The P2Y4 receptor forms homo-oligomeric complexes in several CNS and PNS neuronal cells

It is well established that several cell surface receptors interact with each other to form dimers and oligomers, which are essential for their activation. Since little is known about the quaternary structure of P2Y receptors, in the present work, we investigated the expression of the G-protein-coupled P2Y₄ subunit as monomeric or higher-order complex protein. We examined both endogenously expressed P2Y₄ subtype with the aid of specific anti-P2Y₄ antiserum, and heterologously transfected P2Y₄-tagged receptors with the use of antitag antibodies. In both cases, we found the P2Y₄ receptor displaying molecular masses corresponding to monomeric, dimeric and oligomeric structures. Experiments performed in the absence of reducing agents demonstrated that there is a strict correlation among the multiple protein bands and that the multimeric forms are at least partially assembled by disulphide bonds. The direct demonstration of P2Y₄ homodimerisation comes instead from co–transfection and differential co–immunoprecipitation experiments, with the use of differently tagged P2Y₄ receptors and antitag antibodies. The structural propensity of the P2Y₄ protein to form homo-oligomers may open the possibility of a novel regulatory mechanism of physiopathological functions for this and additional P2Y receptors.     

Analysis of the gliding pattern of the canine flexor digitorum profundus tendon through the A2 pulley

Friction between a tendon and its pulley was first quantified using the concept of the arc of contact. Studies of human tendons conformed closely to a theoretical nylon cable/nylon rod model. However, we observed differences in measured friction that depended on the direction of motion in the canine model. We hypothesized that fibrocartilaginous nodules in the tendon affected the measurements and attempted to develop a theoretical model to explain the observations we made. Two force transducers were connected to each end of the canine flexor digitorum profundus tendon and the forces were recorded when it was moved through the A2 pulley toward a direction of flexion by an actuator and then reversed a direction toward extension. The changes of a force as a function of tendon excursion were evaluated in 20 canine paws. A bead cable/rod model was developed to simulate the canine tendon-pulley complex. To interpret the results, a free-body diagram was developed. The two prominent fibrocartilaginous nodules in the tendon were found to be responsible for deviation from a theoretical nylon cable gliding around the rod model, in a fashion analogous to the effect of the patella on the quadriceps mechanism. A bead cable/rod model qualitatively reproduced the findings observed in the canine tendon-pulley complex. Frictional coefficient of the canine flexor tendon-pulley was 0.016+/-0.005. After accounting for the effect created by the geometry of two fibrocartilaginous nodules within the tendon, calculation of frictional force in the canine tendon was possible.     

The Effect of Lubricin on the Gliding Resistance of Mouse Intrasynovial Tendon

The purpose of this study was to investigate the role of lubricin on the gliding resistance of intrasynovial tendons by comparing lubricin knockout, heterozygous, and wild type mice. A total of thirty-six deep digital flexor (DDF) tendons in the third digits of each hind paw from eighteen adult mice were used, including six lubricin knockout mice (Prg4 –/–), six heterozygous mice (Prg4 +/–), and six wild type mice (Prg4 +/+). The tendon gliding resistance was measured using a custom-made device. Tendon structural changes were evaluated by scanning electron and light microscopy. The gliding resistance of intrasynovial tendons from lubricin knockout mice was significantly higher than the gliding resistance of either wild type or heterozygous mice. The surface of the lubricin knockout tendons appeared to be rougher, compared to the wild type and heterozygous tendons. Synovial hyperplasia was found in the lubricin knockout mice. Cartilage-like tissue was found in the tendon and pulley of the lubricin knockout mice. Our findings confirm the importance of lubricin in intrasynovial tendon lubrication. This knockout model may be useful in determining the effect of lubricin on tendon healing and the response to injury.     

Phylogenetic community structure: temporal variation in fish assemblage

Hypotheses about phylogenetic relationships among species allow inferences about the mechanisms that affect species coexistence. Nevertheless, most studies assume that phylogenetic patterns identified are stable over time. We used data on monthly samples of fish from a single lake over 10 years to show that the structure in phylogenetic assemblages varies over time and conclusions depend heavily on the time scale investigated. The data set was organized in guild structures and temporal scales (grouped at three temporal scales). Phylogenetic distance was measured as the mean pairwise distances (MPD) and as mean nearest‐neighbor distance (MNTD). Both distances were based on counts of nodes. We compared the observed values of MPD and MNTD with values that were generated randomly using null model independent swap. A serial runs test was used to assess the temporal independence of indices over time. The phylogenetic pattern in the whole assemblage and the functional groups varied widely over time. Conclusions about phylogenetic clustering or dispersion depended on the temporal scales. Conclusions about the frequency with which biotic processes and environmental filters affect the local assembly do not depend only on taxonomic grouping and spatial scales. While these analyzes allow the assertion that all proposed patterns apply to the fish assemblages in the floodplain, the assessment of the relative importance of these processes, and how they vary depending on the temporal scale and functional group studied, cannot be determined with the effort commonly used. It appears that, at least in the system that we studied, the assemblages are forming and breaking continuously, resulting in various phylogeny‐related structures that makes summarizing difficult.     

A Canine Non-Weight-Bearing Model with Radial Neurectomy for Rotator Cuff Repair

Background

The major concern of using a large animal model to study rotator cuff repair is the high rate of repair retears. The purpose of this study was to test a non-weight-bearing (NWB) canine model for rotator cuff repair research.

Methods

First, in the in vitro study, 18 shoulders were randomized to 3 groups. 1) Full-width transections repaired with modified Mason-Allen sutures using 3-0 polyglactin suture, 2) Group 1 repaired using number 2 (#2) polyester braid and long-chain polyethylene suture, and 3) Partial-width transections leaving the superior 2 mm infraspinatus tendon intact without repair. In the in vivo study of 6 dogs, the infraspinatus tendon was partially transected as the same as the in vitro group 3. A radial neurectomy was performed to prevent weight bearing. The operated limb was slung in a custom-made jacket for 6 weeks.

Results

In the in vitro study, mean ultimate tensile load and stiffness in Group 2 were significantly higher than Group 1 and 3 (p<0.05). In the in vivo study, gross inspection and histology showed that the preserved superior 2-mm portion of the infraspinatus tendon remained intact with normal structure.

Conclusions

Based on the biomechanical and histological findings, this canine NWB model may be an appropriate and useful model for studies of rotator cuff repair.     

Galectin-3 Impairment of MYCN-Dependent Apoptosis-Sensitive Phenotype Is Antagonized by Nutlin-3 in Neuroblastoma Cells

MYCN amplification occurs in about 20–25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.     

Hyaluronic acid diminishes the resistance to excursion after flexor tendon repair: an in vitro biomechanical study

Adhesion between the tendon and tendon sheath after primary flexor tendon repair is seen frequently, and postoperative finger function is occasionally unsatisfactory. A reduction of the friction may facilitate tendon mobilization, which in turn may reduce the risk of the adhesion and restriction of range of motion. We considered the possibility of utilizing the hyaluronic acid (HA) as a lubricant. To evaluate the effect of HA, the gliding resistance between the canine flexor digitorum profundus tendon repaired by a modified Kessler suture technique with running epitendinous suture and the annular pulley located on the proximal phalanx (corresponding to the A2 pulley in humans) was evaluated and compared before and after administration of HA. The HA solution measurement groups were identified as follows; intact tendon as a control; repaired tendon; tendon soaked in 0.1, 1, and 10 mg/ml HA. The resistance increased after repairing, then it decreased after soaking in 10 mg/ml HA solution. The results of this study revealed that HA diminishes the excursion resistance after flexor tendon repair. We believe that some style of administration of the HA might reduce the excursion resistance and prevent adhesion until the synovial surface is fully developed.     

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle -actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.     

Antifouling activity of enzyme‐functionalized silica nanobeads

The amelioration of biofouling in industrial processing equipment is critical for performance and reliability. While conventional biocides are effective in biofouling control, they are potentially hazardous to the environment and in some cases corrosive to materials. Enzymatic approaches have been shown to be effective and can overcome the disadvantages of traditional biocides, however they are typically uneconomic for routine biofouling control. The aim of this study was to design a robust and reusable enzyme‐functionalized nano‐bead system having biofilm dispersion properties. This work describes the biochemical covalent functionalization of silica‐based nanobeads (hereafter referred to as Si‐NanoB) with Proteinase K (PK). Results showed that PK‐functionalized Si‐NanoB are effective in dispersing both protein‐based model biofilms and structurally altering Pseudomonas fluorescens biofilms, with significant decreases in surface coverage and thickness of 30.1% and 38.85%, respectively, while increasing surface roughness by 19 % following 24 h treatments on bacterial biofilms. This study shows that enzyme‐functionalized nanobeads may potentially be an environmentally friendly and cost effective alternative to pure enzyme and chemical treatments. Biotechnol. Bioeng. 2016;113: 501–512. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.