My Experience in a Clinical Trial for a Rare Gynecologic Cancer.

In this Patient Platform, historian Susan Wade, Ph.D., shares her experience as a participant in an ongoing single-arm phase 2 study of chemoimmunotherapy and radiation for vulvar cancer. She offers her perspective on some of the challenges of having a rare tumor and participating in a clinical trial.

Mice with an RGS-insensitive Gαi2 protein show growth hormone axis dysfunction.

Mice carrying an RGS-insensitive Gαi2 mutation display growth retardation early after birth. Although the growth hormone (GH)-axis is a key endocrine modulator of postnatal growth, its functional state in these mice has not been characterized. The present study was undertaken to address this issue. Results revealed that pituitary mRNA levels for GH, prolactin (PRL), somatostatin (SST), GH-releasing-hormone receptor (GHRH-R) and GH secretagogue receptor (GHS-R) were decreased in mutants compared to controls. These changes were reflected by a significant decrease in plasma levels of GH, IGF-1 and IGF-binding protein-3 (IGFBP-3). Mutants were also less responsive to GHRH and ghrelin (GhL) on GH stimulation of release from pituitary primary cell cultures. In contrast, they were more sensitive to the inhibitory effect of SST. These data provide the first evidence for an alteration of the functional state of the GH-axis in Gαi2G184S mice that likely contributes to their growth retardation.

PARASITES OF THE BLUE IGUANA (CYCLURA LEWISI) FROM GRAND CAYMAN ISLAND.

Feces (n = 226; 2004-2015) from healthy captive and wild blue iguanas (Cyclura lewisi) from Grand Cayman, Cayman Islands, were examined for endoparasites. Parasites identified included Nyctotherus sp. and Entamoeba sp. cysts and trophozoites, trichomonad trophozoites, and oxyurid and trichostrongylid eggs. Endoparasites from postmortem examinations (n = 13) included adult and larval nematodes: Ozolaimus megatyphlon, Ozolaimus monhystera, Alaeuris travassosi, Atractis mega, and an unidentified species of Oswaldocruzia. Entamoeba spp. were more likely in captive juveniles of both sexes than captive or wild adults of either sex; Entamoeba spp. were more likely in captive adult females than captive adult males; trichomonad trophozoites were more likely in adult captive and wild iguanas of both sexes than in captive juveniles of either sex; and Nyctotherus spp. were more likely in juvenile captive males than captive adult males or females and more likely in adult wild males than captive juvenile males. Trichostrongylid eggs were more likely in adult wild females than adult captive females and more likely in captive and wild adults of both sexes than in captive juveniles of both sexes. Oxyurid eggs were more likely in adult captive and wild iguanas of both sexes than captive juveniles of either sex. Blue iguanas have a variety of endoparasites regardless of age, sex, or captive vs wild status, with no type found exclusively in either captive or wild populations. Ectoparasites from wild adults included adult ticks (Amblyomma torrei) and a single adult mite (Hirstiella trombidiformis). All are new host records for this species and Grand Cayman. Knowledge of parasite status of captive and wild populations is important to evaluate the relative risk of introduction of captive animals into wild populations.

Pharmacological Inhibition of Myocardin-related Transcription Factor Pathway Blocks Lung Metastases of RhoC-Overexpressing Melanoma.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR.

Small-Molecule Inhibition of Rho/MKL/SRF Transcription in Prostate Cancer Cells: Modulation of Cell Cycle, ER Stress, and Metastasis Gene Networks.

Metastasis is the major cause of cancer deaths and control of gene transcription has emerged as a critical contributing factor. RhoA- and RhoC-induced gene transcription via the actin-regulated transcriptional co-activator megakaryocytic leukemia (MKL) and serum response factor (SRF) drive metastasis in breast cancer and melanoma. We recently identified a compound, CCG-1423, which blocks Rho/MKL/SRF-mediated transcription and inhibits PC-3 prostate cancer cell invasion. Here, we undertook a genome-wide expression study in PC-3 cells to explore the mechanism and function of this compound. There was significant overlap in the genes modulated by CCG-1423 and Latrunculin B (Lat B), which blocks the Rho/MKL/SRF pathway by preventing actin polymerization. In contrast, the general transcription inhibitor 5,6-dichloro-1-ß-d-ribofuranosyl-1H-benzimidazole (DRB) showed a markedly different pattern. Effects of CCG-1423 and Lat B on gene expression correlated with literature studies of MKL knock-down. Gene sets involved in DNA synthesis and repair, G1/S transition, and apoptosis were modulated by CCG-1423. It also upregulated genes involved in endoplasmic reticulum stress. Targets of the known Rho target transcription factor family E2F and genes related to melanoma progression and metastasis were strongly suppressed by CCG-1423. These results confirm the ability of our compound to inhibit expression of numerous Rho/MKL-dependent genes and show effects on stress pathways as well. This suggests a novel approach to targeting aggressive cancers and metastasis.

Microlichus americanus acariasis in saffron finches (Sicalis flaveola) with dermatitis and feather loss.

Over a 5-year period, 13 saffron finches (Sicalis flaveola) housed in mixed aviaries at the Bronx Zoo (Bronx, New York) were examined with feather loss and dermatitis, primarily affecting the nape, neck, and dorsum. Feather loss, hyperkeratosis, epidermal hyperplasia, and mixed granulocytic and mononuclear inflammation were identified in biopsies from live birds and tissue sections from postmortem specimens. In 10 of 13 cases, sections of arthropod parasites were seen histologically within feather follicles and along the surface of affected skin. Based on morphological characteristics, mites recovered from samples of formalin-fixed skin in 4 birds were identified as Microlichus americanus, an epidermoptid mite infrequently reported from wild birds and hippoboscid flies. Gross and histological lesions strongly implicate M. americanus as the cause of dermatitis affecting practically all saffron finches in the collection.

Selectivity and anti-Parkinson's potential of thiadiazolidinone RGS4 inhibitors.

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein-protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson's disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3ß, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 µM. CCG-203769 enhances Gαq-dependent cellular Ca(2+) signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson's disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson's disease and other neural disorders.

Gastrointestinal Parasites of Ecuadorian Mantled Howler Monkeys (Alouatta palliata aequatorialis) Based on Fecal Analysis.

An analysis of gastrointestinal parasites of Ecuadorian mantled howler monkeys, Alouatta palliata aequatorialis, was conducted based on examination of fecal smears, flotations, and sedimentations. At least 1 type of parasite was detected in 97% of the 96 fecal samples screened across 19 howler monkey groups using these techniques. Samples averaged 3.6 parasite species per individual (±1.4 SD). Parasites included species representing genera of 2 apicomplexans: Cyclospora sp. (18% of individual samples) and Isospora sp. (3%); 6 other protozoa: Balantidium sp. (9%), Blastocystis sp. (60%), Chilomastix sp. (4%), Dientamoeba sp. (3%), Entamoeba species (56%), Iodamoeba sp. (5%); 4 nematodes: Enterobius sp. (3%), Capillaria sp. (78%), Strongyloides spp. (88%) which included 2 morphotypes, Trypanoxyuris sp. (12%); and the platyhelminth Controrchis sp. (15%). A statistically significant positive correlation was found between group size and each of 3 different estimators of parasite species richness adjusted for sampling effort (ICE: r(2) = 0.24, P = 0.05; Chao2: r(2) = 0.25, P = 0.05, and Jackknife: r(2) = 0.31, P = 0.03). Two significant associations between co-infecting parasites were identified. Based on the prevalence data, individuals infected with Balantidium sp. were more likely to also be infected with Isospora sp. (χ(2) = 6.02, P = 0.01), while individuals harboring Chilomastix sp. were less likely to have Capillaria sp. present (χ(2) = 4.03, P = 0.04).

Conditional disruption of interactions between Gαi2 and regulator of G protein signaling (RGS) proteins protects the heart from ischemic injury.

BACKGROUND: Regulator of G protein signaling (RGS) proteins suppress G protein coupled receptor signaling by catalyzing the hydrolysis of Gα-bound guanine nucleotide triphosphate. Transgenic mice in which RGS-mediated regulation of Gαi2 is lost (RGS insensitive Gαi2G184S) exhibit beneficial (protection against ischemic injury) and detrimental (enhanced fibrosis) cardiac phenotypes. This mouse model has revealed the physiological significance of RGS/Gαi2 interactions. Previous studies of the Gαi2G184S mutation used mice that express this mutant protein throughout their lives. Thus, it is unclear whether these phenotypes result from chronic or acute Gαi2G184S expression. We addressed this issue by developing mice that conditionally express Gαi2G184S. METHODS: Mice that conditionally express RGS insensitive Gαi2G184S were generated using a floxed minigene strategy. Conditional expression of Gαi2G184S was characterized by reverse transcription polymerase chain reaction and by enhancement of agonist-induced inhibition of cAMP production in isolated cardiac fibroblasts. The impact of conditional RGS insensitive Gαi2G184S expression on ischemic injury was assessed by measuring contractile recovery and infarct sizes in isolated hearts subjected to 30 min ischemia and 2 hours reperfusion. RESULTS: We demonstrate tamoxifen-dependent expression of Gαi2G184S, enhanced inhibition of cAMP production, and cardioprotection from ischemic injury in hearts conditionally expressing Gαi2G184S. Thus the cardioprotective phenotype previously reported in mice expressing Gαi2G184S does not require embryonic or chronic Gαi2G184S expression. Rather, cardioprotection occurs following acute (days rather than months) expression of Gαi2G184S. CONCLUSIONS: These data suggest that RGS proteins might provide new therapeutic targets to protect the heart from ischemic injury. We anticipate that this model will be valuable for understanding the time course (chronic versus acute) and mechanisms of other phenotypic changes that occur following disruption of interactions between Gαi2 and RGS proteins.

How prepared are college freshmen athletes for the rigors of college strength and conditioning? A survey of college strength and conditioning coaches.

Training programs for high school athletes have changed over the last 20 years. High school physical education classes have transformed into sport-specific conditioning classes with intensities matching college or professional athlete programming. In addition, involvement in private, sport-specific, training increased; but despite these advanced training methods, are high school athletes prepared for collegiate sport competition? An anonymous survey was sent to 195 Division I strength and conditioning coaches (SCC) to discern incoming college freshman athletes' physical and psychological preparedness for the rigors of collegiate training and sport competition. Fifty-seven (29%) responses were received. Strength and conditioning coaches stated that incoming college freshman athletes lack lower extremity strength, overall flexibility, and core strength as well as proper Olympic lifting technique. Strength and conditioning coaches also stated that athletes lacked the mental toughness to endure collegiate sport training in addition to claiming incoming athletes lacked knowledge of correct nutrition and recovery principles. These results suggest a lack of collegiate training/sport preparedness of high school athletes. High school strength and conditioning specialist's goal is to produce better athletes and doing so requires the strength and conditioning coach/trainer to have knowledge of how to train high school athletes. One way to assure adequate knowledge of strength and conditioning training principles is for high school coaches/trainers to be certified in the field. Strength and conditioning certifications among high school strength and conditioning coaches/trainers would encourage developmentally appropriate training and would provide universities with athletes who are prepared for the rigors of collegiate sport training/competition.

Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay.

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100µM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.

Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway.

CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s).

Microfluidic interrogation and mathematical modeling of multi-regime calcium signaling dynamics.

Through microfluidic interrogation we analyzed real-time calcium responses of HEK293 cells stimulated with short pulses of the M3 muscarinic receptor ligand carbachol in two different concentration regimes. Lower ligand concentrations elicit oscillatory calcium signals while higher concentrations trigger a rapid rise that eventually settles down at a steady-state slightly above pre-stimulus levels, referred to as an acute signal. Cells were periodically pulsed with carbachol at these two concentration regimes using a custom-made microfluidic platform, and the resulting calcium signals were measured with a single fluorescent readout. Pulsed stimulations at these two concentration regimes resulted in multiple types of response patterns that each delivered complementary information about the M3 muscarinic receptor signaling pathway. These multiple types of calcium response patterns enabled development of a comprehensive mathematical model of multi-regime calcium signaling. The resulting model suggests that dephosphorylation of deactivated receptors is rate limiting for recovery of calcium signals in the acute regime (high ligand concentration), while calcium replenishment and IP3 production determine signal recovery in the oscillatory regime (low ligand concentration). This study not only provides mechanistic insight into multi-regime signaling of the M3 muscarinic receptor pathway, but also provides a general strategy for analyzing multi-regime pathways using only one fluorescent readout.

Optimization of novel nipecotic bis(amide) inhibitors of the Rho/MKL1/SRF transcriptional pathway as potential anti-metastasis agents.

CCG-1423 (1) is a novel inhibitor of Rho/MKL1/SRF-mediated gene transcription that inhibits invasion of PC-3 prostate cancer cells in a Matrigel model of metastasis. We recently reported the design and synthesis of conformationally restricted analogs (e.g., 2) with improved selectivity for inhibiting invasion versus acute cytotoxicity. In this study we conducted a survey of aromatic substitution with the goal of improving physicochemical parameters (e.g., ClogP, MW) for future efficacy studies in vivo. Two new compounds were identified that attenuated cytotoxicity even further, and were fourfold more potent than 2 at inhibiting PC-3 cell migration in a scratch wound assay. One of these (8a, CCG-203971, IC50=4.2 µM) was well tolerated in mice for 5 days at 100mg/kg/day i.p., and was able to achieve plasma levels exceeding the migration IC50 for up to 3 h.

Characterization of Giardia duodenalis infections in dogs in Trinidad and Tobago.

To our knowledge, the zoonotic potential of Giardia duodenalis has not been assessed in companion animals in Trinidad and Tobago. This report details the first attempt to evaluate the potential zoonotic risk of G. duodenalis in dogs and identify assemblages of G. duodenalis found in dog populations on both islands. Fecal samples were collected from free-roaming dogs and dogs at the Trinidad and Tobago Society for the Prevention of Cruelty to Animals from October 2010 to June 2011. A total of 168 samples were collected of which 104 samples were analyzed for the presence of G. duodenalis by PCR amplification of the ssu-rRNA gene with subsequent assemblage-typing. A subset of samples was also analyzed by ELISA. Twenty-six samples were positive for G. duodenalis by PCR for an overall prevalence of 25%. Four samples were identified as assemblage C (15.4%), 21 as assemblage D (80.8%), and one as assemblage E (3.8%). Puppies were four-times more likely to be infected with G. duodenalis than adult dogs (OR 4.61, 95% CI 1.73-12.2). There was a significant agreement between ELISA and PCR in the detection of the protozoa (κ=0.67). We infer from our results that while the prevalence of G. duodenalis is relatively high in Trinidad and Tobago, the zoonotic risk of infection in humans is low since neither assemblage A nor B was identified in the study population.

Prevalence of antibodies to Toxoplasma gondii in white-tailed deer (Odocoileus virginianus) in New York State, USA.

Sera collected from 299 white-tailed deer (Odocoileus virginianus) harvested in New York State by hunters in November 2010 were assayed for anti-Toxoplasma gondii IgG antibodies. White-tailed deer are a useful sentinel for risk of human and domestic animal exposure to Toxoplasma oocysts and pose a potential risk for infection to humans and other animals by ingestion of the meat. White-tailed deer share grazing space with domestic animals raised for meat and are likely to be exposed by horizontal transmission through oocyst consumption, similar to other grazing species of economic concern. Overall, 42.2% of samples were positive by enzyme-linked immunosorbent assay, indicating a true prevalence of 38.5%, with a significantly higher proportion of adult than immature deer antibody positive. No significant difference in prevalence was found between male and female deer nor was there a significant effect of local human population density on deer antibody prevalence. These results provide insight into the risk of environmental Toxoplasma exposure in New York State and support horizontal transmission through oocyst consumption as the most common mechanism of white-tailed deer infection.

Free-ranging black howler monkeys, Alouatta pigra, in southern Belize are not parasitized by Controrchis biliophilus.

Several coprological studies of Alouatta pigra, the black howler monkey, inhabiting Belize and Mexico have been published in the past several years. Trematodes, specifically Controrchis biliophilus (Dicrocoeliidae), have been detected in A. pigra from all locations in Belize and Mexico examined in those studies. A routine coprological survey of A. pigra was conducted in May and June 2010 as part of baseline data collection for an A. pigra population in Punta Gorda, Toledo District, southern Belize. The 51 fecal samples collected in this area were all negative for C. biliophilus. Subsequently, two additional fecal samples were collected from another population of A. pigra in Toledo District and 25 additional fecal samples were collected from six other areas of Belize during December 2010 and January 2011 and were examined for parasites. To date, C. biliophilus eggs have been detected in fecal samples from A. pigra inhabiting every district of Belize except Toledo District in southern Belize. This finding is notable, for no other population of A. pigra completely free of C. biliophilus infection had been located prior to this study.

Investigation of an outbreak of besnoitiosis in donkeys in northeastern Pennsylvania.

OBJECTIVE: To describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey operations in northeastern Pennsylvania and to report the outcome of attempted treatment of 1 naturally infected individual. DESIGN: Observational study. ANIMALS: 29 donkeys (Equus asinus) in northeastern Pennsylvania. PROCEDURES: Donkeys were examined for lesions suggestive of besnoitiosis in an outbreak investigation. Information was collected regarding the history and signalment of animals on each premises. Rhinolaryngoscopy was performed to identify nasopharyngeal and laryngeal lesions. Serum samples were collected for immunofluorescent antibody testing and immunoblotting for Besnoitia spp. Skin biopsy samples were obtained from 8 animals with lesions suggestive of besnoitiosis for histologic examination. Quantitative real-time PCR assay for Besnoitia spp was performed on tissue samples from 5 animals. RESULTS: Besnoitiosis was confirmed in 6 of the 8 suspected cases. The most common lesion site was the nares, followed by the skin and sclera. Donkeys with clinical signs of disease had higher serum antibody titers and tested positive for a greater number of immunoblot bands than did donkeys without clinical signs of disease. All animals evaluated by PCR assay tested positive. Putative risk factors for disease included age and sex. Ponazuril was not effective at treating besnoitiosis in a naturally infected donkey. CONCLUSIONS AND CLINICAL RELEVANCE: Knowledge of clinical and serologic features of besnoitiosis in donkeys will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Besnoitiosis may be an emerging disease of donkeys in the United States.

Chimeric protein A/G conjugate for detection of anti-Toxoplasma gondii immunoglobulin G in multiple animal species.

Serological testing for toxoplasmosis diagnosis remains the method of choice in human medicine due to the accessibility of the requisite sample, the difficulty in predicting the parasite's location in the host for direct detection, and the availability of established commercial methods. In veterinary medicine, although the first 2 conditions are unchanged, there is a need for commercially produced test methods that are validated for Toxoplasma gondii detection across the range of animal species that can serve as intermediate hosts. The development of such a serological method for animals would allow the diagnosis of toxoplasmosis in individual animals and a higher throughput method for population-level toxoplasmosis surveys. The incorporation of a non-species-specific chimeric protein A/G conjugate into an anti-Toxoplasma immunoglobulin G enzyme-linked immunosorbent assay is described. Serum from potential intermediate hosts was reevaluated using this method and compared with earlier testing using an established agglutination procedure. Very good agreement between the 2 tests was noted (κ = 0.81), establishing the method as a useful option for veterinary diagnostic testing.

Gαi2 signaling: friend or foe in cardiac injury and heart failure?

Receptors coupled to G proteins have many effects on the heart. Enhanced signaling by Gα(s) and Gα(q) leads to cardiac injury and heart failure, while Gα(i2) signaling in cardiac myocytes can protect against ischemic injury and ß-adrenergic-induced heart failure. We asked whether enhanced Gα(i2) signaling in mice could protect against heart failure using a point mutation in Gα(i2) (G184S), which prevents negative regulation by regulators of G protein signaling. Contrary to our expectation, it worsened effects of a genetic dilated cardiomyopathy (DCM) and catecholamine-induced cardiac injury. Gα (i2) (G184S/+) /DCM double heterozygote mice (TG9(+)Gα (i2) (G184S/+)) had substantially decreased survival compared to DCM animals. Furthermore, heart weight/body weight ratios (HW/BW) were significantly greater in TG9(+)Gα (i2) (G184S/+) mice as was expression of natriuretic peptide genes. Catecholamine injury in Gα (i2) (G184S/G184S) mutant mice produced markedly increased isoproterenol-induced fibrosis and collagen III gene expression vs WT mice. Cardiac fibroblasts from Gα (i2) (G184S/G184S) mice also showed a serum-dependent increase in proliferation and ERK phosphorylation, which were blocked by pertussis toxin and a mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor. Gα(i2) signaling in cardiac myocytes protects against ischemic injury but enhancing Gα(i2) signaling overall may have detrimental effects in heart failure, perhaps through actions on cardiac fibroblasts.