Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually.     

GABA Binding to an Insect GABA Receptor: A Molecular Dynamics and Mutagenesis Study

RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors.Abbreviations used: nACh, nicotinic acetylcholine; AChBP, acetylcholine binding protein; GABA, gamma-aminobutyric acid; MD, molecular dynamics; RDL, resistant to dieldrin; RMSD, root mean-square displacement; RMSF, root mean-square fluctuation     

Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.     

Biphasic investigation of tissue mechanical response during freezing front propagation

Cryopreservation of engineered tissue (ET) has achieved limited success due to limited understanding of freezing-induced biophysical phenomena in ETs, especially fluid-matrix interaction within ETs. To further our understanding of the freezing-induced fluid-matrix interaction, we have developed a biphasic model formulation that simulates the transient heat transfer and volumetric expansion during freezing, its resulting fluid movement in the ET, elastic deformation of the solid matrix, and the corresponding pressure redistribution within. Treated as a biphasic material, the ET consists of a porous solid matrix fully saturated with interstitial fluid. Temperature-dependent material properties were employed, and phase change was included by incorporating the latent heat of phase change into an effective specific heat term. Model-predicted temperature distribution, the location of the moving freezing front, and the ET deformation rates through the time course compare reasonably well with experiments reported previously. Results from our theoretical model show that behind the marching freezing front, the ET undergoes expansion due to phase change of its fluid contents. It compresses the region preceding the freezing front leading to its fluid expulsion and reduced regional fluid volume fractions. The expelled fluid is forced forward and upward into the region further ahead of the compression zone causing a secondary expansion zone, which then compresses the region further downstream with much reduced intensity. Overall, it forms an alternating expansion-compression pattern, which moves with the marching freezing front. The present biphasic model helps us to gain insights into some facets of the freezing process and cryopreservation treatment that could not be gleaned experimentally. Its resulting understanding will ultimately be useful to design and improve cryopreservation protocols for ETs.     

Mechanism of loss of consciousness during vascular neck restraint

Vascular neck restraint (VNR) is a technique that police officers may employ to control combative individuals. As the mechanism of unconsciousness is not completely understood, we tested the hypothesis that VNR simply compresses the carotid arteries, thereby decreasing middle cerebral artery blood flow. Twenty-four healthy police officers (age 35 ± 4 yr) were studied. Heart rate (HR), arterial pressure, rate of change of pressure (dP/dt), and stroke volume (SV) were measured using infrared finger photoplethysmography. Bilateral mean middle cerebral artery flow velocity (MCAVmean) was measured by using transcranial Doppler ultrasound. Neck pressure was measured using flat, fluid-filled balloon transducers positioned over both carotid bifurcations. To detect ocular fixation, subjects were asked to focus on a pen that was moved from side to side. VNR was released 1-2 s after ocular fixation. Ocular fixation occurred in 16 subjects [time 9.5 ± 0.4 (SE) s]. Pressures over the right (R) and left (L) carotid arteries were 257 ± 22 and 146 ± 18 mmHg, respectively. VNR decreased MCAVmean (R 45 ± 3 to 8 ± 4 cm/s; L 53 ± 2 to 10 ± 3 cm/s) and SV (92 ± 4 to 75 ± 4 ml; P < 0.001). Mean arterial pressure (MAP), dP/dt, and HR did not change significantly. We conclude that the most important mechanism in loss of consciousness was decreased cerebral blood flow caused by carotid artery compression. The small decrease in CO (9.6 to 7.5 l/min) observed would not seem to be important as there was no change in MAP. In addition, with no significant change in HR, ventricular contractility, or MAP, the carotid sinus baroreceptor reflex appears to contribute little to the response to VNR.     

Pathogen and host genotype differently affect pathogen fitness through their effects on different life-history stages

ABSTRACT: BACKGROUND: Adaptation of pathogens to their hosts depends critically on factorsaffecting pathogen reproductive rate. While pathogen reproduction is the end result of an intricate interaction between host and pathogen, the relative contributions of host and pathogen genotype to variation in pathogen life history within the hostare not well understood. Untangling these contributions allows us to identify traits withsufficient genetic variation for selection to act and to identify mechanisms of coevolution between pathogens and their hosts. We investigated the effects of pathogen and host genotype on three life-history components of pathogen fitness; infection efficiency, latent period, and sporulation capacity, in the oat crown rust fungus, Puccinia coronata f.sp. avenae, as it infects oats (Avena sativa). RESULTS: We show that both pathogen and host genotype significantly affect total spore production butdo so through their effects on different life-history stages. Pathogen genotype has the strongest effect on the early stage of infection efficiency, while host genotype most strongly affects the later life-history stages of latent period and sporulation capacity.In addition, host genotype affected the relationship between pathogen density and the later life-history traits oflatent period and sporulation capacity. We did not find evidence of pathogen-by-host genotypic (GxG) interactions. CONCLUSION: Our results illustrate mechanisms by which variation in host populationswill affect the evolution of pathogen lifehistory. Results show that differentpathogen life-history stages have the potential to respond differently to selection by host or pathogen genotypeand suggest mechanisms of antagonistic coevolution. Pathogen populations may adapt tohost genotype through increased infection efficiency while their plant hosts may adapt by limiting the later stages ofpathogen growthand spore production within the host.     

The evolution of Trypanosoma cruzi: the 'bat seeding' hypothesis

Recent discussions on the evolution of Trypanosoma cruzi have been dominated by the southern super-continent hypothesis, whereby T. cruzi and related parasites evolved in isolation in the mammals of South America, Antarctica and Australia. Here, we consider recent molecular evidence suggesting that T. cruzi evolved from within a broader clade of bat trypanosomes, and that bat trypanosomes have successfully made the switch into other mammalian hosts in both the New and Old Worlds. Accordingly, we propose an alternative hypothesis--the bat seeding hypothesis--whereby lineages of bat trypanosomes have switched into terrestrial mammals, thereby seeding the terrestrial lineages within the clade. One key implication of this finding is that T. cruzi may have evolved considerably more recently than previously envisaged.     

Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

Background

Angiogenesis is widely investigated in conjunction with cancer development, in particular because of the possibility of early stage detection and of new therapeutic strategies. However, such studies are negatively affected by the limitations of imaging techniques in the detection of microscopic blood vessels (diameter 3-5 ??m) grown under angiogenic stress. We report that synchrotron-based X-ray imaging techniques with very high spatial resolution can overcome this obstacle, provided that suitable contrast agents are used.

Results

We tested different contrast agents based on gold nanoparticles (AuNPs) for the detection of cancer-related angiogenesis by synchrotron microradiology, microtomography and high resolution X-ray microscopy. Among them only bare-AuNPs in conjunction with heparin injection provided sufficient contrast to allow in vivo detection of small capillary species (the smallest measured lumen diameters were 3-5 ??m). The detected vessel density was 3-7 times higher than with other nanoparticles. We also found that bare-AuNPs with heparin allows detecting symptoms of local extravascular nanoparticle diffusion in tumor areas where capillary leakage appeared.

Conclusions

Although high-Z AuNPs are natural candidates as radiology contrast agents, their success is not guaranteed, in particular when targeting very small blood vessels in tumor-related angiography. We found that AuNPs injected with heparin produced the contrast level needed to reveal--for the first time by X-ray imaging--tumor microvessels with 3-5 ??m diameter as well as extravascular diffusion due to basal membrane defenestration. These results open the interesting possibility of functional imaging of the tumor microvasculature, of its development and organization, as well as of the effects of anti-angiogenic drugs.