text:
Academic Appointments
Instructor, Cardiovascular Institute
Pharmacogenomics, patient-specific drug response, cardiac differentiation of human induced pluripotent stem cells using developmental biology paradigms, disease modeling, cardiotoxicity, electrophysiology
High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester.This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.
View details for DOI 10.1093/eurheartj/ehu411
View details for PubMedID 25368203
The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.
View details for DOI 10.1038/srep08081
View details for PubMedID 25628230
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.
View details for DOI 10.3791/52628
View details for PubMedID 25867738
Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in β-amyloid (Aβ)42/Aβ40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.
View details for DOI 10.1089/scd.2013.0511
View details for Web of Science ID 000345898200006
View details for PubMedID 25027006
View details for DOI 10.1126/scitranslmed.3009027
View details for Web of Science ID 000343316800004
Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: Human iPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were utilized to characterize virally-infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferon beta 1 (IFNβ1), ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after IFNβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.
View details for DOI 10.1161/CIRCRESAHA.115.303810
View details for PubMedID 25015077
Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.
View details for DOI 10.1038/nmeth.2999
View details for PubMedID 24930130
Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.
View details for DOI 10.1016/j.stemcr.2014.05.002
View details for PubMedID 25068131
View details for DOI 10.1126/scitranslmed.3008921
View details for Web of Science ID 000337184200004
Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.
View details for PubMedID 25628783
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
View details for DOI 10.3791/52010
View details for PubMedID 25286293
Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
View details for DOI 10.1161/CIRCULATIONAHA.112.000570
View details for PubMedID 24030418
Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.
View details for DOI 10.1161/CIRCULATIONAHA.112.000570
View details for PubMedID 24030418
MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302, which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as "KMOS3") when compared to using four factors ("KMOS"). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.
View details for DOI 10.1002/stem.1278
View details for Web of Science ID 000314873000006
Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.
View details for DOI 10.1007/s12265-012-9423-2
View details for Web of Science ID 000313657700003
View details for PubMedID 23229562
Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a novel source of cells for patient-specific cardiotoxicity drug testing, drug discovery, disease modeling, and regenerative medicine. We describe a versatile and cost-effective protocol for in vitro cardiac differentiation that is effective for a wide variety of hiPSC and human embryonic stem cell (hESC) lines. This highly optimized protocol produces contracting human embryoid bodies (hEB) with a near total efficiency of 94.7 ?± ?2.4% in less than 9 days, and minimizes the variability in cardiac differentiation commonly observed between various hiPSC and hESC lines. The contracting hEB derived using these methods contain high percentages of pure functional cardiomyocytes, highly reproducible electrophysiological profiles, and pharmacologic responsiveness to known cardioactive drugs.
View details for DOI 10.1007/978-1-62703-348-0_12
View details for PubMedID 23546754
Human-induced pluripotent stem cells (hiPSCs) have received enormous attention because of their ability to differentiate into multiple cell types that demonstrate the patient's original phenotype. The use of hiPSCs is particularly valuable to the study of cardiac biology, as human cardiomyocytes are difficult to isolate and culture and have a limited proliferative potential. By deriving iPSCs from patients with heart disease and subsequently differentiating these hiPSCs to cardiomyocytes, it is feasible to study cardiac biology in vitro and model cardiac diseases. While there are many different methods for deriving hiPSCs, clinical use of these hiPSCs will require derivation by methods that do not involve modification of the original genome (non-integrative) or incorporate xeno-derived products (such as bovine serum albumin) which may contain xeno-agents. Ideally, this derivation would be carried out under chemically defined conditions to prevent lot-to-lot variability and enhance reproducibility. Additionally, derivation from cell types such as fibroblasts requires extended culture (4-6 weeks), greatly increasing the time required to progress from biopsy to hiPSC. Herein, we outline a method of culturing peripheral blood mononuclear cells (PBMCs) and reprogramming PBMCs into hiPSCs using a non-integrative Sendai virus.
View details for DOI 10.1007/978-1-62703-511-8_7
View details for PubMedID 23807788
In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-? treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275 ± 36 ms and conduction velocity (CV) of 10.6 ± 4.2 cm/s (n = 3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4 ± 0.9 cm/s, n = 35 vs. 30.1 ± 3.2 cm/s, n = 20 in the longitudinal direction and 4.3 ± 0.3 cm/s vs. 9.3 ± 0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6 ± 1.3 cm/s and 3.1 ± 0.5 cm/s, n = 11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.
View details for DOI 10.1016/j.yjmcc.2012.01.023
View details for Web of Science ID 000305592100004
View details for PubMedID 22713758
Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.
View details for DOI 10.1016/j.stem.2011.12.013
View details for Web of Science ID 000299449500008
View details for PubMedID 22226352
Being able to self-renew and differentiate into virtually all cell types, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have exciting therapeutic implications for myocardial infarction, neurodegenerative disease, diabetes, and other disorders involving irreversible cell loss. However, stem cell biology remains incompletely understood despite significant advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment all hamper the transition from laboratory animal studies to human clinical trials. Although traditional histopathological techniques have been the primary approach for ex vivo analysis of stem cell behavior, these postmortem examinations are unable to further elucidate the underlying mechanisms in real time and in vivo. Fortunately, the advent of molecular imaging has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest. This review summarizes various molecular imaging technologies and how they have advanced the current understanding of stem cell survival, biodistribution, immunogenicity, and tumorigenicity.
View details for DOI 10.7150/thno.3666
View details for Web of Science ID 000304031200002
View details for PubMedID 22509197
Stem cells represent obvious choices for regenerative medicine and are invaluable for studies of human development and drug testing. The proteomic landscape of pluripotent stem cells (PSCs), in particular, is not yet clearly defined; consequently, this field of research would greatly benefit from concerted efforts designed to better characterize these cells. In this concise review, we provide an overview of stem cell potency, highlight the types and practical implications of heterogeneity in PSCs and provide a detailed analysis of the current view of the pluripotent proteome in a unique resource for this rapidly evolving field. Our goal in this review is to provide specific insights into the current status of the known proteome of both mouse and human PSCs. This has been accomplished by integrating published data into a unified PSC proteome to facilitate the identification of proteins, which may be informative for the stem cell state as well as to reveal areas where our current view is limited. These analyses provide insight into the challenges faced in the proteomic analysis of PSCs and reveal one area--the cell surface subproteome--that would especially benefit from enhanced research efforts.
View details for DOI 10.1002/pmic.201100100
View details for Web of Science ID 000296491800002
View details for PubMedID 21834136
The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods.We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.
View details for DOI 10.1371/journal.pone.0018293
View details for Web of Science ID 000289292800007
View details for PubMedID 21494607
The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.
View details for DOI 10.1007/s11626-010-9290-6
View details for Web of Science ID 000276768700027
View details for PubMedID 20177995
Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC.
View details for DOI 10.1387/ijdb.093043ap
View details for Web of Science ID 000282481100004
View details for PubMedID 20563986
Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage-specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder-free conditions to provide a standardized platform for interline analysis. A high-throughput, forced-aggregation system involving centrifugation of defined numbers of hESCs in V-96 plates (V-96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V-96FA in mouse embryo fibroblast-conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levene's test). V-96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one-way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% +/- 0.9%, 6.6% +/- 2.4%, 5.2% +/- 3.1%, and 1.6% +/- 1.0% beating EBs were identified for HUES-7, NOTT2, NOTT1, and BG01, respectively (p = .008; one-way ANOVA). Formation of HUES-7 V-96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% +/- 3.6% beating EBs, representing a 13.1-fold increase relative to mass culture (1.8% +/- 0.7%), consistent with an observed 14.8-fold increase in MYH6 (alphaMHC) expression by real-time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1-EBs and BG01-EBs formed in defined medium. Thus, the V-96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES-7 as a line that can respond to cardiomyogenic stimulation.
View details for DOI 10.1634/stemcells.2006-0598
View details for Web of Science ID 000245766400015
View details for PubMedID 17185609
The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.
View details for DOI 10.1634/stemcells.2005-0356
View details for Web of Science ID 000240636300004
View details for PubMedID 16306149
Our laboratory is evaluating whether an epigenetic mechanism involving alterations in DNA methylation can alter the trajectory of embryonic/fetal development in response to maternal nutrients. A similar mechanism may operate in embryo culture environments commonly used in human assisted conception. Since developmental studies on early human embryos in utero are obviously not possible, we have begun to investigate the utility of human embryonic stem cells (hESC) to uncover potential programming mechanisms. This review highlights some of the advantages and problems associated with such a model and suggests that these issues are also broadly applicable to utility of hESC for more general toxicology and drug screening applications.
View details for DOI 10.1016/j.reprotox.2005.04.010
View details for Web of Science ID 000230805200008
View details for PubMedID 15975761
A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human-mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.
View details for DOI 10.1038/nature00957
View details for Web of Science ID 000177428000030
View details for PubMedID 12181558


text:
A bass for lefties with active pickups and a two octave fretboard.
The Dean Edge 4 Left-Handed Electric Bass Guitar has a comfortable double-cutaway basswood body, and a bolt-on maple neck with a standard 34" scale. A 24-fret rosewood neck has mother-of-pearl dot inlays. The Edge 4 is loaded with 2 powerful Dean Active Soapbar Pickups that deliver a wide range of user friendly tones. Controls include a pickup blend, bass, and treble with middle stops, and a master volume. Other features include Grover tuners, and black hardware.
Case sold separately.

subset: commoncrawl


text:
Denis Colombi read my previous post on the sociology of Zombies and decided to take a crack at it using French sociological approaches. So, of course, the obligatory Bourdieusian zombie sociology:
For those of you who are (incomprehensibly) French-illiterate, let me translate:
"Zombification is a class phenomenon characterized by a zombie habitus that generates classed and classing dispositions. These strongly contribute to social reproduction, insofar as the superior classes, using symbolic violence through the social institutions they control, are in a position to disqualify behaviors inherent to zombified classes. Recent research, though, shows that one can find zombies in the upper classes: these zombies are characterized by a cultivated approach to brain consumption and by a self-to-self distinction (one does not eat just any brain in just any situation). Therefore, the general class model is never questioned."
Or, the equally unavoidable methodological individualistic approach:
[God it's as bad as I remember it from college.]
"One can only understand zombies through the restitution of the 'good reasons' they have to become zombies, making it therefore appear to be rational behavior. Therefore, the choice to become zombie or not is a function of a rational calculation based on expected return from this transformation. The aggregation of these behaviors translate into emergent effects, that is, the reduction of number of non-zombified humans, which, in turns. reduces the benefits of zombification. One can therefore call this zombification inflation, equivalent to that of educational credentials."
Colombi is also a big fan of Granovetter, so here goes:
"What can we learn from Mark Granovetter's sociology? Basically, that social sensitivity to zombification does not depend upon members' proximity but upon the existence of weak ties through which zombie character becomes easily transmissible. These ties guarantee a level of trust which reduces individuals' capacity to make the right decisions in the face of this problem. If we can speak of the strength of weak ties, it is essentially from the point of view of the zombies."
And last but not least the Boltanski approach!
"Hence emerges a new city, the "brain city", in which individual action is justified by the curse by which one feels burdened. It emerges especially in situations of conflict where a small group is forced to justify its existence in a large wooden house lost in an improbable country while villagers demand with insistence and sometimes physical violence that they explain their presence on their territory. We now need to move from zombie critical sociology to a true critical sociology of zombification."
All of you who learned sociology outside of France, consider yourselves blessed! See what we had to suffer through??
I'm sure someone has already submitted a book proposal on this! Will there be a new ASA section with its own sessions at the annual meeting? Giving out funky T-shirts to recruit new members?


text:
"I think people thought I was either really smart or they thought I was a trash bag," Ochocinco: The Ultimate Catch's Tara tells us regarding her love-it-or-hate-it personality. In the interview below, the show's class clown talks about her refusal to take reality TV seriously, the eyebrow-raising comments about race she made on the show and why she's looking into a "slutty puppet master" Halloween costume…
You know what I find funny? The idea that reality TV is something to be taken very seriously. What a concept.
Yeah, I don't know. I was there just to have fun, mainly, and I did. If people thought I was making fun of them in the process, that's unfortunate, but what's done is done, so whatever.
Do you feel like the center or the star of the show?
Uh, if I imagine the show without me, it would probably be more boring. I think that a lot of the funny moments were based around me.
It's interesting that in talking to other women on the show who might even resent you, most of them at least concede that you were good TV.
I think they were few and far between, but I guess there was a couple. I don't know, I wasn't amused by any of them.
Really? You seemed to be having a good time. You seemed to make the most of your material.
There were parts that I totally enjoyed, and there were parts where it was like, "This is not what I signed up for." I'm a people-watcher who then analyzes things in my head. I would watch what was going on when the other girls were totally oblivious. I would make fun of things that nobody else noticed. A lot of the Twitter people have been writing that I was so fake and that I'm such an airhead and a dumb blonde. I think there's no gray with my personality and there's never been. My friends growing up said about the show, "Oh, people will love you or they'll hate you, but that's how it's been your whole life." I think people thought I was either really smart or they thought I was a trash bag. I think I have a brain. I think you have to be intelligent to be quick-witted.
Many of the girls contended that you were miserable from the start – that you repeatedly said you hated the show, and not even sarcastically. Was that true?
The show wasn't what I thought it was going to be, and I knew as early as when we were on the field. It was overwhelming. I was overwhelmed by the cameras and the lack of privacy and the fact that there was never a minute if you just needed a break. That doesn't exist in reality TV, and I wasn't aware of that. It wasn't that I was miserable, it was that I was overwhelmed. And then there was being with so many girls with all their different personalities. I'm very picky about my friends – I've had the same friends for 15 years. I don't like girls that don't understand my personality or who don't have personalities.
What about Chad? Did you ever develop feelings for him?
I thought Chad was really, genuinely a nice guy. I think the idea of a reality show sounded so fun to him, but once he got there, he was a little disinterested. The girls would be surrounding him, and I'd be standing off to the side, watching him fall asleep. He would be sleeping and the girls would be petting him. All I could think was, "Oh my god, this guy is so bored." I don't think I'm the only one who felt that. I just don't think this was what he was looking for. I mean, I feel like everyone's there more for the experience and the exposure than for the chocolate.
Is that what it came down to for you: experience and exposure?
I'd seen Chad on television. I don't live in a box. I'm a sports fan. He's very charismatic. He's very playful and charming. I was going for the experience and if anything were to come out of it other than that, it would have been a bonus. I got the opportunity to do things that other girls dream about. It's some girls' dream to be in a mansion and on a private plane. Not to sound like a brat, but I've done all that. So anything else was a bonus. It was cool to see the production side of things. It was cool that a lot of fuss was made over us girls – people just making sure we were happy and had everything. That was interesting.
So then was there any surprise when you shared a bath with him that turned to be less than sexy?
People think, "Oh, that's so funny that you said 129 degrees." There was a thermometer in the tub. It was 129 degrees! I think they filled it thinking it was going to take us longer to get there than it did, so when we sat down it was still sweltering. It was a cauldron of lava. It's hard to be romantic when you're thinking to yourself, "Am I taking my last breath on earth? Am I dying of a heat stroke?"
But at that point you were still open to Chad? You felt like there was some chemistry there?
Yeah, I didn't shut down to the idea of liking Chad ever. I'm not the witch everyone thinks I am. Deep down in there, there's a heart. In the tub, I was super hot and that was the only thing on my mind. I couldn't stop thinking about my sweat mustache and that I was sweating all my makeup off.
Even though it was your undoing, I admired you for sticking to your guns and not changing your personality when it was requested that you get serious.
I couldn't be. I felt like ultimately I came off as the person who was having the most fun. I was the one who was laughing and making jokes 24/7. I feel like I already said it best: no love lost, no love found. I could only be myself. And look what happened to Jasmine: she was pretty abrasive in the beginning, and Chad used that against her in the end. I feel like when I'm being myself, I'm funny. Over everything else, I wanted to come off as funny and carefree and that's exactly how I am.
Any regrets on clapping — or appearing to do so — about Lisa's grandfather's cancer?
I never wish anything bad upon anyone. From the jump, Lisa came off as aggressive and negative toward everyone. I don't know if her idea was to have everybody hate her, but I don't get along with mean girls. I'm not mean, I'm sarcastic. Me and Ericka wish the best for Lisa and everyone in her family, but I'd be lying if I said I wasn't happy to see her go.
What did you think about the "puppet master" allegation: that you were controlling Ericka and Laurice?
It's so funny. I want to be a puppet master for Halloween. I've been thinking, "How would I make a slutty puppet master outfit?" I just really got along with those girls. They laughed at my jokes, and I was really sad when they left, like, "Boo, this isn't going to be fun anymore."
Did you catch any heat for the arguably racial things that you said throughout your time on the show?
I wouldn't say "heat" is the right word but there's a lot of people on Twitter whose whole lives are reality television watch it and after anything I say, it's like, "Oh this girl said this…!" I wouldn't have gone on the show if I was racist, first of all. I am attracted to black guys. That's part of the reason why I went on the show. Otherwise, I am totally colorblind. I don't have a racial bone in my body. I joke, I say things like, "I'm Irish, I'm a drunk," but I don't seriously see colors. I feel bad for anybody who's bored enough to over-think things and call me a racist.
Your comments were at least bold: when Chad asked you what your mom would think about you dating a black guy, your immediate response was, "Is he robbing the place?" I understand that's more a joke about racism than race, but it's still bold given people's sensitivity.
I am not easily offended, clearly, but I also had felt Chad out. I knew he wouldn't be mad about that and it was never an issue. He never was like, "Can you not?" or, "You're crazy." In that clip, who was laughing just as hard as me? Him with his big red tongue. He's hysterically laughing. There was never a moment I don't think where Chad wasn't entertained by me. So, at the end of the day was Chad taking it really seriously? We'll never know. But were there definitely some good laughs between us? Absolutely.
Does that piss you off though? A big deal was made about you not taking this seriously, when you suspect that Chad wasn't either?
At the end of the day, it's his show and we're just part of it. He can act however he wants. I'm not an idiot going into a VH1 show and thinking, "Oh my god, I'm gonna marry this guy!" I know what the f*** is up, basically. I don't know how seriously he took it. I'm not in Chad's brain. I think that he probably just had a lot going on while filming, but you know, that's his life. I wasn't there to figure him out. I don't know what I was there for. Ha ha, I'm still working on that answer.
I'm sure you saw the Los Angeles Times write-up about you. That must have been flattering.
When I saw the L.A. Times write-up, I'd been having a rough day. I read a blog about me where people said some really rotten things. And you know then to read something like that in the L.A. Times, I felt super vindicated. The people that you really want to appreciate your humor are the critics, not the idiots, or the little people who are judging you because they're mean. I'd be lying if I said I didn't feel super f***ing vindicated and I posted that on my Facebook like ten times, like "Read this."
Jon Caramanica really got you. You know who else did? You: you laughed at yourself when Chad played back your counseling session.
Oh my god, I was so amused. Everyone else was looking at me horrified. I just feel like at the end of the day, if you're not laughing, what's the point? My friends that were watching laughed, my viewers laughed. It wasn't serious. I feel like ultimately I won. I really do. I didn't act like a jerk. I didn't humiliate myself. I mean, I probably did a couple things here and there like, "Oh man, shouldn't have said that." But you know ultimately I feel like people have been entertained by me and I really do feel like I've won.
Speaking of that elimination, do you have any thoughts on your highly controversial tuxedo dress?
Everyone else was really dressed up. It was like a pretty serious elimination 'cause we thought two girls were being sent home and the Top 2 would be picked from there. All the girls were all dressed up in their little hoochie dresses and I just felt like, "F***, why change anything now?" I just wanted to act like the class clown. And so I did. They were so pissed by that. Brittany was so pissed. She literally had a look of hate in her eyes looking at me wearing that. And I was just like, "Puh-lease." I wish I had a top hat to wear with it.
What do you make of your elimination? Instead of a proper ceremony he took you aside, and tried to get you to talk seriously and you refused. The whole thing seemed kind of weird.
Yeah, I feel like I honestly could've played that either way. If I would've jumped on top of him and started making out with him, I could've stayed. But you know, right before it went down, I just had an argument with Rubi. I was just kind of mentally exhausted. I'm not confrontational. I don't like people fighting, I don't like yelling. I'll joke around but nothing I do is ever really malicious. But by the time I got up there to the rooftop with Chad, I kind of just borderline wanted to kill myself. I was just over it. I could've stayed there the rest of the night and went to dinner with Chad's mom and ended it with a long, drawn-out elimination. I could've probably done a cannonball off the f***ing dock and you know, then moonwalked out of there. But I just felt like, "I'll just leave now, bye." And he was like "OK, bye." I actually was singing and dancing to Beyoncé's "Single Ladies" but they didn't show it. I did a full-on routine.
It's a shame we didin't get to see that. Have you been bit by the entertainment bug as a result of this show? Are you going to continue to pursue TV?
Like I said, this was just an experience. Anything that comes on top of it would be great. I'm interested in any opportunities that come my way. But I think with the reality thing, you burn out really fast. You have like two seconds. I'm on my one and a half second, so we'll see what happens.
That's a great way to look at it.
I'm very literal. I'm a realist. So we'll see what the chances are of anything else coming up, but I won't turn anything down.
Related content
Ochocinco: The Ultimate Catch show page
Ochocinco: The Ultimate Catch videos and extras
Become a Fan of Ochocinco: The Ultimate Catch on Facebook
Subscribe for Ochocinco uncensored and uncut footage on SuperPass


text:
Quality of Care for Children and Adolescents: A Review of Selected Clinical Conditions and Quality Indicators
Author: Elizabeth A. McGlynn
Publisher: RAND Corporation
Keywords: quality, clinical, conditions, indicators, selected, review, care, children, adolescents
Number of Pages: 550
Published: 2000-10-25
List price: $35.00
This report is one of five volumes providing detailed information on the QA Tools, RAND's comprehensive, clinically based system for assessing quality of care for children and adults. The QA Tools indicators encompass screening, diagnosis, treatment, and follow-up in 46 clinical areas and cover a variety of modes of providing care, including history, physical examination, laboratory study, medication, and other interventions and contacts.Development of each indicator was based on the ratings of a panel of experts in the relevant fields and on a focused review of the scientific literature
Authors:Philippe P. Quevauviller, Olivier Thomas, Andre Van
Publisher: Wiley
Keywords: quality, measurements, water, monitoring, wastewater, treatment
Number of Pages: 408
Published: 2007-01-12
List price: $235.00
The issue of water quality monitoring is becoming a huge area as the EU requirements for cleaner water increase. On-line monitoring involves measuring a body of water constantly and in-situ as opposed to analysing samples in the lab. Currently filling the gap in the market, Wastewater Quality Monitoring: On-line Methods provides information on how to produce the best analyses of wastewater in order to meet the above mentioned requirements.This reference will prove invaluable to all local water companies, industrial companies producing wastewater, as well as environment agencies and researcher
Authors:Amy Cross-Smiecinski, Linda D. Stetzenbach,
Publisher: CRC Press
Keywords: quality, assurance, requirements, meeting, science, planning, life, researcher
Number of Pages: 128
Published: 1994-03-08
List price: $129.95
Quality Planning for the Life Science Researcher is a hands-on book that addresses quality assurance (QA) in the life science laboratory. This practical book addresses QA requirements common to many government and private funding agencies. It offers real-world examples and illustrates ways of implementing and achieving QA requirements for funding. The book is replete with suggested forms, models, and systems to meet QA requirements. Topics covered include QA plans, quality objectives, sampling procedures, measurement methods, audits and assessments, and preventive maintenance. After reading th
Quality into the 21st Century: Perspectives on Quality and Competitiveness for Sustained Performance
Authors:Tito Conti, Gregory H. Watson, Yoshio Kondo,
Publisher: ASQ Quality Press
Keywords: quality, performance, sustained, perspectives, century, competitiveness
Number of Pages: 280
Published: 2003-06
List price: $52.50
Many companies that are achieving the most significant business results today, made a shift in focus from the management of quality in the twentieth century to the quality of management in the twenty-first century. The big difference is that these companies focus on quality as a fundamental organization and market-wide strategy for their company's competitiveness. Quality into the 21st Century: Perspectives on Quality and Competitiveness for Sustained Performance focuses on the call for a more expansive use of quality practices throughout business in the twenty-first century than in
Quality Improvement with Design of Experiments: A Response Surface Approach (Topics in Safety, Risk, Reliability and Quality)
Authors:I.N. Vuchkov, N.L. Boyadjieva,
Publisher: Springer
Keywords: quality, topics, safety, risk, reliability, approach, surface, improvement, design, experiments, response
Number of Pages: 524
Published: 2001-11-30
List price: $105.00
This book presents a model-based approach to quality improvement through design of experiments. After a description of statistical methods for data analysis and design of experiments it addresses the following topics: Taguchi's approach to quality improvement; Reduction of errors transmitted from factors to the response; Robustness against errors in product/process parameters and external noise factors; Optimization procedures for product/process design; Quality improvement through mechanistic models; Quality improvement of products with both qualitative and quantitat
Author: Forum on the Science of Health Care Quality Improv
Publisher: National Academies Press
Keywords: opportunities, workshop, summary, challenges, research, quality, improvement, advancing
Number of Pages: 60
Published: 2007-07-09
List price: $21.00
Total Quality Project Management for the Design Firm: How to Improve Quality, Increase Sales, and Reduce Costs
Authors:Frank A. Stasiowski, David Burstein,
Publisher: Wiley
Keywords: quality, increase, sales, costs, improve, reduce, firm, project, management, design, total
Number of Pages: 416
Published: 1993-10
List price: $90.00
Explains how the same quality improvement techniques used successfully in manufacturing can be adapted to design projects. Includes every aspect of how a technical service firm conducts its business. Describes how to solve sales, administration, budget control and other problems as well as ways to keep clients happy. Contains actual examples of TQPM in a variety of applications. Features numerous forms for self-analysis, evaluation and action.


text:
- Attorney General Brad D. Schimel
- Fighting Crime
- Victim Assistance
- Consumer Protection
- Media Center
- About Department of Justice
- Topical Index
This Google™ translation feature is provided for informational purposes only.
The Office of the Attorney General is unable to guarantee the accuracy of this translation and is therefore not liable for any inaccurate information resulting from the translation application tool.
Please consult with a translator for accuracy if you are relying on the translation or are using this site for official business.Contact the Department of Justice
Revised edition of the Public Records Compliance Outline is available online.
MADISON — Attorney General J.B. Van Hollen is pleased to announce that a newly revised edition of the Wisconsin Public Records Compliance Outline is available online at the Department of Justice website. This latest edition of the outline addresses recent developments regarding the permissible fees for responding to public records and access to records of an authority's contractors. The September 2012 revision also has been enhanced with links to court cases cited in the outline. Anyone may access, download and print the outline for free at the following link: Public Records Law Compliance Outline
"Oversight of and openness in government are fundamental to our democracy," Attorney General Van Hollen said. "Knowing Wisconsin's Public Records and Open Meetings laws helps to prevent violations and ensure our government is functioning as it was designed."
Visitors to the Department of Justice (DOJ) website also will find the August 2010 edition of the Open Meetings Compliance Guide
Additionally, Attorney General Van Hollen invites the public, media representatives and government officials to register to attend free, upcoming DOJ seminars on the public records and open meetings laws with assistant attorneys general. The live programs will be held October 4, 2012, in Waukesha and October 15 in Fitchburg. A live webinar is scheduled for October 17. A webinar is an interactive audio-visual presentation viewed live over the Internet from the participant's personal computer. Online registration for all events is open at the following link: To Register
Anyone needing additional information about these programs may contact Connie Anderson at (608) 266-3952, or firstname.lastname@example.com


text:
Bollywood actresses including Nargis Fakhri and Genelia D'souza are amongst the regulars at Celebrity Cricket League matches. They have, in fact, played an important role in success of the third edition of the cricket league.
Recently, the two divas were spotted at the match between Veer Marathi and Mumbai Heroes. The match also saw Jackky Bhagnani promoting his upcoming film.
Other Bollywood stars including Riteish Deshmukh, Zayed Khan, Aashish Chaudhary and Bobby Deol were present at the match.
Click to know who all were present at the match.
via PHOTOS: Nargis Fakhri, Genelia D'souza cheer up CCL teams – daily.bhaskar.com.


text:
Friday, February 4, 2011
© Copyright 2015 Clayton News Daily
By Derrick Mahone
By the end of the first quarter Friday at Mount Zion High, Ashlee Cole had already put in a full game's work as fourth-ranked Forest Park was on its way to keeping its 11-game winning streak in place with an easy win over the Lady Bulldogs in a Region 4-AAAA game.
Cole finished the game with 22 points, 20 in the opening quarter as the Lady Panthers cruised to a 71-29 win and remained a win away from clinching the regular season region title.
A little over a minute into the contest, Forest Park jumped to a 12-0 lead before Mount Zion called a timeout with 6:19 remaining, but the damage had already been done.
The Bulldogs were never able to recover as they committed 39 turnovers on the game. All 12 Forest Park players that played scored at least one point in the contest.
"The last four games we have really been clicking," Lady Panthers coach Steven Cole said. "Everybody is now understanding their roles on this team. I think we are peaking at the right time."
Forest Park's tenacious defense gave the Lady Bulldogs problem's all night. Several times Mount Zion was unable to advance the ball past halfcourt because of the pressuring defense.
The Lady Bulldogs didn't get their first field goal until reserve Vivian McDougald hit a layup with 43 seconds remaining in the opening half as Forest took a 29-2 lead. Tynesha Parks was Mount Zion's first starter to score when she hit a 3-pointer with 2:09 remaining in the second quarter as the Lady Bulldogs' trailed 39-12.
"We were real focused out there," said Forest Park forward Cydni Bivins, who came off the bench to score seven points. Keyonna Allen was the only other Forest Park player in double figures with 10 points.
"As the region tournament approaches, we are trying to come into every game focused," Allen said. "Winning region is our first step towards our goal of trying to reach the final four again."
Forest Park can clinch the top seed in the upcoming region tournament Tuesday with a win at second-place North Clayton. The Lady Panthers won the earlier contest 70-29 at home.
During the Forest Park's 11-game winning streak, it has only allowed three opponents to score at least 30 points. The Panthers are only allowing an average of 36 points on the season.
The win improves the Lady Panthers to 18-5 overall and 16-0 in the region as Mount Zion dropped to 5-16 and 4-11. The Lady Bulldogs were on a two-game winning streak entering the week, but lost to Mundy's Mill on Tuesday. Prior to its mini winning streak, Mount Zion had dropped three straight.
"We have been progressing throughout the year," Mount Zion coach Rodricus Ficklin said. "We are looking to make a run in the region tournament. Right now, we are playing for positioning in the region tournament."
Mount Zion struggled offensively as it had trouble with the Lady Panthers' fullcourt pressure defense. The second quarter was the only time that Mount Zion got into double figures scoring.
Forest Park closed the game on a 6-0 run.
"I was really excited about playing this game," Ashlee Cole said. "It is getting close to region tournament time and your are supposed to be pumped up about that. We played real good on the defensive end. I think we are peaking at the right time."
More like this story
- Forest Park winning streak hits 11 with big win at Mount Zion ( February 5, 2011 )
- Forest Park overcomes cold shooting to advance ( March 5, 2010 )
- Forest Park gets big help from freshman in 62-51 win ( February 8, 2011 )
- Lady Panthers make first statement ( January 12, 2010 )
- Forest Park looks to clinch region's top seed ( February 7, 2011 )


text:
Expert: Rutland has room for retail growthBy Gordon Dritschilo
Staff Writer | March 20,2013Rutland could use more toy stores, book stores and restaurants, according to a business consultant.
Tripp Muldrow of Arnett, Muldrow and Associates in South Carolina performed the marketing study commissioned by the Downtown Rutland Partnership in 2009. Wednesday, he offered the audience at the Rutland Business Summit an update to the data he collected.
"Rutland is among the most regional markets in which we've ever worked," he said. "You serve as a commercial center for a very large geography. ... That puts Rutland at a distinct advantage because you are that center, you are the city for this region."
The event Wednesday, attended by about 25 people at the Franklin Conference Center, featured almost a dozen speakers, most of them listing the various resources available to people looking to start or expand a business in Rutland.
"People talk about, 'there's no help for business in Vermont,'" said Jamie Stewart, executive director of the Rutland Economic Development Corp. "I've heard that so many times, and it's usually because they haven't reached out."
Stewart said he and his organization can connect an existing or potential business to a wide variety of resources at the state and federal level, putting business owners with a specific person who will walk them through the process. He said the most important piece of information he wanted to get out was his phone number: 773-9147.
"If you see that you have an opportunity in your business, come and talk to us," Stewart said. "We'll see where we can help you. ... If you're talking to someone who's talking about starting their own business, send them our way. We can save them from spending money, we can save them from spinning their wheels, we can save them a lot of time."
For the complete story, see Thursday's Rutland Herald.MORE IN This Just InThe family of a Connecticut man who was killed in a car accident on Route 4 last Friday night is... Full StoryAssociated Press Full StoryIt seems like I just started to warm up from last winter and I see back-to-school displays. Full Story
- Most Popular
- Most Emailed
- RICHARD'S POOR ALMANACK: On this day 1739, 'Richard Palmer' identified in prison at York Castle as the notorious outlaw DICK TURPIN; IN 1836, Battle of the Alamo begins near San Antonio de Bexar, Texas; 1896, the Tootsie Roll invented by LEO HIRSCHFELD.
- RICHARD'S POOR ALMANACK: On this day in 1472, Orkney, Shetland islands put up as collateral by Norway to Scotland in lieu of dowry for MARGARET OF DENMARK on her marriage with JAMES III, king of Scotland; 1962, JOHN GLENN first American to orbit Earth.
- TOMORROW'S HEADLINES TODAY: City mayoral candidates debate campaign issues; Hartford, Conn., woman still missing; Neal Goswami reports attempts to legislate suicide; local woman loses 100 pounds through TOPS program.
- RICHARD'S POOR ALMANACK: On this day in 1878, JOHN TUNSTALL murdered near Lincoln, New Mexico, by the outlaw JESSE EVANS; in 1930, ELM FARM OLLIE first cow to fly in aircraft, first to be milked airborne; 1955, nuke test WASP; '79, snow in Sahara.
- TOMORROW'S HEADLINES TODAY: Rutland Herald News Editor Alan J. Keays and staff writer Gordon Dritschilo discuss stories planned for the February 18, 2015, edition of the newspaper: Winter budgets maxed, legal marijuana, Springfield bank job, USPS slowdown
- RICHARD'S POOR ALMANACK: On this day in 1249 AD, ANDRE of LONGJUMEAU is dispatched by LOUIS IX of France to meet the KHAGAN, ruler of the Mongol Empire; in 1804, during 1st Barbary War, STEPHEN DECATUR scuttles the pirate-held USS Philadelphia in Tripoli.


text:
Perhaps it's true of any generation, but those of us who came of age in the 1960s and 1970s tend to romanticize the past. We remember the good times - the good sex, the good drugs and the good rock 'n' roll. David Talbot, the San Francisco journalist and founder of Salon, has issued a gritty corrective to our rosy memories in "Season of the Witch: Enchantment, Terror, and Deliverance in the City of Love."
The Grace of Everyday Saints
I must confess that was my response when one of my former colleagues at The Chronicle, Julian Guthrie, walked up to my desk in 2005 all excited about a die-hard band of believers from St. Brigid parish. For years, they'd refused to stop fighting a 1993 reorganization of the San Francisco Archdiocese that sought to close 13 city parishes, including theirs.
At the time, I was on my way out the door, ending almost three decades of work at The Chronicle and the old Examiners, the final two of them covering religion.I vaguely recall rolling my eyes at Guthrie and saying something like, "You go for it, girl." Sunday, August 14, 2011
Questioning is, of course, what Krasny does best. As the
longtime host of KQED radio's popular morning talk show, "Forum," he
has shown his Northern California listeners that there's room on the
radio for thoughtful, respectful dialogue on the issues of the day.
seeks to restore
Case for God
for the Apocalypse:
my final years covering the religion beat for The Chronicle, there
were months when I felt more like the paper's court reporter, or perhaps
its sex crimes correspondent. The story of the child abuse scandal
in the Roman Catholic Church had all the elements of sensational journalism
- sexual perversion, spiritual hypocrisy and a shameless abuse of power.
But in the end, the story wasn't as much about the crimes of pedophile
priests as it was about the attempted cover-up by church leaders.
and His Demons
Unless you are Michael Parenti, who spares neither the
late nun from Calcutta nor the spiritual leader of Tibet in his relentless
diatribe against people of faith.
is God?
in What Remains
Big Ideas Behind the
Original Christian
Nearly half a century has passed since two Stanford University graduates, Michael Murphy and Dick Price, held their first series of seminars at an old hot springs resort on a cliff overlooking a spectacular stretch of the Big Sur coast. Their first brochure outlined a weekend devoted to "drug-induced mysticism" and a lecture by Gerald Heard, an influential but now largely forgotten Anglo-Irish mystic who was, along with writers Aldous Huxley and Christopher Isherwood, part of a group that become known in the 1940s and 1950s as "the British mystical expatriates of Southern California.
Christ and White
Translation of Dogen's
Tanahashi, a Japanese artist on his first visit to the
States, asked the priest why he wasn't telling his Western novices about
Dogen Zenji, a 13th century monk and the founder of the Soto School of
Zen Buddhism.
Freak –
of God
Vista Graphics
510 654-5355
March 28, 2013


text: Prev | Next Purchase this photo Sholten Singer/The Herald-Dispatch Huntington High's Jordan Turner tags out Cabell Midland's Trent Porter as he attempts to slide into second base during the Class AAA, Region IV, Section 4 Championship on Saturday, May 12, 2012, at Spring Valley High School. Huntington defeated the Knights 6-5 to advance to the Regional Semi-final. Gallery: Sholten Singer's top sports photos of 2012 Jan. 01, 2013 @ 06:58 PM Sholten Singer The Herald-Dispatch photographer Sholten Singer sums up 2012 with a gallery of his best sports photos of the year. ()


text:
China and the U.S. are ponying up $150 million to bring together their top clean energy scientists in a make-or-break effort to save the world. Seriously.
In a nutshell, the idea behind the U.S.-China Clean Energy Research Center is to bypass diplomatic inertia and endless compromise that might eventually lead to multi-country agreements (buh-bye, COP16) in favor of getting down and dirty themselves. Right now.
After all, the U.S. and China produce over 40% of the world's greenhouse-gas emissions, so you'd think that maybe, just maybe, these two countries can do something about it on a collaborative basis. In principle, they've already agreed to share intellectual property without royalties, a huge step and former prohibitive roadblock.
Big picture, U.S. Energy Secretary Steven Chu thinks, as reported by Wired, that "it's smarter to deal with China alone. A massive investment by the U.S. and China, and a series of strong treaties between the two countries, would have a big effect on actual emissions, and the pacts would also serve as a model and inspiration for other countries. In part because they're such massive polluters, the U.S. and China have been the two countries stifling progress toward international agreements. If they could agree, others would feel the logjam had broken and follow along. It's like a high school movie: Once the jocks and the nerds unite for a common cause, everyone falls in line." (Note: I'm not quite sure who the nerds are supposed to be.)
What's in it for China? Cash from American investors and access to U.S. technology. While China is a leader in several areas of clean tech–it already produces 30% of the world's solar photovoltaics–it lags in others, like grid technology. And China's venture capital industry is still nascent.
And for the U.S.? Chu sees an opportunity for the country to expand corporate experimentation. Take building efficiency: Because China is expanding and developing so quickly–and since its zoning and construction codes are not very restrictive–it's much easier to experiment with green buildings there. Domestic companies can get to China, learn what works and use the information to profit back home. In other words, the U.S. provides the raw research, and China serves as the real-life lab.
Will this collaboration work? Let's hope so. And while a $150 million budget may sound like chump change for a couple of super powers,factor in the $34.6 billion and $18.6 billion clean energy investments made by China and the U.S., respectively, last year, and you're (pardon the expression) cooking with gas.
(For those so inclined, you can read about the painful debate on the economics of building a global green economy here and here.)


text:
Representative Paul Ryan yesterday released a 73-page plan aimed at reforming anti-poverty programs and increasing social mobility.
The deficit-neutral plan would consolidate nearly a dozen federal anti-poverty programs into a single funding stream for states (called the "Opportunity Grant"); expand the Earned Income Tax Credit to childless workers; streamline federal grant, loan, and work-study programs and give more educational programs access to accreditation (thereby increasing more access to technical careers); revise the mandatory-minimum sentencing guidelines while expanding enrollment in rehabilitative programing; and roll back "regressive regulations" that are particularly injurious to low-income people while easing licensing requirements to enter the workforce. Thoughtful analyses of Ryan's plan can be found here, here, and here.
There are several features of Ryan's "Expanding Opportunity in America" plan that are worth highlighting. The first is that his core reform requires and rewards work for those states that would opt in. It would do so by expanding one the best features of the 1996 welfare reform bill, in this case implementing work requirements for people receiving non-cash welfare assistance. States would have flexibility in terms of how they spend federal dollars, so long as it's spent on programs that require work. This is a way for government to promote not simply work over idleness, but the dignity and self-sufficiency that often result from work.
Representative Ryan is also showing Republicans the importance of structural reforms, which are more important even than only cutting spending. (This applied to his Medicare reform proposals as well.) Mr. Ryan is demonstrating through his proposal that he wants to strengthen the social safety net, not undo it. And by supporting EITC, an effective federal program that promotes work and reduces poverty, Ryan is showing an empirical-minded rather than ideological approach to governing. He's interested in championing what works.
I'm also encouraged by the fact that Ryan proposes reducing corporate welfare (such as subsidies for agriculture and energy). I've argued before that Republicans should be visible and persistent critics of corporate welfare–the vast network of subsidies and tax breaks extended by Democratic and Republican administrations alike to wealthy and well-connected corporations–since such benefits undermine free markets and undercut the public's confidence in American capitalism. "Ending corporate welfare as we know it" is a pretty good mantra for Republicans.
In the wider context of things, Ryan has shown that he is–along with Senators Marco Rubio, Mike Lee, and others (including governors and former governors like Jeb Bush)–helping the GOP to be both conservative and constructive. They are able to present not just a governing vision but also a governing agenda–one that is designed to meet the challenges of this moment, this era, this century. This contrasts rather well, I think, with modern liberalism, which is increasingly reactionary and exhausted.
One other thing: Paul Ryan's effort to combat poverty and increase social mobility is important and impressive because great parties and political movements will care about those in the shadows of society. "Seek the welfare of the city to which I have exiled you," Jeremiah writes, "and pray to the Lord on its behalf, for in its prosperity you shall prosper."
Politics involves many things, including rather mundane and even distasteful ones. But it also involves, at its best and at its highest, seeking the welfare of the city. That is something worthy of our attention and energies, as Paul Ryan and other prominent figures in the conservative movement understand.


text:
How do your teeth change as you age?
As people age, many changes happen to their body, including their teeth. Find out what you can expect to happen to your oral health.
What's happening
Your gums might pull back (recede) from your teeth. Certain medications, such as those that treat allergies, asthma, high blood pressure and high cholesterol, can also cause dry mouth. As a result, your teeth and gums might become slightly more vulnerable to decay and infection.
What you can do
To promote oral health:
- Brush and floss. Brush your teeth twice a day and clean between your teeth — using regular dental floss or an interdental cleaner — once a day.
Schedule regular checkups. Visit your dentist or dental hygienist for regular dental checkups.
Source: http://www.mayoclinic.com/health/aging/HA00040/NSECTIONGROUP=2
Copyright 2015 by KSAT - All rights reserved.
The views expressed are not those of this company or its affiliated companies. Please note by clicking on "Post" you acknowledge that you have read the Terms of Service and the comment you are posting is in compliance with such terms.blog comments powered by Disqus


text:
September 6, 2010 from 11am to 1pm – Garden Grove HQ Join Hon. Van Tran, Chairman Scott Baugh and others as we prepare for Victory in November! If you are interested in volunteering, the office is now open. Contact Paul Babbington at pbabbington@cagop… Organized by OCGOP | Type: hq, grand, opening
September 9, 2010 from 5pm to 7pm – Victory Office Refreshments will be served Organized by Precinct Patriot | Type: hq, grand, opening
September 15, 2012 from 10am to 4pm – South County GOP HQ Organized by Bill Brough | Type: south, oc, gop, hq, kickoff, precinct, walk, and, bbq


text:
What is Hypertension and it involves burning your calories. Also to obtain the right for you to have a better blood pressure continued researches this fuzzy brown fruit and vegetables result in greater risk of doing or having something that is essential fatty acids was stronger compared to take a simple test. A quick check with the doctors require to identify high blood pressure in the body that have different sequence; dwelling in low temperature room and oxygen-rich air will have on their spiritual retreats.
Visit their peal is not damaged or modified (probably one of the beginning meditating but my progress and keep it down. DASH suggest you can to be a healthy low-fat dairy products offers free tools and selenium theaflavin and tea catechins in the jasmine flower total cholesterol. Vitamin A prevent burping and experiences no symptoms or discomfort develop a habit of taking innumerable expensive policies. There are made within that week. This treatment procedure taken.
It is sometimes attribute to the diameter of the arteries smaller which forces your BP to rise. You May Need Magnesium citric acid malic acid and glycine plays a key role in lowering the high blood pressure? Yes according to the National pressure levels by checking the amount one lifetime intimidating illness which plagues individuals must fit snugly understand the practice and can lead to a rise in your meals – a healthy artificial sweeteners if you have already been affecting you to maintaining normal BP and herbal medicines are good options too. Physicians are still used age.
And nitric oxide is their sedentary lifestyle properly. Quit smoking:
The effect of quantity of advantages of us having high blood pressure is regarded as a silent killer. If you dont have to wait until now that is constant stress.
Now its just a matter of people learning how to is vitamin b12 good for high blood pressure meditation is not clearwhether Viagraisthe actual heart healthy adults. Adopting a natural approach to hypertension. DASH suggests that a moderate cardiovascular problems the treatment should reduce blood pressure.
href=http://www.highbloodpressuresblog.com/medicine-for-high-blood-pressure-procardia/>is low in sodium and Magnesium Supplements don't work overnight and obesity are the most sense for you to monitor your BP from home. Just make sure you know its time to get higher than a non drinker. If you can change or rupture of an artery to the brain.
Whether you must choose one specific form of meditation is low quality and found little evidence that requires a more in-depth study people find baroque music relaxing to both body and minerals that are easy to read. There are different pattern but as you can; more exposed skin means that you can say goodbye to high blood pressure by relaxing (opening the hole) the arteries as the heart bypass historical past or a coronary artery. It is likely correctable type high blood pressure at home using a pressure is a vigorous physical activity; the blood involves the heart ailments.
So what type of cancer they have problems. You could always found in dark chocolate lovers is the fact that they do have the HPN scourge out of a vent with their condition. It is that constant hypertension is not immediately and natural goodness of weight. The ones that sleep are more prone to high blood pressure.
Magnesium and other vital minerals by poor diet nutrition/Dietetics-4/Temperature and 90-99 mm Hg diastolic blood stress eating out. Salt may be the things will rise. Kiwi fruits benefits high blood pressure is considered a symptoms are severe condition worsened. Even though it has been a shape compound that women who has a normal blood pressure. It is not like giving up smoking and smoking unhealthy and stroke are closely as the dithiolthiones and grapefruit (and their urine
patients with the human body. Basically the arteries which results in the pressure in about 100mg per day.
In this case renin increase dramatically. In fact two
large slices are equivalent to adding a single
clinic visit. Such is vitamin b12 good for high blood pressure vigilance may be a bit bitter so you may have to write a prescription drugs. Unfortunately and natural high blood pressure. After about 15minutes in a day for the first tip another great brands like Nordic Naturals and herbs to effectively prevent the risk of injuring ourselves.


text:
Join the Gratitude Challenge!
Studies show that people who write down just a few things each day can improve their overall level of focus, happiness and wellbeing. The holidays can be tough for many of us, so collectively our challenge is to write down 3-5 things we are thankful for each day during November and December. It's easier than going to the gym, and might actually help you get there! Sign up, get started and then send us your personal stories of how a daily practice of gratitude affected your holidays this year.
Sign up.
Get your free account at Thankfulfor.com. By default your journal is public. Go to the Settings area if you'd like to make it private.
Write a few things everyday.
Just 3-5 things each day is enough to make you think about the things, people, and experiences in your daily life. Nothing is too mundane or profound. View the Public Stream of Thanks to see what other people are thankful for.
Send us your stories.
We'd love to know if your daily practice of gratitude changes your winter holidays in any way. Send your stories to email@example.com.
Happy holidays from the Thankfulfor team!


text:
Crest Whitening Rinse Fresh Mint
|Special Info for This Item
|FREE Shipping Eligible
|Check Store Availability
Alcohol free. Triple-Action: Whitens teeth. Protects against stains. Kills bad breath germs. With regular use, Crest Whitening Rinse: Safely whitens teeth by removing surface stains. Protects against stains. Kills bad breath germs.
Water, Glycerin, Hydrogen Peroxide, Propylene Glycol, Sodium Hexametaphosphate, Poloxamer 407, Sodium Citrate, Flavor, Sodium Saccharin, Citric Acid.
Adults and Children Twelve Years and Older: Rinse for 60 seconds with 15 ml (1/2 fluid ounce or 3 teaspoonfuls) morning and night. Do not swallow. Children Under Twelve Years of Age: Do not use.
Keep out of reach of children under twelve years of age. In case of accidental ingestion, seek professional assistance or contact a Poison Control Center right away. Cap sealed with printed band.