Clinical applications of the quick-freezing and deep-etching method to human diabetic nephropathy

Mesangial expansion and glomerular basement membrane (GBM) thickening were not different between normoalbuminuric (NA) and microalbuminuric (MA) type 2 diabetic patients. The quick-freezing and deep-etching (QF-DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. In the present study, the QF-DE method was applied to the renal biopsy from 6 type 2 diabetic patients without definable renal diseases other than diabetic nephropathy. Four patients were NA and the other two were MA. Three control specimens were normal parts in surgically resected kidneys of renal cell carcinoma. Replica membranes were prepared by the QF-DE method as previously described. By the QF-DE method, both GBM middle layer and mesangial matrix (MM) were composed of polygonal meshwork structures. The mesh pores of GBM and MM were more enlarged in size and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The diameters of mesh pores in the diabetic patients were significantly larger than those in the control subjects. In conclusion, the QF-DE method could be applied to needle renal biopsy and the present study has firstly clarified the difference of ultrastructural changes between NA and MA type 2 diabetic patients, which had not been disclosed by the conventional electron microscopy, were revealed by the QF-DE method.     

Simple spectral stray light correction method for array spectroradiometers

A simple, practical method has been developed to correct a spectroradiometer's response for measurement errors arising from the instrument's spectral stray light. By characterizing the instrument's response to a set of monochromatic laser sources that cover the instrument's spectral range, one obtains a spectral stray light signal distribution matrix that quantifies the magnitude of the spectral stray light signal within the instrument. By use of these data, a spectral stray light correction matrix is derived and the instrument's response can be corrected with a simple matrix multiplication. The method has been implemented and validated with a commercial CCD-array spectrograph. Spectral stray light errors after the correction was applied were reduced by 1-2 orders of magnitude to a level of approximately 10(-5) for a broadband source measurement, equivalent to less than one count of the 15-bit-resolution instrument. This method is fast enough to be integrated into an instrument's software to perform real-time corrections with minimal effect on acquisition speed. Using instruments that have been corrected for spectral stray light, we expect significant reductions in overall measurement uncertainties in many applications in which spectrometers are commonly used, including radiometry, colorimetry, photometry, and biotechnology.     

Headspace GC/MS analysis of residual vinyl chloride and vinylidene chloride in polyvinyl chloride and polyvinylidene chloride products

A headspace GC/MS analysis method for the simultaneous determination of residual vinyl chloride (VC) and vinylidene chloride (VDC) in polyvinyl chloride (PVC) and polyvinylidene chloride (PVDC) products was developed. A test sample was swelled overnight with N,N-dimethylacetamide in a sealed vial. The vial was incubated for 1 hour at 90 degrees C, then the headspace gas was analyzed by GC/MS using a PLOT capillary column. The recoveries from spiked PVC and PVDC samples were 90.0-112.3% for VC and 85.2-108.3% for VDC. The determination limits were 0.01 microg/g for VC and 0.06/microg/g for VDC, respectively. By this method, VC was detected in two PVC water supply pipes at the levels of 0.61 and 0.01 microg/g. On the other hand, VC and VDC were not detected in any of the food container-packages or toys tested.     

Gene expression property of high-density three-dimensional tissue of HepG2 cells formed in radial-flow bioreactor

In our previous study, we examined three-dimensional culture using 5-ml radial-flow bioreactor (RFB) and showed that genes encoding cell cycle related proteins were suppressed in a stable phase. In this study, we analyzed the gene expression profiles of RFB-cultivated HepG2 cells and found that vascular endothelial growth factor (VEGF) production was strongly induced in the stable phase compared with the growth phase or static two-dimensional culture. When human umbilical vein endothelial cells (HUVECs) were grown under the conditioned medium of the stable phase, it was found that the formation of new blood vessels was induced in the angiogenesis model. DNA microarray analysis showed that the expression levels of both genes related to cell cycle arrest and which are known as tumor markers have increased in the stable phase. This result suggests that HepG2 cells in the stable phase maintain an active tumor phenotype. In addition, the expression of genes induced in the hypoxic condition was also induced in the stable phase. When the culture was carried out under a higher dissolved oxygen (DO) concentration, VEGF production did not decrease significantly and the new blood-vessel-forming ability of the conditioned medium was not suppressed. This suggests that the induction of VEGF production in a stable phase is not affected by DO during the tested level. These results suggest that the RFB cell culture system may be used to assess tumor progression mechanism under three-dimensional condition in vitro.     

Bone composite behaviour: effects of mineral-organic bonding

The mechanical properties of a composite material rely not only on the volume fraction, orientation and properties of the individual constituents, but upon their bonding interactions as well. This study examines the role of bonding between the mineral and organic constituents of bovine compact bone. Intact and completely demineralized samples were tested in tension following treatment in varying ionic strength sodium chloride or phosphate ion containing buffers to examine the interfacial bonding forces between bone's constituents. Phosphate ion treatment caused a reduction in the mechanical properties of intact samples but not in the demineralized samples. A sodium chloride solution with ionic strength equal to that of the phosphate ion buffer did not alter the mechanical properties of the intact or demineralized samples. Ash weight analysis, calcium probe measurements and SDS-gel electrophoresis indicated intact samples were not demineralized nor were bone structural proteins removed during treatment. Data suggest that the reduction in the mechanical properties of intact samples with phosphate ion treatment was due to an alteration in the interfacial bonding between the mineral and organic constituents of bone. Phosphate ions can compete with the negative domains of organic constituents for calcium binding sites of bone mineral and thereby interrupt or partially debond the interactions between the mineral and organic constituents of bone.     

Strong and Specific Recognition of CAG/CTG Repeat DNA (5’-dWGCWGCW-3’) by a Cyclic Pyrrole-Imidazole Polyamide

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (T_m) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.