Citations

Abstract BACKGROUND Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

The objective of this study was to assess the potential for noninvasively identifying viscoelastic material properties in arterial vessels using ultrasound radiation force and modern computational inverse problem techniques. A rubber tube embedded in gelatin was excited at different points along the axis using impulsive ultrasound radiation force. The resulting mechanical waves were measured using a laser vibrometer and pulse-echo ultrasound. The inverse problem was cast as an optimization problem, in which the discrepancy between the measured dynamic response and the computed finite element model response was minimized with respect to viscoelastic material parameters. The model was very sensitive to the tube’s viscoelastic parameters and relatively insensitive to those of the gelatin. The viscoelastic properties of the tube obtained from the inverse problem were compared to the results obtained from a dynamic mechanical analyzer (DMA). For the overlapping range of frequencies (130–190 Hz), the results of the inverse problem gave an equivalent modulus that range from 7.3–8.5 MPa, while the DMA values range from 6.0–9.2 MPa. These results have important implications for using ultrasound radiation force in noninvasive characterization of the viscoelastic properties of arteries in vivo. [This work was supported in part by Grant No. EB002640 from NIH.]

The availability of population-based epidemiological data on the incident risk of stroke is very scarce in Argentina and other Latin American countries. In response to the priorities established by the World Health Organization and the United Nations, PREVISTA was envisaged as a population-based program to determine the risk of first-ever and recurrent stroke and transient ischemic attack incidence and mortality in Tandil, Buenos Aires, Argentina. The study will be conducted according to Standardized Tools for Stroke Surveillance (STEPS Stroke) methodology and will enroll all new (incident) and recurrent consecutive cases of stroke and transient ischemic attack in the City of Tandil between May 1st, 2013 and April 30, 2015. The study will include patients with ischemic stroke, non-traumatic primary intracerebral hemorrhage, subarachnoid hemorrhage, and transient ischemic attack. To ensure the inclusion of every cerebrovascular event during an observation period of two years, we will instrument an ‘intensive screening program’, consisting of a comprehensive daily tracking of every potential event of stroke or transient ischemic attack using multiple overlapping sources. Mortality would be determined during follow-up for every enrolled patient. Also, fatal community events would be screened daily through revision of death certificates at funeral homes and local offices of vital statistics. All causes of death will be adjudicated by an ad-hoc committee. The close population of Tandil is representative of a large proportion of Latin-American countries with low- and middle-income economies. The findings and conclusions of PREVISTA may provide data that could support future health policy decision-making in the region.

ABSTRACT Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a+ structures, some of which colocalized with CD63+ crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation.

Key PointsPhenotypic and genotypic profiling of MDM2 in DLBCL. MDM2 as a negative regulator of p53 tumor suppressor function.

Summary Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.

AbstractPlatelet attachment to von Willebrand factor (vWF) requires the interaction between the platelet GP1bα and exposed vWF‐A1 domains. Structural insights into the mechanism of the A1‐GP1bα interaction have been limited to an N‐terminally truncated A1 domain that lacks residues Q1238 − E1260 that make up the linker between the D3 and A1 domains of vWF. We have demonstrated that removal of these residues destabilizes quaternary interactions in the A1A2A3 tridomain and contributes to platelet activation under high shear (Auton et al., J Biol Chem 2012;287:14579–14585). In this study, we demonstrate that removal of these residues from the single A1 domain enhances platelet pause times on immobilized A1 under rheological shear. A rigorous comparison between the truncated A1‐1261 and full length A1‐1238 domains demonstrates a kinetic stabilization of the A1 domain induced by these N‐terminal residues as evident in the enthalpy of the unfolding transition. This stabilization occurs through site and sequence‐specific binding of the N‐terminal peptide to A1. Binding of free N‐terminal peptide to A1‐1261 has an affinity and this binding although free to dissociate is sufficient to suppress the platelet pause times to levels comparable to A1‐1238 under shear stress. Our results support a dual‐structure/function role for this linker region involving a conformational equilibria that maintains quaternary A domain associations in the inactive state of vWF at low shear and an intra‐A1‐domain conformation that regulates the strength of platelet GP1bα‐vWF A1 domain associations in the active state of vWF at high shear.

Transaldolase (TA) exchange overestimates gluconeogenesis measured with deuterated water (2H2O). However, it is unknown whether TA differs in people with type 2 diabetes (T2DM).2H2O was ingested, and [1-13C]acetate and [3-3H]glucose were infused in T2DM ( n = 10) and healthy nondiabetic (ND; n = 8) subjects. TA was assessed from the ratio of13C3 to13C4 glucose enrichment (13C3/13C4) measured by13C NMR. Glucose turnover was measured before (∼16-h fast) and during hyperglycemic (∼10 mM) moderate-dose insulin (∼0.35 mU·kg−1·min−1) clamp.13C3/13C4 in T2DM vs. ND was <1.0 and not different at baseline and clamp, indicating equivalent TA. To determine whether incomplete triose phosphate isomerase (TPI) exchange contributed to asymmetric13C3/13C4, [U-13C]glycerol was infused in lieu of [1-13C]acetate during a separate visit in a subset of ND ( n = 7) subjects. Ratio of13C3/13C4 obtained following either tracer was <1.0 at baseline and during clamp, indicating that TPI exchange was essentially complete and did not contribute to asymmetric glucose enrichment. Uncorrected and corrected rates of gluconeogenesis were no different ( P = not significant) in T2DM vs. ND both at baseline and during clamp. TA correction resulted in equivalent estimates of corrected gluconeogenesis in T2DM and ND that were ∼25–35% lower than uncorrected gluconeogenesis both at baseline and during the clamp. The asymmetric enrichment of glucose from13C-gluconeogenic tracers is attributable to TA exchange and can be utilized to correct for TA exchange. In conclusion, TA exchange does not differ between T2DM and ND under fasting or hyperglycemic clamp conditions, and the2H2O method continues to provide an accurate estimation of gluconeogenesis.