bluuebunny/crossref_metadata_2025 huggingface dataset:
In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects
Authors: Lastella Patrizia , Surdo Nicoletta Concetta , Resta Nicoletta , Guanti Ginevra , Stella Alessandro
Published: September 2006

Background

Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay.

Results

We analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion.

Conclusion

Our results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers.
A microsatellite-based consensus linkage map for species of Eucalyptusand a novel set of 230 microsatellite markers for the genus
Authors: Brondani Rosana PV , Williams Emlyn R , Brondani Claudio , Grattapaglia Dario
Published: September 2006

Background

Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.

Results

The consensus map covers ~90% of the recombining genome of Eucalyptus, involves 234 mapped EMBRA loci on 11 linkage groups, an observed length of 1,568 cM and a mean distance between markers of 8.4 cM. A compilation of all microsatellite linkage information published in Eucalyptus allowed us to establish the homology among linkage groups between this consensus map and other maps published for E. globulus. Comparative mapping analyses also resulted in the linkage group assignment of other 41 microsatellites derived from other Eucalyptus species as well as candidate genes and QTLs for wood and flowering traits published in the literature. This report significantly increases the availability of microsatellite markers and mapping information for species of Eucalyptus and corroborates the high conservation of microsatellite flanking sequences and locus ordering between species of the genus.

Conclusion

This work represents an important step forward for Eucalyptus comparative genomics, opening stimulating perspectives for evolutionary studies and molecular breeding applications. The generalized use of an increasingly larger set of interspecific transferable markers and consensus mapping information, will allow faster and more detailed investigations of QTL synteny among species, validation of expression-QTL across variable genetic backgrounds and positioning of a growing number of candidate genes co-localized with QTLs, to be tested in association mapping experiments.
Support for immunization registries among parents of vaccinated and unvaccinated school-aged children: a case control study
Authors: Linkins Robert W , Salmon Daniel A , Omer Saad B , Pan William KY , Stokley Shannon , Halsey Neal A
Published: September 2006

Background

Immunizations have reduced childhood vaccine preventable disease incidence by 98–100%. Continued vaccine preventable disease control depends on high immunization coverage. Immunization registries help ensure high coverage by recording childhood immunizations administered, generating reminders when immunizations are due, calculating immunization coverage and identifying pockets needing immunization services, and improving vaccine safety by reducing over-immunization and providing data for post-licensure vaccine safety studies. Despite substantial resources directed towards registry development in the U.S., only 48% of children were enrolled in a registry in 2004. Parental attitudes likely impact child participation. Consequently, the purpose of this study was to assess the attitudes of parents of vaccinated and unvaccinated school-aged children regarding: support for immunization registries; laws authorizing registries and mandating provider reporting; opt-in versus opt-out registry participation; and financial worth and responsibility of registry development and implementation.

Methods

A case control study of parents of 815 children exempt from school vaccination requirements and 1630 fully vaccinated children was conducted. Children were recruited from 112 elementary schools in Colorado, Massachusetts, Missouri, and Washington. Surveys administered to the parents, asked about views on registries and perceived utility and safety of vaccines. Parental views were summarized and logistic regression models compared differences between parents of exempt and vaccinated children.

Results

Surveys were completed by 56.1% of respondents. Fewer than 10% of parents were aware of immunization registries in their communities. Among parents aware of registries, exempt children were more likely to be enrolled (65.0%) than vaccinated children (26.5%) (p value = 0.01). A substantial proportion of parents of exempt children support immunization registries, particularly if registries offer choice for participation. Few parents of vaccinated (6.8%) and exempt children (6.7%) were aware of laws authorizing immunization registries. Support for laws authorizing registries and requiring health care providers to report to registries was more common among parents of vaccinated than exempt children. Most parents believed that the government, vaccine companies or insurance companies should pay for registries.

Conclusion

Parental support for registries was relatively high. Parental support for immunization registries may increase with greater parental awareness of the risks of vaccine preventable diseases and utility of vaccination.
The risk of death associated with delayed coronary artery bypass surgery
Authors: Sobolev Boris G , Levy Adrian R , Kuramoto Lisa , Hayden Robert , Brophy James M , FitzGerald J Mark
Published: September 2006

Background

The detrimental effect of delaying surgical revascularization has been estimated in randomized trials and observational studies. It has been argued that the Kaplan-Meier method used in quantifying the hazard of delayed treatment is not appropriate for summarizing the probability of competing outcomes. Therefore, we sought to improve the estimates of the risk of death associated with delayed surgical treatment of coronary artery disease.

Methods

Population-based prospective study of 8,325 patients registered to undergo first time isolated coronary artery bypass grafting (CABG) in any of the four tertiary hospitals that provide cardiac care to adult residents of British Columbia, Canada. The cumulative incidence of pre-operative death, the cumulative incidence of surgery, and the probability that a patient, who may die or undergo surgery, dies if not operated by certain times over the 52-week period after the decision for CABG were estimated. The risks were quantified separately in two groups: high-severity at presentation were patients with either persistent unstable angina or stable angina and extensive coronary artery disease, and low-severity at presentation were stable symptomatic patients with limited disease.

Results

The median waiting time for surgery was 10 weeks (interquartile range [IQR] 15 weeks) in the high-severity group and 21 weeks (IQR 30 weeks) in the low-severity group. One percent of patients died before surgery: 54 in the high-severity and 26 in the low-severity group. For 58 (72.5%) patients, death was related to CVD (acute coronary syndrome, 33; chronic CVD, 16; other CVD, 4; and sudden deaths, 5). The overall death rate from all causes was 0.61 (95% CI 0.48-0.74) per 1,000 patient-weeks, varying from 0.62 (95% CI 0.45-0.78) in the high-severity group to 0.59 (95% CI 0.37-0.82) in the low-severity group. After adjustment for age, sex, and comorbidity, the all-cause death rate in the low-severity group was similar to the high-severity group (OR = 1.02, 95% CI 0.64-1.62). The conditional probability of death was greater in the high-severity group than in the low-severity group both for all-cause mortality (p = 0.002) and cardiovascular deaths (p <0.001).

Conclusion

The probability of death conditional on not having undergone a required CABG increases with time spent on wait lists.
Amount of health care and self-care following a randomized clinical trial comparing flexion-distraction with exercise program for chronic low back pain
Authors: Cambron Jerrilyn A , Gudavalli M Ram , McGregor Marion , Jedlicka James , Keenum Michael , Ghanayem Alexander J , Patwardhan Avinash G , Furner Sylvia E
Published: September 2006

Background

Previous clinical trials have assessed the percentage of participants who utilized further health care after a period of conservative care for low back pain, however no chiropractic clinical trial has determined the total amount of care during this time and any differences based on assigned treatment group. The objective of this clinical trial follow-up was to assess if there was a difference in the total number of office visits for low back pain over one year after a four week clinical trial of either a form of physical therapy (Exercise Program) or a form of chiropractic care (Flexion Distraction) for chronic low back pain.

Methods

In this randomized clinical trial follow up study, 195 participants were followed for one year after a four-week period of either a form of chiropractic care (FD) or a form of physical therapy (EP). Weekly structured telephone interview questions regarded visitation of various health care practitioners and the practice of self-care for low back pain.

Results

Participants in the physical therapy group demonstrated on average significantly more visits to any health care provider and to a general practitioner during the year after trial care (p < 0.05). No group differences were noted in the number of visits to a chiropractor or physical therapist. Self-care was initiated by nearly every participant in both groups.

Conclusion

During a one-year follow-up, participants previously randomized to physical therapy attended significantly more health care visits than those participants who received chiropractic care.
Risk of acute myocardial infarction with nonselective non-steroidal anti-inflammatory drugs: a meta-analysis
Authors: Singh Gurkirpal , Wu Olivia , Langhorne Peter , Madhok Rajan
Published: September 2006

The use of cyclo-oxygenase 2 selective nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with increased risk of acute myocardial infarction (AMI). The association between the risks of AMI with nonselective NSAIDs is less clear. We reviewed the published evidence and assessed the risk of AMI with nonselective NSAIDs. We performed a meta-analysis of all studies containing data from population databases that compared the risk of AMI in NSAID users with that in non-users or remote NSAID users. The primary outcome was objectively confirmed AMI. Fourteen studies met predefined criteria for inclusion in the meta-analysis. Nonselective NSAIDs as a class was associated with increased AMI risk (relative AMI risk 1.19, 95% confidence interval [CI] 1.08 to 1.31). Similar findings were found with diclofenac (relative AMI risk 1.38, 95% CI 1.22–1.57) and ibuprofen (relative AMI risk 1.11, 95% CI 1.06 to 1.17). However, this effect was not observed with naproxen (relative AMI risk 0.99, 95% CI 0.88–1.11). In conclusion, based on current evidence, there is a general direction of effect, which suggests that at least some nonselective NSAIDs increase AMI risk. Analysis based on the limited data available for individual NSAIDs, including diclofenac and ibuprofen, supported this finding; however, this was not the case for naproxen. Nonselective NSAIDs are frequently prescribed, and so further investigation into the risk of AMI is warranted because the potential for harm can be substantial.
Mechanical ventilation interacts with endotoxemia to induce extrapulmonary organ dysfunction
Authors: O'Mahony DShane , Liles WConrad , Altemeier William A , Dhanireddy Shireesha , Frevert Charles W , Liggitt Denny , Martin Thomas R , Matute-Bello Gustavo
Published: September 2006

Introduction

Multiple organ dysfunction syndrome (MODS) is a common complication of sepsis in mechanically ventilated patients with acute respiratory distress syndrome, but the links between mechanical ventilation and MODS are unclear. Our goal was to determine whether a minimally injurious mechanical ventilation strategy synergizes with low-dose endotoxemia to induce the activation of pro-inflammatory pathways in the lungs and in the systemic circulation, resulting in distal organ dysfunction and/or injury.

Methods

We administered intraperitoneal Escherichia coli lipopolysaccharide (LPS; 1 μg/g) to C57BL/6 mice, and 14 hours later subjected the mice to 6 hours of mechanical ventilation with tidal volumes of 10 ml/kg (LPS + MV). Comparison groups received ventilation but no LPS (MV), LPS but no ventilation (LPS), or neither LPS nor ventilation (phosphate-buffered saline; PBS).

Results

Myeloperoxidase activity and the concentrations of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC were significantly increased in the lungs of mice in the LPS + MV group, in comparison with mice in the PBS group. Interestingly, permeability changes across the alveolar epithelium and histological changes suggestive of lung injury were minimal in mice in the LPS + MV group. However, despite the minimal lung injury, the combination of mechanical ventilation and LPS resulted in chemical and histological evidence of liver and kidney injury, and this was associated with increases in the plasma concentrations of KC, MIP-2, IL-6, and TNF-α.

Conclusion

Non-injurious mechanical ventilation strategies interact with endotoxemia in mice to enhance pro-inflammatory mechanisms in the lungs and promote extra-pulmonary end-organ injury, even in the absence of demonstrable acute lung injury.
Insulin Growth Factor-I and Epidermal Growth Factor Receptors Recruit Distinct Upstream Signaling Molecules to Enhance AKT Activation in Mammary Epithelial Cells
Authors: Fleming Jodie M. , Desury Gwenaëlle , Polanco Tiffany A. , Cohick Wendie S.
Published: September 2006

IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth and tumorigenesis. Whereas both growth factors increase DNA synthesis in MECs, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. In the present study, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5–100 ng/ml). IGF-I activated AKT via insulin receptor substrate-1 and p85, the regulatory subunit of phosphatidylinositol-3 kinase. Treatment with EGF had no effect on insulin receptor substrate-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2 and Gab2 with phospho-SHC, phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.
Regulation of Adenosine 5′,Monophosphate-Activated Protein Kinase and Lipogenesis by Androgens Contributes to Visceral Obesity in an Estrogen-Deficient State
Authors: McInnes Kerry J. , Corbould Anne , Simpson Evan R. , Jones Margaret E.
Published: September 2006

Menopause is associated with an accumulation of visceral fat. An emerging concept suggests that relatively elevated levels of circulating androgens, compared with estrogens in postmenopausal women, underlie this shift in body fat distribution. In this study we administered dihydrotestosterone (DHT) to ovariectomized mice to examine the effect of relative androgen excess on adipose tissue distribution and function in estrogen-deficient mice. Compared with controls, DHT-treated mice exhibited increased body weight and visceral fat mass associated with triglyceride accumulation. Phosphorylation of AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase was significantly decreased by DHT in visceral fat. In 3T3-L1 cells, DHT decreased phosphorylation of AMPK in a dose-dependent manner. In addition, DHT increased the expression of lipogenic genes (fatty acid synthase, sterol regulatory element binding protein-2, and lipoprotein lipase) in visceral fat. These data provide the first in vivo evidence that an increased androgen to estrogen ratio can promote visceral fat accumulation by inhibiting AMPK activation and stimulating lipogenesis.
Molecular Genetic Studies of Gene Identification for Osteoporosis: A 2004 Update
Authors: Liu Yong-Jun , Shen Hui , Xiao Peng , Xiong Dong-Hai , Li Li-Hua , Recker Robert R , Deng Hong-Wen
Published: September 2006

This review summarizes comprehensively the most important and representative molecular genetics studies of gene identification for osteoporosis published up to the end of December 2004. It is intended to constitute a sequential update of our previously published review covering the available data up to the end of 2002. Evidence from candidate gene association studies and genome-wide linkage studies in humans, as well as quantitative trait locus mapping animal models are reviewed separately. Studies of transgenic and knockout mice models relevant to osteoporosis are summarized. An important extension of this update is incorporation of functional genomic studies (including DNA microarrays and proteomics) on osteogenesis and osteoporosis, in light of the rapid advances and the promising prospects of the field. Comments are made on the most notable findings and representative studies for their potential influence and implications on our present understanding of genetics of osteoporosis. The format adopted by this review should be ideal for accommodating future new advances and studies.
Leptin Inhibits Bone Formation Not Only in Rodents, but Also in Sheep
Authors: Pogoda Pia , Egermann Marcus , Schnell Johann C , Priemel Matthias , Schilling Arndt F , Alini Mauro , Schinke Thorsten , Rueger Johannes M , Schneider Erich , Clarke Iain , Amling Michael
Published: September 2006

This study examines the effect of long-term ICV administration of leptin in ewes. We found that central application significantly decreased osteoblast activity as measured by serum analysis as well as by histomorphometry, resulting in decreased trabecular bone volume. These data provide additional evidence that bone formation and therefore bone remodeling is at least in part centrally controlled.

Introduction: Genetic studies in mice have identified leptin as a potent inhibitor of bone formation acting through the central nervous system and unraveled the central nature of bone mass control and its disorders. Although these studies have radically enhanced our understanding of skeletal physiology because they have established a hypothalamic regulation of bone remodeling through the sympathetic nervous system, controversy remains about the physiological relevance of these observations because leptin's effect on bone after intracerebroventricular (ICV) application has only been shown in mice. To address whether leptin has a role in regulating bone mass beyond rodents, we treated ewes with long-term ICV application of leptin and analyzed the bone phenotype after a treatment period of 3 months.

Materials and Methods: Three groups of corriedale sheep were compared: (1) control entire (control), (2) ovariectomy (OVX) and ICV application of cerebrospinal fluid (CSF); and (iii) OVX and ICV application of leptin (leptin). Analysis included histomorphometric characterization of iliac crest, spine and femur by histology and biomechanical testing and measurement of bone turnover parameters in serum and urine.

Results: Central application of leptin decreased bone formation by 70% and mineralizing surface (MS/BS, 39.4 ± 3.3% versus 16.1 ± 2.1%) significantly (p < 0.01). Whereas OVX increased osteoclast indices and urinary cross-lap excretion by two and three times, respectively, serum parameters of osteoblast activity were significantly reduced by ICV application of leptin (p < 0.01). Consequently, ewes treated with leptin were osteopenic (iliac crest BV/TV entire, 22.7 ± 1.3%; CSF, 18.9 ± 2.4%; leptin, 12.4 ± 2.6%), whereas bone torsional failure load reflecting the cortex of the tibia was not yet changed after 3 months of treatment (p < 0.01).

Conclusions: Taken together, these data suggest that leptin controls bone formation after ICV application, leading to reduction of trabecular bone mass in sheep. Most importantly, however, they show that the central regulation of bone formation is not limited to rodents, but is also found in large animals, providing further evidence that bone remodeling in vertebrates is centrally controlled.
Bone Sialoprotein Enhances Migration of Bone Marrow Stromal Cells Through Matrices by Bridging MMP-2 to αvβ3-Integrin
Authors: Karadag Abdullah , Fisher Larry W
Published: September 2006

BMSCs migrate through matrix barriers and differentiate into osteoblasts. BSP enhances osteogenic cell migration through basement membrane and collagen matrices in vitro by localizing MMP-2 on the cell surface through αvβ3-integrin.

Introduction: The specific mechanisms by which bone marrow stromal cells (BMSCs) leave their primary sites, move through matrices encountered during homing to their site of final differentiation, and remove preexisting matrices in preparation for bone matrix production are not well understood.

Materials and Methods: The enhanced migration of human osteoblast precursor cells through matrix barriers by bone sialoprotein (BSP) was studied by a modified Boyden-chamber assay. The bridging of normally soluble matrix metalloproteinase 2 (MMP-2) to the cell surface receptor, αvβ3-integrin, by BSP was analyzed by flow cytometry.

Results: BSP enhanced the in vitro passage of BMSCs and pre-osteoblasts through matrix barriers (Matrigel and denatured type I collagen) in a dose-dependent manner. An intact ArgGlyAsp (RGD) was required in the BSP for enhanced migration through the barriers but was not sufficient, as shown by the inactivity of two other SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family members, osteopontin and dentin matrix protein-1. The specificity of the BSP enhancement activity was apparently caused by this molecule's ability to bridge MMP-2 to the cell surfaces.

Conclusions: Pre-osteoblasts and their BMSC precursors may use MMP-2/BSP/integrin complexes to disrupt matrix barriers during migration to their final destinations in vivo.
Quantification of the Roles of Trabecular Microarchitecture and Trabecular Type in Determining the Elastic Modulus of Human Trabecular Bone
Authors: Liu Xiaowei S , Sajda Paul , Saha Punam K , Wehrli Felix W , Guo X Edward
Published: September 2006

The roles of microarchitecture and types of trabeculae in determining elastic modulus of trabecular bone have been studied in μCT images of 29 trabecular bone samples by comparing their Young's moduli calculated by finite element analysis (FEA) with different trabecular type‐specific reconstructions. The results suggest that trabecular plates play an essential role in determining elastic properties of trabecular bone.

Introduction: Osteoporosis is an age‐related disease characterized by low bone mass and architectural deterioration. Other than bone volume fraction (BV/TV), microarchitecture of bone is also believed to be important in governing mechanical properties of trabecular bone. We quantitatively examined the role of microarchitecture and relative contribution of trabecular types of individual trabecula in determining the elastic property of trabecular bone.

Materials and Methods: Twenty‐nine human cadaveric trabecular bone samples were scanned at 21‐μm resolution using a μCT system. Digital topological analysis (DTA) consisting of skeletonization and classification was combined with a trabecular type‐specific reconstruction technique to extract the skeleton and identify topological type of trabeculae of the original trabecular bone image. Four different μCT‐based finite element (FE) models were constructed for each specimen: (1) original full voxel; (2) skeletal voxel; (3) rod‐reconstructed, preserving rod volume and plate skeleton; and (4) plate‐reconstructed, preserving plate volume and rod skeleton. For each model, the elastic moduli were calculated under compression along each of three image‐coordinate axis directions. Plate and rod tissue fractions directly measured from DTA‐based topological classification were correlated with the elastic moduli computed from full voxel model.

Results: The elastic moduli of skeleton models were significantly correlated with those of full voxel models along all three coordinate axes (r2 = 0.38−0.53). The rod‐reconstructed model contained 21.3% of original bone mass and restored 1.5% of elastic moduli, whereas the plate‐reconstructed model contained 90.3% of bone mass and restored 53.2% of elastic moduli. Plate tissue fraction showed a significantly positive correlation (r2 = 0.49) with elastic modulus by a power law, whereas rod tissue fraction showed a significantly negative correlation (r2 = 0.42).

Conclusions: These results quantitatively show that the microarchitecture alone affects elastic moduli of trabecular bone and trabecular plates make a far greater contribution than rods to the bone's elastic behavior.
Component Analysis of HPLC Profiles of Unique Lipoprotein Subclass Cholesterols for Detection of Coronary Artery Disease
Authors: Okazaki Mitsuyo , Usui Shinichi , Fukui Akio , Kubota Isao , Tomoike Hitonobu
Published: September 2006

Background: Patients with coronary artery disease (CAD) are known to have several lipoprotein abnormalities. We examined plasma cholesterol concentrations of major lipoproteins and their subclasses, using a gel permeation HPLC, to establish an association between a lipoprotein subclass pattern and the presence of CAD.

Methods: We performed a simple and fully automated HPLC, followed by mathematical treatment on chromatograms, for measuring cholesterol concentrations of major lipoproteins and their subclasses in 62 male patients (45 with CAD and 17 controls without CAD) who underwent cardiac catheterization.

Results: For major lipoprotein classes, the patient group had a significantly (P <0.05) higher LDL-cholesterol (LDL-C) and lower HDL-cholesterol (HDL-C), but no difference in VLDL-cholesterol (VLDL-C) concentrations. For lipoprotein subclasses, the patient group had a significantly higher small VLDL-C (mean particle diameter of 31.3 nm, P <0.001), small LDL-C (23.0 nm, P <0.05), and very small LDL-C (16.7–20.7 nm, P <0.001), but a significantly lower large HDL-C (12.1 nm, P <0.001) concentrations. Combined variables of “small VLDL-C + small LDL-C + very small LDL-C – large HDL-C” differentiated the patient from the control group more clearly than single-subclass measurements or calculated traditional lipid markers.

Conclusions: These results suggest the usefulness of multiple and simultaneous subclass analysis of proatherogenic and antiatherogenic lipoproteins and indicate that HPLC and its component analysis can be used for easy detection and evaluation of abnormal distribution of lipoprotein subclasses associated with CAD.
Factors Affecting S-Homocysteinylation of LDL Apoprotein B
Authors: Zinellu Angelo , Zinellu Elisabetta , Sotgia Salvatore , Formato Marilena , Cherchi Gian Mario , Deiana Luca , Carru Ciriaco
Published: September 2006

Background: Hyperhomocysteinemia is an important risk factor for vascular disease and atherosclerosis, but the mechanisms by which homocysteine exerts its deleterious effects are not known. Because oxidation and/or homocysteinylation may increase atherogenicity of LDL, we investigated S-homocysteinylation of LDL as a possible contributor to atherosclerosis pathogenesis.

Methods: We used capillary electrophoresis to measure LDL-bound thiols [homocysteine, cysteine (Cys), cysteinylglycine, glutathione, and glutamylcysteine] in 104 healthy study participants We also assessed total plasma thiol concentrations and lipid profiles.

Results: Our data suggest that apoprotein B (apoB)-cysteinylglycine (CysGly), apoB-Hcy, and apoB-Cys concentrations are markedly higher in men than in women. The percentage of CysGly and glutathione on apoB was higher than that of the same thiols in plasma, whereas the other thiols were markedly less prevalent in lipoprotein than in plasma. Pearson correlation showed that among all thiols, only total plasma Hcy is related to apoB-Hcy concentrations. Multiple correlation analysis confirmed that total Hcy was the most important determinant of apoB-Hcy. Age and LDL cholesterol also showed positive associations, but Cys and, mainly, CysGly were negatively associated with apoB-Hcy concentrations.

Conclusions: apoB-Hcy derivative formation is mainly dependent on total homocysteine concentration. Increased cholesterol concentrations are related to increased apoB-Hcy. CysGly seems to compete with Hcy for binding to LDL apoprotein, suggesting that CysGly may protect against atherosclerosis by decreasing the concentrations of Hcy transferred by LDL from plasma to endothelial and subendothelial spaces.
High-Speed Interferometric Detection of Label-Free Immunoassays on the Biological Compact Disc
Authors: Zhao Ming , Nolte David , Cho Wonryeon , Regnier Fred , Varma Manoj , Lawrence Greg , Pasqua John
Published: September 2006

Background: We describe a direct-detection immunoassay that uses high-speed optical interferometry on a biological compact disc (BioCD).

Methods: We fabricated phase-contrast BioCDs from 100-mm diameter 1.1-mm thick borosilicate glass disks coated with a 10-layer dielectric stack of Ta2O5/SiO2 that serves as a mirror with a center wavelength at 635 nm. The final layer is a λ/4 layer of SiO2 onto which protein patterns are immobilized through several different chemical approaches. Protein on the disc is scanned by a focused laser spot as the disc spins. Interaction of the light with the protein provides both a phase-modulated signal and a local reference that are combined interferometrically to convert phase into intensity. A periodic pattern of protein on the spinning disc produces an intensity modulation as a function of time that is proportional to the surface-bound mass. The binding of antigen or antibodies is detected directly, without labels, by a change in the interferometric intensity. The technique is demonstrated with a reverse assay of immobilized rabbit and mouse IgG antigen incubated against anti-IgG antibody in a casein buffer.

Results: The signal increased with increased concentration of analyte. The current embodiment detected a concentration of 100 ng/L when averaged over ∼3000 100-micron-diameter protein spots.

Conclusions: High-speed interferometric detection of label-free protein assays on a rapidly spinning BioCD is a high-sensitivity approach that is amenable to scaling up to many analytes.
Chip Electrophoresis as a Method for Quantifying Total Microalbuminuria
Authors: Chan Owen TM , Herold David A
Published: September 2006

Background: Microalbuminuria is an important prognostic marker in diabetic nephropathy and cardiovascular disease. Initially, most commercial assays used immunoreactivity to quantify microalbuminuria; however, size-exclusion HPLC demonstrated the existence of nonimmunoreactive forms of albumin that may not be detected by immunoassay. Recent liquid chromatography tandem mass spectrometry analyses suggested that size-exclusion HPLC gave higher results attributable to other urine proteins coeluting with albumin. We describe an assay that measures total microalbuminuria (immunoreactive and nonimmunoreactive) without any discernable interference from other common urine proteins.

Methods: We used an automated chip electrophoresis system that utilized microfluidic separation technology and fluorescent sample detection. Each albumin specimen was mixed with the manufacturer’s sample buffer in addition to a chicken albumin internal calibrator and then electrophoresed without additional reducing agents.

Results: With variable concentrations of bovine serum albumin normalized to a chicken albumin internal calibrator, the electrophoresis system was best fit with a polynomial (R2 = 0.9997; concentration range, 5–300 mg/L). The lower limit of detection was 5 mg/L. Interchip and intrachip variation studies conducted on patient urine demonstrated CVs of 3%–13%. The introduction of potentially interfering agents (i.e., molecular analytes, nonalbumin proteins) did not alter precision. Compared with immunoassay, the chip electrophoresis identified higher microalbuminuria concentrations in all urine samples. The method also clearly resolved the albumin peak from interfering proteins.

Conclusions: Unlike immunoassay, chip electrophoresis can detect both immunoreactive and nonimmunoreactive forms of albumin. This system is a simple, robust method to quantify microalbuminuria with good sensitivity, precision, and accuracy.
Association of Adiponectin Gene Variations with Risk of Incident Myocardial Infarction and Ischemic Stroke: A Nested Case-Control Study
Authors: Hegener Hillary H , Lee I-Min , Cook Nancy R , Ridker Paul M , Zee Robert YL
Published: September 2006

Background: Adiponectin (ADIPOQ) gene variations are associated with risk of cardiovascular disease in patients with diabetes. No prospective data are available, however, on the risk of atherothrombotic disorders in persons with ADIPOQ variations who do not have diabetes.

Methods: From a group of DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we assessed the presence of 5 ADIPOQ genetic variants (rs266729, rs182052, rs822396, rs2241766, and rs1501299) in samples from 600 Caucasian men who subsequently suffered an atherothrombotic event (incident myocardial infarction or ischemic stroke) and from 600 age- and smoking-matched Caucasian men who remained free of reported vascular disease during follow-up (controls).

Results: Genotype distributions for the variations tested were in Hardy-Weinberg equilibrium. Marker-by-marker conditional logistic regression analysis, adjusted for potential risk factors, showed an association of rs266729 [recessive: odds ratio (OR), 0.26; 95% confidence interval (CI), 0.10–0.64; P = 0.004] and rs182052 (recessive: OR, 0.40; 95% CI, 0.21–0.76; P = 0.006) with decreased risk of ischemic stroke. These findings remained significant after Bonferroni correction. Haplotype-based (constituted by rs266729, rs182052, and rs822396) conditional logistic regression analysis, adjusted for the same potential risk factors, showed an association of haplotype G-A-G (OR, 0.28; 95% CI, 0.09–0.87; P = 0.03) with decreased risk of ischemic stroke. Prespecified analysis limited to participants without baseline diabetes showed similar significant findings.

Conclusions: The present prospective investigation provides further evidence for a protective role of adiponectin gene variation in the risk of ischemic stroke that was independent of the presence of diabetes.
Measuring Radial Total Stresses on Model Suction Caissons in Clay
Authors: Chen W , Randolph MF
Published: September 2006

Miniature total pressure transducers (TPTs) were incorporated on model suction caissons in order to measure changes in radial stress during installation and loading of the caissons. The accuracy of TPTs in clay was first assessed by calibration tests at both 1g and in a geotechnical centrifuge at up to 120g. Calibration tests in a modified triaxial apparatus showed that the accuracy of the TPTs under loading, unloading, cyclic loading, and sustained loading was better than 92.5%. The initial readings of the TPTs were also found to change only slightly on transfer among air, water, and soil, in spite of differences in the heat exchange environment. The cross-sensitivity of TPTs to an axial load of 300 N on the caisson was insignificant. The accuracy of TPTs in water in the centrifuge was better than 96%. The TPTs also gave reasonable measurements of radial stresses generated on the outer wall of suction caissons during penetration in clay at 120g, as assessed by comparison with stress changes predicted using the strain path method.
Molecular Profiling of CD34+ Cells in Idiopathic Myelofibrosis Identifies a Set of Disease-Associated Genes and Reveals the Clinical Significance of Wilms' Tumor Gene 1 (WT1)
Authors: Guglielmelli Paola , Zini Roberta , Bogani Costanza , Salati Simona , Pancrazzi Alessandro , Bianchi Elisa , Mannelli Francesco , Ferrari Sergio , Le Bousse-Kerdilès Marie-Caroline , Bosi Alberto , Barosi Giovanni , Migliaccio Anna Rita , Manfredini Rossella , Vannucchi Alessandro M.
Published: September 2006

This study was aimed at the characterization of a gene expression signature of the pluripotent hematopoietic CD34+ stem cell in idiopathic myelofibrosis (IM), which would eventually provide novel pathogenetic insights and/or diagnostic/prognostic information. Aberrantly regulated genes were revealed by transcriptome comparative microarray analysis of normal and IM CD34+ cells; selected genes were also assayed in granulocytes. One-hundred seventy four differentially expressed genes were identified and in part validated by quantitative polymerase chain reaction. Altered gene expression was corroborated by the detection of abnormally high CD9 or CD164, and low CXCR4, membrane protein expression in IM CD34+ cells. According to class prediction analysis, a set of eight genes (CD9, GAS2, DLK1, CDH1, WT1, NFE2, HMGA2, and CXCR4) properly recognized IM from normal CD34+ cells. These genes were aberrantly regulated also in IM granulocytes that could be reliably differentiated from control polycythemia vera and essential thrombocythemia granulocytes in 100% and 81% of cases, respectively. Abnormal expression of HMGA2 and CXCR4 in IM granulocytes was dependent on the presence and the mutational status of JAK2V617F mutation. The expression levels of both CD9 and DLK1 were associated with the platelet count, whereas higher WT1 expression levels identified IM patients with more active disease, as revealed by elevated CD34+ cell count and higher severity score. In conclusion, molecular profiling of IM CD34+ cells uncovered a limited number of genes with altered expression that, beyond their putative role in disease pathogenesis, are associated with patients' clinical characteristics and may have potential prognostic application.
Survival of a Genetically Modified Root‐Colonizing Pseudomonad and Rhizobium Strain in an Acidic Soil
Authors: Staley Thomas E. , Brauer David K.
Published: September 2006

Maintaining threshold populations of inoculum microorganisms in the soil environment is important for such practical applications as biocontrol, plant growth‐promotion, bioremediation, and nodulation. However, because of both technical and labor constraints in monitoring bacterial viability in nonsterile soils, few studies have reported on survival kinetics, particularly in relation to subtle alterations in soil acidity‐related factors. A genetically modified strain Pseudomonas putida R20/lacZY or Rhizobium leguminosarum bv. trifolii 162S7a/gusA was introduced into conditioned, nonsterile Gilpin (fine loamy, mixed mesic, Typic Hapludult) silt loam soil, limed at four low levels (pHw 4.71, 4.81, 4.92, and 4.99) or derivative soil solutions with highly correlated (R2 ≥ 0.81) chemical properties. Immediate declines in viability of both strains were found in all soils, reaching 0.1 to 1% initial colony‐forming unit (CFU) g−1 soil in 35 h for P. putida and in 68 h for R. leguminosarum bv. trifolii Death rate constants (kd) for both strains were directly related to lime level (soil pH), although differences were not significant (P > 0.05) for the rhizobium. Use of soil solutions gave similar responses for both strains, but over much shorter incubation times. As with soils, kd values for both strains in soil solutions were directly related to lime level (solution pH). In both soil and soil solution experiments, survival (kd) was negatively correlated (R2 ≥ −0.914) with pH and basic cation (Ca and Mg), and positively correlated (R2 ≥ 0.933) with Al, concentrations. This relationship of viability to soil solution chemistry was broadly confirmed for both bacterial strains by use of fluorescent probes, suggesting increased cell membrane damage at lower pHs. These results demonstrate not only the alternative utility of using soil solutions, rather than nonsterile soils, for bacterial viability assessments, but also the positive effect of low‐level liming (∼0.28 pH unit increase) on survival of beneficial root‐colonizing bacteria in acidic soils.
Tillage and Nitrogen Effects on Soil Organic Matter Fractions in Wheat‐based Systems
Authors: Dou Fugen , Hons Frank M.
Published: September 2006

Management practices that alter plant residue production and distribution influence SOC (soil organic carbon) dynamics. The objectives of this study were to investigate the impacts of tillage, cropping system, and N fertilization on SOC and soil N pools through physical fractionation of a central Texas soil after 20 yr. Nitrogen fertilization and no‐tillage (NT) significantly increased wheat (Triticum aestivumL.) straw yield. Compared with conventional tillage (CT), SOC under NT in surface (0–5‐cm) samples was 38, 69, and 68% greater for continuous wheat (CW), wheat–soybean [Glycine max(L.) Merr.]–sorghum [Sorghum bicolor(L.) Moench] rotation (SWS), and double‐cropped wheat–soybean (WS), respectively. The greatest SOC was observed in WS under NT with N fertilization, and the lowest occurred in CW under CT without N. Increased cropping intensity increased SOC compared with monoculture. Nitrogen fertilization only significantly increased SOC sequestration under NT. No‐tillage increased SOC concentration in all physical size fractions compared with CT. Increased cropping intensity and N fertilization significantly increased SOC sequestration in most size fractions only under NT. Intraparticulate organic matter C (IPOM‐C) was proportionally more affected by tillage than total SOC, indicating that this fraction was more sensitive to management. Carbon concentrations in all size fractions were significantly correlated with each other as well as SOC. Our results indicated that NT associated with enhanced cropping intensity and N fertilization sequestered greater SOC and soil total N.
Influence of Sodicity, Clay Mineralogy, Prewetting Rate, and Their Interaction on Aggregate Stability
Authors: Ruiz-Vera Victor M. , Wu Laosheng
Published: September 2006

Sodicity adversely affects soil physical conditions reflected by weak structural stability. Soil clay mineralogy influences the degree of aggregate disruption induced by sodicity. The main objective of this research was to evaluate the interactive effects of clay mineralogy, prewetting rate (PWR), and sodicity on soil aggregation. Three soils with predominantly clay minerals of smectite, vermiculite‐smectite, and kaolinite were equilibrated with NaCl–CaCl2 solutions having sodium adsorption ratio (SAR) values of 0, 20, and 50 and electrical conductivity (EC) = 3.0 dS m−1 After air‐drying, the treated samples were packed and prewetted at rates of 2 and 30 mm h−1 with the NaCl–CaCl2 solutions. Aggregate‐size distribution, macroscopic swelling, and surface soil dispersion were determined after the packed samples were equilibrated to a matric potential of −0.1 MPa. The kaolinitic soil showed the lowest inherent aggregate stability when subjected to slow PWR and the lowest SAR. Furthermore, aggregate stability of the kaolinitic loam soil was not significantly affected by increasing SAR. The Millox (smectitic) soil, on the other hand, was most susceptible to aggregate slaking, whereas the Malibu (vermiculitic) soil was most susceptible to differential swelling (at slow PWR). When SAR was low, aggregate slaking by fast PWR was the main cause of the aggregate breakdown. At SAR ≥ 20, swelling and dispersion became more important to the structural stability.