Circulating microRNAs (miRNAs) are promising biomarkers for cancer detection. However, multiethnic and multicentric studies of non-small-cell lung cancer (NSCLC) are lacking. We recruited 221 NSCLC patients, 161 controls and 56 benign nodules from both China and America. Initial miRNA screening was performed using the TaqMan Low Density Array followed by confirming individually by RT-qPCR in Chinese cohorts. Finally, we performed a blind trial from an American cohort to validate our findings. RT-qPCR confirmed that miR-483-5p, miR-193a-3p, miR-25, miR-214 and miR-7 were significantly elevated in patients compared to controls. The areas under the curve (AUCs) of the ROC curve of this five-serum miRNA panel were 0.976 (95% CI, 0.939–1.0; P < 0.0001) and 0.823 (95% CI, 0.75–0.896; P < 0.0001) for the two confirmation sets, respectively. In the blind trial, the panel correctly classified 95% NSCLC cases and 84% controls from the American cohort. Most importantly, the panel was capable of distinguishing NSCLC from benign nodules with an AUC of 0.979 (95% CI, 0.959–1.0) in the American cohort and allowed correct prediction of 86% and 95% stage I–II tumors in the Chinese and American cohorts, respectively. This serum miRNA panel holds the potential for diagnosing ethnically diverse NSCLC patients.

SummaryAlthough much has been learned about the molecular basis of immunoglobulin M (IgM) rheumatoid factors (KFs) in healthy individuals and in patients with mixed cryoglobulinemia and rheumatoid arthritis, little is known about the genetic origins of the potentially pathogenic IgG RFs in the inflamed rheumatoid synovia of patients. Recently, we generated from unmanipulated synovium B cells several hybridomas that secreted self-associating IgG KFs. To delineate the genetic origins of such potentially pathogenic RFs, we adapted the anchored polymerase chain reaction to rapidly clone and characterize the expressed Ig V genes for the L1 and the D1 IgG RFs. Then, we identified the germline counterparts of the expressed L1 IgG KF V genes. The results showed that the L1 heavy chain was encoded by a Vh gene that is expressed preferentially during early ontogenic development, and that is probably located within 240 kb upstream of the Jh locus. The overlap between this KF Vh gene and the restricted fetal antibody repertoire is reminiscent of the natural antibody-associated Vh genes, and suggests that at least part of the "potential pathogenic" IgG RFs in rheumatoid synovium may derive from the "physiological" natural antibody repertoire in a normal immune system. Indeed, the corresponding germline Vh gene for L1 encodes the heavy chain of an IgM KF found in a 19-wk-old fetal spleen. Furthermore, the comparisons of the expressed ILF V genes and their germline counterparts reveal that the L1 heavy and light chain variable regions had, respectively, 16 and 7 somatic mutations, which resulted in eight and four amino acid changes. Strikingly, all eight mutations in the complementarity determining regions of the V gene-encoded regions were replacement changes, while only 6 of 11 mutations in the framework regions caused amino acid changes. Combined with Ll's high binding affinity toward the Fc fragment, these results suggest strongly that the L1 IgG KF must have been driven by the Fc antigen.

The gene encoding N,N'-diacetylchitobiase (chitobiase) of the chitinolytic marine bacterium Vibrio harveyi has been isolated. While expression of the chitobiase gene (chb) was inducible by N,N'-diacetylchitobiose in V. harveyi, it was expressed constitutively when cloned in Escherichia coli, suggesting that controlling elements are not closely linked to chb. Chitobiase was found in the membrane fraction of E. coli cells containing plasmids with the cloned V. harveyi chb gene. When membranes of such cells were separated on Osborn gradients, chitobiase activity was found mainly in the outer membrane band. Translocation of the enzyme to the outer membrane was accompanied by cleavage of a signal peptide. A fusion protein, in which 22 amino acids from the amino terminus of prechitobiase were replaced with 21 amino acids from the pUC19 lacZ amino terminus, was not processed, and 99% of the activity was located in the cytoplasmic fraction. A homology to six amino acids surrounding the lipoprotein processing and modification site was found near the amino terminus of prechitobiase.Chitin is an insoluble polysaccharide consisting of ,-(1,4)-linked N-acetylglucosamine (GlcNac) units. Large reservoirs of this polysaccharide in the marine and terrestrial environments are derived from the cell walls of fungi and the cuticles of crustaceans and insects (9, 23). Many bacteria in these environments are able to hydrolyze chitin to GlcNac (16,18,19) by using two enzymes, chitinase and N,N'-diacetylchitobiase (chitobiase). Chitin is first hydrolyzed by chitinase to low-molecular-weight multimers of GlcNac, the dimer N,N'-diacetylchitobiose (chitobiose) being predominant. Chitobiase then hydrolyzes chitobiose to GlcNac.To begin an analysis of these enzymes, we have cloned the genes encoding chitinase and chitobiase from the gramnegative marine bacterium Vibrio harveyi. Chitinase genes have also been cloned from Serratia marcescens (5, 10), in which two chitinase genes encoding two different chitinases have been identified (10). The chitobiase gene has been isolated from S. marcescens (8) and Vibrio vulnificus (25). Here, we show that chitobiase is located in the outer membrane of Escherichia coli cells containing the cloned chitobiase gene (chb). Based on minicell analysis of plasmidencoded proteins, the approximate sizes of the unprocessed and processed proteins are 95.3 kilodaltons (kDa) and 92.1 kDa, respectively. When the first 21 amino acids of lacZ from pUC19 were substituted for the first 22 amino acids of prechitobiase, a hybrid protein resulted that had chitobiase activity, remained unprocessed, and was found only in the cytoplasm, as would be expected if the amino terminus of prechitobiase were involved in processing and translocation to the outer membrane. MATERIALS AND METHODSBacterial strains, plasmids, and media. E. coli K-12 strain LE392 (F-) hsdR lacY galK2 galT22 metBI trpR55 supE44 supF58, E. coli K-12 strain JM109 (F' traD36 proAB+ lacIqZAMJ5) recAl endAl gyrA96 thi hsdR17 supE44 relAl * Corresponding author.A(la...

KEYWORDS: autoantibody, fetal antibody repertoire. antibody netwrk, Ig V genes, B cell malignancyInt Rev Immunol Downloaded from informahealthcare.com by Flinders University of South Australia on 01/08/15For personal use only.