Inna Chervoneva

Type V Collagen Controls the Initiation of Collagen Fibril Assembly

Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113–124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-α1(V) chains were created by homologous recombination. The col5a1–/– animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/– animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.

Decorin regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development

Tendon function involves the development of an organized hierarchy of collagen fibrils. Small leucine‐rich proteoglycans have been implicated in the regulation of fibrillogenesis and decorin is the prototypic member of this family. Decorin‐deficient mice demonstrate altered fibril structure and mechanical function in mature skin and tail tendons. However, the developmental role(s) of decorin needs to be elucidated. To define these role(s) during tendon development, tendons (flexor digitorum longus) were analyzed ultrastructurally from postnatal day 10 to 90. Decorin‐deficient tendons developed abnormal, irregularly contoured fibrils. Finite mixture modeling estimated that the mature tendon was a three‐subpopulation mixture of fibrils with characteristic diameter ranges. During development, in each subpopulation the mean diameter was consistently larger in mutant mice. Also, diameter distributions and the percentage of fibrils in each subpopulation were altered. Biomechanical analyses demonstrated that mature decorin‐deficient tendons had significantly reduced strength and stiffness; however, there was no reduction in immature tendons. Expression of decorin and biglycan, a closely related family member, was analyzed during development. Decorin increased with development while biglycan decreased. Spatially, both had a comparable localization throughout the tendon. Biglycan expression increased substantially in decorin‐deficient tendons suggesting a potential functional compensation. The accumulation of structural defects during fibril growth, a period associated with decorin expression and low biglycan expression, may be the cause of compromised mechanical function in the absence of decorin. Our findings indicate that decorin is a key regulatory molecule and that the temporal switch from biglycan to decorin is an important event in the coordinate regulation of fibrillogenesis and tendon development. J. Cell. Biochem.

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 μl of diluted nipple aspirate fluid over a 5–40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75–84% of samples from women with cancer but in only 0–9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer.

Effects of antiepileptic drugs on lipids, homocysteine, and C-reactive protein†

The widely prescribed anticonvulsants phenytoin and carbamazepine are potent inducers of cytochrome P450 enzymes, which are involved in cholesterol synthesis. We sought to determine whether these drugs have an effect on cholesterol and other serological markers of vascular risk.

Murine Model of the Ehlers-Danlos Syndrome col5a1 HAPLOINSUFFICIENCY DISRUPTS COLLAGEN FIBRIL ASSEMBLY AT MULTIPLE STAGES

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proα1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.

Risk of Major Bleeding with Concomitant Dual Antiplatelet Therapy After Percutaneous Coronary Intervention in Patients Receiving Long-Term Warfarin Therapy

Study Objectives. To characterize the safety of concomitant aspirin, clopidogrel, and warfarin therapy after percutaneous coronary intervention (PCI), and to identify patient characteristics that increase the risk of hemorrhage.

Major and Minor Depression After Traumatic Brain Injury

OBJECTIVE To examine minor as well as major depression at 1 year posttraumatic brain injury (TBI), with particular attention to the contribution of depression severity to levels of societal participation. DESIGN Observational prospective study with a 2-wave longitudinal component. SETTING Inpatient rehabilitation centers, with 1-year follow up conducted primarily by telephone. PARTICIPANTS Persons with TBI (N=1570) enrolled in the TBI Model System database and followed up at 1-year postinjury. INTERVENTIONS Not applicable. MAIN OUTCOME MEASURES FIM, Patient Health Questionnaire-9, Participation Assessment with Recombined Tools-Objective, Glasgow Outcome Scale-Extended, and the Satisfaction With Life Scale. RESULTS Twenty-two percent of the sample reported minor depression, and 26% reported major depression at 1-year post-TBI. Both levels of depression were associated with sex (women), age (younger), preinjury mental health treatment and substance abuse, and cause of injury (intentional). There was a monotonic dose-response relationship between severity of depression and all 1-year outcomes studied, including level of cognitive and physical disability, global outcome, and satisfaction with life. With other predictors controlled, depression severity remained significantly associated with the level of societal participation at 1-year post-TBI. CONCLUSIONS Minor depression may be as common as major depression after TBI and should be taken seriously for its association to negative outcomes related to participation and quality of life. Findings suggest that, as in other populations, minor and major depression are not separate entities, but exist on a continuum. Further research should determine whether people with TBI traverse between the 2 diagnoses as in other patient groups.

Collagen fibril assembly during postnatal development and dysfunctional regulation in the lumican‐deficient murine cornea

The transparent cornea is the outer barrier of the eye and is its major refractive surface. Development of a functional cornea requires a postnatal maturation phase involving development, growth and organization of the stromal extracellular matrix. Lumican, a leucine‐rich proteoglycan, is implicated in regulating assembly of collagen fibrils and the highly organized extracellular matrix essential for corneal transparency. We investigated the regulatory role(s) of lumican in fibril assembly during postnatal corneal development using wild type (Lum+/+) and lumican‐null (Lum−/−) mice. In Lum+/+ mice, a regular architecture of small‐diameter fibrils is achieved in the anterior stroma by postnatal day 10 (P10), while the posterior stroma takes longer to reach this developmental maturity. Thus, the anterior and the posterior stroma follow distinct developmental timelines and may be under different regulatory mechanisms. In Lum−/− mice, it is the posterior stroma where abnormal lateral associations of fibrils and thicker fibrils with irregular contours are evident as early as P10. In contrast, the anterior stroma is minimally perturbed by the absence of lumican. In Lum+/+ mice, lumican is expressed throughout the developing stroma at P10, with strong expression limited to the posterior stroma in the adult. Therefore, the posterior stroma, which is most vulnerable to lumican‐deficiency, demonstrates an early developmental defect in fibril structure and architecture in the Lum−/− mouse. These defects underlie the reported increased light scattering and opacity detectable in the adult. Our findings emphasize the early regulation of collagen structure by lumican during postnatal development of the cornea. Developmental Dynamics 235:2493–2506, 2006.

Selection of optimal reference genes for normalization in quantitative RT-PCR

BackgroundNormalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.ResultsWe propose a robust approach for selecting optimal subset(s) of reference genes with the smallest variance of the corresponding normalizing factors. The normalizing factor variance estimates are based on the estimated unstructured covariance matrix of all available candidate reference genes, adjusting for all possible correlations. Robustness is achieved through bootstrapping all candidate reference gene data and obtaining the bootstrap upper confidence limits for the variances of the log-transformed normalizing factors. The selection of the reference gene subset is optimized with respect to one of the following criteria: (A) to minimize the variability of the normalizing factor; (B) to minimize the number of reference genes with acceptable upper limit on variability of the normalizing factor, (C) to minimize the average rank of the variance of the normalizing factor. The proposed approach evaluates all gene subsets of various sizes rather than ranking individual reference genes by their stability, as in the previous work. In two publicly available data sets and one new data set, our approach identified subset(s) of reference genes with smaller empirical variance of the normalizing factor than in subsets identified using previously published methods. A small simulation study indicated an advantage of the proposed approach in terms of sensitivity to identify the true optimal reference subset in the presence of even modest, especially negative correlation among the candidate reference genes.ConclusionsThe proposed approach performs comprehensive and robust evaluation of the variability of normalizing factors based on all possible subsets of candidate reference genes. The results of this evaluation provide flexibility to choose from important criteria for selecting the optimal subset(s) of reference genes, unless one subset meets all the criteria. This approach identifies gene subset(s) with smaller variability of normalizing factors than current standard approaches, particularly if there is some nontrivial innate correlation among the candidate genes.

Clinical need for continuous glucose monitoring in the hospital.

Automation and standardization of the glucose measurement process have the potential to greatly improve glycemic control, clinical outcome, and safety while reducing cost. The resources required to monitor glycemia in hospitalized patients have thus far limited the implementation of intensive glucose management to patients in critical care units. Numerous available and up-and-coming technologies are targeted for the hospital patient population. Advantages and limitations of these devices are discussed herewith in.