A Lightweight Browser-Based Tool for Collaborative and Blinded Image Analysis.

Collaborative manual image analysis by multiple experts in different locations is an essential workflow in biomedical science. However, sharing the images and writing down results by hand or merging results from separate spreadsheets can be error-prone. Moreover, blinding and anonymization are essential to address subjectivity and bias. Here, we propose a new workflow for collaborative image analysis using a lightweight online tool named Tyche. The new workflow allows experts to access images via temporarily valid URLs and analyze them blind in a random order inside a web browser with the means to store the results in the same window. The results are then immediately computed and visible to the project master. The new workflow could be used for multi-center studies, inter- and intraobserver studies, and score validations.

Autonomic nervous regulation of cellular processes during subchondral bone remodeling in osteoarthritis.

Osteoarthritis (OA) is the most prevalent degenerative joint disease. Besides loss of articular cartilage and synovial inflammation, OA progression is characterized by pathological changes in the subchondral bone. In early OA, subchondral bone remodeling typically shifts to an increased bone resorption. However, as the disease progresses an increased bone formation takes place, leading to higher bone density with subsequent bone sclerosis. These changes can be influenced by different local or systemic factors. Recent evidence suggests that the autonomic nervous system (ANS) plays a role in regulating subchondral bone remodeling in OA. In this review, we 1) introduce bone structure and cellular mechanisms of bone remodeling in general, 2) explain the subchondral bone changes during OA pathogenesis, 3) then describe the contribution of the sympathetic nervous system (SNS) and parasympathetic nervous system (PNS), the two major autonomic branches, to physiological subchondral bone remodeling, 4) followed by the influence of the SNS and PNS on subchondral bone remodeling in OA, and 5) finally, discuss the potential of therapeutic approaches targeting different components of the ANS.NEW & NOTEWORTHY The autonomic nervous system (ANS) with its two major branches, the sympathetic and parasympathetic nervous systems, plays a role in osteoarthritis pathogenesis by influencing bone structure and remodeling. We here review the current knowledge on subchondral bone remodeling with special regard to different bone cell types and underlying mechanisms at the cellular and molecular level. A better understanding of these mechanisms is needed for the development of novel OA treatment strategies targeting the ANS.

Prostate-Specific Membrane Antigen (PSMA)-Positive Extracellular Vesicles in Urine-A Potential Liquid Biopsy Strategy for Prostate Cancer Diagnosis?

All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.

Adrenergic signalling in osteoarthritis.

Adrenoceptors (ARs) mediate the effects of the sympathetic neurotransmitters norepinephrine (NE) and epinephrine (E) in the human body and play a central role in physiologic and pathologic processes. Therefore, ARs have long been recognized as targets for therapeutic agents, especially in the field of cardiovascular medicine. During the past decades, the contribution of the sympathetic nervous system (SNS) and particularly of its major peripheral catecholamine NE to the pathogenesis of osteoarthritis (OA) attracted growing interest. OA is the most common degenerative joint disorder worldwide and a disease of the whole joint. It is characterized by progressive degradation of articular cartilage, synovial inflammation, osteophyte formation, and subchondral bone sclerosis mostly resulting in chronic pain. The subchondral bone marrow, the periosteum, the synovium, the vascular meniscus and numerous tendons and ligaments are innervated by tyrosine hydroxylase-positive (TH+) sympathetic nerve fibers that release NE into the synovial fluid and cells of all abovementioned joint tissues express at least one out of nine AR subtypes. During the past decades, several in vitro studies explored the AR-mediated effects of NE on different cell types in the joint. So far, only a few studies used animal OA models to investigate the contribution of distinct AR subtypes to OA pathogenesis in vivo. This narrative review shortly summarizes the current background knowledge about ARs and their signalling pathways at first. In the second part, we focus on recent findings in the field of NE-induced AR-mediated signalling in different joint tissues during OA pathogenesis and at the end, we will delineate the potential of targeting the adrenergic signalling for OA prevention or treatment. We used the PubMed bibliographic database to search for keywords such as 'joint' or 'cartilage' or 'synovium' or 'bone' and 'osteoarthritis' and/or 'trauma' and 'sympathetic nerve fibers' and/or 'norepinephrine' and 'adrenergic receptors / adrenoceptors' as well as 'adrenergic therapy'.

ß2-Adrenoceptor Deficiency Results in Increased Calcified Cartilage Thickness and Subchondral Bone Remodeling in Murine Experimental Osteoarthritis.

Purpose: Recent studies demonstrated a contribution of adrenoceptors (ARs) to osteoarthritis (OA) pathogenesis. Several AR subtypes are expressed in joint tissues and the ß2-AR subtype seems to play a major role during OA progression. However, the importance of ß2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in ß2-AR-deficient (Adrb2-/- ) mice after surgical OA induction. Methods: OA was induced by destabilization of the medial meniscus (DMM) in male wildtype (WT) and Adrb2-/- mice. Cartilage degeneration and synovial inflammation were evaluated by histological scoring. Subchondral bone remodeling was analyzed using micro-CT. Osteoblast (alkaline phosphatase - ALP) and osteoclast (cathepsin K - CatK) activity were analyzed by immunostainings. To evaluate ß2-AR deficiency-associated effects, body weight, sympathetic tone (splenic norepinephrine (NE) via HPLC) and serum leptin levels (ELISA) were determined. Expression of the second major AR, the α2-AR, was analyzed in joint tissues by immunostaining. Results: WT and Adrb2-/- DMM mice developed comparable changes in cartilage degeneration and synovial inflammation. Adrb2-/- DMM mice displayed elevated calcified cartilage and subchondral bone plate thickness as well as increased epiphyseal BV/TV compared to WTs, while there were no significant differences in Sham animals. In the subchondral bone of Adrb2-/- mice, osteoblasts activity increased and osteoclast activity deceased. Adrb2-/- mice had significantly higher body weight and fat mass compared to WT mice. Serum leptin levels increased in Adrb2-/- DMM compared to WT DMM without any difference between the respective Shams. There was no difference in the development of meniscal ossicles and osteophytes or in the subarticular trabecular microstructure between Adrb2-/- and WT DMM as well as Adrb2-/- and WT Sham mice. Number of α2-AR-positive cells was lower in Adrb2-/- than in WT mice in all analyzed tissues and decreased in both Adrb2-/- and WT over time. Conclusion: We propose that the increased bone mass in Adrb2-/- DMM mice was not only due to ß2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, ß2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues.

Compromised repair of radiation-induced DNA double-strand breaks in Fanconi anemia fibroblasts in G2.

Fanconi anemia (FA) is a rare chromosomal instability syndrome with various clinical features and high cancer incidence. Despite being a DNA repair disorder syndrome and a frequently observed clinical hypersensitivity of FA patients towards ionizing radiation, the experimental evidence regarding the efficiency of radiation-induced DNA double-strand break (DSB) repair in FA is very controversial. Here, we performed a thorough analysis of the repair of radiation-induced DSBs in G1 and G2 in FA fibroblasts of complementation groups A, C, D1 (BRCA2), D2, E, F, G and P (SLX4) in comparison to normal human lung and skin fibroblasts. γH2AX, 53BP1, or RPA foci quantification after X-irradiation was combined with cell cycle markers. Cytogenetic analyses were performed on first metaphases after irradiation in G1 and by premature chromosome condensation after exposure in G2. Furthermore, the role of canonical-NHEJ and alternative-NHEJ for the fidelity of the repair of radiation-induced DSBs was examined. In FA fibroblasts, DSB repair was normal in G1 but compromised and more error-prone in the slow repair component of G2 as suggested by higher yields of radiation-induced γH2AX and 53BP1 foci as well as chromatid exchanges. However, RPA foci quantification in G2 indicated proficiency for homology-directed repair of DSBs in FA except for FA D1 (BRCA2). In lung fibroblasts, DSB repair in G1 was conducted with normal kinetics but elevated chromosome exchanges compared to skin fibroblasts. The overall repair of radiation-induced DSBs and the formation of chromosome exchanges in normal and FA fibroblasts in G1 and G2 were governed by canonical-NHEJ with no contribution of alternative-NHEJ. Together, we show impaired repair of radiation-induced DSBs in various FA complementation groups in the slow repair component of G2 that might promote the formation of potentially oncogenic aberrations and clinical radiation hypersensitivity.