The Effect of Factor VIII Deficiencies and Replacement and Bypass Therapies on Thrombus Formation under Venous Flow Conditions in Microfluidic and Computational Models

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.     

Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAR^Ex8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAR^Ex8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAR^Ex8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAR^Ex8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAR^Ex8, to allow the initial infection the intact epithelium. In addition, CAR^Ex8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.     

Misexpression of Gbx2 throughout the mesencephalon by a conditional gain‐of‐function transgene leads to deletion of the midbrain and cerebellum in mice

The mouse homeobox gene, Gbx2, is expressed in discreet domains in the neural tube and plays a key role in forebrain and hindbrain development. Previous studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which are respectively expressed in the anterior and posterior parts of the neural plate, positions the prospective midbrain–hindbrain junction. We describe here a conditional Gbx2 gain‐of‐function transgenic mouse line, Gbx2‐GOF, which expresses Gbx2 and red fluorescence protein, mCherry, upon Cre‐mediated recombination. In the absence of Cre, β‐galactosidase is broadly expressed in mouse embryos and adult brains carrying the transgene. By combining Gbx2‐GOF and En1^Cre knock‐in allele, we activated expression of Gbx2 and mCherry throughout the mesencephalon (mes) and rhombomere 1 (r1). The ectopic expression of Gbx2 causes an anterior shift of the mes/r1 junction at embryonic day 10.5. Interestingly, we found that persistent expression of Gbx2 throughout the mes/r1 region largely abolishes expression of the isthmic organizer gene Fgf8, leading to deletion of the midbrain and cerebellum at later stages. Our data suggest that the juxtaposition of the expression domains of Gbx2 and Otx2 within the mes/r1 area is essential for the maintenance of Fgf8 expression. Furthermore, the Gbx2‐GOF transgenic line is suitable for functional study of Gbx2 during development. genesis 47:667–673, 2009. © 2009 Wiley‐Liss, Inc.     

Familial longevity is marked by lower diurnal salivary cortisol levels: the Leiden Longevity Study

Background

Reported findings are inconsistent whether hypothalamic-pituitary-adrenal (HPA) signaling becomes hyperactive with increasing age, resulting in increasing levels of cortisol. Our previous research strongly suggests that offspring from long-lived families are biologically younger. In this study we assessed whether these offspring have a lower HPA axis activity, as measured by lower levels of cortisol and higher cortisol feedback sensitivity.

Methods

Salivary cortisol levels were measured at four time points within the first hour upon awakening and at two time points in the evening in a cohort comprising 149 offspring and 154 partners from the Leiden Longevity Study. A dexamethasone suppression test was performed as a measure of cortisol feedback sensitivity. Age, gender and body mass index, smoking and disease history (type 2 diabetes and hypertension) were considered as possible confounding factors.

Results

Salivary cortisol secretion was lower in offspring compared to partners in the morning (Area Under the Curve = 15.6 versus 17.1 nmol/L, respectively; p = 0.048) and in the evening (Area Under the Curve = 3.32 versus 3.82 nmol/L, respectively; p = 0.024). Salivary cortisol levels were not different after dexamethasone (0.5 mg) suppression between offspring and partners (4.82 versus 5.26 nmol/L, respectively; p = 0.28).

Conclusion

Offspring of nonagenarian siblings are marked by a lower HPA axis activity (reflected by lower diurnal salivary cortisol levels), but not by a difference in cortisol feedback sensitivity. Further in-depth studies aimed at characterizing the HPA axis in offspring and partners are needed.     

Sidestream Smoke Exposure Increases the Susceptibility of Airway Epithelia to Adenoviral Infection

Background

Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR.

Methodology and Findings

Cultured human airway epithelial cells (CaLu-3) were used as a model to investigate the effect of sidestream cigarette smoke (SSS), mainstream cigarette smoke (MSS), or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3β (GSK3β) is downregulated by SSS exposure and treatment with a specific GSK3β inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection.

Conclusions

This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3β inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend the use of clinically relevant therapeutics and increase the understanding of potential side effects.     

MACBenAbim: A Multi-platform Mobile Application for searching keyterms in Computational Biology and Bioinformatics

Computational biology and bioinformatics are gradually gaining grounds in Africa and other developing nations of the world. However, in these countries, some of the challenges of computational biology and bioinformatics education are inadequate infrastructures, and lack of readily-available complementary and motivational tools to support learning as well as research. This has lowered the morale of many promising undergraduates, postgraduates and researchers from aspiring to undertake future study in these fields. In this paper, we developed and described MACBenAbim (Multi-platform Mobile Application for Computational Biology and Bioinformatics), a flexible user-friendly tool to search for, define and describe the meanings of keyterms in computational biology and bioinformatics, thus expanding the frontiers of knowledge of the users. This tool also has the capability of achieving visualization of results on a mobile multi-platform context.

Availability

MACBenAbim is available from the authors for non-commercial purposes.     

DNA protection by histone-like protein HU from the hyperthermophilic eubacterium Thermotoga maritima

In mesophilic prokaryotes, the DNA-binding protein HU participates in nucleoid organization as well as in regulation of DNA-dependent processes. Little is known about nucleoid organization in thermophilic eubacteria. We show here that HU from the hyperthermophilic eubacterium Thermotoga maritima HU bends DNA and constrains negative DNA supercoils in the presence of topoisomerase I. However, while binding to a single site occludes approximately 35 bp, association of T. maritima HU with DNA of sufficient length to accommodate multiple protomers results in an apparent shorter occluded site size. Such complexes consist of ordered arrays of protomers, as revealed by the periodicity of DNase I cleavage. Association of TmHU with plasmid DNA yields a complex that is remarkably resistant to DNase I-mediated degradation. TmHU is the only member of this protein family capable of occluding a 35 bp nonspecific site in duplex DNA; we propose that this property allows TmHU to form exceedingly stable associations in which DNA flanking the kinks is sandwiched between adjacent proteins. We suggest that T. maritima HU serves an architectural function when associating with a single 35 bp site, but generates a very stable and compact aggregate at higher protein concentrations that organizes and protects the genomic DNA.     

Pollution characteristics and health risk assessment of potentially toxic elements in school playground soils: A case study of Lagos,Nigeria

Abstract

Soil samples were collected in February 2014 from 25 school playgrounds in Lagos, Nigeria to assess the potential adverse effects of the exposure of children to potentially toxic element (PTE). In each of the playgrounds, about 500?g of bulked soil samples were collected, dried, sieved, acid digested, and analyzed by inductively coupled plasma mass spectrometry (ICP‐MS). Results showed that soils studied were characteristically unpolluted as the average PTE concentration at each site did not exceed the soil guideline values. Considering the pollution assessment tools employed, some soil samples showed some form of anthropogenic input from PTE. Health risk assessment was employed to assess PTE exposure from ingestion, inhalation, and dermal contact. Result indicated that the highest risk is associated with ingestion followed by dermal contact and inhalation. For non‐carcinogenic effects, exposure to school playground soils poses no threat to children as the overall value of hazard index is less than the safe level of 1. For carcinogenic effects, only Cr and Ni were considered and were below the threshold of 1?×?10^–6. This study has demonstrated that minimal risk arises from the investigated playgrounds and that regular monitoring is required to keep the PTE contents low in soils to avoid risk to human health.

Abbreviations: SH, school playground; PTE, potentially toxic elements