Mechanical properties of mineralized collagen fibrils as influenced by demineralization

Dentin and bone derive their mechanical properties from a complex arrangement of collagen type-I fibrils reinforced with nanocrystalline apatite mineral in extra- and intrafibrillar compartments. While mechanical properties have been determined for the bulk of the mineralized tissue, information on the mechanics of the individual fibril is limited. Here, atomic force microscopy was used on individual collagen fibrils to study structural and mechanical changes during acid etching. The characteristic 67 nm periodicity of gap zones was not observed on the mineralized fibril, but became apparent and increasingly pronounced with continuous demineralization. AFM-nanoindentation showed a decrease in modulus from 1.5 GPa to 50 MPa during acid etching of individual collagen fibrils and revealed that the modulus profile followed the axial periodicity. The nanomechanical data, Raman spectroscopy and SAXS support the hypothesis that intrafibrillar mineral etches at a substantially slower rate than the extrafibrillar mineral. These findings are relevant for understanding the biomechanics and design principles of calcified tissues derived from collagen matrices.     

Security Analysis and Improvement of ‘a More Secure Anonymous User Authentication Scheme for the Integrated EPR Information System’

Over the past few years, secure and privacy-preserving user authentication scheme has become an integral part of the applications of the healthcare systems. Recently, Wen has designed an improved user authentication system over the Lee et al.’s scheme for integrated electronic patient record (EPR) information system, which has been analyzed in this study. We have found that Wen’s scheme still has the following inefficiencies: (1) the correctness of identity and password are not verified during the login and password change phases; (2) it is vulnerable to impersonation attack and privileged-insider attack; (3) it is designed without the revocation of lost/stolen smart card; (4) the explicit key confirmation and the no key control properties are absent, and (5) user cannot update his/her password without the help of server and secure channel. Then we aimed to propose an enhanced two-factor user authentication system based on the intractable assumption of the quadratic residue problem (QRP) in the multiplicative group. Our scheme bears more securities and functionalities than other schemes found in the literature.     

Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid

Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.     

Apolipoprotein E levels in cerebrospinal fluid and the effects of ABCA1 polymorphisms

Background

Animal studies suggest that brain apolipoprotein E (apoE) levels influence amyloid-β (Aβ) deposition and thus risk for Alzheimer's disease (AD). We have previously demonstrated that deletion of the ATP-binding cassette A1 transporter (ABCA1) in mice causes dramatic reductions in brain and cerebrospinal fluid (CSF) apoE levels and lipidation. To examine whether polymorphisms in ABCA1 affect CSF apoE levels in humans, we measured apoE in CSF taken from 168 subjects who were 43 to 91 years old and were either cognitively normal or who had mild AD. We then genotyped the subjects for ten previously identified ABCA1 single nucleotide polymorphisms (SNPs).

Results

In all subjects, the mean CSF apoE level was 9.09 μg/ml with a standard deviation of 2.70 μg/ml. Levels of apoE in CSF samples taken from the same individual two weeks apart were strongly correlated (r² = 0.93, p < 0.01). In contrast, CSF apoE levels in different individuals varied widely (coefficient of variation = 46%). CSF apoE levels did not vary according to AD status, APOE genotype, gender or race. Average apoE levels increased with age by ~0.5 μg/ml per 10 years (r² = 0.05, p = 0.003). We found no significant associations between CSF apoE levels and the ten ABCA1 SNPs we genotyped. Moreover, in a separate sample of 1225 AD cases and 1431 controls, we found no association between the ABCA1 SNP rs2230806 and AD as has been previously reported.

Conclusion

We found that CSF apoE levels vary widely between individuals, but are stable within individuals over a two-week interval. AD status, APOE genotype, gender and race do not affect CSF apoE levels, but average CSF apoE levels increase with age. Given the lack of association between CSF apoE levels and genotypes for the ABCA1 SNPs we examined, either these SNPs do not affect ABCA1 function or if they do, they do not have strong effects in the CNS. Finally, we find no evidence for an association between the ABCA1 SNP rs2230806 and AD in a large sample set.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Denitrification and nitric oxide reduction in an aerobic toluene-treating biofilter

The presence of significant denitrification activity in an aerobic toluene-treating biofilter was demonstrated under batch and flow-through conditions. N2O concentrations of 9.2 ppmv were produced by denitrifying bacteria in the presence of 15% acetylene, in a flow-through system with a bulk gas phase O2 concentration of >17%. The carbon source for denitrification was not toluene but a byproduct or metabolite of toluene catabolism. Denitrification conditions were successfully used for the reduction of 60 ppmv nitric oxide to 15 ppmv at a flow rate of 3 L min-1 (EBRT of 3 min) in a fully aerated, 17% v/v O2 (superficially aerobic) biofilter. Higher NO removal efficiency (97%) was obtained by increasing the toluene supply to the biofilter.     

Cervical cancer after multiple negative cytologic tests in long-term members of a prepaid health plan

OBJECTIVE: To estimate squamous cell cervical cancer incidence within 3.5 years of 1, 2 and > or = 3 consecutive, normal cytologic tests. STUDY DESIGN: Data were from a case-control study of cervical cancer screening efficacy. Long-term members of a prepaid health plan diagnosed with cancer from 1983 to 1995 (the cases) were grouped by number of prior conventional cytologic tests and by time from the last negative screening test to the diagnosis date. Women from the population at risk were estimated by multiplying the timated by multiplying the proportions of women without cancer (the matched controls) in each screening category by the numbers of long-term of the numbers of long-term female members enrolled from male members enrolled from 1983 to 1995. RESULTS: Of an estimated 6,802,641 woman-years of observation, 129 cases were diagnosed within 3.5 years of > or = 21 negative screening test. After > or = 3 consecutive negative tests, incidence per 100,000 woman-years grouped by time from the last negative screening test were: 1.43 (26/1,813,552) 0-18 months later, 4.24 (17/400,584) 19-30 months later and 4.73 (10/211,217) 31- 42 months later. CONCLUSION: The first 18 months after the last negative screening Pap test in women with > or = 3 prior negative tests, cancer incidence increases to an estimated 4-5 per 100,000 woman-years in each of the subsequent 2 years.