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Western blot analysis of p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p-MEK1/2 (Ser217/221), p-p90RSK (Ser380), p-MSK1 (Thr581) in JIMT-1 BR-3, MDA-MB-361, MDA-MB-231 BrM2 and BS-004 cell lines treated with GDC-0084 in increasing concentrations for a total of 6 hours

Purpose:Previous studies have shown that the PI3K/Akt/mTOR pathway is activated in up to 70% of breast cancer brain metastases, but there are no approved agents for affected patients. GDC-0084 is a brain penetrant, dual PI3K/mTOR inhibitor that has shown promising activity in a preclinical model of glioblastoma. The aim of this study was to analyze the efficacy of PI3K/mTOR blockade in breast cancer brain metastases models.Experimental Design: The efficacy of GDC-0084 was evaluated in PIK3CA-mutant and PIK3CA wild-type breast cancer cell lines and the isogenic pairs of PIK3CA wild-type and mutant (H1047R/+) MCF10A cells in vitro. In vitro studies included cell viability and apoptosis assays, cell-cycle analysis, and Western blots. In vivo, the effect of GDC-0084 was investigated in breast cancer brain metastasis xenograft mouse models and assessed by bioluminescent imaging and IHC.Results:In vitro, GDC-0084 considerably decreased cell viability, induced apoptosis, and inhibited phosphorylation of Akt and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines. In contrast, GDC-0084 led only to growth inhibition in PIK3CA wild-type cell lines in vitro. In vivo, treatment with GDC-0084 markedly inhibited the growth of PIK3CA-mutant, with accompanying signaling changes, and not PIK3CA wild-type brain tumors.Conclusions:The results of this study suggest that the brain-penetrant PI3K/mTOR targeting GDC-0084 is a promising treatment option for breast cancer brain metastases with dysregulated PI3K/mTOR signaling pathway conferred by activating PIK3CA mutations. A national clinical trial is planned to further investigate the role of this compound in patients with brain metastases.

Photomicrographs illustrating p-MEK1/2 (Ser221) and p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) expression in mouse brains harboring either JIMT-1 BR-3 or MDA-MB-231 BrM2 intracranial tumors, stratified by treatment with sham or GDC-0084.

GDC-0084-induced changes in cell cycle in the PIK3CA-mutant cell line MDA-MB-361 and in the PIK3CA-wildtype cell line BS-004

Autonomic nerves control organ function through the sympathetic and parasympathetic branches, which have opposite effects. In the bone marrow, sympathetic (adrenergic) nerves promote hematopoiesis; however, how parasympathetic (cholinergic) signals modulate hematopoiesis is unclear. Here, we show that B lymphocytes are an important source of acetylcholine, a neurotransmitter of the parasympathetic nervous system, which reduced hematopoiesis. Single-cell RNA sequencing identified nine clusters of cells that expressed the cholinergic α7 nicotinic receptor (Chrna7) in the bone marrow stem cell niche, including endothelial and mesenchymal stromal cells (MSCs). Deletion of B cell-derived acetylcholine resulted in the differential expression of various genes, including Cxcl12 in leptin receptor

We report the discovery and characterization of a nearby (~ 85 pc), older (27 +/- 3 Myr), distributed stellar population near Lower-Centaurus-Crux (LCC), initially identified by searching for stars co-moving with a candidate transiting planet from TESS (HD 109833; TOI 1097). We determine the association membership using Gaia kinematics, color-magnitude information, and rotation periods of candidate members. We measure it's age using isochrones, gyrochronology, and Li depletion. While the association is near known populations of LCC, we find that it is older than any previously found LCC sub-group (10-16 Myr), and distinct in both position and velocity. In addition to the candidate planets around HD 109833 the association contains four directly-imaged planetary-mass companions around 3 stars, YSES-1, YSES-2, and HD 95086, all of which were previously assigned membership in the younger LCC. Using the Notch pipeline, we identify a second candidate transiting planet around HD 109833. We use a suite of ground-based follow-up observations to validate the two transit signals as planetary in nature. HD 109833 b and c join the small but growing population of, 23 pages, 15 figures, Accepted for publication in AJ

A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creERT2; Leprfl/fl mice reveals that leptin’s effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes. The beneficial effects of exercise on cardiovascular disease are linked to decreased inflammation through crosstalk between adipose tissue and hematopoietic progenitor cells in the bone marrow.

Myeloperoxidase (MPO) is a key component of innate immunity but can damage tissues when secreted abnormally. We developed a new generation of a highly efficient MPO-activatable MRI probe (heMAMP) to report MPO activity. heMAMP has improved Gd stability compared to bis-5-HT-Gd-DTPA (MPO-Gd) and demonstrates no significant cytotoxicity. Importantly, heMAMP is more efficiently activated by MPO compared to MPO-Gd, 5HT-DOTA(Gd), and 5HT-DOTAGA-Gd. Molecular docking simulations revealed that heMAMP has increased rigidity via hydrogen bonding intramolecularly and improved binding affinity to the active site of MPO. In animals with subcutaneous inflammation, activated heMAMP showed a 2-3-fold increased contrast-to-noise ratio (CNR) compared to activated MPO-Gd and 4-10 times higher CNR compared to conventional DOTA-Gd. This increased efficacy was further confirmed in a model of unstable atherosclerotic plaque where heMAMP demonstrated a comparable signal increase and responsiveness to MPO inhibition at a 3-fold lower dosage compared to MPO-Gd, further underscoring heMAMP as a potential translational candidate.

Mature super-Earths and sub-Neptunes are predicted to be $\simeq$Jovian radius when younger than 10 Myr. Thus, we expect to find 5-15$R_\oplus$ planets around young stars even if their older counterparts harbor none. We report the discovery and validation of TOI 1227 b, a $0.85\pm0.05R_J$ (9.5$R_\oplus$) planet transiting a very low-mass star ($0.170\pm0.015M_\odot$) every 27.4 days. TOI~1227's kinematics and strong lithium absorption confirm it is a member of a previously discovered sub-group in the Lower Centaurus Crux OB association, which we designate the Musca group. We derive an age of 11$\pm$2 Myr for Musca, based on lithium, rotation, and the color-magnitude diagram of Musca members. The TESS data and ground-based follow-up show a deep (2.5\%) transit. We use multiwavelength transit observations and radial velocities from the IGRINS spectrograph to validate the signal as planetary in nature, and we obtain an upper limit on the planet mass of $\simeq0.5 M_J$. Because such large planets are exceptionally rare around mature low-mass stars, we suggest that TOI 1227 b is still contracting and will eventually turn into one of the more common $, Accepted to the Astronomical Journal. Minor updates during referee process and proofs

Recurrent MI is common in patients with coronary artery disease and associates with high mortality. Here we developed a surgical mouse model in which two subsequent MIs affect different left ventricular regions in the same mouse. Recurrent MI was induced by ligating the left circumflex followed by the left anterior descending branch of the coronary artery. We characterized the resulting ischemia by whole-heart fluorescent coronary angiography after optical organ clearing and by cardiac MRI. We report that a first MI induces bone marrow “memory” via a circulating signal, thereby affecting hematopoietic factor expression in bone marrow macrophages. This altered the organism’s reaction to subsequent events. Inspite at least similar extent of injury reported by blood troponin, recurrent MI caused reduced emergency hematopoiesis and less leukocytosis than a first MI. Consequently, fewer leukocytes migrated to the ischemic myocardium. The hematopoietic response to lipopolysaccharide was also mitigated after a previous MI. Our data suggest that hematopoietic and innate immune responses are shaped by a preceding MI.

Purpose: Previous studies have shown that the PI3K/Akt/mTOR pathway is activated in up to 70% of breast cancer brain metastases, but there are no approved agents for affected patients. GDC-0084 is a brain penetrant, dual PI3K/mTOR inhibitor that has shown promising activity in a preclinical model of glioblastoma. The aim of this study was to analyze the efficacy of PI3K/mTOR blockade in breast cancer brain metastases models. Experimental Design: The efficacy of GDC-0084 was evaluated in PIK3CA-mutant and PIK3CA wild-type breast cancer cell lines and the isogenic pairs of PIK3CA wild-type and mutant (H1047R/+) MCF10A cells in vitro. In vitro studies included cell viability and apoptosis assays, cell-cycle analysis, and Western blots. In vivo, the effect of GDC-0084 was investigated in breast cancer brain metastasis xenograft mouse models and assessed by bioluminescent imaging and IHC. Results: In vitro, GDC-0084 considerably decreased cell viability, induced apoptosis, and inhibited phosphorylation of Akt and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines. In contrast, GDC-0084 led only to growth inhibition in PIK3CA wild-type cell lines in vitro. In vivo, treatment with GDC-0084 markedly inhibited the growth of PIK3CA-mutant, with accompanying signaling changes, and not PIK3CA wild-type brain tumors. Conclusions: The results of this study suggest that the brain-penetrant PI3K/mTOR targeting GDC-0084 is a promising treatment option for breast cancer brain metastases with dysregulated PI3K/mTOR signaling pathway conferred by activating PIK3CA mutations. A national clinical trial is planned to further investigate the role of this compound in patients with brain metastases.

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. Bone-marrow endothelial cells in the haematopoietic stem-cell niche form a network of blood vessels that regulates blood-cell traffic as well as the maintenance and function of haematopoietic stem and progenitor cells. Here, we report the design and in vivo performance of systemically injected lipid–polymer nanoparticles encapsulating small interfering RNA (siRNA), for the silencing of genes in bone-marrow endothelial cells. In mice, nanoparticles encapsulating siRNA sequences targeting the proteins stromal-derived factor 1 (Sdf1) or monocyte chemotactic protein 1 (Mcp1) enhanced (when silencing Sdf1) or inhibited (when silencing Mcp1) the release of stem and progenitor cells and of leukocytes from the bone marrow. In a mouse model of myocardial infarction, nanoparticle-mediated inhibition of cell release from the haematopoietic niche via Mcp1 silencing reduced leukocytes in the diseased heart, improved healing after infarction and attenuated heart failure. Nanoparticle-mediated RNA interference in the haematopoietic niche could be used to investigate haematopoietic processes for therapeutic applications in cancer, infection and cardiovascular disease.

Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.

A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug.

Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746–750, T790M/L858R, and T790M/del746–750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746–750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746–750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo...

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.

CHARLOTTE KEENAN, SUSAN ELMORE, SABINE FRANCKE-CARROLL, RAMON KEMP, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania, USA National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, North Carolina, USA Center for Food Safety and Applied Nutrition (CFSAN), U.S. Food and Drug Administration, College Park, Maryland, USA Merck Research Laboratories, Riom, France Pfizer Inc., Groton, Connecticut, USA NIEHS, Research Triangle Park, North Carolina, USA Charles River, Frederick, Maryland, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, United Kingdom U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.

Glucokinase activators (GKAs) are being developed for the treatment of type 2 diabetes. The toxicity of 4 GKAs (PF-04279405, PF-04651887, piragliatin, and PF-04937319) was assessed in mice, rats, dogs, and/or monkeys. GKAs were administered for 2 to 8 weeks. Standard endpoints, glucose, and insulin were assessed. All compounds produced varying degrees of hypoglycemia in all species. Brain neuronal necrosis and/or peripheral neuropathy were observed with most compounds. These findings are consistent with literature reports linking hypoglycemia with nervous system effects. Arteriopathy, mainly of cardiac vessels, was observed at a low frequency in monkey and/or dog. Arteriopathy occurred only at doses that produced severe and prolonged periods of repeated hypoglycemia. Since this lesion occurred in multiple studies with structurally distinct GKAs, these results suggested arteriopathy was related to GKA pharmacology. The morphological characteristics of the arteriopathy were consistent with that produced by experimental catecholamine administration. We hypothesize that the prolonged periods of hypoglycemia resulted in increased local and/or systemic concentrations of catecholamines via a counterregulatory and/or stress-related mechanism. Alternatively, prolonged hypoglycemia may have resulted in endothelial dysfunction leading to arteriopathy. This risk can be managed in human patients in clinical studies by careful glucose monitoring and intervention to avoid prolonged episodes of hypoglycemia.

In non-rodent toxicity studies supporting pharmaceutical or chemical product registration, the value of histopathology evaluation of all tissues from all animals from all dose groups is an ongoing discussion topic among pathologists. This manuscript documents an examination of this topic through a retrospective review of internal nonclinical study data from non-rodent toxicity studies performed at three pharmaceutical companies (Abbott Laboratories, Eli Lilly, and Pfizer, Inc.) and an informal survey of the current practices within the toxicological pathology community. The retrospective review of 325 non-rodent studies in which all organs in all dose groups were examined revealed no evidence that risk assessment would have changed if only the control and high-dose animals and target organs only in intermediate dose groups had been examined. One study had target tissues in a lower-dose group that were not identified in the high-dose group; however, there was no impact on the overall study interpretation. The recently revised European Medicines Agency guideline regarding repeated-dose toxicity studies encourages the examination of all tissues at all dose levels in non-rodent studies. In conclusion, the evaluation of all tissues from all animals may not be justified as a routine practice; however, regulatory guidance with input from toxicologic pathologists will influence these policy decisions.

Two experiments were conducted to determine the physiological responses of consuming a fungus-free (FF) or fungus-infected (INF) tall fescue diet (Exp. 1) or the FF diet plus ergotamine tartrate at 30 ppm (FF/ET, Exp. 2) in a thermoneutral (21 degrees C) or heat-stressed environment (32 degrees C, dry bulb; 10 degrees C dew point). Treatment periods lasted 28 d after a 10-d adaptation period. Experiment 1 was replicated three times, and Exp. 2 was replicated twice, with eight Holstein steers in each replicate (mean BW = 185 kg). Feed intake (FI), rectal temperature (RT), and respiration rate (RR) were recorded daily, and heart rate (HR) and infrared temperatures at the ear canal (ECT), ear tip (ETT), pastern (PT), coronary band (CBT), and tail tip (TTT) were recorded weekly. Consumption of INF and FF/ET compared with FF diets decreased (P less than .10) FI, HR, ECT, PT, CBT, and TTT and elevated (P less than .10) RT and RR by 2.8, 2.77 kg/d; 17, 23 beats/min; .8, 1.1 degrees C; .9, 1.1 degrees C; .8, .9 degrees C; 1.1, 2.6 degrees C; .3, .5 degrees C; and 8, 8 breaths/min; respectively. Ear-tip temperature was reduced (P less than .10) 1.6 degrees C by consumption of INF but not by consumption of FF/ET. Reduction of peripheral temperatures is indicative of reduced blood flow to peripheral areas as a result of vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

CHARLOTTE KEENAN (CHAIR), SUSAN ELMORE, SABINE FRANCKE-CARROLL, ROY KERLIN, SHYAMAL PEDDADA, JOHN PLETCHER, MATTHIAS RINKE, STEPHEN PETER SCHMIDT, IAN TAYLOR, AND DOUGLAS C. WOLF GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740, USA Pfizer Inc., Groton, Connecticut 06340, USA National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Charles River, Frederick, Maryland 21701, USA Bayer Schering Pharma AG, Wuppertal, Germany Huntingdon Life Sciences, Eye, Suffolk IP23 7PX, UK U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA

u u m l t f F o b A rcheological data and ancient historical records document the presence of cleft lip and palate and even attempts to surgically epair the cleft defect. Current estimates suggest hat as many as 6500 babies are born each year in he United States with orofacial clefts, including left palate and cleft lip with and without cleft alate. The prevalence of orofacial clefts (cleft lip nd palate and cleft lip alone) during the period rom 1999 to 2001 is estimated to be 16.86 per 0,000 live births and that of trisomy 21 was stimated to be 13.65 per 10,000 live births. The are of children with orofacial clefts is complex and team approach to their care is widely recommendd. The American Cleft Palate Association “Paameters for Evaluation and Treatment of Patients ith Cleft Lip/Palate and Other Craniofacial Anomlies” (revised 2004) suggests that teams may inlude professionals from audiology, radiology, geetics and genetic counseling, nursing, pediatrics, ral and maxillofacial surgery, orthodontics, denistry, otolaryngology, psychology, social work, and peech pathology among others. Teams see patients t regular intervals, meet to discuss the needs of the amily, and make recommendations to the family so plan can be formulated for management of the raniofacial defect and associated medical probems.

Since its description by Aladar Aujeszky in 1902, pseudorabies (Aujeszky’s disease) has become recognized as a worldwide disease in which pigs, the natural host, are a source of infection for ruminants. Virus and characteristic microscopic lesions in sheep have been localized to specific neurologic structures. A nonsuppurative encephalitis and a necrotizing encephalitis have been reported from spontaneous pseudorabies outbreaks in sheep flocks; lesions were in the cerebral cortex, cerebellum, and brain stem. In experimental infections, histologic lesions and virus were localized in neural structures associated with the route of inoculation. Nonsuppurative ganglionitis and encephalitis were in the trigeminal ganglia and medulla oblongata of experimental sheep inoculated by the conjunctival, oral, and intranasal routes. A virus-induced neuronal necrosis was found in the cervicothoracic ganglia of sheep in natural and experimental pseudorabies virus (PrV) infections. Sheep inoculated intratracheally with aerosolized PrV had histologic lesions, viral antigen, and virus in the medulla oblongata and in trigeminal, cranial cervical, and cervicothoracic ganglia. 16 This report illustrates the location of neural structures that contain lesions and virus in experimental and natural infections of sheep. Six adult ewes were euthanized and dissected to expose the cranial cervical ganglia, cervicothoracic ganglia, brain stem, and trigeminal ganglia. These tissues were easily obtained with standard necropsy instruments and without significantly altering necropsy protoco1s and prosection time. The cranial cervical ganglia (Figs. l-4) are bilaterally symmetrical sympathetic ganglia at the cranial end of the sympathetic trunk. These grayish to pale brown, fusiform neural structures are approximately 8 mm long and 6 mm wide and are embedded in the adipose tissue of the retropharyngeal area. Each ganglion lies ventral to the posterior foramen lacerum, ventromedial to the tympanic bulla, medial to the jugular process of the occipital bone, and on the lateral aspect of the rectus capitis ventralis muscle.’ The cranial cervical ganglia are excised prior to removing the head. After removing or reflecting the tongue, pharynx, larynx, trachea, and esophagus, the rectis capitis ventralis muscles are identified and then transected at the level of the atlantooccipital articulation. The cranial cervical ganglia are found by first locating the exposed ventral ovoid space at the atlantooccipital articulation. The space is formed cranially by the caudal aspect of the occipital bone at the ventral edge of the foramen magnum and caudally by the ventral edge of the cranial atlas. After this space is identified, the rectis capitis

Altman NH, Street G, Temer J: 1969, Castration and its re11. lation to tumors of the adrenal gland in the goat. Am J Vet Res 30:583-589. Bastianello SS: 1983, A survey of neoplasia in domestic species over a 40-year period from 1935 to 1974 in the republic of 12. South Africa. III. Tumors occurring in pigs and goats. Onderstepoort J Vet Res 50:25-28. 13. Brandly PJ, Migaki G: 1963, Types of tumors found by federal meat inspectors in an eight year survey. Ann NY Acad Sci 108: 872-879. 14. Cordy DR: 1990, Tumors of the nervous system and the eye. In: Tumors of domestic animals, ed. Moulton JE, 3rd ed., pp. 6 52-654. University of California Press, Berkeley, CA. Damodaran S, Parthasarathy KR: 1971, Neoplasms of goats 15 . and sheep. Indian Vet J 49:649-652. Davis CL, Kemper HE: 1936, Common warts (papillomata) in 16. goats. J Am Vet Med Assoc 88: 175-179. Erlandson RA, Woodruff JM: 1982, Peripheral nerve sheath 17. tumors: an electron microscopic study of 43 cases. Cancer 49: 273-287. Ghadially FN: 1985, Is it Schwannoma or a fibroblastic neoplasm? In: Diagnostic electron microscopy of tumors, 2nd ed., 18. pp. 206-227. Butterworth & Co., London, England. Gould VE, Moll R, Moll I, et al.: 1986, The intermediate filament complement of the spectrum of nerve sheath tumors. Lab Invest 55:463474. Hadlow WJ: 1978, High prevalence of thymoma in the dairy goat; report of seventeen cases. Vet Pathol 15: 153-169. Jackson C: 1936, The incidence and pathology of tumors of domesticated animals in South Africa. A study of the Onderstepoort collection of neoplasms with special reference to their histopathology. Onderstepoort J Vet Sci 6:429. Janser RC, Makek M: 1983, Intraoral malignant melanotic Schwannoma. Arch Path01 Lab Med 107:298-30 1. Laber-Laird KE, Jokinen MP, Jerome CP: 1988, Naturally occurring Schwannoma in a Fischer 344 rat. Vet Pathol 25:320322. Matsunou H, Shimoda T, Kakimoto S, et al.: 1985, Histopathologic and immunohistochemical study of malignant tumors of peripheral nerve sheath (malignant Schwannoma). Cancer 56:2269-2279. Richter WR: 1958, Adrenal cortical adenomata in the goat. Am J Vet Res 19:895. Sastry GA: 1959, Neoplasms of animal in India. An account of neoplasms collected in 12 years. Vet Med 54:428-430. Street CS, Altman NH, Temer JY, Berdjis CC: 1968, A series of thymomas in the angora goat. Edgewood Arsenal Special Publication 100-36, Department of the Army, Edgewood Arsenal, MD. Zubaidy AJ: 1976, Caprine neoplasms in Iraq: case reports and review of the literature. Vet Pathol 13:460-461.

Raf kinases (A-Raf, B-Raf, C-Raf) link Ras activation to MEK/ERK pathway activation, and are part of the key MAPK signaling pathway that controls cellular functions such as cell proliferation, migration, differentiation, and survival. Activating mutations in B-Raf occurs in a variety of cancers and inhibition of these signaling proteins in the pathway, therefore, represents a therapeutic targeting strategy for diverse cancers. We developed PF-04880594, an ATP-competitive and reversible inhibitor of B-Raf. To assess safety prior to initiation of clinical trials, repeat dose toxicity studies were performed in the rat and dog. Animals were treated orally at 0, 25, 75, 300→150 mg/kg/day in the rat or 0, 0.5, 6, 15→12 mg/kg/day in the dog for 30 days followed by a 30-day treatment-free period to assess recovery or delayed toxicity. Plasma concentrations of the B-Raf inhibitor were measured on Day 1 and at the end of the dosing phase of the study. Body weight and food consumption were measured at regular intervals and clinical signs were recorded daily. In the rat, dose-related clinical signs, occurring in the skin at ≥ 25 mg/kg included dry skin and/or edema in the paws, erythema and desquamation of tail skin > 75 mg/kg, and erythema of the ear at 300→150 mg/kg. In the dog, clinical signs observed at ≥ 6 mg/kg included desquamation and alopecia of the skin involving the ear, head, eyebrow, muzzle, thorax, lips, nose, scrotum or forepaw; and edema of the ear, muzzle, head, forepaw, hindpaw or scrotum. Erythema was observed at all dose levels on the muzzle, ear or head during life. In both species, these clinical signs correlated microscopically with epithelial hyperplasia in these affected tissues. Hyperplasia of the stratified epithelium of the tongue and esophagus occurred in both species, In the rat, epithelial hyperplasia was additionally observed in the urinary bladder and nonglandular stomach and dose-related proliferation of woven bone was observed primarily on endocortical surfaces in the proximal tibia and /or distal femur. Clinical pathology revealed an inflammatory response indicated by increased fibrinogen and neutrophil counts in dogs at ≥ 6 mg/kg (males > females) and in rats at ≥ 75 mg/kg. At the end of the recovery period, the inflammatory response and the woven bone changes were not evident, but epithelial hyperplasia was only partly resolved. A no-observed-effect-level was not identified in either study due to the epithelial hyperplasia. At low doses, margins of exposure were over 10-fold in the rat and 0.03-fold in the dog of projected clinically efficacious plasma concentration in humans. These studies indicate epithelial hyperplasia is paradoxically induced by this selective B-Raf inhibitor. The occurrence of this change in the rodent and canine suggests that this effect could develop in other mammalian species. Combination studies to test the effects of Raf and MEK inhibition on hyperplasia are currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1681.

To determine the effect of dry (D); reconstituted and ensiled (R); reconstituted and acid-treated (A); and urea-treated, high-moisture (U) sorghum grain on starch digestibility, four Angus x Hereford steers (means BW = 350 kg) with duodenal and ileal cannulas were used in a 4 x 4 Latin square design. Diets consisting of 69% ground sorghum grain were fed every 2 h in equal portions (8.2 kg/d). Diets averaged 46.5% starch and 12% CP, except for U, which averaged 14% CP due to urea treatment. Ytterbium attached to sorghum was used as a particulate marker. Duodenal, ileal, and fecal samples were taken 1 h postfeeding after a 14-d adaption to diets. Whole samples were analyzed. Preduodenal starch digestion (%) was 89, 83, 76, and 70, and starch digestion over the total tract was 99, 97, 95 and 91 for R, U, A, and D, respectively. Starch digestion proximal to each site (duodenum and ileum) was enhanced (P less than .05) by R and U compared with D. Within the small intestine, there was a linear relationship (P less than .003) between starch digestion and daily starch supply. However, digestibility of starch in the small intestine (mean = 45%) was not different among diets. Apparent digestibility of starch in the large intestine was not significantly different from digestibility in the small intestine. Urea-treated sorghum grain was equivalent to reconstituted, ensiled sorghum in digestion characteristics and was superior to dry sorghum.

Effects of amount of feed intake on in vivo remen propionate production and the reproducibility of measurements of propionate production rates were investigated in five dairy steers weighing 142 to 228 kg. Each steer received 275 g of an 80%-grain diet every 2 hours, and three of the five steers later received 500 g/2 hours. Rumen propionate pool sizes and production rates were determined at both intakes by administering a single dose of [1-14C] propionate via rumen fistula and observing decreasing specific activity of rumen propionate for 4 hours. A linear regression equation was calculated from the natural logs of decreasing specific activity. Propionate pool sizes averaged 30.6 and 47.4 g, and production rates averaged 579 and 1032 g/day at feed intakes of 275 and 500 g/2 hours. Thus, propionate production changed in almost direct proportion to feed intake. Acetate to propionate ratios were about 3:1 in rumen fluid. Considerable variability in estimates of propionate pool sizes and production rates was found both within and among steers. The results indicate propionate production is nearly equivalent to blood glucose turnover determined previously; all propionate produced in the rumen, however, is not used for glucose synthesis.

Three Acremonium coenophialum metabolites, ergosterol, ergosterol peroxide, and ergosta-4,6,8. (14);22-tetraen-3-one, were isolated from field-grown, fungus-infected. (toxic) tall fescue and mycelium grown in vitro. Mycelium contained 10 J.Lg of total steroljmg dry weight of tissue, with ergosterol comprising 40% of the total sterols. Mycelium contained EP only when grown in the light. EP was toxic to brine shrimp, two bacterial species, and chick embryos. Whether EP is involved in tall fescue toxicity is not known, but it was the only toxic compound found in chloroform-methanol extracts from both fungus-infected fescue and laboratory-grown mycelium and EP was not found in nontoxic tall fescue. Cattle grazed on Kentucky 31 tall fescue (Festuca arundinaceae Schreb.) during the summer often exhibit symptoms known collectively as "summer syndrome". These symptoms include reduced weight gain, rough hair coat, slightly elevated temperature, excessive salivation, rapid breathing, and excitability (Hoveland et al., 1983). Although summer syndrome was previously associated with hot weather, recent research has shown that poor weight gain occurs throughout the grazing season when cattle feed on tall fescue infected with an endophytic fungus (Hoveland et al., 1983). Bacon et al. (1977) isolated the endophytic fungus Epichloe typhina (Fr.) Tulasne from bent grass. The imperfect (Anamorphic) stage of this fungus is Acremonium typhinum Morgan-Jones and Gams. The endophytic fungus isolated from tall fescue in Alabama, Georgia, Kentucky, Texas, and New Zealand (Morgan-Jones and Gams, 1982; Latch et al., 1984; White and Cole, 1985, 1985b) has been classified as A. coenop­ hialum Morgan-Jones and Gams; a perfect (teleomorphic) state has never been reported on tall fescue. According to Morgan-Jones (personal communication), the endo­ phytic anamorphs from bent grass and tall fescue differ morphologically as well as in host specificity. In our view, the fungus endophyte of tall fescue is A. coenophialum Morgan-Jones and Gams and all other names are errone­ ous. It has often been suggested that alkaloids are a likely cause of fescue foot and perhaps other problems associated

Phosphoproteins were examined by electrophoresis and autoradiography in fractions of tick salivary glands. When whole salivary glands were preincubated in 32Pi, then stimulated by 10 μM dopamine and subsequently fractionated, substantial phosphate was incorporated into 45,000-, 47,000-, and 62,000-dalton proteins of the plasma membrane-rich 11,500g pellet and 100,000g supernatant. When tissue homogenates were incubated in [γ-32P] ATP prior to subcellular fractionation, the 62,000-, 47,000-, and 45,000-dalton proteins were enhanced by cyclic AMP in all fractions and were most prominent in the membrane-rich 11,500g fraction. Phosphoproteins of the same molecular masses were also found in the 11,500g pellet and 100,000g supernatant when labelled with [γ-32P] ATP in the presence of cAMP.

To test the hypothesis that glucose utilization in ruminants is related to energy consumption independent of diet composition or energy content, eight Holstein steers (165-230 kg) were fed either a high grain (70% corn, 20% alfalfa, 8.8% soybean meal) or high hay (70% alfalfa, 28% corn) diet. The two diets were fed in quantities that allowed comparisons to be made either isocalorically based on calculated net energy for gain (NEg) or at identical dry matter intakes. The daily ration was divided into 12 equal portions and dispensed every 2 hours by automatic feeders to achieve steady-state conditions with respect to glucose metabolism. After adaptation to the dietary regimen, rates of glucose irreversible loss were measured following a single injection of 500 muCi of [6-3H]glucose. Glucose irreversible loss also was measured after a 7-day fast. Glucose irreversible loss was equal when both diets were fed isocalorically, was larger among steers fed the grain diet when comparisons were made at equal intakes of dry matter, and increased when the intake of either diet was increased. The increase in glucose irreversible loss with increasing energy intake showed a highly significant linear relationship over the range of intakes for both diets. It was concluded that energy intake, in contrast to diet composition, is responsible for the regulation of glucose irreversible loss.

An experiment was conducted to test the accuracy of the metabolizable protein system in predicting the amount of urea that would be useful in a corn-based cattle diet. Treatment diets included a basal, low-protein (7.8% CP) negative control (NC) with no supplemental N and a positive control (PC) that contained soybean meal. Urea was added to the NC diet in quantities calculated to be either 25% deficient (LU), equal to (MU) or 25% in excess (HU) of the urea fermentation potential ( UFP ). In vitro rumen fermentation studies were used to determine sequential ammonia production and digestible dry matter content of the diets. In a growth trial, 12 individually-fed Angus, Hereford and Angus X Hereford steers weighing an average of 213 kg were assigned randomly to each treatment diet. At the conclusion of the 112-d trial, rumen ammonia and jugular blood urea N (BUN) concentrations were determined on two steers from each treatment before feeding and at 1, 2, 3, 4 and 5 h postfeeding. In vitro ammonia concentrations of the NC and PC treatments were lower (P less than .05) than that of urea containing diets. In vivo rumen ammonia concentrations at 1 h postfeeding and BUN levels at 3 h postfeeding were low for both the NC and PC diets compared with urea-containing diets. Both of these values increased with each successive increase of added urea to the NC diet. During the initial 70 d of the growth trial, daily gains were improved (P less than .05) by addition of urea up to the MU level, which fulfilled the calculated UFP .(ABSTRACT TRUNCATED AT 250 WORDS)

The waxmoth, Galleria mellonella, was injected with radiolabelled acetate, propionate, and oleic acid to study the biosynthesis of n- nonanal and n- undecanal , its sex pheromones. The sex attractants were collected from the moths by condensation in a cold trap immersed in an acetone-dry ice bath, and then isolated by gas-liquid chromatography. The n- nonanal was found to contain 1.23% of the radiolabelled carbon from the 10-14C-oleic acid, 0.13% from the 1-14C-oleic acid, 0.20% from the 3-14C-sodium propionate, and 0.30% from the 1,2-14C-acetic acid. n- Undecanal contained 0.05% of the radiolabelled carbon from the 3-14C-sodium propionate, 0.07% from the 1,2-14C-acetic acid, and 0.09% from both the oleic acids injected. Both acetate and propionate are incorporated into the pheromone components but do not appear to be of primary importance. The incorporation of oleic acid suggests that α- and /or β-oxidation is involved in the biosynthesis of the pheromone fractions. The data show that odd numbered straight-chain aldehydes are synthesized from many different precursors much the same as other lipids.

On etudie la preparation et la dechlorhydratation d'isomeres cis et trans d'acides de chloro-11,12 dihydro-9,10 ethano-9,10 anthracene-carboxyliques-1 et -acetiques-9

1. Enzyme activity, basal or dopamine-stimulated (10 microM), was linear with time to 25 min and with protein concentration to 0.8 mg protein/ml of final assay volume. Activity was maximal between pH 7.0 and 7.5. 2. Mg2+ maximally stimulated basal or dopamine-sensitive adenylate cyclase activity at about 4 mM. 3. Adenylate cyclase had a Km of 0.042 mM for ATP and maximum velocities for basal and dopamine-stimulated activity of 107 and 179 pmol cyclic AMP formed/mg protein per min, respectively. 4. Half-maximal stimulation of the enzyme occurred at about 4.2 x 10(-7) M dopamine with the threshold being less than 10(-9) M. Dopamine increased the Vmax but had no effect on the Km of ATP. 5. Eighty-five to 90% of the adenylate cyclase activity was found in the particulate fraction. 6. Calcium ion produced a marked inhibition of adenylate cyclase activity above 0.04 mM and half-maximal inhibition occurred near 0.1-0.2 mM.

An endophytic fungus found in Kentucky 31 (KY-31) tall fescue has been associated with the "summer syndrome" (fescue toxicity) in grazing cattle. Four experiments were conducted to investigate the use of weanling rats as an animal model to study the fescue toxicity syndrome. When graded levels of fungus-infected (INF) or fungus-free (FF) KY-31 seed were fed, feed intake and growth rates were inversely related to the amount of INF seed in the diet. When diets containing 50% seed were fed, rats eating INF seed had reduced (P less than .05) daily water consumption (9 vs 20 ml) and lowered (P less than .05) body temperatures (36.8 vs 37.7 C) when compared with the FF treatments. Autoclaving the seed increased feed intake and growth rate of rats receiving both INF and FF seed diets, but daily gains continued to be less (P less than .05) in the rats fed autoclaved INF diets when compared with rats fed autoclaved FF diets (2.3 vs 6.8 g). Rats fed diets containing either 40% shredded wheat on which the fescue endophyte had been grown or 40% shredded wheat that was not innoculated did not differ in performance. Crude fiber, nitrogen-free extract, gross energy and dry matter digestibilities were enhanced (P less than .05) by the presence of INF seed in the diets when compared with the FF seed treatments. Crude protein and ether extract digestibilities and apparent biological value of fescue protein were not affected by presence of the fungus. The effect of INF seed on organ weights was inconsistent.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous research has implicated an endophytic fungus as being associated with fescue toxicity (summer syndrome) in cattle grazing Kentucky 31 tall fescue (Festuca arundinacea Schreb.) pastures. Hay and seed were harvested from Kentucky 31 pastures known to be either fungus-free or heavily infested with an endophytic fungus identified as Acremonium coenophialum Morgan-Jones and Gams. Four diets containing either 60% fungus-free seed, 60% fungus-infested seed, 85% fungus-free hay or 85% fungus-infested hay were group-fed to three steers each (avg wt 239 kg) in a 53-d feeding trial. Presence of the fungus reduced (P less than .05) daily gains (kg/d) in steers fed either the seed diets (.96 vs .20) or the hay diets (.66 vs .28). Feed intake was depressed 36% for the seed diets and 8% for the hay diets when the fungus was present. Rectal temperatures were elevated .6 C (P less than .05) for both groups receiving diets containing the fungus, but respiration rate was elevated only in the fungus-infested seed group. In vitro dry matter disappearance was not decreased by presence of the fungus. Steers receiving fungus from either seed or hay were nervous and highly excitable, which resulted in large variations in plasma epinephrine and norepinephrine concentrations. The results of this experiment further implicate an endophytic fungus as being associated with fescue toxicity.