of each kit.DNA was also purified using QIAamp DNA Blood Mini Kit and TSGA10 gene .MeDIP-Real time PCR: To clearly determine the efficiency of each performed protocols in isolation of free fetal DNA, they should be separated from maternal DNA and being amplified. Methylated DNA immunoprecipitation (MeDIP) technique was implicated to at first enrich total methylated maternal and fetal free DNAs. Methylated fetal DNA sequences were then amplified using three specific primer pairs in real time PCR reaction. MeDIP-Real time PCR protocol was performed on all normal samples isolated by each three mentioned protocols according to previously reported method of analysis . . MeDIP-RStandard curve was drawn for three fetal specific sequence reactions using a DNA sample isolated from a whole blood sample with known concentration and copy number. All the three real time PCR programs were optimized as 40 cycles of 10 second at 95ºC following annealing and extension at 60 ºC for 30 seconds. Every Real time PCR reaction was included 2x SYBR Premix Ex Taq master mix  and fetal specific primer pairs adjusted with nuclease free water up to total volume of 10 µl. Amplification was performed in a Rotor Gene 6000 Real time machine . Melt curve analysis was performed following each run of amplification to confirm the specificity of the amplified products and absence of primer dimer. Melting curve analysis was included 57°C for 15 sec which was followed by temperature increase to 95°C for 15 sec at the rate of 1°C per sec with continuous reading of fluorescence.Statistical analysis: The mean of total DNA concentrations and optical density (OD) in absorbance of 260/280 was determined by NanoDrop ND-1000 spectrophotometer and compared with each other using SPSS software . In all the statistical analysis, α was considered to be 0.05 and ρ-value less than 0.05 was significant.Due to the normalized pattern of all data using histogram analysis , parametric tests were used to compare mean of parameters of three protocols with each other.DNA yield and quality: The mean of total DNA concentration, yield and purity (OD ratio of 260/280 nm/nm and OD ratio of 260/230 nm/nm are shown in The mean of DNA yield obtained by our proposed was significantly higher than two kit based protocols (p<0.001). In using QIAamp DNA Blood Mini Kit according to manufacturer’s instruction without any modification, 15 samples failed to be amplified in the PCR reaction . AccuPrepTM Genomic DNA Extraction) Kit  (The best DNA purity was achieved through Bioneer (< 0.001) .Minimum quantity and quality of DNA with significant deviation from other protocols was belonged to the Qiagen kit .PCR amplification: The efficiency of the proposed method of DNA extraction on isolation of small DNA fragments was revealed using four primer pairs designed to amplify different small DNA templates . DNAs frMeDIP Real time PCR: The mean of fetal specific DNA sequences concentrations was presented  of obtaining DNA to the optimal quality in favor of keeping DNA quantity to afford the best protocol for isolation of cffDNA. In addition, although, all the isolation processes for three used protocols were performed on the same place, the effect of isolation site would be tested in different laboratories with different situations and analytical tools. Taken together, our proposed method in the present study, could serve as a high efficiency and cost benefit alternative choice of cffDNA extraction in NIPD tests in all of populations with every financial supports. In addition, the proposed method could be an appropriate option to current methodologies and kit protocols used for isolation of free circulating DNA of cancer patients or viral diseases.Inhibitory effect of NaI on nucleases and double digestion of DNA associated proteins may be the main reasons behind the superiority of suggested protocol. Significantly higher amplification of fetal specific sequences in suggested protocol would be strong evidence on recovery of small fetal fragments as demonstrated with its superior total DNA quantity and its amplification in different PCR reactions.


Artemisinin-based combination therapies (ACTs) have substantially reduced worldwide malaria burden and deaths. But malaria parasites have become resistant to artemisinins. Prior studies suggested two different molecular pathways of artemisinin-resistance. Here we unify recent findings into a single model, where elevation of a lipid, phosphatidylinositol-3- phosphate (PI3P) results in vesicle expansion that increases the engagement with the unfolded protein response (UPR). Vesicle expansion  efficiently induces artemisinin resistance likely by promoting ‘proteostasis’ (protein translation coupled to proper protein folding and vesicular remodeling) to mitigate artemisinin-induced proteopathy . Vesicular amplification engages the host red cell, suggesting that artemisinin resistant malaria may also persist by taking advantage of host niches and escaping the immune response. Plasmodium falciparum, the most virulent of human malaria parasites [P. falciparum resistance to artemisinins and ACTs has emerged, threatening global malaria control and elimination.World-wide levels of malaria have substantially decreased over the last decade. Notably, twenty-one countries are expected to have eliminated the disease by 2020 . Yet a sarasites . ArtemisP. falciparum infection in humans is initiated when an infected mosquito bites to release sporozoites that infect liver cells. In the liver, sporozoites develop into merozoites which emerge into the blood stream to invade erythrocytes (red blood cells) and develop through distinct ring, trophozoite and schizont stages over a 48 hour asexual life cycle (fe cycle . The blofe cycle . Proteompfkelch13 (K13) is the primary marker of artemisinin resistance as defined both clinically and in vitro using the Ring-stage Survival Assay  of P. falciparum exposed to multiple short pulses of artemisinins during the asexual cycle and grown long term (for 24 months). Two isolated clones without mutation in K13 . A normalized gene set enrichment score (GSEA) was used to capture links in the gene sets of the in vitro selected transcriptome to clinical artemisinin resistance at the transcriptional level (in vitro assay where 10 hour parasites were pulsed for 4 hours (thought to mimic in vivo cycles of drug pressure), the in vitro-selected strains showed higher tolerance for artemisinin than parental counterparts. Trans expression of two top hit genes PfTrx1 and PfSpp2 (respectively involved in cellular protection and protein translation) in the parent 3D7 strain, showed a small increase in resistance when measured in 10 hour rings pulsed for 4 hours but did not contribute resistance measurable by RSA  as well as proposed determinants of K13-independent resistance. As shown in panel 2j, transgenic elevation of parasite PI3P (via expression of VPS34) in absence of K13 mutation, induced greater than a log change of resistance in the RSA and comparable to levels induced by K13C580Y. Moreover, expression of VPS34myc amplified a proteome that significantly overlapped with upregulated genes of the in vivo clinical transcriptome  and some association with the in vivo clinical artemisinin resistance transcriptome. But transgenic expression of the high value candidate genes of the oxidative stress and protein damage, did not result in an RSA survival rate of 2%  well- defined effector arms of the UPR of eukaryotes in artemisinin resistance.In sum, amplification of PI3P vesiculation as modeled in vesicles ●●, theirvesicles ●●, proviIn higher eukaryotes, PI3P expansion in the ER stimulates autophagy to efficiently remove misfolded/toxic protein aggregates –19 as weAn unexpected feature of PI3P vesicles is that upon amplification they reach all destinations in mutant parasite . This maOn the heels of the identification of K13 resistance marker, different technological approaches of transcriptomics  versus functional and genetic studies (showing requirement of the lipid PI3P), suggested two distinct processes of artemisinin resistance. However, subsequent work using proteomics as an important bridging technology, suggests that both function in the same pathway. Furthermore, individual ER-UPR components are involved in artemisinin induced latency, but do not induce significant levels of resistance. Other candidate genes have not been tested. However, expansion of PI3P vesicles efficiently induces a log increase in artemisinin resistance. Vesicle formation and turnover are complex processes that may remodel both the parasite and red cell, suggesting that understanding their functions may provide insights about parasite mechanisms to survive exposure to artemisinins as well as host pathogenesis.


Guided by social-cognitive domain specific theory , this study explored the issue of role reversal in the aging parent-adult child relation when parents are experiencing age-related functional limitations. Data was collected from adult children  with a living parent of 70 years old or older who participated in a focus group and were analyzed by a directed analysis . Participants legitimated their intervention into parents’ autonomy when they perceived a potential risk of parents’ health and safety and involvement of those and of others. Eight types of intervention emerged: (1) monitoring and talking with potential risk with parent (2) convincing parents under the name of super power or an authority figure (3) scaffolding parent’s task by teaching skills, (4) scaffolding by sharing role, (5) scaffolding by optimizing environment, (5) overriding parents’ autonomy behind parents, (6) forcefully overriding, (7) giving up parents’ behavioral modification by accepting parents’ lifestyle, and (8) giving up because of discomfort of talking about the issue . Thus, adult children changed their strategies of intervention from monitoring their parents’ behaviors to overriding parents’ autonomy, depending on their appraisal of potential harms of parents’ prudential and moral domains of life and of their own work/family conditions from monitoring to overriding. Adult children’s possible ways of scaffolding in helping their parents accept their children’s interventions as letting parents maintain their psychological autonomy, including communication skills to discuss uncomfortable topics is discussed.


We identified 262 differentially expressed genes (DEGs) in HEU compared to HUU infants. Weighted gene co-expression network analysis (WGCNA) identified six modules that had significant associations with clinical traits. Functional enrichment analysis on both DEGs and the six significantly associated modules revealed an enrichment of G-protein coupled receptors and the immune system, specifically affecting neutrophil function and antibacterial responses. Additionally, malaria pathogenicity genes (thrombospondin 1-(THBS 1), interleukin 6 (IL6), and arginine decarboxylase 2 (ADC2)) were down-regulated. Of interest, the down-regulated immunity genes were positively correlated to the expression of epigenetic factors of the histone family and high-mobility group protein B2 (HMGB2), suggesting their role in the dysregulation of the HEU transcriptional landscape. Overall, we show that genes primarily associated with neutrophil mediated immunity were repressed in the HEU infants. Our results suggest that this could be a contributing factor to the increased susceptibility to bacterial infections associated with higher morbidity and mortality commonly reported in HEU infants.HIV-exposed uninfected (HEU) infants are disproportionately at a higher risk of morbidity and mortality, as compared to HIV-unexposed uninfected (HUU) infants. Here, we used transcriptional profiling of peripheral blood mononuclear cells to determine immunological signatures of  The higher mortality is often due to the HEU infants’ increased susceptibility to infectious diseases, many of which prove difficult to treat3. Although the rates of diarrheal infections in HEU infants are similar to those in infants born to HIV negative mothers, commonly referred to as HIV-unexposed uninfected (HUU) infants, they are often more severe requiring hospitalization4. Furthermore, life-threatening bacterial infections with encapsulated pathogens, including Haemophilus influenza and Streptococcus pneumonia, are more common during the first twelve months of life in HEU infants7. The increased susceptibility of HEU infants to infections can partly be attributed to the lower levels of transferred protective maternal antibodies, inadequate parental care or exposure to maternal ailments such as tuberculosis8. Moreover, in utero exposure to HAART and HIV antigens may also be associated with adverse health outcomes such as mitochondrial dysfunction10, cardiac function and growth12 and an altered cytokine milieu leading to poor immune cell development and immune responses after birth14.Highly active antiretroviral therapy (HAART), is currently the most promising intervention for HIV control, particularly in the prevention of mother to child transmission (PMTCT). PMTCT has increased the number of HIV infected women who can safely have HIV uninfected infants. Despite escaping viral infection, the HIV exposed but uninfected (HEU) infants, have been shown to have an increased risk in mortality and morbidity15. In addition, HEU’s have higher numbers of CD3+ cells16, an intricate pattern of defects in CD4+ and CD8+ T-lymphocyte subpopulations, , altered dendritic cells19, a reduction in the proportion of circulating follicular helper T-cells20 and impaired progenitor T-cell function that leads to reduced thymic output and results in lower naïve CD4 counts21. Some of these T-cell parameters that are altered at birth are known to persist beyond the first year of life18. The B-cell compartment is also affected in HEU infants, albeit more subtly. Some studies have reported an increase in cord blood B-lymphocytes marked by higher numbers of CD19+/CD5+ cells16, a reduction in the resting memory B-cells (primarily due to changes in the unswitched memory B-cell subset22) and poorer humoral responses to a wide range of vaccines17.In comparison to HUU infants, HEU infants have previously been shown to have an enhanced expression of CD40L on activated T-lymphocytesThese phenotypic, functional and clinical observations highlight a compromised immune system in HEU infants. Comparisons at the transcriptomic level can provide a robust and sensitive approach to identify subtle changes underlying biological and immune mechanism differences between HEU and HUU infants. In this study, we performed transcriptional analyses of peripheral blood mononuclear cells (PBMCs) from HEU and HUU infants using an RNAseq approach. We uncovered several HEU transcriptome markers and showed that the down-regulated genes in HEU infants are functionally related to diverse biological pathways with an over-representation of pathways associated with immunity.23. A total of 19 HEU and 15 HUU infants were analysed. The median age of the HEU infants sampled at the early time point was 12.13 months (IQR [12.07–12.60]) and for the late time point, 18.9 months (IQR [17.95–21.13]). On the other hand, the median age of the HUU infants was 12.58 months (IQR [12.21–13.03]) and 16.56 (IQR [15.18–22.18]) for the early and late time points, respectively. There was no statistical difference (Mann Whitney nonparametric test) between the median age of the HUU and HEU infants at both the early (p = 0.60) and late (p = 0.20) time points, respectively. A comparison of the haematological parameters, in HEU and HUU infants, taken at the time of sample collection, showed no statistical differences in white and red blood cell counts, lymphocyte, platelets, neutrophil, monocyte and eosinophil counts  and twenty-four months (n = 14) after birth from HEU infants and in HUU infants (n = 15). After quality control filtering, 47 transcriptomes with an average read depth of 30 million per sample were obtained  followed by H1FX , TNFSF14 and CISH. The down-regulated gene clusters consisted of: five epigenetic factors , genes that function in transcriptional regulation , genes involved in the malaria pathway  and genes involved in neutrophil-mediated immunity   Fig. .Figure 225. In the analysis of the up-regulated gene set, two-thirds of all DEGs surprisingly collapsed into one GO term of plasma membrane, particularly the hydroxycarboxylic acid-binding receptors that belong to the G- protein-coupled receptor family and tumor necrosis factor that is involved in systemic inflammation  and thrombospondin-1 (THBS1) DEGS that were enriched in the down-regulated gene set. Malaria is endemic in the study region, and thus our data suggests immune responses to the disease may be affected in HEU infants. To test this hypothesis, we analysed antibody responses to Plasmodium falciparum apical membrane antigen (PfAMA) 1, a leading malaria vaccine candidate, and responses to Plasmodium schizont extract, a crude marker of exposure to malaria parasites during the blood stage infection, in the larger cohort from where the HEU and HUU infants included in the transcriptomic analysis had been sampled. A comparison between the HEU and HUU infants’ antibody titers confirmed the hypothesis. HEU infants residing in a high malaria transmission region, had significantly lower antibodies specific for Plasmodium schizont extract  and black  were significant within the HIV exposure trait (HEU vs HUU) whereas modules brown , purple  and green/yellow  were significant within the hematocrit, neutrophils and eosinophils traits, respectively  (Supplementary Table RETN), that plays a crucial role in neutrophil activation and the formation of neutrophil extracellular traps (NETs)26.Of the five significant modules, modules brown, purple and green/yellow were enriched in immunological GO terms Fig. . Of inteH1FX and HMGB2 genes, were top amongst the significantly down-regulated genes in HEU infants. Interestingly, on performing a correlation analysis between the 262 DEGs and individual genes from the chromatin hub, a strong positive correlation with all the downregulated genes and a strong negative association with the upregulated genes was observed  ATP1B3-AS1, in HEU infants. ATP1B3-AS1 is an antisense transcript of ATP1B3 gene, which belongs to the Na+/K+ ATPase family that catalyzes the hydrolysis of ATP. The ATP1B3 gene has recently been shown to be a cofactor of the bone marrow stromal cell antigen 2 (BST-2) protein. BST-2 is highly expressed on the surface of HIV infected cells and has been demonstrated to stop the release of newly produced virions by blocking virus escape from infected cells. Knockdown of ATP1B3 enhances the expression of BST-2, in turn modulating the restriction of HIV-1 virion production. The high expression of the antisense transcript, ATP1B3-AS1, may therefore be an indicator of exposure to viral particles and the priming of BST-2 dependent anti-HIV responses in HEU infants as previously suggested28.It is speculated that viral particles may traverse the placental membrane, most likely due to the trans-placental diffusion of HIV soluble proteins as opposed to live and replication-competent HIV. Exposure to HIV soluble proteins may trigger immune responses to HIV. Previous reports showed that more than 20% of HEU infants possess immunological memory to HIV despite not being infectedTNFSF14, CISH, FFAR2, HCAR2, and HCAR3 in HEU infants, highlights a role for immune processes involving cells such as dendritic cells (DCs), neutrophils and macrophages. TNFSF14 is expressed on activated T lymphocytes, preferentially CD8+29, and the upregulation of TNFSF14 could be as a result of increased CD8+ activation as previously reported in HEU infants30. CISH is expressed during the development of DCs and regulates DC-mediated cytotoxic T-lymphocyte activation32. Its expression is associated with the negative regulation of cytokine signaling, controlling inflammation during infection, and susceptibility to infectious diseases such as bacteria and malaria33. High expression of CISH in HEU infants may similarly predispose them to severe bacterial infection and may be potentially associated with the high inflammation observed in HEU infants34. HCAR2 and HCAR3, encode for receptors expressed on various immune cells such as neutrophils and macrophages, which have been shown to have anti-inflammatory effects38. FFAR2, on the other hand, regulates the differentiation and activation of macrophages and leukocytes39.The high expression of genes primarily associated with the plasma membrane identified a number of receptor genes including those on the surface of immune cells. The high transcription levels of LUCAT1 and LINC01531 were among the DEGs that could potentially be involved in transcriptional regulation.Further analysis of the downregulated genes revealed a high concentration of DEGs related to immune processes associated with neutrophils. Our data has demonstrated the down-regulation of several genes coding for proteins involved in chromatin structure and regulation of the global transcription program. The strong co-regulation of expression between the DEGs and the chromatin genes, implies that they both contribute to the observed transcriptome changes in HEU infants and identified biological pathways. However, we acknowledge that there are likely to be additional players to the chromatin genes that may contribute to the observed changes. Indeed, several non-coding RNA species including 40. Neutrophils function in both the innate and adaptive immune system42 attacking microorganisms by phagocytosis, degranulation or via the generation of NETs45. Genes primarily associated with neutrophil mediated immunity  were repressed in the HEU infants and could be a contributing factor to the increased susceptibility of HEU infants to bacterial infections48. RETN, found in specific neutrophil granules and cell membranes49 plays a role in the formation of NETs and the activation of neutrophils26, whereas BPI is bactericidal and stimulates phagocytosis by neutrophils through the complement system50. LCN2 encodes for lipocalin 2 , a protein mostly released by neutrophils. This protein is known for its bacteriostatic role46 and enhancement of phagocytosis by macrophages51. The downregulation of HP which encodes for an acute phase bacteriostatic protein that is released during inflammation and injury by activated neutrophils52, may also interfere with neutrophil function. CAMP encodes for cathelicidin antimicrobial peptides which not only have direct antimicrobial53 properties, but they also affect neutrophil activity through neutrophil recruitment55 and inhibition of neutrophil apoptosis57.HEU infants have been reported to have normal neutrophil counts with an impaired neutrophil functionPneumocystis jirovecii pneumonia and they had regular clinical visits, which ruled out on-going febrile infections during blood sample collection. Whether daily cotrimoxazole is beneficial and reduces invasive bacterial infections58 is unclear as evidence for this is weak and current guidelines have been debated60. Some studies have reported little to no effect of cotrimoxazole on mortality, LRTIs and diarrheal infections62, while others have observed a significant reduction in hospitalizations63. Cotrimoxazole not only acts as an antimicrobial agent, it may also directly interfere with the host’s immune system64. Therefore, we cannot rule out the immunomodulatory role of cotrimoxazole on the gene expression profiles observed in HEU infants and a repeat of the analysis post cotrimoxazole prophylaxis, that is, after two years of life, may be important for future work. Daily cotrimoxazole may also explain the lower expression profiles for genes associated with malaria pathogenesis on the KEGG pathway. Although not the primary aim, daily cotrimoxazole does protect against malarial disease as previously shown67. In the wider cohort, HEU infants residing in a high malaria transmission area had lower antibody levels to Plasmodium schizont extract and PfAMA1 compared to the community controls living in the same region, implying that they had experienced fewer malaria episodes, in agreement with the gene expression profile.It is worth noting that the HEU infants in this study were on daily cotrimoxazole prophylaxis in line with the recommended policy to protect them from 20, as well as revealing new insights into the potential pathways involved in the global dysregulation of immune development in this group of infants. We observed changes in genes involved in innate immune responses, specifically neutrophil function. It is possible that HEU infants have an ineffective neutrophil response that would predispose them to severe bacterial infections and subsequently increased morbidity and mortality, and further studies are warranted to address this.To our knowledge, this is the first study that has used a transcriptomic approach to describe the gene expression profile of PBMCs from HEU infants. Our data confirm previous reports indicating reduced immune responses in HEU infantsAll methods were performed in accordance with the Kenya Medical Research Institute Scientific and Ethics Review Unit guidelines and regulations for handling and analyzing human-derived biological samples. The Kenya Medical Research Institute Scientific and Ethics Review Unit provided ethical approval (SSC2085) and written informed consent was obtained from mothers of the infants recruited into the study.23. HIV care was provided as per the World Health Organization recommendation at the time of the study. Briefly, HEU infants born to mothers, not on HAART were prescribed nevirapine prophylaxis at birth. Treatment was continued until one week after the complete cessation of breastfeeding, while for those with mothers on HAART, nevirapine prophylaxis was stopped at six weeks of life. Infant’s HIV-1 status was determined by polymerase chain reaction (PCR) at six weeks of age or at first contact, and repeated by a rapid antibody test at nine and 18 months of age. Infants suffering from an acute infection or malnutrition at the time of recruitment were excluded from the study. The infants were followed up longitudinally every three months until they were 24 months of age, with an upper age limit of up to 30 months being used to cover late follow-up visits. All HEU infants were given prophylactic cotrimoxazole during the first 18 months of life. Age-matched community control HUU infants were recruited from three localities within the catchment area of Kilifi County with varying malaria endemicity and sampled at a single time point22. From the parent study, we used samples from 19 HEU infants that contributed to the 12 and 24-month time points. However, one and five infant samples were not available at the 12 and 24-month time points, respectively. Also, we obtained control samples from 15 HUU infants specifically from the low malaria transmission region, to avoid any malaria interference on gene expression profiles. Due to ethical reasons, we could not get blood samples from healthy HUU infants more than once. Samples from selected age-matched infants at 12 months or 24 months were therefore analyzed. In total 34 individuals (19 HEU infants matched to 15 HUU infants) were used for RNA sequencing and the gene expression profiling reported here. During the parent study, 5 ml whole blood was drawn and processed within 4 hours. From this, 300 µl of whole blood was used for RNA isolation by first depleting the red blood cells through lysis and storing the PBMCs at −80 °C until ready for RNA isolation.This study used samples previously collected from a longitudinal cohort of HEU infants recruited between 2011 and 2012 at the comprehensive care and research clinic (CCRC), Kilifi County hospital, previously describedThe frozen PBMCs were thawed in bulk and RNA extracted as per the Qiagen RNeasy kit protocol. The quality of the extracted RNA was confirmed using Agilent Bioanalyzer and messenger RNA (mRNA) enrichment done. mRNA sequencing libraries for 200 bp short-inserts were prepared according to the manufacturer’s recommendations. RNA sequencing was done using the Hi-Seq Illumina platform using nine lanes of single-ended reads at a read length of 50 base-pairs (50 bp). RNA extraction, Library preparation, and sequencing were performed at the Beijing Genomics Institute (BGI), China.68 and bowtie2 v2.2.55269 software, the high quality reads were aligned against the human reference genome GRCh38vs86 obtained from Ensembl70 using the file transfer protocol (FTP). The estimated abundance levels were then quantified at the exon level using HTSeq v0.6.15471. The output of abundance estimation was in form of text files for each sample. Text files from all samples were combined into a count matrix for differential expression analysis using Bioconductor packages in R.High quality sequenced data was obtained through quality control which involved removing the adapter sequences, low quality reads and unknown sequences. Using Tophat2 v2.1.05172. A threshold of Padj < 0.05, after adjusting for multiple testing using Benjamini-Hochberg, and an absolute log2 fold change of 1 was used to identify differentially expressed genes (DEGs). Hierarchical clustering analysis was then done on the DEGs.The quantified abundance estimates, in the form of a count matrix, were normalized and analyzed for differential gene expression using the geometric mean method and negative binomial distribution incorporated in the DESeq2 v1.22.2 package73. As input data, variance stabilizing transformation was applied to the expression dataset using the variance stabilization transformation function in the DESeq2 package. Genes with read counts less than 10 and “NA” as a p-value were filtered out and using a heuristic cut-off, the top 8000 most variant genes were selected. Correlational analysis of the selected genes was done using the Pearson correlation co-efficient to create a similarity matrix. Power adjacency function (β) was used for soft thresholding to convert the similarity matrix into an adjacency matrix. Scale free topology criterion was used to obtain the power adjacency function and linear regression model fitting index R2 used to choose the optimum power for our analysis, β = 20  R packageThe module-trait relationship was determined by relating eigengenes from each module to clinical traits using pearson correlation. Associations were deemed significant if the eigengene module membership had a p-value of ≤0.001 and the eigengene-trait absolute cor was ≥0.25. The significant modules were further analysed for functional enrichment.24. DEGs were divided into two gene sets, up-regulated and down-regulated, as input and an evidence score of 0.7 selected to increase stringency in the analysis. Enrichment analysis was performed on the significant modules using the GOenrichmentanalysis function of WGCNA R package and visualized using Cytoscape74.We conducted enrichment analysis on the significant DEGs using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) v11.0 softwarePlasmodium falciparum schizont extracts and PfAMA1 had been measured using previously reported protocols76. Plasma samples from HEU (n = 31) and HUU (n = 57) infants were analysed at 18 months of age . Samples were classified based on malaria exposure, that is, HEU and HUU infants residing in high malaria transmission region  or HEU and HUU infants residing in low malaria incidence region in Kilifi . Of the infants whose antibody levels had been determined, HEU (n = 6) and HUU (n = 5) were infants included in the current transcriptome analysis. Only HUU infants from the low malaria transmission region were included in the transcriptome analysis to avoid potential impact of malaria exposure on gene expression profiles. To determine antibody titers, plates pre-coated with 20 ng/µl of recombinant- PfAMA1 or crude Plasmodium falciparum schizont extract were probed with sera from HEU infants or controls (HUU) and IgG antibody levels against PfAMA1 or schizont extract measured in optical density (OD) values. Hyperimmune serum serially diluted and included on each plate was assigned antibody arbitrary units and used to convert OD values of the samples into relative arbitrary units. The arbitrary units were then log transformed to normalize the antibody titres and the non-parametric Mann-Whitney test used to determine differences in median antibody titres between HEU and HUU infants in low, and similarly between HEU and HUU infants in high malaria exposure regions. These data were used to test the observed KEGG pathway enriched in the down-regulated gene set in HEU infants.During the parent study, antibody levels against crude Supplementary dataSupplementary table S3Supplementary table S4