Selective requirements for NRP1 ligands during neurovascular patterning

Blood vessels and neurons share several types of guidance cues and cell surface receptors to control their behaviour during embryogenesis. The transmembrane protein NRP1 is present on blood vessels and nerves. NRP1 binds two structurally diverse ligands, the semaphorin SEMA3A and the VEGF164 isoform of vascular endothelial growth factor. SEMA3A was originally identified as a repulsive cue for developing axons that acts by signalling through receptor complexes containing NRP1 and plexins. In vitro, SEMA3A also inhibits integrin function and competes with VEGF164 for binding to NRP1 to modulate the migration of endothelial cells. These observations resulted in a widely accepted model of vascular patterning in which the balance of VEGF164 and SEMA3A determines endothelial cell behaviour. However, we now demonstrate that SEMA3A is not required for angiogenesis in the mouse, which instead is controlled by VEGF164. We find that SEMA3A, but not VEGF164, is required for axon patterning of limb nerves, even though the competition between VEGF164 and SEMA3A for NRP1 affects the migration of neuronal progenitor cells in vitro and has been hypothesised to control axon guidance. Moreover, we show that there is no genetic interaction between SEMA3A and VEGF164 during vasculogenesis, angiogenesis or limb axon patterning, suggesting that ligand competition for NRP1 binding cannot explain neurovascular congruence, as previously suggested. We conclude that NRP1 contributes to both neuronal and vascular patterning by preferentially relaying SEMA3A signals in peripheral axons and VEGF164 signals in blood vessels.     

Glioma-associated endothelial cells show evidence of replicative senescence

The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-beta-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.     

Automated Protein Turnover Calculations from 15N Partial Metabolic Labeling LC/MS Shotgun Proteomics Data

Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS ¹⁵N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (¹⁵N) to the intensity sum of all light (¹⁴N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover.     

Retention of solutes and different-sized particles in the digestive tract of the ostrich (Struthio camelus massaicus), and a comparison with mammals and reptiles

Ostriches (Struthio camelus) achieve digesta retention times, digesta particle size reduction and digestibilities equal to similar-sized herbivorous mammals, in contrast to some other avian herbivores. The sequence of digestive processes in their gastrointestinal tract, however, is still unexplored. Using two groups of four ostriches (mean body mass 75.1 ± 17.3 kg) kept on fresh alfalfa, we tested the effect of two intake levels (17 and 42 g dry matter kg(-0.75)d(-1)) on the mean retention time (MRT) of a solute and three different-sized (2, 10, 20 mm) particle markers, mean faecal particle size (MPS), and digestibility. Intake level did not affect MRT, but MPS (0.74 vs. 1.52 mm) and dry matter digestibility (81 vs. 78%). The solute marker (MRT 22-26 h) was excreted faster than the particle markers; there was no difference in the MRT of 10 and 20 mm particles (MRT 28-32 h), but 2mm particles were retained longer (MRT 39-40 h). Because the solute marker was not selectively retained, and wet-sieving of gut contents of slaughtered animals did not indicate smaller particles in the caeca, the long MRT of small particles is interpreted as intermittent excretion from the gizzard, potentially due to entrapment in small grit. The marker excretion pattern also showed intermittent peaks for all markers in five of the animals, which indicates non-continuous outflow from the gizzard. When adding our data to literature data on avian herbivores, a dichotomy is evident, with ostrich and hoatzin (Opisthocomus hoazin) displaying long MRTs, high digestibilities, and gut capacities similar to mammalian herbivores, and other avian herbivores such as grouse, geese or emus with shorter MRTs, lower fibre digestibilities and lower gut capacities. In the available data for all avian herbivores where food intake and MRTs were measured, this dichotomy and food intake level, but not body mass, was related to MRT, adding to the evidence that body mass itself may not be sole major determinant of digestive physiology. The most striking difference between mammalian and avian herbivores from the literature is the fundamentally lower methane production measured in the very few studies in birds including ostriches, which appears to be at the level of reptiles, in spite of general food intake levels of a magnitude as in mammals. Further studies in ostriches and other avian herbivores are required to understand the differences in digestive mechanisms between avian and mammalian herbivores.     

Angiogenic alterations associated with circulating neoplastic DNA in ovarian carcinoma

OBJECTIVES: Forty percent of women with ovarian carcinoma have circulating free neoplastic DNA identified in plasma. Angiogenesis is critical in neoplastic growth and metastasis. We sought to determine whether circulating neoplastic DNA results from alterations in the balance of angiogenesis activators and inhibitors. METHODS: Sixty patients with invasive ovarian carcinomas with somatic TP53 mutations that had been characterized for circulating neoplastic DNA had carcinoma analyzed for microvessel density using immunohistochemistry with CD31 and for the expression of VEGF, ANGPT1, ANGPT2, PTGS2, PLAU, THBS1, CSF1, PIK3CA, HIF1A, IL8, MMP2, and MMP9 message by real-time quantitative polymerase chain reaction. The expression of each gene was calculated relative to GAPDH expression for each neoplasm. Patient plasma had been tested for circulating neoplastic DNA using a ligase detection reaction. RESULTS: MMP2 expression was significantly correlated with free plasma neoplastic DNA (P = .007). Microvessel density was not correlated with plasma neoplastic DNA or BRCA1/2 mutation status. The expression pattern of other angiogenic factors did not correlate with plasma neoplastic DNA but correlated with each other. BRCA1/2 mutated carcinomas had significantly different expression profiles of angiogenesis activators and inhibitors in comparison to sporadic carcinomas. CONCLUSIONS: MMP2 expression is associated with the presence of circulating neoplastic DNA in women with ovarian carcinoma. These data are consistent with the proinvasive properties of MMP2 and suggest that the presence of circulating neoplastic DNA indicates a more aggressive malignant phenotype. Carcinomas with germ line BRCA1/2 mutations had a lower angiogenic profile than those without mutations.     

Measuring health-related quality of life of HIV-positive adolescents in resource-constrained settings

Background and Objectives

Access to antiretroviral treatment among adolescents living with HIV (ALH) is increasing. Health-related quality of life (HRQOL) is relevant for monitoring the impact of the disease on both well-being and treatment outcomes. However, adequate screening tools to assess HRQOL in low-resource settings are scarce. This study aims to fill this research gap, by 1) assessing the psychometric properties and reliability of an Eastern African English version of a European HRQOL scale for adolescents (KIDSCREEN) and 2) determining which version of the KIDSCREEN (52-, 27- and 10-item version) is most suitable for low-resource settings.

Methods

The KIDSCREEN was translated into Eastern African English, Luganda (Uganda) and Dholuo (Kenya) according to standard procedures. The reconciled version was administered in 2011 to ALH aged 13–17 in Kenya (n = 283) and Uganda (n = 299). All three KIDSCREEN versions were fitted to the data with confirmatory factor analysis (CFA). After comparison, the most suitable version was adapted based on the CFA outcomes utilizing the results of previous formative research. In order to develop a general HRQOL factor, a second-order measurement model was fitted to the data.

Results

The CFA results showed that without adjustments, the KIDSCREEN cannot be used for measuring the HRQOL of HIV-positive adolescents. After comparison, the most suitable version for low-resource settings - the 27-item version - was adapted further. The introduction of a negative wording factor was required for the Dholuo model. The Dholuo (CFI: 0.93; RMSEA: 0.039) and the Luganda model (CFI: 0.90; RMSEA: 0.052) showed a good fit. All cronbach’s alphas of the factors were 0.70 or above. The alpha value of the Dholuo and Lugandan HRQOL second-order factor was respectively 0.84 and 0.87.

Conclusions

The study showed that the adapted KIDSCREEN-27 is an adequate tool for measuring HRQOL in low-resource settings with high HIV prevalence.     

Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina

Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.     

Characterization and DNA interaction of the Pt(II)(pq)(bdt) complex: A theoretical and experimental research

A mononuclear complex Pt(pq)(bdt) (1) (where pq = 2-(2′pyridyl)quinoxaline and bdt = benzene-1,2-dithiolate) has been prepared and characterized by NMR spectroscopy, ES mass spectroscopy and elemental analysis. Furthermore, its molecular and electronic structure has been fully elucidated by means of the density functional theory (DFT) and time-dependent density functional theory (TDDFT). The former reveals an extensive distortion of the planarity of complex 1, while the latter an intense mixed metal ligand to ligand charge transfer (MM′LLCT) transition in the visible region of the spectrum. Interactions of complex 1, the free ligands pq and bdt with double stranded calf thymus DNA before and after illumination were studied by UV-spectrophotometric (melting curves) and circular dichroism (CD) measurements, indicating that complex 1 is able to form adducts with DNA and to distort the double helix by changing the base stacking. Under our experimental conditions, it is unclear that complex 1 can photocleave DNA. Viscosity changes of calf thymus DNA (CT-DNA) in the presence of an incremental amount of complex 1 demonstrate that in very low ratios, [1]/[DNA]  0.02, this complex binds intercalatively to the DNA, while in higher ratios a partial or a non-classical interacalation should occur due to the tetrahedral distortion of the molecule and the existence of the dithiolato-ligand.     

Neuropilin 1 and 2 control cranial gangliogenesis and axon guidance through neural crest cells

Neuropilin (NRP) receptors and their class 3 semaphorin (SEMA3) ligands play well-established roles in axon guidance, with loss of NRP1, NRP2, SEMA3A or SEMA3F causing defasciculation and errors in growth cone guidance of peripherally projecting nerves. Here we report that loss of NRP1 or NRP2 also impairs sensory neuron positioning in the mouse head, and that this defect is a consequence of inappropriate cranial neural crest cell migration. Specifically, neural crest cells move into the normally crest-free territory between the trigeminal and hyoid neural crest streams and recruit sensory neurons from the otic placode; these ectopic neurons then extend axons between the trigeminal and facioacoustic ganglia. Moreover, we found that NRP1 and NRP2 cooperate to guide cranial neural crest cells and position sensory neurons; thus, in the absence of SEMA3/NRP signalling, the segmentation of the cranial nervous system is lost. We conclude that neuropilins play multiple roles in the sensory nervous system by directing cranial neural crest cells, positioning sensory neurons and organising their axonal projections.     

RASSF1C oncogene elicits amoeboid invasion,cancer stemness,and extracellular vesicle release via a SRC/Rho axis

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.