Collagen and non-collagenous proteins molecular crosstalk in the pathophysiology of osteoporosis.

Abstract Collagenous and non-collagenous proteins (NCPs) in the extracellular matrix, as well as the coupling mechanisms between osteoclasts and osteoblasts, work together to ensure normal bone metabolism. Each protein plays one or more critical roles in bone metabolism, sometimes even contradictory, thus affecting the final mechanical, physical and chemical properties of bone tissue. Anomalies in the amount and structure of one or more of these proteins can cause abnormalities in bone formation and resorption, which consequently leads to malformations and defects, such as osteoporosis (OP). The connections between key proteins involved in matrix formation and resorption are far from being elucidated. In this review, we resume knowledge on the crosstalk between collagen type I and selected NCPs (Transforming Growth Factor-β, Insulin-like Growth Factor-1, Decorin, Osteonectin, Osteopontin, Bone Sialoprotein and Osteocalcin) of bone matrix, focusing on their possible involvement and role in OP. The different elements of this network can be pharmacologically targeted or used for the design/development of innovative regenerative strategies to modulate a feedback loop in bone remodelling

Otolith sectioning reveals higher maximum age in greater weever (Trachinus draco)

Growth and maximum age are two key parameters that inform resilience of fish populations to exploitation. Existing information on those for greater weever inhabiting the eastern North Sea is based on the analysis of whole otoliths. Here, we present a reanalysis using sectioned otoliths. The results reveal a different growth pattern and a higher maximum age than that previously reported. The higher maximum age makes greater weever populations more vulnerable to exploitation. Such information can serve as a basis for the estimation of the growth curve that can be used for future assessment of the species

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis

Analysis of multiple protein detection methods in human osteoporotic bone extracellular matrix: From literature to practice

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One pot-like regiospecific access to 1-aryl-1H-pyrazol-3(2H)-one derivatives and evaluation of the anticancer activity

A set of variously substituted 1-arylpyrazol-3-one derivatives, including the di-ortho-aryl substituted ones, was synthesized as new potential anticancer compounds. To fulfill this aim, herein a regiospecific synthesis was proposed utilizing a new revisited one pot procedure, starting from commercial anilines and easily accessible 2,5-dimethyl-furan-3-one. In the course of the sequential ordered steps, in some cases, a nitro group displacement by chlorine took place to a minor extent. The in vitro screening against the full panel of ~60 human cancer cell lines (NCI) showed a moderate, but promising selective antiproliferative activity against the UO31 renal tumor cell line, only in compounds with the introduction on the phenyl moiety of a -CF3 or two Cl groups

BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly, rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases

Injectable thermosensitive formulation based on polyurethane hydrogel/mesoporous glasses for sustained co-delivery of functional ions and drugs

Mini-invasively injectable hydrogels are widely attracting interest as smart tools for the co-delivery of therapeutic agents targeting different aspects of tissue/organ healing (e.g., neo-angiogenesis, inflammation). In this work, copper-substituted bioactive mesoporous glasses (Cu-MBGs) were prepared as nano- and micro-particles and successfully loaded with ibuprofen through an incipient wetness method (loaded ibuprofen approx. 10% w/w). Injectable hybrid formulations were then developed by dispersing ibuprofen-loaded Cu-MBGs within thermosensitive hydrogels based on a custom-made amphiphilic polyurethane. This procedure showed almost no effects on the gelation potential (gelation at 37 °C within 3–5 min). Cu2+ and ibuprofen were co-released over time in a sustained manner with a significantly lower burst release compared to MBG particles alone (burst release reduction approx. 85% and 65% for ibuprofen and Cu2+, respectively). Additionally, released Cu2+ species triggered polyurethane chemical degradation, thus enabling a possible tuning of gel residence time at the pathological site. The overall results suggest that hybrid injectable thermosensitive gels could be successfully designed for the simultaneous localized co-delivery of multiple therapeutics

cAMP and Pyk2 interact to regulate prostate cell proliferation and function.

In cultured prostate cancer cells cAMP blocks proliferation and induces neuroendocrine differentiation. Pyk2 expression inversely correlates with malignancy of prostate cancer. The aim of this study was to investigate the interaction between cAMP and Pyk2 in the prostate. EPN cells, a line derived from human normal prostate expressing Pyk2, and EPN-PKM3 cells, an EPN clone bearing a Pyk2 kinase-negative mutant, were adopted as model system. cAMP inhibited cell growth in both prostate cell lines, and activated Pyk2, but not ERK1/2, in EPN cells. cAMP treatment, abolished the activation of AKT1, an important component of the pro-survival pathway, in the EPN cells but not in EPN-PKM3 cells. Finally, upon cAMP treatment, EPN and EPN-PKM3 cells exhibited different expression patterns of HOX genes, an important network controlling cell identity. These data demonstrated for the first time that Pyk2 and cAMP interact in regulating prostate cell functions and in "keeping" prostate identity

Different ability of three Mediterranean oak species to tolerate progressive water stress

Inter-comparisons in the gas exchange patterns and root characteristics under both well-watered and drought conditions were done in three-years-old seedlings of three oak species (Quercus cerris L., Q. frainetto Ten., and Q. ilex L.) growing in controlled environment. Well-watered Q. cerris had greater physiological performances than other oaks, but under drought it was not able to face the water stress showing also structural modifications such as reduction of root length and average diameter. On the other hand, Q. ilex maintained root growth both in drought or well-watered soils. Moreover, it was able to keep open stomata also under water stress, although stomatal conductance (g s) was low. Q. frainetto had an intermediate position in regard to its physiological and root structural characteristics between Q. cerris and Q. ilex under drought stress. For all oaks the relationship between g s and the ratio of sub-stomatal and ambient CO2 concentration (C i/C a) highlighted the dynamic adaptation of g s to the increase of hydraulic resistances of leaf, stem, and roots portions, more evident during the air humidity change and progressive soil dehydration. This suggests a well-triggered above-and under-ground mechanism to endure the drought stress