Daehee Hwang

Generation of T Follicular Helper Cells Is Mediated by Interleukin-21 but Independent of T Helper 1, 2, or 17 Cell Lineages

After activation, CD4(+) helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor beta (TGF-beta) or Th17-specific orphan nuclear receptors RORalpha and RORgamma in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-beta signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage.

Direct Transfer of α-Synuclein from Neuron to Astroglia Causes Inflammatory Responses in Synucleinopathies

Abnormal neuronal aggregation of α-synuclein is implicated in the development of many neurological disorders, including Parkinson disease and dementia with Lewy bodies. Glial cells also show extensive α-synuclein pathology and may contribute to disease progression. However, the mechanism that produces the glial α-synuclein pathology and the interaction between neurons and glia in the disease-inflicted microenvironment remain unknown. Here, we show that α-synuclein proteins released from neuronal cells are taken up by astrocytes through endocytosis and form inclusion bodies. The glial accumulation of α-synuclein through the transmission of the neuronal protein was also demonstrated in a transgenic mouse model expressing human α-synuclein. Furthermore, astrocytes that were exposed to neuronal α-synuclein underwent changes in the gene expression profile reflecting an inflammatory response. Induction of pro-inflammatory cytokines and chemokines correlated with the extent of glial accumulation of α-synuclein. Together, these results suggest that astroglial α-synuclein pathology is produced by direct transmission of neuronal α-synuclein aggregates, causing inflammatory responses. This transmission step is thus an important mediator of pathogenic glial responses and could qualify as a new therapeutic target.

Trifurcate Feed-Forward Regulation of Age-Dependent Cell Death Involving miR164 in Arabidopsis

Aging induces gradual yet massive cell death in higher organisms, including annual plants. Even so, the underlying regulatory mechanisms are barely known, despite the long-standing interest in this topic. Here, we demonstrate that ORE1, which is a NAC (NAM, ATAF, and CUC) transcription factor, positively regulates aging-induced cell death in Arabidopsis leaves. ORE1 expression is up-regulated concurrently with leaf aging by EIN2 but is negatively regulated by miR164. miR164 expression gradually decreases with aging through negative regulation by EIN2, which leads to the elaborate up-regulation of ORE1 expression. However, EIN2 still contributes to aging-induced cell death in the absence of ORE1. The trifurcate feed-forward pathway involving ORE1, miR164, and EIN2 provides a highly robust regulation to ensure that aging induces cell death in Arabidopsis leaves.

Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells

BackgroundVarious cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation. These microvesicles can mediate communication between cells and affect various tumor-related processes in their target cells.ResultsWe present potential roles of CRC cell-derived microvesicles in tumor progression via a global comparative microvesicular and cellular transcriptomic analysis of human SW480 CRC cells. We first identified 11,327 microvesicular mRNAs involved in tumorigenesis-related processes that reflect the physiology of donor CRC cells. We then found 241 mRNAs enriched in the microvesicles above donor cell levels, of which 27 were involved in cell cycle-related processes. Network analysis revealed that most of the cell cycle-related microvesicle-enriched mRNAs were associated with M-phase activities. The integration of two mRNA datasets showed that these M-phase-related mRNAs were differentially regulated across CRC patients, suggesting their potential roles in tumor progression. Finally, we experimentally verified the network-driven hypothesis by showing a significant increase in proliferation of endothelial cells treated with the microvesicles.ConclusionOur study demonstrates that CRC cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells, suggesting that microvesicles of cancer cells can be involved in tumor growth and metastasis by facilitating angiogenesis-related processes. This information will help elucidate the pathophysiological functions of tumor-derived microvesicles, and aid in the development of cancer diagnostics, including colorectal cancer.

Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia

Abnormal aggregation of α-synuclein and sustained microglial activation are important contributors to the pathogenic processes of Parkinsons disease. However, the relationship between disease-associated protein aggregation and microglia-mediated neuroinflammation remains unknown. Here, using a combination of in silico, in vitro and in vivo approaches, we show that extracellular α-synuclein released from neuronal cells is an endogenous agonist for Toll-like receptor 2 (TLR2), which activates inflammatory responses in microglia. The TLR2 ligand activity of α-synuclein is conformation-sensitive; only specific types of oligomer can interact with and activate TLR2. This paracrine interaction between neuron-released oligomeric α-synuclein and TLR2 in microglia suggests that both of these proteins are novel therapeutic targets for modification of neuroinflammation in Parkinsons disease and related neurological diseases.

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.

Proteomic analysis of microvesicles derived from human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSCs self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.

Determination of minimum sample size and discriminatory expression patterns in microarray data

MOTIVATION Transcriptional profiling using microarrays can reveal important information about cellular and tissue expression phenotypes, but these measurements are costly and time consuming. Additionally, tissue sample availability poses further constraints on the number of arrays that can be analyzed in connection with a particular disease or state of interest. It is therefore important to provide a method for the determination of the minimum number of microarrays required to separate, with statistical reliability, distinct disease states or other physiological differences. RESULTS Power analysis was applied to estimate the minimum sample size required for two-class and multi-class discrimination. The power analysis algorithm calculates the appropriate sample size for discrimination of phenotypic subtypes in a reduced dimensional space obtained by Fisher discriminant analysis (FDA). This approach was tested by applying the algorithm to existing data sets for estimation of the minimum sample size required for drawing certain conclusions on multi-class distinction with statistical reliability. It was confirmed that when the minimum number of samples estimated from power analysis is used, group means in the FDA discrimination space are statistically different. CONTACT gregstep@mit.edu

The Rice Oligonucleotide Array Database: an atlas of rice gene expression

BackgroundMicroarray technologies facilitate high-throughput gene expression analysis. However, the diversity of platforms for rice gene expression analysis hinders efficient analysis. Tools to broadly integrate microarray data from different platforms are needed.ResultsIn this study, we developed the Rice Oligonucleotide Array Database (ROAD,http://www.ricearray.org) to explore gene expression across 1,867 publicly available rice microarray hybridizations. The ROAD’s user-friendly web interface and variety of visualization tools facilitate the extraction of gene expression profiles using gene and microarray element identifications. The ROAD supports meta-analysis of genes expressed in different tissues and at developmental stages. Co-expression analysis tool provides information on co-regulation between genes under general, abiotic and biotic stress conditions. Additionally, functional analysis tools, such as Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology, are embedded in the ROAD. These tools facilitate the identification of meaningful biological patterns in a list of query genes.ConclusionsThe Rice Oligonucleotide Array Database provides comprehensive gene expression profiles for all rice genes, and will be a useful resource for researchers of rice and other grass species.

Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin‐digested using a gel‐assisted protocol, and quantified by label‐free LC‐MS/MS, using an MSE mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α‐1‐antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi‐quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.