Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues

Inhibiting endothelin receptors with macitentan strengthens the bone protective action of RANKL inhibition and reduces metastatic dissemination in osteosarcoma

Current treatments for osteosarcoma, combining conventional polychemotherapy and surgery, make it possible to attain a five-year survival rate of 70% in affected individuals. The presence of chemoresistance and metastases significantly shorten the patient’s lifespan, making identification of new therapeutic tools essential. Inhibiting bone resorption has been shown to be an efficient adjuvant strategy impacting the metastatic dissemination of osteosarcoma, tumor growth, and associated bone destruction. Unfortunately, over-apposition of mineralized matrix by normal and tumoral osteoblasts was associated with this inhibition. Endothelin signaling is implicated in the functional differentiation of osteoblasts, raising the question of the potential value of inhibiting it alone, or in combination with bone resorption repression. Using mouse models of osteosarcoma, the impact of macitentan, an endothelin receptor inhibitor, was evaluated regarding tumor growth, metastatic dissemination, matrix over-apposition secondary to RANKL blockade, and safety when combined with chemotherapy. The results showed that macitentan has no impact on tumor growth or sensitivity to ifosfamide, but significantly reduces tumoral osteoid tissue formation and the metastatic capacity of the osteosarcoma. To conclude, macitentan appears to be a promising therapeutic adjuvant for osteosarcoma alone or associated with bone resorption inhibitors

Advances in osteosarcoma

Purpose of Review This article gives a brief overview of the most recent developments in osteosarcoma treatment, including targeting of signaling pathways, immune checkpoint inhibitors, drug delivery strategies as single or combined approaches, and the identification of new therapeutic targets to face this highly heterogeneous disease. Recent Findings Osteosarcoma is one of the most common primary malignant bone tumors in children and young adults, with a high risk of bone and lung metastases and a 5-year survival rate around 70% in the absence of metastases and 30% if metastases are detected at the time of diagnosis. Despite the novel advances in neoadjuvant chemotherapy, the effective treatment for osteosarcoma has not improved in the last 4 decades. The emergence of immunotherapy has transformed the paradigm of treatment, focusing therapeutic strategies on the potential of immune checkpoint inhibitors. However, the most recent clinical trials show a slight improvement over the conventional polychemotherapy scheme. Summary The tumor microenvironment plays a crucial role in the pathogenesis of osteosarcoma by controlling the tumor growth, the metastatic process and the drug resistance and paved the way of new therapeutic options that must be validated by accurate pre-clinical studies and clinical trials

Proteoglycans and osteolysis.

Osteolysis is a complex mechanism resulting from an exacerbated activity of osteoclasts associated or not with a dysregulation of osteoblast metabolism leading to bone loss. This bone defect is not compensated by bone apposition or by apposition of bone matrix with poor mechanical quality. Osteolytic process is regulated by mechanical constraints, by polypeptides including cytokines and hormones, and by extracellular matrix components such as proteoglycans (PGs) and glycosaminoglycans (GAGs). Several studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial: some studies showed a repressive effect of GAGs on osteoclastic differentiation, whereas others described a stimulatory effect. The controversy also affects osteoblasts which appear sometimes inhibited by polysaccharides and sometimes stimulated by these compounds. Furthermore, long-term treatment with heparin leads to the development of osteoporosis fueling the controversy. After a brief description of the principal osteoclastogenesis assays, the present chapter summarizes the main data published on the effect of PGs/GAGs on bone cells and their functional incidence on osteolysis

Synthesis of Novel Mannoside Glycolipid Conjugates for Inhibition of HIV-1 Trans-Infection

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