Biomedical subjects

Ronghai Bo

Publications and source records attributed to Ronghai Bo.

3 recordsLinked to original sources

PubMed2004

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML.

The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC(50) 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.

Adult↗

PubMed2004

A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens.

Gene silencing through RNA interference (RNAi) has been established as a means of conducting reverse genetic studies. In order to better understand the determinants of short interfering RNA (siRNA) knockdown for use in high-throughput cell-based screens, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. The extent of RNA knockdown was measured for 22 genes by quantitative real-time PCR. Analysis of the parameters correlating with effective knockdown showed that (i) duplexes targeting the middle of the coding sequence silenced significantly poorer, (ii) silencing by duplexes targeting the 3'UTR was comparable with duplexes targeting the coding sequence, (iii) pooling of four or five duplexes per gene was remarkably efficient in knocking down gene expression and (iv) among duplexes that achieved a >70% knockdown of the mRNA there were strong nucleotide preferences at specific positions, most notably positions 11 (G or C) and 19 (T) of the siRNA duplex. Finally, in a proof-of-principle pathway-wide cell-based genetic screen, conducted to detect negative genetic regulators of Akt S473 phosphorylation, both known negative regulators of this phosphorylation, PTEN and PDK1, were found. These data help to lay the foundation for genome-wide siRNA screens in mammalian cells.

Cell Line↗

PubMed2003

Pathogen discovery from human tissue by sequence-based computational subtraction.

We have recently reported a new pathogen discovery approach, "computational subtraction". With this approach, non-human transcripts are detected by sequencing cDNA libraries from infected tissue and eliminating those transcripts that match the human genome. We show now that this method is experimentally feasible. We generated a cDNA library from a tissue sample of post-transplant lymphoproliferative disorder (PTLD). 27,840 independent cDNA sequences were filtered by computational subtraction against the known human sequence to identify 32 nonmatching transcripts. Of these, 22 (0.1%) were found to be amplifiable from both infected and noninfected samples and were inferred to be human DNA not yet contained in the available human genome sequence. The remaining 10 sequences could be amplified only from Epstein-Barr virus (EBV)-infected tissues. All 10 corresponded to the known EBV sequence. This proof-of-principle experiment demonstrates that computational subtraction can detect pathogenic microbes in primary human-diseased tissue.

DNA, Complementary↗