Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.     

A Cellular Screening Assay Using Analysis of Metal-Modified Fluorescence Lifetime

Current methods for screening cell receptor internalization often require complex image analysis with limited sensitivity. Here we describe a novel bioassay based on detection of changes in global fluorescence lifetime above a gold substrate, with superresolution axial sensitivity and no need for image analysis. We show that the lifetime of enhanced green fluorescent protein expressed in a cellular membrane is greatly reduced in close proximity to the gold, resulting in a distance-dependent lifetime distribution throughout the cell. We demonstrate the application of this phenomenon in a screening assay by comparing the efficacies of two small molecule inhibitors interfering with the internalization process of a G protein-coupled receptor.     

Deleted in Liver Cancer 1 (DLC1) Utilizes a Novel Binding Site for Tensin2 PTB Domain Interaction and Is Required for Tumor-Suppressive Function

Background

Deleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently deleted and underexpressed in hepatocellular carcinoma (HCC) as well as in other cancers. Recent independent studies have shown interaction of DLC1 with members of the tensin focal adhesion protein family in a Src Homology 2 (SH2) domain-dependent mechanism. DLC1 and tensins interact and co-localize to punctate structures at focal adhesions. However, the mechanisms underlying the interaction between DLC1 and various tensins remain controversial.

Methodology/Principal Findings

We used a co-immunoprecipitation assay to identify a previously undocumented binding site at 375–385 of DLC1 that predominantly interacted with the phosphotyrosine binding (PTB) domain of tensin2. DLC1-tensin2 interaction is completely abolished in a DLC1 mutant lacking this novel PTB binding site (DLC1ΔPTB). However, as demonstrated by immunofluorescence and co-immunoprecipitation, neither the focal adhesion localization nor the interaction with tensin1 and C-terminal tensin-like (cten) were affected. Interestingly, the functional significance of this novel site was exhibited by the partial reduction of the RhoGAP activity, which, in turn, attenuated the growth-suppressive activity of DLC1 upon its removal from DLC1.

Conclusions/Significance

This study has provided new evidence that DLC1 also interacts with tensin2 in a PTB domain-dependent manner. In addition to properly localizing focal adhesions and preserving RhoGAP activity, DLC1 interaction with tensin2 through this novel focal adhesion binding site contributes to the growth-suppressive activity of DLC1.     

Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF(120/120) mice. VEGF(120/120) mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.     

Measurement of Pulmonary Flow Reserve and Pulmonary Index of Microcirculatory Resistance for Detection of Pulmonary Microvascular Obstruction

Background

The pulmonary microcirculation is the chief regulatory site for resistance in the pulmonary circuit. Despite pulmonary microvascular dysfunction being implicated in the pathogenesis of several pulmonary vascular conditions, there are currently no techniques for the specific assessment of pulmonary microvascular integrity in humans. Peak hyperemic flow assessment using thermodilution-derived mean transit-time (T_mn) facilitate accurate coronary microcirculatory evaluation, but remain unvalidated in the lung circulation. Using a high primate model, we aimed to explore the use of T_mn as a surrogate of pulmonary blood flow for the purpose of measuring the novel indices Pulmonary Flow Reserve [PFR = (maximum hyperemic)/(basal flow)] and Pulmonary Index of Microcirculatory Resistance [PIMR = (maximum hyperemic distal pulmonary artery pressure)×(maximum hyperemic T_mn)]. Ultimately, we aimed to investigate the effect of progressive pulmonary microvascular obstruction on PFR and PIMR.

Methods and Results

Temperature- and pressure-sensor guidewires (TPSG) were placed in segmental pulmonary arteries (SPA) of 13 baboons and intravascular temperature measured. T_mn and hemodynamics were recorded at rest and following intra-SPA administration of the vasodilator agents adenosine (10–400 µg/kg/min) and papaverine (3–24 mg). Temperature did not vary with intra-SPA sensor position (0.010±0.009 v 0.010±0.009°C; distal v proximal; p = 0.1), supporting T_mn use in lung for the purpose of hemodynamic indices derivation. Adenosine (to 200 µg/kg/min) & papaverine (to 24 mg) induced dose-dependent flow augmentations (40±7% & 35±13% T_mn reductions v baseline, respectively; p<0.0001). PFR and PIMR were then calculated before and after progressive administration of ceramic microspheres into the SPA. Cumulative microsphere doses progressively reduced PFR (1.41±0.06, 1.26±0.19, 1.17±0.07 & 1.01±0.03; for 0, 10⁴, 10⁵ & 10⁶ microspheres; p = 0.009) and increased PIMR (5.7±0.6, 6.3±1.0, 6.8±0.6 & 7.6±0.6 mmHg.sec; p = 0.0048).

Conclusions

Thermodilution-derived mean transit time can be accurately and reproducibly measured in the pulmonary circulation using TPSG. Mean transit time-derived PFR and PIMR can be assessed using a TPSG and adenosine or papaverine as hyperemic agents. These novel indices detect progressive pulmonary microvascular obstruction and thus have with a potential role for pulmonary microcirculatory assessment in humans.     

Enterovirus‐A71 exploits peripherin and Rac1 to invade the central nervous system

Enterovirus‐A71 (EV‐A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV‐A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV‐A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV‐A71, PRPH co‐localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron‐like and neuroblastoma cell lines, surface‐expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non‐structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV‐A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV‐A71 also exploits some of the many PRPH‐interacting partners. Of these, small GTP‐binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV‐A71.     

Prevalence of simian malaria parasites in macaques of Singapore

Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth causative agent of human malaria. Recently, naturally acquired P. cynomolgi infection in humans was also detected in Southeast Asia. The main reservoir of both parasites is the long-tailed and pig-tailed macaques, which are indigenous in this region. Due to increased urbanization and changes in land use, there has been greater proximity and interaction between the long-tailed macaques and the general population in Singapore. As such, this study aims to determine the prevalence of simian malaria parasites in local macaques to assess the risk of zoonosis to the general human population. Screening for the presence of malaria parasites was conducted on blood samples from 660 peridomestic macaques collected between Jan 2008 and Mar 2017, and 379 wild macaques collected between Mar 2009 and Mar 2017, using a Pan-Plasmodium-genus specific PCR. Positive samples were then screened using a simian Plasmodium species-specific nested PCR assay to identify the species of parasites (P. knowlesi, P. coatneyi, P. fieldi, P. cynomolgi, and P. inui) present. All the peridomestic macaques sampled were tested negative for malaria, while 80.5% of the 379 wild macaques were infected. All five simian Plasmodium species were detected; P. cynomolgi being the most prevalent (71.5%), followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed. The study revealed that Singapore’s wild long-tailed macaques are natural hosts of the five simian malaria parasite species, while no malaria was detected in all peridomestic macaques tested. Therefore, the risk of simian malaria transmission to the general human population is concluded to be low. However, this can be better demonstrated with the incrimination of the vectors of simian malaria parasites in Singapore.