Engineering human cell-based, functionally integrated osteochondral grafts by biological bonding of engineered cartilage tissues to bony scaffolds

In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone) using a fibrin gel (Tisseel). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and--since it is based on clinically compliant cells and materials--is amenable to be readily tested in the clinic

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p63$_{2}$/p73$_{2}$ hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p63$_{2}$/p73$_{2}$ hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma

A necroptosis-independent function of RIPK3 promotes immune dysfunction and prevents control of chronic LCMV infection

Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damageand impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic andpersistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated becauseproteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like(MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining therole of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributesto the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing thatnecroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docilestrain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads arenot altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, weidentified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. Thiswas not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function ofRIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to animpaired antiviral immune response and abrogated clearance of chronic LCMV infectio

The consequences of a new software package for the quantification of gated-SPECT myocardial perfusion studies

Semiquantitative analysis of myocardial perfusion scintigraphy (MPS) has reduced inter- and intraobserver variability, and enables researchers to compare parameters in the same patient over time, or between groups of patients. There are several software packages available that are designed to process MPS data and quantify parameters. In this study the performances of two systems, quantitative gated SPECT (QGS) and 4D-MSPECT, in the processing of clinical patient data and phantom data were compared. The clinical MPS data of 148 consecutive patients were analysed using QGS and 4D-MSPECT to determine the end-diastolic volume, end-systolic volume and left ventricular ejection fraction. Patients were divided into groups based on gender, body mass index, heart size, stressor type and defect type. The AGATE dynamic heart phantom was used to provide reference values for the left ventricular ejection fraction. Although the correlations were excellent (correlation coefficients 0.886 to 0.980) for all parameters, significant differences (p < 0.001) were found between the systems. Bland-Altman plots indicated that 4D-MSPECT provided overall higher values of all parameters than QGS. These differences between the systems were not significant in patients with a small heart (end-diastolic volume < 70 ml). Other clinical factors had no direct influence on the relationship. Additionally, the phantom data indicated good linear responses of both systems. The discrepancies between these software packages were clinically relevant, and influenced by heart size. The possibility of such discrepancies should be taken into account when a new quantitative software system is introduced, or when multiple software systems are used in the same institution.Vascular Biology and Interventio

Consensus guidelines for management of hyperammonaemia in paediatric patients receiving continuous kidney replacement therapy.

Hyperammonaemia in children can lead to grave consequences in the form of cerebral oedema, severe neurological impairment and even death. In infants and children, common causes of hyperammonaemia include urea cycle disorders or organic acidaemias. Few studies have assessed the role of extracorporeal therapies in the management of hyperammonaemia in neonates and children. Moreover, consensus guidelines are lacking for the use of non-kidney replacement therapy (NKRT) and kidney replacement therapies (KRTs, including peritoneal dialysis, continuous KRT, haemodialysis and hybrid therapy) to manage hyperammonaemia in neonates and children. Prompt treatment with KRT and/or NKRT, the choice of which depends on the ammonia concentrations and presenting symptoms of the patient, is crucial. This expert Consensus Statement presents recommendations for the management of hyperammonaemia requiring KRT in paediatric populations. Additional studies are required to strengthen these recommendations

Інформаційна технологія для класифікації наукових текстів на основі методу модифікованої логістичної регресії

Об’єкт дослідження: процес класифікації наукових текстів та практичне використання технологій обробки природної мови в освітніх додатках, з метою підвищення ефективності освітнього процесу. Предмет дослідження: методи, моделі машинного навчання та обробки природньої мови у задачах класифікації наукових текстів. Мета магістерської роботи: вдосконалення та пришвидшення процесу класифікації текстів з допомогою моделі логістичної регресії, з метою застосування її у освітніх додатках для покращення освітнього процесу. Методи дослідження. Для створення рекомендаційного та навчального асистента були використані засоби та методи машинного навчання, теорії множин, лінейної алгебри й обробки природної мови. Наукова новизна полягає у тому, що вдосконалено та розширено можливості методу логістичної регресії на основі комбінування його з методом ранжування, що в результаті дозволило використати метод логістичної регресії для навчального асистенті. Практична цінність полягає у тому, що в результаті роботи, було створено прототип навчального асистента, що використовує скомбіновану з методом ранжування модель логістичної регресії для класифікації текстів. Використані методи та підходи у прототипі можуть застосовуватись як при розробці «інтелектуальних» навчальних систем, так й в практиці викладання дисциплін, пов’язаних з обробкою природної мови

Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells

© 2018, American Society for Clinical Investigation. This article has been published in final form at https://doi.org/10.1172/JCI120612First-generation immune checkpoint inhibitors, including anti-CTLA-4 and anti-programmed death 1 (anti-PD-1) antibodies, have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) are pattern-recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not until recently considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs, including Siglec-9, on tumor-infiltrating T cells from non-small cell lung cancer (NSCLC), colorectal, and ovarian cancer patients. Siglec-9-expressing T cells coexpressed several inhibitory receptors, including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anticancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with reduced survival, and Siglec-9 polymorphisms showed association with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as a potential target for improving T cell activation for immunotherapy.Peer reviewe

Strategic and practical guidelines for successful structured illumination microscopy

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d