Optimization of computer software settings improves accuracy of pulsed-field gel electrophoresis macrorestriction fragment pattern analysis

Computer-assisted analysis of pulsed-field gel electrophoresis (PFGE) libraries can facilitate comparisons of fragment patterns present on multiple gels. We evaluated the ability of the Advanced Analysis (version 4.01) and Database (version 1.12) modules of the Phoretix gel analysis software package (Nonlinear USA, Inc., Durham, N.C.) to accurately match DNA fragment patterns. Two gels containing 38 lanes of SmaI-digested Enterococcus faecalis OG1RF DNA were analyzed to assess the impact of (i) varying the lane position of the standards, (ii) using gel plugs made at different times, and (iii) normalizing the fragment patterns by using molecular weight (MW) algorithms versus retardation factor (R(f)) algorithms. Two sets of PFGE libraries (one containing SmaI restriction patterns from 62 Enterococcus faecium isolates and the other containing SmaI restriction patterns of 89 Staphylococcus aureus isolates) were analyzed to assess the impact of varying the matching tolerance algorithm (designated as the vector box setting [VBS]) in the Phoretix software. Varying the lane position of standards on a gel and using gel plugs made on different days resulted in different VBSs, although it was not possible to judge whether those differences were statistically significant. Normalization of E. faecalis OG1RF fragment patterns by R(f) and MW methodology yielded no statistically significant differences in variability between the same fragment on different lanes. Suboptimal VBSs decreased the specificity with which related isolates were grouped together in dendrograms. The optimal VBS for analysis of PFGE fragment patterns from E. faecalis isolates differed from that for S. aureus isolates and sometimes was not that recommended by the manufacturer. Thus, computer-assisted analysis of PFGE patterns seemed to compensate for the intra- and intergel variation evaluated in the present study, and optimizing the software for the species to be tested was a critical preliminary step before further PFGE library analysis.     

Polymer latexes for cell-resistant and cell-interactive surfaces

Novel polymer latexes were prepared that can be applied in several ways for the control and study of cell behavior on surfaces. Acrylic latexes with glass transitions ranging from -30 to 100 degrees C were synthesized by dispersion polymerization in a water and alcohol solution using an amphiphilic comb copolymer as a stabilizing agent. The comb had a poly(methyl methacrylate) backbone and hydrophilic poly(ethylene glycol) (PEG) side chains, which served to stabilize the dispersion and create a robust hydrophilic coating on the final latex particles. The end groups of the comb stabilizer can be selectively functionalized to obtain latex particles with a controlled density of ligands tethered to their surfaces. Latexes were prepared with adhesion peptides (RGD) linked to the surface of the acrylic beads to induce attachment and spreading of cells. Coalesced films obtained from the RGD-bearing latex particles promoted attachment of WT NR6 fibroblasts, while films from unmodified latex particles were resistant to these cells. Additionally, RGD-linked beads were embedded in cell-resistant comb polymer films to create cell-interactive surfaces with discrete clustered-ligand domains. Cell attachment and morphology were seen to vary with the surface density of the RGD-bearing latex beads.     

Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies

Summary Ten monoclonal antibodies (MAbs) were raised against dengue 1 (DEN 1, Hawaii) virus E glycoprotein. Specificity of the MAbs was tested by ELISA and immunofluoresence. Eight were DEN 1 type-specific, one was DEN group-reactive (DGR) and one was flavivirus cross-reactive (FCR). Two of these type specific MAbs exhibited haemagglutination-inhibition (HI) and neutralized (N) DEN 1 virus in vivo (HS). These two MAbs showed 100% protection against a challenge of 100 LD₅₀ of DEN 1 virus in adult Swiss albino mice. The remaining six MAbs were HI negative, N negative and non-protective against challenge (NHS). Of these only three were reactive in the CF test. The DGR, FCR and one of the NHS MAbs (NHS-3) did not react with DEN 1 virus grown in Vero cells, whereas they reacted with DEN 1 virus grown in LLC-MK2 and C6/36 cells in immunofluorescence, probably indicating a difference in the synthesis/processing of viral proteins in these different cell lines. An epitope map of the E gp was drawn using a computer programme based on the additivity index values. The epitope map delineated five domains, a) S-I representing type-specific, HI positive, N positive and protecting MAbs. b) S-II representing type-specific, HI negative, N negative MAbs. c) S-III representing type-specific HI/N negative MAb, but distinct from S-II. d) DGR representing HI/N negative DEN group reactive MAb. e) FCR representing HI/N negative flavivirus cross-reactive MAb. Epitope analysis of a number of different DEN 1 strains isolated in India over a period of 30 years showed that the domains S-II and S-III which react with HI negative, DEN-1 specific MAbs were variable. The DGR domain and the S-I domains were conserved.     

Identification of some antigenically related outer-membrane proteins of strains of Vibrio cholerae O1 and non-O1 serovars involved in intestinal adhesion and the protective role of antibodies to them

Outer-membrane proteins (OMPs) of Vibrio cholerae strains of O1 and non-O1 serovars were studied. Marked similarity was found in the OMP profiles of different V. cholerae O1 strains but the OMP profile of a non-O1 strain was somewhat different. Antigenic relatedness between the OMPs of different V. cholerae strains was established by enzyme-linked immunosorbent assay (ELISA). Immunoblotting experiments demonstrated that at least two OMPs of 36 and 25-26 Kda were immunogenic and common to strains of O1 and non-O1 serovars. Antiserum raised against the outer membrane of a V. cholerae strain, and rendered specific for its OMP by absorption with lipopolysaccharide, inhibited in vitro the intestinal adhesion of the homologous and heterologous strains of V. cholerae irrespective of their biotype, serotype and serovar. Furthermore, antiserum to OMPs induced passive protection against vibrio challenge in rabbit ileal loop experiments. These results suggest that the OMPs may be useful in immunoprophylaxis against cholera.     

Primary gastric T-cell lymphoma of suppressor-cytotoxic (CD8+) phenotype: discordant expression of T-cell receptor subunit beta F1, CD7, and CD3 antigens.

Primary gastric T-cell lymphomas are rare neoplasms, and all but one of the previously phenotyped cases have shown a helper-inducer phenotype. The present case is the second reported case of a primary gastric T-cell lymphoma of suppressor-cytotoxic phenotype. The tumor histology was similar to that described in some forms of node-based peripheral T-cell lymphomas. Phenotypic analysis revealed low expression of pan-T marker CD7, reduced expression of CD3, but higher density and frequency of expression of CD8 antigens that could be predicted on the basis of the pan-T markers. Natural killer cell (NK) related markers CD16, HNK-1 and NKH-1 were not expressed by the neoplastic cells. T-cell receptor (TCR) beta subunit expression was detected on fewer cells than would have been predicted on the basis of CD3 and CD8 expression, and TCR delta chain expression was undetectable.     

Novel Semi‐Fluorinated Poly(ether imide)s Derived From 4‐(p‐Aminophenoxy)‐3‐trifluoromethyl‐4′‐aminobiphenyl

An unsymmetrical diamine monomer 4‐(p‐aminophenoxy)‐3‐trifluoromethyl‐4′‐aminobiphenyl has been synthesized successfully. This monomer leads to the synthesis of different novel poly(ether imide)s when reacted with different dianhydrides like pyromellatic dianhydride (PMDA), benzophenone tetracarboxylic acid dianhydride (BTDA), 2,2‐bis(3,4‐dicarboxyphenyl) hexafluoropropane (6FDA), and oxy diphthalic anhydride (ODA). The poly(ether imide) prepared from this monomer on reaction with 6FDA is soluble in several organic solvents such as N‐methylpyrolidinone (NMP), dimethylformamide (DMF), N,N‐dimethylacetamide (DMAc), tetrahydrofuran (THF), and CHCl₃. The poly(ether imide)s prepared from BTDA and ODA are soluble in NMP, DMF, and DMAc but not in THF or CHCl₃, whereas the polymer prepared from PMDA is soluble only in NMP. The water uptake value for these poly(ether imide) films is very low (0.2–0.5%), and exhibited low dielectric constants (2.81 at 1 MHz). The polymers exhibited high thermal stability up to 532 °C in air for 5% weight loss, and high glass transition temperatures up to 288 °C. The polymer exhibited high tensile strength up to 135 MPa, modulus 3.2 GPa, and elongation at break up to 25%, depending on the exact polymer structure.

The structure of the poly(ether imide) synthesised from 4‐(p‐aminophenoxy)‐3‐trifluoromethyl‐4′‐aminobiphenyl and 2,2‐bis(3,4‐dicarboxyphenyl) hexafluoropropane. This polymer was soluble in many organic solvents.     

Application of validated mapping algorithms between generic PedsQL scores and utility values to individuals with sickle cell disease

Quality of Life Research - There is a paucity of empirically estimated health state utility (HSU) values to estimate health-related quality of life among individuals with sickle cell disease (SCD)....