Comparison of pharmaceutical properties and biological activities of prednisolone, deflazacort, and vamorolone in DMD disease models.

Duchenne muscular dystrophy (DMD) is a progressive disabling X-linked recessive disorder that causes gradual and irreversible loss of muscle, resulting in early death. The corticosteroids prednisone/prednisolone and deflazacort are used to treat DMD as the standard of care; however, only deflazacort is FDA approved for DMD. The novel atypical corticosteroid vamorolone is being investigated for treatment of DMD. We compared the pharmaceutical properties as well as the efficacy and safety of the three corticosteroids across multiple doses in the B10-mdx DMD mouse model. Pharmacokinetic studies in the mouse and evaluation of p-glycoprotein (P-gP) efflux in a cellular system demonstrated that vamorolone is not a strong P-gp substrate resulting in measurable central nervous system (CNS) exposure in the mouse. In contrast, deflazacort and prednisolone are strong P-gp substrates. All three corticosteroids showed efficacy, but also side effects at efficacious doses. After dosing mdx mice for two weeks, all three corticosteroids induced changes in gene expression in the liver and the muscle, but prednisolone and vamorolone induced more changes in the brain than did deflazacort. Both prednisolone and vamorolone induced depression-like behavior. All three corticosteroids reduced endogenous corticosterone levels, increased glucose levels, and reduced osteocalcin levels. Using micro-computed tomography, femur bone density was decreased, reaching significance with prednisolone. The results of these studies indicate that efficacious doses of vamorolone, are associated with similar side effects as seen with other corticosteroids. Further, because vamorolone is not a strong P-gp substrate, vamorolone distributes into the CNS increasing the potential CNS side-effects.

Single Crystal X-ray Diffraction Studies of Two Polymorphic Modifications of the Dicarbonyl-o-Semiquinonato Rhodium Complex at Different Temperatures. Destruction Stimulated by Cooling Versus Stability.

It was found that the dicarbonyl-rhodium-o-semiquinonate complex (which thread-like crystals can bend reversibly under light/warm activation) can form two polymorphic modifications: isometric prisms (1) and sticks (2). Some thin sticks can bend as mentioned above. X-ray diffraction studies of polymorphic modifications at different temperatures were carried out. It was found that crystals 1 are destructed after cooling to 110 K as opposed to crystals 2. In turn, the reversible phase transition is detected in 2. In both polymorphic modifications, stack packaging motifs through the direct Rh-Rh bond are observed. The principal difference between packages of polymorphic modifications is that molecules 1 in the adjacent stacks are shifted relative to each other along the stack, in contrast to crystal 2. It was found that different packing of stacks leads to different anisotropic compression of crystals 1 and 2 during cooling, which is a key factor of their stability. Using the molecular invariom approach, the nature of the chemical bonds and charge distribution was investigated; the energy of the Rh-Rh bonds was estimated.

Novel dioxolene nickel complexes with sterically hindered diazabutadienes. Coupling of aza-ligands coordinated to nickel.

A square-planar bis-o-semiquinonato nickel complex interacts with N,N'-disubstituted 1,4-diazabutadienes-1,3 forming six-coordinate compounds. The X-ray structural study indicates complex geometry to be close to the octahedral. Magnetic properties are determined by metal-ligand ferromagnetic exchange interactions which are promoted by complex geometry. In polar solvents (THF, CH2Cl2, and CHCl3) complexes are partly dissociated into corresponding diazabutadiene-nickel catecholate and free o-quinone. In the case of the most sterically hindered 1,4-bis-(2,6-di-iso-propylphenyl)-2,3-dimethyl-1,4-diazabutadiene-1,3 in n-hexane or toluene the above-mentioned reaction is accompanied by the coupling through the back-bonded methyl groups of diazabutadiene. The organic product of the coupling was eliminated from the complex, isolated and structurally characterized. Taking into account the quantitative yield the coupling reaction is the actual procedure for the synthesis of new potential nitrogen ligands.

Ferromagnetic Coupling in the Heterospin Bis-Catecholato-Manganese(IV) Complex with Pyridine Substituted by Nitronyl-nitroxide.

A new bis(3,6-di-tert-butyl-catecholato)manganese complex with two 4-NIT-Py ligands was synthesized and characterized [4-NIT-Py = pyridine substituted at position 4 with nitronyl-nitroxide radical, 2-(pyridin-4-yl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl]. X-ray diffraction indicated an octahedral environment of the manganese atom with a trans arrangement of ligands. Bonds lengths in the inner coordination core of the metal and in the chelate cycles that are representative of the charge distribution between the metal and ligands displayed a Mn(IV)(Cat2-)2 charge distribution. Variable-temperature magnetic susceptibility measurements detected intramolecular ferromagnetic coupling between the Mn(IV) S = 3/2 spins and spins of nitronyl-nitroxyls and intermolecular ferromagnetic interactions of spins of adjacent nitronyl-nitroxide fragments in a chain of molecules at low temperatures. The last phenomenon is revealed by short contacts between nitronyl-nitroxide radicals of adjacent complex molecules.

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss.

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1(+/-) mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1(+/+) and Becn1(+/-) immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.

Autophagy opposes p53-mediated tumor barrier to facilitate tumorigenesis in a model of PALB2-associated hereditary breast cancer.

Hereditary breast cancers stem from germline mutations in susceptibility genes such as BRCA1, BRCA2, and PALB2, whose products function in the DNA damage response and redox regulation. Autophagy is an intracellular waste disposal and stress mitigation mechanism important for alleviating oxidative stress and DNA damage response activation; it can either suppress or promote cancer, but its role in breast cancer is unknown. Here, we show that similar to Brca1 and Brca2, ablation of Palb2 in the mouse mammary gland resulted in tumor development with long latency, and the tumors harbored mutations in Trp53. Interestingly, impaired autophagy, due to monoallelic loss of the essential autophagy gene Becn1, reduced Palb2-associated mammary tumorigenesis in a Trp53-wild-type but not conditionally null background. These results indicate that, in the face of DNA damage and oxidative stress elicited by PALB2 loss, p53 is a barrier to cancer development, whereas autophagy facilitates cell survival and tumorigenesis.

ATG proteins mediate efferocytosis and suppress inflammation in mammary involution.

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1(+/-) and Atg7-deficient mammary epithelial cells (MECs) produced 'competent' apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment. Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.

Autophagy regulates keratin 8 homeostasis in mammary epithelial cells and in breast tumors.

Autophagy is activated in response to cellular stressors and mediates lysosomal degradation and recycling of cytoplasmic material and organelles as a temporary cell survival mechanism. Defective autophagy is implicated in human pathology, as disruption of protein and organelle homeostasis enables disease-promoting mechanisms such as toxic protein aggregation, oxidative stress, genomic damage, and inflammation. We previously showed that autophagy-defective immortalized mouse mammary epithelial cells are susceptible to metabolic stress, DNA damage, and genomic instability. We now report that autophagy deficiency is associated with endoplasmic reticulum (ER) and oxidative stress, and with deregulation of p62-mediated keratin homeostasis in mammary cells, allograft tumors, and mammary tissues from genetically engineered mice. In human breast tumors, high phospho(Ser73)-K8 levels are inversely correlated with Beclin 1 expression. Thus, autophagy preserves cellular fitness by limiting ER and oxidative stress, a function potentially important in autophagy-mediated suppression of mammary tumorigenesis. Furthermore, autophagy regulates keratin homeostasis in the mammary gland via a p62-dependent mechanism. High phospho(Ser73)-K8 expression may be a marker of autophagy functional status in breast tumors and, as such, could have therapeutic implications for breast cancer patients.

Pathogen Phytosensing: Plants to Report Plant Pathogens.

Real-time systems that provide evidence of pathogen contamination in crops can be an important new line of early defense in agricultural centers. Plants possess defense mechanisms to protect against pathogen attack. Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-regulatory elements corresponding to key genes involved in defense, and pathogen-specific responses. Identified inducible promoters and cis-acting elements could be utilized in plant sentinels, or 'phytosensors', by fusing these to reporter genes to produce plants with altered phenotypes in response to the presence of pathogens. Here, we have employed cis-acting elements from promoter regions of pathogen inducible genes as well as those responsive to the plant defense signal molecules salicylic acid, jasmonic acid, and ethylene. Synthetic promoters were constructed by combining various regulatory elements supplemented with the enhancer elements from the Cauliflower mosaic virus (CaMV) 35S promoter to increase basal level of the GUS expression. The inducibility of each synthetic promoter was first assessed in transient expression assays using Arabidopsis thaliana protoplasts and then examined for efficacy in stably transgenic Arabidopsis and tobacco plants. Histochemical and fluorometric GUS expression analyses showed that both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohormone treatments with increased GUS expression when compared to untreated plants. Pathogen-inducible phytosensor studies were initiated by analyzing the sensitivity of the synthetic promoters against virus infection. Transgenic tobacco plants infected with Alfalfa mosaic virus showed an increase in GUS expression when compared to mock-inoculated control plants, whereas Tobacco mosaic virus infection caused no changes in GUS expression. Further research, using these transgenic plants against a range of different pathogens with the regulation of detectable reporter gene could provide biological evidence to define the functional differences between pathogens, and provide new technology and applications for transgenic plants as phytosensors.

Generation of active pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis.

A noninvasive, cell-autonomous reporter system was developed to monitor the generation and distribution of physiologically active pools of abscisic acid (ABA). ABA response (abi1-1) and biosynthesis (aba2-1) mutants of Arabidopsis (Arabidopsis thaliana) were used to validate the system in the presence and absence of water stress. In the absence of water stress, low levels of ABA-dependent reporter activation were observed in the columella cells and quiescent center of the root as well as in the vascular tissues and stomata of cotyledons, suggesting a nonstress-related role for ABA in these cell types. Exposure of seedlings to exogenous ABA resulted in a uniform pattern of reporter expression. In marked contrast, reporter expression in response to drought stress was predominantly confined to the vasculature and stomata. Surprisingly, water stress applied to the root system resulted in the generation of ABA pools in the shoot but not in the root. The analysis of the response dynamics revealed a spread of physiologically active ABA from the vascular tissue into the areoles of the cotyledons. Later, ABA preferentially activated gene expression in guard cells. The primary sites of ABA action identified by in planta imaging corresponded to the sites of ABA biosynthesis, i.e. guard cells and cells associated with vascular veins. Hence, water stress recognized by the root system predominantly results in shoot-localized ABA action that culminates in a focused response in guard cells.