Long-term arsenic exposure impairs differentiation in mouse embryonal stem cells.

Arsenic is a contaminant found in many foods and drinking water. Exposure to arsenic during development can cause improper neuronal progenitor cell development, differentiation, and function, while in vitro studies have determined that acute arsenic exposure to stem and progenitor cells reduced their ability to differentiate. In the current study, P19 mouse embryonal stem cells were exposed continuously to 0.1-µM (7.5 ppb) arsenic for 32 weeks. A cell lineage array examining messenger RNA (mRNA) changes after 8 and 32 weeks of exposure showed that genes involved in pluripotency were increased, whereas those involved in differentiation were reduced. Therefore, temporal changes of select pluripotency and neuronal differentiation markers throughout the 32-week chronic arsenic exposure were investigated. Sox2 and Oct4 mRNA expression were increased by 1.9- to 2.5-fold in the arsenic-exposed cells, beginning at Week 12. Sox2 protein expression was similarly increased starting at Week 16 and remained elevated by 1.5-fold to sixfold. One target of Sox2 is N-cadherin, whose expression is a hallmark of epithelial-mesenchymal transitions (EMTs). Exposure to arsenic significantly increased N-cadherin protein levels beginning at Week 20, concurrent with increased grouping of N-cadherin positive cells at the perimeter of the embryoid body. Expression of Zeb1, which helps increase the expression of Sox2, was also increased started at Week 16. In contrast, Gdf3 mRNA expression was reduced by 3.4- to 7.2-fold beginning at Week 16, and expression of its target protein, phospho-Smad2/3, was also reduced. These results suggest that chronic, low-level arsenic exposure may delay neuronal differentiation and maintain pluripotency.

Effect of melatonin on bovine theca cells in vitro.

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8-22mm) follicles and treated with various hormones in serum-free medium for 24h or 48h. Melatonin caused a dose-dependent inhibition (P<0.05) of LH+insulin-like growth factor 1 (IGF1)-induced androstenedione and progesterone production. Also, melatonin inhibited (P<0.05) LH+IGF1-induced expression of steroidogenic acute regulatory protein (StAR) mRNA (via real-time polymerase chain reaction) in theca cells, but it had no effect (P>0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P<0.05) the number of bovine theca cells in both LH- and LH+IGF1-treated cultures. In conclusion, melatonin may act as an endocrine regulator of ovarian function in cattle by stimulating theca cell proliferation and inhibiting differentiation via inhibition of hormone-induced steroidogenesis.

Influence of block copolymer feature size on reactive ion etching pattern transfer into silicon.

A successful realisation of sub-20 nm features on silicon (Si) is becoming the focus of many technological studies, strongly influencing the future performance of modern integrated circuits. Although reactive ion etching (RIE), at both micrometric and nanometric scale has already been the target of many studies, a better understanding of the different mechanisms involved at sub-20 nm size etching is still required. In this work, we investigated the influence of the feature size on the etch rate of Si, performed by a cryogenic RIE process through cylinder-forming polystyrene-block-polymethylmethacrylate (PS-b-PMMA) diblock copolymer (DBC) masks with diameter ranging between 19-13 nm. A sensible decrease of the etch depth and etch rate was observed in the mask with the smallest feature size. For all the DBCs under investigation, we determined the process window useful for the correct transfer of the nanometric cylindrical pattern into a Si substrate. A structural and physicochemical investigation of the resulting nanostructured Si is reported in order to delineate the influence of various RIE pattern effects. Feature-size-dependent etch, or RIE-lag, is proved to significantly affect the obtained results.

Neutral wetting brush layers for block copolymer thin films using homopolymer blends processed at high temperatures.

Binary homopolymer blends of two hydroxyl-terminated polystyrene (PS-OH) and polymethylmethacrylate (PMMA-OH) homopolymers (Mn âˆ¼ 16000 g mol(-1)) were grafted on SiO2 substrates by high-temperature (T > 150 °C), short-time (t < 600 s) thermal treatments. The resulting brush layer was tested to screen preferential interactions of the SiO2 substrate with the different symmetric and asymmetric PS-b-PMMA block copolymers deposited on top of the grafted molecules. By properly adjusting the blend composition and the processing parameters, an efficient surface neutralization path was identified, enabling the formation, in the block copolymer film, of homogeneous textures of lamellae or cylinders perpendicularly oriented with respect to the substrate. A critical interplay between the phase segregation of the homopolymer blends and their grafting process on the SiO2 was observed. In fact, the polar SiO2 is preferential for the PMMA-rich phase that forms a homogeneous layer on the substrate, while the PS-rich phase is located at the polymer-air interface. During the thermal treatment, phase segregation and grafting proceed simultaneously. Complete wetting of the PS rich phase on the PMMA rich phase leads to the formation of a PS/PMMA bilayer. In this case, the progressive diffusion of PS chains toward the polymer-SiO2 interface during the thermal treatment allows tuning of the brush layer composition.

Solid-state dewetting of ultra-thin Au films on SiO2 and HfO2.

Ultra-thin Au films with thickness (h) ranging from 0.5 to 6.0 nm were deposited at room temperature (RT) by means of e-beam evaporation on SiO2 and HfO2. Due to the natural solid-state dewetting (SSD) of the as-deposited films, Au nanoparticles (NPs) were formed on the substrates. By properly adjusting the h value, the size and the density of the Au NPs can be finely tuned. For h = 0.5 nm, spherical-like Au NPs with diameter below 5 nm and density in the order of 10(12) Au NPs cm(-2) were obtained without any additional thermal treatment independently from the substrate. The dependence of the Au NPs characteristics on the substrate starts to be effective for h ≥ 1.0 nm where the Au NPs diameter is in the 5-10 nm range and the density is around 10(11) Au NPs cm(-2). The effect of a subsequent high temperature (400-800 °C) annealing in N2 atmosphere on the Au NPs was investigated as well. For h ≤ 1.0 nm, the Au NPs characteristics evidenced an excellent thermal stability. Whereas the thermal treatment affects the cristallinity of the Au NPs. For the thicker films (2.0 ≤ h ≤ 6.0 nm), the thermal treatment becomes effective to induce the SSD. The proposed methodology can be exploited for the synthesis of Au NPs with diameter below 10 nm on different substrates at RT.

Use of combined cutting balloon and high-pressure balloon technique for the treatment of double-chambered right ventricle or primary infundibular stenosis: a case series.

Five dogs and two cats with a diagnosis of double-chambered right ventricle or primary infundibular stenosis were referred to undergo a combined cutting balloon and high-pressure balloon technique. At admission five cases were asymptomatic, one had a history of syncope and one had signs of right-sided congestive heart failure. Each patient underwent a complete transthoracic echocardiogram, thoracic radiographs, an angiogram and the combined interventional procedure. Median diameter of the right mid-ventricular stenosis was 4 mm (range 2-8.7 mm) in dogs, and it measured 1.9 and 2 mm in cats. Under general anesthesia initial dilation with an 8-mm × 2-cm cutting balloon was performed from a left external jugular vein approach followed by dilation with a high-pressure balloon (1.5:1 balloon diameter-right outflow tract diameter ratio). In one dog and the two cats the procedure was not completed due to technical issues. In the other four dogs the median intracavitary proximal chamber pressure decreased from 100 mmHg (range 70-150 mmHg) before the procedure to 57 mmHg (range 45-70 mmHg) post-dilation. Long-term follow-up (from six months to two years) showed complete or partial reverse remodeling of the proximal chamber with a median residual pressure gradient below 80 mmHg (range 46-75 mmHg) for all four dogs. This case series shows that this procedure should be considered in dogs with right ventricular outflow tract obstruction. In cats, the procedure might be feasible, if additional guidewire inventory were available.

In vitro effects of two environmental toxicants, beauvericin and glyphosate in Roundup, on cell numbers and steroidogenesis of bovine ovarian cells.

Beauvericin is an emerging Fusariotoxin naturally occurring in cereal grains throughout the world whereas glyphosate (N-phosphonomethyl-glycine) is a non-selective systemic herbicide used worldwide. The purpose of this study is to evaluate a newly developed ovarian cell culture system (that includes both granulosa and theca cells) as an in vitro model for toxicological studies. Specifically, the effects of beauvericin and glyphosate in formulation with Roundup on ovarian cell numbers and steroid production were evaluated. Ovaries collected from cattle without luteal structures were sliced into 30-70 pieces each, and granulosa and theca cells were collected. Harvested cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 48 h in serum-free medium containing testosterone (500 ng/mL; as an estrogen precursor) with the following eight treatments: (1) controls, (2) FSH (30 ng/mL) alone, (3) FSH plus insulin-like growth factor-1 (IGF1; 30 ng/mL), (4) FSH plus IGF1 plus beauvericin (3 µM), (5) FSH plus IGF1 plus glyphosate in Roundup (10 µg/mL), (6) FSH plus IGF1 plus fibroblast growth factor 9 (FGF9, 30 ng/mL), (7) a negative control without added testosterone, and (8) IGF1 plus LH (30 ng/mL) with basal medium without added testosterone. In the presence of FSH, IGF1 significantly increased cell numbers, estradiol and progesterone production by severalfold. Glyphosate in Roundup formulation significantly inhibited IGF1-induced cell numbers and estradiol and progesterone production by 89-94%. Beauvericin inhibited IGF1-induced cell numbers and estradiol and progesterone by 50-97% production. LH plus IGF1 significantly increased androstenedione secretion compared with controls without added testosterone indicating the presence of theca cells. In conclusion, the present study demonstrates that toxicological effects of beauvericin and glyphosate in Roundup formulation are observed in a newly developed ovarian cell model system and further confirms that both glyphosate and beauvericin may have the potential to impair reproductive function in cattle.

Gene-expression profiling of endomyocardial biopsies from dogs with dilated cardiomyopathy phenotype.

INTRODUCTION: The employment of advanced molecular biology technologies has expanded the diagnostic investigation of cardiomyopathies in dogs; these technologies have predominantly been performed on postmortem samples, although the recent use of endomyocardial biopsy in living dogs has enabled a better premortem diagnostic approach to study the myocardial injury. ANIMALS, MATERIALS, AND METHODS: Endomyocardial biopsies were collected in nine dogs with a dilated cardiomyopathy phenotype (DCM-p) and congestive heart failure and submitted to histologic examination, next-generation sequencing (NGS), and polymerase chain reaction analysis. Data from three healthy dogs (Fastq files) were retrieved from a previously approved study and used as a control group for ribonucleic acid sequencing. RESULTS: Histologic examination revealed endocardial fibrosis in six of nine dogs, whereas lymphocytic interstitial infiltrates were detected in two of nine dogs, and lymphoplasmacytic and macrophage infiltrates were detected in one of nine dogs. On polymerase chain reaction analysis, two dogs tested positive for canine parvovirus two and one dog for canine distemper virus. Gene-expression pathways involved in cellular energy metabolism (especially carbohydrates-insulin) and cardiac structural proteins were different in all DCM-p dogs compared to those in the control group. When dogs with lymphocytic interstitial infiltrates were compared to those in the control group, NGS analysis revealed the predominant role of genes related to inflammation and pathogen infection. CONCLUSIONS: Next-generation sequencing technology performed on in vivo endomyocardial biopsies has identified different molecular and genetic factors that could play a role in the development and/or progression of DCM-p in dogs.

Collective behavior of block copolymer thin films within periodic topographical structures.

We perform a systematic study of the effect of adjacent nanostructures on the confinement of block copolymers (BCP) within pre-patterned trenches in 100 nm thick SiO2 films. Asymmetric PS-b-PMMA BCP with a styrene fraction of 0.71, Mn = 67100 are used. When deposited in the form of thin film, these BCP naturally self-organize upon annealing and form a PS matrix with hexagonally packed PMMA cylinders perpendicularly oriented with respect to the substrate. An accurate study of the confinement of this BCP thin film within isolated trenches is performed as a function of their width (80-260 nm). In this specific configuration the confinement of the BCP thin film within the pre-patterned structures has only been partially achieved. The effect of adjacent trenches on the arrangement of the BCP thin film is investigated using parallel trenches periodically distributed on the surface. The effective confinement of the BCP film is strongly modified by the periodicity of the pre-patterned structures.

In-plane organization of silicon nanocrystals embedded in SiO2 thin films.

Nanofabrication of buried structures with dimensions below 5 nm and with controlled 3D-positioning at the nanoscale was attempted to open new routes to future nanodevices where single nanostructures could be systematically interfaced. A typical example is ultralow-energy ion beam synthesis where already the depth positioning of embedded arrays of silicon nanocrystals can be finely controlled with nanometric precision. In this study, we investigated for the first time the control of the in-plane organization of the nanocrystals using a legitimate patterning option for microelectronic industries, self-assembled block-copolymer. The compatibility with the ultralow-energy ion beam synthesis process of polymeric nanoporous films used as mask was demonstrated together with the capability to control in 3D the organization of Si nanocrystals. The resulting nano-organization consists in a hexagonal array of 20 nm wide nanovolumes containing on average 8 nanocrystals embedded at a controlled depth within a silica matrix.

Rapid thermal processing of self-assembling block copolymer thin films.

Self-assembling block copolymers generate nanostructured patterns which are useful for a wide range of applications. In this paper we demonstrate the capability to control the morphology of the self-assembling process of PS-b-PMMA diblock copolymer thin films on unpatterned surfaces by means of fast thermal treatment performed in a rapid thermal processing machine. The methodology involves the use of radiation sources in order to rapidly drive the polymeric film above the glass transition temperature. Highly ordered patterns were obtained for perpendicular-oriented cylindrical and lamellar PS-b-PMMA block copolymers in less than 60 s. This approach offers the unprecedented opportunity to investigate in detail the kinetics of the block copolymer self-assembly during the early stages of the process, providing a much deeper understanding of the chemical and physical phenomena governing these processes.

Arsenic impairs stem cell differentiation via the Hippo signaling pathway.

Arsenic is a ubiquitous toxic metalloid, with over 150 million people exposed to arsenic concentrations above the current 10 ppb drinking water standard through contaminated food and water. Arsenic is a known developmental toxicant as neuronal and muscle development are disrupted following arsenic exposure during embryogenesis. In this study, murine embryonic stem cells were chronically exposed to 0.1 µM (7.5 ppb) arsenic for 32 weeks. RNA sequencing showed that the Hippo signaling pathway, which is involved in embryonic development and pluripotency maintenance, is impaired following arsenic exposure. Thus, temporal changes in the Hippo pathway's core components and its downstream target genes Ctgf and c-Myc were investigated. Protein expression of the pathway's main effector YAP in its active form was significantly upregulated by 3.7-fold in arsenic-exposed cells at week 8, while protein expression of inactive phosphorylated YAP was significantly downregulated by 2.5- and 2-fold at weeks 8 and 16. Exposure to arsenic significantly increased the ratio between nuclear and cytoplasmic YAP by 1.9-fold at weeks 16 and 28. The ratio between nuclear and cytoplasmic transcriptional enhancer factor domain was similarly increased in arsenic-treated samples by 3.4- and 1.6-fold at weeks 16 and 28, respectively. Levels of Ctgf and c-Myc were also upregulated following arsenic exposure. These results suggest that chronic exposure to an environmentally relevant arsenic concentration might hinder cellular differentiation and maintain pluripotency through the impairment of the Hippo signaling pathway resulting in increased YAP activation.

Arsenic Impairs Differentiation of Human Induced Pluripotent Stem Cells into Cholinergic Motor Neurons.

Arsenic exposure during embryogenesis can lead to improper neurodevelopment and changes in locomotor activity. Additionally, in vitro studies have shown that arsenic inhibits the differentiation of sensory neurons and skeletal muscle. In the current study, human-induced pluripotent stem (iPS) cells were differentiated into motor neurons over 28 days, while being exposed to up to 0.5 µM arsenic. On day 6, neuroepithelial progenitor cells (NEPs) exposed to arsenic had reduced transcript levels of the neural progenitor/stem cell marker nestin (NES) and neuroepithelial progenitor marker SOX1, while levels of these transcripts were increased in motor neuron progenitors (MNPs) at day 12. In day 18 early motor neurons (MNs), choline acetyltransferase (CHAT) expression was reduced two-fold in cells exposed to 0.5 µM arsenic. RNA sequencing demonstrated that the cholinergic synapse pathway was impaired following exposure to 0.5 µM arsenic, and that transcript levels of genes involved in acetylcholine synthesis (CHAT), transport (solute carriers, SLC18A3 and SLC5A7) and degradation (acetylcholinesterase, ACHE) were all downregulated in day 18 early MNs. In day 28 mature motor neurons, arsenic significantly downregulated protein expression of microtubule-associated protein 2 (MAP2) and ChAT by 2.8- and 2.1-fold, respectively, concomitantly with a reduction in neurite length. These results show that exposure to environmentally relevant arsenic concentrations dysregulates the differentiation of human iPS cells into motor neurons and impairs the cholinergic synapse pathway, suggesting that exposure impairs cholinergic function in motor neurons.

Noninvasive electrocardiographic parameters to assess interventricular dyssynchrony in dogs with bundle branch blocks.

OBJECTIVES: To define electrocardiographic features of complete left bundle branch block (LBBB) and right bundle branch block (RBBB), and the use of R-peak time (RPT) to identify interventricular dyssynchrony in dogs with BBB. ANIMALS, MATERIALS AND METHODS: Twelve-lead ECG tracings of 20 dogs with RBBB, 20 with LBBB, and 60 healthy dogs were retrospectively analyzed and RPT was measured in precordial leads. Interventricular dyssynchrony index (IDI) was than calculated. RESULTS: In RBBB, mean electrical axis (MEA) was -111° [-120/-100°], V1RPT was significantly longer (61 ms [55-72 ms]) than left precordial leads RPT (V2:25 ms [22-30 ms]; V3:25 ms [22-29 ms]; V4:24 ms [21-29 ms]; V5:25 ms [22-29 ms]; V6:25 ms [22-29 ms]) and when compared to normal dogs (P < 0.001). In LBBB, MEA was 76° [70/81°], RPT in left precordial leads was significantly longer (V2:49 ms [34-58 ms]; V3:49 ms [43-57 ms]; V4:52 ms [45-62 ms]; V5:53 ms [45-63 ms]; V6:55 ms [45-63 ms]) than V1RPT (17 ms [15-20 ms]) and when compared to normal dogs (P < 0.001). V1RPT > 28 ms and V5RPT > 36 ms were found to predict the presence of RBBB and LBBB with a sensitivity of 100% and 96.7%, and a specificity of 96.7% and 99.5%, respectively. The IDI was 23% [16-29%] in normal dogs and significantly greater in dogs with RBBB (33% [30-38%]; P < 0.001) and LBBB (32% [23-41%]; P = 0.006). CONCLUSIONS: This study defines ECG features and RPT in dogs with BBB. Electrical interventricular dyssynchrony can be defined using IDI in dogs with BBB.

The effect of random copolymer on the characteristic dimensions of cylinder-forming PS-b-PMMA thin films.

The block copolymer self-assembly approach has received great attention in recent years as a possible way to overcome the limits of conventional lithography and to fabricate sub-22 nm structures. At this level, precise nanometric control is crucial for technological applications and the search for a flexible and reproducible protocol is a great challenge. The polystyrene-b-poly(methylmethacrylate) (PS-b-PMMA) system, with a styrene fraction of 0.71, spontaneously separates into a periodic array of hexagonally packed PMMA cylinders embedded in a matrix of PS and, under suitable processing conditions, this is perpendicularly oriented with respect to the underlying substrate. The selective removal of the PMMA allows us to obtain a nanoporous PS matrix with well-defined pore dimensions. Perpendicular orientation of the PMMA cylinders requires surface neutralization by means of a suitable PS-r-PMMA random copolymer. The choice of the random copolymer is not trivial, because different PS-r-PMMA copolymers strongly affect the characteristics of the PS-b-PMMA film deposited on it. In this paper the effects of the selected PS-r-PMMA on the arrangement as well as on the peculiar dimensions (pore diameter, pore to pore distance) of the final nanoporous PS thin film are studied. Reliable protocols for the fabrication of a disposable polymeric mask are proposed in view of its application in advanced lithographic processes.

The fabrication of tunable nanoporous oxide surfaces by block copolymer lithography and atomic layer deposition.

Patterned nanoscale materials with controllable characteristic feature sizes and periodicity are of considerable interest in a wide range of fields, with various possible applications ranging from biomedical to nanoelectronic devices. Block-copolymer (BC)-based lithography is a powerful tool for the fabrication of uniform, densely spaced nanometer-scale features over large areas. Following this bottom-up approach, nanoporous polymeric films can be deposited on any type of substrate. The nanoporous periodic template can be transferred to the underlying substrate by dry anisotropic etching. Nevertheless the physical sizes of the polymeric mask represent an important limitation in the implementation of suitable lithographic protocols based on BC technology, since the diameter and the center-to-center distance of the pores cannot be varied independently in this class of materials. This problem could be overcome by combining block copolymer technology with atomic layer deposition (ALD): by means of BC-based lithography a nanoporous SiO2 template, with well-reproducible characteristic dimensions, can be fabricated and subsequently used as a backbone for the growth of perfectly conformal thin oxide films by ALD. In this work polystyrene-b-poly(methylmethacrylate) (PS-b-PMMA) BC and reactive ion etching are used to fabricate hexagonally packed 23 nm wide nanopores in a 50 nm thick SiO2 matrix. By ALD deposition of Al2O3 thin films onto the nanoporous SiO2 templates, nanostructured Al2O3 surfaces are obtained. By properly adjusting the thickness of the Al2O3 film the dimension of the pores in the oxide films is progressively reduced, with nanometer precision, from the original size down to complete filling of the pores, thus providing a simple and fast strategy for the fabrication of nanoporous Al2O3 surfaces with well-controllable feature size.

Radiofrequency catheter ablation of cranial vena cava flutter in four dogs.

Four dogs were referred to our institution for incessant supraventricular tachycardias causing weakness; congestive heart failure was present in one dog. At admission, all dogs had a surface electrocardiogram showing a narrow QRS complex tachycardia with a ventricular rate ranging from 80 to 300 bpm, variable atrioventricular conduction ratio from 1:1 to 3:1, and positive atrial depolarizations in inferior leads (II, II, III, and aVF), with isoelectric lines between them. Three of four dogs had a dilated cardiomyopathy phenotype; one dog had a heart base tumor involving the cranial vena cava wall. According to the electrocardiographic findings, a presumptive diagnosis of reverse typical or atypical atrial flutter was considered, and endocardial mapping was planned for each dog. During the electrophysiologic study, continuous atrial activation compatible with atypical atrial flutter was observed in all dogs, with concealed entrainment obtained at the level of the isthmus located at the distal portion of the cranial vena cava, close to the entrance into the right atrium. A linear radiofrequency catheter ablation was performed from the right atrial wall to the distal part of the cranial vena cava with a permanent interruption of the isthmic conduction in all dogs at a 6-month follow-up.

Heart rhythm characterization during sudden cardiac death in dogs.

INTRODUCTION/OBJECTIVES: Inherited or acquired arrhythmic disorders and cardiac disease have been associated with sudden cardiac death (SCD) in dogs. The electrical mechanism related to death in most of these cases is unknown. This retrospective study aimed to describe arrhythmic events in dogs that experienced SCD during Holter monitoring. ANIMALS, MATERIALS AND METHODS: Nineteen client-owned dogs that experienced SCD during Holter examination were included. Clinical records from a Holter service database were reviewed, and both the rhythm preceding death and the dominant rhythm causing SCD were analysed. Clinical data, Holter diaries and echocardiographic diagnosis were also evaluated. RESULTS: Structural heart disease was identified in 12/19 dogs (dilated cardiomyopathy in five dogs, arrhythmogenic right ventricular cardiomyopathy in four dogs, myxomatous mitral valve disease in two dogs, and suspected myocarditis in one dog), five of which had concurrent congestive heart failure. Sudden cardiac death was related to ventricular premature complexes or monomorphic ventricular tachycardia degenerating into ventricular fibrillation in 42% of dogs, polymorphic ventricular tachycardia, or torsade de pointes-like inducing ventricular fibrillation in 21%, and asystole or presumptive agonal pulseless electrical activity triggered by malignant bradyarrhythmias in 37%. CONCLUSIONS: The most common rhythm associated with SCD in our population of dogs was ventricular tachycardia leading to ventricular fibrillation, although bradyarrhythmia-related SCD, possibly related to inappropriate vagal reflexes, was also a notable cause.

R-peak time in clinically healthy dogs with different thoracic conformations.

R-peak time (RPT) is an electrocardiographic parameter that represents the time taken for electrical activation to spread from the endocardium to the epicardium. In human medicine, right ventricular RPT is measured from lead V1 to lead V2, and left ventricular RPT from lead V5 to lead V6. The aim of the present study was to define RPT duration in a group of clinically healthy dogs with different thoracic conformations. Sixty clinically healthy dogs underwent a 12-lead electrocardiogram recorded using a previously described precordial system. The dogs were allocated into three morphologic groups. In the brachymorphic group, the median and 25th-75th percentiles for RPT in V1 were 10.5 ms (10-12 ms); V2, 18 ms (16.5-20 ms); V3, 19 ms (18-22 ms); V4, 20 ms (17-23.5 ms); V5, 21 ms (18.5-24 ms); and V6: 22 ms (18.5-25.5 ms). In the mesomorphic group, RPT in V1 was 16 ms (14-18 ms); V2, 22 ms (20-24 ms); V3, 23 ms (21-25 ms); V4, 23 ms (22-25 ms); V5, 25 ms (23-27 ms); and V6, 28 ms (25-30 ms). In the dolichomorphic group, RPT in V1 was 15 ms (13-17 ms); V2, 29 ms (26-32.5 ms); V3, 30 ms (27-33.5 ms); V4, 29.5 ms (26-35 ms); V5, 30 ms (28-34 ms); and V6, 31.5 ms (28-35 ms). RPT in V1 was significantly shorter than RPT in V2 to V6 in all morphotypes (P < 0.05). In all precordial leads, RPT was significantly different between morphotypes (P < 0.05). These results are in agreement with previous findings in humans and with the observation that V1 reads the right ventricle and V2 to V6 read the left ventricle. These preliminary data provide RPT ranges in clinically healthy dogs of different morphotypes.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro.

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.