Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 μm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.  In a typical experiment, siRNA-lipid microarrays are covered with adherent cells in a culture medium for reverse transfection-mediated uptake of the siRNA [2 are routinely achieved on this platform so that responses to thousands of siRNAs can be robustly assessed in a single culture [etc.) much as one would stain cells grown on a coverslip  is a key technique for this purpose. Large-scale nucleic acid synthesis techniques now enable convenient and low cost synthesis of thousands of RNAis so that assessment of the effects of manipulating the expression levels of thousands of genes is possible. Initial efforts in RNAi screening in mammalian cells were accomplished using automated strategies in which the effects of RNAi knockdown were assessed in multiwell format . This required substantial laboratory instrumentation and automation, while the costs of RNAi reagent libraries were high. A strategy to miniaturize this process was first suggested by Sabatini et al.  and subset al. ,3,4,5 behe siRNA  reagentsThe quantitative assessment of the impact of high-throughput RNAi knockdown on immunofluorescently stained molecular features—the subject of the present article—has already been applied in several cell biological studies, and the potential of the approach is only beginning to be realized. Early applications include analysis of the impact of specific RNAi-induced knockdowns on cellular abundance of protein complexes , regulatThe advantages of assessing RNAi-induced changes using image cytometry following immunofluorescence staining—whether in CSMAs or in multiwell cultures—are substantial compared to strategies that assess bulk changes in cell number or metabolic activity  or thatIn the following sections, we describe fundamental aspects of image cytometry-based RNAi screening applications on CSMAs and review some of the basic methods. We also discuss in detail the custom assays established in our laboratory for analysis of quantitative cancer cell phenotypes. Finally, we conclude with an assessment of future developments of imaging-based RNAi analyses.l-glutamine. HaCat, MFC7 and MDA-MB-231 cells were grown in DMEM (Gibco) supplemented with 10% FBS, 10 µg/mL penicillin and streptomycin and 2 mM L-glutamine. RWPE-1 (ATCC) cells were grown in Keratinocyte Serum Free Medium  supplemented with 0.05 mg/mL BPE (bovine pituitary extract) and 5 ng/mL EGF. SKBR3 cells (ATCC) were grown in McCoy’s 5A medium (Gibco) supplemented with 10% FBS, 10 µg/mL penicillin and streptomycin, and 2 mM l-glutamine.All cell lines grown on CSMAs were cultured according to the protocols recommended for the cell line. Primary kidney tumor cells were isolated from fresh patient surgical specimens obtained under an Institutional Review Board-approved protocol at Oregon Health and Science University (OHSU). These primary kidney cells, along with BT20, HCC1569, HCC1954, MDA-MB-468, U-2OS, KFr13, VCaP, and 22RV1 cells  were grown in RPMI-1640  supplemented with 10% FBS, 10 µg/mL penicillin and streptomycin and 2 mM 6 cells in 4.5 mL of growth medium were added to each array well (4-well plates) and allowed to adhere at +37 °C for 5–15 min. Cells were dispersed with non-trypsin cell detachment reagent HyQtase  prior to seeding on the CSMAs since this dispersal method enabled rapid adhesion to the array spots. Non-adherent cells were washed off and 4.5 mL of fresh medium was added per array well. siRNA transfer to the adherent cells took place during growth periods ranging from 48 to 144 h prior to staining and imaging.Transfections with siRNAs and cell culture on the CSMAs were carried out as described previously . siRNAs 4Cl to quench any remainder of the paraformaldehyde fixative and washed 5 min with PBS. Cells were permeabilized with 0.3% Triton-X100 in PBS for 15 min at room temperature, washed once with PBS and blocked with 2% filtered BSA in PBS for 60 min at room temperature. After blocking, the arrays were washed 2 × 5 min with PBS, rinsed briefly with distilled H2O and air-dried. Array areas were inscribed with a hydrophobic border using a PAP-pen (Sigma-Aldrich) to reduce the amount of antibody used during staining. Cells were rinsed with 0.05% PBS-Tween 20 and stained with primary antibody in 2% BSA-PBS (100 μL per 20 × 20 mm array surface) for 1 h at room temperature or overnight at 4 °C. These arrays were washed 2 × 5 min with PBS and for 5 min with 0.05% PBS-Tween 20 and then stained with 100 μL of diluted Alexa fluorochrome-conjugated (Life Technologies) secondary antibodies. Secondary antibody incubation and parallel DAPI counterstaining was performed for 1 h at room temperature, followed by washing as described for primary antibodies. The stained arrays were then rinsed with distilled H2O, air-dried and stored for imaging. The cells were rehydrated for imaging by covering the arrays with PBS or by mounting under a coverslip using ProLong Gold anti-fade reagent (Life Technologies).Cells transfected with siRNAs during growth on CSMAs were immunofluorescently stained according to the following protocol. First the culture medium was aspirated carefully from each array well and the cells were fixed with 2% paraformaldehyde  in PBS for 15 min at room temperature. Cells were then rinsed once briefly with 50 mM NHCells grown on CSMAs were fixed in 4% formaldehyde in PBS for 10 min, permeabilized with 70% ethanol at 4 °C for 1 h, and prehybridized with wash buffer  at room temperature for 10 min. Cells were then incubated in parallel with cyclin D1 antisense-oligonucleotide probe-sets labeled with Cy-5 and anti-Ki67 antibody  in hybridization solution  at 37 °C overnight in a dark/humid chamber, and incubated with wash buffer at 37 °C for 30 min twice. Secondary antibody incubation (Alexa488-conjugated goat anti-rabbit antibody in the hybridization solution) was carried out at room temperature for 1 h, followed by incubation with wash buffer for 30 min, with DAPI for nuclear staining, and with 2× SSC. Cells on arrays were mounted in Prolong Gold anti-fade reagent (Life Technologies). Probe sets for CCND1 mRNA detection were designed using a Stellaris™ Probe Designer version 1.0 . They weAfter transfection, cells grown on CSMA were fixed and stained according to the manufacturer instructions for the DuoLink II PLA kit  with a minor change in PLA probe dilution. The primary antibodies were diluted 1:200 in 2% BSA-PBS and incubated overnight at 4 °C . The PLA probe antibodies were diluted 1:20 in 2% BSA-PBS supplemented with the PLA blocking concentrate and incubated for 2 h at 37 °C. The array surfaces were circumscribed with a hydrophobic border drawn with a PAP-pen (Sigma-Aldrich) to minimize antibody and PLA detection reagent consumption. Cells were counterstained for filamentous actin using fluorescently labeled phalloidin  and DNA (DAPI). Images for each spot in the CSMAs were acquired automatically using an Olympus scan^R imager and image analysis software  was used to quantify PLA levels in individual cells.Western blot analysis of total cell lysates prepared from cells transfected on CSMAs with 384 replicate spots of a single control or targeting siRNA were fractionated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes . The membranes were blocked against non-specific binding using 5% skim milk. Membranes were probed with primary antibodies  overnight at 4 °C. Equal loading was confirmed by probing the same filter with a specific antibody for β-tubulin or β-actin . Signals were revealed with horseradish peroxidase-coupled secondary anti-mouse or anti-rabbit IgG antibodies .2 CCD digital camera . The Olympus scan^R system is an inverted microscope designed for fully automated image acquisition of biological samples in high-density sample platforms such as CSMA plus image analysis algorithms for feature quantification. Each spot on a CSMA was imaged individually with a 20× LUCPLFLN NA 0.40 objective using specific filter sets for DAPI, Alexa488, Alexa568 and Alexa647 dyes . The scan^R image analysis software suite was also used for quantitative analysis of image features. Analysis capabilities included cell/particle counting, protein expression analysis with immunofluorescence quantitation, subcellular particle quantitation assays, cell cycle analysis, and protein localization and co-localization assays. Image features quantified for cell populations using the scan^R software were further analyzed using FCS Express 3.0 software . The effects of each siRNA knockdown on each specific response endpoint were assessed by comparing image parameters  with comparable image parameters measured for cells transfected with a non-targeting scrambled control siRNA.Array imaging was performed using an Olympus scan^R integrated imager and image analysis suite  equipped with a Hamamatsu ORCA-R2-M-phase of the cell cycle , an established marker for heterochromatin formation. Epithelial-to-mesenchymal transition is one of the fundamental cellular phenotypes associated with the course of cancer pathogenesis. qCPs for differentiation status report the intensity of staining for differentiation associated proteins including Beta-Catenin, Cytokeratins-8/14/19, E-Cadherin, EpCAM, Fibronectin and Vimentin [We have now developed immunofluorescent staining and quantitative image analysis procedures for many of the key molecular features associated with the cancer hallmarks to facilitate use of RNAi screening for in-depth biological discovery in the context of cancer research. Measures of RNAi-induced changes in these features—designated herein as quantitative cancer phenotypes (qCPs)—enables accurate identification of aberrant genes or networks that are causal for many aspects of cancer. This approach assesses many more aspects of cancer physiology than are routinely assessed in screening strategies that are sensitive only to events that alter proliferation or immortalization. We are now applying this approach to systematic assessment of oncogenic events, signal transduction programs and associated deregulated gene networks that are postulated to contribute to the hallmarks of cancer by the Cancer Genome Atlas (TCGA)  and relaVimentin . Loss ofVimentin . The qCPVimentin . Figure Vimentin ,11,16,17in situ Hybridization (FISH) for genome number (DNA-FISH) [in situ detection of specific DNA or RNA sequences in fixed cell samples using probe oligonucleotides that are complementary to the specific sequence(s) of interest. We tested the performance of RNA-FISH for quantification of RNA levels altered during RNAi screening by assessing CCND1 (Cyclin D1) mRNA levels. We use the Stellaris FISH method (Biosearch Technologies) in which detection probes are comprised of multiple short, fluorescently labeled oligonucleotides targeting one mRNA transcript [2 cells in comparison to the other groups  , or abunanscript . The higanscript  and nuclWe anticipate that use of immuno-FISH as described coupled with high-throughput RNAi screens will enable coordinate analysis of the impact RNAi knockdown on both transcriptional and translational regulation of target genes and proteins. This will allow discrimination between RNAi effects that directly alter expression of the target genes and those that affect post-translational mechanisms regulating the protein functions. in situ analysis of protein–protein interactions on cells grown on CSMAs using an antibody-based proximity ligation assay (PLA) [Interactions between proteins enable and regulate a wide range of cellular processes. Protein–protein interactions typically are studied using methods such as mass spectrometry of immunoprecipitated protein complexes or via analysis in yeast two hybrid systems. These methods reveal a broad range of interacting proteins. However, they do not reveal where within cells these proteins interact nor do they assess the impact of RNAi knockdown on these interactions. This can only be accomplished using image-based analysis strategies. To this end, we have developed a strategy for ay (PLA) . The PLAay (PLA) . The PLAay (PLA) . This drin situ. Analysis of protein proximity after PLA staining is accomplished by segmenting the individual PLA signals and quantifying the number of individual PLA signals per cell. In in situ hybridization or a combination of both. Analyses can be carried out for cells growing in diverse culture conditions and during treatment with therapeutic compounds or other siRNAs. Assays for >25 response endpoints already have been developed for the CSMA platform. Automated, image analysis allows images of cells on individual array spots to be acquired and analyzed the amount and spatial distribution of molecular features associated with several cancer hallmarks. We have demonstrated applications of the CSMA technology ranging from general cancer target gene discovery to detailed analyses of specific cellular processes including regulation of integrin cell-adhesion receptors [The CSMA analysis platform represents a technological advance in RNAi screening. It enables assessment of the functional impact of large-scale gene knockdown in many different cell types quickly and at low reagent cost. The impact of siRNA knockdown on multiple molecular and cellular response endpoints can be revealed using immunofluorescent staining or fluorescence eceptors ,11. The The ability to assess changes induced by targeted RNAis using image cytometry assays enables assessment of the functional importance of a large number of disease-linked genomic aberrations and aberrant regulatory networks. Loss-of-function genetic screening has been used widely to assess the impact of RNAi knockdown on cell viability and/or immortalization. More recently, imaging-based screening strategies, coupled with immunofluorescent staining for cancer hallmarks has expanded the range of biological endpoints that can be assessed during RNAi screening. The ability to simultaneously assess the levels of specific proteins and RNA species, their spatial locations and the proximities of specific proteins, greatly increases the information that can be gained from RNAi screening experiments. This powerful form of high-throughput gene function assessment at cellular and subcellular resolution has already proven useful for functional annotation of normal and aberrant human genes. Several emerging imaging techniques such as automated confocal microscopy, super-resolution fluorescence microscopy and integrated light and electron microscopy will likely lead to further developments in the field of cell biological gene function assessment by enabling quantitative analysis of RNAi-induced changes in cellular function and structure. Understanding of human genetic and cell biological functions is increasing as the range of biomarkers for specific cellular processes increases. Multiplex analysis on CSMA carrying RNAis allows correlative analyses of the impact of gene perturbations on the molecular and cellular features they regulate in individual cells. In the future, RNAi screening experiments will likely shift from general cell biological discovery with established model cell lines to clinical applications where RNAi screening is performed directly with patient derived cells .


Circulating tumor cells (CTCs) play a crucial role in cancer dissemination and provide a promising source of blood-based markers. Understanding the spectrum of transcriptional profiles of CTCs and their corresponding regulatory mechanisms will allow for a more robust analysis of CTC phenotypes. The current challenge in CTC research is the acquisition of useful clinical information from the multitude of high-throughput studies. To gain a deeper understanding of CTC heterogeneity and identify genes, pathways and processes that are consistently affected across tumors, we mined the literature for gene expression profiles in CTCs. Through in silico analysis and the integration of CTC-specific genes, we found highly significant biological mechanisms and regulatory processes acting in CTCs across various cancers, with a particular enrichment of the leukocyte extravasation pathway. This pathway appears to play a pivotal role in the migration of CTCs to distant metastatic sites. We find that CTCs from multiple cancers express both epithelial and mesenchymal markers in varying amounts, which is suggestive of dynamic and hybrid states along the epithelial-mesenchymal transition (EMT) spectrum. Targeting the specific molecular nodes to monitor disease and therapeutic control of CTCs in real time will likely improve the clinical management of cancer progression and metastases. Nearly 90% of all cancer deaths are attributed to distant tumor metastasis46It has been suggested that cancer cell extravasation may have been co-opted from leukocyte extravasationCarcinoma cell invasion and migration during metastasis is also influenced by the transition of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT)1516192021In the present study, we collected, reanalyzed and mapped the gene expression data from several published studies in CTCs to the signaling pathways and networks available to date in an attempt to reveal new insights into CTC signaling. We integrated the transcriptomic changes observed in CTCs across various cancer types to identify with high confidence putative candidate markers involved in extravasation of CTCs. The EMT spectrum was scored for the first time in CTCs from transcriptome datasets to reveal their EMT profile across cancers. The study unfolds some key biological processes that are inherent to these circulating cells.CTC phenotying and genotyping studies have expanded our knowledge on tumor progression and tumor evolution2419202728n = 3,251) in the CTC_ALL dataset showed significant enrichment of the ‘Leukocyte Extravasation Pathway’ at 44.9% . The top canonical pathways with a positive z-score included ‘Integrin Signaling’ , ‘ILK Signaling’ , ‘Colorectal Cancer Metastasis Signaling’ , ‘Glioma Invasiveness Signaling’ , ‘Gα12/13 Signaling’  and ‘B Cell Receptor Signaling’  pathways. These pathways are involved in cell migration, adhesion, growth and differentiation, inhibiting apoptosis and enabling cell survival303132333435TGFβ1(p = 2.99E-39), and the IPA gene view indicated TGFβ1 as the upstream regulator of many genes that play a vital role in cancer progression, such as SERPINE1, ACTA2, CDKN1A, SMAD3, SMAD2, CTGF, FN1, CDH1, SMAD7, COL1A1, FOXP3, CDKN2B, COL1A2, VIM. The top diseases were ‘Cancer’  and ‘Organismal Injury and Abnormalities’ , both with a p-value range of 2.53E-08 to 5.08E-33. The top molecular functions were ‘Cell Death and Survival’  with a predicted decrease in cell death and predicted increase in cell survival, and ‘Cellular Growth and Proliferation’ . Similarly, IPA analysis of the CTC_FC dataset of overexpressed genes  positioned the leukocyte extravasation pathway as third in the rank of enrichment . The top ten enriched canonical pathways associated with each dataset are illustrated in Ingenuity Pathway Analysis (IPA) of the overexpressed genes (CXCL12) and C-X-C motif chemokine receptor 4 (CXCR4)] and cytokines [suppressor of cytokine signaling 1 (SOCS1)], to guide them to distant metastatic sites to establish new tumors. Furthermore, the expression of these markers has been correlated with the presence of CTCs in peripheral blood and metastasis3940Although comparatively less is known about cancer cell extravasation as compared with that of leukocytes, previous studies suggest a similar process might be operating in CTCs937The leukocyte extravasation pathway (LEP) is a reasonably well characterized, multi-step process of leukocyte emigration from the blood into tissues through the vascular endothelium and is vital for immune surveillance and inflammationITGB1, ITGB2, ITGAL, ITGAM and ITGA4 and cell adhesion markers such as ICAM3, PECAM1, JAM3 and F11R. The signal then travels via a chain of phosphorylation events that ultimately lead to phosphorylation and activation of the myosin light chain (MLC), and this causes retraction of the actin cytoskeleton in the tail-end of the migrating leukocyte or CTCExtravasation occurs via three sequential steps: (1) rolling, docking and adhesion via endothelial cell adhesion molecules (CAMs); (2) transmigration (extravasation) of leukocytes/CTCs; and (3) the subsequent extravasation of cells across vascular tight junctions9LGALS3), known to be overexpressed in circulation of cancer patients, interacts with MUC1 resulting in exposure of cell surface adhesion molecule such as CD44 enhancing cancer cell adhesionICAM1 signals activate calcium flux and protein kinase C (PKC), which are required for leukocyte migration. Similarly, vascular cell adhesion molecule (VCAM1) ligation is required to open the “endothelial passage” by activating the production of reactive oxygen species (ROS) by NADPH oxidase. This in turn activates MMPs, which causes a loss of VE-cadherin-mediated adhesion and allows leukocytes or CTCs to extravasateCancer cells and leukocytes both use a group of adhesion receptors called galectins. Galectin-3 (P2Y2) on cancer cells to mediate their transendothelial migrationANGPTL4) instigates vascular ‘leakiness’ and endothelial disruption by binding to integrins and TGFβ via the SMAD signaling pathway47ANGPTL2 and ANGPTL4. In sum, the enrichment observed in our analyses of the pan-cancer datasets will advance our current understanding of the biological mechanisms behind cancer cell dissemination and allow for a more targeted validation, with the hope to improve the management and prevention of metastatic progression.Additionally, recent evidence suggests that platelets contribute to the survival and dissemination of cancer cells by interacting and aggregating around them, which allows cancer cells to evade immune assaults from natural killer cells44http://string-db.org/) to identify functional hubs with the most number of interactions [data not shown]. Some of the highly connected proteins included SRC, CDC42, PTK2, PXN, PIK3CA, MAPK14, VAV1, ACTB and ITGB2 among others. Earlier network studies on adhesome and EMT showed similar hubs of activity that can be used for targeted studies4951Often pathways do not depict all the interconnections and cross-talk, hence in addition to pathways, we constructed a LEP-related network using available protein-protein interactions in STRING database  and principal component analysis (using GeneSpring GX software version 13.0) on differentially expressed gene datasets from the CTC_FC list (SPARC), codes for an important ECM protein reported to be differentially expressed and a modulator of several cellular processes required for metastasis initiation in the tumor microenvironment64SPARC acts as a tumor suppressor by repressing angiogenesis by inhibiting vascular endothelial growth factor (VEGF); whereas, in pancreatic, breast, melanoma and glioblastoma cancers, its overexpression leads to the activation of MMPs, which in turn promote EMTSPARC has been shown to be necessary in pancreatic cancer metastasisSPARC overexpression is associated with poor survival68HINT1) in colorectal, pancreatic and breast CTCs , breast (BCLAF1) and melanoma (FAM162A) CTCs.To understand the gene expression profiles observed in CTCs among various cancers and identify signatures, we performed unsupervised clustering  . The EMT score spectrum of CTCs exhibit similarities with our publishedhttp://www.cytoscape.org/) by including key EMT regulators. The differential gene expression data from CTCs were then overlaid on this network. We noted an upregulation in SPARC, MYLK, MYL6, GG11, F13A1 and FHL1  and CD9, LCN2, and ABCC3  in CTCs of at least two cancers. SPARC  is a well-known marker associated with invasion and an important mediator of bone metastasis89MYLK and CD9 have been implicated in lymph node metastasis in different cancers91ABCC3 overexpression is reportedly involved in glycolysis, drug resistance and overall poor prognosis in bladder cancer [not in our dataset]89FHL1 has been linked with increased metastasis in esophageal and gastric cancers93LCN2), a tissue-specific factor not found in peripheral blood leukocytes, was reported in a CTC-derived cell line (BHGc7) from relapsed lung cancer patientsConsidering the disparate nature of the CTC_FC dataset, we built a gene interaction-based functional EMT network using a selected subset of gene expression data from colorectal, pancreatic and breast CTCs (DEGs when compared to PBMCs); this was done to acquire an accurate comparison of differential values and allow us to segregate high-confident EMT hub molecules from the network. The EMT network  was builThis study has uncovered promising CTC-driven signaling nodes in the extravasation pathway and identified the key contributors that may determine favorable clinical outcomes. These CTC-associated signaling profiles will provide a valuable resource to identify downstream events crucial to the functioning of CTCs in the metastatic cascade. These findings bring us further toward understanding the mechanisms of various biological processing pathways within the circulating cell and pave the way for the identification of appropriate therapeutic targets against CTC, metastasis and cancer.We employed a multi-stage screening and filtering strategy to identify molecular signatures in CTCs. Briefly, we screened the literature for articles published on CTC expression. A list of all differentially expressed genes was prepared and used to identify significantly altered pathways and networks.p < 0.05) gene list constituted genes reported to be overexpressed or downregulated in CTCs when compared to primary tumors, cancer cell lines, peripheral blood mononuclear cells (PBMCs), or normal tissue (each classified as CTC sub-group in the manuscript). Gene expression data were taken from studies that evaluated cancer patient CTC levels, cancer cells cultured ex vivo and spiked into the blood collected from healthy individuals, or CTCs isolated from blood of orthotopically grafted tumors in mice. From this extensive dataset compiled from 98 papers, data of differentially expressed genes from five large-scale transcriptomics studies across five different cancers was collated separately to generate a unique gene list with fold change values. Two of the five studies used RNA sequencing experiments and three used cDNA microarray to capture expression data. Details of the source for datasets are provided in PubMed was searched using keywords ‘circulating tumor cells signaling’  which resulted in 1,073 publications. These publications were reviewed at the abstract level and 153 papers were narrowed down for further screening. Another PubMed search was carried out using keywords ‘circulating tumor cells gene expression’ , which resulted in 2,169 publications. Further review narrowed this down to 78 publications. Of the 231 (153 + 78) papers, 98 (42.4%) were thoroughly annotated for gene expression in CTCs [see Additional https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31023 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18670) were reanalyzed using GEO2R (http://www.ncbi.nlm.nih.gov/geo/geo2r/) package to obtain the set of DEGs in CTCs. RNA-Seq data from the prostate cancer study were also reanalyzed to obtain genes that were differentially expressed with log2 fold change of 1.5 or higher using an in-house bioinformatics pipeline. Genes were filtered to include only the protein-coding genes for pathway analysis. Any pseudogenes, uncharacterized genes, non-coding RNA gene annotation were excluded. Entrez Gene IDs and gene biotype information were retrieved using the Ensemble Biomart (http://asia.ensembl.org/biomart/martview/) tool for human genome assembly; GRCh38p.5. Genes from various datasets were aggregated to form a master list of non-redundant DEGs.Transcriptome datasets were compiled from both high-throughput RNA-Seq experiments and microarray experiments (whichever available) related to CTCs. Supplementary results were compiled where available; otherwise data were processed to obtain the final set of genes. From the semi-quantitative experiments, sets of statistically significant, differentially expressed genes (DEG) with a log2 fold change cutoff of 1.5 and p-value < 0.05 were considered for downstream analysis. The colorectal and pancreatic microarray gene expression data  and compared them to previously computed EMT scores for each tumor type and its cancer cell line collectionTwo sets of data were used for pathway analysis: (1) the lenient gene list collated from 98 studies and (2) the differentially expressed genes list from the five high-throughput transcriptomics studies. (Denoted as CTC_ALL and CTC_FC (FC = fold-change), respectively for the purpose of differentiating between the datasets.)http://www.ingenuity.com/) tool was used for pathway analysis. Relationships of molecules differentially expressed in our dataset were inspected using the Core Analysis module in IPA Ingenuity Knowledge Base reference repository. Only the overexpressed genes from the CTC_ALL and CTC_FC datasets were uploaded into IPA, separately. Canonical pathway analyses identified the top canonical pathways that were significant in our datasets.The Ingenuity pathway analysis , a free, open-source software for drawing and editing biological pathways, was used to manually depict the enrichment for Leukocyte Extravasation Signaling. Molecular interaction data were sourced from the KEGG pathway database (http://www.genome.jp/kegg/) and IPA pathways . Literature mining was done for reported signaling patterns in CTCs, and only a handful of publications were available. Molecular interactions were annotated from these publications and appended onto the depicted pathway. An internal pathway review system was followed to carefully inspect for erroneous annotations. The pathway map was uploaded to GeneSpring software provided by Agilent Technologies  for enrichment. The gene list used for enrichment was identical to that used for IPA analysis .PathVisio  database was used for generating protein-protein interactions and Cytoscape , an open-source platform, was used for visualizing the EMT biological pathways and molecular interaction network, and integrating the CTC gene expression data. In-house python scripts and the Adobe Illustrator (version CS5.1) graphics editor were used to draw or edit the figures.STRING .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The primary cilium, a sensory organelle, regulates cell proliferation and neuronal development of dentate granule cells in the hippocampus. However, its role in the function of mature dentate granule cells remains unknown. Here we specifically depleted and disrupted ciliary proteins IFT20 and Kif3A (respectively) in mature dentate granule cells and investigated hippocampus-dependent contextual memory and long-term plasticity at mossy fiber synapses. We found that depletion of IFT20 in these cells significantly impaired context-dependent fear-related memory. Furthermore, we tested synaptic plasticity of mossy fiber synapses in area CA3 and found increased long-term potentiation upon depletion of IFT20 or disruption of Kif3A. Our findings suggest a role of primary cilia in the memory function of mature dentate granule cells, which may result from abnormal mossy fiber synaptic plasticity. A direct link between the primary cilia of mature dentate granule cells and behavior will require further investigation using independent approaches to manipulate primary cilia. Cilia are hair-like structures composed of microtubules, which can be classified as either motile or primary based on their movement and microtubule structure245Moreover, mutations in ciliary proteins that cause structural or functional abnormalities are implicated in a wide variety of diseases known as ciliopathies, and defects in primary cilia can lead to neurological impairments such as intellectual developmental disorders and cognitive deficits1011513Here, we examined the physiological and behavioral function of primary cilia in mature DGCs. We conditionally ablated intraflagellar transport (IFT) genes, IFT20 or expressed a dominant negative form of Kinesin family member 3A (Kif3A) under the control of the CaMKII promoter using adeno-associated viruses (AAV). We found that ablation of IFT20 in mature DGCs led to impaired hippocampus-dependent contextual memory. We further found that IFT20 ablation or Kif3A disruption in mature DGCs enhanced long-term potentiation (LTP) induced at mossy fiber (MF) synapses of these cells.Conditional IFT20 mutant mice were kindly provided by Dr. Gregory J Pazour at the University of Massachusetts Medical SchoolHigh titer engineered AAV9 under the CaMKII promoter was purchased from the inventory of vector core facility from University of Pennsylvania. AAV9-DIO-dnKif3A-dTomato was produced as described previouslyMice were anesthetized and transcardially perfused with PBS and 4% PFA. Fixed brains were sliced by a sliding microtome, creating 40 μm thick coronal brain sections.Prior to primary antibody incubation, brain sections were blocked with 10% donkey serum in 0.25% Triton-PBS for 1 hour. Next, sections were incubated overnight at 4 °C with diluted primary antibodies . The next day, brain sections were incubated in diluted secondary antibodies solution  for 2 hours at room temperature. Sections were mounted on slides using DAPI-containing mounting media to stain nuclei. All images were acquired on an Olympus FV1000 confocal system. For DCX + cell quantification, randomly selected, 40 μm-thick, z-stacked images were collected across the entire anterior/posterior axis of the dentate gyrus  mice), and 2 μm guard zones were used to avoid edge artifacts. Dendrite morphology was analyzed using the Sholl analysis function of the NeuronJ plug-in for ImageJ.Cilia were identified and related to their cell of origin for quantification using an anatomical- and landmark-based approach4Analysis of parvalbumin and GFP co-localization was conducted by taking 40 μm-thick, z-stacked images across the entire anterior/posterior axis of the dentate gyrus (field side 320 μm × 320 μm). Co-localization was confirmed by overlaying GFP in 3-D reconstructed images of parvalbumin + cells in subgranular zone and hilus of the dentate gyrus.We conducted the procedure for CFC as we have previously describedPECFC was conducted to probe pattern memory encoding on the basis of partial cue information from a fear context to which an animal was pre-exposed. We used an experimental paradigm based on previous workWe used a contextual fear discrimination test adapted from previous workFor the next 8 consecutive days, mice were exposed to context A (fear context) and a similar context, context B (safe context in which no shock was delivered). Context B consisted of a stainless steel grid floor as in context A, but contained black and white striped front and back walls and lacked an olfactory cue. This context was cleaned with odorless non-alcoholic antiseptic solution before each mouse was placed in the context. Mice were allowed to explore freely for 180 seconds without any shock. The order of exposure on each day was AB/AB/BA/BA/AB/BA/AB and there was a 4-hour gap between exposures to each context.All mice movements were recorded through FreezeFrame software and percentage of freezing was analyzed by FreezeView software with 1 second of minimum bout duration. Freezing levels during the first 180 seconds of each session were averaged for each group and used to calculate discrimination ratios;Discrimination Ratio = (Freezing Context A – Freezing Context B)/(Freezing Context A + Freezing Context B).3 opaque box) lacking objects. On day 4, mice were exposed to two identical objects arranged in one positioning pattern for 10 minutes. On day 5, one of the two objects was moved to a novel location. Object location zones were defined and animal paths were recorded using EthoVision XT video tracking software.We performed the spatial novelty recognition test similar to what has been previously describedThe EPM is a paradigm used for testing anxiety-like behavior in rodentsThe light/dark transition test measures anxiety-like behavior associated with rodents’ innate light avoidance. The apparatus has two connecting compartments- a light compartment that has transparent plastic walls and was illuminated during experiment, and a dark compartment that is covered with black and opaque plastic walls. Mice were placed in the dark compartment and allowed to freely explore both compartments for 10 minutes. Latency to enter the light compartment and time spent in each compartment were measured.Preparation of brain slices and electrophysiological recordings was performed as previously described16Data were analyzed using two-tailed unpaired Student t-tests, repeated measures ANOVAs, and two-way ANOVAs. The cut-off for statistical significance was set at a p value of less than 0.05. All data are presented as mean ± SEM.We set out to examine the physiological and behavioral role of primary cilia in mature DGCs by specifically depleting IFT20 in these cells. AAV9 expressing Cre recombinase under the control of the CaMKII promoter (or eGFP under the CaMKII promoter as control) was stereotaxically injected into the dentate gyrus of adult floxed IFT20 micemDGCs) versus eGFP-controls  and enzymes including ACIII, and signaling pathways downstream of these could be another possible cause. Indeed, in SSTR3 or ACIII-knockout animals, several hippocampus-dependent behavioral defects have been identified13per se. Likewise, more work will be needed to definitively confirm that the behavioral effects observed in the current study are not the result of non-ciliary functions of the proteins we disrupted.Though the major downstream signaling pathways of primary cilia that lead to the observed disruptions remain to be determined, the Wnt/β-catenin pathway is a possible candidate, given previous findings by others and us9Learning and memory generally occur via a three-step process: encoding, consolidation, and retrieval. The DG-CA3 juncture has been found to be heavily involved in the formation of a contextual memory representation294748Our finding that depletion of IFT20 in mature DGCs leads to impairments in both CFC and PECFC suggest that primary cilia are important for proper rapid memory encoding. A potential model consistent with these findings is that primary cilia in mature DGCs may regulate the activity of mature DGCs and their CA3 outputFinally, one potential limitation that we should point out is the viral vector-based approach for targeting a specific cell population, although this approach is still currently widely used in opto- and chemo-genetic gene delivery. In this study, we expressed Cre recombinase under the CaMKII promoter to target DGCs. Although we observed robust targeting of CaMKII-driven Cre expression to DGCs and noted no overlap between PV+ interneurons and CaMKII-driven GFP expression in the dentate gyrus, others have reported limited specificity of this promoter to excitatory neurons in cortical layer V and hippocampal area CA1, labeling some inhibitory PV+ interneurons as well52mDGCs and dnKif3A-expressing mice.In the adult brain, new DGCs are continuously generated2324182831In summary, we demonstrated a physiological function of the neuronal primary cilium. Our findings implicate primary cilia of mature DGCs in hippocampus-associated contextual memory. It will be interesting to further test the cellular mechanisms by which primary cilia regulate the activity of the dentate granule neurons, and also to examine possible effects of primary cilia in other brain regions.How to cite this article: Rhee, S. et al. Depletion of primary cilia from mature dentate granule cells impairs hippocampus-dependent contextual memory. Sci. Rep.6, 34370; doi: 10.1038/srep34370 (2016).


Levothyroxine (LT4) and rifampin (RIF) are sometimes used together; however, no clinical studies have assessed the effects of these drugs on thyroid function or the need to adjust LT4 dose.We retrospectively reviewed the records of 71 Korean patients who started RIF during LT4 treatment. Clinically relevant cases that required dose adjustment according to the American Thyroid Association (ATA)/American Association of Clinical Endocrinologists (AACE) guidelines were identified, and risk factors of increased LT4 dose were analyzed.After administering RIF, median serum thyroid-stimulating hormone (TSH) level  was significantly higher than that before RIF . An increased LT4 dose was required for 50% of patients in the TSH suppression group for thyroid cancer and 26% of patients in the replacement group for hypothyroidism. Risk factor analysis showed that remaining thyroid gland , the time interval between starting RIF and TSH measurement , and baseline LT4 dose per kg body weight  were clinically relevant variables.In patients receiving LT4, serum thyroid function test should be performed after starting RIF treatment. For patients with no remnant thyroid gland and those receiving a lower LT4 dose, close observation is needed when starting RIF and TB medication. Levothyroxine (LT4) is widely used to replace thyroid hormone in patients with hypothyroidism and to suppress serum thyroid-stimulating hormone (TSH) for the treatment of differentiated thyroid cancer (DTC). The primary pathway of LT4 metabolism is sequential deiodination. The main site of deiodination is the liver, although several other organs including the kidneys also metabolize LT4. LT4 is a substrate for CYP3A4, a member of the hepatic cytochrome P450 family of enzymes. Approximately 80% of LT4 is eliminated by the kidneys, and the remaining 20% is eliminated in the stool .Mycobacterium avium complex. These mycobacterial diseases require long-term RIF treatment. RIF is a potent inducer of hepatic cytochrome P450 enzymes, especially CYP3A4, thus increasing the metabolism of many drugs that are metabolized by the liver [Rifampin (RIF) is used to treat active tuberculosis (TB) and latent TB infection (LTBI), as well as disease caused by nontuberculous mycobacteria (NTM) such as he liver ,3.In some patients undergoing LT4 treatment, elevated TSH leads to an increase in LT4 dose after administration of RIF. The American Thyroid Association (ATA)/American Association of Clinical Endocrinologists (AACE) hypothyroidism guidelines recommend measuring TSH within 4–8 weeks after starting a drug that might influence LT4 dose, such as RIF –7. FurthIn this study, we investigated the effect of RIF on thyroid function in patients undergoing LT4 therapy. We also analyzed risk factors of a clinically relevant change in thyroid function.This study was a retrospective cohort study of adults who started RIF treatment during LT4 treatment between November 1994 and June 2014 at Samsung Medical Center . Patients 18 years or younger and those who took RIF for diseases other than TB, LTBI, or NTM infection were not included. We also excluded patients administered RIF for less than 4 weeks and patients with an obvious cause for a change in thyroid function, including LT4 discontinuation due to poor compliance or in preparation for radioiodine treatment. Seventy-one Korean patients were enrolled in this study. The study population was divided into two groups based on the reason for LT4 treatment: LT4 replacement to manage hypothyroidism and TSH suppression to manage DTC.The primary outcome of this study was change in thyroid function as measured by serum TSH and free thyroxine fT4) after RIF administration. The secondary outcome was the percentage of patients with a clinically relevant change in thyroid function leading to a need for LT4 dose adjustment. A clinically relevant change in thyroid function was defined as a change that required LT4 dose adjustment according to the ATA/AACE guidelines. In the LT4 replacement group, a serum TSH level that exceeded the upper normal limit after starting RIF was classified as clinically relevant by 2012 ATA/AACE hypothyroidism guidelines  after RI,9.The following factors were investigated for possible biological and/or pharmacological associations with LT4 dose adjustment: age, sex, reason for LT4 treatment, LT4 dose, reason for RIF treatment, RIF dose, time interval between starting RIF and serum TSH measurement, underlying liver disease, and underlying renal disease ,11.U test for continuous variables not following a normal distribution, Chi-square test for categorical variables, and Fisher’s exact test for categorical variables with an expected frequency less than five. Age, sex, liver disease, renal disease, RIF-TSH duration, remnant thyroid, etiology of RIF, RIF dose, and LT4 dose were used as covariates of multivariable analysis. Binary logistic regression with likelihood ratio test and backward elimination was used for multivariable analysis. All statistical analyses were performed with SPSS Statistical Software package version 22.All variables including baseline characteristics are presented as number with percentage for categorical variables, mean ± standard deviation (SD) for continuous variables following a normal distribution, and median with interquartile range (IQR) for continuous variables not following a normal distribution. Changes in serum TSH and serum free T4 were evaluated using the Wilcoxon signed rank test (Wilcoxon matched pairs test). Bivariate analysis of variables between clinically relevant and clinically irrelevant changes in thyroid function were performed with the Student’s t-test for continuous variables following a normal distribution, Mann-Whitney The data used in this study were made available in an anonymized format, and the need for informed consent was waived. This retrospective observational study was approved by the Institutional Review Board of Samsung Medical Center (IRB No. 2015-08-101), and full permission was granted to review and publish information obtained from patient records.The study population (N = 71) was divided into a TSH suppression group who received LT4 to suppress DTC recurrence (N = 46) and a TSH replacement group who took LT4 to replace thyroid hormone (N = 25). Baseline characteristics of the study population are presented in U test) were significantly different , reason for LT4 treatment , and time between starting RIF and TSH measurement  is added, the new equilibrium depends on the second drug, an effect known as drug-drug interaction (DDI). For example, a combination of steroid and RIF requires 2 weeks to reach a new equilibrium. Dynamic DDI modeling with RIF revealed that the new equilibrium time is proportional to the half-life of the substrate ,13. The Past studies have documented that isoniazide (INH) is the substrate of N-acetyltransferase. The slow-acetylator phenotype (poor metabolizers) is prone to INH side effects due to increased blood INH concentration. INH inhibits CYP3A, contrary to RIF, and thus RIF might have a weaker effect on CYP3A in slow acetylators. In this study, most patients took INH and RIF together, but all patients were Korean and only 10–15% of Koreans are expected to be slow acetylators –16. In cThe small study population from a single tertiary referral center is a limitation of this study, and a further study in a larger population is warranted. In addition, quality of life  was not investigated because of the weakness of the retrospective design.In conclusion, serum TSH increased and fT4 decreased significantly after the start of RIF treatment in patients taking LT4 for either hormone replacement for hypothyroidism or TSH suppression to manage DTC. Therefore, clinicians should closely monitor thyroid function when RIF is administered, particularly in patients without remaining thyroid gland and those taking a low dose of LT4. Further studies are needed to confirm these results, especially in populations where slow acetylators are more prevalent, such as in Caucasians and African Americans.S1 Table(DOCX)Click here for additional data file.


Saccostrea glomerata, Ostrea angasi, Crassostrea gigas, Mytilus galloprovincialis and Anadara trapezia. Band width selection was undertaken using Fourier wavelet transformation coupled with a genetic algorithm (GA) to aggregate adjacent wavelet bands to select the minimum number of IR bands that were consistently identified in the majority of individual spectra.Data presented in this article are related to the research article entitled “Near Infra-red spectroscopy quantitative modelling of bivalve protein, lipid and glycogen composition using single-species versus multi-species calibration and validation sets” [1]. Band width selections were determined using a data-driven approach to modelling Near Infra-red Spectra (NIRS) of protein, lipid and glycogen content in bivalves. Models were produced for single species and combined species of  Specifications TableValue of the data•Data provides example of different bandwidth selections associated with energy stores in bivalve species when using a data-driven approach to NIRS quantitative modelling.•Data provides first steps to allowing potential comparisons with other NIRS bandwidth selection processes.•Bandwidth selection was undertaken for individual species and pooled to generate 3-oyster and 5-bivalve species models.1In the near infra-red range of light, absorptions correspond to overtones and combinations of fundamental bands of molecular vibrations Saccostrea glomerata, Ostrea angasi, Mytilus galloprovincialis and Anadara trapezia. Multi-species models were developed for 3 oyster species  and all 5-bivalve species.The data provided are the results of a data-driven bandwidth selection process implemented when undertaking quantitative modelling of bivalve energy storage components of protein, lipid and glycogen in whole animals. Single species models were developed for each energy storage component for 2Bivalve species used in this modelling were collected across 8 sites in New South Wales (NSW), Victoria and South Australia (SA) in Australia across four seasons to provide a wide range of samples.−1 (32 scans) measured as absorbance at a resolution of 16 cm−1 with data intervals of 2 cm−1. NIR capture was undertaken in triplicate and samples rotated up to 120° between each image capture. Spectra were captured using Perkin Elmer Spectrum software, v.10.4.3.339 and corrected for stray light and reference corrected.NIR spectra were collected with a Perkin Elmer Frontier FT-IR Spectrometer using the NIR spectral unit. Samples added to the dish were gently pressed into the dish (30 mm) then tapped three times with a spatula to ensure even packing. NIR spectra were captured at wavelengths 10,000–4000 cmData-driven software was developed to undertake all pre-processing and predictive NIR spectra model generation. Data was pre-processed by applying multiplicative scatter correction and second derivative using the mean of the triplicate NIRS scans to normalise the data. Samples were then screened for outliers with samples where the Mahalanobis distance between individual analyte concentration values and the median analyte concentration for the entire sample dataset is > 3.0 with outliers being excluded from the model datasets −1 and 50 cm−1. Wavelet peaks between 2 and 100 cm−1 were tested, with the range of 5 to 50 cm−1 obtaining the most robust models.Band width selection was undertaken using Fourier wavelet transformation coupled with a GA to aggregate adjacent wavelet bands to select the minimum number of IR bands that were consistently identified in the majority of individual spectra. The wavenumber search range used to identify the wavelet peaks in the pre-processed spectra was between 5 cm


The aims of this study were: (1) to test whether oscillatory shear stress further exacerbates endothelial dysfunction in patients with moderate-severe COPD, and (2) to test whether low flow oxygen administration improves endothelial function and is protective against oscillatory shear stress-induced endothelial dysfunction in patients with moderate-severe COPD. In 17 patients and 10 age-matched non-smoking control subjects we examined brachial artery flow-mediated dilation (FMD) and circulating microparticles before and after 20 minutes of experimentally-induced oscillatory shear stress. COPD patients performed this intervention a second time following a 20-minute wash in period of low flow supplemental oxygen to normalize arterial oxygen saturation. COPD patients had ~six-fold greater baseline retrograde shear rate (P < 0.05) and lower FMD (P < 0.05). The oscillatory shear stress intervention induced significant decreases in brachial artery FMD of all groups (P < 0.05). Oscillatory shear stress elevated circulating markers of endothelial cell apoptosis (CD31+/CD41b− microparticles) in COPD patients, but not age-matched controls. Supplemental oxygen administration abrogated the oscillatory shear stress-induced increase in CD31+/CD41b− microparticles, and improved FMD after accounting for the shear stress stimulus. We have demonstrated that acutely disturbed blood flow with increased retrograde shear stress further deteriorates the already impaired endothelial function with attendant endothelial apoptosis in patients with moderate-severe COPD. Patients with COPD present with elevated levels of circulating endothelial-derived microparticles (EMPs) and impaired flow-mediated dilation (FMD), indicating a pro-atherogenic endothelial cell phenotype; however, whether the endothelium of patients with COPD remains sensitive to oscillatory shear stress-induced dysfunction remains unknown.Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the lungs that exerts complications systemically, highlighted by an increased risk of cardiovascular disease. Accordingly, the main cause of death among these patients is cardiovascular events8. Studies in young, healthy humans indicate that 20–30 minutes of experimentally-induced oscillatory shear stress increases EMP markers of endothelial cell activation and apoptosis, and decreases FMD10. The influence of disturbed hemodynamics on endothelial function has never been assessed in patients with COPD. Further, acute hypoxemia increases circulating MPs, while superimposing oscillatory shear stress impairs FMD in young, healthy humans11. Hypoxemia is a characteristic of moderate-severe COPD; however, the potential link between chronic hypoxemia and EMPs in COPD has yet to be investigated.The magnitude and direction of shear stress exerted upon the endothelium mediates gene expression; laminar, pulsatile shear stress induces a quiescent endothelial cell phenotype that is anti-inflammatory, vasodilatory, and antithrombotic, whereas oscillatory, low time-averaged mean shear stress is pro-atherogenicThe primary aims of this study were two-fold: 1) to test whether oscillatory shear stress further exacerbates endothelial dysfunction in patients with COPD compared to age-matched controls, and 2) to test whether low flow oxygen administration, to alleviate hypoxemia, improves endothelial function and is protective against oscillatory shear stress-induced endothelial dysfunction in patients with COPD.Characteristics of the study population are given in Table Baseline systolic, diastolic and mean blood pressures were not different between the two interventions, or between the two groups Table .Table 2B2 reversal ( P< 0.05) , with OSI increasing in all groups following cuff inflation (P < 0.01). At rest, FMD was 36% lower in the COPD patients than in controls . Mean arterial pressure (MAP) as a covariate had no effects on baseline FMD . The covariates ratio of forced expiratory volume in the first second and forced vital capacity (FEV1/FVC ratio) and oxygen saturation (SaO2) had significant effects on baseline FMD . The retrograde intervention induced significant decreases in brachial artery FMD (absolute terms) and FMD% (relative terms) in the experimental arm of all groups, including COPD following O2 normalization ( P< 0.05)  and SaO2 . MAP and FEV1/FVC ratio had no significant effects on the FMD response to low flow oxygen supplementation in COPD patients , but SaO2 did have a significant effect . In COPD patients a 15% decrease in FMD% due to oscillatory shear stimulus during breathing room air and 12% during breathing O2 had moderate effect size . The decrease in FMD% of about 37% in controls had an effect size of 1.69. There was a moderate correlation between baseline FMD and retrograde shear in COPD patients  but no correlation was observed in controls . Further, after inclusion of baseline diameter and SRAUC as covariates, FMD was greater during O2 normalization than room air breathing in patients with COPD .Pre-intervention, 10-minute, and 20-minute shear rate patterns were significantly different between COPD and controls, with COPD patients displaying greater retrograde shear rate both before and during O2 normalization. There was no correlation between the change in CD31+/41− EMPs and FMD in the COPD patients – for both room air and O2 breathing. There were no changes in MP levels in the control arm of COPD patients. In control subjects there was no impact of retrograde shear stress (P > 0.05) or experimental vs. control arm (P > 0.05) on any MP sub-type levels.Plasma MP changes for the experimental arm in COPD patients are shown in Fig. 2 abrogates the oscillatory shear stress-induced increase in CD31+/41b− and improves endothelium-dependent vasodilation for a given stimulus. However, the mechanisms underlying these changes seem to be different in COPD patients compared to age-matched controls. In patients with moderate-severe COPD, the oscillatory shear stress induces increases in circulating markers associated with endothelial apoptosis that is paralleled by a modest attenuation of endothelial function (measured via FMD). Moreover, at rest the lower FMD of COPD patients compared to controls is accompanied by a correspondingly elevated baseline retrograde shear rate. This indicates that disturbed blood flow may contribute to increased cardiovascular risk in this population by exacerbating endothelial dysfunction12. In apparently healthy older adults, a large attenuation in FMD following oscillatory shear stress is not coupled with an increase in CD31+/41b− MP, suggesting mechanisms unrelated to EMP release or clearance. Collectively, these data for the first time provide in vivo evidence that links the detrimental role of pro-atherogenic events  with endothelial dysfunction in moderate-severe COPD patients.The main novel findings of this study are: (1) in patients with moderate-severe COPD, acute increases in retrograde shear stress and/or reductions in mean shear stress further deteriorate the already impaired endothelial function with attendant endothelial apoptosis, and (2) that supplemental O13. Given its presence in COPD patients, the main question of this study was if disturbed blood flow, which is a pro-atherogenic stimulus, would further deteriorate endothelial function14. We have shown that acute periods of oscillatory shear stress lead to further endothelial damage, decreasing brachial FMD while elevating CD31+/CD41b− MP, which is indicative of endothelial apoptosis. Although all measures have been undertaken to limit bias based upon image interpretation/discrimination  the delta of less than 1% in FMD reduction in COPD patients following the acute intervention of retrograde shear stress manipulations requires discussion of data with caution. However, this small reduction in FMD may be quite important, as it occurred in spite of previous data demonstrating that individuals with a priori low FMD do not undergo a further reduction in FMD following a retrograde shear stress stimulus15. This may reflect an augmented susceptibility to endothelial damage/dysfunction in COPD, suggesting that despite COPD patients having dysfunctional endothelium are still susceptible to further deterioration. Interestingly, an increase in circulating CD31+/CD41b− expressing MP and decrease in FMD manifests during COPD exacerbations16, suggesting a potential mechanism linking the increased risk of cardiovascular event following exacerbation1. Further, although a notably small change, it may also carry a significant weight with regards to cardiovascular risk17.Endothelial dysfunction is regarded as an early manifestation of atherogenic events and vascular injurya priori reduced endothelial function, COPD patients in our study had higher resting retrograde shear rate as compared to healthy controls. To our knowledge this is the first study to examine shear patterns and retrograde shear rate in moderate-severe COPD patients. A laminar, pulsatile shear pattern in conduit arteries mainly presents as blood flow in the anterograde direction but increased retrograde flow patterns may manifest from increased vascular tone in the downstream resistance vessel18. This is commonplace in advanced age, obesity, and hypertension20. We have now demonstrated that retrograde shear rate may contribute to endothelial dysfunction in patients with COPD.Concurrent to the et al.15. One possible explanation could be the presence of a threshold shear rate stimulus, which has dose-dependent effects on reducing FMD%9. This has implications considering the protocol implemented by Schreuder et al. did not use cuff inflation above 60 mmHg, nor did they employ a second cuff on the upper arm15. In a control experiment, Jenkins et al. found that the proximal 40 mmHg did not produce changes in MP, but there were slight increases in retrograde shear rate10, which may have contributed to the reduction in FMD observed in the current study. In control subjects, the 20-minute retrograde shear stress intervention did not cause any significant endothelial damage (no increase in MP) yet there was a large decrease in the subsequent FMD. Therefore, while retrograde shear stress impairs the endothelium in COPD patients at the cellular and functional level, other mechanisms downstream of retrograde shear stress manipulation must impair FMD in healthy controls, such as a decrease in endothelial NO synthase expression21 and/or elevated oxidative stress22.We have observed a decrease in FMD% in apparently healthy aged controls after generating retrograde shear stress that is not in accordance to previous findings of Schreuder 10, which lead to endothelial cell apoptosis  but not activation .The retrograde shear stress intervention induced elevations in markers of endothelial cell apoptosis, CD31+/41b− MP. Unlike the Jenkins study, where complete distal occlusion (220 mmHg) was applied to increase retrograde shear stress, we used only 75 mmHg21. However, in this study we did not observe shear stress-induced elevations of MPs bearing markers from leukocytes, neutrophils, or platelet-endothelial adhesion molecules in COPD patients.The specific cellular mechanisms by which systemic inflammation plays an important role in the pathogenesis of cardiovascular disease in COPD are complex. The atherosclerotic process starts with the injury of the vascular endothelium that is even more exposed to damage in the presence of increased retrograde shear stress . Oscillatory shear stress with elevations in retrograde shear showed increased expression of adhesion molecules and elevated endothelin-1 expression, which would allow circulating leukocytes to adhere to the damaged endothelial surface23 and sympathetic outflow to vascular beds24, we reasoned that normalization of oxygen saturation would reduce sympathetic outflow24 and alleviate the FMD impairments associated with hypoxia-mediated sympathoexcitation25. A previous investigation demonstrated that in healthy individuals sympathetic outflow remains elevated for ≥20-minutes following 20 minutes of moderate (80% SaO2) hypoxia26, however, this elevation in sympathetic outflow is mitigated with hyperoxia24. Despite this rationale for low-flow oxygen therapy, the authors are fully aware that correcting hypoxemia with 20-minutes of supplemental oxygen is not likely to fully reverse the vascular effects of chronic  hypoxemia. However, short-term application (20 minutes) of nasal oxygen has previously been demonstrated to elicit a modest decrease in muscle sympathetic nerve activity27. We found no related influence on the retrograde shear pattern and we observed that FMD at rest was unchanged despite a smaller stimulus, which may suggest some improvement in endothelial function. Another consideration is that endothelial dysfunction is initiated early in COPD when airflow obstruction is mild to moderate but arterial hypoxemia is absent, indicating that hypoxia may not be directly related to the observed reductions in FMD in moderate-severe COPD patients, however, our investigation is the first to explore this role in moderate-severe patients. In agreement with the present study, Dinh-Xuan et al. demonstrated correlation, albeit not causation, of impaired endothelium-dependent dilation and reduced PaO2 values in vitro, suggesting that chronic hypoxemia contributes to the impairment in endothelial function in COPD patients28. Of note, oxidative stress has been implicated as a contributor to endothelial dysfunction in COPD patients29; however, 20-minutes of oxygen therapy does not change plasma levels of oxidative and inflammatory markers30. Therefore, it is unlikely the changes observed in the current study are due to altered redox balance.Administration of low-flow supplemental oxygen appeared to improve FMD after covarying for changes in SRAUC in COPD patients. As hypoxia increases carotid body output8. Thus, in patients with COPD, we propose that hypoxemia contributes to endothelial dysfunction by promoting endothelial dysfunction that can be amplified by oscillatory shear stress. After normalizing FMD for baseline diameter and SRAUC, endothelial function was significantly improved during O2 administration in patients with COPD. The improvement in FMD may implicate a role for hypoxia in restricting endothelial sensitivity to increases in shear stress, supporting previous in vitro findings28. Thus, it appears as though both mechanical (oscillatory shear stress) and chemical (hypoxia) insults may contribute to and worsen endothelial dysfunction in patients with COPD.It is intriguing that the oscillatory shear stress-induced elevation in CD31+/41b− was abolished in the oxygen normalization trial  oxygen to achieve a SaO2 of 98% in our COPD patients. This allowed for comparisons with controls who posses a SaO2 of 98% at rest. As controls were already normoxic, no intervention was necessary to “restore” an already intact peripheral oxyhemoglobin saturation. Indeed, superimposing oxygen supplementation onto a healthy SaO2 (~98%) can lead to multiple physiological consequences such as altered sympatheticsMethodological considerations. Clinically, the aim of oxygen therapy is to increase PaO34, oxidative stress35, nitric oxide bioavailability36, and peripheral vascular function37. This would have rendered a comparison between O2 supplemented COPD patients and O2 supplemented controls highly confounded, which rationalized the exclusion of such a trial from the present study.In COPD endothelial dysfunction presents alongside elevated levels of resting retrograde shear rate. Acutely disturbed blood flow characterized by an oscillatory shear stress pattern deteriorates endothelial function in both patients and controls, however in moderate-severe COPD patients such dysfunction is compounded with attendant increases in circulating MPs indicative of endothelial cell apoptosis, and is of greater potential consequence given the already impaired vasculature of this population. Administration of oxygen abolishes the oscillatory shear stress-induced increase in circulating MP, and appears to improve FMD for a given stimulus. Examining whether eliminating retrograde shear and augmenting mean shear (i.e. via limb heating) improves endothelial function in patients with COPD may provide additional support that disturbed blood flow contributes to endothelial dysfunction in this population.2 in a concentration higher than 90% with a flow 1–2 L/min. Inclusion criteria for patients were defined as moderate-to-severe airflow obstruction on post-bronchodilator spirometry and arterial oxyhemoglobin saturation (SaO2) below 90% at rest. COPD was diagnosed and classified according to the Global Initiative for Obstructive Lung Disease (GOLD). All procedures were performed in accordance with the Declaration of Helsinki and the study was approved by the Ethics Committee of the University of Split School of Medicine. Written, informed consent was obtained from all participants. The study was registered at ClinicalTrials.gov under NCT03004352 identifier on December 22nd, 2016. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.The study population consisted of 17 patients (69 ± 8 years) with stable COPD (no exacerbations in the previous six weeks), and 10 age-matched non-smoking control subjects without airflow obstruction (65 ± 7 years). The 14 patients who were on home oxygen therapy used stationary oxygen concentrators that deliver Oet al.10 was modified to induce retrograde flow within the experimental arm lasting for 20 minutes. Briefly, a pneumatic cuff placed immediately distal to the epicondyles was inflated to 75 mmHg to provoke low time-averaged shear stress with a high retrograde component. A second pneumatic cuff was placed close to the axilla and inflated to 40 mmHg to facilitate trapping of circulating MPs. The contralateral (control) arm was cuff-free during this intervention. Measures of brachial artery shear rate were taken 10 and 20 minutes into the intervention and blood samples were taken from the experimental and control arms just before cuff deflation. Following a five-minute recovery after cuff release another FMD measurement was made.Prior to their arrival at the laboratory, participants were instructed to abstain from exercise, alcohol and caffeine for a minimum of 12 hours and were fasted for 4 hours prior to the testing session. Current smokers had their last cigarette ≥8 hours before the measurements. Patients and controls remained on their prescribed medication during the study. All experimental measures took place in a quiet, temperature-controlled (20–22 °C) room in the Laboratory for Integrative Physiology, University of Split School of Medicine. Venous catheters were placed in the superficial antecubital vein of the right  and left (control) arms while participants were rested supine. Following 20-minutes rest, blood samples were taken from both arms, and measures of brachial artery hemodynamics and FMD were acquired. Immediately after this assessment, an occlusion model adopted by Jenkins 2). The normalized arterial oxygen saturation was maintained throughout the second set of tests. Control subjects were tested only while breathing room air.COPD patients underwent the testing as outlined at rest breathing room air and following 20-minutes of low flow supplemental oxygen (100%) administered through a nasal cannula in order to normalize arterial oxygen saturation  with a 10-MHz multifrequency linear array probe. Images of the brachial artery were acquired about 5 cm proximal to the antecubital fossa. Anterograde and retrograde shear rate  was calculated from anterograde and retrograde area under the curve data that were subsequently averaged from positive and negative data points, respectively.Brachial artery blood velocity and diameter were measured using duplex ultrasound , and the same experienced ultrasonographer (OFB) was involved in each assessment for a given subject. The same custom-designed edge-detection and wall-tracking software as used for the Brachial Artery Blood Flow Measures was utilised for offline measurements of baseline diameter, blood flow, peak diameter and SR. Peak diameter after cuff deflation was automatically detected according to an algorithm that identified the maximum bracket of data subsequent to a moving window-smoothing function39. The time to peak diameter (s) was calculated from the point of cuff deflation to the maximum post-deflation diameter39. Diameter data are presented as absolute  and relative  rise of peak diameter from the preceding baseline diameter. In accordance with procedural recommendations40, we also measured the post-deflation area under curve for the shear rate response in order to best interpret any changes in FMD.Endothelium-dependent vasodilator function of the brachial artery of the experimental arm was assessed before and after each retrograde shear stress intervention according to international guidelines42. Microparticle characteristics remain unchanged when samples stored at either 4 °C or at room temperature are processed in a time span of 3 weeks from time of collection42. The blood was centrifuged for 5 minutes at 1500 g, the supernatant was made 12.5 mM EDTA and then centrifuged at 15000 g for 30 minutes. Aliquots of the 15,000 g supernatant were stained with antibodies for MPs analysis by flow cytometry. Endothelial microparticles (EMP) were defined as those that bound annexin V and expressed CD31  but were negative for CD41 , and those that bound annexin V and positive for CD62E . Other MPs were characterized as those binding annexin V and expressing CD41, CD142 (tissue factor), CD66b , and von Willebrand factor (vWF). Flow cytometry analysis of MPs was performed with an 8-color, triple laser MACSQuant  using the manufacturers’ acquisition software. All reagents and solutions used for MPs analysis were sterile and filtered (0.2 μm filter). MPs were stained with annexin V and analyzed as previously described, including micro-beads with diameters of 0.3 μm , 1.0 μm, and 3.0 μm  to assess the size of particles42. Surface markers were evaluated with use of the “Fluorescence-Minus-One Control Test”. We define MPs as annexin V-positive particles with diameters up to 1 µm as previously described42. In previous analyses the variability between repeated measures in the same samples was below 5%. We have also run a control sample to assure we use the correct gating protocol and a suspension of polystyrene fluorescent beads to be sure the particle counts are correct.Blood samples were obtained into 8 mL Cyto-chex tubes , coded and sent by express mail to the University of Maryland where all analyses were performed within 48 hours after arrival, blinded to the laboratory, as described in detail elsewhereth and 20th minute of intervention).All data are reported as mean ± SD with statistical significance set at P < 0.05. For shear rate analysis, we used a 3-way ANOVA to determine the difference between COPD patients and control subjects, room air and 100% oxygen condition and the impact of cuff inflation on shear rate  in COPD subjects was assessed using a 2-way repeated measures ANOVA (factors: room air vs. oxygen breathing and pre vs. post retrograde shear). The impact of elevated retrograde shear stress and oxygen normalization on MPs , and MPs (control arm) were assessed using a Friedman repeated measures ANOVA on ranks. Post-hoc t–tests were performed if a main effect was found using Tukey’s HSD correction for multiple comparisons. The relationship between baseline FMD and baseline retrograde shear in patients and controls was tested with Pearson’s correlation test.2 administration) in COPD patients with logarithmically-transformed baseline diameter and shear rate area under the curve (SRAUC) as covariates. Post-hoc t–tests were performed if a main or interaction effect was found using a Bonferroni correction for multiple comparisons. The effects of mean arterial pressure (MAP), oxygen saturation (SaO2) and the ratio of forced expiratory volume in the first second and forced vital capacity (FEV1/FVC ratio) on baseline FMD, FMD response to oscillatory shear and low flow oxygen supplementation were tested as covariates. Cohen’s d was used to compute the effect size of the difference between the two population means.The impact of retrograde shear stress on FMD in control subjects breathing room air was assessed using a two-tailed paired t-test. The impact of retrograde shear stress on MP levels in controls was analysed using a 2-way mixed design ANOVA . To account for stimulus differences, FMD was compared using a 2-way ANCOVA (pre- and post-intervention during room air and O


Naturally occurring antibodies against amyloid-β peptides have been found in human cerebrospinal fluid and in the plasma of healthy individuals, but were significantly lower in Alzheimer's disease (AD) patients, suggesting that AD may be an immunodeficient disorder. The performance of anti-amyloid-β antibodies in transgenic mice models of AD showed that they are delivered to the central nervous system, preventing and dissolving amyloid-β plaques. Moreover, these antibodies protected the mice from learning and age-related memory deficits. Active and/or passive immunization against the amyloid-β peptide has been proposed as a method for preventing and/or treating AD. Immunotherapy represents fascinating ways to test the amyloid hypothesis and offers genuine opportunities for AD treatment, but requires careful antigen and antibody selection to maximize efficacy and minimize adverse events.


Increasing rice yield with fewer external inputs is critical to ensuring food security, reducing environmental costs, and improving returns. Use of hybrid rice has expanded greatly in China due to its higher yield potential. Meanwhile, large and increasing amounts of nitrogen (N) fertilizers have been used for expanding rice production in China. It is not clear to what extent the success of hybrid rice in China is associated with N fertilizer inputs.We observed that the higher grain yield with N fertilizer in hybrid rice was driven more by a higher yield without N fertilizer than by increases in grain yield with N fertilizer under moderate to high soil fertility conditions.Our results suggest that greater application of N fertilizers is not needed to benefit from hybrid rice production under moderate to high soil fertility conditions, and that improving and maintaining soil fertility should be a focus for sustaining hybrid rice production. Moreover, our study also indicates that zero-N testing may be a potentially useful tool to develop hybrid rice with high yield and without requirement of greater external N inputs under moderate to high soil fertility conditions.The online version of this article (10.1186/s12284-017-0182-1) contains supplementary material, which is available to authorized users. Rice is a staple food for over half of the world’s population , has imposed substantial environmental costs, including increased greenhouse gas emissions Davidson , enhanceThe development of hybrid cultivars has made a major contribution to increasing the yield potential of rice crops Yuan . Hybrid −1, about 75% higher than the world average  with the grain yield obtained without N fertilizer (Y0), also comparing differences between hybrid and inbred rice cultivars in terms of the increases in grain yield attained with N fertilizer (∆YN). Then, in another field experiment , we evaluated the trends in YN, Y0 and ∆YN in representative hybrid rice cultivars selected to represent different phases of hybrid development since 1996.To address the question, two field experiments were conducted during 2012 to 2016  and Y-liangyou 1 (YLY1) was 22% and 16% higher than the YN for the inbred cultivars Huanghuangzhan (HHZ) and Yuxiangyouzhan (YXYZ) in 2012 and 2013, respectively Additional file 2:Table S1.Grain yield in rice cultivars grown under two N fertilizer rates in Xingyi, Guizhou Province, China in 2012–2014 (Experiment I), and in Ningxiang, Hunan Province, China in 2015 and 2016 (Experiment II). (DOC 53 kb)Additional file 3:Table S2.Indigenous soil N uptake and labeled-N uptake in rice cultivars in a micro-plot experiment with application of 15 N labeled urea in Changsha, Hunan Province in China in 2013. (DOC 31 kb)


Neonatal nephron loss may follow hypoxic-ischemic events or nephrotoxic medications. Its long-term effects on the kidney are still unclear. Unlike term infants, preterm neonates less than 36 weeks gestational age show ongoing nephrogenesis. We hypothesized that nephron loss during nephrogenesis leads to more severe renal sequelae than nephron loss shortly after the completion of nephrogenesis. Rats show nephrogenesis until day 10 of life resembling the situation of preterm infants. Animals were uninephrectomized at day 1 (UNX d1) resulting in nephron reduction during nephrogenesis and at day 14 of life (UNX d14) inducing nephron loss after the completion of nephrogenesis. 28 days after uninephrectomy the compensatory renal growth was higher in UNX d1 compared to UNX d14. Nephrin was reduced and collagen deposition increased in UNX d1. At 1 year of age, glomerulosclerosis and markers of tubulointerstitial damage were most prevalent in UNX d1. Moreover, the number of desmin-positive podocytes was higher and nephrin was reduced in UNX d1 indicating podocyte damage. Infiltration of inflammatory cells was heightened after UNX d1. Uninephrectomized animals showed no arterial hypertension. We conclude that neonatal nephron loss during active nephrogenesis leads to more severe glomerular and tubulointerstitial damage, which is not a consequence of compensatory arterial hypertension. Moreover it could be shown that nephron number at birth and the development of chronic kidney disease as well as cardiovascular disease later in life correlate inversely5. In this context Brenner et al. postulated that in individuals with a reduced nephron number sufficient glomerular ultrafiltration is achieved by compensatory glomerular hyperperfusion based on secondary arterial hypertension6. In the long term this chronic mechanical burden leads to manifest glomerular and tubulointerstitial damage. Chronic arterial hypertension in turn induces pathologies of the cardiovascular system and the kidneys8. On the other hand, a reduced nephron number does not necessarily result in hypertension9. Thus, the interdependency of reduced renal mass, the development of hypertension and renal damage still remains unclear.Low nephron number is a risk factor for an adverse outcome after renal damage. Clinical and animal studies revealed that an early loss of nephrons during the neonatal period is particularly detrimental to renal outcome10. In contrast to term infants, preterm infants prior to 36 weeks of gestation reveal a still ongoing nephrogenesis11. At the same time, these individuals are highly susceptible to renal injury and concomitant nephron loss: Moreover, preterm infants more frequently suffer from neonatal morbidities like neonatal sepsis or patent ductus arteriosus. Acute kidney injury and nephron loss may occur during hypoxic-ischemic events as a consequence of cardiocirculatory decompensation or as side-effects of necessary medications  frequently resulting in a reduced number of functional nephrons12.Preterm birth is associated with a higher morbidity and mortality due to the immaturity and higher vulnerability of organs13. Uninephrectomy guarantees a defined nephron reduction and induces acute and chronic compensatory mechanisms in the remaining kidney. In this study we tested the hypothesis that acute nephron loss during active nephrogenesis leads to different adaptive changes in the contralateral kidney compared to a nephron loss immediately after termination of nephrogenesis. We further hypothesized that a nephron loss during ongoing nephrogenesis might predispose to more severe renal damage later in life.In rats a physiologically active nephrogenesis persists until day 10 of life, resembling the renal situation of preterm infantsIn an animal model of neonatal unilateral nephrectomy (UNX) at day 1 (ongoing nephrogenesis) and at day 14 (nephrogenesis terminated) of life, renal alterations were studied 4 weeks after UNX intervention and at 1 year of age.Body weights and heart weights were not influenced by UNX Table . Four weAt 52 weeks of age body weights were not significantly different between controls, UNX d1 and UNX d14 animals  expression) did not reveal significant differences in the kidneys of UNX d1 and UNX d14 , or the use of different rat strains (Wistar vs. Sprague Dawley). Sprague Dawley rats were found to be more likely to develop hypertension than Wistar rats15. Woods et al. observed an age-dependent increase of arterial hypertension in animals after neonatal uninephrectomy preceding renal damage4. Similar findings of increased blood pressure were made in an ovine model of nephron loss during renal development: Female and male sheep developed higher basal blood pressure after fetal uninephrectomy compared to controls and subsequently a decrease in renal function19. This would argue for different underlying pathomechanisms compared to our study .The main finding of this study is that neonatal nephron loss during ongoing nephrogenesis results in more severe glomerular and tubulointersitial damage than nephron loss after termination of nephrogenesis. These renal changes are not a consequence of arterial hypertension. This is in contrast to the findings of Woods 21. On the other hand, there are also several clinical studies, which describe an increased risk for the development of hypertension in children with a solitary kidney22 or in kidney donors23. The reasons for the discrepancies in study outcomes regarding blood pressure are still unclear, but might reflect different study designs or differences in the selection of the study collective.There are conflicting data concerning the development of hypertension in humans with reduced renal mass: Our observations of arterial normotension after early uninephrectomy are in line with several clinical studies showing no significantly increased incidence of arterial hypertension in individuals born with a solitary kidney or in kidney donorset al., who described no detectable de novo nephrogenesis in mice after neonatal uninephrectomy24. Therefore, we do not believe that differences in nephron numbers can account for the observed differences in renal outcome in our experimental groups. In contrast, Douglas-Denton et al. found a significant increase of nephron number after fetal uninephrectomy in the ovine fetus25. Different effects of uninephrectomy might not only be due to species differences, but also to the difference between intrauterine/prenatal vs. extrauterine/postnatal conditions. Nevertheless, we also detected early renal adaptations after uninephrectomy: In both uninephrectomized groups glomerular size and cell number increased. An increase in glomerular size is a common feature in many models of reduced renal mass27. Acute nephron reduction, e.g. uninephrectomy, commonly results in compensatory renal growth26. Compensatory renal growth was, however, most prominent in rats after uninephrectomy during ongoing nephrogenesis. An influence of age on compensatory renal growth was described in several studies. Uninephrectomy at a young age resulted in more compensatory renal growth than uninephrectomy at an older age28. In our study, this compensatory renal growth was accompanied by increased glomerular collagen deposition and some loss of podocyte integrity only in rats uninephrectomized during ongoing nephrogenesis. This is in accordance with findings by Okuda et al.30 and Celsi et al.31, who described a declining incidence of glomerulosclerosis in rats with increasing age at the time of uninephrectomy.What might account for the differences in renal outcome of rats uninephrectomized during ongoing nephrogenesis versus rats uninephrectomized shortly after the completion of nephrogenesis? We do not have any evidence that uninephrectomy at day 1 of life results in a higher nephron number compared to uninephrectomy later in life  activity, however, was only minor and not associated with overt changes in the animals’ blood pressure. Reduced RAAS activity is a typical feature of the aging kidney42. Magro et al. described a decrease in plasma renin activity preceding glomerulosclerosis in aging rats43. Plasma renin activity was also reduced in long-term observations in sheep after fetal uninephrectomy44. This is consistent with our findings of a somewhat decreased plasma renin activity in aging rats after uninephrectomy during ongoing nephrogenesis, which develop more prominent glomerulosclerosis than rats uninephrectomized after completion of nephrogenesis.After uninephrectomy a dysregulation of the renin-angiotensin system is frequently observed: The activity of angiotensin converting enzyme (ACE) was significantly augmented in rats after uninephrectomy compared to controls, which indicates that activation of RAS might contribute to the maintenance of the homeostasis of glomerular filtration rate after nephron reduction18.Finally, as cardiovascular and renal outcome is known to be sex-dependent, a limitation of our study has to be mentioned: Our study is limited in that we only studied male individuals and therefore cannot appraise the effects of early nephron loss in females, which might differ from the effects seen in males, as reviewed by Lankadeva and colleaguesTaken together, our data suggests that nephron loss during ongoing nephrogenesis results in glomerular and tubulointerstitial damage in the absence of arterial hypertension later in life. In contrast, a nephron loss after completion of nephrogenesis results in more subtle renal alterations. Our animal model of uninephrectomy during ongoing nephrogenesis or after completion of nephrogenesis reflects the situation of an acute nephron loss in human preterm or term neonates, respectively. Thus, preterm neonates who suffer from medication-induced nephron loss during their neonatal period might be of a particular risk to develop renal disease later in life. Our observations highlight the urgent need to avoid neonatal renal damage with acute nephron loss in preterm infants and may help to develop adapted procedures of prevention and aftercare for this patient cohort.46.All procedures performed on animals were approved by the local government authorities  and were done in compliance with the DIRECTIVE 2010/63/EU of the European Parliament. Pregnant female Wistar rats were kept on a standard diet  with free access to tap water in a room maintained at 22 ± 2 °C with a 12 h dark/light cycle. After spontaneous delivery male pups were divided into two groups: Neonatal left sided uninephrectomy (UNX) was performed on day 1 (UNX d1) and day 14 (UNX d14) of life under isoflurane anesthesia. More detailed information on the periinterventional and surgical procedures is available in the supplementary information . Histological analyses were performed after 4 weeks of recovery  and at the age of one year . For each group, animals were used from 4 litters, with UNX and controls deriving from the same litters. Controls of the 1 year time point were combined (4 controls for UNX d1 and 4 controls for UNX d14) as they did not differ in their outcome. Animals of each group obtained the standard rat chow . One week before sacrifice, the rats were kept in metabolic cages for 24 hours to collect urine. Blood samples were obtained at sacrifice after isoflurane anesthesia. Rats were sacrificed and kidneys and adrenal glands were weighed. For kidney analyses, tissue samples were prepared for histology, RNA isolation and immunohistochemistry as describedBlood pressure values were obtained by intra-arterial blood pressure measurements. At the day of sacrifice, rats were anesthetized with isoflurane. Catheters were implanted in the right femoral artery and tunneled subcutaneously. After a recovery phase of 2 hours mean arterial blood pressure was recorded by a polygraph  in conscious animals for 30 minutes.46 In short, Score 0 reflects an unaffected glomerulus, score 1 mesangial expansion or sclerosis involving <25% of the glomerular tuft; score 2 equals a sclerosis 25–50%; score 3 represents a sclerosis 50–75%, and/or segmental extracapillary fibrosis or proliferation; score 4 indicates a global sclerosis >75% or global extracapillary fibrosis of proliferation, or complete collapse of the glomerular tuft. All histological evaluations were performed by an investigator blinded to the group assignment.Glomerular cell number and glomerulosclerosis were assessed in kidney sections stained with periodic acid-Schiff’s (PAS) reagent. For glomerular cell number, nuclei were counted in 50 glomeruli per renal cross section. Quantification of glomerulosclerosis was performed by using a semiquantitative score of of 0 to 4 as described beforeSerum cholesterol, triglycerides, creatinine and urea were measured using an automatic analyser Integra 800 . Plasma renin activity was measured with a commercially available radioimmunoassay kit .2O2. Primary antibodies were incubated overnight at the following dilutions: Rat endothelial cell antigen  in a dilution of 1:20. Proliferating cell nuclear antigen (PCNA) was used for detection of proliferating cells  1:50, ED-1 for macrophages  1:50, CD3 for T-cells  1:300, CD8a for cytotoxic T-cells  1:50. Morevover the following antibodies were used at the respective dilutions: Collagen IV  1:500, α-SMA  1:50, desmin (DAKO) 1:50, renin  1:100, nephrin  1:50, WT-1  1:50. Appropriate secondary antibodies (Vector) were diluted 1:500, before avidin D peroxidase (Vector) was applied at a dilution of 1:2000. Finally DAB (Vector) was added, sections were counterstained with hematoxylin and covered with entellan.For immunohistochemical staining kidneys were fixed in methyl carnoy’s solution and embedded in paraffin. 2 µm sections were stained as described below. Sections were blocked with 3% HWT-1 and PCNA-positive glomerular cells were counted in 50 glomeruli/renal section. RECA-positive capillaries were counted in 20 non-overlapping high-power cortical views. Interstitial smooth muscle actin-positive cells (activated fibroblasts), CD3-positive cells (T-cells), CD8a-positive cells (cytotoxic T-cells) and ED-1-positive cells (macrophages) were counted in 5 non-overlapping medium-power cortical views and presented as positive cells/cortical view. Renin-positive juxtaglomerular apparatus were counted in renal sections and were expressed as % of the total number of glomeruli in a renal section. Glomerular collagen IV expression was quantified in 20 glomeruli/renal section, as a ratio of stained area/total area of glomerular cross sections, using MetaVue software  software.47: Score 0: absent staining or staining less than 5% of the assessed area; Score I: 5–25% stained area; Score II: 25–50% stained area; Score III: 50–75% stained area; Score IV: > 75% stained area. Glomerular desmin expression was quantified in 20 glomeruli/renal section, as a ratio of stained area/total area of glomerular cross sections, using MetaVue software. All immunohistochemical evaluations were done in a blinded manner.Glomerular desmin staining in podocytes was assessed using a semiquantitative scoring system as described2O. Subsequently, real-time PCR was accomplished with an ABI PRISM 7000 Sequence Detector System and SYBR Green (Applied Biosystems) or TaqMan reagents (Applied Biosystems) according to the manufacturer’s protocol. The relative amount of the specific mRNA was normalized with respect to 18S rRNA. See supplementary information  was homogenized in 500 µl RLT buffer reagent  with an Ultraturrax for 30 s and total RNA was extracted with RNeasy® Mini columns (Qiagen) according to the manufacturer’s instructions. TaqMan reverse transcription reagents  with random hexamers as primers were used to obtain first-strand cDNA. Final RNA concentration in the reaction mixture was adjusted to 0.1 ng/µl. To test for genomic DNA contamination, reactions without Multiscribe reverse transcriptase were performed as negative controls. Reverse transcription products were diluted 1:1 with dH0 mg was post hoc test or, where appropriate, Kruskal-Wallis, followed by Dunn’s test were performed to assess significant differences between the groups using the GraphPad Prism software . Results were considered significant at p < 0.05.Data are expressed as mean ± standard error of the mean (SEM). Student’s t-test was used to assess differences between UNX and their age-matched controls at the four weeks time point. To assess differences between UNX d1, UNX d14 and controls of the 52 weeks time point, first normality was tested using the Shapiro-Wilk test. One-way analysis of variance (one-way ANOVA), followed by Bonferroni The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information