Yulin Shou

The mammalian metabolite, 2-methoxyestradiol, affects P53 levels and apoptosis induction in transformed cells but not in normal cells.

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1-2 microM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37 degrees C. The effects included enhanced apoptosis and a modest G2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30 degrees C marked increases in apoptosis and a prominent G2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.

Beryllium sensitivity is linked to HLA-DP genotype.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.

ABSTRACT A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.

Effects of gamma-linolenic acid and arachidonic acid on cell cycle progression and apoptosis induction in normal and transformed cells

The effects of arachidonic acid (AA) and gamma-linolenic acid (GLA) on cell cycle progression and apoptosis induction, using flow cytometry, were compared on normal human skin fibroblasts, strain HSF43 with wild type (wt) p53, large T antigen transformed HSF43 cells (line E8T4) with non functional p53, and on two lymphoblast cell lines, TK6 with wt p53 and WTK1 with mutant p53. AA and GLA caused similar dose (50, 75 and 100 microg/ml AA and GLA) and time dependent (24 and 48 h) induction of apoptosis in each cell line. The degrees of the response of the four cell lines were, however, different. The normal HSF43 cells were most resistant against apoptosis induction and the WTK1 cells most susceptible. The apoptosis induction appeared to be independent of functional p53. Cell cycle progression was also similarly affected by AA and GLA in the two cell types. In the fibroblast type cells (HSF43 and E8T4) S- and G2/M-phase arrests were evident after 48 h exposure to AA and GLA, and in the lymphoblast cell lines (TK6 and WTK1) the cells were arrested in the G1-phase.

Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: Individual variability and repeatability

T-cell lymphocyte populations can be delineated into subsets based on expression of cell surface proteins that can be measured in peripheral blood by monoclonal antibodies and flow cytometry percentages of the lymphocyte subpopulations. In order to accurately assess immunocompetence in birds, natural variability in both avian immune function and the methodology must be understood. Our objectives were to (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate repeatability and variability in the methodology with respect to poultry in a free-range and environmentally diverse situation, and (3) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations. This work demonstrated the repeatability of using flow cytometry for measurements of peripheral blood in chickens using anti-chicken antibodies for lymphocyte subpopulations. Immunofluorescence staining of cells isolated from peripheral blood revealed that the CD3(+) antibodies reacted with an average of approximately 12-24% of the lymphoid cells in the blood, depending on the fluorescence type. The CD4(+) and CD8(+) molecules were expressed in a range of 4-31% and 1-10% of the lymphoid cells in the blood, respectively. Both fluorescence label and antibody company contribute to the variability of results and should be considered in future flow cytometry studies in poultry.

Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis

ABSTRACT A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.

Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis

ABSTRACT The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ± 2%) and FCM (4% ± 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSDPFGE ] = 3% ± 2% and RSDFCM = 1.2% ± 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods.

Differential expression of small RNAs from Burkholderia thailandensis in response to varying environmental and stress conditions

BackgroundBacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology.ResultsWe profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors. In particular, the trans-encoded BTH_s1 and s39 exhibited differential expression profiles dependent on growth phase and cell stimuli, such as antibiotics and serum. Furthermore, knockdown of the highly-expressed BTH_s39 by antisense transcripts reduced B. thailandensis cell growth and attenuated host immune response upon infection, indicating that BTH_s39 functions in bacterial metabolism and adaptation to the host. In addition, expression of cis-encoded BTH_s13 and s19 found in the 5′ untranslated regions of their cognate genes correlated with tight regulation of gene transcript levels. This sRNA-mediated downregulation of gene expression may be a conserved mechanism of post-transcriptional gene dosage control.ConclusionsThese studies provide a broad analysis of differential Burkholderia sRNA expression profiles and illustrate the complexity of bacterial gene regulation in response to different environmental stress conditions.

Clinical and acquired immunologic responses to West Nile virus infection of domestic chickens (Gallus gallus domesticus)

Numerous bird species are highly susceptible to North American strains of West Nile virus (WNV), and although domestic chickens are relatively resistant to WNV-associated disease, this species currently represents the most practical avian model for immune responses to WNV infection. Knowledge of the immunomodulation of susceptibility to WNV in birds is important for understanding taxonomic differences in infection outcomes. While focusing on immunophenotyping of CD3(+), CD4(+), CD8(+), and CD45(+) lymphocyte subpopulations, we compared lymphocyte subpopulations, blood chemistries, cloacal temperatures, IgM and IgG antibody titers, and differential whole-blood cell counts of WNV-infected and uninfected hens. Total blood calcium and lymphocyte numbers were lower in WNV-infected chickens compared with uninfected chickens. The heterophil-to-lymphocyte ratio increased over time from 2 to 22 d postinoculation (DPI) in uninfected chickens and from 2 to 8 DPI in WNV-infected chickens, although levels declined from 8 to 22 DPI in the latter group. No significant differences were found in the remaining immunological and hematological variables of the WNV-infected and uninfected groups. Our results reaffirm that chickens are resistant to WNV infection, and demonstrated that the heterophil-to-lymphocyte ratio differed between groups, allowing for sorting of infection status. Similar patterns in immune responses over time in both infected and uninfected hens may be related to age (i.e., 10 wk) and associated immune development.

Evaluation of Bacillus anthracis and Yersinia pestis sample collection from nonporous surfaces by quantitative real-time PCR.

Aim:  We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment.