搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Alternative splicing is a major contributor of transcriptomic complexity,but the extent to which transcript isoforms are translated into stable,functional protein isoforms is unclear. Furthermore,detection of relatively scarce isoform-specific peptides is challenging,with many protein isoforms remaining uncharted due to technical limitations. Recently,a family of advanced targeted MS strategies,termed internal standard parallel reaction monitoring (IS-PRM),have demonstrated multiplexed,sensitive detection of pre-defined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here,we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides,which are specific to individual gene product isoforms,serve as “triggers” and “targets” in the IS-PRM method,Tomahto. Using the model human stem cell line WTC11,LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic “trigger” peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest,predicted to contain corresponding endogenous “target” peptides. Compared to DDA mode,Tomahto increased detectability of isoforms by 3.6-fold,resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach,called LRP-IS-PRM,is a new modality for generating protein-level evidence of alternative isoforms – a critical first step in designing functional studies and eventually clinical assays. View Publication

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically,it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose,a large set of proteins representing diverse structures and functions,some of which are known or potential drug targets,were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86{\%} of the targets,as determined by luminescence-based plate assays,blotting,and imaging. In order to determine whether endogenously tagged proteins yield more representative models,cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases,only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. View Publication

The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme,named apopain,is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3,the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro,suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death. View Publication

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development,cell growth,and migration,as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers,and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here,we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands,[(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)-phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist),was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1),an antagonist,did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay,SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists SANT-1 and SANT-2 bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway." View Publication

Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease,and offer potentialbackslashrbackslashnfor patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function,and to gain insight into cardiac growth and disease,tissue engineers must quantify the contractile forces of these single cells. Currently,axial contractile forces of isolated adult heart cells can only be measuredbackslashrbackslashnby two-point methods such as carbon fiber techniques,which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificialbackslashrbackslashnsupport layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput,is functionally analogous to current gold-standard axial force assays for adult heart cells,and prescribes elongated cell shapes without protein patterning. Finally,we calibrate these force posts withbackslashrbackslashnpiezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far betterbackslashrbackslashnthan that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culturebackslashrbackslashnplatforms will enable improved cell placement and the complex suspension of cells across 3D constructs. View Publication

The human naive pluripotent stem cell (PSC) state,corresponding to a pre-implantation stage of development,has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate,a normal karyotype,the ability to form teratomas,transcriptional similarities to human pre-implantation embryos,reduced heterochromatin levels,and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP-/- cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state,with implications for early human embryology. View Publication

Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin,which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2,ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation,target gene transcription and osteogenic differentiation. Using dorsomorphin,we examined the role of BMP signaling in iron homeostasis. In vitro,dorsomorphin inhibited BMP-,hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin,which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo,systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver,whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation,normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis. View Publication

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry,we sorted cells with bright (ALDH(br)) and dim (ALDH(lo)) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH(br) cells are indeed a different clone,but when left in normal culture conditions will give rise to ALDH(lo) cells. Furthermore,the ALDH(br) cells grow slower,have low clonal efficiency,and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH(br) and ALDH(lo) cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension,measure ALDH activity,and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH(br) cells grew much slower in primary recipient mice. Histologically,there was no significant difference in the expression of ALDH in primary tumors originating from ALDH(br) or ALDH(lo) cells. Secondary and tertiary xenografts originating from ALDH(br) grew faster and bigger than those formed by ALDH(lo) cells. In conclusion,ALDH(br) cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide,but require support of other cells (ALDH(lo)) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer. View Publication

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers,potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach,particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival,rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623,a clinically viable,highly brain-penetrant LXRα-partial/LXRβ-full agonist selectively kills GBM cells in an LXRβ- and cholesterol-dependent fashion,causing tumor regression and prolonged survival in mouse models. Thus,a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS. View Publication

The purpose of this study is to review the development of BRAF inhibitors,with emphasis on the trials conducted with dabrafenib (GSK2118436) and the evolving role of dabrafenib in treatment for melanoma patients. Fifty percent of cutaneous melanomas have mutations in BRAF,resulting in elevated activity of the mitogen-activated protein kinase signaling pathway. Dabrafenib inhibits the mutant BRAF (BRAF(mut)) protein in melanomas with BRAF(V600E) and BRAF(V600K) genotypes. BRAF(V600E) metastatic melanoma patients who receive dabrafenib treatment exhibit high clinical response rates and compared with dacarbazine chemotherapy,progression-free survival. Efficacy has also been demonstrated in BRAF(V600K) patients and in those with brain metastases. Dabrafenib has a generally mild and manageable toxicity profile. Cutaneous squamous cell carcinomas and pyrexia are the most significant adverse effects. Dabrafenib appears similar to vemurafenib with regard to efficacy but it is associated with less toxicity. It is expected that new combinations of targeted drugs,such as the combination of dabrafenib and trametinib (GSK1120212,a MEK inhibitor),will provide higher response rates and more durable clinical benefit than dabrafenib monotherapy. View Publication

SUMMARYUnderstanding how human gene expression is coordinately regulated by functional units of proteins across the genome remains a major biological goal. Here,we present COMET,a high-throughput screening platform for combinatorial effector targeting for the identification of transcriptional modulators. We generate libraries of combinatorial dCas9-based fusion proteins,containing two to six effector domains,allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription. Importantly,we keep full proteins or domains intact,maintaining catalytic cores and surfaces for protein-protein interactions. We observe more than 5800 significant hits that modulate transcription,we demonstrate cell type specific transcriptional modulation,and we further investigate epistatic relationships between our effector combinations. We validate unexpected combinations as synergistic or buffering,emphasizing COMET as both a method for transcriptional effector discovery,and as a functional genomics tool for identifying novel domain interactions and directing locus-specific biochemistry. View Publication

Single-cell technologies can measure the expression of thousands of molecular features in individual cells undergoing dynamic biological processes. While examining cells along a computationally-ordered pseudotime trajectory can reveal how changes in gene or protein expression impact cell fate,identifying such dynamic features is challenging due to the inherent noise in single-cell data. Here,we present DELVE,an unsupervised feature selection method for identifying a representative subset of molecular features which robustly recapitulate cellular trajectories. In contrast to previous work,DELVE uses a bottom-up approach to mitigate the effects of confounding sources of variation,and instead models cell states from dynamic gene or protein modules based on core regulatory complexes. Using simulations,single-cell RNA sequencing,and iterative immunofluorescence imaging data in the context of cell cycle and cellular differentiation,we demonstrate how DELVE selects features that better define cell-types and cell-type transitions. DELVE is available as an open-source python package: https://github.com/jranek/delve. Characteristic genes or proteins driving continuous biological processes are difficult to uncover from noisy single-cell data. Here,authors present DELVE,an unsupervised feature selection method to identify core molecular features driving cell fate decisions. View Publication