Standardization of surface electromyography utilized to evaluate patients with dysphagia

<p>Abstract</p> <p>Backgorund</p> <p>Patients suspected of having swallowing disorders, could highly benefit from simple diagnostic screening before being referred to specialist evaluations. We introduce surface electromyography (sEMG) to carry out rapid assessment of such patients and propose suggestions for standardizing sEMGs in order to identify abnormal deglutition.</p> <p>Methods</p> <p>Specifics steps for establishing standards for applying the technique for screening purposes (e.g., evaluation of specific muscles), the requirements for diagnostic sEMG equipment, the sEMG technique itself, and defining the tests suitable for assessing deglutition (e.g., saliva, normal, and excessive swallows and uninterrupted drinking of water) are presented in detail. A previously described normative database for single swallowing and drinking and standard approach to analysis was compared to data on the duration and electric activity of muscles involved in deglutition and with sEMG recordings in order to estimate stages of a swallow.</p> <p>Conclusion</p> <p>SEMG of swallowing is a simple and reliable method for screening and preliminary differentiation among dysphagia and odynophagia of various origins. This noninvasive radiation-free examination has a low level of discomfort, and is simple, timesaving and inexpensive to perform. With standardization of the technique and an established normative database, sEMG can serve as a reliable screening method for optimal patient management.</p

Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line’s species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification

Efficient Differentiation of Embryonic Stem Cells into Mesodermal Precursors by BMP, Retinoic Acid and Notch Signalling

The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

Differentiating Embryonic Stem Cells Pass through ‘Temporal Windows’ That Mark Responsiveness to Exogenous and Paracrine Mesendoderm Inducing Signals

BACKGROUND: Mesendoderm induction during embryonic stem cell (ESC) differentiation in vitro is stimulated by the Transforming Growth Factor and Wingless (Wnt) families of growth factors. PRINCIPAL FINDINGS: We identified the periods during which Bone Morphogenetic Protein (BMP) 4, Wnt3a or Activin A were able to induce expression of the mesendoderm marker, Mixl1, in differentiating mouse ESCs. BMP4 and Wnt3a were required between differentiation day (d) 1.5 and 3 to most effectively induce Mixl1, whilst Activin A induced Mixl1 expression in ESC when added between d2 and d4, indicating a subtle difference in the requirement for Activin receptor signalling in this process. Stimulation of ESCs with these factors at earlier or later times resulted in little Mixl1 induction, suggesting that the differentiating ESCs passed through 'temporal windows' in which they sequentially gained and lost competence to respond to each growth factor. Inhibition of either Activin or Wnt signalling blocked Mixl1 induction by any of the three mesendoderm-inducing factors. Mixing experiments in which chimeric EBs were formed between growth factor-treated and untreated ESCs revealed that BMP, Activin and Wnt signalling all contributed to the propagation of paracrine mesendoderm inducing signals between adjacent cells. Finally, we demonstrated that the differentiating cells passed through 'exit gates' after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression. CONCLUSIONS: These studies suggest that differentiating ESCs are directed by an interconnected network of growth factors similar to those present in early embryos and that the timing of growth factor activity is critical for mesendoderm induction

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity

The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment