How Homocysteine Modulates the Function of Osteoblasts and Osteocytes

Over the years, numerous mechanisms have been identified through which homocysteine affects osteoblast functioning. These include alterations in collagen structure, epigenetic modifications and changes in RANKL-OPG production by osteoblasts. These mechanisms are reviewed in this chapter. We have also herein discussed how homocysteine affects osteocyte behavior. With onset of hyperhomocysteinemia induction of osteocyte specific genes particularly the mineralization genes like Dmp1 and Sost is facilitated producing untoward mineralization, osteocyte apoptosis, deviations from regular bone remodeling process and onset of targeted remodeling in bone. These modifications have immense effect on the overall mechanical stability of bone. Homocysteine thus represents an independent risk factor for bone fragility

Exploring Goal Conflicts and How They Are Managed in a Biomedical Laboratory Using Rasmussen’s Model of Boundaries

Introduction: Occupational health and safety management systems are widely used as a systematic approach to managing occupational health and safety. However, sometimes they are restrictive and underspecified to deal with dynamic workplace demands. Rasmussen used a model of boundaries to conceptualize this dynamic model of safety, where the space of possibilities lay within 3 boundaries and workers used various means to stay within the boundaries to remain both productive and safe at work. Methods: This study applied the Rasmussen model of boundaries to understand the factors that formed the boundaries, the gradients, and countergradients in a biomedical laboratory. Results: The most central goal was to be the first to publish, and this formed the boundary to scientific output failure; the boundary to unacceptable workload and boundary to functionally acceptable performance were the other 2 boundaries in line with the Rasmussen model. The workers had developed methods (mental risk assessment, teamwork, and experience and familiarity) of working, which ensured they remained productive and safe. This can be described as resilient performance, where resilience is not something that a system has but something it does to adjust their performance when faced with expected or unexpected changes. Discussion and Conclusion: A customized portfolio of rule-based non negotiable instructions and a risk assessment–based approach would be best suited for a biomedical laboratory. The workers have learned resilient performance on their own and unknowingly are already practicing this. It is now time to formally incorporate such practices into the safety systems of biomedical laboratories

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2 A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1 alpha, IL-1 beta, G-CSF, GM-CSF, MIP-1 alpha, IFN-gamma, IL-17, and TNF-alpha. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality (C) 2013 Elsevier Inc. All rights reserved

Development and characterization of supramolecular calcitonin assembly and assessment of its interactions with the bone remodelling process

Osteoporosis is the most common metabolic bone disease, which poses an immense socio-economic burden on the society. Human calcitonin, though safe, is not considered as a therapeutic option because of its high tendency to self-associate to form amyloid fibrils thereby affecting its potency. To circumvent this issue we harnessed the inherent capacity of aggregation and developed an assemblage of human calcitonin monomers, Supramolecular Calcitonin Assembly (SCA-I)], which releases biologically active calcitonin monomers in a sustained manner for a period of at least three weeks. AFM and FT-IR analysis showed that SCA-I is amorphous aggregates of calcitonin monomers. Both SCA-I and monomer released from it demonstrated superior anti-osteoclast activity and proteolytic stability in-vitro. SCA-I upon single injection significantly improved bone formation markers and reduced bone resorption markers in ovariectomized (OVX) rat model of postmenopausal osteoporosis. Micro-CT analysis revealed that calcitonin released from SCA-I exhibits its beneficial effect on cortical bone more profoundly compared to trabecular bone. This study demonstrates that SCA-I is more effective compared to the human calcitonin monomers on osteoclasts and has site-specific effect on bone in a model of post-menopausal osteoporosis. This approach opens up an innovative way to use and study the function of human calcitonin

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field