P = 0.04). We also observed that a higher proportion of histologic grade 1 and 2 cases had high functional impact variants in the SCYL2 gene compared with grade 3 cases. Our findings indicate that individual breast cancers harbor a substantial number of potentially functionally important nucleotide variations in kinase genes, most of which are present in unique combinations and include both somatic and germline functional variants.Kinases play a key role in cancer biology, and serve as potential clinically useful targets for designing cancer therapies. We examined nucleic acid variations in the human kinome and several known cancer-related genes in breast cancer. DNA was extracted from fine needle biopsies of 73 primary breast cancers and 19 metastatic lesions. Targeted sequencing of 518 kinases and 68 additional cancer related genes was performed using the SOLiD sequencing platform. We detected 1561 unique, non-synonymous variants in kinase genes in the 92 cases, and 74 unique variants in 43 kinases that were predicted to have major functional impact on the protein. Three kinase groups—CMGC, STE and TKL—showed greater mutational load in metastatic compared to primary cancer samples, however, after correction for multiple testing the difference was significant only for the TKL group ( The kinome refers to a collection of 518 kinases encoded in the human genome that correspond to approximately 1.7% of the human protein-coding genesNumerous direct and indirect functional interactions exist between kinases and inhibition of a particular kinase may be rendered therapeutically ineffective by compensatory activation of other kinases346Several studies described a variable pattern of kinome gene expression in breast cancer and suggested that kinase expression patterns have prognostic value and could also be used to guide new therapeutic strategies91010The goal of this study was to assess the type and prevalence of potentially functional nucleotide variants (NVs) in the kinome of human breast cancers. We examined if the variants with potential functional impact are uniformly distributed across the kinome or cluster in certain kinase families, and assessed the correlation between variants and different breast cancer subtypes. We did not exclude known, germ line, single nucleotide polymorphisms (SNP) from our analysis because we hypothesized that functional interactions may exist between germ line and somatic events in the kinome. The current study examines the kinase mutation landscape of breast cancer at substantially greater sequencing depth than previous studies, in particular the The Cancer Genome Atlas (TCGA) effortCancer biopsies from 73 patients with primary stage I-III breast cancer who subsequently received neoadjuvant chemotherapy  and 19 patients with metastatic disease were included in this study. Biopsies were obtained prospectively for biomarker and genomic analysis of cancer in a study approved by the Institutional Review Board at the MD Anderson Cancer Center and all patients signed informed consent. All methods and experiments involving human subjects and tissues were performed in accordance with the institutional guidelines approved by the Yale University School of Medicine and the MD Anderson Cancer Center.For patients with stage I-III breast cancer, fine needle aspiration of the primary tumor was performed before neoadjuvant chemotherapy and pathologic response was available for all patients and were classified as either pathologic compete response (pCR) or residual cancer (RD). For patients with metastatic cancer, the most safely accessible lesion was biopsied, all patients had multiple prior lines of chemotherapy for metastatic cancer before the biopsy were obtained. Patient characteristics are presented in www.agilent.com/genomics/sureselect). SOLiD paired end sequencing kinome multiplexing libraries were constructed from the equal pooled, individual, barcoded kinome library. Batches of 16 barcoded, individual kinome libraries were pooled in equal molar ratio as one multiplexed library and sequenced in one session.For target capture and enrichment we used the Agilent SureSelect Human Kinome XT Kit and Target Enrichment system . The enrhttp://mutationassessor.org/)1615All sequenced paired end reads from each barcoded sample were processed by the Applied Biosystems BioScope™ software (version 1.3) using the diBayes and small.indel models to call single nucleotide variants (SNVs) and small insertions or deletions (indels). The reference genome used in this analysis was GRCh37 (hg19). We used ANNOVARWe used the Duplication Gene Database (DGD) to ensure that none of the HFI NVs we found were in co-located or duplicated genesStatistical analysis of the data generated by BioScope was carried out in statistical language RGene expression data used in this study were generated with Affymetrix HG-U133A gene chips as described before and were previously published (GSE20194)21http://kinase.com/human/kinome). We could map 428 of the 529 known kinases to 783 probe sets on the Affymetrix HG-U133A chip using Gene ID annotation . When multiple probe sets targeted the same gene, we retained only the probe set with the highest average expression value and greatest variance.Gene expression array data were normalized using MAS5 with median target array intensity of 600 using R/Bioconductorhttps://tcga-data.nci.nih.gov/docs/publications/brca_2012/)We selected all HER2-negative samples from the TCGA dataset that had mutation data available (56 ER-negative and 211 ER-positive). Datasets were downloaded from the TCGA breast carcinoma web site .We compiled a previously unpublished cohort of 470 breast cancer cases from Foundation Medicine, Inc. This cohort contained 185 metastatic breast cancer samples (39%). The mean age was 54.8 years (standard deviation 12.7) and 49 (10%) of the cancers were Approximately 40 million nucleotide reads were acquired per sample, of which 61% could be mapped to the targeted kinase coding regions, resulting in a mean nucleotide coverage in the targeted regions of 369x. The mean number of nucleotide variants per sample was 1052, ranging from 493 to 1536 in individual cases, 97% of which were already cataloged in dbSNP138 or COSMIC databases. The remaining 3% of the variants were novel. The median number of synonymous variants per sample was 273 (range 119–396) and of non-synonymous variants 153 (range 75–223) . The rawThe three different methods to predict functional importance yielded different numbers of functionally important variants for the same data, but each method predicted that only a small fraction of the detected variants are likely to be functionally important. In all, 3203 unique, non-synonymous variants were detected accross the 92 cases. Of these, 1561 (49%) had a high functional impact score by two or more functional predictors . FurtherN = 295).We examined the reproducibility of findings in replicate experiments. Aliquots of the same tumor DNA were sequenced twice in 17 of the samples. The overall concordance rate for all nucleic acid calls was very high ranging from 97 to 99%. However, concordance for variant calls was substantially lower ranging from 75% to 54%. Reproducibility was close to 80% with coverage ≥20x but it did not improve with further increasing coverage above 20x. For all subsequent analysis, therefore we used only variants that had >20 fold coverage and were predicted to be of high functional impact by 2 or more methods , and found no evidence for variant detection difference between samples in different sequencing batches. Categorizing the samples based on primary or metastatic tumor origin also yielded similar results, indicating that the number of HFI NVs per gene per sample was not influenced substantially by sequencing batch or sample type (i.e. primary versus metastasis) .The mean number of HFI variants per sample was 24 (range 5 to 40), with each tumor appearing to harbor a unique assortment of variants . These vP-unadjusted = 0.06). TESK2 is a dual specificity protein kinase that phosphorylates both serine/threonine and tyrosine residues and has not previously been implicated in cancer but plays a role in spermatogenesisThe top 10 genes by p-value, with the most frequent HFI variants, are presented in P-unadjusted = 0.04). Of note, SCYL2 is expressed in breast cancer and is involved with clathrin-dependent vesicle traffickingWe also tested for significant co-occurrence of any pair of known somatic (defined as present in COSMIC) and germ line variants (defined as present in dbSNP) using the Fisher Exact test but observed no significant co-occurrences or mutual exclusions. Classifying the patients according to PAM50, and the variants according to the resulting subtypes, resulted in few mutational load differences at a gene level between the different sub-types, none of which were statistically significant. The small sample size, however, limits the power of these analyses. Comparisons of gene level variant frequencies by response to neoadjuvant therapy and by histologic grade are summarized in P = 0.04). The TK (Tyrosine Kinase) group had a significantly lower mean variant burden in HER2-positive cases compared with triple negative (P = 0.01) and ER-positive cancers (P = 0.04) (When we compared HFI variant frequencies across the 10 kinase groups = 0.04) . The kinP = 0.009), confirming the results from our cohort  was present in 46% of the triple negative breast cancer (TNBC) cases compared to 27% of the ER-positive/HER2-negative (ER+) cases (r cohort . Also a P = 0.05) and a higher CNA load compared to primary tumors  had the highest mutation frequently in both primary (88%) and metastatic cancers (96%), followed by genes in the STE kinase group  which were mutated in 83% of the primary and 81% of the metastatic cancers. Genes in the CMCG group  had the most frequent copy number alterations in 86% of the primary and 65% of the metastatic cancers. However, neither difference was statistically significant. Interestingly, both primary and metastatic cancers had at least 1 mutation per patient on average in genes in the Atypical, STE and TK kinase groups , suggesty tumors . OverallUnderstanding the cancer kinome is of interest due to the important regulatory role of these molecules and their potential therapeutic role. The purpose of this analysis was to assess the functional variant landscape of the kinome in breast cancer. We hoped to identify novel high frequency recurrent variants that might represent potential new drug targets. This targeted sequencing study had higher coverage than previous reports including the TCGA; however, we could not identify any novel, high frequency variants. One important observation that we made is that the largest constituent of the high functional impact variants is germline polymorphisms already included in the dbSNP138 and 1000 Human Genomes databases.In addition to the already known TP53 and PI3K mutations, we detected frequent variants in BRCA1 (20%) and observed several predicted high impact SNVs in many MAPK family enzymes. Predicted HFI SNVs were not distributed evenly across disease subsets; SNVs in ULK4, BMP2K, PALB2, ALPK3 were more frequent in triple negative cancers (TNBC) whereas SNVs in EPHA2 was more common in ER positive cancers. For ER negative patients, we also observed variants in several genes that were not reported in TCGA, including TESK2, EIF2AK4 and CSNK1A1L(P = 0.04). This finding suggests a role for mutations in TKL kinases in cancer metastasis.Another novel implication of our study is the association between variants in SCYL2 with histologic grade, though the association remains weak and statistically insignificant after correction for multiple testing. We observed that a higher proportion of grade 1/2 cases had a functional variant in SCYL2 compared with grade 3 cases. SCYL2 regulates clathrin-dependent vesicle trafficking and contributes to cell migration and invasionOur findings suggest a paucity of shared, high frequency “driver mutations” in kinase genes in breast cancers. Each cancer appears to harbor an individual combination of genomic events that together may constitute the tumor “driver”. Many of the predicted HFI variants are known germ line polymorphisms, which raise the possibility that these may interact with, and could even determine what somatic mutations are functionally important for a given cancer. Depending on the germ line context, different somatic events may constitute cancer driver events in different individuals.Examining the presumed somatic (in COSMIC) and germline variants (in dbSNP) in individual patients suggests the possibility of functional interactions between the germline and somatic mutations. For example, patient M9  had a missense germline variant, rs45445894, in the Insulin-like growth factor 1 receptor (IGF1R) and a somatic mutation  in PI3K catalytic subunit type 2 alpha (PIK3C2A). RNAi-based silencing of PIK3C2A in a large set cancer cell lines showed a critical role for this gene in maintaining the neoplastic phenotype293033While the somatic-germline hypothesis might be a possibility, that kinases that need not be mutated may be activated by overexpression, and might also be able to explain the paucity of kinase variants. Our results are largely observational and do not point to new recurrent HFI variants; however, overall, our study provides supportive evidence for potentially unique combinations of kinase and other gene variants in breast cancer. The variants of unknown significance identified here would need to be validated externally and by functional assays to strengthen the claim for their functional significance.In conclusion, we found that individual breast cancers harbor a large number of potentially functionally important nucleotide variations in kinase genes (the mean number of kinase variants was 24 per case). Most of the HFI SNVs include previously described germline variants with experimentally validated or suspected impact on protein function. Individual variants are rare and are present in unique combinations in each case. We hypothesize that any particular somatic mutation may cause different biological consequences depending on the constellation of functionally active germline SNPs that are present in the same cell. An important future challenge is to experimentally assess the functional interaction between germline and somatic variants and determine how, if at all, they contribute to the biological behavior of cancer.How to cite this article: Agarwal, D. et al. Characterization of DNA variants in the human kinome in breast cancer. Sci. Rep.5, 14736; doi: 10.1038/srep14736 (2015).


Evidence for selecting the same total knee arthroplasty prosthesis whether the posterior cruciate ligament (PCL) is retained or resected is rarely documented. This study reports prospective midterm clinical, radiographic, and functional outcomes of a fixed-bearing design implanted using two different surgical techniques. The PCL was completely retained in 116 knees and completely resected in 43 knees. For the entire cohort, clinical knee (96 ± 7) and function (92 ± 13) scores and radiographic outcomes were good to excellent for 84% of patients after 5–10 years in vivo. Range of motion averaged 124° ± 9°, with 126 knees exhibiting ≥120° flexion. Small differences in average knee flexion and function scores were noted, with the PCL-resected group exhibiting an average of 5° more flexion but an average function score that was 7 points lower compared to the PCL-retained group. Fluoroscopic analysis of 33 knees revealed stable tibiofemoral translations. This study demonstrates that a TKA articular design with progressive congruency in the lateral compartment can provide for femoral condyle rollback in maximal flexion activities and achieve good clinical and functional performance in patients with PCL-retained and PCL-resected TKA. This TKA design proved suitable for use with either surgical technique, providing surgeons with the choice of maintaining or sacrificing the PCL.  Contemporary total knee arthroplasty (TKA) provides reliable pain relief and restoration of moderate function for patients suffering from severe joint degeneration. Outcomes are typically very good , 2, but Traditionally, orthopaedic surgeons have been trained to execute surgical techniques that depend on the integrity of the posterior cruciate ligament by utilizing one of two basic TKA designs, the cruciate-retaining type (CR) or the posterior-stabilized type (PS). However, joint laxity can vary widely after TKA due to common variations in these surgical techniques, including whether the PCL is preserved with a tibial bone block at the insertion site, recessed to the level of the tibial bone cut, or completely resected –29. TKA The current study was initiated in order to gather evidence documenting the performance of a new TKA articular design concept that incorporates progressive congruency in the lateral compartment Figures  and 2. TTwo clinical sites in the United States participated in this prospective IRB-approved study to record clinical results of a fixed-bearing TKA design  implanted using two different surgical techniques; either the PCL was retained with a bone block or the PCL was completely resected. Patients with surgery dates between January, 2002 and December, 2006, were identified from TKA surgical databases provided by the two surgeons, providing for evaluation at a minimum follow-up of five years. Inclusion criteria for this study required that patients be older than 21 years, demonstrate severe arthritis with bone to bone disease in at least one knee compartment, have varus or valgus deformity not exceeding 15 degrees, not have had prior knee sepsis, and not have undergone prior knee arthoplasty for any reason. Patients were required to provide informed consent and to be willing to travel to the clinical office to undergo physical evaluation and a radiological exam. Eligible patients were contacted by telephone to schedule clinical visits and were enrolled sequentially until the enrollment target of 150 TKA was achieved.During the four-year operative period, a total of 251 TKA were implanted by the two participating surgeons using the same implant design. A total of 134 patients (159 TKA) met the inclusion criteria and were willing to participate in the study, including 116 of 193 (60%) TKA implanted by a surgeon who preserved the PCL with a bone block and 43 of 58 (74%) TKA implanted by a surgeon who completely resected the PCL. There were 84 female patients (98 TKA) and 50 male patients (61 TKA) with an average age of 69 ± 9  years at the time of index TKA. Average duration of function for all TKA was 8.0 ± 1.1  years.Surgical exposure for both surgeons at index TKA was a mid-vastus approach through a standard midline skin incision. Handling of the PCL followed the surgical techniques routinely used by each participating surgeon. Preservation of the PCL was accomplished with a bone block surgical technique, whereas resection of the PCL was accomplished through complete ligament removal and insertion of a Hohmann retractor for anterior tibial dislocation. All knees in both groups were implanted with the same fixed-bearing TKA design (3D Knee), including patellar resurfacing and cement fixation of all three components. All modular tibial inserts and domed patellar components were machined from compression molded polyethylene and were sterilized using gamma radiation and packaged in nitrogen gas.Patients were followed up prospectively to record clinical and radiological outcomes. The physical evaluation included measurement of maximum knee flexion using a hand-held goniometer and assessment of clinical outcome using the Knee Society Score (KSS knee and function scores) . The radIn addition to the clinical and radiological outcomes described above, some patients were requested to participate in a quantitative analysis of two deep flexion activities in the early follow-up period (3 to 13 months), including 20 patients from the PCL-retained group and 13 patients from the PCL-resected group. Similar to previous fluoroscopy studies , 38–40, P = 0.002) but an average function score that was 7 points lower (P = 0.003) compared to the PCL-retained group (P < 0.001), which were generally representative of the diverse geographic locations of the two clinical sites.Prospectively measured clinical scores and radiographic outcomes were generally good to excellent after five to ten years of in vivo function . Among aed group . The cliRadiological assessments by a nonauthor third party orthopaedic surgeon were completed on 141 TKA that had sufficient follow-up films for examination. Restoration of suitable limb alignment was noted, recorded as part of the KSS knee score. Narrow (less than 2 mm), nonprogressive radiolucent lines were noted on 19% of the TKA, including 27% and 2% of the TKA in the PCL-retained and PCL-resected groups, respectively. None showed progression on the yearly radiographs. The largest proportion of radiolucent lines were noted in two femoral regions of dense cortical bone on the anterior femur (zone 1) or in sclerotic bone on the posterior femur (zone 4) and one tibial region on the medial tibial pleateau (zone 1). Three TKA in the PCL-retained group exhibited wide radiolucent lines (more than 2 mm) in one zone and were also classified as nonprogressive on serial radiographs. None of the TKA in the PCL-resected group had wide radiolucent lines.P = 0.15) between the PCL-retained group measuring an average of 131° ± 13° and the PCL-resected group averaging 124° ± 11°. Tibial axial rotation averaged 10° for both groups during kneeling. Lateral femoral rollback in kneeling averaged 10 mm in the PCL-retained group, which was significantly larger (P = 0.01) than the average 5 mm in the PCL-resected group. Maximum skeletal flexion for the lunge activity averaged 120° ± 11° and 123° ± 17° for the PCL-retained and PCL-resected groups, respectively. In the PCL-resected group during the lunge, three patients showed slight articular lift-off in the medial compartment at flexion angles greater than 139°, consistent with observations from normal knees in deep flexion [Detailed fluoroscopic analysis revealed few significant differences between the PCL-retained and PCL-resected groups . Maximum flexion . Tibial Much of the clinical success of TKA is partially dependent upon using TKA designs with suitable congruity and constraint to provide adequate knee joint stability throughout the full range of motion –17. AP sPhysical examinations and in vivo fluoroscopic analysis showed reasonably consistent flexion kinematics in knees with or without a PCL Tables  and 2. AWe evaluated TKA performance using clinical outcome scores, in which stability was assessed using the KSS knee score (25 points are assessed for stability), and functional fluoroscopic assessments during deep flexion activities. This combination was selected because previous studies have been unable to differentiate the performance of different TKA designs using only clinical outcomes or gait analysis, even when comparing vastly different articular geometries such as cruciate-retaining or posterior-stabilized TKA , 42–45. A critical question for TKA designs intended for use with or without the PCL is whether the tibiofemoral articulation provides adequate knee stability to eliminate or reduce unproductive AP sliding during dynamic activities like gait and stair climbing. The larger condylar translations (approximately 10 mm) recorded during the deep flexion activities in the current study are consistent with the TKA achieving femoral rollback essential for accomplishing deep flexion kneeling, lunge, and step up/down activities , 40. TheThis new TKA design concept was originally introduced in 2001 and was based on new knowledge from studies of in vivo kinematics and retrieved implant analysis . Based oThe most widely used design concept for providing AP stability after resection of both cruciate ligaments is to incorporate a femoral cam and tibial post articulation . HoweverSeveral limitations are recognized. Outcomes were assessed solely using KSS knee and function scores, as was the clinical standard for the involved clinics at the start of this study. KSS are known to be weak predictors of differences between patients. While other clinical outcome measures may have detected outcome differences between the study groups, the current study incorporated quantitative radiographic assessments and functional fluoroscopic analysis to supplement the KSS. Regarding surgical technique, one of the known differences between PCL-retaining and PCL-sacrificing surgical techniques is the potential for an increased flexion gap in the latter case. When noted intraoperatively, this was accommodated through the use of thicker tibial inserts in the latter case. The proportion of TKA having nonprogressive radiolucent lines was greater for the PCL-retained group than for the PCL-resected group. This is likely due to technicians who were more demanding in aligning the beam with the prostheses and newer radiography equipment in use at the clinic where the PCL-retained group were followed up. These factors combined to produce better views of the interface between the bone and cement on higher resolution images, aiding detection and follow-up of any progression of observed radiolucent lines at that clinical site.Limitations related to the limited size of patient cohorts are noted. Dissimilar surgical volume at the two involved clinical sites led to unequally sized patient cohorts with suitable follow-up data acquired within the timeframe of this study. While such dissimilarity can lead to overly broad population variance, and contributed to the differences in patient ages for each group, the authors chose to err on inclusion rather than reduce the PCL-retaining group from 116 TKA down to 43 TKA. The follow-up rate was less than 75% because of the decision to capture an initial group of 150 TKA in patients who met the inclusion criteria and were willing to come to the clinic for follow-up, rather than trying to follow up all patients, many of whom maintain only seasonal residency in the geographic regions surrounding the clinical sites. Studies involving a sequential series of TKA patients operated with this TKA are needed to fully characterize outcomes from patients who are not motivated to return for follow-up or have reduced willingness to participate in clinical studies. The fluoroscopic analysis included only 33 patients who all had high outcome scores. Such selection criteria have been used in many previously published fluoroscopic studies and have proven to yield significant results –40.This novel TKA design showed excellent functional performance with or without the posterior cruciate ligament over the intermediate time period of five to ten years. The standard implantation technique provided surgeons with the option of using one implant solution when dealing with variable posterior cruciate ligament integrity. The 3D Knee provided the required stability to achieve excellent clinical and radiographic outcomes and consistent kinematic results in both the PCL-retained and PCL-resected patient cohorts. Continued multicentered studies of this TKA design will be necessary to produce the evidence-based data to ultimately prove long-term clinical success.


Despite its ability to infect DC and dampen their functions, MCMV induces very robust, efficient and long-lasting CD8 T cell responses. Their priming may rely on the unique ability of uninfected XCR1+ DC to cross-present engulfed viral antigens and thus to counter MCMV interference with antigen presentation. A balance appears to have been reached during co-evolution, allowing controlled replication of the virus for horizontal spread without pathological consequences for the immunocompetent host. We will discuss the role of the interplay between the virus and DC in setting this balance, and how advancing this knowledge further could help develop better vaccines against other intracellular infectious agents.Infection of mice with murine cytomegalovirus (MCMV) recapitulates many physiopathological characteristics of human CMV infection and enables studying the interactions between a virus and its natural host. Dendritic cells (DC) are mononuclear phagocytes linking innate and adaptive immunity which are both necessary for MCMV control. DC are critical for the induction of cellular immunity because they are uniquely efficient for the activation of naïve T cells during their first encounter with a pathogen. DC are equipped with a variety of innate immune recognition receptors (I2R2) allowing them to detect pathogens or infections and to engulf molecules, microorganisms or cellular debris. The combinatorial engagement of I2R2 during infections controls DC maturation and shapes their response in terms of cytokine production, activation of natural killer (NK) cells and functional polarization of T cells. Several DC subsets exist which express different arrays of I2R2 and are specialized in distinct functions. The study of MCMV infection helped deciphering the physiological roles of DC subsets and their molecular regulation. It allowed the identification and first  Human cytomegalovirus (HCMV) is DNA β-herpes virus extremely prevalent word wild and establishing a persistent latent infection in immunocompetent hosts. HCMV infection is generally asymptomatic even during primary infection. However, HCMV is an important opportunistic agent causing severe morbidity or death in immunocompromised hosts such as AIDS patients, fetuses and newborns  or intravenous (i.v.) routes are the main experimental models used. These experimental settings are highly reproducible and allow using many mutant mouse strains or viruses with reasonably low amounts of animals per group. This enabled to precisely decipher the cellular and molecular mechanisms regulating immune responses to MCMV. A parallel can be drawn for key pathological features and protection mechanisms between experimental MCMV infection in mice and in natura HCMV infection in humans. Hence, most of the observations gathered with experimental MCMV infection in mice should apply to some extent to in natura immune responses against MCMV infection in mice and most importantly against HCMV infection in humans.Mouse cytomegalovirus (MCMV) is a natural rodent pathogen, establishing life-long persistent latent infections in mice. + DC play a key role in setting this balance, in particular by promoting the induction of protective inflationary effector memory CD8 T cell responses. This characteristic of adaptive immune responses to CMV infections is being used to help develop better vaccines against other intracellular infectious agents, notably Human Immunodeficiency Virus (HIV).During the acute phase of the infection, MCMV can infect hematopoietic cells including macrophages and dendritic cells (DC) but also many non-hematopoietic cells such as hepatocytes, endothelial cells or epithelial cells , which is devoid of any other differentiation potential. In contrast, Langerhans cells (LC), which are exclusively found in the epidermis, and monocyte derived DC (MoDC), which develop only during inflammation, belong to the monocytic lineage. Mouse pDC can be unequivocally identified as CD11b−CD11cint and Bst2hi or SiglecH+. They are specialized in rapid and high level production of the antiviral cytokines type I interferon (IFN-I), including during MCMV infection (Table +-type DC and CD11b+ DC exist both as lymphoid tissue-resident DC (LT-DC) and as sentinel immune cells surveying all non-lymphoid tissues and migrating to their draining lymph nodes upon activation (MigDC). CD11b+ cDC are identified as Lineage −CD11c+/highLy6C−CD64−MerTK− cells, to distinguish them from macrophages which express MerTK and from MoDC which derive from Ly6Chi  monocytes and are Ly6G−CD11b+CCR2+CD64lowMerTK− + cDC which both specifically express the chemokine receptor XCR1 under control of the native immediate-early 1/3 promoter  , also NK cells (3–7%) but not much B and T cells (<2%) , in the marginal zone, at 6–8 h after inoculation. By 17 h, MCMV has disseminated in the red pulp, and by 48 h to the white pulp including in DC  monocytes to infection site where they become infected. These cells then serve as immune vehicles to disseminate the virus to distal organs, especially to the SG. Non-classical monocytes are also essential for the establishment of latent infection   and non-infected (EGFP−) splenic DC subsets from the same mice and examine their gene expression profiling in order to get an unbiased and global view of how the infection of DC in vivo modulates their biology. In any case, in vivo, most DC are not infected and thus not susceptible to the cell-intrinsic action of viral immune evasion genes. In addition, there is no dominant negative effect of the DC that are eventually paralyzed or polarized toward immunosuppressive functions due to their infection by MCMV. On the contrary, in vivo, most of the DC are uninfected, mature and immunogenic  Figure . Moreoveo Figure . pDC arey Figure . The expc Figure .in vitro vs. in vivo are differences in the microenvironment in which the interaction between the virus and the DC is taking place. We will discuss in particular the role of the inflammatory milieu and of cross-talk with other immune cell types in promoting protective functions of DC in MCMV infected mice  harbor a 7-fold increase in the fraction of infected DC, showing that IFN-I efficiently protects DC from MCMV infection in vivo  or for the downstream signaling adaptor MyD88, as well as to a lesser extent in mice deficient for TLR3 or for the downstream signaling adaptor TRIF . Even with viruses defective for MHC-I immune evasion genes, no efficient CD8 T cell priming was observed against antigens that could only be presented directly  enhancement of the priming of CD8 T cells, by facilitating cross-presenting DC physical access to viral antigens, and by limiting the negative feedback caused by the killing of infected cells  (Hansen et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A lack of cell surface markers for the specific identification, isolation and subsequent analysis of living prostate tumor cells hampers progress in the field. Specific characterization of tumor cells and their microenvironment in a multi-parameter molecular assay could significantly improve prognostic accuracy for the heterogeneous prostate tumor tissue. Novel functionalized gold-nano particles allow fluorescence-based detection of absolute mRNA expression levels in living cells by fluorescent activated flow cytometry (FACS). We use of this technique to separate prostate tumor and benign cells in human prostate needle biopsies based on the expression levels of the tumor marker alpha-methylacyl-CoA racemase (AMACR). We combined RNA and protein detection of living cells by FACS to gate for epithelial cell adhesion molecule (EPCAM) positive tumor and benign cells, EPCAM/CD45 double negative mesenchymal cells and CD45 positive infiltrating lymphocytes. EPCAM positive epithelial cells were further sub-gated into AMACR high and low expressing cells. Two hundred cells from each population and several biopsies from the same patient were analyzed using a multiplexed gene expression profile to generate a cell type resolved profile of the specimen. This technique provides the basis for the clinical evaluation of cell type resolved gene expression profiles as pre-therapeutic prognostic markers for prostate cancer. In men, prostate cancer is the most commonly diagnosed cancer and is the second leading cause of cancer related deaths . For 201in vitro culture assays after fixation.Current pre-therapeutic risk assessment is based on limited clinical parameters such as histological grading of needle biopsies by the Gleason score, clinical TNM classification and blood serum-based PSA levels . The neeUntil recently, isolation of living cells was primarily limited to detection based on the expression of cell surface proteins. Novel, functionalized gold nano-particles allow for the isolation of living cells based on absolute mRNA expression levels of a specific target . Alpha-mIn this study, we present a technique to characterize a prostate tumor by cell type resolved gene expression profiling from low input material such as needle biopsies. Distinct cell types were isolated simultaneously from needle biopsies. These cells were viable and were either used for in vitro culture or for multiplex gene expression analysis. Multiple biopsies were analyzed to cover different sections of the tumor.Two independent human prostate cancer RNA-seq studies with cancer and matched benign samples from 10 patients per study were analysed , 43. BotCells were sorted directly into 5μl 2x reaction mix . Cells were frozen at –80°C for efficient lysis for 2 h. RT/TAQ polymerase, polyT primer and all specific TaqMan assays (Life Technologies) were added (0.2x) for reverse transcription and 22 cycles of pre-amplification . Pre-amplified samples were diluted 1:5 with DEPC water and stored at −20°C.For gene expression analysis, 1μl of pre-amplified sample was used for qRT-PCR. Specific TaqMan assays  and TaqMan Fast Universal RNX 2x were used in 20μl total volume for amplification .Preamplified cDNA and TaqMan assays were mixed with appropriate loading buffer and loaded onto a 48.48. dynamic array for gene expression (Fluidigm) according to the manufacturer's instructions. Amplification was performed on a Biomark analyzer (Fluidigm) using a standard protocol .TM script (Fluidigm) for R. A detection limit was defined and CT values were converted into  for easier visualization. Negative values are represented as zero. Euclidean hierarchical clustering was performed. Values range from 0 to 12 with increasing expression level and are color-coded from red to white.Results from multiplex analysis on 48.48 dynamic array were exported as heatmaps and analyzed using the SINGuLARIn the standard qRT-PCR experiments, expression values were normalized to AMACR low expressing populations. Considerable variation in the stochastic expression levels of housekeeping genes - and all other genes - at the single cell level makes the use of housekeeping genes not feasible . We expeWritten consent was obtained from patients prior to any treatment including radical prostatectomy. The work was approved by a local ethics committee. Radical prostatectomy specimens were received and processed within 10 minutes after the dissection by a surgical-pathologist. Specimens were incised dorso-ventrally and needle biopsies were retrieved by conventional punch technique. Histological validation using H&E staining was performed on cross sections from the area where needle biopsies were retrieved. Needle cores were cut into 1×1mm pieces and incubated with 1:10 Collagenase (10mg/ml) in DMEM supplemented medium overnight at 37°C. Cores were washed and incubated with 5% Trypsin EDTA for 10 minutes at 37°C. Cores were separated into single cells by mechanical force (18 and 20G needles) and filtration (40μm). Cells were resuspended in 1ml DMEM and either used for analysis or frozen in liquid nitrogen.TM (BD Biosciences). 500 ml PREGM Medium includes the following factors: BPE, 2 ml; Hydrocortisone, 0.5 ml; hEGF, 0.5 ml; Epinephrine, 0.5 ml; Transferrin, 0.5 ml; Insulin, 0.5 ml; Retinoic Acid, 0.5 ml; Triiodothyronine, 0.5 ml; GA-1000, 0.5 ml. Matrigel is a commercially available extracellular matrix analoga. 15% matrigel provided highest highest culturing success.Primary epithelial cells were sorted directly into PREGM medium (Lonza) supplemented with 15% MatrigelIn brief, a 27 bp complementary target sequence is conjugated to a gold nanoparticle. A shorter complementary reporter strand binds to the target strand and is conjugated with a fluorophore (Cy5). The fluorophore is quenched by the gold nanoparticle when in the bound state. An endogenous target sequence with a higher binding affinity (due to its increased length) will replace the reporter strand and lead to the emission of a fluorescent signal, which can be detected by conventional FACS. No toxic or immunologic reactions induced by the gold nanoparticles have been reported to date . The endCells were incubated with 1:20 diluted (PBS) SmartFlare (AMACR-Cy5 (SF985) or Scramble-Cy5 (SF102)) for 6 h in 1ml DMEM at 37°C (light protected).Cell sorting was conducted with a FACSAria III cell sorter (BD Biosciences).We used the following antibodies:Anti-Human CD326 (Ep-CAM), Brilliant Violet 421, clone 9C4 , Anti-Human CD45, PerCP-Cy5.5, clone HI30 YOPRO-1 iodide , Anti-Human p63 / P504S, Klon: BC4A4, polyclonal (DCS diagnostics)n = 20). A total of 322 genes were identified as significantly differentially expressed in both data sets at a false discovery rate (FDR) of 0.1. The fold change in both data sets is highly correlated (Spearman's correlation coefficient ρ = 0.9037)  . A spear 0.9037) . Finallyin vivo validation of the AMACR SmartFlare specificity. LNCAP and RWPE-1 cells were incubated with 20μl of 1:20 diluted AMACR or a scrambled SmartFlare oligo as a control and monitored over 24 h for the absolute fluorescent intensity signal per cell using fluorescent time-lapse microscopy. Cells were automatically tracked by additional nuclear staining with Hoechst dye. Figure p < 0.0005). The scramble signal is detectable at comparable levels in both cell lines and significantly lower compared to the AMACR signal (p < 0.005).AMACR was among the 5 most up-regulated genes of the 322 genes identified and therefore was selected for customized SmartFlare design. The target sequence was directed against nucleotide 97–123 (according to the manufacturer). In a preceding experiment we confirmed the intracellular uptake of AMACR SmartFlare and a scramble control using confocal microspcopy in LNCAP cells after 4 h of incubation . The intp < 0.05). We also showed that the AMACR and p63 protein staining pattern in prostate tumors and benign glands mirrors the mRNA expression pattern in LNCAP cells and RWPE-1 cells. Tumor glands lack a basal cell layer and therefore do not stain positive for p63. In contrast, luminal benign epithelial cells show weak or absent staining for AMACR, whereas epithelial tumor cells show in most cases strong staining for AMACR instead , CD45 (lymphocytes) and Epcam . We confirmed that the sorted cell populations were identified by the expression of specific marker genes Fig. . ExpressTo account for the multifocal occurrence of prostate cancer, four separate needle biopsies  from another prostatectomy specimen were analyzed using the same setup as before Fig. . DistribWe can conclude that AMACR mRNA expression level-based separation of epithelial cells generates cell populations that closely resemble tumor and benign prostate cells, with respect to their gene expression profile. Even representing the expected heterogeneity of AMACR expression within the same specimen.10 step of fluorescence intensity. These findings most likely represent a continuous regulation of AMACR expression within the epithelial cell population. Technical explanations such as hydrolysis of the SmartFlare reporter oligo or detection of splice variants could account for such a signal distribution. Unspecific signals induced by hydrolysis are controlled by scramble SmartFlares, which showed significantly lower fluorescent signals than those detected in the AMACR high gate. The AMACR SmartFlare was designed to detect exon one, which is included in all known splice variants. No single cell analysis data regarding AMACR expression in living human prostate tumor cells are available for comparison.It is the first time that this technology has been applied to primary prostate tissue and small input material such as a needle biopsy. We selected AMACR as a target in this study because of its routine use in cancer diagnostics and the good correlation of expression at the mRNA and protein level , 13. HowA limitation of the expression profiles of the separated populations occurs when attempting to correlate the data with detailed histological analysis. The cross section of the prostatectomy sample showed a heterogeneous tumor tissue with tumor and benign glands and proportions of mesenchymal cells and inflammatory cells such lymphocytes. However, no exact estimation can be made to analyze the cellular composition along the needle biopsy. For future studies, improved biopsy technologies are needed, which would allow for simultaneous analysis of living cells and histological validation. We currently explore the use of a needle punch system to generate two tissue cylinders in very close proximity (< 1mm). One cylinder can be used for the analysis of living cells and the other one for standard histological analysis. A second method we currently explore is to cut one tissue biopsy cylinder longitudinally and analyze the two halfs separately. The latter methods would then even further reduce the input material. However we could already recover sufficient number of cells and paraffin embedded material for downstream analysis in a feasibility experiment (data not shown).The use of 200 cells from each population allows for the detection of a large number of transcripts and samples in a cost efficient way. As yet, this does not allow resolution at the single cell level. The number of detectable genes per cell decreases at the single cell level, which is mainly attributed to highly stochastic expression of certain transcripts in a single cell at a given time point , 17. FroThe multiplex gene expression analysis on a 48.48 dynamic array is limited to 48 genes and 48 samples. However, it represents a cost efficient technology for analyzing a large number of samples with minimal reagent input and high precision due to the microfluidic driven assembly of the nano liter scaled reaction volumes. To further increase the power of this system in order to reveal novel interaction networks, potentially to be used as prognostic criteria, RNA-seq technology has to be applied via adopted single cell protocols , 47.Cell populations referred to as mesenchymal cells in this work are known to consist of further subpopulations such as stromal cells, endothelial cells, myofibroblast cells and cancer associated fibroblasts (CAFs) . The samUsing a cell type resolved analytical method of living cells to dissect prostate tumor tissue can overcome several previous limitations and improve the quality of tissue analysis post-biopsy: Living cells from primary tissue can be analyzed for a gene expression signature and, from the same population of cells, could be used for functional assays such as drug screening, murine xenografts or three-dimensional tissue cultures to examine drug response within the complex interaction of different cell types. Such analyses cannot be performed with fixed tissue biopsies. Further more, potential “omics” analyses from living cells provides a higher quality of input material compared to formalin or alcohol fixed tissue.A commercially available gene expression signature assay for prostate cancer has been recently validated in several patient cohorts and showed superior prediction for prostate cancer disease recurrence after the initial treatment when compared to clinical and histological variables . HoweverIn summary, an integrated cell type resolved gene expression analysis derived from multiple histological validated needle biopsies from the same organ provides the basis to further analyze novel interaction networks, define prognostic signatures and identify therapeutic targets in heterogeneous tumor tissue. Such tools will help to identify prostate cancer patients at high risk for tumor progression or death from cancer and treat them effectively as well as to prevent unnecessary treatment of patients with indolent prostate cancer.


As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in  The Notch signaling pathway plays an evolutionarily conserved role in regulating interactions between adjacent cells in a wide range of developmental contexts  and its Delta, Serrate, Lag2) ligand, expressed on the surface of an adjacent cell, “activates” the Notch receptor. This interaction facilitates removal of the NECD fragment, which allows two sequential cleavage events that release the Notch Intracellular Domain (NICD) from the cell surface whereupon it translocates to the nucleus. There, NICD forms a complex with CSL  proteins and a number of other factors together with which it can drive transcription of target genes recognized by the CSL DNA-binding domain 5 K [X]2 N (shown in bold above). The broader functional significance of this conserved sequence will need to be explored in future studies.We have identified a putative Mib1-interacting domain (MID) in the intracellular domain of DeltaD and have shown that it is required for both interaction and ubqiuitylation by Mib1. Similar analysis has been done to identify the Mib1-interacting domain in  by Mib1 . SimilarNN) at position 591–592 in DeltaD within the MID. This di-asparagine motif appears related to a previously identified larger conserved domain in DSL ligands . The study by Glittenburg et al showed that Serrate/Jagged homologues have a conserved motif [E/D][E/D]X[–]NNX5NX[–]NP[L/I] and a mutant form of Serrate that lacks this motif lose the ability to trans-activate Notch. Delta homologues have a related motif [E/D]X[–]NN[L/I]. Our study now shows that the conserved di-asparagines within this broader motif are required for effective interaction, ubiquitylation and internalization of DeltaD by Mib1. Together these observations suggest that despite poor overall similarity in the intracellular domain of DSL ligands short conserved domains that are functionally important have now been identified. We expect the identification of these critical Mind bomb interaction domains will provide an important starting point for future studies examining the mechanisms that regulate interaction of Mib1 and Delta in both vertebrates and invertebrates.Our study also identified a conserved di-asparagine pair  phenotype, or having no suppression, weak suppression or strong suppression of neurogenesis.(TIFF)Click here for additional data file.


Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and  Elucidating the relevant mechanisms is important for understanding normal stem cell and tissue biology, and also because of the potential for developing therapies that can target stem-like cells in cancer.Stem cells of the adult mammary gland are predicted to have a capacity for self-renewal and to give rise to the two major epithelial cell lineages of mammary ducts: luminal and basal. Substantial progress has been made towards characterizing mouse mammary stem cell populations, both in vivo lineage tracing experiments have challenged some of these conclusions Evidence that adult mammary tissue contains multipotent self-renewing stem cells was first provided by classical transplantation studies in which a normal epithelial ductal tree, comprising both basal and luminal cell lineages, could be regenerated from small tissue fragments or individual cells Ex vivo assays of mammary epithelial cell sphere formation in suspension culture, mammospheres, offer a complementary approach to stem cell studies that is amenable to signaling pathway analysis. Originally developed for analysis of neuronal precursors, the ability of cells to form spheroids has been used as a stem cell assay for several other tissue types, including prostate and mesenchymal stem cells The canonical Wnt/β-catenin signaling pathway is one of the principal signaling mechanisms associated with regulation of stem cell behavior in numerous tissues Wnt1 mouse strain is a well characterized model for the studying the consequences of Wnt signaling in the mammary gland and its effects on stem cells Wnt1 transgene predominantly in luminal epithelium, and results in activation of canonical Wnt/β-catenin signaling in the basal layer Wnt1 mice display widespread mammary epithelial hyperplasia and are predisposed to carcinomas with nearly 100% penetrance Wnt1 was reported to contain larger numbers of CD24+ CD29HI cells, which are enriched for stem cell activity, in comparison to wild-type glands + CD29HI immunophenotype showed increased colony formation in response to purified Wnt3a The MMTV-Intracellular signaling elicited by members of the Wnt family of secreted ligands can be broadly classified into two modes: canonical and non-canonical. In the canonical pathway, Wnt ligand binding to receptor complexes containing Frizzled and Lrp5/6 proteins results in stabilization of cytoplasmic β-catenin and transcriptional activation mediated by β-catenin/TCF complexes While there is strong support for Wnt/β-catenin signaling promoting mammary stem cell properties, as described above, the roles of non-canonical signaling in the mammary gland are much less clear. Moreover, apparently conflicting data exists concerning functional interactions between canonical and non-canonical Wnt signaling in other experimental systems. Thus Wnt5a has been reported to act either act in opposition to, in concert with, or independently of Wnt/β-catenin signaling To elucidate the effects of canonical and non-canonical Wnt signaling on stem cell properties of mouse mammary epithelium, here we test the consequence of altered Wnt signaling activity on mammosphere cultures, specifically quantifying the number of secondary mammosphere-forming units (MFUs)et al. Mammospheres culture methods were adapted from Dontu Primary mammosphere cultures were disassociated with trypsin for 30 minutes in the presence of a vital cell labeling dye, . Cell suspensions were filtered through a 40 µM cell strainer and then diluted in mammosphere medium with 1% methylcellulose to limit cell aggregation, and 10 nM dexamethasone to maintain transcription from the MMTV promoter. Cells were plated in 96-well low adherence plates using 24 wells per treatment, and were scored for mammosphere formation after one week of growth. In all secondary mammosphere assays, cells were plated at 1000 cells per well except in lentiviral shRNA knockdown experiments, in which they were plated at 2000 cells per well. All exogenous treatments were given as a single dose to the dissociated cells at the time of secondary assay plating. Mammospheres were defined as colonies in methylcellulose suspension culture that contained 10 or more cells visible under phase contrast, of which fewer than 50% still retained the cell tracking dye, consistent with the dye being diluted out upon successive cell divisions.Secondary mammosphere cultures were harvested and washed with PBS. Mammospheres were fixed and permeabilized with a 1∶1 acetone:methanol fixation solution for 30 minutes at −20°C, and PBS was used to rehydrate the mammospheres for 10 minutes at room temperature. Non-specific antibody binding was blocked with a 3% BSA/PBS solution for 1 hour at room temperature. Primary antibodies to cytokeratin 8 , and cytokeratin 14 , both at a 1∶200 dilution in 3% BSA/PBS, were applied overnight at 4°C. Mammospheres were then washed twice in PBS. Cells were incubated in a 1∶1000 dilution of Alexafluor-conjugated secondary antibodies (Life technologies) in 3% BSA/PBS for 4 hours. Cells were washed twice with PBS and mounted in Vectashield mounting media (Vector laboratories) including 0.1 µg/ml DAPI.Numbers of mammospheres reported are the mean of 24 wells per treatment from representative experiments. All experiments were repeated 3 or more times, demonstrating consistent statistical relationship patterns. Comparisons between treatments were made using Student's t test with p<.05 required for significance.Mammospheres were prepared as for secondary mammosphere assays, except cells were plated in 6-well low adherence plates. After one week cells were harvested by centrifugation and total RNA was extracted using RNeasy mini kits (Qiagen). cDNA was produced using the iScript cDNA synthesis kit (Bio-Rad). Quantitech primers (Qiagen) were used with SYBR-Green mastermix (Quanta) for quantitative PCR using an MJ Opticon2 system (BioRad) according to manufacturer's instructions.Ror2 mRNA were obtained from Dr. T. Stappenbeck To infect mammosphere cultures with lentivirus, primary mammospheres were disassociated as for secondary mammosphere assays. Before plating, single cell suspensions in DMEM/F12 media were mixed with lentiviral particles in PBS at a multiplicity of infection of greater than 5∶1, plus a 1∶200 dilution of Transdux reagent (System Biosciences) for 30 minutes at 37°C. After infection, the cell suspension was plated as for secondary sphere assays. The 7TGC lentiviral reporter vector was obtained from Addgene (Plasmid#24304) In experiments using inhibitors and recombinant proteins in conjunction with secondary mammosphere assays, agents were added to single cell suspensions before plating. Recombinant Wnt3a (Peprotech), and Recombinant Wnt5a (R&D systems) were used at 200 ng/ml, except when noted. Recombinant Dkk1 (R&D systems) was used at 200 ng/ml. JNK inhibitor SP600125  was used at 10 µM, and iCRT3  This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committees of Weill Cornell Medical College (Protocol Number: 0052–11), and the New York Blood Center (Protocol Number: 267).ex vivo mammosphere cultures derived from primary mouse mammary epithelium. Dissociated epithelial cells were obtained from wild-type mice and from MMTV-Wnt1 transgenic animals. Such mammosphere cultures provide an assay system for stem cell-initiated sphere growth, independent of previously identified stem cell enrichment markers Wnt1 derived mammospheres Wnt1 and wild-type mammospheres have similar capacity to produce progeny cells expressing differentiation markers during mammosphere growth in vitro. In addition, wild-type and MMTV-Wnt1 mammospheres exhibited similar morphology in both shape and size  to suppress Ror2 expression, and validated its ability to knock down Ror2 mRNA in mammospheres by qRT-PCR , who observed that treatment of human breast epithelial cells with Wnt5a, in conjunction with activation of Wnt/β-catenin and TGFβ pathways, enhanced the efficiency of mammosphere formation as well inducing the expression of EMT markers in vitro, suggesting that its positive effect on mammospheres is not unique to breast tissues While elevated levels of canonical Wnt/β-catenin signaling are clearly associated with promoting hyperplasia and tumorigenesis in the mammary gland, both in mice and humans Ror2, a receptor tyrosine kinase that binds Wnt5a and transduces a non-canonical Wnt signal that includes activation of JNK Ror2 involvement, we found that Wnt5a-induced mammosphere formation was abolished by inhibition of JNK. We conclude that while Wnt3a promotes mammosphere formation via canonical Wnt signaling, Wnt5a does so by a non-canonical mechanism. Moreover, our data particularly implicate a Ror2-JNK pathway among the numerous other pathways that have been ascribed to non-canonical Wnt signaling Although Wnt5a typically signals via β-catenin-independent mechanisms, there are special circumstances in which it has been found to activate the canonical Wnt/β-catenin pathway in vivo have yielded data that may be inconsistent with those from classical mammary reconstitution assays in vivo, mammosphere assays provide a promising system for dissecting the responses of individual self-renewing cells to defined factors in vitro.It remains to be determined whether the independent and additive effects of canonical and non-canonical Wnt signaling on mammosphere formation are caused by both signals operating on the same population of mammosphere-forming cells, or whether they act on distinct target populations. Recent studies aimed at identifying mouse mammary stem cells by lineage tracing Figure S1Wild-type mammospheres can be serially passaged for multiple generations. Wild-type mammosphere cultures were serially passaged weekly. The number of mammospheres formed per 2000 cells plated was assayed at each passage. Passage one represents the number of secondary mammospheres resulting from passage from primary to secondary culture. Error bars show 95% confidence intervals.(TIF)Click here for additional data file.


In this way, we recently found that entorhinal theta-frequency input to the DG most effectively passes filter mechanisms of the trisynaptic circuit network, generating activity waves which propagate across the entire DG-CA axis. These “trisynaptic circuit waves” involve high-frequency firing of CA3 pyramidal neurons, leading to a rapid induction of classical NMDA receptor-dependent long-term potentiation (LTP) at CA3-CA1 synapses (CA1 LTP). CA1 LTP has been substantially evidenced to be essential for some forms of explicit learning in mammals. Here, we review data with particular reference to whole network-level approaches, illustrating how activity propagation can take place within the trisynaptic circuit to drive formation of CA1 LTP.Decades of brain research have identified various parallel loops linking the hippocampus with neocortical areas, enabling the acquisition of spatial and episodic memories. Especially the hippocampal trisynaptic circuit [entorhinal cortex layer II → dentate gyrus (DG) → cornu ammonis (CA)-3 → CA1] was studied in great detail because of its seemingly simple connectivity and characteristic structures that are experimentally well accessible. While numerous researchers focused on functional aspects, obtained from a limited number of cells in distinct hippocampal subregions, little is known about the neuronal network dynamics which drive information across multiple synapses for subsequent long-term storage. Fast voltage-sensitive dye imaging  The hippocampal formation is critically involved in several cognitive functions like formation of spatial and episodic memories and spatial navigation.KEY CONCEPT 3Complex spatio-temporal patterns of electrical activity in large/meso-scale neuronal networks.voltage-sensitive dye imaging (VSDI) proved themselves to be instrumental in covering the methodologically demanding “mesoscale” of neuroscience research  or theta-burst stimulation of CA3-CA1 projections, possesses the properties of “cooperativity,” “associativity,” and “input-specificity,” and predominantly results from a recruitment of additional AMPA receptors to the postsynaptic membrane.in vivo and in vitro studies using local field potential or single-cell recording in area CA1, excluding the detection of neuronal activity in upstream regions  synapses  of a particular hippocampal pathway, several groups deactivated specific fiber tracts To overcome the limited spatial scale of single-cell and local field potential recordings, several recent studies applied VSDI to hippocampal slice preparations. Most of these investigations examined neuronal network activity in distinct regions of the hippocampal formation like the EC  leads to pronounced neuronal activity in the DG, but only feeble or no activation of CA areas  EC/DG-input is very effective at generating trisynaptic circuit waves, whereas 1 and 20 Hz EC/DG-input evokes weaker ones. Moreover, we revealed that trisynaptic circuit waves critically depend on frequency facilitation of mossy fiber synaptic transmission onto CA3 pyramidal neurons and observed that DG activity increasingly declines during 0.2, 1, 5, and 20 Hz EC/DG-input, respectively  synapse formed by dentate gyrus granule cell axons and CA3 neurons. Mossy fiber synaptic transmission onto CA3 pyramidal neurons exhibits prominent frequency facilitation, a form of presynaptic short-term plasticity which is strongly pronounced in the theta-frequency range and can potentiate the otherwise weak neurotransmission by up to ~1200%.KEY CONCEPT 7Neural substrate which only generates significant output in response to an input signal if the input signal ranges in a certain frequency band. If the filter displays smooth output attenuation characteristics, it represents a “low-order band-pass filter.”KEY CONCEPT 8Frequency band covering ~4–12 Hz of rhythmical (oscillatory) neural activity. Theta activity occurs in various brain structures, including the hippocampal formation, and is tightly linked to mechanisms of learning and memory.The remarks given above are in line with previous work proposing that the DG acts as a filter to prevent the hippocampus from sensory overflow or runaway excitation as seen in pathological brain states like schizophrenia or temporal lobe epilepsy, respectively  becomes forceful enough to fire the target cell (Jonas et al., B receptors (Teyler and Alger, Although the situation in the DG is presumably more complicated Hsu, , severalAs stated in the introduction, much evidence speaks in favor for an essential role of CA1 LTP in the formation of some explicit memories in mammals. If so, induction of CA1 LTP should be triggered by sensory information transfer to the hippocampus. How could this take place at the level of the trisynaptic circuit network? We propose that synchronous theta-rhythmical spiking in EC layer II cell ensembles, which occurs during EC theta oscillations (Mizuseki et al., The remarks given above suggest that trisynaptic circuit waves and induction of CA1 LTP by them also occur naturally at the level of sparse numbers of trisynaptic interconnections and associated microcircuits (Jung and McNaughton, Prior to our work, “polysynaptic LTP” in area CA1 has already been described by Buzsáki  and NakaFinally, it is important to mention that LTP in the hippocampus has been evidenced to be related to hippocampal theta and gamma oscillations (Bikbaev and Manahan-Vaughan, Based on our recent work (Stepan et al., in vitro (Airan et al., A comprehensive elucidation of the mechanisms and physiological functions of hippocampal network dynamics does not only require sophisticated recording methods like fast VSDI or multi-electrode array techniques, but also tools which allow a temporally precise activation or silencing of specific types of neurons. The latter is now provided by the optogenetic toolbox (e.g., Zhang et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP The metabolome reflects the current biological state of an organism and is the endpoint of all interactions between environment, genome, transcriptome and proteome. The metabolome and its response to different conditions deliver valuable mechanistic insights into as diverse topics as nutrition, diseases, biomarkers, toxicity, crop traits, aging, and stress among others –7. In biThe current major bottleneck for wide spread use of metabolomics and delivery of highly reproducible and reliable results is the sampling of suspension cultured cells while maintaining the levels of intracellular physiological metabolites unperturbed. Many protocols have been suggested and recently tremendous progress towards sophisticated sampling has been made for various types of organisms such as bacteria –16, yeasAn optimal sampling should simultaneously (i) remove extracellular metabolite contaminations from culture medium, (ii) avoid loss of intracellular metabolites through leakage, export, unspecific adsorption, interconversions or degradation, (iii) be widely applicable to all types of cells with little adaptation effort, and (iv) ensure optimal subsequent extraction with high recovery rates and broad metabolite coverage . In the Sampling strategies consist in either “quench first” (cold/solvent) or “separate first” (filtration/centrifugation). The obvious advantage of “quench first” is the earliest possible stop of metabolism avoiding degradation and interconversion, but literature has shown that not all of the other prerequisites can be achieved for an optimal method. In many cell types quenching leads to considerable leakage, which was reported for the most common quenching method with either solvents  ,21, low Escherichia coli) and mammalian cells .The obvious advantage of “separate first” is the leakage-free removal of extracellular contaminations and the importance of the contamination removal cannot be overemphasized  (DSMZ) were cultivated in shaker flasks on 4.5 g/L glucose in LB medium (Sigma Aldrich) under aerobic conditions at 37°C and 220 rpm on an orbital shaker  until late exponential phase.2 at 36.5°C with constant stirring at 150 rpm. Cells were harvested on day 5, cell numbers were 9.0∙106 cells/mL with 93.1% viability as was determined with a Vi-Cell analyzer (Beckman Coulter Inc.) based on trypan-blue exclusion. Medium osmolality on day 5 was 269 mosmol/kg and was measured with a freezing point osmometer .CHO-DG44 cells expressing an antibody were cultivated in 160 mL proprietary PBG-CD-C4 media supplemented with 6 mM L-glutamine and start feed in 500 mL Erlenmeyer flasks in an Infors HT multitron cell incubation shaker under 8% CONS0 cells expressing an IgG antibody were cultivated in a 5 L bioreactor  in a proprietary chemically defined medium, consisting of all proteinogenic amino acids, glucose, myo-inositol, O-phosphoethanolamine, pyruvate, putrescine, pantothenic acid, folic acid, glutathione (GSH), nicotinamide, riboflavin, sulfate salts, phosphate salts and trace elements. The production bioreactor process was a 10 day fed-batch process with daily addition of feed starting on day 2. Cells were harvested daily from day 3 on, cell density and viability were determined with a Cedex automated cell counter . The extracellular content of glucose, glutamate, glutamine, lactate and ammonia were monitored daily with a Bioprofile Flex .Cell suspension was transferred into standard vials and cells were pelleted by centrifugation for 1 min with a standard table top centrifuge . Supernatant was carefully removed, samples were snap frozen in liquid nitrogen, stored at -80°C and shipped on dry ice.13C6] D-glucose in isotonic NaCl solution (room temperature) for MxP® Broad Profiling samples and with 4.5 g/L 12C-glucose in isotonic NaCl solution (room temperature) for MxP® Energy samples. Once the washing solution flowed through the filtered, liquid nitrogen was immediately added to completely stop metabolism, which was typically around 20 s after withdrawal of cell culture suspensions. Filters were folded and inserted into standard polypropylene vials with forceps and precooled (dry ice) quenching solution was added immediately. Quenching solutions for MxP® Broad Profiling was dichloromethane (DCM) /Ethanol (EtOH) (9:11) and for MxP® Energy DCM/EtOH (2:1). Tubes were frozen in liquid nitrogen and stored at -80°C or shipped on dry ice.Cell culture suspensions were pipetted onto ethanol pre-wetted standard PTFE filter  placed on a filter funnel  fitted on a filtering manifold . Suspensions were immediately filtered with 35 mbar, applied by a standard vacuum pump (Büchi V-710). Cells were washed with 1 mL of 4.5 g/L uniformly labeled  D-glucose in yeast nitrogen base amino acid-free medium (Y1250 Sigma–Aldrich). Cells were cultured under aerobic conditions at 28°C on an orbital shaker at 180 rpm and harvested at an optical density of 4  by short centrifugation at 4°C. Cells were washed twice with 10 g/L [U13C6] D-glucose in 0.15 M ammonium acetate by centrifugation. The cell pellet was quenched with DCM/EtOH (2/1) and extracted as described for the metabolite profiling MxP® Energy. The polar phase containing the labeled metabolites was stored at -80°C until further use as internal standard.For production of ® Energy method. Metabolites were quantified via external calibration standards using the relative peak areas to the spiked 13C-labeled yeast extract as internal standard which was added to the samples directly before extraction. Linearity was above 0.99 (Pearson R2) for ranges from 50 ng/mL to 5 μg/mL, except for pyruvate and ADP where linearity was given in the range of 1 μg/mL to 5 μg/mL.For selected metabolites with either high turnover rates or of paramount biological interest (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-diphosphate , glutathione (GSH), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), glutamine (Gln), glutamate (Glu), pyruvate) quantitative measurements were established based on the MxP® Broad Profiling and values normalized against the corresponding 13C-analyte for MxP® Energy) within each analytical sample. This method assumes that the majority of metabolite levels are similarly influenced by cell densities, which was sufficiently so in this study. In order to keep the high difference between the metabolite levels measured in the two sampling methods an adjustment parameter  samples divided by median of all centrifuged (C) samples) was introduced to all C samples.Metabolite profiling data were normalized to the median of all metabolites from one platform  model  was useFor multivariate analysis data was further centered and scaled to unit variance. This introduces a common scale for all metabolites independent of their absolute variance, so that models cannot be dominated by few high-variance metabolites. Multivariate statistical analysis including PCA  were performed by using Simca P+ software .1. draw the sample, 2. separate, 3. wash, 4. quench, 5.  store, 6. extract and 7. measure  covering broadly both lipid and polar metabolites. A sampling method, which is compatible with such an extraction has a very high probability of being well compatible with similar or milder conditions as well as focused metabolite investigations. Filter stability with other typical extraction solvents can be taken for granted for filters stable in DCM mixtures, ball or bead milling and liquid nitrogen. General metabolite coverage of extractions with multiple solvents is higher than with single-solvent extractions [While good solutions exist for the steps ractions .13C-labeled carbon source to the washing solution was found to avoid metabolic changes due to nutritional stress.Manual sample drawing was used as it is the most readily available and easily implemented method without need for special equipment. If an automated sample drawing equipment exists, its use is strongly encouraged and will likely be compatible with the method proposed here. In order to stop all metabolic activity and to minimize impact of subsequent handling variations, a quick freeze in liquid nitrogen was introduced directly in the filter funnel. This is the fastest, most robust and widely available possibility for stopping metabolism. The addition of the TM filters were used, as they met all criteria and are world-wide available.The filter itself is one of the most crucial parameters in filtration sampling methods and must simultaneously be (i) solvent resistant, (ii) contamination-free during sampling and extraction, (iii) mechanically stable in liquid nitrogen and bead mill extraction, and (iv) able to retain all possible types and sizes of cells. In order to retain even small cell types such as bacteria, pore size as routinely used for sterile filtration, which is around 0.2 μm, is necessary. However, small pore sizes can be more demanding towards the filter with higher risk of clogging and rupturing. Therefore, initially also pore sizes up to 3 μm were tested but discarded later on in the qualification process (see further section filtration). The suitability of several commercially available filters were tested according to the above-mentioned criteria in the order of increasing effort. Filters were excluded from subsequent analysis if they disintegrated during extraction, broke after liquid nitrogen application or too inflexible for folding into tubes, left visible residues after GC derivatization of a blank extraction (which would risk clogging the columns or too high blanks) or had high blank levels in either of the applied measurement platforms. Different filter types such as depth or membrane filters as well as different filter materials such as cellulose mixed ester (cellulose nitrate and–acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon or glass fiber were tested. Hydrophilic filters were activated in distilled water and hydrophobic filters in EtOH to enable quick filtration of cell suspensions  depositing on the filter as filter cake from the fluid (e.g. cell culture medium) which passes through the filter and becomes the filtrate . For samAlterations of biological parameters such as cell size, cell form, cell density, cell age, pH, salinity, viscosity, temperature, extracellular matrix or protein content, to increase filtrate flux  directly6 of eukaryotic cells  to 1-10∙109 of prokaryotic cells (e.g. E. coli) per measured sample.However here, the technical parameters pore size, filter type and filter material are fixed by the need to (i) retain all possible cell types (pore size around 0.2 μm) and (ii) compatibility with subsequent extraction and measurements (hydrophobic PTFE membrane filter). Filtrate flux increases with decreasing ratio of cells per filter area, but the increase is confined by the need for sufficient cell mass for measurements and still good to handle filter sizes. In order to achieve some safety margin allowing also filtration of non-ideal cell suspensions relatively large filters with a 47 mm diameter were selected. These filter sizes with the small pore size of 0.2 μm worked very well with the required cell numbers ranging from 1-10∙10The increase of pressure difference increases the filtrate flux but at the same time intensifies filter cake compression and thus mechanical strain on cells. Additionally, filter cake compression increases filter cake resistance so that filtrate flux improvement is finite. Impact of vacuum strength ranging from strongest vacuum with 20 mbar to weakest with 250 mbar was tested on cell integrity with CHO cells, which are typical eukaryotic cell and mechanically least stable compared to other cell types such as yeast or bacteria. Within the applied vacuum range metabolite levels in cells and in filtrate remained unchanged suggesting sufficient cell integrity. The slight decrease of some metabolite levels from 35 mbar to 20 mbar  could inWeaker vacuum was investigated to avoid unnecessary strain on cells and values ≥70 mbar resulted in high residual fluid in the filter cakes. The residual fluid formed a rigid ice layer during quenching with liquid nitrogen preventing subsequent folding without material losses. Additionally, sufficient filtrate flux allowing filtration of sufficient cell numbers in less than 5 s was only achieved with vacuum strengths <70 mbar. In conclusion, a vacuum strength of 35 mbar was selected as it was easily reproducible in other laboratories and was the best compromise between avoiding leakage and sufficiently fast as well as dry filtration of diverse cell suspensions.Washing steps aim at the removal of contaminations of the cells from the culture medium. The washing step also increases the overall sampling time and thus the risk for interconversions of metabolites with high turnover rates ,22,24–27Composition and temperature of the washing solution is important in order to minimize stress-induced metabolite leakage or release from cells. Cold washing solutions reduce cellular metabolism but also induce the cold shock phenomenon, which is the release or leakage of metabolites due to rapid temperature reduction. The cold shock can occur in any cell type  and is wE. coli. Washing with 13C-glucose leads to metabolization of 13C-glucose during washing and consequently increases 13C glycolysis metabolites with very high turnover rates. The 13C metabolization simultaneously stabilized intracellular levels of the corresponding 12C metabolites for up to 60 s . Therefore, addition of a 13C carbon source to the washing solution in equal concentrations as culture medium is recommended to preserve 12C-metabolite levels especially when fast turnover rates are expected.Often the metabolites adjacent to the main carbon source are the ones with the highest turnover rates, such as glycolysis intermediates glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and fructose-1,6-diphosphate  for glucose. Stabilizing these metabolites during the washing is of high interest, especially for cell types with known fast metabolizations rates such as  to 60 s  and did After washing, the quickest and most efficient way to stop metabolic activity is to snap freeze with liquid nitrogen by pouring it directly on the cells immediately after filtering the washing solution. This also eliminates influence of possible time variations from the subsequent folding of the filter into tubes and addition of quenching solution, which are comparatively the most variable and lengthy steps (sampling in teams of two persons is easier). After filters were folded into tubes, dry-ice precooled solvents were added to the cells to protect metabolites from oxidation, avoid further metabolic reactions during storage and stabilize metabolites against possible temperature fluctuations during sample shipment. The addition of solvents prior to shipment was found to be far more practical than with inert gas as it also risks accidental loss of cells due to the gas stream. The solvents were selected to correspond to the extraction solvents so that subsequent extraction could be performed without any additional transferring steps such as evaporation and resuspension.® FastQuench (FQ) was applied successfully, with only some sporadic clogging observed towards the end of culture when product titer increased and total cell numbers were high (>20∙106), which is in agreement with the literature [The transferability of the method was tested with NS0 cells in a different laboratory with personnel following the protocol described above and without extra training in the method. NS0 cells producing an antibody were selected as an example for a typical production system and were grown in a chemically defined medium for ten days with a daily feed starting on day 2 in 4 replicate reactors. MxPterature . For com® Broad Profiling and MxP® Energy) for both sampling methods from which 180 were known metabolites and 75 known unknown metabolites. The metabolites covered most important biological classes such as amino acids, energy metabolites, carbohydrates, vitamins, cofactors, nucleotides, complex lipids and fatty acids.A total of 255 metabolites were found at concentration levels above the limit of quantification  and two essential vitamins  with significantly lower values than in FQ samples . Thus, CE. coli due to the higher cell death rates for NS0 cells.The fact that most of the measured metabolites were significantly higher in C samples than in FQ samples also shows how complex the supernatant of cells grown in chemically defined medium can become within three days of inoculation. Cell death and active secretion of intracellular metabolites has been discussed previously in literature ,25 and tThe metabolites detected with lower levels in C than in FQ samples also suggest that these metabolites may be preferentially used by the cells and are not adequately fortified in the medium, thus making them for supplementation potentially increasing cell growth. Some of these metabolites are already well known to be beneficial when supplemented such as α-ketoglutarate  or lipidThe contamination with cell culture medium of centrifuged samples results in very misleading interpretations, often not only metabolite levels between sampling methods differed but also the progression of metabolite levels with culture duration differed considerably .For example, intracellular glucose and lactate levels in C samples would seem to be rather constant and only decrease slightly in the later days. The lactate to glucose ratio remained constant in the range 0.4 to 0.6. In contrast, FQ samples suggest lower glucose and lactate levels at day 3  indicating efficient glycolysis into lactate paired with efficient secretion. Accordingly the lactate to glucose ratio of 0.3 is very low and increases with culture duration up to 0.9 which correspond better to expectations and observed extracellular levels of glucose and lactate . FQ sampThus, contamination with cell culture medium has a high impact and risk misleading results as early as day 3 of cell culture, even when the cell viability is greater than 95%. Special care and caution should be taken in very rapid sampling in the sub-second range where washing has to be omitted to achieve sufficient speed, e.g. by determination of extracellular levels and mass balancing for every investigated metabolite.® FastQuench was developed.In this study the most critical factors for each step during sampling for metabolomics analysis were determined  and accoE. coli) and eukaryotic (CHO) cells was successfully applied for NS0 cells at a different laboratories with staff who was not familiar with the method. Cell size and form are the main factors determining the retention of cells on filters [The sampling method–established and validated for prokaryotic  and 1-10∙109 for prokaryotic cells (e.g. E. coli) per filter are a good working range and for less cells filter diameter can be accordingly reduced. Second, we recommend performing once the full sampling and measurement of 2 blanks, 5x replicates with isotonic washing and 5x replicates with isotonic washing plus 13C carbon source, measuring both cells and final filtrates.As test experiments we recommend to determine, e.g. with 5 different cell numbers, the optimal amount of cells that can be well filtered with the selected filtration assembly, applied vacuum (50 mbar to 35 mbar) and cell culture type without risking clogging. For 47 mm filters cell numbers ranging from 1-10∙10Other extraction protocols can be expected to work well as long as care is taken to avoid unnecessary metabolite degradation from elevated temperatures, prolonged handling or repeated evaporations  and alloAutomatization of fast filtration would further improve sampling speed and results. Although automation is always preferable, the instrumental demand is much higher and may not be worthwhile especially for sporadic users.® FastQuench, is compatible with high-throughput, routine extraction and measurement protocols used for sample types such as plasma, blood, or urine, thereby leading to higher comparability of results between these different matrices.The sampling method presented here is straightforward and relies on readily available equipment allowing its use in different labs. Additionally, the developed fast filtration method, MxPS1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


LV diastolic vortex formation has been suggested to critically contribute to efficient blood pumping function, while altered vortex formation has been associated with LV pathologies. Therefore, quantitative characterization of vortex flow might provide a novel objective tool for evaluating LV function. The objectives of this study were 1) assess feasibility of vortex flow analysis during both early and late diastolic filling in vivo in normal subjects using 4D Flow cardiovascular magnetic resonance (CMR) with retrospective cardiac gating and 3D vortex core analysis 2) establish normal quantitative parameters characterizing 3D LV vortex flow during both early and late ventricular filling in normal subjects.With full ethical approval, twenty-four healthy volunteers (mean age: 20±10 years) underwent whole-heart 4D Flow CMR. The Lambda2-method was used to extract 3D LV vortex ring cores from the blood flow velocity field during early (E) and late (A) diastolic filling. The 3D location of the center of vortex ring core was characterized using cylindrical cardiac coordinates , Radial (R)). Comparison between E and A filling was done with a paired T-test. The orientation of the vortex ring core was measured and the ring shape was quantified by the circularity index (CI). Finally, the Spearman’s correlation between the shapes of mitral inflow pattern and formed vortex ring cores was tested.Distinct E- and A-vortex ring cores were observed with centers of A-vortex rings significantly closer to the mitral valve annulus , closer to the ventricle’s long-axis  and more elliptical in shape  compared to E-vortex. The circumferential location and orientation relative to LV long-axis for both E- and A-vortex ring cores were similar. Good to strong correlation was found between vortex shape and mitral inflow shape through both the annulus (r=0.66) and leaflet tips (r=0.83).Quantitative characterization and comparison of 3D vortex rings in LV inflow during both early and late diastolic phases is feasible in normal subjects using retrospectively-gated 4D Flow CMR, with distinct differences between early and late diastolic vortex rings.The online version of this article (doi:10.1186/s12968-014-0078-9) contains supplementary material, which is available to authorized users. Vortex formation within the left ventricular (LV) blood flow has been suggested to critically contribute to efficient blood pumping function . A vortein vivo and in vitro studies have reported vortex ring formation during early diastolic filling, originating at the distal tip of the mitral valve (MV) leaflets , with μ as the λ2 average of voxels with λ2 < 0) allowed identification of a separate compact vortex ring core (when detected) in all subjects. The strong inter- and intra-observer agreements  at the late filling peak compared to early filling, which can be attributed to the lower velocity and shorter length of inflow jet during late filling compared to early filling. The relatively closer position of the vortex ring core to the LV long-axis  at the late filling, can be explained using the confirmed correlation between shapes of the vortex ring core and the mitral valve opening, where a restricted opening of the mitral valve during late filling results in a vortex core center closer to the long-axis compared to full valvular opening at the early filling. Since vortex ring originates from the inlet jet at the distal tip of the mitral valve (MV) leaflets ,10,13, vThe relatively small variation between normal subjects in derived parameters Table  indicateThe suggested LV-normalized vortex parameters might help to provide more insights about the normal vortex formation and provide normative parameters to compare the 3D vortex flow between controls and patients. This could help to understand the hemodynamics of patients with impaired LV relaxation and restrictive filling, where the E/A ratio is abnormal. In addition, the close correlation found between vortex formation and the flow at the tip of the opened mitral valve leaflets suggests that patients with impaired leaflet function, as can be seen in patients with left ventricular dysfunction  after mi3 was used. However, it was our aim to evaluate large scale vortex ring cores which are expected to have volumes significantly larger than the MR voxel size. In three volunteers, a higher resolution of 2.3×2.3×3 mm3 was used, which did not result in significant different findings from the other subjects. Further methodological and quantitative analysis on the effect of acquisition resolution may be helpful but was beyond the scope of this work. Identified Lambda2-based vortex cores could not be used for volumetric measurements (e.g. vortex volume or size) as applying different Lambda2 isovalue thresholds can result in different volumes for the same vortex core. Therefore, the vortex parameters derived in this study were chosen as not to be dependent on vortex volume. 4D Flow data was acquired using free breathing scans and no motion compensation was applied. Nevertheless, no motion artifacts were visually observed in the velocity data, and since all subjects underwent the same scan protocol, potential inter- and intra-subject effects on the measurements might be assumed to be similar among all subjects.Limitations of this study include a relatively small number of healthy subjects and lack of comparison with patients. However, an objective detection of possible anomalies in the vortex flow of patients should be preceded by finding reliable quantitative measures defining the reference normal vortex flow. The current study was performed in a relatively young population (age range 9 – 44 years). Global diastolic function parameters, as the E/A ratio remain relatively stable during the second, third and fourth decade of life , which ein vivo study using 4DFlow CMR to confirm previous CFD findings of vortex ring formation during late filling and to provide standardized parameters that allow quantitative characterization of vortex flow during both early and late left ventricular filling. The derived quantitative parameters provided consistent measurements within the studied population and strong correlation was found between the shape of the formed vortices and the shape of the inflow area at the level of both the mitral annulus and the tip of the opened MV leaflets. This study provides reference parameters defining normal vortex flow, which may allow objective quantitative evaluation of vortex flow in patients with cardiac disease.In summary, this is the first