Methods Mol Biol Research Articles

A Novel Mechanism of Megakaryopoiesis from Pre-Adipocytes: Involvement of Transferrin/ CD71/ TPO Pathways

Platelets are released from terminally differentiated megakaryocytes (MKs) through lineage commitment, although the underlying mechanisms of megakaryopoiesis and subsequent thrombopoiesis are incompletely understood. MKs were reportedly generated from not only hematopoietic stem cells (HSCs), but also pre-adipocytes, without any gene transfer in an in vitro culture system (Matsubara et al, 2012 Methods Mol Biol). Because of high efficiency of platelet production from pre-adipocytes, much interest has been attracted to clarify the mechanisms of megakaryopoiesis from pre-adipocytes. Here we demonstrate a novel mechanism that megakaryopoiesis from pre-adipocytes is regulated by the inducible production of endogenous TPO via transferrin receptor CD71. NF-kB pathway in pre-adipocytes might modulate the TPO production through the stimulation of transferrin.Previously, we reported that pre-adipocyte cell line OP9 has the gene expressions of thrombopoietin (TPO) and its receptor c-MPL. This study first examined the TPO protein levels in supernatant from human (Cell Applications) and mouse pre-adipocytes cultured with MK lineage induction (MKLI) media (Ono et al, 2012 Blood, Matsubara et al, 2012 Methods Mol Biol) in the absence of recombinant TPO (rTPO). The TPO levels were 36.6 ± 9.2 pg/mL in mouse pre-adipocytes on Day 8 and 36.1 ± 5.8 pg/mL in human pre-adipocytes on Day 8, and undetectable levels of TPO were observed in both cells before MK induction. Differentiation efficiency (approximately 20%) of MKs from pre-adipocytes cultured with rTPO was similar to that without rTPO. The function of pre-adipocyte-derived platelets cultured with rTPO was similar to that without rTPO when assessed by incorporation of platelets into thrombus formation on the collagen-coated surface under flow conditions. The number of MKs produced from pre-adipocytes was markedly decreased in the presence of anti-c-MPL blocking antibody, AMM2. Regarding the MK differentiation from HSCs, it is well known that HSCs do not differentiate into MKs in the absence of rTPO. We did not observe the TPO secretion from human bone marrow CD34(+) cells and mouse bone marrow Lin(-)Sca1(+)c-kit(+) cells in a culture condition using MKLI media in the absence of rTPO. The observations suggest that the MK differentiation from pre-adipocytes has a distinct mechanism of megakaryopoiesis from HSCs. These findings indicate the unique mechanism of megakaryopoiesis from pre-adipocytes; the endogenous TPO stimulation via c-MPL in pre-adipocytes has activity for promoting MK differentiation and subsequent production of functional platelets.To elucidate the molecular mechanism of TPO production during MK differentiation from pre-adipocytes, we examined which components of MKLI media were responsible for TPO production. We found iron-saturated transferrin, but not apo-transferrin, included in MKLI media is the critical factor to induce TPO production and MK differentiation from pre-adipocytes. The effects of transferrin were not observed when pre-adipocytes were cultured in the presence of anti-CD71 blocking antibody, 8D3, or deferoxamine mesilate, an iron-chelating agent. The effects of transferrin on TPO production and MK differentiation were also abolished in pre-adipocytes transfected with siRNA-CD71, and the TPO levels were 44.8 ± 15.9 pg/mL for siRNA-control and undetectable levels for siRNA-CD71 on Day 6. We next investigated a mechanism of inducing TPO production via CD71 in pre-adipocytes. The 5’ region of human TPO gene contains transcription factor-binding sequences, such as GATA1, GATA2 and NF-kB. Since NF-kB is a downstream target of CD71, we then examined whether NF-kB involved in MK differentiation derived from pre-adipocytes. Nuclear translocation of NF-kB in pre-adipocytes was observed when cells were stimulated with transferrin. Both TPO secretion and MK production were decreased in the presence of a peptide to inhibit nuclear translocation of NF-kB. Taken together, it is concluded that NF-kB pathway in pre-adipocytes modulates megakaryopoiesis through endogenous TPO production via CD71. The present findings suggest that pre-adipocytes have a unique mechanism of megakaryopoiesis through lineage commitment. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Value of Sequencing-Based Minimal Residual Disease Detection in Multiple Myeloma

▪Background: Although molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual, which is laborious and time-consuming. We utilized the LymphoSIGHTTM platform, which employs consensus primers and next-generation sequencing (NGS) to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone, to assess mCR. This technique has been shown to have 1-2 logs greater sensitivity compared to ASO-PCR and flow cytometry, respectively (Faham et al, Blood 2012). Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. We compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts and bone marrow (BM) in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting.Methods: One hundred and nine Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or better after ASCT. BM slides from 84 MM patients and fresh/frozen BM cells from 25 MM patients at diagnosis, as well as autografts/post-ASCT BM cells from each patient, were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using NGS. Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples.Results : Myeloma clonotypes could be identified in autografts/post-ASCT BM cells in 98 of 109 patients (90%) and by ASO-qPCR in 63 of 101 patients (62%). MRD by NGS was assessed in autografts of 89 patients. 70 of 89 patients (79%) were positive by NGS; 28 of 62 patients (45%) were positive by ASO-qPCR. Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, the sensitivity of ASO-PCR was 10-4-10-5, whereas that of NGS was 10-6 or lower when a sufficient amount of DNA was available for analysis. Eight cases where MRD was not detected in the autograft by NGS (MRDNGS(-)) and 38 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide, while 11 MRDNGS(-) cases and 32 MRDNGS(+) cases were followed without post-ASCT therapy. The MRDNGS(-) cases without post-ASCT therapy showed significantly better progression-free survival (PFS) than the MRDNGS(+) cases without post-ASCT therapy (P = 0.012) (Figure 1A) although overall survival rates were comparable between these groups. To investigate the value of sensitive detection by NGS, we compared PFS in 11 MRDNGS(-) cases (Group 1) with the 12 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 showed significantly better PFS than Group 2 (P = 0.027) (Figure 1B). Furthermore, 9 MRDNGS(-) in post-ASCT BM cases tended to show a better PFS than 18 MRDNGS(+) in post-ASCT BM cases (P = 0.075) (Figure 1C). In a multivariate analysis, post-ASCT therapy using novel agents (P <0.001) and MRDNGS(-) in autograft (P=0.025) were independently associated with superior PFS while ISS I/II vs III (P = 0.387) and MRDASO(-) in autograft (P=0.174) were not.Conclusions: In this study, we showed the prognostic value of MRD detection using the NGS-based LymphoSIGHT platform in autografts of patients with MM who received and in those who did not receive post-ASCT therapy with novel agents. The NGS platform has improved sensitivity compared with ASO-qPCR in detecting MRD in autografts. Patients with low level MRD detected by NGS but not by ASO-qPCR have worse prognosis compared to patients who are MRD negative by sequencing, which underscores the need for sensitive detection. [Display omitted] DisclosuresZheng:Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Identification of Novel Methylation Biomarkers to Predict Clinical Response to SGI-110, a Second Generation Hypomethylating Agent (HMA), in Patients with Acute Myeloid Leukaemia (AML)

Introduction: SGI-110 is second generation HMA formulated as a dinucleotide of decitabine (DAC) and deoxyguanosine delivered as a small volume, pharmaceutically stable SQ injection allowing longer half-life and more extended decitabine exposure than DAC IV infusion. SGI-110 differentiated pharmacokinetic profile resulted in potent hypomethylation and clinical responses in previously treated MDS and AML patients in the phase 1 trial (Kantarjian et al. 2012).Here, we have identified novel DNA-methylation biomarker candidates that may be predictive of response to SGI-110 using Differential Methylation Hybridisation (DMH) profiling of the NCI-60 cell line panel (Fassbender A et al, Methods Mol Biol 2010). Cell lines were stratified based on SGI-110 EC50 values from Colony Forming Assay and degree of LINE-1 (Long Interspersed Nucleotide Elements) demethylation post-SGI-110 treatment. The stratification was used to classify cell lines into SGI-110 sensitive and resistant and to generate 249 genomic methylation sites as candidate biomarkers of response to SGI-110. Candidate markers from this analysis will now be compared in DNA samples derived from whole blood from treatment naïve and relapsed/refractory AML patients that were classified into responders and non-responders after treatment with SGI-110 in our phase 2 clinical study.Methods: DMH was used for unbiased profiling of methylated loci throughout the genome. Briefly, the genome is cleaved into a defined fragment library using methylation unspecific restriction enzymes, followed by PCR-adapter ligation. In a second step, the fragments are digested by methylation-sensitive restriction enzymes cutting only at unmethylated restriction sites, while methylated fragments and fragments that do not have any restriction site were protected from the restriction digestion. The methylated fragments and the control fragments are then selectively amplified by PCR and the PCR product is labeled and hybridized to Epigenomics’ proprietary DMH microarray. Direct Bisulfite Sequencing (DBS) is used to assess the DNA-methylation pattern of marker candidates at CpG level. Regions with CpGs of interest are amplified to generate PCR products that are directly sequenced using a 4 dye labelled ddNTP Sanger sequencing. Quantitative DNA methylation of CpG region with a lengths of up to 550 bp are derived from the resulting trace files using the ESME software (Lewin J et al, Bioinformatics. 2004 Nov 22;20(17):3005-12).Results: Using this method, we have seen a clear correlation of high sensitivity to SGI-110 with high LINE-1 methylation level. Five (leukemia) cell lines classified as sensitive to SGI-110 by EC50 values were among the 6 cell lines with the highest LINE-1 methylation (> 73% methylation) while resistant cell lines showed a LINE-1 methylation < 70%.A high degree of correlation was observed between LINE-1 methylation and genome-wide DNA-Methylation measured by DMH in over 50k genomic sites and supported the role of LINE-1 as a useful indicator for sensitivity to SGI-110 and global DNA methylation. Fifty genomic fragments that better characterized sensitive and resistant cell lines were selected for further validation.The differential DNA-methylation discriminating between SGI-110 sensitive and resistant cancer cell lines (n=18) was validated by DBS analysis at CpG level in genomic fragments. We will present data obtained from the DBS evaluation of methylation in these validated methylation marker candidates in up to 45 AML blood samples that were classified into responders and non-responders after treatment with SGI-110 in a phase 2 clinical trial.Conclusions: We identified DNA methylation patterns associated to sensitivity and resistance to the novel HMA SGI-110. The validated methylation biomarker discovery process based on DMA and DBS approaches may help to identify and characterise specific subgroups of AML patients that could preferentially respond to SGI-110. DisclosuresJueliger:Astex Pharmaceuticals: Employment. Lyons:Astex Pharmaceuticals: Employment. Lewin:Epitenomics AG: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Taverna:Astex Pharmaceuticals, Inc.: Employment.

Genome-wide analysis of methylation in bovine clones by methylated DNA immunoprecipitation (MeDIP).

Methylated DNA immunoprecipitation (MeDIP), when coupled to high-throughput sequencing or microarray hybridization, allows for the identification of methylated loci at a genome-wide scale. Genomic regions affected by incomplete reprogramming after nuclear transfer can potentially be delineated by comparing the MeDIP profiles of bovine clones and non-clones. This chapter presents a MeDIP protocol largely inspired from Mohn and colleagues (Mohn et al., Methods Mol Biol 507:55-64, 2009), with PCR primers specific for cattle, and when possible, overviews of experimental designs adapted to the comparison between clones and non-clones.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Insights from Genetic Models of Melanoma in Fish

Fish melanoma models are increasingly used in studies of both spontaneous and induced melanoma formation. The melanoma Xiphophorus model was the first genetic model of melanoma available to researchers since the 1920s (Klin Wochenschrift 7:1561–1562, 1928; Z Indukt Abstammungs 44:253–257, 1927). Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research (Methods Mol Biol 461:521–539, 2008). These models are now starting to produce a wealth of novel information on the genetics of melanoma (including somatic mutations), signaling pathways and molecular mechanisms, immune responses, and determinants of metastasis. Moreover, the models are extremely well suited for drug/small-molecule screens aimed at discovering new therapeutic strategies in general treatment and personalized medicine and for the study of cancer-immune cell interactions. This review provides a summary of current and prospective studies in fish models of melanoma and pinpoints the translational potentials of melanoma models in fish.

Folding and Lipid Membrane Interactions of BamD, An Essential Component of the β-Barrel Assembly Machine from Escherichia Coli

BamD (YfiO) [1] is an essential component of the β-barrel assembly machine of the outer membrane of Escherichia coli. BamD is synthesized in the cytosol and translocated across the cytopasmic membrane in unfolded form for processing and transport to the outer membrane by the LOL system [2].To examine the interactions of BamD with lipid membranes and BamD folding, we have over-expressed BamD into the periplasm and isolated it from membrane fractions in unfolded form in 8 M urea. Circular dichorism spectroscopy indicated secondary structure formation in BamD upon rapid dilution of the denaturant urea in aqeuous buffer. In the absence of lipid bilayers, BamD displayed a lower content of α-helix structure. The CD-data indicated that native-like folding of BamD required the presence of lipid-membranes of phosphatidylglycerol and phosphatidylcholine. An increased content of phosphatidylglycerol facilitated BamD folding. Fluorescence spectroscopy demonstrated BamD binding to both zwitterionic and negatively charged lipid. The lipid/BamD stoichiometry was estimated to 17 phosphatidylglycerol/BamD and to 31 phosphatidylcholine/BamD.[1] Malinverni JC, Werner J, Kim S, Sklar JG, Kahne D, Misra R, Silhavy TJ (2006) YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli. Mol Microbiol 61:151-164.[2] Narita S, Tokuda H (2010) Sorting of bacterial lipoproteins to the outer membrane by the Lol system. Methods Mol Biol 619:117-129.

A Comparison Between Next-Generation Sequencing and ASO-qPCR For Minimal Residual Disease Detection In Multiple Myeloma: The Clinical Value In ASCT Setting

BackgroundAlthough molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. We utilized a sequencing method, termed the LymphoSIGHT™ platform, which employs consensus primers and high-throughput sequencing to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone. The sequencing method is quantitative at frequencies above 10-5 and the lower limit of detection is below 10-6. Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. MethodsWe compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Because myeloma cells exist patchily in bone marrow (BM), myeloma cells in PBSC autografts may reflect the whole amount of tumor in vivo. Thirty-six Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or complete response (CR) after ASCT. BM slides from 28 MM patients and fresh BM cells from 8 MM patients at diagnosis as well as autografts were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using next-generation sequencing (NGS). Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples (Faham et al, Blood 2012). ResultsMRD in autografts could be assessed 36 of 36 (100%) by NGS and 30 of 36 (83%) by ASO-qPCR. MRD in autografts was detected in 27 of 36 (75%) by NGS and 11 of 30 (37%) by ASO-qPCR (Figure 1A). Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, ASO-qPCR could not detect myeloma cells at MRD levels of 10-5 or lower. Two cases where MRD was not detected by NGS (MRDNGS(-)) and 14 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide while 7 MRDNGS(-) cases and 13 MRDNGS(+) cases were followed without post-ASCT therapy. The best post-ASCT responses were as follows: 6 (67%) mCR, 1 (11%) sCR and 2 (22%) VGPR in 9 MRDNGS(-) cases; 2 (14%) mCR, 2 (14%) sCR, 2 (14%) CR, 8 (58%) VGPR in 14 MRDNGS(+) cases with post-ASCT therapy; 2 (15%) sCR, 10 (78%) VGPR and 1 (7%) PR in 13 MRDNGS(+) cases without post-ASCT therapy. The MRDNGS(-) cases tended to show a better PFS than the MRDNGS(+) cases with post-ASCT therapy (P = 0.400) and showed a significantly better PFS than those without post-ASCT therapy (P = 0.032) (Figure 1B) although overall survival rates were comparable among the three groups. To investigate the value of sensitive detection by NGS, we compared PFS in 7 MRDNGS(-) cases (Group 1) with the 6 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 tended to show a better PFS than Group 2 (P = 0.091) (Figure 1C). This underscores the value of sensitive detection of MRD in MM. [Display omitted] ConclusionsA high correlation between NGS and PCR MRD results was observed, and MRD-negativity in PBSC autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR. Disclosures:Zheng:Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment.

Chronic Infection, a Neglected Cause Of Development Of Monoclonal Gammopathy Of Undetermined Significance (MGUS) and Myeloma

IntroductionChronic infection can lead to B-cell malignancy via the direct transformation of infected B-lymphocytes or indirectly via cell transformation consecutive to chronic antigen-driven stimulation; the two mechanisms may occur simultaneously (Seifert et al. Methods Mol Biol 2013; 971:1; Hermouet et al. New Engl J Med 2003; 348:178; de Martel et al. Lancet Oncol 2012). Numerous studies have established that viruses (Epstein-Barr virus (EBV), human herpes virus 8, hepatitis C virus (HCV)) or bacteria (H. pylori) can induce lymphoma and chronic lymphocytic leukemia (CLL). In contrast, the role of chronic infection in the pathogenesis of myeloma is rarely investigated. Yet monoclonal immunoglobulins (mc Ig) that arise in HCV-positive myeloma patients typically target the virus (Bigot-Corbel et al. Blood 2008). Thus HCV infection may lead to monoclonal gammopathy of undetermined significance (MGUS) and eventually, myeloma. The present study shows that mc Ig also frequently target EBV, and less frequently, H. pylori. MethodsIn order to study the specificity of mc Ig, we designed a new assay based on a multiplexed infectious protein (MIP) microarray that combines representative panels of epitopes of a panel of germs that included HCV, EBV, H. pylori and 5 other germs, spotted in triplicate on nitrocellulose-coated slides (Feron et al. Anal Biochem 2013; 433:202). Slides were first incubated with either serum or purified mc Ig, then with an infrared dyed (IRD)-labeled secondary antibody, and fluorescence signals were detected and quantified. ResultsThe specificity of purified mc Ig from 101 patients diagnosed with MGUS (n=34) or myeloma (n=67) was analysed using the 8-germ MIP microarray assay. We found that 20.6% of MGUS and 24.2% of myeloma patients (p=0.810) presented with a mc Ig that was specific for an antigen from HCV, EBV, or H. pylori. Indeed for 23/101 patients (22.7%), the purified mc Ig recognized HCV (n=10), EBV (n=11) or H. pylori (n=2). In contrast, none of the 101 mc Ig studied targeted CMV, a virus against which a majority of individuals possess antibodies.Interestingly, HCV- and EBV-specific mc Ig all targeted a single antigen. For 10/11 (91%) of the HCV-positive patients, the mc Ig was directed against HCV core protein. Regarding EBV-positive patients, the mc Ig specifically recognized EBV in 23% cases (11/59 tested), and in all cases the mc Ig targeted Epstein Barr nuclear antigen (EBNA). For 8% (2/25 tested) of H. pylori-positive patients, the mc Ig targeted different antigens of H. Pylori. The specificity of each mc Ig was confirmed by Western Blot analysis. ConclusionAltogether, 22.7% of the 101 MGUS and myeloma patients examined presented a mc Ig specific for HCV, EBV or H. pylori. Thus beside lymphoma and CLL, chronic infection by these 3 germs can also induce MGUS and eventually, trigger the pathogenic processes that lead to myeloma. Efforts should be made to identify the subsets of patients with mc Ig specific for HCV, EBV and H. pylori, preferably at the MGUS stage, as anti-infection treatment is expected to cure MGUS and prevent progression toward myeloma. Disclosures:No relevant conflicts of interest to declare.

Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Investigation of the novel pharmacology of Neuropeptide Y (NPY) receptor dimers constrained by bimolecular fluorescence complementation (BiFC)

Dimerisation of GPCRs potentially influences their pharmacology via binding site cooperativity or modified signalling. We have used bimolecular fluorescence complementation (BiFC; Kilpatrick et al. (2012) Methods Mol Biol 897) to constrain Neuropeptide Y (NPY) Y1 receptor (Y1R) homo and heterodimers of known composition. Using quantitative platereader imaging we measured the internalisation of BiFC receptor dimers simultaneously with endocytosis of SNAP tagged Y1R protomer to compare the pharmacology of Y1/Y1, Y1/Y4 or Y1/Y5 BiFC dimers.Y1/Y1 and Y1/Y4 BiFC dimers internalised in response to NPY or PP (Y1/Y4), with potencies comparable to individually expressed YR subtypes. Endocytosis of Y1/Y1 or Y1/Y4 dimers to NPY was also inhibited by the Y1 antagonist BIBO3304, with expected pKb values ((8.2–8.5) from NPY concentration‐response curve shifts). However, the Y5 selective agonist PP(1–17)(Ala31, Aib32) NPY(18– 36) showed reduced potency for constrained Y1/Y5 BiFC dimers, compared to Y5‐GFP internalisation. BIBO3304 demonstrated a lower apparent affinity (pKb 7.4 ± 0.1) for Y1/Y5 BiFC dimers compared to the SNAP‐Y1 sub‐population (pKb 8.2 ± 0.4). This suggests an interaction between Y1 and Y5 orthosteric binding sites of Y1/Y5 BiFC dimers, with implications for selective targeting of this dimer as a mediator of central NPY appetite responses. LK is supported by the British Pharmacological Society.

Optimization of Mitochondrial and Cytosolic pH Determination in Madin Darby Kidney Cells using CLSM Images

Reactive Oxygen Species (ROS) production and impairment of mitochondrial functioning is a central research topic. A recent review for Multiple Sclerosis1. Investigation of mitochondrial functioning in living cells under normal physiological conditions and metabolic inhibition provides biologically relevant information using confocal cell imaging2,3 Cytosolic calcium clearance does not necessarily lead to mitochondrial dysfunction as long as the mitochondrial matrix pH stayed acidified compared to the cytosol.2 Studying intra-mitochondrial versus cytosolic electrolyte homeostasis in living cells under metabolic stress might be crucial to understand the conditions where mitochondria become detrimental by producing ROS. Caveats are presented to properly obtain information about mitochondrial and cellular pH, structure and properties of the mitochondrial network under normal conditions and metabolic inhibition, by using optimized protocols4. The method critically depends on availability and selection of nuclear areas with lowest background fluorescence contribution while keeping experiment conditions rigorously similar. We present the influence of factors such as cell type, plating number, age of the cells, attachment surface properties, cell confluency, loading protocol of the dyes, background area selection on the values obtained. Optimization of parameters such as objective choice, detector voltage stability, fluorescence intensity saturation, illumination power and bleaching, imaging depth, point spread function (PSF) and reduction of cell motility are elucidated. Confluent Madin Darby Canine Kidney (MDCK) cells were loaded with mitotracker green and pH indicator SNARF. Images were collected with a Zeiss LSM 510 Meta CLSM. Data were analyzed with ImageJ4, and Matlab.1. Lassmann, H., and van Horssen, J., FEBS Lett. 20112. Smets, I. et al., Am. J. Physiol. Renal Physiol., 20043. Baron, Sz. et al., JASN, 20054. vandeVen, M, & Balut, C. et al. Methods Mol Biol. 2010

Peptide Microarrays with Near Zero background enable high quality and massive data production from seroscreening of autoimmune diseases

Peptide Microarrays with Near Zero background enable high quality and massive data production from seroscreening of autoimmune diseases

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential, non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity-based search with the suicide inhibitor haemagglutinin (HA)-SUMO-vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin-specific protease-like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l--an essential but distant zebrafish homologue of USPL1--also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low-abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.

Imaging and Genomics: Is There a Synergy?

One promising route for imaging is exemplified by the article by Gevaert et al in this issue: That study, which analyzed features extracted from non–small cell lung cancer CT and PET cases, offers an original approach to exploring the clinical prognostic value of imaging-genomics.

Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine

Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403–2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161–171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12–17, 2007; Sunami et al. in Anal Biochem 357(1):128–136, 2006; Ishikawa et al. in FEBS Lett 576(3):387–390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238–241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations.

High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum-derived vesicles to Legionella-containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4-phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P-binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.

Nature’s Genetic Gradients and the Clinical Phenotype

When God created the earth, he placed (modern) mankind in East Africa ≈200 000 years ago, as suggested by the phylogenetic and archeological studies.1,2 These hunter-gatherers populated the land and started to migrate, first to the west and southern tip of Africa and then north toward North Africa. Mankind gradually crossed the Arabian Peninsula to the Middle East and Eurasia. Then, over thousands of years of northerly movement, man crossed the Bering land bridge that connected Siberia to Alaska and migrated down to the Americas. This out-of-Africa migration took thousands of generations. It was interrupted by the Ice Age, which led to depopulation and isolation of the surviving population followed by the new waves of migrations, resulting in multiple founding populations.3 Articles see pages 552 and 558 At the genetic level, the tree of life was produced through replication of the genome. For the genome to replicate, cells are equipped with DNA polymerases to copy the parental DNA, which they do at blazing speeds.4 However, “no protein is perfect,” and neither are the DNA polymerases. So, the DNA polymerases commit occasional errors in copying the template (parental) DNA and incorporate wrong nucleotides into the new DNA strands. To correct the errors, nature afforded the cells with DNA repair enzymes, which are assigned the task of maintaining the integrity of the nucleotide pools before replication.5 Nevertheless, the process of DNA replication and editing, although exceedingly precise, is imperfect. The error rate of the DNA replication and editing machinery is estimated to be at 10−6 to 10−10 nucleotides incorporated into the new strand of DNA.6,7 The inheritance is forward. Therefore, the misincorporated nucleotides are passed on to the next generation, and with each generation, new errors in DNA replication are introduced. The occasional “errors of …

Vascular Gap Junctions in Hypertension

The proteins that form the gap junctions (connexins) are widely expressed in organs that are central to the development of hypertension: endocrine organs, kidney, brain, heart, and vasculature (Figure 1). Surprisingly, there is little information on the modification of connexins in hypertension in any of these organs except the vasculature, the subject of our review, but it would be hoped that this lack of information might spur research on these organs. Figure 1. Possible roles for the connexins in the control of blood pressure. A, Electron micrograph of a longitudinal view of an arteriole. Vascular connexins can play multiple roles in regulating function and vasomotor tone. Cell–cell communication, both radial and longitudinal, links the cells of the arteriolar wall. B, Most every organ involved in the control of blood pressure abundantly expresses ≥1 of the connexins. Essentially no experimental evidence exists regarding a possible link between connexins and hypertension in nonvascular tissues. Augmented vasomotor tone typically plays a key role in the development of hypertension, and tone depends on cell–cell communication established by paracrine molecules and, in addition, gap junctions. Paracrine-based linkages between cells of the vasculature are well known, and the possible roles of such linkages in the genesis of hypertension have been extensively explored. Much less is known of the roles of gap junctional communication in establishing vasomotor tone and of the effects of modification of gap junctions on hypertension. The gap junctions are formed by joining 2 hexameric assemblies of connexin protein monomers, that is, hemichannels (1 in Figure 2A). In the vasculature, assays of message and protein show that the hemichannels can be assembled from combinations of 4 connexins, named according to their apparent molecular weight: Cx37, Cx40, Cx43, and Cx45. Two hemichannels are linked to form a gap junction, which can provide both homocellular …

Endocytosis pathways in endothelium: how many?

vascular endothelium is a cellular monolayer with the organization of a simple squamous epithelium that lines the entire cardiovascular system and constitutes a regulatable barrier between blood and tissues ([9][1], [17][2]). By its location, it is easily accessible to blood-borne drugs or imaging