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1.  Androgen receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters 
Cell Death & Disease  2013;4(11):e907-.
The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. However, the molecular mechanisms that underlie the ligand-independent apoptosis, including the activity of AR in testicular stem cells, are not completely understood. In the present study, we generated induced pluripotent stem cells (iPSCs) from bovine testicular cells by electroporation of octamer-binding transcription factor 4 (OCT4). The cells were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4, which maintained and stabilized the expression of stemness genes and pluripotency. The iPSCs were used to assess the apoptosis activity following exposure to phthalate esters, including di (2-ethyhexyl) phthalates, di (n-butyl) phthalate, and butyl benzyl phthalate. Phthalate esters significantly reduced the expression of AR in iPSCs and induced a higher ratio of BAX/BCL-2, thereby favoring apoptosis. Phthalate esters also increased the expression of cyclin-dependent kinase inhibitor 1 (p21Cip1) in a p53-dependent manner and enhanced the transcriptional activity of p53. The forced expression of AR and knockdown of p21Cip1 led to the rescue of the phthalate-mediated apoptosis. Overall, this study suggests that testicular iPSCs are a useful system for screening the toxicity of environmental disruptors and examining their effect on the maintenance of stemness and pluripotency, as well as for identifying the iPSC signaling pathway(s) that are deregulated by these chemicals.
PMCID: PMC3847308  PMID: 24201806
environmental hormone; nuclear reprogramming; p53; testis cells; toxicity screening
3.  Dynamics of maternal and paternal effects on embryo and seed development in wild radish (Raphanus sativus) 
Annals of Botany  2010;106(2):309-319.
Background and Aims
Variability in embryo development can influence the rate of seed maturation and seed size, which may have an impact on offspring fitness. While it is expected that embryo development will be under maternal control, more controversial hypotheses suggest that the pollen donor and the embryo itself may influence development. These latter possibilities are, however, poorly studied. Characteristics of 10-d-old embryos and seeds of wild radish (Raphanus sativus) were examined to address: (a) the effects of maternal plant and pollen donor on development; (b) the effects of earlier reproductive events (pollen tube growth and fertilization) on embryos and seeds, and the influence of embryo size on mature seed mass; (c) the effect of water stress on embryos and seeds; (d) the effect of stress on correlations of embryo and seed characteristics with earlier and later reproductive events and stages; and (e) changes in maternal and paternal effects on embryo and seed characteristics during development.
Eight maternal plants (two each from four families) and four pollen donors were crossed and developing gynoecia were collected at 10 d post-pollination. Half of the maternal plants experienced water stress. Characteristics of embryos and seeds were summarized and also compared with earlier and later developmental stages.
Key Results
In addition to the expected effects of the maternal plants, all embryo characters differed among pollen donors. Paternal effects varied over time, suggesting that there are windows of opportunity for pollen donors to influence embryo development. Water-stress treatment altered embryo characteristics; embryos were smaller and less developed. In addition, correlations of embryo characteristics with earlier and later stages changed dramatically with water stress.
The expected maternal effects on embryo development were observed, but there was also evidence for an early paternal role. The relative effects of these controls may change over time. Thus, there may be times in development when selection on the maternal, paternal or embryo contributions to development are more and less likely.
PMCID: PMC2908165  PMID: 20519237
Raphanus sativus; embryo development; maternal effects; paternal effects; seed development; seed size; water stress; wild radish
4.  Speeded gradual lengthening and secondary angled blade plate stabilisation for proximal tibial shaft non-union with shortening 
International Orthopaedics  2007;32(5):693-696.
Eighteen patients with proximal tibial shaft non-union and shortening were treated. In each patient, the non-union area was débrided, realigned and stabilised with an Ilizarov lengthening frame. The tibia was gradually lengthened by 1–1.5 mm per day. After achieving the desired length, external fixation was converted to an angled blade plate and packed with cancellous bone graft. Follow-up of 16 patients for a median of 2.4 (1.2–4.5) years revealed satisfactory outcomes in all. No wound infections were noted. The described technique has a high success rate, a short treatment course and reduces patient discomfort. This method may be considered preferential treatment for all patients with the specified indications.
PMCID: PMC2551709  PMID: 17492448
5.  Effects of Growth Conditions on Structural Properties of ZnO Nanostructures on Sapphire Substrate by Metal–Organic Chemical Vapor Deposition 
Nanoscale Research Letters  2009;4(4):377-384.
ZnO was grown on sapphire substrate by metal–organic chemical vapor deposition using the diethylzinc (DEZn) and oxygen (O2) as source chemicals at 500 °C. Influences of the chamber pressure and O2/DEZn ratio on the ZnO structural properties were discussed. It was found that the chamber pressure has significant effects on the morphology of ZnO and could result in various structures of ZnO including pyramid-like, worm-like, and columnar grain. When the chamber pressure was kept at 10 Torr, the lowest full width at half-maximum of ZnO (002) of 175 arc second can be obtained. On the other hand, by lowering the DEZn flow rate, the crystal quality of ZnO can be improved. Under high DEZn flow rate, the ZnO nanowall-network structures were found to grow vertically on the sapphire substrate without using any metal catalysts. It suggests that higher DEZn flow rate promotes three-dimensional growth mode resulting in increased surface roughness. Therefore, some tip on the ZnO surface could act as nucleation site. In this work, the growth process of our ZnO nanowall networks is said to follow the self-catalyzed growth mechanism under high-DEZn flow rate.
PMCID: PMC2894013  PMID: 20596413
ZnO; Chamber pressure; O2/DEZn ratio; Nanowall networks; Self-catalyzed
6.  EP6 Quantitative Proteomics 
There are numerous approaches to study the proteome in a quantitative manner. All rely heavily on optimized sample preparation and appropriate statistical analysis of resulting datasets. This session will cover the following aspects of quantitative proteomics approaches:
Quantitative profiling of the membrane proteome requires special considerations not addressed in typical mass-spectrometry analyses. Optimized sample preparation and separation strategies will be discussed in the context of enriched membrane fractions and a quantitative proteomics platform using stable isotopes.In shotgun proteomics, a complex protein mixture is first digested to peptides, which are then analyzed by a combination of nanoflow chromatography and tandem mass spectrometry. The effects of subtle changes in sample preparation and chromatographic conditions in the characterization of complex mixtures will be presented. A discovery-based mass spectrometry approach using a bench-top LTQ linear ion trap and in-house written software for label-free differential protein profiling will be presented. This approach is quite comprehensive and is compatible with even the most inexpensive mass spectrometers. For proteins not detected routinely using our discovery-based approaches, we have applied selected reaction monitoring using a TSQ Quantum Ultra. This approach has been used to identify and quantify proteins at the low ng/mL level in plasma without any prior fractionation. A software pipeline has been developed to go from hypothesized proteins of interest derived from the literature to predicted hSRM transitions, collision offsets, and predicted chromatographic retention times. The combination of both discovery- and hypothesis-driven proteomics using nanoflow separations and tandem mass spectrometry provides us with unparalleled sensitivity and dynamic range in characterizing complex mixtures.Spectrum counting is an appealing and relatively straightforward approach for quantitative proteomics. Since the spectrum count of a protein in a proteomic analysis is the total number of peptides, not just unique peptides detected and identified for a given protein, searching criteria and false-positive minimization is important. There are several different versions of spectral counting currently in use, but each approach has shared core characteristics. An additional important consideration for quantitative proteomic analysis is the use of replicates for statistical analysis and determining the proper statistical test to use based on the overall structure of the datasets. This presentation will describe the foundation of spectral counting and the modifications to this approach used by different researchers. In addition, selected examples of the biological implementation of these approaches will be described.
PMCID: PMC2292016
7.  Mycobacterium avium subsp. paratuberculosis Fibronectin Attachment Protein Facilitates M-Cell Targeting and Invasion through a Fibronectin Bridge with Host Integrins  
Infection and Immunity  2004;72(7):3724-3732.
Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the α5, αV, β1, and β3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.
PMCID: PMC427427  PMID: 15213112
8.  Prevalence, Risk Factors, and Genetic Diversity of Bartonella henselae Infections in Pet Cats in Four Regions of the United States 
Journal of Clinical Microbiology  2004;42(2):652-659.
Blood was collected from a convenience sample of 271 pet cats aged 3 months to 2 years (mean age, 8 months, median and mode, 6 months) between May 1997 and September 1998 in four areas of the United States (southern California, Florida, metropolitan Chicago, and metropolitan Washington, D.C.). Sixty-five (24%) cats had Bartonella henselae bacteremia, and 138 (51%) cats were seropositive for B. henselae. Regional prevalences for bacteremia and seropositivity were highest in Florida (33% and 67%, respectively) and California (28% and 62%, respectively) and lowest in the Washington, D.C. (12% and 28%, respectively) and Chicago (6% and 12%, respectively) areas. No cats bacteremic with B. clarridgeiae were found. The 16S rRNA type was determined for 49 B. henselae isolates. Fourteen of 49 cats (28.6%) were infected with 16S rRNA type I, 32 (65.3%) with 16S rRNA type II, and three (6.1%) were coinfected with 16S rRNA types I and II. Flea infestation was a significant risk factor for B. henselae bacteremia (odds ratio = 2.82, 95% confidence interval, 1.1 to 7.3). Cats ≥13 months old were significantly less likely to be bacteremic than cats ≤6 months old (odds ratio = 0.18, 95% confidence interval, 0.05 to 0.61). Flea infestation, adoption from a shelter or as a stray cat, hunting, and being from Florida or California were significant risk factors for B. henselae seropositivity. DNA fingerprint was significantly associated with region (P = 0.03) and indoor/outdoor status of cats (P = 0.03).
PMCID: PMC344466  PMID: 14766832
9.  Fibronectin Attachment Protein Is Necessary for Efficient Attachment and Invasion of Epithelial Cells by Mycobacterium avium subsp. paratuberculosis  
Infection and Immunity  2002;70(5):2670-2675.
Attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by two epithelial cell lines were enhanced by soluble fibronectin (FN). Peptide blocking of the FN attachment protein (FAP-P) inhibited the internalization of M. avium subsp. paratuberculosis. Disruption of FAP-P expression significantly reduced attachment and ingestion of M. avium subsp. paratuberculosis by T-24 and Caco-2 cells. The results indicate that the interaction between FN and FAP-P facilitates attachment and internalization of M. avium subsp. paratuberculosis by epithelial cells.
PMCID: PMC127902  PMID: 11953410
10.  Standardization of Broth Microdilution and Disk Diffusion Susceptibility Tests for Actinobacillus pleuropneumoniae and Haemophilus somnus: Quality Control Standards for Ceftiofur, Enrofloxacin, Florfenicol, Gentamicin, Penicillin, Tetracycline, Tilmicosin, and Trimethoprim-Sulfamethoxazole 
Journal of Clinical Microbiology  2001;39(12):4283-4287.
Quality control (QC) standards for the in vitro antimicrobial susceptibility testing of two fastidious veterinary pathogens, Actinobacillus pleuropneumoniae and Haemophilus somnus, were developed in a multilaboratory study according to procedures established by the National Committee for Clinical Laboratory Standards for broth microdilution and disk diffusion testing. The medium recommended for the broth microdilution testing is cation-adjusted Mueller-Hinton broth supplemented with 2% lysed horse blood, 2% yeast extract, and 2% supplement C. This medium has been designated veterinary fastidious medium. The medium recommended for the disk diffusion testing is chocolate Mueller-Hinton agar. The recommended QC organisms are A. pleuropneumoniae ATCC 27090 and H. somnus ATCC 700025. The QC MICs of ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole were determined for each isolate, as were the zone size ranges. Of the results from the participating laboratories, 94.0% of the zone diameter results and 97.0% of the MIC results fell within the suggested QC ranges for all compounds. These QC guidelines should allow greater accuracy in interpreting results when testing these antimicrobial agents against fastidious pathogens.
PMCID: PMC88537  PMID: 11724833
11.  Fibronectin Attachment Protein Homologue Mediates Fibronectin Binding by Mycobacterium avium subsp. paratuberculosis 
Infection and Immunity  2001;69(4):2075-2082.
Attachment of Mycobacterium avium subsp. paratuberculosis to host tissue and penetration of mucosal surfaces are pivotal events in the pathogenesis of Johne's disease. Fibronectin (FN) binding is required for attachment and internalization of several mycobacteria by epithelial cells in vitro. The objective of this study was to further characterize the FN binding activity of M. avium subsp. paratuberculosis. Although the bacteria bound FN poorly at pH above 7, brief acid pretreatment greatly enhanced FN binding within the pH range (3 to 10) studied. A 4.6-kbp fragment from an M. avium subsp. paratuberculosis genomic library was found to contain a 1,107-bp open reading frame that shows very high nucleotide sequence identity with that of the FN attachment protein (FAP) gene of M. avium subsp. avium. Pretreatment of FN with an FN-binding peptide from M. avium subsp. avium FAP abolished FN binding, indicating that M. avium subsp. paratuberculosis binds FN in a FAP-dependent manner. Pretreatment of M. avium subsp. paratuberculosis with anti-FAP immunoglobulin G did not abrogate FN binding; blocking occurred only when anti-FAP was added together with FN. FAP was detected by immunofluorescence only in lipid-extracted M. avium subsp. paratuberculosis. Western blotting and immunoelectron microscopy revealed that FAP is located near the interior of the cell envelope of M. avium subsp. paratuberculosis. The results indicate that a FAP homologue mediates the attachment of FN to M. avium subsp. paratuberculosis. Further, given the subcellular location of FAP, it is considered that this protein operates at the terminus of a coordinated FN binding system in the cell envelope of M. avium subsp. paratuberculosis.
PMCID: PMC98132  PMID: 11254560

Results 1-11 (11)