Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved

Consequences of Daily Administered Parathyroid Hormone on Myeloma Growth, Bone Disease, and Molecular Profiling of Whole Myelomatous Bone

Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions

Archetypal autophagic players through new lenses for bone marrow stem/mature cells regulation

The bone marrow landscape consists of specialized and stem/progenitor cells, which coordinate important tissue-related and systemic physiological features. Within the marrow cavity, stem/progenitor and differentiated hematopoietic and skeletal cells congregate into dynamic functional assemblies throughout specific anatomical regions, termed niches. There is a need for better understanding of the bone marrow microareas, through exploration of the intramural physical and molecular interactions of the distinctive cell populations. The elective liaisons established among the mesenchymal/stromal stem cell and hematopoietic stem cell lineage trees play a key role in orchestrating the stem/mature cell behavior and customized hierarchies within bone marrow cell populations. Recently, the autophagic apparatus has been discovered to be an important feature of bone marrow homeostasis. Autophagy-related factors involved in the labyrinthic and highly dynamic bone marrow workshop redesign the niche framework by coordinating the operational schedule of pluripotent stem and mature cells. The following report summarizes the most recent breakthroughs in our understanding of the intramural relationships between bone marrow cells and key autophagic mediators. Doubtless, the consideration of the autophagy-related and unrelated functions of main players, such as p62, Atg7, Atg5, and Beclin-1 remains a compelling task to thoroughly understand the complex relations between the heterogenic cell types that populate bone marrow

Multihormonal control of vitellogenin mRNA expression in the liver of frog, Rana esculenta

In Rana esculenta in an in vitro system, hepatic vitellogenin synthesis can be induced by growth hormone in both sexes. In this study: (1) the ability of this hormone to induce transcription of the VTG gene was determined, and (2) this ability was compared with that of estradiol-17β. The results indicate that growth hormone stimulates VTG mRNA transcription both in vivo and in vitro, in both sexes. The levels of mRNA are related to protein levels in the medium. In addition, seasonal variation occurs in the VTG gene transcription under growth hormone and estradiol-17β; indeed the more active inducer was growth hormone during the reproductive period and estradiol-17β during the prereproductive phase

P62/SQSTM1 beyond Autophagy: Physiological Role and Therapeutic Applications in Laboratory and Domestic Animals

Inflammation is the preceding condition for the development of mild and severe pathological conditions, including various forms of osteopenia, cancer, metabolic syndromes, neurological disorders, atherosclerosis, cardiovascular, lung diseases, etc., in human and animals. The inflammatory status is induced by multifarious intracellular signaling cascades, where cytokines, chemokines, arachidonic acid metabolites, adhesion molecules, immune cells and other components foster a "slow burn" at a local or systemic level. Assuming that countering inflammation limits the development of inflammation-based diseases, a series of new side-effects-free therapies was assessed in experimental and domestic animals. Within the targets of the drug candidates for quenching inflammation, an archetypal autophagic gear, the p62/sqstm1 protein, has currently earned attention from researchers. Intracellular p62 has been recently coined as a multi-task tool associated with autophagy, bone remodeling, bone marrow integrity, cancer progression, and the maintenance of systemic homeostasis. Accordingly, p62 can act as an effective suppressor of inflamm-aging, reducing oxidative stress and proinflammatory signals. Such an operational schedule renders this protein an effective watchdog for degenerative diseases and cancer development in laboratory and pet animals. This review summarizes the current findings concerning p62 activities as a molecular hub for cell and tissues metabolism and in a variety of inflammatory diseases and other pathological conditions. It also specifically addresses the applications of exogenous p62 (DNA plasmid) as an anti-inflammatory and homeostatic regulator in the treatment of osteoporosis, metabolic syndrome, age-related macular degeneration and cancer in animals, and the possible application of p62 plasmid in other inflammation-associated diseases

NOVEL INJECTABLE HYBRID HYDROGELS AS BIOCOMPATIBLE AND BIODEGRADABLE MATRICES FOR PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS

Hydrogels are cross-linked 3-dimensional polymeric networks displaying viscoelastic behavior and tissue-like mechanical properties1. Thermosensitive hydrogels are particularly promising materials as they allow for minimally invasive administration2. In this work, a new biodegradable and thermosensitive hydrogel as drug delivery and tissue engineering system was developed.Methods. Hydrogels were tested for gelation kinetics by vial tilting method, mechanical properties by rheology (Physica – MCR 101 (Anton Paar) rheometer, 1Hz and 1% strain), degradation and release behavior by tests in phosphate buffer (PBS) pH 7.4 at 37°C. Hydrogel cytocompatibility with mouse bone marrow stromal cell (BMSCs) and NIH 3T3 mouse fibroblasts was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Biocompatibility in vivo was assessed upon subcutaneous administration of placebo hydrogels in Balb/C mice. Rapidly gelling thermosensitive hydrogels with good cyto/biocompatibility and capability to controllably release active peptides were developed. This hydrogel technology holds great potential in the pharmaceutical and biomedical fields

Thermosensitive hybrid hyaluronan/p(HPMAm‐lac)‐PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis

Osteoarthritis (OA), due to cartilage degeneration, is one of the leading causes of disability worldwide. Currently, there are not efficacious therapies to reverse cartilage degeneration. In this study we evaluated the potential of hybrid hydrogels, composed of a biodegradable and thermosensitive triblock copolymer cross‐linked via Michael addition to thiolated hyaluronic acid, in contrasting inflammatory processes underlying OA. Hydrogels composed of different w/w % concentrations of hyaluronan were investigated for their degradation behavior and capacity to release the polysaccharide in a sustained fashion. It was found that hyaluronic acid was controllably released during network degradation with a zero‐order release kinetics, and the release rate depended on cross‐link density and degradation kinetics of the hydrogels. When locally administered in vivo in an OA mouse model, the hydrogels demonstrated the ability to restore, to some extent, bone remineralization, proteoglycan production, levels of Sox‐9 and Runx‐2. Furthermore, the downregulation of proinflammatory mediators, such as TNF‐α, NFkB, and RANKL and proinflammatory cytokines was observed. In summary, the investigated hydrogel technology represents an ideal candidate for the potential encapsulation and release of drugs relevant in the field of OA. In this context, the hydrogel matrix could act in synergy with the drug, in reversing phenomena of inflammation, cartilage disruption, and bone demineralization associated with OA

The effects of 808-nm near-infrared laser light irradiation on actin cytoskeleton reorganization in bone marrow mesenchymal stem cells

Tailoring the cell organelles and thus changing cell homeostatic behavior has permitted the discovery of fascinating metabolic features enabling enhanced viability, differentiation, or quenching inflammation. Recently, photobiomodulation (PBM) has been accredited as an effective cell manipulation technique with promising therapeutic potential. In this prospective, in vitro results revealed that 808-nm laser light emitted by a hand-piece with a flat-top profile at an irradiation set up of 60 J/cm2 (1 W, 1 W/cm2; 60 s, continuous wave) regulates bone marrow stromal cell (BMSC) differentiation toward osteogenesis. Considering the importance of actin cytoskeleton reorganization, which controls a range of cell metabolic activities, comprising shape change, proliferation and differentiation, the aim of the current work is to assess whether PBM therapy, using a flat-top hand-piece at higher-fluence irradiation on BMSCs, is able to switch photon signals into the stimulation of biochemical/differentiating pathways involving key activators that regulate de novo actin polymerization. Namely, for the first time, we unearthed the role of the flat-top hand-piece at higher-fluence irradiation on cytoskeletal characteristics of BMSCs. These novel findings meet the needs of novel therapeutically protocols provided by laser treatment and the manipulation of BMSCs as anti-inflammatory, osteo-inductive platforms

The 808 nm and 980 nm infrared laser irradiation affects spore germination and stored calcium homeostasis: A comparative study using delivery hand-pieces with standard (Gaussian) or flat-top profile

Photobiomodulation relies on the transfer of energy from incident photons to a cell photoacceptor. For many years the concept of photobiomodulation and its outcome has been based upon a belief that the sole receptor within the cell was the mitochondrion. Recently, it has become apparent that there are other photoacceptors operating in different regions of the electromagnetic spectrum. Alternative photoacceptors would appear to be water and mechanisms regulating calcium homeostasis, despite a direct effect of laser photonic energy on intracellular calcium concentration outwith mitochondrial activity or influence, have not been clearly demonstrated. Therefore, to increase the knowledge of intracellular‑calcium and laser photon interaction, as well as to demonstrate differences in irradiation profiles with modern hand-pieces, we tested and compared the photobiomodulatory effect of 808 nm and 980 nm diode laser light by low- and higher-energy (60s, 100 mW/cm2, 100 mW/cm2, 500 mW/cm2, 1000 mW/cm2, 1500 mW/cm2, 2000 mW/cm2) irradiated with a "standard" (Gaussian fluence distribution) hand-piece or with a "flat-top" (uniform fluence) hand-piece. For this purpose, we used the eukaryote unicellular-model Dictyostelium discoideum. The 808 nm and 980 nm infrared laser light, at the energy tested directly affect the stored Ca2+ homeostasis, independent of the mitochondrial respiratory chain activities. From an organism perspective, the effect on Ca2+-dependent signal transduction as the regulator of spore germination in Dictyostelium, demonstrates how a cell can respond quickly to the correct laser photonic stimulus through a different cellular pathway than the known light-chromophore(mitochondria) interaction. Additionally, both hand-piece designs tested were able to photobiomodulate the D. discoideum cell; however, the hand-piece with a flat-top profile, through uniform fluence levels allows more effective and reproducible effects

The 808 nm and 980 nm infrared laser irradiation affects spore germination and stored calcium homeostasis: A comparative study using delivery hand-pieces with standard (Gaussian) or flat-top profile

reserved11Photobiomodulation relies on the transfer of energy from incident photons to a cell photoacceptor. For many years the concept of photobiomodulation and its outcome has been based upon a belief that the sole receptor within the cell was the mitochondrion. Recently, it has become apparent that there are other photoacceptors operating in different regions of the electromagnetic spectrum. Alternative photoacceptors would appear to be water and mechanisms regulating calcium homeostasis, despite a direct effect of laser photonic energy on intracellular calcium concentration outwith mitochondrial activity or influence, have not been clearly demonstrated. Therefore, to increase the knowledge of intracellular‑calcium and laser photon interaction, as well as to demonstrate differences in irradiation profiles with modern hand-pieces, we tested and compared the photobiomodulatory effect of 808 nm and 980 nm diode laser light by low- and higher-energy (60s, 100 mW/cm2, 100 mW/cm2, 500 mW/cm2, 1000 mW/cm2, 1500 mW/cm2, 2000 mW/cm2) irradiated with a "standard" (Gaussian fluence distribution) hand-piece or with a "flat-top" (uniform fluence) hand-piece. For this purpose, we used the eukaryote unicellular-model Dictyostelium discoideum. The 808 nm and 980 nm infrared laser light, at the energy tested directly affect the stored Ca2+ homeostasis, independent of the mitochondrial respiratory chain activities. From an organism perspective, the effect on Ca2+-dependent signal transduction as the regulator of spore germination in Dictyostelium, demonstrates how a cell can respond quickly to the correct laser photonic stimulus through a different cellular pathway than the known light-chromophore(mitochondria) interaction. Additionally, both hand-piece designs tested were able to photobiomodulate the D. discoideum cell; however, the hand-piece with a flat-top profile, through uniform fluence levels allows more effective and reproducible effects.mixedFerrando S.; Agas D.; Mirata S.; Signore A.; De Angelis N.; Ravera S.; Utyuzh A.S.; Parker S.; Sabbieti M.G.; Benedicenti S.; Amaroli A.Ferrando, S.; Agas, D.; Mirata, S.; Signore, A.; De Angelis, N.; Ravera, S.; Utyuzh, A. S.; Parker, S.; Sabbieti, M. G.; Benedicenti, S.; Amaroli, A