Evidence for negative binding cooperativity within CCR5-CCR2b heterodimers

It is well established that most G protein-coupled receptors are able to form homo- and heterodimers, although the functional consequences of this process often remain unclear. CCR5 is a chemokine receptor that plays an important role in inflammatory diseases and acts as a major coreceptor for human immunodeficiency viruses. CCR5 was previously shown to homodimerize and heterodimerize with CCR2b, a closely related receptor. In the present study, we have analyzed the functional consequences of this dimerization process, in terms of ligand binding, stimulation of intracellular cascades, and internalization. Bioluminescence resonance energy transfer and coimmunoprecipitation assays demonstrated that CCR5 and CCR2b heterodimerize with the same efficiency as they homodimerize. In contrast to what has been reported previously, no cooperative signaling was observed after costimulation of the two receptors by their respective ligands. However, we observed that CCR5-specific ligands that are unable to compete for monocyte chemoattractant protein (MCP-1) binding on cells expressing CCR2b alone efficiently prevented MCP-1 binding when CCR5 and CCR2b were coexpressed. The extent of this cross-competition was correlated with the amount of CCR5 expressed in cells, as determined by fluorescence-activated cell sorting analysis. Similar observations were made for the CCR2b-selective ligand MCP-1 that competed efficiently for macrophage inflammatory protein-1beta binding on cells expressing both receptors. Internalization assays did not allow us to demonstrate cointernalization of the receptors in response to agonist stimulation. Together, our observations suggest that CCR5 and CCR2b form homo- and heterodimers with similar efficiencies and that a receptor dimer can only bind a single chemokine.     

The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 microM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry (microLC-microESI-MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH+ to [MH - 129]+ transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH+ to [MH - 129]+ transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen-GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).     

Limb salvage in bone sarcomas in patients younger than age 10: a 20-year experience

The authors present their experience over the last 20 years in limb salvage procedures of a consecutive series of 40 children under 10 years of age (range 2-10 years) with bone sarcomas. Nineteen were osteogenic sarcomas and 21 were Ewing sarcomas. Only one case, located in the distal phalanx of the toe, was treated by straightforward amputation. Intercalary allografts and Canadell's technique were used to preserve joints whenever possible, and prosthesis or osteoarticular allografts were used when the joint surface was involved. Survival rate in this series was 75%. There were four local recurrences. At the last follow-up (mean 11.2 years, range 5-19 years postop), 90% of the patients preserved their limbs. Eighty percent of the authors' results were excellent or good according to the Musculoskeletal Tumor Society Scale. Limb salvage is a real possibility even in young children with bone sarcomas. The age of the patient itself is not a contraindication for limb salvage.     

Framework for verification of positional tolerances with a 3D non-contact measurement method

New methods of data acquisition such as 3D scanners have become increasingly necessary for the verification of the geometric dimensioning and tolerancing (GD&T) specifications in the manufacture of industrial parts. This paper proposes a method for the verification of a positional tolerance in two cylindrical features in an industrial part digitized using a 3D non-contact scanner. Apart from enabling an adequate quantification of the obtained results, this method also permits the displaying of different ways of visualizing the results. The tolerance zone within which the used scanner carries out the analysis was measured and quantified. Some aspects taken into consideration were the geometric quality of the piece and the repeatability, the reproducibility and the uncertainty of the digital process, which comprises the acquisition and processing of the data. This research work concludes that the digital method presented hereby, with its corresponding virtual inspection, is valid for the verification of positional tolerances, even when it is required to maintain the GD&T specification regardless of the feature size. In addition, this article raises the discussion over the best way to compute the traditional datum referencing rules to the new data acquisition methods.     

3D-printed implantable devices with biodegradable rate-controlling membrane for sustained delivery of hydrophobic drugs

Implantable drug delivery systems offer an alternative for the treatments of long-term conditions (i.e. schizophrenia, HIV, or Parkinson’s disease among many others). The objective of the present work was to formulate implantable devices loaded with the model hydrophobic drug olanzapine (OLZ) using robocasting 3D-printing combined with a pre-formed rate controlling membrane. OLZ was selected as a model molecule due to its hydrophobic nature and because is a good example of a molecule used to treat a chronic condition schizophrenia. The resulting implants consisted of a poly(ethylene oxide) (PEO) implant coated with a poly(caprolactone) (PCL)-based membrane. The implants were loaded with 50 and 80% (w/w) of OLZ. They were prepared using an extrusion-based 3D-printer from aqueous pastes containing 36–38% (w/w) of water. The printing process was carried out at room temperature. The resulting implants were characterized by using infrared spectroscopy, scanning electron microscopy, thermal analysis, and X-ray diffraction. Crystals of OLZ were present in the implant after the printing process. In vitro release studies showed that implants containing 50% and 80% (w/w) of OLZ were capable of providing drug release for up to 190 days. On the other hand, implants containing 80% (w/w) of OLZ presented a slower release kinetics. After 190 days, total drug release was ca. 77% and ca. 64% for implants containing 50% and 80% (w/w) of OLZ, respectively. The higher PEO content within implants containing 50% (w/w) of OLZ allows a faster release as this polymer acts as a co-solvent of the drug.     

Imaging the role of toll-like receptor 4 on cell proliferation and inflammation after cerebral ischemia by positron emission tomography

The influence of toll-like receptor 4 on neurogenesis and inflammation has been scarcely explored so far by using neuroimaging techniques. For this purpose, we performed magnetic resonance imaging and positron emission tomography with 3′-deoxy-3′-[¹⁸F]fluorothymidine and [¹¹C]PK11195 at 2, 7, and 14 days following cerebral ischemia in TLR4^+/+ and TLR4^−/− mice. MRI showed similar infarction volumes in both groups. Despite this, positron emission tomography with 3′-deoxy-3′-[¹⁸F]fluorothymidine and [¹¹C]PK11195 evidenced an increase of neurogenesis and a decrease of inflammation in TLR4^−/− mice after ischemia. These results evidence the versatility of neuroimaging techniques to monitor the role of toll-like receptor 4 after cerebral ischemia.     

Accuracy of digitization obtained from scannable and nonscannable elastomeric impression materials

Statement of problemElastomeric impression materials have been marketed for optimizing direct digital acquisition without requiring a stone cast. The trueness and precision of the digitization of these new elastomeric impression materials are unclear.PurposeThe purpose of this in vitro study was to compare the trueness and precision of digital dental casts obtained from the direct digitization of 2 types of vinylsiloxanether (VSXE) impression materials by using a laboratory laser scanner.Material and methodsThirty-eight elastomeric impressions were made of a master die with a similar morphology to a premolar crown preparation. The impression materials were Identium (IDE) and Identium Scan (SCAN), designed for direct digitalization. Each impression was digitalized by using an optical scanner to create digital casts. A computer-aided design (CAD) reference model of trueness (CRM) was created and aligned to each digital cast for digital 3-dimensional discrepancy analysis.ResultsThe mean ±standard deviation global trueness of IDE was 53 ±16 μm and that of SCAN was 46 ±3 μm. SCAN digital casts showed higher precision (58 ±5 μm) than IDE (69 ±18 μm) (P<.05). At the margin of the preparation and at the axial surfaces, SCAN models showed higher trueness (3 ±6 μm and 1 ±5 μm) than IDE (15 ±10 μm and 2 ±37 μm), respectively.ConclusionsScannable impressions could be digitalized with higher global precision than conventional elastomeric materials. Higher trueness could be achieved in specific impression locations such as gingival areas or axial walls of preparations, where the light emitted by the scanner was not blocked.     

A Study of Criminal History among Young Adults with Co-occurring Mental Health and Substance Use Disorders in Psychiatry Outpatient

ABSTRACT

The goal of this study was to examine risk/protective factors to criminality in an adolescent population. Retrospective chart review was utilized to gather data on psychiatric history, substance use, and criminality among adolescent patients within a psychiatric outpatient clinic at a university school of medicine. An earlier age of psychiatric diagnosis and comorbid substance use was found to be a marked risk factor for criminality, whereas female gender and education were shown to be protective factors. These findings can aid in intervention strategies targeted at adolescents who are most at risk for criminality.