The kinase DOUBLETIME is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of DOUBLETIME interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knock-down of CG17282 produced behavioral arrhythmicity and long periods, high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME. Overexpression of DOUBLETIME in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances DOUBLETIME-dependent PERIOD degradation, indicating that CG17282 stimulates DOUBLETIME’s circadian function. In photoreceptors, CG17282 accumulates rhythmically in PERIOD- and DOUBLETIME-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds DOUBLETIME and tetratricopeptide repeats that may promote assembly of larger protein complexes. We have named CG17282 Bride of Doubletime and established it as a mediator of DOUBLETIME’s effects on PERIOD, most likely in cytosolic foci that regulate PERIOD nuclear accumulation.
The autoimmune regulator (AIRE) is essential for prevention of autoimmunity; its role is best understood in the thymus where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)hi, CD80lo, CD86lo, epithelial cell adhesion molecule (EpCAM)hi, CD45lo bone marrow-derived peripheral antigen presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4+ T cells through a mechanism that does not require regulatory T cells (Treg), and is resistant to innate inflammatory stimuli. Together these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.
The neurite outgrowth inhibitor, Nogo-A, has been shown to be overexpressed in skeletal muscle in amyotrophic lateral sclerosis (ALS); it is both a potential biomarker and therapeutic target. We performed a double-blind, two-part, dose-escalation study, in subjects with ALS, assessing safety, pharmacokinetics (PK) and functional effects of ozanezumab, a humanized monoclonal antibody against Nogo-A. In Part 1, 40 subjects were randomized (3∶1) to receive single dose intravenous ozanezumab (0.01, 0.1, 1, 5, or 15 mg/kg) or placebo. In Part 2, 36 subjects were randomized (3∶1) to receive two repeat doses of intravenous ozanezumab (0.5, 2.5, or 15 mg/kg) or placebo, approximately 4 weeks apart. The primary endpoints were safety and tolerability (adverse events [AEs], vital signs, electrocardiogram (ECG), and clinical laboratory tests). Secondary endpoints included PK, immunogenicity, functional endpoints (clinical and electrophysiological), and biomarker parameters. Overall, ozanezumab treatment (0.01–15 mg/kg) was well tolerated. The overall incidence of AEs in the repeat dose 2.5 mg/kg and 15 mg/kg ozanezumab groups was higher than in the repeat dose placebo group and repeat dose 0.5 mg/kg ozanezumab group. The majority were considered not related to study drug by the investigators. Six serious AEs were reported in three subjects receiving ozanezumab; none were considered related to study drug. No study drug-related patterns were identified for ECG, laboratory, or vital signs parameters. One subject (repeat dose 15 mg/kg ozanezumab) showed a weak, positive anti-ozanezumab-antibody result. PK results were generally consistent with monoclonal antibody treatments. No apparent treatment effects were observed for functional endpoints or muscle biomarkers. Immunohistochemical staining showed dose-dependent co-localization of ozanezumab with Nogo-A in skeletal muscle. In conclusion, single and repeat dose ozanezumab treatment was well tolerated and demonstrated co-localization at the site of action. These findings support future studies with ozanezumab in ALS.
ClinicalTrials.gov NCT00875446 GSK-ClinicalStudyRegister.com GSK ID 111330
Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca2+ indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca2+]i at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca2+ dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background.
Sleep is important for maintenance of normal physiology in animals. In mammals, neuropeptide Y (NPY), a homolog of Drosophila neuropeptide F (NPF), is involved in sleep regulation, with different effects in human and rat. However, the function of NPF on sleep in Drosophila melanogaster has not yet been described. In this study, we investigated the effects of NPF and its receptor-neuropeptide F receptor (NPFR1) on Drosophila sleep. Male flies over-expressing NPF or NPFR1 exhibited increased sleep during the nighttime. Further analysis demonstrated that sleep episode duration during nighttime was greatly increased and sleep latency was significantly reduced, indicating that NPF and NPFR1 promote sleep quality, and their action on sleep is not because of an impact of the NPF signal system on development. Moreover, the homeostatic regulation of flies after sleep deprivation was disrupted by altered NPF signaling, since sleep deprivation decreased transcription of NPF in control flies, and there were less sleep loss during sleep deprivation and less sleep gain after sleep deprivation in flies overexpressing NPF and NPFR1 than in control flies, suggesting that NPF system auto-regulation plays an important role in sleep homeostasis. However, these effects did not occur in females, suggesting a sex-dependent regulatory function in sleep for NPF and NPFR1. NPF in D1 brain neurons showed male-specific expression, providing the cellular locus for male-specific regulation of sleep by NPF and NPFR1. This study brings a new understanding into sleep studies of a sexually dimorphic regulatory mode in female and male flies.
Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3′,5′-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.
Blood vessel formation is important for many physiological and pathological processes, and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumor or promoting “normalization” of tumor blood vessels in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows investigating cell-cell and cell-matrix interactions in a controlled environment, and is thereby a crucial step in developing high content (HC) and high throughput (HT) screening assays to search for modulators of blood vessel formation. Human umbilical vein endothelial cells (HUVECs) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by 6–8 passages in culture, making stable integration of fluorescent markers and large-scale expansion for high throughput screening problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized human microvascular endothelial cells (HMEC-1, hereafter HMECs). We then evaluated HMEC cultures, both alone and co-cultured with an epicardial mesothelial cell (EMC) line that contributes vascular smooth muscle cells, to determine suitability for HTS or HCS.
The endothelial and epicardial lines were engineered to express a panel of nuclear- and cytoplasm-localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 hours upon plating on Matrigel, the cells migrated and coalesced into networks of vessel-like structures. If co-cultured with EMCs, the branches formed cylindrical-shaped structures of HMECs surrounded by EMC-derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds.
HMEC-based lines retain most of the angiogenic features of primary endothelial cells, yet possess long-term stability and ease of culture, making them intriguing candidates for large-scale primary HC and HT screening (of ~10,000–1,000,000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial-derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.
Angiogenesis; microvascular; fluorescent proteins; time-lapse microscopy of live cells in culture
A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.
diabetes; chlorpromazine; ethopropazine
Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z′ values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.
Acquisition of invasive cell behavior underlies tumor progression and metastasis. To define in more molecular detail the mechanisms underlying invasive behavior, we developed a high throughput screening strategy to quantitate invadopodia; actin-rich membrane protrusions of cancer cells which contribute to tissue invasion and matrix remodeling. We developed a high content, imaged-based assay, and tested the LOPAC 1280 collection of pharmacologically active agents. We found compounds that potently inhibited invadopodia formation without overt toxicity, as well as compounds that increased invadopodia number. One of the two compounds that increased both invadopodia number and invasive behavior was the chemotherapeutic agent paclitaxel, which has potential clinical implications for its use in the neoadjuvant and resistance settings. Several of the invasion inhibitors were annotated as cyclin-dependent kinase (cdk) inhibitors. Loss-of-function experiments determined that Cdk5 was the relevant target. We further determined that the mechanism by which Cdk5 promotes both invadopodia formation and cancer invasion is by phosphorylation and down regulation of the actin regulatory protein caldesmon.
Cdk5; caldesmon; paclitaxel; cancer; Src
Understanding the regulation of human immune responses is critical for vaccine development and treating infectious diseases. We have previously shown that simultaneous engagement of the T cell receptor (TCR) and complement regulator CD46 on human CD4+ T cells in the presence of interleukin-2 (IL-2) induces potent secretion of the immunomodulatory cytokine IL-10. These T cells mediate IL-10-dependent suppression of bystander CD4+ T cells activated in vitro with anti-CD3 and anti-CD28 costimulation, reflecting a T regulatory type 1 (Tr1)-like phenotype. However, CD46-mediated negative regulation of pathogen-specific T cells has not been described. Therefore, we studied the ability of CD46-activated human CD4+ T cells to suppress T cell responses to Mycobacterium bovis BCG, the live vaccine that provides infants protection against the major human pathogen Mycobacterium tuberculosis. Our results demonstrate that soluble factors secreted by CD46-activated human CD4+ T cells suppress mycobacterium-specific CD4+, CD8+, and γ9δ2 TCR+ T cells. Dendritic cell functions were not downregulated in our experiments, indicating that CD46-triggered factors directly suppress pathogen-specific T cells. Interestingly, IL-10 appeared to play a less pronounced role in our system, especially in the suppression of γ9δ2 TCR+ T cells, suggesting the presence of additional undiscovered soluble immunoregulatory factors. Blocking endogenous CD46 signaling 3 days after mycobacterial infection enhanced BCG-specific T cell responses in a subset of volunteers. Taken together, these results indicate that CD46-dependent negative regulatory mechanisms can impair T cell responses vital for immune defense against mycobacteria. Therefore, modulating CD46-induced immune regulation could be integral to the development of improved tuberculosis therapeutics or vaccines.
Microtiter plate (MTP) assays often exhibit distortions, such as caused by edge-dependent drying and robotic fluid handling variation. Distortions vary by assay system but can have both systematic patterns (predictable from plate to plate) and random (sporadic and unpredictable) components. Random errors can be especially difficult to resolve by assay optimization alone, and postassay algorithms reported to date have smoothing effects that often blunt hits. We implemented a 5 × 5 bidirectional hybrid median filter (HMF) as a local background estimator to scale each data point to the MTP global background median and compared it with a recently described Discrete Fourier Transform (DFT) technique for correcting errors on computationally and experimentally generated MTP datasets. Experimental data were generated from a 384-well format fluorescent bioassay using cells engineered to express eGFP and DsRED. MTP arrays were produced with and without control treatments used to simulate hits in random wells. The HMF demonstrated the greatest improvements in MTP coefficients of variation and dynamic range (defined by the ratio of average hit amplitude to standard deviation, SD) for all synthetic and experimental MTPs examined. After HMF application to a MTP of eGFP signal from mouse insulinoma (MIN6) cells obtained by a plate-reader, the assay coefficient of variation (CV) decreased from 8.0% in the raw dataset to 5.1% and the hit amplitudes were reduced by only 1% while the DFT method increased the CV by 36.0% and reduced the hit amplitude by 21%. Thus, our results show that the bidirectional HMF provides superior corrections of MTP data distortions while at the same time preserving hit amplitudes and improving dynamic range.
The software to perform hybrid median filter MTP corrections is available at http://bccg.burnham.org/HTS/HMF_Download_Page.aspx, password is pbushway.
Image-based autofocus determines focus directly from the specimen (as opposed to reflective surface positioning with an offset), but sequential acquisition of a stack of images to measure resolution/sharpness and find best focus is slower than reflective positioning. Simultaneous imaging of multiple focal planes, which is also useful for 3D imaging of live cells, is faster but requires complicated optics.
With color CCD cameras and white light sources common, we asked if axial chromatic aberration can be utilized to acquire multiple focal planes simultaneously, and if it can be controlled through a range sufficient for practical use. For proof of concept, we theoretically and experimentally explored the focal differences between three narrow wavelength bands on a 3-chip color CCD camera with and without glass inserts of various thicknesses and dispersions.
Ray tracing yielded changes in foci of 0.65–0.9 µm upon insertion of 12.5-mm thick glass samples for green (G, 522 nm) vs. blue (B, 462 nm) and green vs. red (G-R, 604 nm). On a microscope: 1) With no glass inserts, the differences in foci were 2.15 µm (G-B) and 0.43 µm (G-R); 2) With glass inserts, the maximum change in foci for G vs. B was 0.44 µm and for G vs. R was 0.26 µm; and 3) An 11.3-mm thick N-BK7 glass insert shifted the foci 0.9 µm (R), 0.6 µm (G), 0.35 µm (B), such that the B and R foci were farther apart (2.1 µm vs. 1.7 µm) and the R and G foci were closer together (0.25 µm vs. 0.45 µm). The slopes of the differences in foci were dependent on thickness, index of refraction and dispersion.
The measured differences in foci are comparable to the axial steps of 0.1–0.24 µm commonly used for autofocus, and focal plane separation can be altered by inserting optical elements of various dispersions and thicknesses. By enabling acquisition of multiple, axially offset images simultaneously, chromatic aberration, normally an imaging pariah, creates a possible mechanism for efficient multiplanar imaging of multiple spectral bands from white light illumination.
automated cytometry; 3D optical volume; simultaneous multiple focal planes; multifocal; on-the-fly autofocus
Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease, and infectious diseases (eg, HCV and tuberculosis). To develop high-content analysis (HCA) techniques to analyze lipid droplets and associated proteins, primary human preadipocytes were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPAR-© agonist that promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone-sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc’s CyteSeer® image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z′ values of 0.54–0.71). Regarding colocalization with lipid droplets, Pearson’s correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and −0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, HuH-7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin C on lipid droplets (Z′s > 0.50) and ADFP expression/colocalization. Oleic acid-induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.
Intracellular lipid droplets are associated with a myriad of afflictions including obesity, fatty liver disease, coronary artery disease and infectious diseases (e.g., HCV and tuberculosis). To develop high content assay (HCA) techniques to analyze lipid droplets and associated proteins, primary human pre-adipocytes, were plated in 96-well dishes in the presence of rosiglitazone (rosi), a PPARγ agonist which promotes adipogenesis. The cells were then labeled for nuclei, lipid droplets, and proteins such as perilipin, protein kinase C (PKC), and hormone sensitive lipase (HSL). The cells were imaged via automated digital microscopy and algorithms were developed to quantify lipid droplet (Lipid Droplet algorithm) and protein expression and colocalization (Colocalization algorithm). The algorithms, which were incorporated into Vala Science Inc’s CyteSeer® image cytometry program, quantified the rosi-induced increases in lipid droplet number, size, and intensity, and the expression of perilipin with exceptional consistency (Z’ values of 0.54 to 0.71). Regarding colocalization with lipid droplets, Pearson’s Correlation coefficients of 0.38 (highly colocalized), 0.16 (moderate), and − 0.0010 (random) were found for perilipin, PKC, and HSL, respectively. For hepatocytes (AML12, Huh7, and primary cells), the algorithms also quantified the stimulatory and inhibitory effect of oleic acid and triacsin c on lipid droplets (Z’s > 0.50) and ADFP expression/colocalization. Oleic-acid induced lipid droplets in HeLa cells and macrophages (THP-1) were also well quantified. The results suggest that HCA techniques can be utilized to quantify lipid droplets and associated proteins in many cell models relevant to a variety of diseases.
Circadian clocks keep time via gene expression feedback loops that are controlled by time-of-day-specific changes in the synthesis, activity, and degradation of transcription factors. Within the Drosophila melanogaster circadian clock, DOUBLETIME (DBT) kinase is necessary for the phosphorylation of PERIOD (PER), a transcriptional repressor, and CLOCK (CLK), a transcriptional activator, as CLK-dependent transcription is being repressed. PER- and DBT-containing protein complexes feed back to repress CLK-dependent transcription, but how DBT promotes PER and CLK phosphorylation and how PER and CLK phosphorylation contributes to transcriptional repression have not been defined. Here, we show that DBT catalytic activity is not required for CLK phosphorylation or transcriptional repression and that PER phosphorylation is dispensable for repressing CLK-dependent transcription. These results support a model in which DBT plays a novel noncatalytic role in recruiting additional kinases that phosphorylate CLK, thereby repressing transcription. A similar mechanism likely operates in mammals, given the conserved activities of PER, DBT, and CLK orthologs.
We evaluated the performance of two plate readers (the Beckman Coulter [Fullerton, CA] DTX and the PerkinElmer [Wellesley, MA] EnVision™) and a plate imager (the General Electric [Fairfield, CT] IN Cell 1000 Analyzer™) in a primary fluorescent cellular screen of 10,000 Molecular Libraries Screening Center Network library compounds for up- and down-regulation of vascular cell adhesion molecule (VCAM)-1, which has been shown to be up-regulated in artherothrombotic vascular disease and is a general indicator of chronic inflammatory disease. Prior to screening, imaging of a twofold, six-step titration of fluorescent cells in a 384-well test plate showed greater consistency, sensitivity, and dynamic range of signal detection curves throughout the detection range, as compared to the plate readers. With the same 384-well test plate, the detection limits for fluorescent protein-labeled cells on the DTX and EnVision instruments were 2,250 and 560 fluorescent cells per well, respectively, as compared to 280 on the IN Cell 1000. During VCAM screening, sensitivity was critical for detection of antagonists, which reduced brightness of the primary immunofluorescence readout; inhibitor controls yielded Z′ values of 0.41 and 0.16 for the IN Cell 1000 and EnVision instruments, respectively. The best 1% of small molecule inhibitors from all platforms were visually confirmed using images from the IN Cell 1000. The EnVision and DTX plate readers mutually identified approximately 57% and 21%, respectively, of the VCAM-1 inhibitors visually confirmed in the IN Cell best 1% of inhibitors. Furthermore, the plate reader hits were largely exclusive, with only 6% agreement across all platforms (three hits out of 47). Taken together, the imager outperformed the plate readers at hit detection in this bimodal assay because of superior sensitivity and had the advantage of speeding hit confirmation during post-acquisition analysis.
Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.
Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and α-myosin heavy chain (αMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.
The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.
A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBTK/R) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBTK/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered “set point” for period length that is not altered by overexpression. Overexpression of the DBTK/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.
In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbtS) and long-period (dbtL) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbtS and dbtL mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBTS, and DBTL proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbtS and dbtL mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKIδ expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbtS and dbtL mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.