Evaluation of a DNA microarray for the rapid detection of extended-spectrum β-lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and NDM).

J Antimicrob Chemother. 2012 May 17;

Authors: Cuzon G, Naas T, Bogaerts P, Glupczynski Y, Nordmann P

Abstract
OBJECTIVES: Carbapenem-resistant Gram-negative bacilli are reported increasingly and represent an emerging public health concern. Laboratory detection of extended-spectrum β-lactamase (ESBL), plasmid-mediated cephalosporinase (pAmpC) and carbapenemase producers remains a challenge for microbiology laboratories and is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. We evaluated a novel microarray, the 'Check-MDR CT103 array' test (Check-Points, Wageningen, The Netherlands), that employs highly specific DNA markers to identify the β-lactamase genes of ESBLs (TEM, SHV and CTX-M, and discriminates between ESBL and non-ESBL TEM and SHV variants), of pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and of carbapenemases (KPC, OXA-48, VIM, IMP and NDM). METHODS: One-hundred-and-eighty-seven well-characterized Gram-negative bacilli isolates possessing different bla genes were tested. Total DNAs were extracted using a Qiagen DNA mini kit. The 'Check-MDR CT103 array' was used as recommended by the manufacturer. RESULTS: The system correctly identified representatives of the three ESBL gene families tested, including differentiation between non-ESBL and ESBL TEM and SHV variants. All bla(CTX-M) genes were classified into the appropriate family group (i.e. CTX-M-1 group, CTX-M-2 group, CTX-M-9 group and CTX-M-8/25/26 group). In addition, the clinically relevant plasmid-encoded cephalosporinase and carbapenemase genes were also reliably detected. Specificities and sensitivities of 100% were recorded for most bla genes. CONCLUSIONS: The 'Check-MDR CT103 array' is a powerful high-throughput tool for rapid identification of ESBL, pAmpC and carbapenemase producers in culture. Because of its rapid performance, this platform is a valuable tool for epidemiological or infection control studies.

PMID: 22604450 [PubMed - as supplied by publisher]

Non-Uniform Distribution Pattern for Differentially Expressed Genes of Transgenic Rice Huahui 1 at Different Developmental Stages and Environments.

PLoS One. 2012;7(5):e37078

Authors: Liu Z, Zhao J, Li Y, Zhang W, Jian G, Peng Y, Qi F

Abstract
DNA microarray analysis is an effective method to detect unintended effects by detecting differentially expressed genes (DEG) in safety assessment of genetically modified (GM) crops. With the aim to reveal the distribution of DEG of GM crops under different conditions, we performed DNA microarray analysis using transgenic rice Huahui 1 (HH1) and its non-transgenic parent Minghui 63 (MH63) at different developmental stages and environmental conditions. Considerable DEG were selected in each group of HH1 under different conditions. For each group of HH1, the number of DEG was different; however, considerable common DEG were shared between different groups of HH1. These findings suggested that both DEG and common DEG were adequate for investigation of unintended effects. Furthermore, a number of significantly changed pathways were found in all groups of HH1, indicating genetic modification caused everlasting changes to plants. To our knowledge, our study for the first time provided the non-uniformly distributed pattern for DEG of GM crops at different developmental stages and environments. Our result also suggested that DEG selected in GM plants at specific developmental stage and environment could act as useful clues for further evaluation of unintended effects of GM plants.

PMID: 22606331 [PubMed - as supplied by publisher]

Repressive processing of antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in Lactobacillus helveticus fermented milk by added peptides.

J Biosci Bioeng. 2012 May 14;

Authors: Wakai T, Yamaguchi N, Hatanaka M, Nakamura Y, Yamamoto N

Abstract
Lactobacillus helveticus can release the antihypertensive peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), from casein in fermented milk by a specific proteolytic system. To better understand the regulation of gene expression of the proteolytic enzymes thought to link to the processing of both antihypertensive peptides in L. helveticus, microarray analysis for whole gene expression in the presence and absence of added peptides in the fermented milk was studied. The productivity of both VPP and IPP in L. helveticus CM4 fermented milk was repressed by adding 2% quantity of Peptone as peptide mixture to the milk. Among the selected 13 amino acids, Gly, Ile, Leu, Phe, Met, Ser and Val were effective in the repression of the productivity of VPP and IPP in the fermented milk. The activity of the cell wall-associated proteinase, which may play a key role in the processing of the two antihypertensive peptides, was significantly repressed by the addition of the 2% quantity of Peptone into the fermented milk. By DNA microarray analysis it was found that prtH2 corresponding to the cell wall-associated proteinase gene, most of the endopeptidase genes such as pepE, pepO1, pepO2 and pepO3, most of the oligopeptide transporter genes, such as dppA2, dppB, dppC, dppD and dppF, most likely involved in the processing of VPP and IPP were down-regulated. These results suggest that amino acids released from milk peptides in the fermented milk might down-regulate the gene expressions of some of the proteolytic enzymes and may cause repression of the release of VPP and IPP in L. helveticus fermented milk.

PMID: 22591842 [PubMed - as supplied by publisher]

State-of-the art data normalization methods improve NMR-based metabolomic analysis.

Metabolomics. 2012 Jun;8(Suppl 1):146-160

Authors: Kohl SM, Klein MS, Hochrein J, Oefner PJ, Spang R, Gronwald W

Abstract
Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0350-z) contains supplementary material, which is available to authorized users.

PMID: 22593726 [PubMed - as supplied by publisher]

Amplified Protein Detection and Identification through DNA-Conjugated M13 Bacteriophage.

ACS Nano. 2012 May 15;

Authors: Lee JH, Domaille DW, Cha JN

Abstract
Sensitive protein detection and accurate identification continues to be in great demand for disease screening in clinical and laboratory settings. For these diagnostics to be of clinical value, it is necessary to develop sensors that have high sensitivity but favorable cost-to-benefit ratios. However, many of these sensing platforms are thermally unstable or require significant materials synthesis, engineering, or fabrication. Recently, we demonstrated that naturally occurring M13 bacteriophage can serve as biological scaffolds for engineering protein diagnostics. These viruses have five copies of the pIII protein, which can bind specifically to target antigens, and thousands of pVIII coat proteins, which can be genetically or chemically modified to react with signal-producing materials, such as plasmon-shifting gold nanoparticles (Au NPs). In this report, we show that DNA-conjugated M13 bacteriophage can act as inexpensive protein sensors that can rapidly induce a color change in the presence of a target protein yet also offer the ability to identify the detected antigen in a separate step. Many copies of a specific DNA oligonucleotide were appended to each virus to create phage-DNA conjugates that can hybridize with DNA-conjugated gold nanoparticles. In the case of a colorimetric positive result, the identity of the antigen can also be easily determined by using a DNA microarray. This saves precious resources by establishing a rapid, quantitative method to first screen for the presence of antigen followed by a highly specific typing assay if necessary.

PMID: 22587008 [PubMed - as supplied by publisher]

Anti-angiogenesis effect of 3'-sulfoquinovosyl-1'-monoacylglycerol (SQMG) via upregulation of thrombospondin 1.

Cancer Sci. 2012 May 16;

Authors: Matsuki K, Tanabe A, Hongo A, Sugawara F, Sakaguchi K, Takahashi N, Sato N, Sahara H

Abstract
We previously reported that 3'-sulfoquinovosyl-1'-monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti-angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real-time PCR. Consequently, upregulation of thrombospondin 1 (TSP-1) in SQMG-treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP-1 upregulation by SQMG, we established stable TSP-1-knockdown transformants (TSP1-KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti-tumor assay. On the reporter assay, transcriptional upregulation of TSP-1 in TSP1-KT could not be induced by SQMG, thus suggesting that TSP-1 upregulation by SQMG occurred via TSP-1 molecule. In addition, growth of TSP1-KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation.

PMID: 22587436 [PubMed - as supplied by publisher]

Capturing the Finer Points of Gene Expression in Psoriasis: Beaming in on the CCL19/CCR7 Axis.

J Invest Dermatol. 2012 Jun;132(6):1535-8

Authors: Rittié L, Elder JT

Abstract
Laser capture microdissection-coupled complementary DNA microarray analysis is a powerful tool for studying minor cell populations in tissues. In this issue, Mitsui et al. use this method to characterize the immune infiltrates that localize in the dermis of psoriatic skin. They identify the T-cell activation regulators C-C chemokine ligand 19 and C-C chemokine receptor 7 as potential mediators of immune organization in psoriasis.

PMID: 22584500 [PubMed - in process]

Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling.

J Cell Physiol. 2012 May 14;

Authors: Ogasawara T, Ohba S, Yano F, Kawaguchi H, Chung UI, Saito T, Yonehara Y, Nakatsuka T, Mori Y, Takato T, Hoshi K

Abstract
How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control GFP gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

PMID: 22585661 [PubMed - as supplied by publisher]

Interaction of Tmem119 and the bone morphogenetic protein pathway in the commitment of myoblastic into osteoblastic cells.

Bone. 2012 May 2;

Authors: Tanaka KI, Inoue Y, Hendy GN, Canaff L, Katagiri T, Kitazawa R, Komori T, Sugimoto T, Seino S, Kaji H

Abstract
Bone morphogenetic proteins (BMPs) are critical for bone regeneration and induce ectopic bone formation in vivo. The constitutively activating mutation (R206H) of the BMP type 1 receptor, activin A type 1 receptor/activin-like kinase 2 (ACVR1/ALK2), underlies the molecular pathogenesis of fibrodysplasia ossificans progressiva (FOP) in which heterotopic ossification occurs in muscle tissue. In the present study, we performed a comparative DNA microarray analysis between stable empty vector- and ALK2(R206H)-transfected mouse myoblastic C2C12 cells. Forty genes were identified whose expression was increased >3.5 times in the experimental group versus the control. The bone formation-related factor, Tmem119, was included in this group. Osteoblast differentiation markers and mineralization were enhanced in C2C12 cells stably expressing Tmem119. Differentiation of myoblastic cells into myotubes was suppressed but differentiation into chondrocytes was little affected. Transcriptional activity of the BMP-2 signaling molecules, Smad1/5, was increased even in the absence of exogenous BMP-2. Endogenous BMP-2 levels positively correlated with Tmem119 levels. A BMP-2/4 neutralizing antibody and dorsomorphin, an ALK2 inhibitor, antagonized Tmem119-enhanced alkaline phosphatase (ALP) levels. Tmem119 siRNA antagonized the BMP-2-induced ALP and osteocalcin, but not Runx2 and Osterix, mRNAs, in C2C12 cells. In conclusion, Tmem119 levels were increased by the FOP-associated constitutively activating ALK2 mutation in myoblasts. The data show that Tmem119 promotes the differentiation of myoblasts into osteoblasts and the interaction with the BMP signaling pathway likely occurs downstream of Runx2 and Osterix in myoblasts. Tmem119 may play a critical role in the commitment of myoprogenitor cells to the osteoblast lineage.

PMID: 22579779 [PubMed - as supplied by publisher]

Menthol induces cell-cycle arrest in PC-3 cells by down-regulating G2/M genes, including polo-like kinase 1.

Biochem Biophys Res Commun. 2012 May 9;

Authors: Kim SH, Lee S, Piccolo SR, Allen-Brady K, Park EJ, Chun JN, Kim TW, Cho NH, Kim IG, So I, Jeon JH

Abstract
Menthol, a naturally occurring monoterpene, is used in foods, cosmetic products, and topical therapeutic preparations. It also exerts cytotoxic activity against several cancer cell types, including prostate cancer cells. However, little is known about the mechanism of menthol action against prostate cancer cells. In this study, we investigated the effect of menthol on the gene expression profile of PC-3 prostate cancer cells using DNA microarray analyses. Gene set enrichment analysis revealed that menthol primarily affects the expression of cell cycle-related genes. Subsequent experimental analyses validated that menthol induces G2/M arrest. Particularly, menthol markedly down-regulated polo-like kinase 1 (PLK1), a key regulator of G2/M phase progression and inhibited its downstream signaling. Our computational analyses and experimental validation provide a basis for future investigations that are aimed at elucidating the action of menthol on cell cycle control in prostate cancer cells.

PMID: 22580005 [PubMed - as supplied by publisher]