Cyclic strain and motion control produce opposite oxidative responses in two human endothelial cell types

The phenotype of endothelial cells (ECs) is specific to the vascular bed from which they originate. To examine how mechanical forces alter the phenotype of different ECs, we compared the effects of cyclic strain and motion control on reactive oxygen species (ROS) production and metabolism and cell adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) vs. human aortic endothelial cells (HAEC). HUVEC and HAEC were subjected to cyclic strain (10% or 20%, 1 Hz), to a motion control that simulated fluid agitation over the cells without strain, or to static conditions for 24 h. We measured H₂O₂ production with dichlorodihydrofluorescein acetate and superoxide with dihydroethidium fluorescence changes; superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activities spectrophotometrically; and vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 protein expression with Western blot analyses. HUVEC under cyclic strain showed 1) higher intracellular H₂O₂ levels, 2) increased SOD, catalase, and GPx activities, and 3) greater VCAM-1 and ICAM-1 protein expression, compared with motion control or static conditions. However, in HAEC, motion control induced higher levels of ROS, enzyme activities associated with ROS defense, and VCAM-1 and ICAM-1 expression than cyclic strain. The opposite responses obtained with these two human EC types may reflect their vessels of origin, in that HAEC are subjected to higher cyclic strain deformations in vivo than HUVEC. phenotype; reactive oxygen species; inflammation; shear stress     

Detection of Clostridium difficile and Salmonella in feral swine population in North Carolina

We sampled 161 feral pigs in eastern North Carolina, USA, to determine the prevalence and antimicrobial resistance profile of Clostridium difficile and Salmonella. Seven (4.4%) and eight (5.0%) pigs tested positive for C. difficile and Salmonella, respectively, highlighting the importance of determining the epidemiology of these pathogens in feral pigs.     

Inhibition of the M. tuberculosis 3β-hydroxysteroid dehydrogenase by azasteroids

The cholesterol metabolism pathway in Mycobacterium tuberculosis (M. tb) is a potential source of energy as well as secondary metabolite production that is important for survival of M. tb in the host macrophage. Oxidation and isomerization of 3β-hydroxysterols to 4-en-3-ones is requisite for sterol metabolism and the reaction is catalyzed by 3β-hydroxysteroid dehydrogenase (Rv1106c). Three series of 6-azasteroids and 4-azasteroids were employed to define the substrate preferences of M. tb 3β-hydroxysteroid dehydrogenase. 6-Azasteroids with large, hydrophobic side chains at the C17 position are the most effective inhibitors. Substitutions at C1, C2, C4 and N6 were poorly tolerated. Our structure-activity studies indicate that the 6-aza version of cholesterol is the best and tightest binding competitive inhibitor (K_i = 100 nM) of the steroid substrate and are consistent with cholesterol being the preferred substrate of M. tb 3β-hydroxysteroid dehydrogenase.     

Inhibition of Homologous Recombination by the Plasmid MucA′B Complex

By its functional interaction with a RecA polymer, the mutagenic UmuD′C complex possesses an antirecombination activity. We show here that MucA′B, a functional homolog of the UmuD′C complex, inhibits homologous recombination as well. In F⁻ recipients expressing MucA′B from a P_tac promoter, Hfr × F⁻ recombination decreased with increasing MucA′B concentrations down to 50-fold. In damage-induced pKM101-containing cells expressing MucA′B from the native promoter, recombination between a UV-damaged F lac plasmid and homologous chromosomal DNA decreased 10-fold. Overexpression of MucA′B together with UmuD′C resulted in a synergistic inhibition of recombination. RecA[UmuR] proteins, which are resistant to UmuD′C inhibition of recombination, are inhibited by MucA′B while promoting MucA′B-promoted mutagenesis efficiently. The data suggest that MucA′B and UmuD′C contact a RecA polymer at distinct sites. The MucA′B complex was more active than UmuD′C in promoting UV mutagenesis, yet it did not inhibit recombination more than UmuD′C does. The enhanced mutagenic potential of MucA′B may result from its inherent superior capacity to assist DNA polymerase in trans-lesion synthesis. In the course of this work, we found that the natural plasmid pKM101 expresses around 45,000 MucA and 13,000 MucB molecules per lexA(Def) cell devoid of LexA. These molecular Muc concentrations are far above those of the chromosomally encoded Umu counterparts. Plasmid pKM101 belongs to a family of broad-host-range conjugative plasmids. The elevated levels of the Muc proteins might be required for successful installation of pKM101-like plasmids into a variety of host cells.     

Effects of a reimbursement incentive on enrollment in a weight control program

Objective: The objective was to examine the effect of offering a reimbursement incentive on the percentage of inquirers who enrolled in a weight control program and on weight loss and program attendance among enrollees. Research Methods and Procedures: We used a sequential control‐intervention design to observe how inquirers of the University of Alabama at Birmingham EatRight Lifestyle Program responded to an enrollment incentive for potential 50% ($150) reimbursement of the total program fee if they attended 10 of 12 classes and lost at least 6% of their current body weight. Inquirers had to be adults with a BMI ≥30 kg/m², seeking information about a weight control program, and informed of the program cost. Outcomes included proportion of inquirers enrolled, overall number of classes attended, and weight loss. Results: Of the 401 people who inquired during the study periods, 24.5% and 25.0% enrolled in the intervention and control periods, respectively. There was a trend toward higher attendance in the intervention group, compared with the control group; there were no differences in percentage of weight loss. The odds of attending ≥10 classes were 2.4 times as high, and both losing >6% body weight and attending ≥10 classes were three times as high in the intervention subjects compared with controls, although non‐significant. Discussion: The potential of earning a performance‐based reimbursement incentive did not affect enrollment in the EatRight Lifestyle Program. Performance‐based incentives may be an ideal mechanism for extending coverage of weight‐loss interventions by insurers because of limited financial risk and improved adherence.     

Settling into an increasingly hostile world: the rapidly closing "recruitment window" for corals

Free space is necessary for larval recruitment in all marine benthic communities. Settling corals, with limited energy to invest in competitive interactions, are particularly vulnerable during settlement into well-developed coral reef communities. This situation may be exacerbated for corals settling into coral-depauperate reefs where succession in nursery microhabitats moves rapidly toward heterotrophic organisms inhospitable to settling corals. To study effects of benthic organisms (at millimeter to centimeter scales) on newly settled corals and their survivorship we deployed terra-cotta coral settlement plates at 10 m depth on the Mesoamerican Barrier Reef in Belize and monitored them for 38 mo. During the second and third years, annual recruitment rates declined by over 50% from the previous year. Invertebrate crusts (primarily sponges) were absent at the start of the experiment but increased in abundance annually from 39, 60, to 73% of the plate undersides by year three. Subsequently, substrates hospitable to coral recruitment, including crustose coralline algae, biofilmed terra-cotta and polychaete tubes, declined. With succession, substrates upon which spat settled shifted toward organisms inimical to survivorship. Over 50% of spat mortality was due to overgrowth by sponges alone. This result suggests that when a disturbance creates primary substrate a "recruitment window" for settling corals exists from approximately 9 to 14 mo following the disturbance. During the window, early-succession, facilitating species are most abundant. The window closes as organisms hostile to coral settlement and survivorship overgrow nursery microhabitats.     

Nucleosomes in the neighborhood: New roles for chromatin modifications in replication origin control

The importance of local chromatin structure in regulating replication initiation has become increasingly apparent. Most recently, histone methylation and nucleosome positioning have been added to the list of modifications demonstrated to regulate origins. In particular, the methylation states of H3K4, H3K36 and H4K20 have been associated with establishing active, repressed or poised origins depending on the timing and extent of methylation. The stability and precise positioning of nucleosomes has also been demonstrated to affect replication efficiency. Although it is not yet clear how these modifications alter the behavior of specific replication factors, ample evidence establishes their role in maintaining coordinated replication. This review will summarize recent advances in understanding these aspects of chromatin structure in DNA replication origin control.Key words: chromatin, histone methylation, nucleosome positioning, nucleosome stability, origin, post-translational modification, replication     

Detection of Rapalog-Mediated Therapeutic Response in Renal Cancer Xenografts Using 64Cu-bevacizumab ImmunoPET

The importance of neovascularization for primary and metastatic tumor growth fostered numerous clinical trials of angiogenesis inhibitors either alone or in combination with conventional antineoplastic therapies. One challenge with the use of molecularly targeted agents has been the disconnection between size reduction and tumor biologic behavior, either when the drug is efficacious or when tumor resistance emerges. Here, we report the synthesis and characterization of ⁶⁴Cu-NOTA-bevacizumab as a PET imaging agent for imaging intratumoral VEGF content in vivo. ⁶⁴Cu-NOTA-bevacizumab avidly accumulated in 786-O renal carcinoma xenografts with lower levels in host organs. RAD001 (everolimus) markedly attenuated ⁶⁴Cu-NOTA-bevacizumab accumulation within 786-O renal carcinoma xenografts. Tumor tissue and cellular molecular analysis validated PET imaging, demonstrating decreases in total and secreted VEGF content and VEGFR2 activation. Notably, ⁶⁴Cu-NOTA-bevacizumab PET imaging was concordant with the growth arrest of RAD001 tumors. These data suggest that immunoPET targeting of angiogenic factors such as VEGF could be a new class of surrogate markers complementing the RECIST criteria in patients receiving molecularly targeted therapies.     

Estimating Genetic Effects and Quantifying Missing Heritability Explained by Identified Rare-Variant Associations

Next-generation sequencing has led to many complex-trait rare-variant (RV) association studies. Although single-variant association analysis can be performed, it is grossly underpowered. Therefore, researchers have developed many RV association tests that aggregate multiple variant sites across a genetic region (e.g., gene), and test for the association between the trait and the aggregated genotype. After these aggregate tests detect an association, it is only possible to estimate the average genetic effect for a group of RVs. As a result of the "winner’s curse," such an estimate can be biased. Although for common variants one can obtain unbiased estimates of genetic parameters by analyzing a replication sample, for RVs it is desirable to obtain unbiased genetic estimates for the study where the association is identified. This is because there can be substantial heterogeneity of RV sites and frequencies even among closely related populations. In order to obtain an unbiased estimate for aggregated RV analysis, we developed bootstrap-sample-split algorithms to reduce the bias of the winner’s curse. The unbiased estimates are greatly important for understanding the population-specific contribution of RVs to the heritability of complex traits. We also demonstrate both theoretically and via simulations that for aggregate RV analysis the genetic variance for a gene or region will always be underestimated, sometimes substantially, because of the presence of noncausal variants or because of the presence of causal variants with effects of different magnitudes or directions. Therefore, even if RVs play a major role in the complex-trait etiologies, a portion of the heritability will remain missing, and the contribution of RVs to the complex-trait etiologies will be underestimated.     

Circulatory Responses to Asphyxia Differ if the Asphyxia Occurs In Utero or Ex Utero in Near-Term Lambs

Background

A cornerstone of neonatal resuscitation teaching suggests that a rapid vagal-mediated bradycardia is one of the first signs of perinatal compromise. As this understanding is based primarily on fetal studies, we investigated whether the heart rate and blood pressure response to total asphyxia is influenced by whether the animal is in utero or ex utero.

Methods

Fetal sheep were instrumented at ∼139 days of gestation and then asphyxiated by umbilical cord occlusion until mean arterial blood pressure decreased to ∼20 mmHg. Lambs were either completely submerged in amniotic fluid (in utero; n = 8) throughout the asphyxia or were delivered and then remained ex utero (ex utero; n = 8) throughout the asphyxia. Heart rate and arterial blood pressure were continuously recorded.

Results

Heart rate was higher in ex utero lambs than in utero lambs. Heart rates in in utero lambs rapidly decreased, while heart rates in ex utero lambs initially increased following cord occlusion (for ∼1.5 min) before they started to decrease. Mean arterial pressure initially increased then decreased in both groups.

Conclusions

Heart rate response to asphyxia was markedly different depending upon whether the lamb was in utero or ex utero. This indicates that the cardiovascular responses to perinatal asphyxia are significantly influenced by the newborn''s local environment. As such, based solely on heart rate, the stage and severity of a perinatal asphyxic event may not be as accurate as previously assumed.