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The objective of the study was to retrospectively evaluate the short-term safety of intrathecal administration of cytosine arabinoside alone or in combination with methotrexate in dogs and cats. One hundred and twelve dogs and eight cats admitted between September 2008 and December 2013, diagnosed with suspected inflammatory (meningoencephalomyelitis of unknown aetiology) or neoplastic disease affecting brain or spinal cord and treated with an intrathecal administration of cytosine arabinoside alone or in combination with methotrexate were included in the study. Recorded information regarding possible adverse events during administration while recovering from anaesthesia and during hospitalization period were evaluated. The results showed that one patient developed generalized tonic-clonic seizure activity after administration of cytosine arabinoside and methotrexate during recovery from anaesthesia, however responded to intravenous administration of diazepam. On the base of our results we can conclude that intrathecal administration of cytosine arabinoside alone or in combination with methotrexate is a safe procedure in dogs and cats. [ABSTRACT FROM AUTHOR]

Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.

Five dogs, four small mixed breed and a Doberman Pinscher, presented in our clinic with hemivertebra. Complete physical, radiological and neurological examinations were done and the spinal deformities were characterized in accord with the Nasca classification used in human medicine. Two dogs had multiple hemivertebrae (round, oval or wedge-shaped: Type 3) in the thoracic region; one dog had an individual surplus half vertebral body (Type 1) plus a wedge-shaped hemivertebra (Type 2b) in the lumbar region; one dog had multiple hemivertebrae which were fused on one side (Type 4a) in the thoracic region; and one dog had a wedge-shaped hemivertebra (Type 2a) in the cervical region.

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200-500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets., Methods and Results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins ( e.g. , TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2 , Fbln3 , Pcoc1 , Col1a1 and Col3a1 . To examine the role of miR-199a-3p in vivo , we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts., Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo , using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin ( Tcap S157/161A ). Methods and Results: Tcap S157/161A and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, Tcap S157/161A mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from Tcap S157/161A mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin-proteasome system in the regulation of telethonin protein expression level. Tcap S157/161A mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in Tcap S157/161A mice. Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lewis, Eminaga, Gautel and Avkiran.)

Background: Apart from the absence of nociception, there is no readily available prognostic test for dogs presenting with paraplegia secondary to acute intervertebral disc extrusion (IVDE)., Objective: To assess if serum C-reactive protein (CRP) can predict the postoperative outcome in paraplegic dogs undergoing surgery for IVDE and to assess the association between serum CRP and presence/absence of nociception on admission, and serum CRP and presence/absence of intramedullary changes seen on magnetic resonance imaging (MRI)., Animals: One hundred dogs that underwent surgery at our hospital between 2018 and 2020 because of acute paraplegia secondary to IVDE and in which serum CRP was measured., Methods: Retrospective observational cohort study. Dogs were classified as 4 or 5 according to the modified Frankel score (MFS) depending on presence/absence of nociception, respectively. MRI images were reviewed and the T2-weighted hyperintensity: L2 vertebral body length was measured. Postoperative outcome was defined as positive if nociception, ambulation or both returned after decompressive surgery., Results: The median (95% CI) serum CRP was 4 (4-5) and 6 (4-7) mg/L in MSF4 and MSF5, respectively (P = .03). A weak linear relationship (R 2  = 0.049, P = .03) was found between CRP and the T2-weighted hyperintensity: L2 vertebral length. Outcome data was available for 85 dogs: CRP was 4 (4-5) and 5 (4-10) mg/L in positive and negative outcome dogs, respectively (P = .32)., Conclusion and Clinical Importance: Serum CRP did not predict outcome after surgery in dogs with paraplegia secondary to IVDE., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

An 8-year-old neutered female English Pointer was referred to a veterinary referral center (southwest of England) with a 4-5-month history of fecal incontinence and no evidence of urinary incontinence. Blood and free-catch urine samples were collected and sent to an off-site laboratory. Further investigations were postponed until laboratory results were available. Blood results showed a mild leukopenia, mild nonregenerative anemia, moderate to marked thrombocytopenia, and a mild increase in ALT and ALP activities. The primary veterinarian and client did not proceed with any further investigations for thrombocytopenia. Three weeks after the initial presentation, there was considerable clinical deterioration and progression of neurologic signs. Thoracic radiographs and an abdominal ultrasonographic examination were unremarkable. Magnetic resonance imaging (MRI) of the brain and spinal cord revealed an intramedullary lesion at the level of the C7 vertebra, a cystic lesion in the forebrain, and a bilateral lesion in the thalamus. A lumbar cerebrospinal fluid (CSF) was collected. CSF analysis showed a robustly increased protein concentration and marked pleocytosis. The cytologic evaluation revealed a mixed cellular population. Occasional neutrophils and monocytoid cells showed purple spherical intracellular inclusions, resembling Ehrlichia morulae. An aliquot of CSF was used off-label with a dot ELISA test, which showed a strong positive result for antibodies against Ehrlichia canis/Ehrlichia ewingii. PCR identified these morulae to be E canis. To best of the authors' knowledge, this is the first case of ehrlichial infection in canine CSF where Ehrlichia sub-species morulae present within neutrophils were confirmed to be Ehrlichia canis using PCR., (© 2020 American Society for Veterinary Clinical Pathology.)

Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 μM ouabain 100 nM blebbistatin) or chronic myocardial Na i load (PLM 3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic 'fingerprint'. Control (PLM WT ), transgenic (PLM 3SA ), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23 Na, 31 P, 13 C NMR followed by 1 H-NMR metabolomic profiling. Elevated Na i leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Na i overload or inhibition of Na/Ca mito may be a new approach to ameliorate metabolic dysregulation in heart failure.

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21-null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21-null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.

This unit describes a step-by-step protocol to detect and quantify proliferating cells in paraffin-embedded tissue sections. Two well-established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after antigen-retrieval and subsequent immunofluorescence staining and confocal microscopy. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLN R9C/+ ). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT, P = 8 × 10 -4 ) and activation of proinflammatory signaling with notable cardiomyocyte-specific induction of a subset of profibrotic cytokines including TGFβ2 and TGFβ3. These changes progressed through DCM and HF, resulting in substantial fibrosis (17.6% of left ventricle [LV] vs. WT, P = 6 × 10 -33 ). Cardiomyocytes displayed a marked shift in metabolic gene transcription: downregulation of aerobic respiration and subsequent upregulation of glucose utilization, changes coincident with attenuated expression of PPARα and PPARγ coactivators -1α (PGC1α) and -1β, and increased expression of the metabolic regulator T-box transcription factor 15 ( Tbx15 ). Comparing DCM transcriptional profiles with those in hypertrophic cardiomyopathy (HCM) revealed similar and distinct molecular mechanisms. Our data suggest that cardiomyocyte-specific cytokine expression, early fibroblast activation, and the shift in metabolic gene expression are hallmarks of cardiomyopathy progression. Notably, key components of these profibrotic and metabolic networks were disease specific and distinguish DCM from HCM.

A ten-year-old male neutered Rhodesian ridgeback cross dog was presented for the investigation of abnormal bilateral protrusion of the third eyelid when chewing. Physical, ophthalmological, and neurological examinations were unremarkable. Thoracic radiographs, abdominal ultrasound, and magnetic resonance of the brain and orbits failed to reveal any abnormalities. Cerebrospinal fluid analysis revealed elevated protein, but the nucleated cell count was normal. trigemino-abducens synkinesis was presumptively diagnosed. Aetiopathogenesis of this condition is discussed. To the authors' knowledge, this is the first report of presumed trigemino-abducens synkinesis in a dog., (© 2014 American College of Veterinary Ophthalmologists.)

Five cats presented with acute-onset neurological signs. Magnetic resonance imaging in four cats showed a T2-weighted hyperintense spinal cord lesion that was mildly contrast-enhancing in three cats. Owing to inflammatory cerebrospinal fluid changes three cats were treated with immunosuppression. One cat was treated with antibiotics. All cats improved initially, but were eventually euthanased owing to the recurrence of neurological signs. Histopathology in all cats showed hyaline degeneration of the ventral spinal artery, basilar artery or associated branches with aneurysmal dilation, thrombosis and ischemic degeneration and necrosis of the spinal cord and brain. Two cats also had similar vascular changes in meningeal vessels. Vascular hyaline degeneration resulting in vascular aneurysmal dilation and thrombosis should be a differential diagnosis in cats presenting with acute central nervous system signs., (© ISFM and AAFP 2014.)

The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5' start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5' start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5'RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X-encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5'RNA-Seq has the potential to identify genome-wide changes in 5' start-site usage that are associated with pathogenic phenotypes.

Rapid advancement of next-generation sequencing technologies has made it possible to study expression profiles of microRNAs (miRNAs) comprehensively and efficiently. Multiplexing miRNA libraries by barcoding can significantly reduce sequencing cost per sample without compromising library quality. This unit provides a step-by-step protocol for isolating miRNAs and constructing multiplexed miRNA libraries. Also described is a custom computational pipeline for analyzing the multiplexed miRNA library sequencing reads generated by Illumina-based technology., (© 2013 by John Wiley & Sons, Inc.)

Next-generation sequencing offers many advantages over other methods of microRNA (miRNA) expression profiling, such as sample throughput and the capability to discover novel miRNAs. As the sequencing depth of current sequencing platforms exceeds what is necessary to quantify miRNAs, multiplexing several samples in one sequencing run offers a significant cost advantage. Although previous studies have achieved this goal by adding bar codes to miRNA libraries at the ligation step, this was recently shown to introduce significant bias into the miRNA expression data. This bias can be avoided, however, by bar coding the miRNA libraries at the PCR step instead. Here, we describe a user-friendly PCR bar-coding method of preparing multiplexed microRNA libraries for Illumina-based sequencing. The method also prevents the production of adapter dimers and can be completed in one day., (© 2012 by John Wiley & Sons, Inc.)

Practical Relevance: Acute spinal conditions are a common emergency presentation in general veterinary practice and have the potential to cause devastating spinal cord injury (SCI) and consequent severe neurological deficits. SCI can be divided into two subgroups: exogenous SCI (vertebral fracture and/or luxation/subluxation) and endogenous SCI (intervertebral disc extrusion and ischaemic myelopathy)., Clinical Challenges: The majority of cats with SCI have concurrent injuries. The clinician must perform a thorough physical examination and prioritise and then stabilise the life-threatening problems before focusing on the neurological examination. The possibility of multiple sites of SCI and spinal shock can make interpretation of the neurological examination challenging. While plain radiographs or myelography are usually diagnostic, they do not give direct information about the integrity of the spinal cord parenchyma or the severity of any damage. If facilities or experienced staff capable of performing the necessary surgery are not available, or advanced imaging is indicated, referral to a specialist veterinary institution should be considered., Audience: This review is aimed at clinicians dealing with feline SCI in the emergency setting or at first-opinion level, and discusses causes, initial management, specific treatment and prognosis., Patient Group: While any cat may potentially be affected by SCI, there is a tendency for exogenous SCI to be more common in younger individuals and, in the authors' experience, pure-breed cats are very rarely presented. Endogenous SCI can be seen in any breed and is typically a condition of adult cats., (Copyright © 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.)

Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number.

Increased circulating levels of resistin have been proposed as a possible link between obesity and insulin resistance; however, many of the potential metabolic effects of resistin remain to be investigated, including systemic versus local resistin action. We investigated potential autocrine effects of resistin on lipid and glucose metabolism in 2- and 16-mo-old transgenic spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin under control of the aP2 promoter. To search for possible molecular mechanisms, we compared gene expression profiles in adipose tissue in 6-wk-old transgenic SHR versus control rats, before development of insulin resistance, by digital transcriptional profiling using high-throughput sequencing. Both young and old transgenic rats showed moderate expression of the resistin transgene in adipose tissue but had serum resistin levels similar to control SHR and undetectable levels of transgenic resistin in the circulation. Young transgenic rats exhibited mild glucose intolerance. In contrast, older transgenic rats displayed marked glucose intolerance in association with near total resistance of adipose tissue to insulin-stimulated glucose incorporation into lipids (6 ± 2 vs. 77 ± 19 nmol glucose·g(-1)·2 h(-1), P < 0.00001). Ingenuity Pathway Analysis of differentially expressed genes revealed calcium signaling, Nuclear factor-erythroid 2-related factor-2 (NRF2)-mediated oxidative stress response, and actin cytoskeletal signaling canonical pathways as those most significantly affected. Analysis using DAVID software revealed oxidative phosphorylation, glutathione metabolism, pyruvate metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling as top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These results suggest that with increasing age autocrine effects of resistin in fat tissue may predispose to diabetes in part by impairing insulin action in adipose tissue.

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC(403/+)) and an Mef2-dependent β-galactosidase reporter transgene. In young, prehypertrophic MHC(403/+) mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC(403/+) myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (βMHC), a molecular marker of hypertrophy, although MHC(403/+) myocytes with or without βMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC(403/403) mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC(403/403) hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC(403/+) and MHC(403/403) hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die.

Mutations in sarcomere protein genes can cause hypertrophic cardiomyopathy (HCM), a disorder characterized by myocyte enlargement, fibrosis, and impaired ventricular relaxation. Here, we demonstrate that sarcomere protein gene mutations activate proliferative and profibrotic signals in non-myocyte cells to produce pathologic remodeling in HCM. Gene expression analyses of non-myocyte cells isolated from HCM mouse hearts showed increased levels of RNAs encoding cell-cycle proteins, Tgf-β, periostin, and other profibrotic proteins. Markedly increased BrdU labeling, Ki67 antigen expression, and periostin immunohistochemistry in the fibrotic regions of HCM hearts confirmed the transcriptional profiling data. Genetic ablation of periostin in HCM mice reduced but did not extinguish non-myocyte proliferation and fibrosis. In contrast, administration of Tgf-β-neutralizing antibodies abrogated non-myocyte proliferation and fibrosis. Chronic administration of the angiotensin II type 1 receptor antagonist losartan to mutation-positive, hypertrophy-negative (prehypertrophic) mice prevented the emergence of hypertrophy, non-myocyte proliferation, and fibrosis. Losartan treatment did not reverse pathologic remodeling of established HCM but did reduce non-myocyte proliferation. These data define non-myocyte activation of Tgf-β signaling as a pivotal mechanism for increased fibrosis in HCM and a potentially important factor contributing to diastolic dysfunction and heart failure. Preemptive pharmacologic inhibition of Tgf-β signals warrants study in human patients with sarcomere gene mutations.

Noonan syndrome (NS) is an autosomal dominant disorder that is associated with multiple developmental abnormalities. Activated mutations of the protein-tyrosine phosphatase, SHP-2/PTPN11, have been reported in approximately 50% of NS cases. Despite being activated, NS-associated SHP-2 mutants require plasma membrane proximity to evoke disease-associated signaling. Here we show that NS-associated SHP-2 mutants induce hypertyrosyl phosphorylation of the transmembrane glycoproteins, SIRPalpha (signal-regulatory protein alpha) and PZR (protein zero-related), resulting in their increased association with NS-associated SHP-2 mutants. NS-associated SHP-2 mutants enhanced SIRPalpha and PZR tyrosyl phosphorylation either by impairing SIRPalpha dephosphorylation or by promoting PZR tyrosyl phosphorylation. Importantly, during embryogenesis in a mouse model of NS, SIRPalpha and PZR were hypertyrosyl-phosphorylated and bound increased levels of the NS-associated SHP-2 mutant. SIRPalpha and PZR have been implicated in extracellular matrix-dependent signaling. Mouse embryonic fibroblasts derived from a mouse model of NS displayed enhanced ERK activation in response to fibronectin plating. Knockdown of SIRPalpha and PZR in these cells attenuated the enhanced activation of ERK following fibronectin plating. Thus, SIRPalpha and PZR serve as scaffolds that facilitate plasma membrane recruitment and signaling of NS-associated SHP-2 mutants.

This study reports the diagnosis and the surgical management of atlanto-axial instability with an oblique axial fracture in a dog. The fracture was diagnosed by radiography and Computed Tomography (CT). In the CT views, the appearance of the distortion of the atlantoaxial articular surface was interpreted as instability. The stabilization was achieved with the cancellous screws. Following the surgery, the neurological status improved and the dog no longer had marked neurological deficits.

The objective of the study was to determine the effect of the dispersed or nondispersed form of the extruded disk material (EDM) on the neurological status and surgical outcomes in Hansen thoracolumbar intervertebral disk disease Type I (IVDD-I). Medical records of 40 dogs with IVDD-I were reviewed, including neurologic status on admission, findings on magnetic resonance imaging (MRI), intraoperative findings, and surgical outcomes. In MRI evaluations, EDM was on the right in 16, on the left in 18, and centrally in 6 cases; in all cases, findings were confirmed by surgery. Extruded disk material was localized and classified as dispersed disk (DD) or nondispersed disk (NDD) according to its dispersion in the epidural space on MRI. Twenty-five dogs had DD and 15 had NDD on both MRI and surgery. There was no significant difference between DD and NDD in preoperative neurological status and surgical outcomes (P > 0.05).

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.