Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

Theoretical analysis of the effects of a radial activation wave and twisting motion on the mechanics of the left ventricle

The mechanical effects resulting from the normal transmural delay of electrical depolarization of the myocardium are investigated. An activation sequence having a finite radial propagation velocity is introduced into the equations of ventricular mechanics. The resulting system of coupled integral equations is solved using a perturbation method based on the small ratio of transmural propagation time to cardiac period. Numerical calculations are performed using cavity pressure and volume waveforms characteristic of the canine left ventricle (LV), for both simultaneous and delayed activation of fiber layers. The results show that a finite transmural electrical propagation velocity tends to: (i) equalize the transmural distribution of sarcomere length during systole; (ii) equalize the transmural distribution of fiber external work/vol; and (iii) insignificantly affect myocardial tissue pressure. Calculations are also performed to investigate the mechanical effects resulting from the application of an externally applied moment that prevents LV torsion. Those results are highly dependent on the transmural distribution of sarcomere length in the stress-free reference state (unloaded diastole). When we assume a uniform distribution, then normal torsion acting with normal activation delay tends to: (i) increase the magnitude of fiber strain in the subendocardium and decrease it in the subepicardium; (ii) equalize the transmural distribution of fiber external work/vol; and (iii) lower myocardial tissue pressure. The normally occurring transmural delay of activation tends to lessen endocardial O2 demand, while the normally occurring torsion further lessens that demand and improves O2 supply.     

Possible antiestrogenic activity of lindane in female rats

During chronic peroral (PO) treatment of weanling, female Fischer 344 rats with daily injections (0.069 mmol/kg) of either 1,1′-(2,2,2-trichloroethylidene) bis [4-chlorobenzene] (p,p′-DDT), 2,4-dichlorophenoxy acetic acid (2,4-D), or γ-hexachlorocyclohexane (lindane), the lindane treatment induced a significant 20% increase in body weight after 110 days. Further investigation with 0,5,10,20, and 40 mg/kg lindane confirmed a significant increase in average body weight gain at the two highest doses after ten weeks of treatment. Significantly greater food consumption was observed, and the Lee index indicated that lindane treatment induced obesity. In addition to obesity, lindane caused a delay in vaginal opening, disrupted estrous cycling, reduced pituitary and uterine weight, and elevated food consumption during proestrus (when appetite is normally suppressed by estradiol). These responses suggest that, by inducing alterations in the reproductive function of the female rat and by interfering with hormonal regulation of energy balance, lindane may be antiestrogenic rather than estrogenic as previously proposed.     

Structural and functional characterization of an inositol polyphosphate receptor from cerebellum.

An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three different polypeptides with Mr of 111,000, 102,000, and 52,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds inositol 1,3,4,5-tetrakisphosphate, inositol hexaphosphate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IP6, and IP3, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 microM, respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are PK+/PNa+ approximately 5 and PK+/PCl approximately 19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 microM IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 microM IP4. IP6 and IP3 did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.     

Variation between European eel <Emphasis Type="Italic">Anguilla anguilla</Emphasis> (L.) stocks in five marshes of the Thames Estuary (United Kingdom)

The European eel (Anguilla anguilla, L.) was historically widely distributed throughout the United Kingdom, in coastal waters, lakes, rivers and wetlands. Recruitment has declined in recent decades and the species is now listed as ‘Critically Endangered’ on the International Union for Conservation of Nature and Natural Resources (IUCN) Red List. Management of suitable wetland habitats may contribute to species recovery; however, little is known about the stocks in these areas. In this study, yellow (adult stage?>?300 mm) eels were sampled in ditches in five marshes bordering the Thames Estuary in England, UK. Ecological variables, including ditch characteristics, invertebrate abundance and water quality parameters were measured. Habitat features were also observed and recorded, including access, land use and water management regimes. Eels were found in all marshes, but at varying catch-per-unit-effort (CPUE). There were no significant correlations between CPUE and the ecological variables, except ditch width. However, a significant difference in CPUE was found between two of the marshes, which may be explained by variations in local habitat management. Mean lengths showed a high proportion of females and mean body condition of four of the marshes was also found to be greater than in three rivers in the same region. These findings suggest that the marshes are potentially favourable eel habitats and that factors influencing habitat quality, such as land use and water management, may affect eel abundance, production of females and body condition. Effective management of such wetlands may therefore contribute to the conservation of European eel.     

Differential susceptibility of naive and memory CD4+ T cells to the cytopathic effects of infection with human immunodeficiency virus type 1 strain LAI.

CD4+ T lymphocytes of individuals infected with human immunodeficiency virus type 1 (HIV-1) exhibit a qualitative defect in their ability to mount memory responses to previously encountered antigens although their responses to mitogens remain normal. T cells responsible for memory responses can be distinguished from naive T cells based on differential expression of isoforms of the tyrosine phosphatase CD45. It has been suggested that memory CD4+ T cells from infected individuals have a greater virus burden than naive CD4+ T cells and that this accounts for the loss of recall responses in infected individuals. However, it has been unclear whether naive and memory T cells are equally susceptible to infection and to the cytopathic effects of the virus. We therefore infected highly purified resting naive and memory CD4+ T cells from HIV-1-seronegative individuals with HIV-1(LAI). Infected cells were then stimulated with phytohemagglutinin to render them permissive for viral replication. Cell viability and growth rate were monitored for 8 to 10 days as indicators of cytopathic effects induced by HIV-1(LAI). Our results indicated that naive and memory CD4+ T cells display marked differences in susceptibility to the cytopathic effects induced by HIV-1(LAI), infection. The cytopathic effects induced by HIV-1(LAI) were much more severe in memory CD4+ T cells than in naive CD4+ T cells. Differential cytopathic effects in naive and memory T cells were not due to differences in virus entry into and replication in these cell populations. Rather, memory cells were more susceptible to cytopathic effects. Pronounced cytopathic effects in memory cells were clearly detectable at 7 day postinfection. Cell death occurred at the single-cell level and was not accompanied by syncytium formation. The growth rate of infected memory CD4+ T cells was also severely compromised compared to that of naive CD4+ T cells, whereas the growth rates of both uninfected naive and memory CD4+ T cells were approximately the same. At least a portion of the dying cells exhibited biochemical changes characteristic of apoptosis. These results suggest that the selective functional defects present in the memory CD4+ T-cell subset of HIV-1-infected individuals may in part be the result of the greater susceptibility of memory T cells to cytopathic effects induced by HIV-1.