Chondrocyte responses to neurovascular peptides, cytokines, and a 3D environment: focus on ADAMs

Chondrocyte exposure to inflammatory stimuli in several arthritic conditions, including osteoarthritis, results in the well-characterised induction of extracellular matrix (ECM) degrading proteinases, notably members of a disintegrin and metalloproteinase with thrombospondin domains (ADAMTS) and matrix metalloproteinase (MMP) families. Here we briefly review the less-studied a disintegrin and metalloproteinase (ADAM) family of proteinases in chondrocyte and cartilage biology. Following damage, cartilage is exposed to neurovascular peptides, and in this study we hypothesised that substance P and bradykinin, alongside inflammatory cytokines, may modulate chondrocyte steady state messenger RNA levels for the proteolytic ADAM family members as well as for key cytokines and neuropeptides. We compared chondrocytes cultured in both 2-dimensional (2D) and 3D environments and found that 3D culture generally resulted in repression of expression of the genes under investigation, with the exception of anti-inflammatory interleukin 10 (IL10) which was markedly up-regulated in a 3D environment. Substance P and bradykinin had little effect on ADAM family expression but further investigation revealed that a combination of bradykinin and cytokines led to enhanced expression of ADAM28 and a synergistic up-regulation of IL6, also observed under hypoxic conditions. Overall this data reveals wider chondrocyte responses to neurovascular peptides which may have an impact in an osteoarthritis context

Imaging of compartmentalised intracellular nitric oxide, induced during bacterial phagocytosis, using a metalloprotein–gold nanoparticle conjugate

Nitric oxide (NO) plays an essential role within the immune system since it is involved in the break-down of infectious agents such as viruses and bacteria. The ability to measure the presence of NO in the intracellular environment would provide a greater understanding of the pathophysiological mechanism of this important molecule. Here we report the detection of NO from the intracellular phagolysosome using a fluorescently tagged metalloprotein–gold nanoparticle conjugate. The metalloprotein cytochrome c, fluorescently tagged with an Alexa Fluor dye, was self-assembled onto gold nanoparticles to produce a NO specific nanobiosensor. Upon binding of NO, the cytochrome c protein changes conformation which induces an increase of fluorescence intensity of the tagged protein proportional to the NO concentration. The nanobiosensor was sensitive to NO in a reversible and selective manner, and exhibited a linear response at NO concentrations between 1 and 300 μM. In RAW264.7γ NO− macrophage cells, the nanobiosensor was used to detect the presence of NO that had been endogenously generated upon stimulation of the cells with interferon-γ and lipopolysaccharide, or spontaneously released following treatment of the cells with a NO donor. Significantly, the nanobiosensor was shown to be taken up by the macrophages within phagolysosomes, i.e., the precise location where the NO, together with other species, destroys bacterial infection. The nanobiosensor measured, for the first time, increasing concentrations of NO produced during combined stimulation and phagocytosis of Escherichia coli bacteria from within localised intracellular phagolysosomes, a key part of the immune system

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.Published as part of The Journal of Physical Chemistry virtual special issue “W. E. Moerner Festschrift”

Synthesis, structure and spectroscopic properties of luminescent GdVO4:Dy3+ and DyVO4 particles

Part of this research was done during visit of D.J. to IFN-CNR CSMFO Lab. and FBK Photonics Unit, Povo-Trento, Italy, in the framework of the STSM (Grant No. 38223) from the project: COST Action MP 1401 Advanced Fibre Laser and Coherent Source as tools for Society, Manufacturing and Lifescience” (2014e2018). The authors from Vinca Institute of Nuclear Sciences acknowledge the financial support of the Ministry of Education, Science and Tech-nological Development of the Republic of Serbia (Project No: 45020 and 172056). L.T.N. Tran acknowledges the scholarship of the Ministry of Education and Training, Vietnam International Education Development. T. G. acknowledges the ERDF PostDoc project No. 1.1.1.2/VIAA/1/16/215 (1.1.1.2/16/I/001).In this work, we focused on the syntheses, structure and spectroscopic properties of GdVO4:Dy3+ and DyVO4 (nano)particles of different sizes and shapes (spherical nanoparticles of 2 nm, 4 nm, and 20 nm in size, nanorods with a few nanometers in diameter and up to 10–20 nm in length and microparticles of 1–8 μm) obtained by four synthetic methods. The size effect on the structure, Raman active modes, and photoluminescence emission intensities was analyzed by X-ray diffraction, Raman and photoluminescence spectroscopy, scanning and transmission electron microscopy, and diffuse reflection spectroscopy. All X-ray diffraction patterns clearly indicated presence of a single tetragonal zircon-type phase; absence of impurity phases indicate that the dopant Dy3+ ions were successfully and uniformly incorporated into the GdVO4 host lattice due to the equal valence and similar ionic radii. Micro-Raman measurements support the XRD measurements and showed Raman-active modes of the REVO4 systems (RE = Gd, Dy). The difference between the two hosts in the diffuse reflectance spectra was observed and it could be attributed to more effective Gd3+ ions on the charge transfer bands and different polarization (compared to bulk material) in smaller nanoparticles. Photoluminescence spectroscopy showed several bands in the visible and near-infrared regions which can be exclusively attributed to the f–f transitions of Dy3+ ions.STSM (Grant No. 38223); COST Action MP 1401 (2014e2018); Ministry of Education, Science and Tech-nological Development of the Republic of Serbia (Project No: 45020 and 172056); ERDF PostDoc project No. 1.1.1.2/VIAA/1/16/215 (1.1.1.2/16/I/001); Institute of Solid State Physics, University of Latvia as the Center of Excellence has received funding from the European Union’s Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamingPhase2 under grant agreement No. 739508, project CAMART

Coping strategies in civilians during air attacks

Background Coping strategies may influence the psychological outcome after a stressful event, both as coping at the time of the event and as strategies of dealing with its consequences after the event. The aim of the study was to investigate coping strategies used by civilians during the air attacks in Yugoslavia in 1999, and their association with the level of exposure, gender and psychological symptoms 1 year later. Method The sample is a non-selective group of 139 medical students from the University of Belgrade, Yugoslavia. Open questions and content analysis were used to assess coping strategies. Symptoms of intrusion and avoidance were assessed, as well as general psychological symptoms. Results Content analysis of answers to open questions revealed nine categories of coping strategies (sport and walks, leisure activities, talking and gathering, humor, avoidance, philosophical approach, getting information, work, and substance abuse). A cluster analysis identified three groups of students with different styles of coping. Students that used dominantly 'talking and gathering' had the highest, and the ones that mostly used 'leisure activities' the lowest scores on intrusion. There were significant gender differences in how coping strategies were associated with intrusive symptoms. Conclusion The type of coping strategies used during the air attacks may contribute to the level of intrusive symptoms 1 year after the event. Different coping strategies might be effective in men and women to reduce intrusive symptoms. Longitudinal and prospective studies are needed to draw definite conclusions on causal relationships between coping strategies and levels of posttraumatic stress

Framing Cutting-Edge Integrative Deep-Sea Biodiversity Monitoring via Environmental DNA and Optoacoustic Augmented Infrastructures

Deep-sea ecosystems are reservoirs of biodiversity that are largely unexplored, but their exploration and biodiscovery are becoming a reality thanks to biotechnological advances (e.g., omics technologies) and their integration in an expanding network of marine infrastructures for the exploration of the seas, such as cabled observatories. While still in its infancy, the application of environmental DNA (eDNA) metabarcoding approaches is revolutionizing marine biodiversity monitoring capability. Indeed, the analysis of eDNA in conjunction with the collection of multidisciplinary optoacoustic and environmental data, can provide a more comprehensive monitoring of deep-sea biodiversity. Here, we describe the potential for acquiring eDNA as a core component for the expanding ecological monitoring capabilities through cabled observatories and their docked Internet Operated Vehicles (IOVs), such as crawlers. Furthermore, we provide a critical overview of four areas of development: (i) Integrating eDNA with optoacoustic imaging; (ii) Development of eDNA repositories and cross-linking with other biodiversity databases; (iii) Artificial Intelligence for eDNA analyses and integration with imaging data; and (iv) Benefits of eDNA augmented observatories for the conservation and sustainable management of deep-sea biodiversity. Finally, we discuss the technical limitations and recommendations for future eDNA monitoring of the deep-sea. It is hoped that this review will frame the future direction of an exciting journey of biodiscovery in remote and yet vulnerable areas of our planet, with the overall aim to understand deep-sea biodiversity and hence manage and protect vital marine resources

Tumor response and patient outcome after preoperative radiotherapy in locally advanced non-inflammatory breast cancer patients

Purpose: The purpose of this analysis was to assess the tumor response and long-term outcome in patients treated with preoperative radiotherapy (PRT) without systemic therapy. Methods: Between 1997 and 2000, 134 patients with non-inflammatory locally advanced breast cancer (LABC) were treated with PRT. The tumor dose was 45 Gy in 15 fractions to the breast and to regional lymph nodes over 6 weeks. Radical mastectomy was performed 6 weeks after PRT to all patients and adjuvant systemic therapy was administered as per protocol. The measures of disease outcome were overall survival (OS) and disease-free survival (DFS) which estimated using the Kaplan-Meier method. Results: Median follow-up was 74 months (range 4-216). Objective clinical tumor response after PRT was observed in 77.6% of the patients. Clinical complete tumor response (cCR) was achieved in 21.6% of the patients. Pathological CR in the breast was achieved in 15% of the patients. The 5- and 10-year OS were 55.1 and 37.8%, respectively. The 5- and 10-year DFS were 39.2 and 27%, respectively. Patients who achieved cCR had significantly longer OS in comparison with patients achieving clinical partial response (cPR) and clinical stable disease (cSD). Similarly, DFS of patients in the cCR group was longer compared with patients with cPR and cSD, yet without statistical significance. Conclusions: Our results showed that local control in LABC patients achieved by primary PRT, followed by radical mastectomy was comparable with the results reported in the literature. Complete pathologic response to PRT identified a subgroup of patients with a trend toward better DFS and OS