Development of technologies aiding large-tissue engineering

Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.     

Inhibition of neuronal calcium channels by a novel peptide spider toxin, DW13.3

Peptide toxins have proved to be useful agents, both in discriminating between different components of native calcium channel currents and in the molecular isolation and designation of their cloned channel counterparts. Here, we describe the isolation and characterization of the biochemical and physiological properties of a novel 74-amino acid peptide toxin (DW13.3) extracted from the venom of the spider Filistata hibernalis. The subtype specificity of DW13.3 was investigated using calcium channel currents recorded from two separate expression systems and several different cultured mammalian cell preparations. Overall, DW13.3 potently blocked all native calcium channel currents studied, with the exception of T-type currents recorded from GH3 cells. Examination of transiently expressed calcium channels in oocytes showed that DW13.3 had the highest affinity for alpha1A, followed by alpha1B > alpha1C > alpha1E. The affinity of DW13.3 for alpha1B N-type currents varied by 10-fold between expressed channels and native currents. Although block occurred in a similar 1:1 manner for all subtypes, DW13.3 produced a partial block of both alpha1A currents and P-type currents in cerebellar Purkinje cells. Selective occlusion of the P/Q-type channel ligand omega-conotoxin MVIIC (but not omega-agatoxin IVA) from its binding site in Purkinje neurons suggests that DW13.3 binds to a site close to the pore of the channel. The inhibition of different subtypes of calcium channels by DW13.3 reflects a common "macro" binding site present on all calcium channels except T-type.     

Reconstruction of cellular biological structures from optical microscopy data

Developments in optical microscopy imaging have generated large high-resolution data sets that have spurred medical researchers to conduct investigations into mechanisms of disease, including cancer at cellular and subcellular levels. The work reported here demonstrates that a suitable methodology can be conceived that isolates modality-dependent effects from the larger segmentation task and that 3D reconstructions can be cognizant of shapes as evident in the available 2D planar images. In the current realization, a method based on active geodesic contours is first deployed to counter the ambiguity that exists in separating overlapping cells on the image plane. Later, another segmentation effort based on a variant of Voronoi tessellations improves the delineation of the cell boundaries using a Bayesian formulation. In the next stage, the cells are interpolated across the third dimension thereby mitigating the poor structural correlation that exists in that dimension. We deploy our methods on three separate data sets obtained from light, confocal, and phase-contrast microscopy and validate the results appropriately.     

Synthetic substrates for human factor VIIa and factor VIIa-tissue factor

A series of 100 tripeptide fluorogenic substrates has been synthesized. These substrates contain Arg in the P1 position, various amino acids in the P2 and P3 positions, and different 6-amino-1-naphthalenesulfonamides (ANSN) as the detecting group (P'). The 38 compounds possessing the highest initial rates of factor VIIa hydrolysis were evaluated for substrate kinetic parameters in the presence and absence of tissue factor (TF) and by factor Xa. Most of these substrates had a higher kcat/KM (keff) value for the factor VIIa-TF complex than for factor Xa. Substitution of different amino acids in the P2 position showed that substrates with bulkier amino acids such as Leu, Pro, and Val have higher values for KM and kcat than those with smaller amino acids (Gly or Ser). The highest second-order rate constants were found for substrates with Val or Pro in the P2 position. A decrease or increase in volume of the P2 substituent (Gly, Ser, or Leu) resulted in a decrease in this constant. Substrates with the highest keff values have Phe in the P3 position. As the hydrophobicity and volume of the amino acid in the P3 position decreased, the keff was reduced. The efficiency of substrates for hydrolysis by factor VIIa was enhanced by an increase of hydrophobicity in the P' structure. TF enhanced the amidolytic activity of the "family" of 38 substrates with ANSN in the P' position on an average of 58-fold.     

Changes of bone-perfusion in osteosynthesis of the femur with a marrow-nail (author's transl)

The perfusion of the bone in the hind leg after osteosynthesis (nailing of the bone-marrow) was studied. In 11 shepherd dogs (bastards) an osteotomy of the femur was done; it was treated with a marrow-nail without boring the marrow-cavity. With the "tracer-microsphere"-method the perfusion of femur, tibia and talus of both hind legs was measured. Measurements were performed before and after surgery, in 10 dogs 2 weeks and in 8 dogs 6 weeks after surgery. Immediately after the operation the perfusion was reduced considerably in all the examined bones of the operated leg. Two weeks later the perfusion was increased in all bones of both hind limbs. In the cancellous bone of the femur the perfusion reached the original preoperative values after 6 weeks; in cortical bone a further increase of the perfusion was noted. This increase was most marked in the cortical bone of the operated femur; it was less in the cortical bone of the other bones.     

Harnessing power from sea water using nano material as photocatalyst and solar energy as light source: the role of hydrocarbon as dual agent

The splitting of water in the presence of ordinary and nano TiO₂ was carried out using hydrocarbon as a dual agent and solar energy as a light source for these experiments. The hydrogen gas evolved was tested and measured using downward displacement of water. The observed results show that more hydrogen was evolved when nano TiO₂ was used as catalyst due to the larger surface area of the nano material. The splitting of sea water yields more hydrogen compared with ordinary water due to the presence of electron donating sodium ions in water. The added hydrocarbon plays a dual role as electron donor and as a trapping agent, which enhances the production of hydrogen to a greater extent compared with the regular donors such as olefin. Copyright © 2013 John Wiley & Sons, Ltd.     

Verification of the applicability of lattice model to concrete fracture by AE study

Notched three-point bend specimens (TPB) were tested under crack mouth opening displacement (CMOD) control at a rate of 0.0004 mm/s and the entire fracture process was simulated using a regular triangular two-dimensional lattice network only over the expected fracture process zone width. The rest of the beam specimen was discretised by a coarse triangular finite element mesh. The discrete grain structure of the concrete was generated assuming the grains to be spherical. The load versus CMOD plots thus simulated agreed reasonably well with the experimental results. Moreover, acoustic emission (AE) hits were recorded during the test and compared with the number of fractured lattice elements. It was found that the cumulative AE hits correlated well with the cumulative fractured lattice elements at all load levels thus providing a useful means for predicting when the micro-cracks form during the fracturing process, both in the pre-peak and in the post-peak regimes.     

Sleep Disturbance and Metabolic Dysfunction: The Roles of Adipokines

Adipokines are a growing group of peptide or protein hormones that play important roles in whole body metabolism and metabolic diseases. Sleep is an integral component of energy metabolism, and sleep disturbance has been implicated in a wide range of metabolic disorders. Accumulating evidence suggests that adipokines may play a role in mediating the close association between sleep disorders and systemic metabolic derangements. In this review, we briefly summarize a group of selected adipokines and their identified function in metabolism. Moreover, we provide a balanced overview of these adipokines and their roles in sleep physiology and sleep disorders from recent human and animal studies. These studies collectively demonstrate that the functions of adipokine in sleep physiology and disorders could be largely twofold: (1) adipokines have multifaceted roles in sleep physiology and sleep disorders, and (2) sleep disturbance can in turn affect adipokine functions that likely contribute to systemic metabolic derangements.