Lihan Zhou

Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

BackgroundAccurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli.ResultsIn this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression.ConclusionThis study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.

Identification and validation of reference genes for expression studies in a rat model of neuropathic pain.

Neuropathic pain is triggered by damage to or as a result of the dysfunction of the somatosensory nervous system. Gene expression profiling using DNA microarray and real-time PCR have emerged as powerful tools for the elucidation of pain-specific pathways and identification of candidate biomarkers and therapeutic targets. Proper normalization of the gene expression data with stable reference genes is a prerequisite to obtaining accurate gene expression changes. We have evaluated the stability of six candidate reference genes which include three commonly used housekeeping genes (ACTB, GAPDH and HMBS) and three ribosomal protein genes (RPL3, RPL19 and RPL29) using real-time PCR in a rat model of neuropathic pain. Unexpectedly, ACTB but not GAPDH was stably expressed. In addition, we have identified RPL29 and RPL3 as novel reference genes. Normalization of expression data using GAPDH or HMBS led to overestimation of transcriptional changes. Using RPL29/RPL3/ACTB as reference genes, a number of transcripts were found to be specifically and significantly regulated in injured dorsal root ganglia. These genes may contribute to the development of neuropathic pain pathology and may serve as candidate biomarkers for potential diagnosis.

Thermoporometry characterization of silica microparticles and nanowires.

We present the results of a systematic study on the porosity of silica microparticles and nanowires prepared by glancing angle deposition-metal-assisted chemical etching (GLAD-MACE) and interference lithography-metal-assisted chemical etching (IL-MACE) techniques using the thermoporometry (TPM) method. Good agreement was obtained between our TPM results and published data provided by the suppliers of silica microparticles. TPM characterization of the GLAD-MACE and IL-MACE nanowires was carried out on the basis of parameters obtained from TPM experiments on microparticles. Our nanowires showed a similar trend but lower values of the pore volume and surface area than nanowires prepared by MACE with AgNO3 solution. We attribute the enhanced bioanalysis performance of the GLAD-MACE nanowires based devices to the increased pore volume and total surface area of the nanowires.

Nano-topology guided neurite outgrowth in PC12 cells is mediated by miRNAs

UNLABELLEDnMicroRNAs (miRNAs) are master regulators of gene expression at post-transcriptional level. The present study investigated the involvement of miRNAs in topological guidance of neurite outgrowth in an NGF treated PC12 cell model cultured on nano-patterned polyethylene terephthalate (PET) substrates fabricated with interference lithography. The expressions of 38 neuronal miRNAs were measured and 3 were found to be differentially regulated during topological guidance of neurite outgrowth. Altering the intracellular levels of these miRNAs disrupted the orderly growth of neurite along nano-patterned substrate. Our results showed miRNAs to be versatile regulators and their involvement should be thoroughly investigated for better understanding of biological processes.nnnFROM THE CLINICAL EDITORnIn this basic science study, strong evidence was found that topological guidance is only one factor, and miRNA-s regulate axonal outgrowth from neurites. Nano-patterned polyethylene terephthalate substrates were used for the study, fabricated using interference lithography. Further studies of this biologically relevant process may pave the way to clinically useful axonal regrowth and axonal guidance methods.

GDNF family ligand dependent STAT3 activation is mediated by specific alternatively spliced isoforms of GFRα2 and RET.

Neurturin (NRTN), a member of the GDNF family of ligands (GFL), is currently investigated in a series of clinical trials for Parkinsons disease. NRTN signals through its cognate receptor GFRα2 and co-receptor RET to induce neurite outgrowth, but the underlying mechanism remains to be better understood. STAT3 was previously shown to be activated by oncogenic RET, independent of ligand and GFRα. In this study, we demonstrated that NRTN induced serine(727) but not tyrosine(705) phosphorylation of STAT3 in primary cortical neuron and neuronal cell lines. Remarkably, STAT3 phosphorylation was found to be mediated specifically by GFRα2c and RET9 isoforms. Furthermore, serine but not tyrosine dominant negative mutant of STAT3 impaired NRTN induced neurite outgrowth, indicative of the role of STAT3 as a downstream mediator of NRTN function. Similar to NGF, the NRTN induced P-Ser-STAT3 was localized to the mitochondria but not to the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NRTN induced neurite outgrowth. Collectively, these findings demonstrated the hitherto unrecognized and novel role of specific GFRα2 and RET isoforms in mediating NRTN activation of STAT3 and the transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 mediated NRTN induced neurite outgrowth.

Establishing multiple omics baselines for three Southeast Asian populations in the Singapore Integrative Omics Study

The Singapore Integrative Omics Study provides valuable insights on establishing population reference measurement in 364 Chinese, Malay, and Indian individuals. These measurements include >u20092.5 millions genetic variants, 21,649 transcripts expression, 282 lipid species quantification, and 284 clinical, lifestyle, and dietary variables. This concept paper introduces the depth of the data resource, and investigates the extent of ethnic variation at these omics and non-omics biomarkers. It is evident that there are specific biomarkers in each of these platforms to differentiate between the ethnicities, and intra-population analyses suggest that Chinese and Indians are the most biologically homogeneous and heterogeneous, respectively, of the three groups. Consistent patterns of correlations between lipid species also suggest the possibility of lipid tagging to simplify future lipidomics assays. The Singapore Integrative Omics Study is expected to allow the characterization of intra-omic and inter-omic correlations within and across all three ethnic groups through a systems biology approach.The Singapore Genome Variation projects characterized the genetics of Singapore’s Chinese, Malay, and Indian populations. The Singapore Integrative Omics Study introduced here goes further in providing multi-omic measurements in individuals from these populations, including genetic, transcriptome, lipidome, and lifestyle data, and will facilitate the study of common diseases in Asian communities.

c-Jun N-terminal kinase in synergistic neurite outgrowth in PC12 cells mediated through P90RSK.

BackgroundSynergistic multi-ligand treatments that can induce neuronal differentiation offer valuable strategies to regulate and modulate neurite outgrowth. Whereas the signaling pathways mediating single ligand-induced neurite outgrowth, such as Akt, extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (P38), have been extensively studied, the mechanisms underlying multi-ligand synergistic neurite outgrowth are poorly understood. In an attempt to gain insight into synergistic neurite outgrowth, PC12 cells were treated with one of three combinations: pituitary adenylate cyclase-activating peptide (PACAP) with epidermal growth factor (EP), basic fibroblast growth factor (FP), or nerve growth factor (NP) and then challenged with the appropriate kinase inhibitors to assess the signaling pathways involved in the process.ResultsResponse surface analyses indicated that synergistic neurite outgrowth was regulated by distinct pathways in these systems. Synergistic increases in the phosphorylation of Erk and JNK, but not Akt or P38, were observed with the three growth factor-PACAP combinations. Unexpectedly, we identified a synergistic increase in JNK phosphorylation, which was involved in neurite outgrowth in the NP and FP, but not EP, systems. Inhibition of JNK using the SP600125 inhibitor reduced phosphorylation of 90 kDa ribosomal S6 kinase (P90RSK) in the NP and FP, but not EP, systems. This suggested the involvement of P90RSK in mediating the differential effects of JNK in synergistic neurite outgrowth.ConclusionsTaken together, these findings reveal the involvement of distinct signaling pathways in regulating neurite outgrowth in response to different synergistic growth factor-PACAP treatments. Our findings demonstrate a hitherto unrecognized mechanism of JNK-P90RSK in mediating synergistic neurite outgrowth induced by the co-treatment of growth factors and PACAP.

Photo-attachment of biomolecules for miniaturization on wicking Si-nanowire platform.

We demonstrated the surface functionalization of a highly three-dimensional, superhydrophilic wicking substrate using light to immobilize functional biomolecules for sensor or microarray applications. We showed here that the three-dimensional substrate was compatible with photo-attachment and the performance of functionalization was greatly improved due to both increased surface capacity and reduced substrate reflectivity. In addition, photo-attachment circumvents the problems induced by wicking effect that was typically encountered on superhydrophilic three-dimensional substrates, thus reducing the difficulty of producing miniaturized sites on such substrate. We have investigated various aspects of photo-attachment process on the nanowire substrate, including the role of different buffers, the effect of wavelength as well as how changing probe structure may affect the functionalization process. We demonstrated that substrate fabrication and functionalization can be achieved with processes compatible with microelectronics processes, hence reducing the cost of array fabrication. Such functionalization method coupled with the high capacity surface makes the substrate an ideal candidate for sensor or microarray for sensitive detection of target analytes.

112 Identification and Validation of Serum MicroRNA Biomarker for Early Detection of Gastric Cancer

stage (p<0.01), lymph node (p=0.0004) and liver metastasis (p = 0.02) in CRC. Furthermore, Kaplan-Meier curves revealed that high serum ANGPTL2 was significantly associated with poor disease free survival (p=0.01) and overall survival (p=0.03) in CRC. Similarly, serum ANGPTL2 levels in GC patients were also significantly higher compared to NC (p<0.0001), had a promising AUC (0.83) to distinguish GC from NC. In addition, serum ANGPTL2 levels also could discriminate early GC patients (stage I) from NC (AUC=0.80). High ANGPTL2 in serum was significantly associated tumor progression, consequently emerged as an independent predicting recurrence marker (HR=5.05, p=0.0004) and prognostic marker (HR=3.6, p=0.01) in GC. Conclusion: Our study first reports that serum ANGPTL2 was a novel biomarker for diagnosis in patients with GC and CRC. In addition, serum ANGPTL2 levels were significantly associated with tumor progression and prognostic outcomes in both GC and CRC. In particular, serum ANGPTL2 was an independent predictive biomarker of recurrence and prognosis in patients with GC.