Gamma-tubulin in chicken erythrocytes: changes in localization during cell differentiation and characterization of cytoplasmic complexes.

The mechanism of marginal band (MB) formation in differentiating erythroid cells is not fully understood, and the proteins involved in nucleation of MB microtubules are largely unknown. To gain insights into the function of gamma-tubulin in MB formation, we have followed its distribution in developing chicken erythrocytes and characterized soluble forms of the protein. In early stages of erythroid cells differentiation, gamma-tubulin was present in microtubule-organizing centers, mitotic spindles, as well as on MB. Its subcellular localization changed in the course of differentiation, and in postnatal peripheral erythrocytes gamma-tubulin was found only in soluble forms. After cold-induced depolymerization gamma-tubulin in erythroid cells formed large clusters that were not observed in matured cells, and re-growth experiments demonstrated that gamma-tubulin was not present in distinct nucleation structures at the cell periphery. Soluble gamma-tubulin formed complexes of various size and large complexes were prone to dissociation in the presence of high salt concentration. Interaction of gamma-tubulin with tubulin dimers was revealed by precipitation experiments. gamma-Tubulin occurred in multiple charge variants whose number increased in the course of erythrocyte differentiation and corresponded with decreased binding to MB. The presented data demonstrate for the first time that gamma-tubulin is a substrate for developmentally regulated posttranslational modifications and that the binding properties of gamma-tubulin or its complexes change during differentiation events.     

Flow-controlled fluoroscopic angiography for the assessment of vascular integrity in bioengineered kidneys

Perfusion decellularization has been proposed as a promising method for generating nonimmunogenic organs from allogeneic or xenogeneic donors. Several imaging modalities have been used to assess vascular integrity in bioengineered organs with no consistency in the methodology used. Here, we studied the use of fluoroscopic angiography performed under controlled flow conditions for vascular integrity assessment in bioengineered kidneys. Porcine kidneys underwent ex vivo angiography before and after perfusion decellularization. Arterial and venous patencies were defined as visualization of contrast medium (CM) in distal capillaries and renal vein, respectively. Changes in vascular permeability were visualized and quantified. No differences in patency were detected in decellularized kidneys compared with native kidneys. However, focal parenchymal opacities and significant delay in CM clearance were detected in decellularized kidneys, indicating increased permeability. Biopsy-induced leakage was visualized in both groups, with digital subtraction angiography revealing minimal CM leakage earlier than nonsubtracted fluoroscopy. In summary, quantitative assessment of vascular permeability should be coupled with patency when studying the effect of perfusion decellularization on kidney vasculature. Flow-controlled angiography should be considered as the method of choice for vascular assessment in bioengineered kidneys. Adopting this methodology for organs premodified ex vivo under normothermic machine perfusion settings is also suggested.     

Inhibition of calcineurin combined with dasatinib has direct and indirect anti‐leukemia effects against BCR‐ABL1+ leukemia

Treatment of BCR‐ABL1⁺ leukemia has been revolutionized with the development of tyrosine kinase inhibitors. However, patients with BCR‐ABL1⁺ acute lymphoblastic leukemia and subsets of patients with chronic myeloid leukemia are at high risk of relapse despite kinase inhibition therapy, necessitating novel treatment strategies. We previously reported synthetic lethality in BCR‐ABL1⁺ leukemia cells by blocking both calcineurin/NFAT signaling and BCR‐ABL1, independent of drug efflux inhibition by cyclosporine. Here, using RNA‐interference we confirm that calcineurin inhibition sensitizes BCR‐ABL1⁺ cells to tyrosine kinase inhibition in vitro. However, when we performed pharmacokinetic and pharmacodynamic studies of dasatinib and cyclosporine in mice, we found that co‐administration of cyclosporine increases peak concentrations and the area under the curve of dasatinib, which contributes to the enhanced disease control. We also report the clinical experience of two subjects in whom we observed more hematopoietic toxicity than expected while enrolled in a Phase Ib trial designed to assess the safety and tolerability of adding cyclosporine to dasatinib in humans. Thus, the anti‐leukemia benefit of co‐administration of cyclosporine and dasatinib is mechanistically pleiotropic, but may not be tolerable, at least as administered in this trial. These data highlight some of the challenges associated with combining targeted agents to treat leukemia. Am. J. Hematol. 89:896–903, 2014. © 2014 Wiley Periodicals, Inc.     

Three-dimensional imaging improves surgical skill performance in a laparoscopic test model for both experienced and novice laparoscopic surgeons

Study objective

The objective of this study was to evaluate and compare the impact of three-dimensional (3D) imaging system on the performance of basic laparoscopic tasks in a test model by novice and experienced surgeons.

Design

Three tasks were performed in a test model by 30 surgeons, 15 experienced surgeons, and 15 with minimal laparoscopic experience. The tasks were performed using 2D and 3D vision systems.

Design classification

Canadian Task Force II-1.

Subjects

Fifteen experienced laparoscopic surgeons and fifteen novices with minimal laparoscopic experience.

Measurements

Performance times were recorded using both two-dimensional and 3D imaging system for each task.

Main results

Performance time for all skills was significantly (P < 0.02) shorter when using 3D imaging system. Performance times were reduced by 18–31 % using 3D imaging for all participants. Experienced surgeons performed the tasks faster and showed similar improvement while using 3D imaging system.

Conclusion

3D vision systems allow for significant improvement in performance times of basic laparoscopic tasks in a test model for both inexperienced and advanced laparoscopic surgeons. Experienced surgeons benefit as much as novices from 3D imaging system. This benefit should be weighed against the disadvantages of the 3D vision systems, mainly cost, decreased light, eye strain, headaches, and shorter focal lengths.     

Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1–S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca²⁺-activated K⁺ channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its “decentralized” distribution of voltage-sensing residues, we probed the BK_Ca VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.     

Manufacturing Nanosized Fenofibrate by Salt Assisted Milling

Purpose  The aim of this study is to develop a new process for manufacturing a nano-sized form of the popular cholesterol-reducing drug fenofibrate which can be implemented on industrial scale with minimal changes of currently used production schemes. Methods  Salt-assisted milling was used to reduce particle size of commercial fenofibrate from micron-sized particles to nanometer domains. Results  The optimal parameters for the salt milling are reported, allowing one to reduce the particle size from tens of micrometers to a hundred of nanometers. Dissolution of nano-sized fenofibrate was studied in various formulations and compared against the micron-sized commercially available fenofibrate. Conclusions  The nano-sized fenofibrate demonstrates faster dissolution kinetics in aqueous media, simulating stomach environment, within the first 60 min as compared to the micronized form. The highest dissolution rate is achieved with the nano-sized fenofibrate when surfactants, such as sodium dodecyl sulfate or inclusion complex forming agents such as alpha-cyclodextrin, are used.