Inyoul Lee

Extracellular microRNA: a new source of biomarkers

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.MicroRNAs (miRNAs) are a recently discovered class of small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes, including differentiation, neoplastic transformation, and cell replication and regeneration. Because of these regulatory roles, it is not surprising that aberrant miRNA expression has been implicated in several diseases. Recent studies have reported significant levels of miRNAs in serum and other body fluids, raising the possibility that circulating miRNAs could serve as useful clinical biomarkers. Here, we provide a brief overview of miRNA biogenesis and function, the identification and potential roles of circulating extracellular miRNAs, and the prospective uses of miRNAs as clinical biomarkers. Finally, we address several issues associated with the accurate measurement of miRNAs from biological samples.

A systems approach to prion disease

Prions cause transmissible neurodegenerative diseases and replicate by conformational conversion of normal benign forms of prion protein (PrPC) to disease‐causing PrPSc isoforms. A systems approach to disease postulates that disease arises from perturbation of biological networks in the relevant organ. We tracked global gene expression in the brains of eight distinct mouse strain–prion strain combinations throughout the progression of the disease to capture the effects of prion strain, host genetics, and PrP concentration on disease incubation time. Subtractive analyses exploiting various aspects of prion biology and infection identified a core of 333 differentially expressed genes (DEGs) that appeared central to prion disease. DEGs were mapped into functional pathways and networks reflecting defined neuropathological events and PrPSc replication and accumulation, enabling the identification of novel modules and modules that may be involved in genetic effects on incubation time and in prion strain specificity. Our systems analysis provides a comprehensive basis for developing models for prion replication and disease, and suggests some possible therapeutic approaches.

Comparative Genomics of the Human and Mouse T Cell Receptor Loci

The availability of the complete genomic sequences of the human and mouse T cell receptor loci opens up new opportunities for understanding T cell receptors (TCRs) and their genes. The full complement of TCR gene segments is finally known and should prove a valuable resource for supporting functional studies. A rational nomenclature system has been implemented and is widely available through IMGT and other public databases. Systematic comparisons of the genomic sequences within each locus, between loci, and across species enable precise analyses of the various diversification mechanisms and some regulatory signals. The genomic landscape of the TCR loci provides fundamental insights into TCR evolution as highly localized and tightly regulated gene families.

Systems Biology and the Discovery of Diagnostic Biomarkers

Systems biology is an approach to the science that views biology as an information science, studies biological systems as a whole and their interactions with the environment. This approach, for the reasons described here, has particular power in the search for informative diagnostic biomarkers of diseases because it focuses on the fundamental causes and keys on the identification and understanding of disease- perturbed molecular networks. In this review, we describe some recent developments that have used systems biology to address complex diseases – prion disease and drug induced liver injury- and use these as examples to illustrate the importance of understanding network structure and dynamics. The knowledge of network dynamics through in vitro experimental perturbation and modeling allows us to determine the state of the networks, to identify molecular correlates, and to derive new disease treatment approaches to reverse the pathology or prevent its progress into a more severe state through the manipulation of network states. This general approach, including diagnostics and therapeutics, is becoming known as systems medicine.

Principal network analysis

MOTIVATION Systems biology attempts to describe complex systems behaviors in terms of dynamic operations of biological networks. However, there is lack of tools that can effectively decode complex network dynamics over multiple conditions. RESULTS We present principal network analysis (PNA) that can automatically capture major dynamic activation patterns over multiple conditions and then generate protein and metabolic subnetworks for the captured patterns. We first demonstrated the utility of this method by applying it to a synthetic dataset. The results showed that PNA correctly captured the subnetworks representing dynamics in the data. We further applied PNA to two time-course gene expression profiles collected from (i) MCF7 cells after treatments of HRG at multiple doses and (ii) brain samples of four strains of mice infected with two prion strains. The resulting subnetworks and their interactions revealed network dynamics associated with HRG dose-dependent regulation of cell proliferation and differentiation and early PrPSc accumulation during prion infection. AVAILABILITY The web-based software is available at: http://sbm.postech.ac.kr/pna.

Issues and Prospects of microRNA-Based Biomarkers in Blood and Other Body Fluids

Cell-free circulating microRNAs (miRNAs) in the blood are good diagnostic biomarker candidates for various physiopathological conditions, including cancer, neurodegeneration, diabetes and other diseases. Since their discovery in 2008 as blood biomarkers, the field has expanded rapidly with a number of important findings. Despite the initial optimistic views of their potential for clinical application, there are currently no circulating miRNA-based diagnostics in use. In this article, we review the status of circulating miRNAs, examine different analytical approaches, and address some of the challenges and opportunities.

Down-Regulation of Shadoo in Prion Infections Traces a Pre-Clinical Event Inversely Related to PrPSc Accumulation

During prion infections of the central nervous system (CNS) the cellular prion protein, PrPC, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp a and Prnp b mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.

Systems Approach to Neurodegenerative Disease Biomarker Discovery

Biomarkers are essential for performing early diagnosis, monitoring neurodegenerative disease progression, gauging responses to therapies, and stratifying neurodegenerative diseases into their different subtypes. A wide range of molecular markers are under investigation in tissues and biofluids as well as through imaging; moreover, many are prominent proteins present in cerebrospinal fluid. However, in more frequently and easily collected fluids such as plasma, these proteins show only a modest correlation with disease and thus lack the necessary sensitivity or specificity for clinical use. High-throughput and quantitative proteomic technologies and systems-driven approaches to biofluid analysis are now being utilized in the search for better biomarkers. Biomarker discovery involves many critical steps including study design, sample preparation, protein and peptide separation and identification, and bioinformatics and data integration issues that must be carefully controlled before independent confirmation and validation. In this review, we summarize current proteomic and nucleic acid technologies involved in the discovery of biomarkers of neurodegenerative diseases, particularly Alzheimers, Parkinsons, Huntingtons, and prion diseases.

Characterization of the Japanese pufferfish (Takifugu rubripes) T-cell receptor α locus reveals a unique genomic organization

Abstract. Polymerase chain reactions with degenerate V gene segment primers were used to isolate the putative T-cell receptor α-chain gene (TCRA) from Japanese pufferfish (Takifugu rubripes). The putative TCRA chain cDNA is composed of an N-terminus leader peptide followed by the variable region and the constant region. The variable portion of the TCRA gene is encoded by V and J gene segments separated in the germline. As in mammals, the V-J junction sequences are GC rich and highly diversified. Amino acid residues that are required to maintain the function and structural integrity of the TCRA polypeptide, including the conserved Trp-Tyr-Lys and Tyr-Tyr-Cys motifs in the V gene segments, the Lys-Leu-X-Phe-Gly-X-Gly-Thr-X-Leu motif in the J gene segment, the three cysteine residues in the constant region and the charged residues in the transmembrane region are all preserved in the pufferfish. These conserved features suggest that the TCRA gene families in fish and mammals have evolved from a common ancestor.

Molecular evidence of stress-induced acute heart injury in a mouse model simulating posttraumatic stress disorder

Significance Exposure to extremely stressful conditions is common, and the effect of such exposure on neuropsychiatric function is well-documented with posttraumatic stress disorder (PTSD). Epidemiological studies reveal a higher risk for cardiovascular conditions among individuals exposed to traumatic events. However, the underlying molecular mechanism for ailments associated with stress exposure is yet to be fully understood. Our study with animal models revealed genetically associated stress-induced tissue injuries on peripheral organs, including the heart. Longitudinal transcriptomics studies uncovered detailed molecular events involved in stress-related heart damage followed immediately by tissue-repairing processes; whether this injury and repairing process causes long-term effects is uncertain. Our findings on heart injury in a PTSD mouse model clearly indicate physiological changes arising from stress. Posttraumatic stress disorder (PTSD) is a common condition induced by life-threatening stress, such as that experienced by soldiers under battlefield conditions. Other than the commonly recognized behavioral and psychological dysfunction, epidemiological studies have also revealed that PTSD patients have a higher risk of other diseases, such as cardiovascular disorders. Using a PTSD mouse model, we investigated the longitudinal transcriptomic changes in heart tissues after the exposure to stress through intimidation. Our results revealed acute heart injury associated with the traumatic experience, reflecting the underlying biological injury processes of the immune response, extracellular matrix remodeling, epithelial-to-mesenchymal cell transitions, and cell proliferation. Whether this type of injury has any long-term effects on heart function is yet to be determined. The differing responses to stress leading to acute heart injury in different inbred strains of mice also suggest that this response has a genetic as well as an environmental component. Accordingly, the results from this study suggest a molecular basis for the observed higher risk of cardiovascular disorders in PTSD patients, which raises the likelihood of cardiac dysfunction induced by long-term stress exposures.