Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271⁺, CD271⁻ mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271⁺ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271⁺ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.     

Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM? Platform

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.     

Characterization of Apo-Form Selective Inhibition of Indoleamine 2,3-Dioxygenase**

Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As it is an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo-form-binding IDO1 inhibitor (GSK5628) to the heme-coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo-binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1), whereas epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis-à-vis specificity and pharmacokinetic parameters.     

Chloroplastic glycolipids fuel aldehyde biosynthesis in the marine diatom Thalassiosira rotula

Enzymatic preparations and specialized analytical tools have shown that chloroplast-derived glycolipids are the main substrates for the biosynthetic pathway that produces antiproliferative polyunsaturated aldehydes in broken cells of the marine diatom Thalassiosira rotula. This process, which is associated with the formation of free fatty acids and lyso compounds from polar lipids but not triglycerides, is largely dependent on glycolipid hydrolytic activity, rather than phospholipase A(2) as previously suggested. Preliminary characterization of lipolytic enzymes has revealed protein bands of 40-45 kDa. Under native conditions these proteins seem to be associated with soluble aggregates that have an apparent molecular weight of approximately 200 kDa. The biochemical process, which is similar to that described in the algal-bloom forming diatom Skeletonema costatum, suggests a mechanism based on decompartmentalization and mixing of preexisting enzymes and substrates.     

Development of spontaneous classificatory behavior in chimpanzees (Pan troglodytes).

Investigated the development of spontaneous classificatory behavior in 5 chimpanzees (Pan troglodytes) tested at different ages and analyzed Ss' spontaneous constructive interactions with sets of logically structured objects. The results show that chimpanzees possess a natural capacity to react to similarities and differences among test stimuli and construct classes. The general progression of their classificatory development is very similar to that reported for human infants from 6 to 24 mo. In both species, classification progresses from constructing single classes by different properties of objects to constructing single classes by similar or identical properties of objects. In addition, like humans, older chimpanzees spontaneously coordinate relations of similarities between sets and construct 2 class-consistent groupings. Chimpanzees' results are compared with those from a similar study with capuchins and macaques. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Influence of Nano,Micro, and Macro Topography of Dental Implant Surfaces on Human Gingival Fibroblasts

Current research on dental implants has mainly focused on the influence of surface roughness on the rate of osseointegration, while studies on the development of surfaces to also improve the interaction of peri-implant soft tissues are lacking. To this end, the first purpose of this study was to evaluate the response of human gingival fibroblasts (hGDFs) to titanium implant discs (Implacil De Bortoli, Brazil) having different micro and nano-topography: machined (Ti-M) versus sandblasted/double-etched (Ti-S). The secondary aim was to investigate the effect of the macrogeometry of the discs on cells: linear-like (Ti-L) versus wave-like (Ti-W) surfaces. The atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis showed that the Ti-S surfaces were characterized by a significantly higher micro and nano roughness and showed the 3D macrotopography of Ti-L and Ti-W surfaces. For in vitro analyses, the hGDFs were seeded into titanium discs and analyzed at 1, 3, and 5 days for adhesion and morphology (SEM) viability and proliferation (Cck-8 and MTT assays). The results showed that all tested surfaces were not cytotoxic for the hGDFs, rather the nano-micro and macro topography favored their proliferation in a time-dependent manner. Especially, at 3 and 5 days, the number of cells on Ti-L was higher than on other surfaces, including Ti-W surfaces. In conclusion, although further studies are needed, our in vitro data proved that the use of implant discs with Ti-S surfaces promotes the adhesion and proliferation of gingival fibroblasts, suggesting their use for in vivo applications.     

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/w_PLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 μm and its internal diameter was 2.61 ± 0.05 mm. The graft’s antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.     

Quality Evaluation of Shelled and Unshelled Macadamia Nuts by Means of Near‐Infrared Spectroscopy (NIR)

The quality of shelled and unshelled macadamia nuts was assessed by means of Fourier transformed near‐infrared (FT‐NIR) spectroscopy. Shelled macadamia nuts were sorted as sound nuts; nuts infected by Ecdytolopha aurantiana and Leucopteara coffeella; and cracked nuts caused by germination. Unshelled nuts were sorted as intact nuts (<10% half nuts, 2014); half nuts (March, 2013; November, 2013); and crushed nuts (2014). Peroxide value (PV) and acidity index (AI) were determined according to AOAC. PCA‐LDA shelled macadamia nuts classification resulted in 93.2% accurate classification. PLS PV prediction model resulted in a square error of prediction (SEP) of 3.45 meq/kg, and a prediction coefficient determination value (R_p²) of 0.72. The AI PLS prediction model was better (SEP = 0.14%, R_p² = 0.80). Although adequate classification was possible (93.2%), shelled nuts must not contain live insects, therefore the classification accuracy was not satisfactory. FT‐NIR spectroscopy can be successfully used to predict PV and AI in unshelled macadamia nuts, though.