Fiber optic probe for polarized reflectance spectroscopy in vivo: design and performance

We present the design and construction of a fiber optic probe for elastic light scattering spectroscopy in vivo with polarized excitation and polarization sensitive detection. The performance of the fiber probe is evaluated using a suspension of polystyrene spheres placed atop a diffusely scattering substrate, and it demonstrates that the size-dependent characteristics of the scatterers can be extracted in the presence of a highly diffusely scattering background using a linear combination of forward and backward Mie scattering components of the scatterers. Subsequently, Mie theory calculations are performed over a broad range of diagnostically relevant parameters of nuclei-mean diameter, size distribution, and relative refractive index-to understand how the polarized reflectance measurements with the fiber probe can be used to extract morphological information about epithelial tissue. Finally, the feasibility of in vivo measurements with the fiber optic based polarization sensitive light scattering spectroscopy is demonstrated.     

Analysis of the genetic diversity of genes 5 and 6 among group C rotaviruses using cDNA probes

Summary Two partial cDNA clones of genes 5 (encoding the major inner capsid protein VP 6) and 6 (encoding a nonstructural protein) of the porcine group (Gp) C rotavirus (Cowden strain) were radiolabeled with³²P and used individually as probes in Northern and dot blot hybridization assays. The specificity of each probe was tested against genomic dsRNA from: (1) porcine Gp A, B, and C rotaviruses; (2) Gp C rotaviruses from different species; and (3) porcine Gp C rotavirus field strains with varying electropherotype patterns. Neither probe hybridized with ds RNA from the porcine Gp A and B strains under the stringency conditions employed in the study. However, the gene 5 probe hybridized with the corresponding gene from the homologous porcine and the heterologous human and bovine Gp C rotaviruses tested. The gene 6 probe hybridized with the corresponding gene from the homologous Cowden strain, but hybridized weakly with gene 6 from the human and bovine Gp C rotaviruses. Both probes recognized all six different porcine Gp C field strains, although with varying intensities. Our results demonstrate that the gene 5 and 6 probes used in this study are specific for Gp C rotaviruses. However, evidence for greater genetic variation in the gene 6 among porcine, bovine and human Gp C strains suggested that the gene 5 probe may prove more broadly reactive among Gp C strains from different species. cDNA probes used in our study should prove useful for the detection of Gp C rotaviruses in feces and facilitate epidemiologic studies.     

Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

Alternative methods to analyse the impact of HIV mutations on virological response to antiviral therapy

Background  

Principal component analysis (PCA) and partial least square (PLS) regression may be useful to summarize the HIV genotypic information. Without pre-selection each mutation presented in at least one patient is considered with a different weight. We compared these two strategies with the construction of a usual genotypic score.     

The synthesis of coliphage T1 DNA: Requirement for host dna genes involved in elongation

Available Escherichia coli mutants with temperature-sensitive mutations in genes essential for DNA replication have been used to investigate the host protein involvement in the DNA synthesis of bacteriophage T1. This study has shown that T1 is independent of the E. coli initiation gene products dnaA, dnaC, dnaI, dnaP, and dnaT. The gene products of the polC, dnaG, and dnaZ loci, know to be involved in the elongation events of replication, are required for a productive T1 infection. T1 was found to be independent of the functioning of the dnaB gene product in the four dnaB mutants tested.     

The importance of chronicity and controllability of stress in the context of stress-illness relationships

The primary purpose of the present study was to increase our understanding of the roles of chronicity and controllability in the measurement of stress within the context of stress-illness relationships. Controllability and chronicity were assessed directly using a modified version of the Everyday Problems Scale. In addition to this scale, measures of depression, psychosomatic symptoms, and social support were administered to 128 women and 100 men. The results indicated that, for both men and women, the number of stressors was the best single predictor of symptoms. However, for women, chronicity and controllability of the stressors accounted for a significant amount of the variance in health outcomes over and above that accounted for by the number of stressors endorsed; for men, the addition of neither chronicity nor controllability consistently increased the strength of the association. While available social support was not found to influence the stress-illness relationships, greater willingness to utilize social support was associated with lower levels of depression.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22

Genetic linkage analysis has been instrumental in mapping thegene for X-linked agammaglobulinemia (XLA) to the proximal longarm of the human X chromosome, to Xq22. Due to the relativerarity of this disease the localization of the gene within Xq22has remained imprecise. We have investigated twenty-nine familiesaffected by XLA and have found no recombinants with the DXS178locus in over 30 informative meioses. DXS178 is now the mostreliable and informative locus for use in pre-natal diagnosisand carrier detection of XLA. In addition, we have identifiednew closely linked proximal and distal flanking markers forXLA, DXS442 and DXS101, respectively. These loci are separatedby 2cM, considerably reducing the extent of DNA within whichthe XLA locus can be contained. This will open up the way formore directed positional cloning efforts for the isolation ofthe XLA gene.     

Psychophysiological correlates of induced headaches

The psychophysiological correlates of induced headaches were examined. Subjects with frequent muscle-contraction headaches, frequent migraine headaches, or infrequent headaches were exposed to a 1-h cognitive stressor while multiple electromyographic and cardiovascular measures were monitored. The stressor was associated with significant changes in all psychophysiological measures. Eleven of 16 headache subjects and 2 of 8 control subjects developed headaches. Significant associations between several psychophysiological variables and headache occurrence were found and individual differences in the association between psychophysiological variables and headaches intensity were examined. The results were consistent with the hypothesized role of cardiovascular factors in the pathophysiology of muscle-contraction headache. This was the first controlled demonstration of headaches occurring as a function of environmental stressors.This research was supported by Grant 5-R01-NS-1875 from the National Institute of Health.