K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule cross-linker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.     

Parallel synthesis of 9-aminoacridines and their evaluation against chloroquine-resistant Plasmodium falciparum

A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented. The method features a new route to 9-chloroacridines that utilizes triflates of salicylic acid derivatives, which are commercially available in a variety of substitution patterns. The route allows ready variation of the two diversity elements present in this class of molecules: the tricyclic aromatic heterocyclic core, and the disubstituted diamine sidechain. In this study, a library of 175 compounds was designed, although only 93 of the final products had purities acceptable for screening. Impurity was generally due to incomplete removal of 9-acridones (18), a degradation product of the 9-chloroacridine synthetic intermediates. The library was screened against two strains of Plasmodium falciparum, including a model of the drug-resistant parasite, and six novel compounds were found to have IC(50) values in the low nanomolar range.     

Thermal Tolerance of Diploid Versus Triploid Rainbow Trout and Brook Trout Assessed by Time to Chronic Lethal Maximum

Synopsis An effect of ploidy on thermal tolerance in juvenile trout was assessed in a series of tests comparing time to chronic lethal maximum (CLMax). Diploid and triploid fish were produced from a common spawn for three different groups each of brook trout Salvelinus fontinalis and of rainbow trout Oncorhynchus mykiss. One or two CLMax tests were performed per group, on between 15 and 50 individuals per ploidy within groups. The tests involved exposure of fish to a progressive 2°C day⁻¹ water temperature increase and recording of the time at which each individual fish reached loss of equilibrium (LE). The time to LE data were rank transformed and analyzed as a randomized complete block design. Although relative performance varied among trials, the analysis indicated overall differences due to ploidy were small and nonsignificant among both brook trout and rainbow trout. Size proved to be significantly correlated with time to LE in the brook trout trials, but not in the rainbow trout trials. Two of the six groups included a large proportion of fish which had received a heat shock following fertilization, but were not successfully triploidized. In both cases, thermal tolerance of the heat-shocked diploids was similar to that of the non-heat shocked control diploids, indicating no persistent effect of the heat shock on thermal tolerance.     

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.     

Salivary alpha amylase and salivary cortisol response to fluid consumption in exercising athletes

The objective of the study was to examine salivary biomarker response to fluid consumption in exercising athletes. Exercise induces stress on the body and salivary alpha amylase (sAA) and salivary cortisol are useful biomarkers for activity in the sympathoadrenal medullary system and the hypothalamic pituitary adrenal axis which are involved in the stress response. Fifteen college students were given 150 ml and 500 ml of water on different days and blinded to fluid condition. The exercise protocol was identical for both fluid conditions using absolute exercise intensities ranging from moderate to high. Saliva was collected prior to exercise, post moderate and post high intensities and analyzed by Salimetrics assays. Exercise was significant for sAA with values different between pre-exercise (85 ± 10 U · ml⁻¹) and high intensity (284 ± 30 U · ml⁻¹) as well as between moderate intensity (204 ± 32 U · ml⁻¹) and high intensity. There was no difference in sAA values between fluid conditions at either intensity. Exercise intensity and fluid condition were each significant for cortisol. Cortisol values were different between pre-exercise (0.30 ± 0.03 ug · dL⁻¹) and high intensity (0.45 ± 0.05 ug · dL⁻¹) as well as between moderate intensity (0.33 ± 0.04 ug · dL⁻¹) and high intensity. Moderate exercise intensity cortisol was lower in the 500 ml condition (0.33 ± 0.03 ug · dL⁻¹) compared with the 150 ml condition (0.38 ± 0.03 ug · dL⁻¹). This altered physiological response due to fluid consumption could influence sport performance and should be considered. In addition, future sport and exercise studies should control for fluid consumption.     

A third base pair for the polymerase chain reaction: inserting isoC and isoG

Two additional bases (isoguanosine and isocytosine), generating a third base pair, have been implemented in PCR. Enzyme fidelity for the third base pair is demonstrated using molecular thermodynamic melting, chemical cleavage and molecular beacons. When amplifying as few as 15 targets containing multiple non-natural base pairs with 40 cycles of amplification, our results confirm sequence conservation. The additional sequence space provided by three base pairs allows for the construction of molecular tools that achieve higher complexity and better discrimination than those possible with natural DNA alone.