Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor

Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Ras. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.     

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.     

Optimal immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase study of various tumours using polyclonal and monoclonal antibodies

The effect of preliminary trypsinization on the immunoreactivity of keratin proteins in formalin-fixed, paraffin-embedded tissues of a variety of tumors (squamous cell carcinomas, adenocarcinomas, mesotheliomas, and transitional cell carcinomas) was evaluated. Three types of trypsin (Type II and Type IX porcine trypsin and Type III bovine trypsin) and varying concentrations of trypsin were assessed. Immunoreactivity of keratin proteins was determined using rabbit anti-keratin antibodies and monoclonal antibodies (combination of AE1 and AE3) and immunoperoxidase techniques. Preliminary trypsinization was mandatory for optimal immunoreactivity of keratin proteins using either polyclonal or monoclonal antibodies. Excellent results were obtained using Type II porcine trypsin at concentrations of 25 mg/dl for 30-45 min or 50 mg/dl for 20 min, at 37 degrees C. Trypsin treatment with excessive concentrations of enzyme and/or extended incubation times promoted tissue digestion and in some cases, yielded decreased immunoreactivity and altered staining patterns.     

Long Noncoding RNAs AC009014.3 and Newly Discovered XPLAID Differentiate Aggressive and Indolent Prostate Cancers

INTRODUCTION: The molecular mechanisms underlying aggressive versus indolent disease are not fully understood. Recent research has implicated a class of molecules known as long noncoding RNAs (lncRNAs) in tumorigenesis and progression of cancer. Our objective was to discover lncRNAs that differentiate aggressive and indolent prostate cancers. METHODS: We analyzed paired tumor and normal tissues from six aggressive Gleason score (GS) 8-10 and six indolent GS 6 prostate cancers. Extracted RNA was split for poly(A)+ and ribosomal RNA depletion library preparations, followed byRNA sequencing (RNA-Seq) using an Illumina HiSeq 2000. We developed an RNA-Seq data analysis pipeline to discover and quantify these molecules. Candidate lncRNAs were validated using RT-qPCR on 87 tumor tissue samples: 28 (GS 6), 28 (GS 3+4), 6 (GS 4+3), and 25 (GS 8-10). Statistical correlations between lncRNAs and clinicopathologic variables were tested using ANOVA. RESULTS: The 43 differentially expressed (DE) lncRNAs between aggressive and indolent prostate cancers included 12 annotated and 31 novel lncRNAs. The top six DE lncRNAs were selected based on large, consistent fold-changes in the RNA-Seq results. Three of these candidates passed RT-qPCR validation, including AC009014.3 (P < .001 in tumor tissue) and a newly discovered X-linked lncRNA named XPLAID (P = .049 in tumor tissue and P = .048 in normal tissue). XPLAID and AC009014.3 show promise as prognostic biomarkers. CONCLUSIONS: We discovered several dozen lncRNAs that distinguish aggressive and indolent prostate cancers, of which four were validated using RT-qPCR. The investigation into their biology is ongoing.     

Biomechanical response of the pubic symphysis in lateral pelvic impacts: a finite element study

Automotive side impacts are a leading cause of injuries to the pubic symphysis, yet the mechanisms of those injuries have not been clearly established. Previous mechanical testing of isolated symphyses revealed increased joint laxity following drop tower lateral impacts to isolated pelvic bone structures, which suggested that the joints were damaged by excessive stresses and/or deformations during the impact tests. In the present study, a finite element (FE) model of a female pelvis including a previously validated symphysis sub-model was developed from computed tomography data. The full pelvis model was validated against measured force-time impact responses from drop tower experiments and then used to study the biomechanical response of the symphysis during the experimental impacts. The FE models predicted that the joint underwent a combination of lateral compression, posterior bending, anterior/posterior and superior/inferior shear that exceeded normal physiological levels prior to the onset of bony fractures. Large strains occurred concurrently within the pubic ligaments. Removal of the contralateral constraints to better approximate the boundary conditions of a seated motor vehicle occupant reduced cortical stresses and deformations of the pubic symphysis; however, ligament strains, compressive and shear stresses in the interpubic disc, as well as posterior bending of the joint structure remained as potential sources of joint damage during automotive side impacts.     

A biomechanical study of periacetabular defects and cement filling

Periacetabular bone metastases cause severe pain and functional disability in cancer patients. Percutaneous acetabuloplasty (PCA) is a minimally invasive, image-guided procedure whereby cement is injected into lesion sites. Pain relief and functional restoration have been observed clinically; however, neither the biomechanical consequences of the lesions nor the effectiveness of the PCA technique are well understood. The objective of this study was to investigate how periacetabular lesion size, cortex involvement, and cement modulus affect pelvic bone stresses and strains under single-legged stance loading. Experiments were performed on a male cadaver pelvis under conditions of intact, periacetabular defect, and cement-filling with surface strains recorded at three strain gage locations. The experimental data were then employed to validate three-dimensional finite element models of the same pelvis, developed using computed tomography data. The models demonstrated that increases in cortical stresses were highest along the posterior column of the acetabulum, adjacent to the defect. Cortical stresses were more profoundly affected in the presence of transcortical defects, as compared to those involving only trabecular bone. Cement filling with a modulus of 2.2 GPa was shown to restore cortical stresses to near intact values, while a decrease in cement modulus due to inclusion of BaSO(4) reduced the restorative effect. Peak acetabular contact pressures increased less than 15% for all simulated defect conditions; however, the contact stresses were reduced to levels below intact in the presence of either cement filling. These results suggest that periacetabular defects may increase the vulnerability of the pelvis to fracture depending on size and cortical involvement and that PCA filling may lower the risk of periacetabular fractures.     

Non‐breeding areas and timing of migration in relation to weather of Scottish‐breeding common sandpipers Actitis hypoleucos

The number of breeding common sandpipers has declined in Britain due to poorer return rates from non‐breeding areas. To investigate little known aspects of their annual cycle, breeding common sandpipers were fitted with geolocators to track their migrations and determine their non‐breeding areas. Ten tagged birds left Scotland on 9 July (median dates and durations are given throughout the abstract). Short‐term staging was carried out by some birds in England and Ireland, then for longer by most birds in Iberia before continuing to West Africa, arriving on 28 July. Six birds spent most of the non‐breeding season (October–February) on the coast of Guinea‐Bissau, suggesting that this is a key area. Single birds occurred in Sierra Leone, Guinea, the Canary Islands and western Sahara. The southward migration from Scotland took 17.5 d (range 1.5–24 d), excluding the initial fuelling period. The first northward movement from Africa was on 12 April. Staging occurred in either Morocco, Iberia or France. Arrival in Scotland was on 2 May. The northward migration took 16 d (range 13.5–20.5 d). The main migration strategy involved short‐ and medium‐range flights, using tail‐winds in most cases. Variation in strategy was associated with departure date; birds that left later staged for shorter durations. Coastal West Africa provides two major habitats for common sandpipers: mudflats associated with mangroves and rice fields. Although the area of mangrove has been depleted, the scale of loss has probably been insufficient to account for the decline in sandpiper numbers. Rice fields are expanding, providing feeding areas for water‐birds. Meteorological data during the migrations suggest that the weather during the southward migration is unlikely to contribute to a population decline but strong cross‐winds or head‐winds during the northward migration to the breeding grounds may do so.     

Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-''light-sheet'', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-''light-sheet'' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.