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author:("Chen, fengxin")
1.  Proteomic Basis of the Antibody Response to Monkeypox Virus Infection Examined in Cynomolgus Macaques and a Comparison to Human Smallpox Vaccination 
PLoS ONE  2010;5(12):e15547.
Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92–95% (166–192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.
PMCID: PMC3012712  PMID: 21209900
2.  Identification of Synaptic Targets of Drosophila Pumilio 
PLoS Computational Biology  2008;4(2):e1000026.
Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage.
Author Summary
The Drosophila Pumilio (Pum) protein was originally identified as a translational control factor for embryo patterning. Subsequent studies have identified Pum's role in multiple biological processes, including the maintenance of germline stem cell, the proliferation and migration of primordial germ cells, olfactory leaning and memory, and synaptic plasticity. Pum is highly conserved across phyla, i.e., from worm to human; however, the mRNA targets of Pum within each tissue and organism are largely unknown. On the other hand, the prediction of RNA binding sites remains a hard question in the computational field. We were interested in finding Pum targets in the nervous system using fruit flies as a model organism. To accomplish this, we used the few Pum binding sequences that had previously been shown in vivo as “training sequences” to construct bioinformatic models of the Pum binding site. We then predicted a few Pum mRNA targets among the genes known to function in neuronal synapses. We then used a combination of “golden standards” to verify these predictions: a biochemical assay called gel shifts, and in vivo functional assays both in embryo and neurons. With these approaches, we successfully confirmed one of the targets as Dlg, which is the Drosophila ortholog of human PSD95. Therefore, we present a complete story from computational study to real biological functions.
PMCID: PMC2265480  PMID: 18463699
3.  Genome-wide promoter extraction and analysis in human, mouse, and rat 
Genome Biology  2005;6(8):R72.
An investigation of how to improve mammalian promoter prediction by incorporating both transcript and conservation information leads to the creation of CSHLmpd, a mammalian promoter database.
Large-scale and high-throughput genomics research needs reliable and comprehensive genome-wide promoter annotation resources. We have conducted a systematic investigation on how to improve mammalian promoter prediction by incorporating both transcript and conservation information. This enabled us to build a better multispecies promoter annotation pipeline and hence to create CSHLmpd (Cold Spring Harbor Laboratory Mammalian Promoter Database) for the biomedical research community, which can act as a starting reference system for more refined functional annotations.
PMCID: PMC1273639  PMID: 16086854
4.  Transcription factor binding element detection using functional clustering of mutant expression data 
Nucleic Acids Research  2004;32(8):2362-2371.
As a powerful tool to reveal gene functions, gene mutation has been used extensively in molecular biology studies. With high throughput technologies, such as DNA microarray, genome-wide gene expression changes can be monitored in mutants. Here we present a simple approach to detect the transcription-factor-binding motif using microarray expression data from a mutant in which the relevant transcription factor is deleted. A core part of our approach is clustering of differentially expressed genes based on functional annotations, such as Gene Ontology (GO). We tested our method with eight microarray data sets from the Rosetta Compendium and were able to detect canonical binding motifs for at least four transcription factors. With the support of chromatin IP chip data, we also predict a possible variant of the Swi4 binding motif and recover a core motif for Arg80. Our approach should be readily applicable to microarray experiments using other types of molecular biology techniques, such as conditional knockout/overexpression or RNAi-mediated ‘knockdown’, to perturb the expression of a transcription factor. Functional clustering included in our approach may also provide new insights into the function of the relevant transcription factor.
PMCID: PMC419446  PMID: 15115798
5.  Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity 
British Journal of Haematology  2013;164(2):233-244.
Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.
PMCID: PMC4253085  PMID: 24206017
immunomodulatory drugs; cereblon; myeloma; cullin 4 ring ligase complex; DNA damage binding protein 1

Results 1-5 (5)