Severity of Mitral Valve Degeneration Is Associated with Chromosome 15 Loci in Whippet Dogs

Mitral valve degeneration (MVD) is the most common form of heart disease in dogs, frequently leading to left-sided congestive heart failure and cardiac mortality. Although breed-specific disease characteristics and overrepresentation point towards a genetic origin for MVD, a causative mutation and complete molecular pathogenesis are unknown. Whippet dogs are overrepresented in incidence of MVD, suggesting an inherited component in this breed. Expressivity of this condition is variable with some dogs showing evidence of more severe disease at earlier ages than other dogs. This phenomenon makes a traditional case versus control genetic study prone to phenotyping error. This study sought to avoid these common pitfalls by identifying genetic loci associated with severity of MVD in Whippets through a genome-wide association study (GWAS). 138 Whippet dogs were characterized for MVD by echocardiographic examination and a novel disease severity score was developed and adjusted for age in each subject. Single nucleotide polymorphism (SNP) genotype data (170k Illumina CanineHD SnpChip) was obtained for DNA isolated from blood of each study subject. Continuous variable genome wide association was performed after correction for population stratification by efficient mixed model association expedited (EMMAX) in 130 dogs. A genome wide significant association was identified on chromosome 15 (peak locus 57,770,326; P_adj = 0.049) and secondary loci of suggestive association were identified on chromosome 2 (peak locus 37,628,875; P_adj = 0.079). Positional candidate genes were identified within the primary and secondary loci including follistatin-related protein 5 precursor (FSTL5) and Rho GTPase-activating protein 26 (ARHGAP26). These results support the hypothesis that severity of MVD in whippets has a genetic basis and warrants further study by either candidate gene sequencing or next-generation techniques.     

Kinematics and kinetics of the lower extremities of young and elder women during stairs ascent while wearing low and high-heeled shoes

The effect of the heel height on the temporal, kinematic and kinetic parameters was investigated in 16 young and 11 elderly females. Kinematic and kinetic data were collected when the subjects ascended stairs with their preferred speed in two conditions: wearing low-heeled shoes (LHS), and high-heeled shoes (HHS). The younger adults showed more adjustments in forces and moments at the knee and hip in frontal and transverse planes. Besides a few significantly changes in joint forces and moments, the elder group demonstrated longer cycle duration and double stance phase, larger trunk sideflexion and hip internal rotation, less hip adduction while wearing HHS. Most differences in joint motions between two groups were found at the hip and knee either in LHS or HHS condition. Instead, the differences in moment occurred at the hip joint and only in HHS. The interaction of the heel height and age showed the influences of heel height on trunk rotation, hip abduction/adduction, and knee and hip force and moment at the frontal plane depended on age. These phenomena suggest that younger and elderly women adapt their gait and postural control differently during stair ascent (SA) while wearing HHS.     

Characterization of an essential RNA secondary structure in the 3' untranslated region of the murine coronavirus genome

We have previously identified a functionally essential bulged stem-loop in the 3' untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication.     

Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment

The human immunodeficiency virus (HIV)-mediated immune response may be beneficial or harmful, depending on the balance between expansion of HIV-specific T cells and the level of generalized immune activation. The current study utilizes multicolor cytokine flow cytometry to study HIV-specific T cells and T-cell activation in 179 chronically infected individuals at various stages of HIV disease, including those with low-level viremia in the absence of therapy ("controllers"), low-level drug-resistant viremia in the presence of therapy (partial controllers on antiretroviral therapy [PCAT]), and high-level viremia ("noncontrollers"). Compared to noncontrollers, controllers exhibited higher frequencies of HIV-specific interleukin-2-positive gamma interferon-positive (IL-2(+) IFN-gamma(+)) CD4(+) T cells. The presence of HIV-specific CD4(+) IL-2(+) T cells was associated with low levels of proliferating T cells within the less-differentiated T-cell subpopulations (defined by CD45RA, CCR7, CD27, and CD28). Despite prior history of progressive disease, PCAT patients exhibited many immunologic characteristics seen in controllers, including high frequencies of IL-2(+) IFN-gamma(+) CD4(+) T cells. Measures of immune activation were lower in all CD8(+) T-cell subsets in controllers and PCAT compared to noncontrollers. Thus, control of HIV replication is associated with high levels of HIV-specific IL-2(+) and IFN-gamma(+) CD4(+) T cells and low levels of T-cell activation. This immunologic state is one where the host responds to HIV by expanding but not exhausting HIV-specific T cells while maintaining a relatively quiescent immune system. Despite a history of advanced HIV disease, a subset of individuals with multidrug-resistant HIV exhibit an immunologic profile comparable to that of controllers, suggesting that functional immunity can be reconstituted with partially suppressive highly active antiretroviral therapy.     

Characterization of the RNA components of a putative molecular switch in the 3' untranslated region of the murine coronavirus genome

RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3' untranslated region (3' UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3' UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.     

Pollen Morphology of Craigia with Reference to Its Systematic Position

Due to the unique floral and pollen morphology of the genus Craigia its systematic position, whether in the family Sterculiaceae or in the family Tiliaceae, has often been debated. In order to add more evidence for solving this problem, a comparative study on pollen morphology of the genus and 15 species of 3 tiliaceous genera, viz.: Tilia Linn., Hainania Merr. and Excentrodendron Chang. et Miau. was carried out under the light and scanning electron microscope. The result shows that the polarity, the structure and position of the aperture of Craigia pollen differ from those in Tiliaceae but are similar to those in Sterculiaceae. From palynology, it is more appropriate to place the genus under discussion in Sterculiaceae than in Tiliaceae.     

Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli

A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

HIV-Infected Individuals with Low CD4/CD8 Ratio despite Effective Antiretroviral Therapy Exhibit Altered T Cell Subsets,Heightened CD8+ T Cell Activation,and Increased Risk of Non-AIDS Morbidity and Mortality

A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm³) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28− and CD57+CD28−), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.