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.     

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.     

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.     

AIDS in obstetric practice

After a brief review of the AIDS virus, its transmission, and clinical aspects, the obstetric implications of HIV infections for a developing country like India are summarized. HIV, the virus that causes AIDS, is transmissible in utero with a 66% risk of infecting the fetus, and may cause intrauterine growth retardation or prematurity. HIV positive pregnant women may become immunosuppressed, so they should be offered pregnancy termination. A woman in labor infected with HIV should be managed like a woman with hepatitis B: intrauterine catheters, fetal scalp electrodes, and fetal blood sampling are contraindicated. Forceps and episiotomy should be used only if needed. Cesarean section to prevent intrapartum infection of the fetus is controversial. While breastfeeding is allowed, breast milk should not be donated to other infants. Nursing staff should be informed that HIV is much less transmissible than hepatitis.     

Phosphorus-Containing Fluoro-Sulfonated Polytriazole Membranes with High Proton Conductivity: Understanding Microstructural and Thermomechanical Behaviors as a Function of Degree of Sulfonation

The current article describes the design and synthesis of a new series of phosphorus-containing fluoro-sulfonated polytriazoles through click polymerization. The synthesized copolytriazoles (PTPFDSH-70 to 90) with different degrees of sulfonation (DS) from 70% to 90% are structurally interpreted by various spectroscopic techniques (¹H, ¹³C, and FTIR). The high molecular weight (weight average molecular weight as high as 77 500 g mol⁻¹ with polydispersity index of 2.29) polymers exhibits excellent mechanical (elongation at break up to 95%), thermal (10% decomposition temperature: 266–317 °C), and oxidative (>14.5 h) stability. The PTPFDSH-70 to 90 possess outstanding water-holding ability in hydrated conditions (swelling ratio [in-plane]: 6.2–7.3% at 80 °C). The microstructural alterations by their thermal relaxations and transitions with increasing DS in the polymers have been thoroughly investigated by dynamic mechanical analysis. The atomic force microscopy and transmission electron microscope images of the PTPFDSH-70 to 90 polymer membranes demonstrated the phase segregated interconnected ionic cluster-like morphology between hydrophilic and hydrophobic domains. The PTPFDSH-90 (DEB:PFAZ:DSAZ = 100:10:90) polymer membrane displays the proton conductivity (176 and 190 ms cm⁻¹ at 80 and 90 °C, respectively) higher than Nafion117 under similar test conditions.     

Image alignment by integrated rotational and translational transformation matrix

Presents an algorithm to align 2D images with similar densitometric patterns in the spatial domain. It performs automatic identification of control points through local maxima in normalized image cross correlations computed through projections of an image function on 2D orthonormal bases over a predefined circular domain. The image is subsequently subjected to translational and rotational corrections through an integrated transformation matrix obtained by the least-squares minimization technique. The algorithm restricts the corrections required for alignment to rotation and translation only, and care is taken to see that the image does not go through scaling (isotropic or anisotropic) or nonlinear distortions. The least-squares method results in implicit equations in three variables, theta , h, upsilon , representing rotation, and translations along x and y axes. The present algorithm, on the other hand, is based on an approximation that results in a set of explicit equations that are easy to solve. The mathematical validity of this approximation is proved and the results obtained show that the algorithm performs well with similar biological (histological, autoradiographic, and tomographic) images. The algorithm is iterative, and its computer implementation is discussed.