Delayed astrocytic contact with cerebral blood vessels in FGF‐2 deficient mice does not compromise permeability properties at the developing blood‐brain barrier

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood‐brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor‐2 (FGF‐2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF‐2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild‐type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight‐junction proteins and we observed no difference in the ultrastructure of the tight‐junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight‐junctions or for basic permeability properties and function of the blood‐brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201–1212, 2016     

Triage of women with equivocal or low-grade cervical cytology results: a meta-analysis of the HPV test positivity rate

Consistent evidence underlines the utility of human papillomavirus (HPV) DNA testing in the management of women with equivocal cervical cytological abnormalities, but not in case of low-grade lesions. We performed a meta-analysis including studies where the high-risk probe of the Hybrid Capture-II is used to triage these two cytological categories. The triage test-positivity rate reflects the colposcopy referral workload.Data were pooled on the HPV test positivity rate in women with atypical squamous cells of undetermined significance (ASCUS/ASC-US) or low-grade squamous intraepithelial lesions (LSIL), derived from different cytological classification systems. The meta-analysis was restricted to studies, published between 1991 and 2007. A random-effect model was applied for meta-analytical pooling and the influence of covariates on the HPV positivity rate was analyzed by meta-regression. The variation by age was assessed within individual studies since age strata were not defined uniformly. On an average, 43% (95% CI: 40–46%) of women with ASCUS/ASC-US were high-risk HPV positive (range 23–74%). In women with LSIL, the pooled positivity rate was 76% (95% CI: 71–81%; range 55–89%). In spite of considerable inter-study heterogeneity, the difference in HPV positivity between the two triage groups was large and highly significant: 32% (95% CI: 27–38%). HPV rates dropped tremendously as age and cutoffs of test positivity increased. Other factors (cytological classification system, country, continent, collection method and year of publication) had no statistically significant impact, except in LSIL triage where HPV positivity was significantly lower in European compared to American studies. Women with LSIL, especially younger women, have high HPV positivity rates suggesting limited utility of reflex HPV triaging these cases. Research is needed to identify more specific methods to triage women with low-grade squamous cervical lesions.     

Type-Specific Human Papillomavirus Biological Features: Validated Model-Based Estimates

Infection with high-risk (hr) human papillomavirus (HPV) is considered the necessary cause of cervical cancer. Vaccination against HPV16 and 18 types, which are responsible of about 75% of cervical cancer worldwide, is expected to have a major global impact on cervical cancer occurrence. Valid estimates of the parameters that regulate the natural history of hrHPV infections are crucial to draw reliable projections of the impact of vaccination. We devised a mathematical model to estimate the probability of infection transmission, the rate of clearance, and the patterns of immune response following the clearance of infection of 13 hrHPV types. To test the validity of our estimates, we fitted the same transmission model to two large independent datasets from Italy and Sweden and assessed finding consistency. The two populations, both unvaccinated, differed substantially by sexual behaviour, age distribution, and study setting (screening for cervical cancer or Chlamydia trachomatis infection). Estimated transmission probability of hrHPV types (80% for HPV16, 73%-82% for HPV18, and above 50% for most other types); clearance rates decreasing as a function of time since infection; and partial protection against re-infection with the same hrHPV type (approximately 20% for HPV16 and 50% for the other types) were similar in the two countries. The model could accurately predict the HPV16 prevalence observed in Italy among women who were not infected three years before. In conclusion, our models inform on biological parameters that cannot at the moment be measured directly from any empirical data but are essential to forecast the impact of HPV vaccination programmes.     

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

Background  

Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells.     

The wobble hypothesis revisited: uridine-5-oxyacetic acid is critical for reading of G-ending codons

According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo⁵U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo⁵U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo⁵U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo⁵U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo⁵U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo⁵U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.     

Towards a computational method for imaging the extracellular potassium concentration during regional ischemia

We investigate the possibility of using body surface potential maps to image the extracellular potassium concentration during regional ischemia. The problem is formulated as an inverse problem based on a linear approximation of the bidomain model, where we minimize the difference between the results of the model and observations of body surface potentials. The minimization problem is solved by a one-shot technique, where the original PDE system, an adjoint problem, and the relation describing the minimum, are solved simultaneously. This formulation of the problem requires the solution of a 5 × 5 system of linear partial differential equations. The performance of the model is investigated by performing tests based on synthetic data. We find that the model will in many cases detect the correct position and approximate size of the ischemic regions, while some cases are more difficult to locate. It is observed that a simple post-processing of the results produces images that are qualitatively very similar to the true solution.     

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.     

Mutations in the Fatty Acid Transport Protein 4 Gene Cause the Ichthyosis Prematurity Syndrome

Ichthyosis prematurity syndrome (IPS) is an autosomal-recessive disorder characterized by premature birth and neonatal asphyxia, followed by a lifelong nonscaly ichthyosis with atopic manifestations. Here we show that the gene encoding the fatty acid transport protein 4 (FATP4) is mutated in individuals with IPS. Fibroblasts derived from a patient with IPS show reduced activity of very long-chain fatty acids (VLCFA)-CoA synthetase and a specific reduction in the incorporation of VLCFA into cellular lipids. The human phenotype is consistent with Fatp4 deficiency in mice that is characterized by a severe skin phenotype, a defective permeability barrier function, and perturbed VLCFA metabolism. Our results further emphasize the importance of fatty acid metabolism for normal epidermal barrier function illustrated by deficiency of a member in the FATP family of proteins.