Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo (p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction ( p ! 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis ( annexin V staining, p ! 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n=3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth. Copyright (C) 2007 S. Karger AG, Basel

Fever as a Cause of Hypophosphatemia in Patients with Malaria

Hypophosphatemia occurs in 40 to 60% of patients with acute malaria, and in many other conditions associated with elevations of body temperature. To determine the prevalence and causes of hypophosphatemia in patients with malaria, we retrospectively studied all adults diagnosed with acute malaria during a 12-year period. To validate our findings, we analyzed a second sample of malaria patients during a subsequent 10-year period. Serum phosphorus correlated inversely with temperature (n = 59, r = −0.62; P<0.0001), such that each 1°C increase in body temperature was associated with a reduction of 0.18 mmol/L (0.56 mg/dL) in the serum phosphorus level (95% confidence interval: −0.12 to −0.24 mmol/L [−0.37 to −0.74 mg/dL] per 1°C). A similar effect was observed among 19 patients who had repeat measurements of serum phosphorus and temperature. In a multiple linear regression analysis, the relation between temperature and serum phosphorus level was independent of blood pH, PCO2, and serum levels of potassium, bicarbonate, calcium, albumin, and glucose. Our study demonstrates a strong inverse linear relation between body temperature and serum phosphorus level that was not explained by other factors known to cause hypophosphatemia. If causal, this association can account for the high prevalence of hypophosphatemia, observed in our patients and in previous studies of patients with malaria. Because hypophosphatemia has been observed in other clinical conditions characterized by fever or hyperthermia, this relation may not be unique to malaria. Elevation of body temperature should be added to the list of causes of hypophosphatemia

Quantifying the Effects of Elastic Collisions and Non-Covalent Binding on Glutamate Receptor Trafficking in the Post-Synaptic Density

One mechanism of information storage in neurons is believed to be determined by the strength of synaptic contacts. The strength of an excitatory synapse is partially due to the concentration of a particular type of ionotropic glutamate receptor (AMPAR) in the post-synaptic density (PSD). AMPAR concentration in the PSD has to be plastic, to allow the storage of new memories; but it also has to be stable to preserve important information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which AMPAR can enter, leave and remain in the synapse are unclear. We used Monte Carlo simulations to determine the influence of PSD structure and activity in maintaining homeostatic concentrations of AMPARs in the synapse. We found that, the high concentration and excluded volume caused by PSD molecules result in molecular crowding. Diffusion of AMPAR in the PSD under such conditions is anomalous. Anomalous diffusion of AMPAR results in retention of these receptors inside the PSD for periods ranging from minutes to several hours in the absence of strong binding of receptors to PSD molecules. Trapping of receptors in the PSD by crowding effects was very sensitive to the concentration of PSD molecules, showing a switch-like behavior for retention of receptors. Non-covalent binding of AMPAR to anchored PSD molecules allowed the synapse to become well-mixed, resulting in normal diffusion of AMPAR. Binding also allowed the exchange of receptors in and out of the PSD. We propose that molecular crowding is an important biophysical mechanism to maintain homeostatic synaptic concentrations of AMPARs in the PSD without the need of energetically expensive biochemical reactions. In this context, binding of AMPAR with PSD molecules could collaborate with crowding to maintain synaptic homeostasis but could also allow synaptic plasticity by increasing the exchange of these receptors with the surrounding extra-synaptic membrane

Whole-Genome Sequencing of a Single Proband Together with Linkage Analysis Identifies a Mendelian Disease Gene

Although more than 2,400 genes have been shown to contain variants that cause Mendelian disease, there are still several thousand such diseases yet to be molecularly defined. The ability of new whole-genome sequencing technologies to rapidly indentify most of the genetic variants in any given genome opens an exciting opportunity to identify these disease genes. Here we sequenced the whole genome of a single patient with the dominant Mendelian disease, metachondromatosis (OMIM 156250), and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of PTPN11, which alters frame, results in premature translation termination, and co-segregates with the phenotype. In a second metachondromatosis family, we confirmed our result by identifying a nonsense mutation in exon 4 of PTPN11 that also co-segregates with the phenotype. Sequencing PTPN11 exon 4 in 469 controls showed no such protein truncating variants, supporting the pathogenicity of these two mutations. This combination of a new technology and a classical genetic approach provides a powerful strategy to discover the genes responsible for unexplained Mendelian disorders

Optimization of Cell Morphology Measurement via Single-Molecule Tracking PALM

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments

Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a “second hit,” that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome

How land use/land cover changes can affect water, flooding and sedimentation in a tropical watershed: a case study using distributed modeling in the Upper Citarum watershed, Indonesia

[EN] Human activity has produced severe LULC changes within the Upper Citarum watershed and these changes are predicted to continue in the future. With an increase in population parallel to a 141% increment in urban areas, a reduction of rice fields and the replacement of forests with cultivations have been found in the past. Accordingly, LCM model was used to forecast the LULC in 2029. A distributed model called TETIS was implemented in the Upper Citarum watershed to assess the impact of the different historical and future LULC scenarios on its water and sediment cycles. This model was calibrated and validated with different LULCs. For the implementation of the sediment sub-model, it was crucial to use the bathymetric information of the reservoir located at the catchment's outlet. Deforestation and urbanization have been shown to be the most influential factors affecting the alteration of the hydrological and sedimentological processes in the Upper Citarum watershed. The change of LULC decreases evapotranspiration and as a direct consequence, the water yield increased by 15% and 40% during the periods 1994-2014 and 2014-2029, respectively. These increments are caused by the rise of three components in the runoff: overland flow, interflow and base flow. Apart from that, these changes in LULC increased the area of non-tolerable erosion from 412 km(2) in 1994 to 499 km(2) in 2029. The mean sediment yield increased from 3.1 Mton -yr(-1) in the 1994 LULC scenario to 6.7 Mton-yr(-1) in the 2029 LULC scenario. An increment of this magnitude will be catastrophic for the operation of the Saguling Dam.This study was partially funded by the Spanish Ministry of Economy and Competitiveness through the research projects TETISMED (CGL2014-58,127-C3-3-R) and TETISCHANGE (RTI2018-093717-B-I00). The authors are also thankful to the Directorate General of Higher Education of Indonesia (DIKTI) for the Ph.D. funding of the first author.Siswanto, SY.; Francés, F. (2019). How land use/land cover changes can affect water, flooding and sedimentation in a tropical watershed: a case study using distributed modeling in the Upper Citarum watershed, Indonesia. Environmental Earth Sciences. 78(17):1-15. https://doi.org/10.1007/s12665-019-8561-0S115781

Stakeholder communication in 140 characters or less: a study of community sport foundations

Community sport foundations (CSFs), like other non-profit organizations, are increasingly employing social media such as Twitter to communicate their mission and activities to their diverse stakeholder groups. However, the way these CSFs utilize social media for communicating such practices remains unclear. Through a mixed-method approach of content analysis of tweets from 22 CSFs established by English professional football clubs and interviews with key individuals within these CSFs (n = 7), this study examines the extent to which CSFs’ core activities are being communicated through Twitter and identifies the strategies employed for doing so. Reflecting the target audiences CSFs are seeking to reach through Twitter and the challenges associated with communication about projects involving marginalized groups, tweets largely concern programs related to sports participation and education. The most frequently employed communication strategy is to inform, rather than interact or engage with stakeholders. However, CSFs with higher organizational capacity attempt to go beyond mere informing towards engaging with stakeholder groups that relate to their social agenda, highlighting the importance of trained and dedicated social media personnel in optimizing CSFs’ use of Twitter for communication