ABSTRACT
Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.
Subject(s)
Melanoma , Skin Neoplasms , Uveal Neoplasms , Animals , Mice , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/therapeutic use , Drug Evaluation, Preclinical , Uveal Neoplasms/drug therapy , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacologyABSTRACT
G protein-coupled estrogen receptor (GPER) is an estrogen receptor expressed in the cardiovascular system. G1, a selective GPER ligand, exerts cardiovascular effects through activation of the PI3K-Akt pathway and Notch signaling in normotensive animals. Here, we investigated whether the G1/GPER interaction is involved in the limitation of infarct size, and improvement of post-ischemic contractile function in female spontaneous hypertensive rat (SHR) hearts. In this model, we also studied Notch signaling and key components of survival pathway, namely PI3K-Akt, nitric oxide synthase (NOS) and mitochondrial K+-ATP (MitoKATP) channels. Rat hearts isolated from female SHR underwent 30 min of global, normothermic ischemia and 120 min of reperfusion. G1 (10 nM) alone or specific inhibitors of GPER, PI3K/NOS and MitoKATP channels co-infused with G1, just before I/R, were studied. The involvement of Notch1 was studied by Western blotting. Infarct size and left ventricular pressure were measured. To confirm endothelial-independent G1-induced protection by Notch signaling, H9c2 cells were studied with specific inhibitor, N-[N-(3,5 difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 5 µM), of this signaling. Using DAPT, we confirmed the involvement of G1/Notch signaling in limiting infarct size in heart of normotensive animals. In the hypertensive model, G1-induced reduction in infarct size and improvement of cardiac function were prevented by the inhibition of GPER, PI3K/NOS, and MitoKATP channels. The involvement of Notch was confirmed by western blot in the hypertensive model and by the specific inhibitor in the normotensive model and cardiac cell line. Our results suggest that GPERs play a pivotal role in mediating preconditioning cardioprotection in normotensive and hypertensive conditions. The G1-induced protection involves Notch1 and is able to activate the survival pathway in the presence of comorbidity. Several pathological conditions, including hypertension, reduce the efficacy of ischemic conditioning strategies. However, G1-induced protection can result in significant reduction of I/R injury also female in hypertensive animals. Further studies may ascertain the clinical translation of the present results.
ABSTRACT
The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose-limiting cardiotoxicity in approximately 20% of patients. The G-protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G-1 reduced myocardial inflammation, improved immunosuppression, triggered pro-survival signaling cascades, improved myocardial mechanical performance, and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand-activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G-1 (50 µg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF-α, IL-1ß, LDH, and ROS levels. Western blotting analysis of cardiac homogenates indicated that G-1 prevents the increase in p-c-jun, BAX, CTGF, iNOS, and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK, and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G-1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G-1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox-induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. J. Cell. Physiol. 232: 1640-1649, 2017. © 2016 Wiley Periodicals, Inc.
Subject(s)
Cardiotonic Agents/therapeutic use , Cardiotoxicity/drug therapy , Doxorubicin/adverse effects , Quinolines/therapeutic use , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , Biomarkers/metabolism , Blood Pressure/drug effects , Cardiotonic Agents/pharmacology , Cardiotoxicity/blood , Cardiotoxicity/pathology , Cardiotoxicity/physiopathology , Diastole/drug effects , Heart Function Tests/drug effects , Humans , Inflammation/pathology , Interleukin-1beta/blood , L-Lactate Dehydrogenase/blood , Ligands , Male , Myocardial Ischemia/blood , Myocardial Ischemia/pathology , Myocardial Ischemia/physiopathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Oxidative Stress/drug effects , Quinolines/pharmacology , Rats, Wistar , Reactive Oxygen Species/blood , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Tumor Necrosis Factor-alpha/blood , Ventricular Function/drug effectsABSTRACT
Neuroimaging research has identified category-specific neural response patterns to a limited set of object categories. For example, faces, bodies, and scenes evoke activity patterns in visual cortex that are uniquely traceable in space and time. It is currently debated whether these apparently categorical responses truly reflect selectivity for categories or instead reflect selectivity for category-associated shape properties. In the present study, we used a cross-classification approach on functional MRI (fMRI) and magnetoencephalographic (MEG) data to reveal both category-independent shape responses and shape-independent category responses. Participants viewed human body parts (hands and torsos) and pieces of clothing that were closely shape-matched to the body parts (gloves and shirts). Category-independent shape responses were revealed by training multivariate classifiers on discriminating shape within one category (e.g., hands versus torsos) and testing these classifiers on discriminating shape within the other category (e.g., gloves versus shirts). This analysis revealed significant decoding in large clusters in visual cortex (fMRI) starting from 90 ms after stimulus onset (MEG). Shape-independent category responses were revealed by training classifiers on discriminating object category (bodies and clothes) within one shape (e.g., hands versus gloves) and testing these classifiers on discriminating category within the other shape (e.g., torsos versus shirts). This analysis revealed significant decoding in bilateral occipitotemporal cortex (fMRI) and from 130 to 200 ms after stimulus onset (MEG). Together, these findings provide evidence for concurrent shape and category selectivity in high-level visual cortex, including category-level responses that are not fully explicable by two-dimensional shape properties.
Subject(s)
Brain Mapping , Pattern Recognition, Visual , Visual Cortex/physiology , Adult , Discrimination, Psychological , Female , Humans , Magnetic Resonance Imaging , Magnetoencephalography , MaleABSTRACT
The authors present a case of parathyroid carcinoma in a patient with primary hyperparathyroidism. Following a literature review, the clinical and diagnostic profile, treatment and prognosis of this rare disease are discussed.
Subject(s)
Adenocarcinoma , Parathyroid Neoplasms , Adenocarcinoma/diagnosis , Adenocarcinoma/surgery , Adult , Humans , Male , Parathyroid Neoplasms/diagnosis , Parathyroid Neoplasms/surgeryABSTRACT
We report a case of pancreatic pseudocyst secondary to acute necrotizing pancreatitis treated with open cystogastrostomy. Following a literature review, we stress the enormous benefits offered by modern diagnostic techniques, and especially imaging techniques, for the diagnosis and monitoring of this disease. Treatment should be delayed for at least six weeks, following which the drainage by open surgery offers the best results and lowest morbidity and mortality, followed by laparoscopy and endoscopy, indicated in particular cases and in patients where open surgery is contraindicated.
Subject(s)
Gastrostomy , Pancreatic Pseudocyst/surgery , Pancreatitis, Acute Necrotizing/surgery , Cholecystectomy, Laparoscopic , Drainage , Duodenoscopy , Female , Follow-Up Studies , Humans , Laparoscopy , Middle Aged , Pancreatic Pseudocyst/diagnosis , Pancreatic Pseudocyst/etiology , Pancreatitis, Acute Necrotizing/complications , Pancreatitis, Acute Necrotizing/diagnosis , Treatment OutcomeABSTRACT
We investigated the influence of bud position, cultivar, tree age, tree carbohydrate status, sampling date, drought and light exposure on the number of leaf primordia formed in dormant vegetative peach buds (Prunus persica (L.) Batsch) relative to the number of primordia formed after bud break (neoformed). During winter dormancy, vegetative peach buds from California and Italy were dissected and the number of leaf primordia recorded. Between leaf drop and bud break, the number of leaf primordia doubled from about five to about 10. Parent shoot length, number of nodes on the parent shoot, cross-sectional area of the parent shoot, bud position along the parent shoot and bud cross-sectional area were correlated with the number of leaf primordia. Previous season light exposure, drought and tree carbohydrate status did not affect the number of leaf primordia present. The number of leaf primordia differed significantly among peach varieties and tree ages at leaf drop, but not at bud break. Our results indicate that neoformation accounted for all shoot growth beyond about 10 nodes. The predominance of neoformed shoot growth in peach allows this species great plasticity in its response to current-season conditions.
Subject(s)
Meristem/growth & development , Plant Leaves/growth & development , Prunus/growth & development , California , Carbohydrate Metabolism/radiation effects , Disasters , Italy , Light , Meristem/metabolism , Meristem/physiology , Plant Leaves/metabolism , Plant Leaves/physiology , Prunus/metabolism , Prunus/physiology , Seasons , Trees/growth & development , Trees/metabolism , Trees/physiologyABSTRACT
BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.
Subject(s)
Genetic Therapy/methods , Retinal Neovascularization/prevention & control , Retinopathy of Prematurity/therapy , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/therapeutic use , Adenoviridae/genetics , Animals , Disease Models, Animal , Genetic Vectors/therapeutic use , Humans , Infant, Newborn , Rats , Retina/pathology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Vitreous Body/metabolismABSTRACT
A protocol for Agrobacterium-mediated transformation of leaf pieces from two almond cultivars has been developed. This protocol gave a transformation percentage of 48.6 for the cultivar MN51 and 27.9 for the cultivar 'Supernova'. The Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector plasmid pBinGUSint has been used. A critical step for optimal transformation was the pre-culture period of leaf pieces prior selection on medium containing kanamycin. Both cultivars showed an almost doubled transformation percentage when the selection was started at day 6 compared to day 0. The presence of neomycin phosphotransferase II and ß-glucuronidase genes carried by pBinGUSint was confirmed in transformed expiants both by Southern blotting analysis and enzyme activity assays.
ABSTRACT
Se realizó un estudio prospectivo durante 4 años de cepas de estreptococos "viridans" o no agrupables. Se estudiaron 100 cepas clínicamente significativas aisladas de hemocultivos (dos o más muestras) y de otros materiales clínicos (cuando se visualizaban al exame directo, en presencia de reacción inflamatória). Cincuenta y ocho cepas fueron identificadas con 10 reacciones bioquímicas y en 42 fue necesario realizar determinaciones adicionales. Solamente 16 cepas resultaron no identificables. Los setreptococos aislados correspondieron a infecciones piógenas (53%) (en especial S. milleri y S. mitior); endocarditis (27%) (S.bovis I. S. sanguis, S. mutans y S. bovis II); infecciones urinarias (10%) (S. bovis II, S. milleri)
Subject(s)
Humans , Endocarditis, Bacterial/microbiology , In Vitro Techniques , Streptococcus/isolation & purification , Urinary Tract Infections/microbiologyABSTRACT
Se realizó un estudio prospectivo durante 4 años de cepas de estreptococos "viridans" o no agrupables. Se estudiaron 100 cepas clínicamente significativas aisladas de hemocultivos (dos o más muestras) y de otros materiales clínicos (cuando se visualizaban al exame directo, en presencia de reacción inflamatória). Cincuenta y ocho cepas fueron identificadas con 10 reacciones bioquímicas y en 42 fue necesario realizar determinaciones adicionales. Solamente 16 cepas resultaron no identificables. Los setreptococos aislados correspondieron a infecciones piógenas (53%) (en especial S. milleri y S. mitior); endocarditis (27%) (S.bovis I. S. sanguis, S. mutans y S. bovis II); infecciones urinarias (10%) (S. bovis II, S. milleri) (AU)
Subject(s)
Humans , In Vitro Techniques , Streptococcus/isolation & purification , Endocarditis, Bacterial/microbiology , Urinary Tract Infections/microbiologyABSTRACT
In a 4 year prospective study of "viridans" or non-groupable streptococci 100 clinically significant strains were isolated. Strains were defined as significant when found in two or more samples of blood cultures, or when seen on Gram staining together with inflammatory reaction. Fifty-two strains were speciated by 10 physiological characteristics. In 42 cases additional tests were performed. Only 16 isolates were unidentifiable by the techniques used. Sources of isolates included purulent lesions (53%) (mainly S. milleri and S. mitior); endocarditis (27%) (S. bovis I, S. sanguis, S. mutans, S. bovis II) and urinary tract infections (10%) (S. bovis II, S. milleri).
Subject(s)
Streptococcal Infections/microbiology , Streptococcus/classification , Endocarditis, Bacterial/microbiology , Humans , Prospective Studies , Streptococcus/isolation & purificationABSTRACT
In a 4 year prospective study of [quot ]viridans[quot ] or non-groupable streptococci 100 clinically significant strains were isolated. Strains were defined as significant when found in two or more samples of blood cultures, or when seen on Gram staining together with inflammatory reaction. Fifty-two strains were speciated by 10 physiological characteristics. In 42 cases additional tests were performed. Only 16 isolates were unidentifiable by the techniques used. Sources of isolates included purulent lesions (53
) (mainly S. milleri and S. mitior); endocarditis (27
) (S. bovis I, S. sanguis, S. mutans, S. bovis II) and urinary tract infections (10
) (S. bovis II, S. milleri).
ABSTRACT
Data on methicillin-resistant Staphylococcus aureus have not been presented before in Argentina. Due to non adequate microbiologic techniques, this phenomenon may be overlooked. Heterogeneous strains are only detected with 5% NaCl agar, low temperature (30 degrees C) or prolonged incubation and/or high inocula (10(6) C.F.U.). Minimal inhibitory concentrations (MICs) of methicillin were studied by the agar dilution technique in antibiotic medium number 4 with and without 5% salt. This study was performed for 107 strains at 37 degrees C, after 24 and 48 h incubation, with an inoculum size of 10(6) CFU. S. aureus strains were considered resistant whenever MICs were greater than or equal to 8 mg/l. Forty seven (44%) methicillin-resistant strains were detected. Thirty one were high level resistant mutants and sixteen showed the heterogeneous phenomenon. Due to the high frequency of resistant strains among clinical specimens in this country, the routine use of elaborate screening tests with special media and or prolonged incubation time is justified.
Subject(s)
Methicillin/pharmacology , Argentina , Bacteriological Techniques , Humans , Penicillin Resistance , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purificationABSTRACT
Data on methicillin-resistant Staphylococcus aureus have not been presented before in Argentina. Due to non adequate microbiologic techniques, this phenomenon may be overlooked. Heterogeneous strains are only detected with 5
NaCl agar, low temperature (30 degrees C) or prolonged incubation and/or high inocula (10(6) C.F.U.). Minimal inhibitory concentrations (MICs) of methicillin were studied by the agar dilution technique in antibiotic medium number 4 with and without 5
salt. This study was performed for 107 strains at 37 degrees C, after 24 and 48 h incubation, with an inoculum size of 10(6) CFU. S. aureus strains were considered resistant whenever MICs were greater than or equal to 8 mg/l. Forty seven (44
) methicillin-resistant strains were detected. Thirty one were high level resistant mutants and sixteen showed the heterogeneous phenomenon. Due to the high frequency of resistant strains among clinical specimens in this country, the routine use of elaborate screening tests with special media and or prolonged incubation time is justified.
ABSTRACT
Data on methicillin-resistant Staphylococcus aureus have not been presented before in Argentina. Due to non adequate microbiologic techniques, this phenomenon may be overlooked. Heterogeneous strains are only detected with 5
NaCl agar, low temperature (30 degrees C) or prolonged incubation and/or high inocula (10(6) C.F.U.). Minimal inhibitory concentrations (MICs) of methicillin were studied by the agar dilution technique in antibiotic medium number 4 with and without 5
salt. This study was performed for 107 strains at 37 degrees C, after 24 and 48 h incubation, with an inoculum size of 10(6) CFU. S. aureus strains were considered resistant whenever MICs were greater than or equal to 8 mg/l. Forty seven (44
) methicillin-resistant strains were detected. Thirty one were high level resistant mutants and sixteen showed the heterogeneous phenomenon. Due to the high frequency of resistant strains among clinical specimens in this country, the routine use of elaborate screening tests with special media and or prolonged incubation time is justified.
