Raman Spectroscopic Imaging for Quantification of Depth-Dependent and Local Heterogeneities in Native and Engineered Cartilage

Articular cartilage possesses a remarkable, mechanically-robust extracellular matrix (ECM) that is organized and distributed throughout the tissue to resist physiologic strains and provide low friction during articulation. The ability to characterize the make-up and distribution of the cartilage ECM is critical to both understand the process by which articular cartilage undergoes disease-related degeneration and to develop novel tissue repair strategies to restore tissue functionality. However, the ability to quantitatively measure the spatial distribution of cartilage ECM constituents throughout the tissue has remained a major challenge. In this experimental investigation, we assessed the analytical ability of Raman micro-spectroscopic imaging to semi-quantitatively measure the distribution of the major ECM constituents in cartilage tissues. Raman spectroscopic images were acquired of two distinct cartilage tissue types that possess large spatial ECM gradients throughout their depth: native articular cartilage explants and large engineered cartilage tissue constructs. Spectral acquisitions were processed via multivariate curve resolution to decompose the “fingerprint” range spectra (800–1800 cm−1) to the component spectra of GAG, collagen, and water, giving rise to the depth dependent concentration profile of each constituent throughout the tissues. These Raman spectroscopic acquired-profiles exhibited strong agreement with profiles independently acquired via direct biochemical assaying of spatial tissue sections. Further, we harness this spectroscopic technique to evaluate local heterogeneities through the depth of cartilage. This work represents a powerful analytical validation of the accuracy of Raman spectroscopic imaging measurements of the spatial distribution of biochemical components in a biological tissue and shows that it can be used as a valuable tool for quantitatively measuring the distribution and organization of ECM constituents in native and engineered cartilage tissue specimens

The role of complementary learning systems in learning and consolidation in a quasi-regular domain

We examine the role of off-line memory consolidation processes in the learning and retention of a new quasi-regular linguistic system similar to the English past tense. Quasi-regular systems are characterized by a dominance of systematic, regular forms (e.g., walk-walked, jump-jumped) alongside a smaller number of high frequency irregulars (e.g., sit-sat, go-went), and are found across many cognitive domains, from spelling-sound mappings to inflectional morphology to semantic cognition. Participants were trained on the novel morphological system using an artificial language paradigm, and then tested after different delays. Based on a complementary systems account of memory, we predicted that irregular forms would show stronger off-line changes due to consolidation processes. Across two experiments, participants were tested either immediately after learning, 12 h later with or without sleep, or 24 h later. Testing involved generalization of the morphological patterns to previously unseen words (both experiments) as well as recall of the trained words (Experiment 2). In generalization, participants showed 'default' regularization across a range of novel forms, as well as irregularization for previously unseen items that were similar to unique high-frequency irregular trained forms. Both patterns of performance remained stable across the delays. Generalizations involving competing tendencies to regularize and irregularize were balanced between the two immediately after learning. Crucially, at both 12-h delays the tendency to irregularize in these cases was strengthened, with further strengthening after 24 h. Consolidated knowledge of both regular and irregular trained items contributed significantly to generalization performance, with evidence of strengthening of irregular forms and weakening of regular forms. We interpret these findings in the context of a complementary systems model, and discuss how maintenance, strengthening, and forgetting of the new memories across sleep and wake can play a role in acquiring quasi-regular systems

Prevalence of Dal blood type and dog erythrocyte antigens (DEA) 1, 4, and 7 in canine blood donors in Italy and Spain

Background: The aim of this study was to determine the prevalence of Dal, and DEA 1, 4, 7 blood types, in a population of canine blood donors from Italy and Spain. Three hundred and twenty blood donor dogs receiving an annual health evaluation were included in the study. DEA 1 blood type was determined using an immunochromatographic strip technique while Dal, DEA 4 and 7 blood types were determined with polyclonal antisera using agglutination on gel columns. Results: Out of 320 dogs blood typed 7 (2 Cane Corso and 5 Doberman Pinschers) (2.2%) were Dal negative; 137 (42.8%) were positive for DEA 1; 320 (100%) were positive for DEA 4 and 43 (13.4%) were positive for DEA 7. Conclusion: This study showed a similar prevalence of DEA 1, 7 and 4 to that reported in previous studies in the same, and in different, geographic areas, and provides new data on the prevalence of the Dal blood group in Italy and Spain. There was no significant difference (P = 0.8409) between prevalence of Dal negative blood types found in our population (2.2%) and the prevalence reported in a canine blood donor population from the USA (2.5%). Our study identified Dal negative dogs in a previously tested breed i.e. Doberman Pinschers, but also the Cane Corso breed was found to have Dal negative dogs

PTEN mediates Notch-dependent stalk cell arrest in angiogenesis

Coordinated activity of VEGF and Notch signals guides the endothelial cell (EC) specification into tip and stalk cells during angiogenesis. Notch activation in stalk cells leads to proliferation arrest via an unknown mechanism. By using gain- and loss-of-function gene-targeting approaches, here we show that PTEN is crucial for blocking stalk cell proliferation downstream of Notch, and this is critical for mouse vessel development. Endothelial deletion of PTEN results in vascular hyperplasia due to a failure to mediate Notch-induced proliferation arrest. Conversely, overexpression of PTEN reduces vascular density and abrogates the increase in EC proliferation induced by Notch blockade. PTEN is a lipid/protein phosphatase that also has nuclear phosphatase-independent functions. We show that both the catalytic and non-catalytic APC/C-Fzr1/Cdh1-mediated activities of PTEN are required for stalk cells' proliferative arrest. These findings define a Notch-PTEN signalling axis as an orchestrator of vessel density and implicate the PTEN-APC/C-Fzr1/Cdh1 hub in angiogenesis

Quaternary structure of a G-protein coupled receptor heterotetramer in complex with Gi and Gs

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function

How Well Can We Assess the Validity of Non-Randomised Studies of Medications? A Systematic Review of Assessment Tools

Objective To determine whether assessment tools for non-randomised studies (NRS) address critical elements that influence the validity of NRS findings for comparative safety and effectiveness of medications. Design Systematic review and Delphi survey. Data sources We searched PubMed, Embase, Google, bibliographies of reviews and websites of influential organisations from inception to November 2019. In parallel, we conducted a Delphi survey among the International Society for Pharmacoepidemiology Comparative Effectiveness Research Special Interest Group to identify key methodological challenges for NRS of medications. We created a framework consisting of the reported methodological challenges to evaluate the selected NRS tools. Study selection Checklists or scales assessing NRS. Data extraction Two reviewers extracted general information and content data related to the prespecified framework. Results Of 44 tools reviewed, 48% (n=21) assess multiple NRS designs, while other tools specifically addressed case–control (n=12, 27%) or cohort studies (n=11, 25%) only. Response rate to the Delphi survey was 73% (35 out of 48 content experts), and a consensus was reached in only two rounds. Most tools evaluated methods for selecting study participants (n=43, 98%), although only one addressed selection bias due to depletion of susceptibles (2%). Many tools addressed the measurement of exposure and outcome (n=40, 91%), and measurement and control for confounders (n=40, 91%). Most tools have at least one item/question on design-specific sources of bias (n=40, 91%), but only a few investigate reverse causation (n=8, 18%), detection bias (n=4, 9%), time-related bias (n=3, 7%), lack of new-user design (n=2, 5%) or active comparator design (n=0). Few tools address the appropriateness of statistical analyses (n=15, 34%), methods for assessing internal (n=15, 34%) or external validity (n=11, 25%) and statistical uncertainty in the findings (n=21, 48%). None of the reviewed tools investigated all the methodological domains and subdomains. Conclusions The acknowledgement of major design-specific sources of bias (eg, lack of new-user design, lack of active comparator design, time-related bias, depletion of susceptibles, reverse causation) and statistical assessment of internal and external validity is currently not sufficiently addressed in most of the existing tools. These critical elements should be integrated to systematically investigate the validity of NRS on comparative safety and effectiveness of medications

Probing proton halo effects in the 8B+64Zn collision around the Coulomb barrier

Proton halo effects in the 8B+64Zn reaction at an energy around 1.5 times the Coulomb barrier have been studied at HIE-ISOLDE CERN using, for the first time, the only existing postaccelerated 8B beam. This, together with the use of a high granularity and large solid angle detection system, allowed for a careful mapping of the elastic angular distribution, especially in the Coulomb-nuclear interference region. Contrary to what is observed for the one-neutron halo nucleus 11Be on the same target in a similar energy range, the analysis of the elastic scattering angular distribution shows only a modest suppression of the Coulomb-nuclear interference peak, with no remarkable enhancement of the total reaction cross-section. Inclusive angular and energy distributions of 7Be produced in direct reaction processes have also been measured. The comparison of these data with the results of theoretical calculations for the elastic and non-elastic breakup contributions indicate that both processes are important. Overall, the experimental data suggest a 8B collision dynamics at the barrier very different from the one of neutron halo nuclei, showing only modest effects of coupling to continuum. This behaviour can be interpreted as due to the presence of the additional Coulomb interactions halo-core and halo-target together with the presence of the centrifugal barrier felt by the valence proton of 8B