P7C3 ameliorates barium chloride-induced skeletal muscle injury activating transcriptomic and epigenetic modulation of myogenic regulatory factors.

Skeletal muscle injury affects the quality of life in many pathologies, including volumetric muscle loss, contusion injury, and aging. We hypothesized that the nicotinamide phosphoribosyltransferase (Nampt) activator P7C3 improves muscle repair following injury. In the present study, we tested the effect of P7C3 (1-anilino-3-(3,6-dibromocarbazol-9-yl) propan-2-ol) on chemically induced muscle injury. Muscle injury was induced by injecting 50 µL 1.2% barium chloride (BaCl2) into the tibialis anterior (TA) muscle in C57Bl/6J wild-type male mice. Mice were then treated with either 10 mg/kg body weight of P7C3 or Vehicle intraperitoneally for 7 days and assessed for histological, biochemical, and molecular changes. In the present study, we show that the acute BaCl2-induced TA muscle injury was robust and the P7C3-treated mice displayed a significant increase in the total number of myonuclei and blood vessels, and decreased serum CK activity compared with vehicle-treated mice. The specificity of P7C3 was evaluated using Nampt+/- mice, which did not display any significant difference in muscle repair capacity among treated groups. RNA-sequencing analysis of the injured TA muscles displayed 368 and 212 genes to be exclusively expressed in P7C3 and Veh-treated mice, respectively. There was an increase in the expression of genes involved in cellular processes, inflammatory response, angiogenesis, and muscle development in P7C3 versus Veh-treated mice. Conversely, there is a decrease in muscle structure and function, myeloid cell differentiation, glutathione, and oxidation-reduction, drug metabolism, and circadian rhythm signaling pathways. Chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) and reverse transcription-qPCR analyses identified increased Pax7, Myf5, MyoD, and Myogenin expression in P7C3-treated mice. Increased histone lysine (H3K) methylation and acetylation were observed in P7C3-treated mice, with significant upregulation in inflammatory markers. Moreover, P7C3 treatment significantly increased the myotube fusion index in the BaCl2-injured human skeletal muscle in vitro. P7C3 also inhibited the lipopolysaccharide-induced inflammatory response and mitochondrial membrane potential of RAW 264.7 macrophage cells. Overall, we demonstrate that P7C3 activates muscle stem cells and enhances muscle injury repair with increased angiogenesis.

Development and testing of nanoparticles delivery for P7C3 small molecule using injury models.

The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3 nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72 h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.

Evaluation of improvements in plan quality with Photon Optimizer v16.1 for single brain lesion SRS treatment.

Background: The purpose of this study is to compare the performance of the Photon Optimizer (PO) version 16.1 algorithm with its earlier version PO v13.6 and with Progressive Resolution Optimizer (PRO) version 13.6 algorithms. Materials and methods: 20 patients with single brain lesions treated with the stereotactic radiosurgery (SRS) technique were retrospectively selected for this study. Initially, for all patients volumetric modulated arc therapy (VMAT) SRS plans were generated with the PRO v 13.6 algorithm. Then, all the plans were re-generated with two versions 13.6 and 16.1 of PO algorithm using the same setup and dose-volume optimization objectives as that of PRO with a similar planning approach. The quality of the generated plans was analysed using ICRU 91 plan evaluation parameters and also using dice similarity co-efficient (DSC), centre of mass distance (CMD) between target and prescription isodose line, Monitor units (MU) and brain-gross tumor volume (GTV) 12 Gy volume. Paired Student t-test was used for statistical analysis with 0.05 as a significant value. Results: PO v16.1 improved all the dosimetric parameters studied compared to PO 13.6, the difference is statistically significant for all the parameters (p < 0.05), except for median dose and brain-GTV 12 Gy volume. PO v16.1 also showed statistically significant improvement for all the dosimetric parameters evaluated, except DSC and conformity index (CI), compared to PRO v13.6. Conclusion: The PO v16.1 generated plans are dosimetrically superior to PO v13.6 and PRO v13.6 in terms of target dose coverage and dose gradient with lesser beam modulation and plan complexity for single brain lesion SRS.

Cardioprotective Effects of 1-(3,6-Dibromo-carbazol-9-yl)-3-Phenylamino-Propan-2-Ol in Diabetic Hearts via Nicotinamide Phosphoribosyltransferase Activation.

Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD+ salvage pathway and is dysregulated in diabetes. Nampt activation results in rescued NADH/NAD+ ratios and provides pharmacological changes necessary for diabetic cardioprotection. Computer docking shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) allows for enhanced Nampt dimerization and association. To test the pharmacological application, we used male leptin receptor-deficient (db/db) mice and treated them with Nampt activator P7C3. The effects of 4-week P7C3 treatment on cardiac function were evaluated along with molecular signaling changes for phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS), and sirtuin 1 (SIRT1). The cardiac function evaluated by ECG and echocardiography were significantly improved after 4 weeks of P7C3 treatment. Biochemically, higher NADH/NAD+ ratios in diabetic hearts were rescued by P7C3 treatment. Moreover, activities of Nampt and SIRT1 were significantly increased in P7C3-treated diabetic hearts. P7C3 treatment significantly decreased the blood glucose in diabetic mice with 4-week treatment as noted by glucose tolerance test and fasting blood glucose measurements compared with vehicle-treated mice. P7C3 activated Nampt enzymatic activity both in vitro and in the 4-week diabetic mouse hearts, demonstrating the specificity of the small molecule. P7C3 treatment significantly enhanced the expression of cardioprotective signaling of p-AKT, p-eNOS, and Beclin 1 in diabetic hearts. Nampt activator P7C3 allows for decreased infarct size with decreased Troponin I and lactose dehydrogenase (LDH) release, which is beneficial to the heart. Overall, the present study shows that P7C3 activates Nampt and SIRT1 activity and decreases NADH/NAD+ ratio, resulting in improved biochemical signaling providing cardioprotection. SIGNIFICANCE STATEMENT: This study shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) is effective in treating diabetes and cardiovascular diseases. The novel small molecule is antiarrhythmic and improves the ejection fraction in diabetic hearts. The study successfully demonstrated that P7C3 decreases the infarct size in hearts during myocardial infarction and ischemia-reperfusion injury. Biochemical and cellular signaling show increased NAD+ levels, along with Nampt activity involved in upregulating protective signaling in the diabetic heart. P7C3 has high therapeutic potential for rescuing heart disease.

NAD+ centric mechanisms and molecular determinants of skeletal muscle disease and aging.

The nicotinamide adenine dinucleotide (NAD+) is an essential redox cofactor, involved in various physiological and molecular processes, including energy metabolism, epigenetics, aging, and metabolic diseases. NAD+ repletion ameliorates muscular dystrophy and improves the mitochondrial and muscle stem cell function and thereby increase lifespan in mice. Accordingly, NAD+ is considered as an anti-oxidant and anti-aging molecule. NAD+ plays a central role in energy metabolism and the energy produced is used for movements, thermoregulation, and defense against foreign bodies. The dietary precursors of NAD+ synthesis is targeted to improve NAD+ biosynthesis; however, studies have revealed conflicting results regarding skeletal muscle-specific effects. Recent advances in the activation of nicotinamide phosphoribosyltransferase in the NAD+ salvage pathway and supplementation of NAD+ precursors have led to beneficial effects in skeletal muscle pathophysiology and function during aging and associated metabolic diseases. NAD+ is also involved in the epigenetic regulation and post-translational modifications of proteins that are involved in various cellular processes to maintain tissue homeostasis. This review provides detailed insights into the roles of NAD+ along with molecular mechanisms during aging and disease conditions, such as the impacts of age-related NAD+ deficiencies on NAD+-dependent enzymes, including poly (ADP-ribose) polymerase (PARPs), CD38, and sirtuins within skeletal muscle, and the most recent studies on the potential of nutritional supplementation and distinct modes of exercise to replenish the NAD+ pool.

PPARs and Microbiota in Skeletal Muscle Health and Wasting.

Skeletal muscle is a major metabolic organ that uses mostly glucose and lipids for energy production and has the capacity to remodel itself in response to exercise and fasting. Skeletal muscle wasting occurs in many diseases and during aging. Muscle wasting is often accompanied by chronic low-grade inflammation associated to inter- and intra-muscular fat deposition. During aging, muscle wasting is advanced due to increased movement disorders, as a result of restricted physical exercise, frailty, and the pain associated with arthritis. Muscle atrophy is characterized by increased protein degradation, where the ubiquitin-proteasomal and autophagy-lysosomal pathways, atrogenes, and growth factor signaling all play an important role. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family of transcription factors, which are activated by fatty acids and their derivatives. PPARs regulate genes that are involved in development, metabolism, inflammation, and many cellular processes in different organs. PPARs are also expressed in muscle and exert pleiotropic specialized responses upon activation by their ligands. There are three PPAR isotypes, viz., PPARα, -ß/δ, and -γ. The expression of PPARα is high in tissues with effective fatty acid catabolism, including skeletal muscle. PPARß/δ is expressed more ubiquitously and is the predominant isotype in skeletal muscle. It is involved in energy metabolism, mitochondrial biogenesis, and fiber-type switching. The expression of PPARγ is high in adipocytes, but it is also implicated in lipid deposition in muscle and other organs. Collectively, all three PPAR isotypes have a major impact on muscle homeostasis either directly or indirectly. Furthermore, reciprocal interactions have been found between PPARs and the gut microbiota along the gut-muscle axis in both health and disease. Herein, we review functions of PPARs in skeletal muscle and their interaction with the gut microbiota in the context of muscle wasting.

Depletion of Gram-Positive Bacteria Impacts Hepatic Biological Functions During the Light Phase.

Living organisms display internal biological rhythms, which are an evolutionarily conserved adaptation to the environment that drives their rhythmic behavioral and physiological activities. The gut microbiota has been proposed, in association with diet, to regulate the intestinal peripheral clock. However, the effect of gut dysbiosis on liver remains elusive, despite that germfree mice show alterations in liver metabolic functions and the hepatic daily rhythm. We analyzed whether the disruption of gut microbial populations with various antibiotics would differentially impact liver functions in mice. Our results support the notion of an impact on the hepatic biological rhythm by gram-positive bacteria. In addition, we provide evidence for differential roles of gut microbiota spectra in xenobiotic metabolism that could protect against the harmful pharmacological effects of drugs. Our results underscore a possible link between liver cell proliferation and gram-positive bacteria.

Metronidazole Causes Skeletal Muscle Atrophy and Modulates Muscle Chronometabolism.

Antibiotics lead to increased susceptibility to colonization by pathogenic organisms, with different effects on the host-microbiota relationship. Here, we show that metronidazole treatment of specific pathogen-free (SPF) mice results in a significant increase of the bacterial phylum Proteobacteria in fecal pellets. Furthermore, metronidazole in SPF mice decreases hind limb muscle weight and results in smaller fibers in the tibialis anterior muscle. In the gastrocnemius muscle, metronidazole causes upregulation of Hdac4, myogenin, MuRF1, and atrogin1, which are implicated in skeletal muscle neurogenic atrophy. Metronidazole in SPF mice also upregulates skeletal muscle FoxO3, described as involved in apoptosis and muscle regeneration. Of note, alteration of the gut microbiota results in increased expression of the muscle core clock and effector genes Cry2, Ror-ß, and E4BP4. PPARγ and one of its important target genes, adiponectin, are also upregulated by metronidazole. Metronidazole in germ-free (GF) mice increases the expression of other core clock genes, such as Bmal1 and Per2, as well as the metabolic regulators FoxO1 and Pdk4, suggesting a microbiota-independent pharmacologic effect. In conclusion, metronidazole in SPF mice results in skeletal muscle atrophy and changes the expression of genes involved in the muscle peripheral circadian rhythm machinery and metabolic regulation.

Inactivation of PPARß/δ adversely affects satellite cells and reduces postnatal myogenesis.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a ubiquitously expressed gene with higher levels observed in skeletal muscle. Recently, our laboratory showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that PPARß/δ modulates myostatin activity to induce myogenesis in skeletal muscle. In the present study, we show that PPARß/δ-null mice display reduced body weight, skeletal muscle weight, and myofiber atrophy during postnatal development. In addition, a significant reduction in satellite cell number was observed in PPARß/δ-null mice, suggesting a role for PPARß/δ in muscle regeneration. To investigate this, tibialis anterior muscles were injured with notexin, and muscle regeneration was monitored on days 3, 5, 7, and 28 postinjury. Immunohistochemical analysis revealed an increased inflammatory response and reduced myoblast proliferation in regenerating muscle from PPARß/δ-null mice. Histological analysis confirmed that the regenerated muscle fibers of PPARß/δ-null mice maintained an atrophy phenotype with reduced numbers of centrally placed nuclei. Even though satellite cell numbers were reduced before injury, satellite cell self-renewal was found to be unaffected in PPARß/δ-null mice after regeneration. Previously, our laboratory had showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that inactivation of PPARß/δ increases myostatin signaling and inhibits myogenesis. Our results here indeed confirm that inactivation of myostatin signaling rescues the atrophy phenotype and improves muscle fiber cross-sectional area in both uninjured and regenerated tibialis anterior muscle from PPARß/δ-null mice. Taken together, these data suggest that absence of PPARß/δ leads to loss of satellite cells, impaired skeletal muscle regeneration, and postnatal myogenesis. Furthermore, our results also demonstrate that functional antagonism of myostatin has utility in rescuing these effects.

Effect of Detector Orientation and Influence of Jaw Position in the Determination of Small-field Output Factor with Various Detectors for High-energy Photon Beams.

Background: Accurate dose measurements are difficult in small fields due to charge particle disequilibrium, partial source occlusion, steep dose gradient, and the finite size of the detector. Aim: The study aims to determine the output factor using various detectors oriented in parallel and perpendicular orientations for three different tertiary collimating systems using 15 MV photon beams. In addition, this study analyzes how the output factor could be affected by different configurations of X and Y jaws above the tertiary collimators. Materials and Methods: Small field output factor measurements were carried out with three detectors for different tertiary collimating systems such as BrainLab stereotactic cones, BrainLab mMLC and Millennium MLC namely. To analyze the effect of jaw position on output factor, measurements have been carried out by positioning the jaws at the edge, 0.25, 0.5, and 1.0 cm away from the tertiary collimated field. Results: The data acquired with 15 MV photon beams show significant differences in output factor obtained with different detectors for all collimating systems. For smaller fields when compared to microDiamond, the SRS diode underestimates the output by up to -1.7% ± 0.8% and -2.1% ± 0.3%, and the pinpoint ion chamber underestimates the output by up to -8.1% ± 1.4% and -11.9% ± 1.9% in their parallel and perpendicular orientation respectively. A large increase in output factor was observed in the small field when the jaw was moved 0.25 cm symmetrically away from the tertiary collimated field. Conclusion: The investigated data on the effect of jaw position inferred that the position of the X and Y jaw highly influences the output factors of the small field. It also confirms that the output factor highly depends on the configuration of X and Y jaw settings, the tertiary collimating system as well as the orientation of the detectors in small fields.

Genetic deletion of Kvß2 (AKR6) causes loss of muscle function and increased inflammation in mice.

The voltage-gated potassium channels (Kv) are complex ion channels with distinct roles in neurotransmission, electrical conductivity of the heart, and smooth and striated muscle functions. Previously, we demonstrated that deletion of Kvß2 in mice results in decreased Pax7 protein levels, hindlimb muscles and body weights, and fiber type switching. In the present study, we tested the hypothesis that Kvß2 regulates skeletal muscle function in mice. The young and old Kvß2 knockout (KO) and wildtype (WT) mice were utilized to test the aging phenotype and skeletal muscle function. Consistent with our previous finding, we found a significant reduction in hindlimb skeletal muscles mass and body weight in young Kvß2 KO mice, which was also significantly reduced in old Kvß2 KO mice compared with age-matched WT mice. Forelimb grip strength, and the hindleg extensor digitorum longus (EDL) muscles force-frequency relations were significantly decreased in young and old Kvß2 KO mice compared to age-matched WT mice. Analysis of transmission electron microscopy images of EDL muscles in young mice revealed a significant reduction in the sarcomere length for Kvß2 KO vs. WT. Hematoxylin and eosin-stained tibialis anterior muscles cryosections displayed a significant decrease in the number of medium (2,000-4,000 µm2) and largest (>4,000 µm2) myofibers area in young Kvß2 KO vs. WT mice. We also found a significant increase in fibrotic tissue area in young Kvß2 KO mice compared with age-matched WT mice. Analysis of RNA Seq data of the gastrocnemius muscles (GAS) identified significant increase in genes involved in skeletal muscle development, proliferation and cell fate determination, atrophy, energy metabolism, muscle plasticity, inflammation, and a decrease in circadian core clock genes in young Kvß2 KO vs. WT mice. Several genes were significantly upregulated (384 genes) and downregulated (40 genes) in young Kvß2 KO mice compared to age-matched WT mice. Further, RT-qPCR analysis of the GAS muscles displayed a significant increase in pro-inflammatory marker Il6 expression in young Kvß2 KO mice compared to age-matched WT mice. Overall, the present study shows that deletion of Kvß2 leads to decreased muscles strength and increased inflammation.

Comparison of individual and composite field analysis using array detector for Intensity Modulated Radiotherapy dose verification.

AIM: To compare the measured and calculated individual and composite field planar dose distribution of Intensity Modulated Radiotherapy plans. MATERIALS AND METHODS: The measurements were performed in Clinac DHX linear accelerator with 6 MV photons using Matrixx device and a solid water phantom. The 20 brain tumor patients were selected for this study. The IMRT plan was carried out for all the patients using Eclipse treatment planning system. The verification plan was produced for every original plan using CT scan of Matrixx embedded in the phantom. Every verification field was measured by the Matrixx. The TPS calculated and measured dose distributions were compared for individual and composite fields. RESULTS AND DISCUSSION: The percentage of gamma pixel match for the dose distribution patterns were evaluated using gamma histogram. The gamma pixel match was 95-98% for 41 fields (39%) and 98% for 59 fields (61%) with individual fields. The percentage of gamma pixel match was 95-98% for 5 patients and 98% for other 12 patients with composite fields. Three patients showed a gamma pixel match of less than 95%. The comparison of percentage gamma pixel match for individual and composite fields showed more than 2.5% variation for 6 patients, more than 1% variation for 4 patients, while the remaining 10 patients showed less than 1% variation. CONCLUSION: The individual and composite field measurements showed good agreement with TPS calculated dose distribution for the studied patients. The measurement and data analysis for individual fields is a time consuming process, the composite field analysis may be sufficient enough for smaller field dose distribution analysis with array detectors.

Nampt activator P7C3 ameliorates diabetes and improves skeletal muscle function modulating cell metabolism and lipid mediators.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6-dibromo-α-[(phenylamino)methyl]-9H-carbazol-9-ethanol) ameliorates diabetes and muscle function. METHODS: We assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/- mice were utilized to test the specificity of P7C3. RESULTS: Insulin resistance increased 1.6-fold in diabetic mice compared with wild-type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db-P7C3 mice fasting blood glucose levels decreased to 0.96-fold compared with C57Bl/6J wild-type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6-fold and 0.54-folds, respectively, at 120 min along with an increase in insulin secretion (1.76-fold) and pancreatic ß-cells (3.92-fold) in db-P7C3 mice. The fore-limb and hind-limb grip strengths were increased to 1.13-fold and 1.17-fold, respectively, together with a 14.2-fold increase in voluntary running wheel distance in db-P7C3 mice. P7C3 treatment resulted in a 1.4-fold and 7.1-fold increase in medium-sized and larger-sized myofibres cross-sectional area, with a concomitant 0.5-fold decrease in smaller-sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67-fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9-fold decrease in mitochondrial area in db-P7C3 mice compared with db-Veh mice. The number of SDH positive myofibres were increased to 1.74-fold in db-P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46-fold) and immunostaining (6.4-fold), respectively. qPCR analysis displayed a 2.9-fold and 1.3-fold increase in Pdk4 and Cpt1, and 0.55-fold and 0.59-fold decrease in Fgf21 and 16S in db-P7C3 mice. There was also a 3.3-fold and 1.9-fold increase in Fabp1 and CD36 in db-Veh mice. RNA-seq differential gene expression volcano plot displayed 1415 genes to be up-regulated and 1726 genes down-regulated (P < 0.05) in db-P7C3 mice. There was 1.02-fold increase in serum HDL, and 0.9-fold decrease in low-density lipoprotein/very low-density lipoprotein ratio in db-P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n-6; AA (0.83-fold), and n-3; DHA (0.69-fold) and EPA (0.81-fold), and a 0.66-fold decrease in endocannabinoid 2-AG and 2.0-fold increase in AEA in db-P7C3 mice. CONCLUSIONS: Overall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.

Activation of the aryl hydrocarbon receptor during pregnancy in the mouse alters mammary development through direct effects on stromal and epithelial tissues.

Activation of the aryl hydrocarbon receptor (AHR), an environment-sensing transcription factor, causes profound impairment of mammary gland differentiation during pregnancy. Defects include decreased ductal branching, poorly formed alveolar structures, suppressed expression of milk proteins, and failure to nutritionally support offspring. AHR is activated by numerous environmental toxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and plays an as yet poorly understood role in development and reproduction. To better understand how AHR activation affects pregnancy-associated mammary gland differentiation, we used a combination of ex vivo differentiation, mammary epithelial transplantation, and AHR-deficient mice to determine whether AHR modulates mammary development through a direct effect on mammary epithelial cells (MECs) or by altering paracrine or systemic factors that drive pregnancy-associated differentiation. Studies using mutant mice that express an AHR protein lacking the DNA-binding domain show that defects in pregnancy-associated differentiation require AHR:DNA interactions. We then used fluorescence-based cell sorting to compare changes in gene expression in MECs and whole mammary tissue to gain insight into affected signaling pathways. Our data indicate that activation of the AHR during pregnancy directly affects mammary tissue development via both a direct effect on MECs and through changes in cells of the fat pad, and point to gene targets in MECs and stromal tissues as putative AHR targets.

Comparison of four commercial devices for RapidArc and sliding window IMRT QA.

For intensity-modulated radiation therapy, evaluation of the measured dose against the treatment planning calculated dose is essential in the context of patient-specific quality assurance. The complexity of volumetric arc radiotherapy delivery attributed to its dynamic and synchronization nature require new methods and potentially new tools for the quality assurance of such techniques. In the present study, we evaluated and compared the dosimetric performance of EDR2 film and three other commercially available quality assurance devices: IBA I'MatriXX array, PTW Seven29 array and the Delta4 array. The evaluation of these dosimetric systems was performed for RapidArc and IMRT deliveries using a Varian NovalisTX linear accelerator. The plans were generated using the Varian Eclipse treatment planning system. Our results showed that all four QA techniques yield equivalent results. All patient QAs passed our institutional clinical criteria of gamma index based on a 3% dose difference and 3 mm distance to agreement. In addition, the Bland-Altman analysis was performed which showed that all the calculated gamma values of all three QA devices were within 5% from those of the film. The results showed that the four QA systems used in this patient-specific IMRT QA analysis are equivalent. We concluded that the dosimetric systems under investigation can be used interchangeably for routine patient specific QA.

Plan evaluation and dosimetric comparison of IMRT using AAPM TG119 test suites and recommendations.

In order to verify intensity modulated radiotherapy quality assurance procedure and to establish the practical base line commissioning, American Association of Physicists in Medicine-Task Group 119 test suite DICOM-RT images and structure were downloaded for planning and dosimetric comparison. The square slab phantom of water equivalent plastic was used for the measurement. This phantom can permit point dose measurement with ionization chamber by placing the chamber at 7.5 cm depth in the slab phantom. The planar dose measurements were carried out by positioning the Matrixx detector at 10 cm depth. The planning and measurements were performed as per AAPM TG119 guidelines. The test suite includes AP:PA field, band test, multitarget, prostate, head and neck and C-shape. The ion chamber measurements were within 3% of the planned dose for target and avoidance structure region. The ion chamber measurement results are in good agreement with the TG119 recommendation of ±3% for all the test suites. The planar dose measurements were performed with Matrixx for individual fields at the planned gantry angle. The results show that the pass criteria for γ ≤ 1 were between 93 to 97% for all the test cases. Our results are in good agreement with the TG119 recommendation. The present study aimed to compare the measured dose with the planned dose using computer planning system. The test suites were used to assess the planning and delivery systems so as to provide the basis for IMRT commissioning and QA.

Comparison of dosimetric characteristics of physical and enhanced dynamic wedges.

BACKGROUND: Wedge filters can be used as missing tissue compensators or wedge pairs to alter the shape of isodose curves so that two beams can be angled with a small hinge angle at a target volume without creating a hotspot. AIM: In this study the dosimetric properties of Varian Enhanced Dynamic Wedge (EDW) and physical wedges (PW) were analyzed and compared. MATERIALS AND METHODS: Ionometric measurements of open field output factor, physical wedge output factor, physical wedge factor and EDW factor for photon beams were carried out. A 3D scanning water phantom was used to scan depth dose and profiles for open and PW fields. The 2D ionization matrix was used to measure profiles of physical and EDW wedges. The isodose curves of physical and EDW angles were obtained using a therapy verification film. RESULTS AND DISCUSSION: The PW output factors of photons were compared with the open field output factors. The physical and EDW factors were compared. The difference in percentage depth dose for open and PW fields was observed for both photon beams. The measured isodose plots for physical and EDW were compared. CONCLUSION: The wedge field output factor increases with field size and wedge angle compared to that of the open field output factor. The number of MU to deliver a particular dose with the EDW field is less than that of the PW field due to a change in wedge factor. The dosimetric characteristics, like profile and isodose of EDW, closely match with that of the PW.

Deletion of Kvß2 (AKR6) Attenuates Isoproterenol Induced Cardiac Injury with Links to Solute Carrier Transporter SLC41a3 and Circadian Clock Genes.

Kvß subunits belong to the aldo-keto reductase superfamily, which plays a significant role in ion channel regulation and modulates the physiological responses. However, the role of Kvß2 in cardiac pathophysiology was not studied, and therefore, in the present study, we hypothesized that Kvß2 plays a significant role in cardiovascular pathophysiology by modulating the cardiac excitability and gene responses. We utilized an isoproterenol-infused mouse model to investigate the role of Kvß2 and the cardiac function, biochemical changes, and molecular responses. The deletion of Kvß2 attenuated the QTc (corrected QT interval) prolongation at the electrocardiographic (ECG) level after a 14-day isoproterenol infusion, whereas the QTc was significantly prolonged in the littermate wildtype group. Monophasic action potentials verified the ECG changes, suggesting that cardiac changes and responses due to isoproterenol infusion are mediated similarly at both the in vivo and ex vivo levels. Moreover, the echocardiographic function showed no further decrease in the ejection fraction in the isoproterenol-stimulated Kvß2 knockout (KO) group, whereas the wildtype mice showed significantly decreased function. These experiments revealed that Kvß2 plays a significant role in cardiovascular pathophysiology. Furthermore, the present study revealed SLC41a3, a major solute carrier transporter affected with a significantly decreased expression in KO vs. wildtype hearts. The electrical function showed that the decreased expression of SLC41a3 in Kvß2 KO hearts led to decreased Mg2+ responses, whereas, in the wildtype hearts, Mg2+ caused action potential duration (APD) shortening. Based on the in vivo, ex vivo, and molecular evaluations, we identified that the deletion of Kvß2 altered the cardiac pathophysiology mediated by SLC41a3 and altered the NAD (nicotinamide adenine dinucleotide)-dependent gene responses.

Dosimetric study of 2D ion chamber array matrix for the modern radiotherapy treatment verification.

Intensity-modulated radiotherapy treatment demands stringent quality assurance and accurate dose determination for delivery of highly conformal dose to the patients. Generally 3D dose distributions obtained from a treatment planning system have to be verified by dosimetric methods. Mainly, a comparison of two-dimensional calculated and measured data in several coplanar planes is performed. In principle, there are many possibilities to measure two-dimensional dose distributions such as films, flat-panel electronic portal imaging devices (EPID), ion chambers and ionization chamber arrays, and radiographic and radiochromic films. The flat-panel EPIDs show a good resolution and offer a possibility for real-time measurements: however to convert the signal into dose, a separate commercial algorithm is required. The 2D ion chamber array system offers the real-time measurements. In this study, dosimetric characteristics of 2D ion chamber array matrix were analyzed for verification of radiotherapy treatments. The dose linearity and dose rate effect of the I'matriXX device was studied using 6 MV, 18 MV photons and 12 MeV electrons. The output factor was estimated using I'matriXX device and compared with ion chamber measurements. The ion chamber array system was found to be linear in the dose range of 2-500 cGy and the response of the detector was found to be independent of dose rate between 100 MU/min to 600 MU/min. The estimated relative output factor with I'matriXX was found to match very well with the ion chamber measurements. To check the final dose delivered during IMRT planning, dose distribution patterns such as field-in-field, pyramidal, and chair tests were generated with the treatment planning system (TPS) and the same was executed in the accelerator and measured with the I'matriXX device. The dose distribution pattern measured by the matrix device for field-in-field, pyramidal, and chair test were found to be in good agreement with the calculated dose distribution by TPS both for 6 and 18 MV photons (gamma < or = 1: 96%, criteria 3%, 3 mm). Two 7-field IMRT plans (one prostate, one head and neck) dose distribution patterns were also measured with I'matriXX device and compared with film dosimetry. The measurements and evaluation proves that I'matriXX can be used for quantifying absolute dose. Moreover, using I'matriXX as absolute dosimeter in IMRT field verification, avoids the time-consuming procedure of making ionometric measurement for absolute dose estimation and film for dose distribution verification. The I'matriXX can also used for routine quality assurance checks like flatness, symmetry, field width, and penumbra of the linear accelerator beam.

Consistency and reproducibility of the VMAT plan delivery using three independent validation methods.

The complexity of VMAT delivery requires new methods and potentially new tools for the commissioning of these systems. It appears that great consideration is needed for quality assurance (QA) of these treatments since there are limited devices that are dedicated to the QA of rotational delivery. In this present study, we have evaluated the consistency and reproducibility of one prostate and one lung VMAT plans for 31 consecutive days using three different approaches: 1) MLC DynaLog files, 2) in vivo measurements using the multiwire ionization chamber DAVID, and 3) using PTWseven29 2D ARRAY with the OCTAVIUS phantom at our Varian Clinac linear accelerator. Overall, the three methods of testing the reproducibility and consistency of the VMAT delivery were in agreement with each other. All methods showed minimal daily deviations that contributed to clinically insignificant dose variations from day to day. Based on our results, we conclude that the VMAT delivery using a Varian 2100CD linear accelerator equipped with 120 MLC is highly reproducible.