Clinical integration of next-generation sequencing technology.

Recent advances in next-generation sequencing (NGS) methods and technology have substantially reduced costs and operational complexity leading to production of benchtop sequencers and commercial software solutions for implementation in small research and clinical laboratories. This article addresses requirements and limitations to successful implementation of these systems, including (1) calibration and validation of the instrumentation, experimental paradigm, and primary readout, (2) secure data transfer, storage, and secondary processing, (3) implementation of software tools for targeted analysis, and (4) training of research and clinical personnel to evaluate data fidelity and interpret the molecular significance of the genomic output.

Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

Acute molecular response of mouse hindlimb muscles to chronic stimulation.

Stimulation of the mouse hindlimb via the sciatic nerve was performed for a 4-h period to investigate acute muscle gene activation in a model of muscle phenotype conversion. Initial force production (1.6 +/- 0.1 g/g body wt) declined 45% within 10 min and was maintained for the remainder of the experiment. Force returned to initial levels upon study completion. An immediate-early growth response was present in the extensor digitorum longus (EDL) muscle (FOS, JUN, activating transcription factor 3, and musculoaponeurotic fibrosarcoma oncogene) with a similar but attenuated pattern in the soleus muscle. Transcript profiles showed decreased fast fiber-specific mRNA (myosin heavy chains 2A and 2B, fast troponins T(3) and I, alpha-tropomyosin, muscle creatine kinase, and parvalbumin) and increased slow transcripts (myosin heavy chain-1beta/slow, troponin C slow, and tropomyosin 3y) in the EDL versus soleus muscles. Histological analysis of the EDL revealed glycogen depletion without inflammatory cell infiltration in stimulated versus control muscles, whereas ultrastructural analysis showed no evidence of myofiber damage after stimulation. Multiple fiber type-specific transcription factors (tea domain family member 1, nuclear factor of activated T cells 1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha and -beta, circadian locomotor output cycles kaput, and hypoxia-inducible factor-1alpha) increased in the EDL along with transcription factors characteristic of embryogenesis (Kruppel-like factor 4; SRY box containing 17; transcription factor 15; PBX/knotted 1 homeobox 1; and embryonic lethal, abnormal vision). No established in vivo satellite cell markers or genes activated in our parallel experiments of satellite cell proliferation in vitro (cyclins A(2), B(2), C, and E(1) and MyoD) were differentially increased in the stimulated muscles. These results indicated that the molecular onset of fast to slow phenotype conversion occurred in the EDL within 4 h of stimulation without injury or satellite cell recruitment. This conversion was associated with the expression of phenotype-specific transcription factors from resident fiber myonuclei, including the activation of nascent developmental transcriptional programs.

The regulatory role of nitric oxide in apoptosis.

Nitric oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology. The complexity of its biological effects is a consequence of its numerous potential interactions with other molecules such as reactive oxygen species (ROS), metal ions, and proteins. The effects of NO are modulated by both direct and indirect interactions that can be dose-dependent and cell-type specific. For example, in some cell types NO can promote apoptosis, whereas in other cells NO inhibits apoptosis. In hepatocytes, NO can inhibit the main mediators of cell death-caspase proteases. Moreover, low physiological concentrations of NO can inhibit apoptosis, but higher concentrations of NO may be toxic. High NO concentrations lead to the formation of toxic reaction products like dinitrogen trioxide or peroxynitrite that induce cell death, if not by apoptosis, then by necrosis. Long-term exposure to nitric oxide in certain conditions like chronic inflammatory states may predispose cells to tumorigenesis through DNA damage, inhibition of DNA repair, alteration in programmed cell death, or activation of proliferative signaling pathways. Understanding the regulatory mechanisms of NO in apoptosis and carcinogenesis will provide important clues to the diagnosis and treatment of tissue damage and cancer. In this article we have reviewed recent discoveries in the regulatory role of NO in specific cell types, mechanisms of pro-apoptotic and anti-apoptotic induction by NO, and insights into the effects of NO on tumor biology.

Muscle tissue engineering.

Only recently have scientists come to appreciate that many tissues and cell populations that were formerly considered to be in a terminally differentiated state are capable of division and or dedifferentiation. It is the goal of the tissue engineer to understand and redirect this potential. Muscle tissue-engineering efforts will be directed toward building in vitro replacements for in vivo problems. Tissue-engineering advances will be interdependent with advances in gene therapy techniques to restore function at a cellular level.

Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.

Control of attachment, morphology, and proliferation of skeletal myoblasts on silanized glass.

Generating skeletal muscle in vitro is an attractive approach to overcome problems associated with autologous transfer of muscle and donor site morbidity during plastic surgery. Such tissue engineering requires application of biomaterials that selectively control the attachment, morphology, and proliferation of muscle progenitor ("satellite") cells. This study examined the initial attachment, morphological characteristics, and proliferative behavior of murine C2C12 myoblasts on glass substrata derivatized with self-assembled monolayers (SAMs) of the organosiloxanes N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane (EDA) and tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosil ane (13F). The fraction of myoblasts resisting detachment upon rinsing was greater on EDA than on 13F. Application of a quantitative moments-based analysis of cell morphology demonstrated that projected area and two size-invariant metrics of shape (extension and dispersion) for these cells were greater for EDA than for 13F. Myoblasts also proliferated faster on EDA than on 13F. These data indicate that EDA-derivatized glass provides a superior substratum for myoblast culture compared to 13F-derivatized glass. Understanding myoblast behavior on these biomaterials that promotes contrasting cellular responses is the first step toward using patterned SAMs to control myotube alignment for tissue engineering skeletal muscle.

Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol.

For cardiovascular biomaterials, thrombosis, thromboembolism and vascular graft occlusion are believed to be precipitated by the adsorption of proteins containing adhesive ligands for platelets. Polyethylene-glycol-diisocyanate(PEG-diisocyanate, 3400 MW) may potentially react with protein amines to form molecular barriers on adsorbed proteins on biomaterials, thereby masking adhesive ligands and preventing acute surface thrombosis. To test this notion, PE, PTFE, and glass microconduits were pre-adsorbed with fibrinogen and treated with PEG-diisocyanate, non-reactive PEG-dihydroxyl, or remained untreated. Following perfusion of 111In-labeled platelets in whole human blood for 1 min (wall shear rate = 312 s(-1)), PEG-diisocyanate treated surfaces experienced 96%(PE), 97%(PTFE) and 94% (glass) less platelet deposition than untreated surfaces. Similar reductions were seen for PEG-diisocyanate versus PEG-dihydroxyl treatment. Low shear perfusions of plasma for one hour prior to blood contact did not reduce the inhibitory effect of PEG-diisocyanate. Platelet adhesion onto collagen coated glass coverslips and platelet deposition onto preclotted Dacron was also reduced by treatment with PEG-diisocyanate (93 and 91%, respectively). Protein-reactive PEG may thus have utility in forming molecular barriers on surface associated proteins to inhibit acute thrombosis on cardiovascular biomaterials.

Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol.

For cardiovascular biomaterials, thrombosis, thromboembolism and vascular graft occlusion are believed to be precipitated by the adsorption of proteins containing adhesive ligands for platelets. Polyethylene-glycol-diisocyanate (PEG-diisocyanate, 3400 MW) may potentially react with protein amines to form molecular barriers on adsorbed proteins on biomaterials, thereby masking adhesive ligands and preventing acute surface thrombosis. To test this notion, PE, PTFE, and glass microconduits were pre-adsorbed with fibrinogen and treated with PEG-diisocyanate, non-reactive PEG-dihydroxyl, or remained untreated. Following perfusion of 111In-labeled platelets in whole human blood for 1 min (wall shear rate = 312 s(-1)), PEG-diisocyanate treated surfaces experienced 96% (PE), 97% (PTFE) and 94% (glass) less platelet deposition than untreated surfaces. Similar reductions were seen for PEG-diisocyanate versus PEG-dihydroxyl treatment. Low shear perfusions of plasma for 1 h prior to blood contact did not reduce the inhibitory effect of PEG-diisocyanate. Platelet adhesion onto collagen-coated glass coverslips and platelet deposition onto preclotted Dacron were also reduced by treatment with PEG-diisocyanate (93 and 91%, respectively). Protein-reactive PEG may thus have utility in forming molecular barriers on surface-associated proteins to inhibit acute thrombosis on cardiovascular biomaterials.