Patterns of genetic,phenotypic, and acoustic variation across a chiffchaff (Phylloscopus collybita abietinus/tristis) hybrid zone

Characterizing patterns of evolution of genetic and phenotypic divergence between incipient species is essential to understand how evolution of reproductive isolation proceeds. Hybrid zones are excellent for studying such processes, as they provide opportunities to assess trait variation in individuals with mixed genetic background and to quantify gene flow across different genomic regions. Here, we combine plumage, song, mtDNA and whole‐genome sequence data and analyze variation across a sympatric zone between the European and the Siberian chiffchaff (Phylloscopus collybita abietinus/tristis) to study how gene exchange between the lineages affects trait variation. Our results show that chiffchaff within the sympatric region show more extensive trait variation than allopatric birds, with a large proportion of individuals exhibiting intermediate phenotypic characters. The genomic differentiation between the subspecies is lower in sympatry than in allopatry and sympatric birds have a mix of genetic ancestry indicating extensive ongoing and past gene flow. Patterns of phenotypic and genetic variation also vary between regions within the hybrid zone, potentially reflecting differences in population densities, age of secondary contact, or differences in mate recognition or mate preference. The genomic data support the presence of two distinct genetic clades corresponding to allopatric abietinus and tristis and that genetic admixture is the force underlying trait variation in the sympatric region—the previously described subspecies (“fulvescens”) from the region is therefore not likely a distinct taxon. In addition, we conclude that subspecies identification based on appearance is uncertain as an individual with an apparently distinct phenotype can have a considerable proportion of the genome composed of mixed alleles, or even a major part of the genome introgressed from the other subspecies. Our results provide insights into the dynamics of admixture across subspecies boundaries and have implications for understanding speciation processes and for the identification of specific chiffchaff individuals based on phenotypic characters.     

Mechanical strain increases cell stiffness through cytoskeletal filament reorganization

We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G'). G' was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10-12 days. G' was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G' Para was 24 +/- 5% (+/- SE) greater compared with Perp (P < 0.05), and 35% +/- 6 greater compared with control (Cont, P < 0.01). The difference between strained and control cells was enhanced by KCl, increasing G' 171 +/- 7% Para compared with 125 +/- 6% Perp and 129 +/- 8% Cont (P < 10-5 both cases). The decrease in G' from baseline due to relaxant agonists isoproterenol and dibutyryl cAMP was similar in all groups. Long-term oscillatory loading of airway smooth muscle (ASM) cells caused stiffness to increase and become anisotropic. These findings are consistent with the hypothesis that cytoskeletal reorganization can enhance ASM stiffness and contractility. They imply, furthermore, that oscillatory loading of ASM may contribute to airway narrowing and failure of airway dilation in asthma.     

Beneficial and harmful effects of oscillatory mechanical strain on airway smooth muscle

Airway smooth muscle (ASM) cells are constantly under mechanical strain as the lung cyclically expands and deflates, and this stretch is now known to modulate the contractile function of ASM. However, depending on the experimental conditions, stretch is either beneficial or harmful limiting or enhancing contractile force generation, respectively. Stretch caused by a deep inspiration is known to be beneficial in limiting or reversing airway constriction in healthy individuals, and oscillatory stretch lowers contractile force and stiffness or lengthens muscle in excised airway tissue strips. Stretch in ASM culture has generally been reported to cause increased contractile function through increases in proliferation, contractile protein content, and organization of the cell cytoskeleton. Recent evidence indicates the type of stretch is critically important. Growing cells on flexible membranes where stretch is non-uniform and anisotropic leads to pro-contractile changes, whereas uniform biaxial stretch causes the opposite effects. Furthermore, the role of contractile tone might be important in modulating the response to mechanical stretch in cultured cells. This report will review the contrasting evidence for modulation of contractile function of ASM, both in vivo and in vitro, and summarize the recent evidence that mechanical stress applied either acutely within 2 h or chronically over 11 d is a potent stimulus for cytoskeletal remodelling and stiffening. We will also point to new data suggesting that perhaps some of the difference in response to stretch might lie with one of the fundamental differences in the ASM environment in asthma and in culture--the presence of elevated contractile tone.     

A distributed nonlinear model of lung tissue elasticity

Maksym, Geoffrey N., and Jason H. T. Bates. Adistributed nonlinear model of lung tissue elasticity.J. Appl. Physiol. 82(1): 32-41, 1997.- We present a theory relating the static stress-strainproperties of lung tissue strips to the stress-bearing constituents,collagen and elastin. The fiber pair is modeled as a Hookean spring(elastin) in parallel with a nonlinear string element (collagen), whichextends to a maximum stop length. Based on a series of fiber pairs, wedevelop both analytical and numerical models with distributedconstituent properties that account for nonlinear tissue elasticity.The models were fit to measured stretched stress-strain curves of fiveuniaxially stretched tissue strips, each from a different dog lung. Wefound that the distributions of stop length and spring stiffness followinverse power laws, and we hypothesize that this results from thecomplex fractal-like structure of the constituent fiber matrices inlung tissue. We applied the models to representative pressure-volume(PV) curves from patients with normal, emphysematous,and fibrotic lungs. The PV curves were fit to theequation V = A  Bexp(KP),where V is volume, P is transpulmonary pressure, andA, B, andK are constants. Our models lead to apossible mechanistic explanation of the shape factorK in terms of the structuralorganization of collagen and elastin fibers.

     

Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry

Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.     

Nanoparticles as a novel class of autophagy activators

Nano-sized objects exist as engineered tools as well as natural or anthropogenic environmental factors. Recent progress in the field of nanotechnology allows for a deeper understanding of their impact on organisms. Recently, we showed that the size-dependent cell interaction with quantum dots is autophagy-mediated. The potential role of other endo- and exogenous nanoparticles in terms of autophagy is discussed here. Their physical properties should be taken into consideration while constructing delivery systems. Furthermore, we propose several models of targeted nanoparticles delivery. Autophagy can be considered as an additional mechanism providing intracellular selectivity for introduced nanoparticles.     

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.