Multisensory gamma stimulation mitigates the effects of demyelination induced by cuprizone in male mice.

Demyelination is a common pathological feature in a wide range of diseases, characterized by the loss of myelin sheath and myelin-supporting oligodendrocytes. These losses lead to impaired axonal function, increased vulnerability of axons to damage, and result in significant brain atrophy and neuro-axonal degeneration. Multiple pathomolecular processes contribute to neuroinflammation, oligodendrocyte cell death, and progressive neuronal dysfunction. In this study, we use the cuprizone mouse model of demyelination to investigate long-term non-invasive gamma entrainment using sensory stimulation as a potential therapeutic intervention for promoting myelination and reducing neuroinflammation in male mice. Here, we show that multisensory gamma stimulation mitigates demyelination, promotes oligodendrogenesis, preserves functional integrity and synaptic plasticity, attenuates oligodendrocyte ferroptosis-induced cell death, and reduces brain inflammation. Thus, the protective effects of multisensory gamma stimulation on myelin and anti-neuroinflammatory properties support its potential as a therapeutic approach for demyelinating disorders.

Oxygen-releasing nanodroplets relieve intratumoral hypoxia in head and neck cancer spheroids.

Hypoxia in solid tumors, including head and neck cancer (HNC), contributes to treatment resistance, aggressive phenotypes, and poor clinical outcomes. Perfluorocarbon nanodroplets have emerged as promising oxygen carriers to alleviate tumor hypoxia. However, a thorough characterization of the hypoxia alleviation effects in terms of sustenance of oxygenated environments have not been thoroughly studied. In this study, we developed and characterized perfluoropentane nanodroplets (PFP NDs) for co-delivery of oxygen and the photoactivatable drug or photosensitizer benzoporphyrin derivative (BPD) to hypoxic HNC spheroids. The PFP NDs exhibited excellent stability, efficient oxygen loading/release, and biocompatibility. Using 3D multicellular tumor spheroids of FaDu and SCC9 HNC cells, we demonstrated the ability of oxygenated PFP NDs to penetrate the hypoxic core and alleviate hypoxia, as evidenced by reduced fluorescence of a hypoxia-sensing reagent and downregulation of hypoxia-inducible factors HIF-1α and HIF-2α. BPD-loaded PFP NDs successfully delivered the photosensitizer into the spheroid core in a time-dependent manner. These findings highlight the potential of PFP NDs as a co-delivery platform to overcome hypoxia-mediated treatment resistance and improve therapy outcomes in HNC.

Defining the Role of the miR-145-KLF4-αSMA Axis in Mitral Valvular Interstitial Cell Activation in Myxomatous Mitral Valve Prolapse Using the Canine Model.

Mitral valve prolapse (MVP) is a common valvular disease, affecting 2-3% of the adult human population and is a degenerative condition. A total of 5-10% of the afflicted will develop severe mitral regurgitation, cardiac dysfunction, congestive heart failure, and sudden cardiac death. Naturally occurring myxomatous MVP in dogs closely resembles MVP in humans structurally, and functional consequences are similar. In both species, valvular interstitial cells (VICs) in affected valves exhibit phenotype consistent with activated myofibroblasts with increased alpha-smooth muscle actin (αSMA) expression. Using VICs collected from normal and MVP-affected valves of dogs, we analyzed the miRNA expression profile of the cells and their associated small extracellular vesicles (sEV) using RNA sequencing to understand the role of non-coding RNAs and sEV in MVP pathogenesis. miR-145 was shown to be upregulated in both the affected VICs and sEV, and overexpression of miR-145 by mimic transfection in quiescent VIC recapitulates the activated myofibroblastic phenotype. Concurrently, KLF4 expression was noted to be suppressed by miR-145, confirming the miR-145-KLF4-αSMA axis. Targeting this axis may serve as a potential therapy in controlling pathologic abnormalities found in MVP valves.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

A novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of nonalcoholic steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver diseases worldwide. There is a pressing clinical need to identify potential therapeutic targets for NASH treatment. Thioredoxin interacting protein (Txnip) is a stress responsive gene that has been implicated in the pathogenesis of NASH, but its exact role is not fully understood. Here, we investigated the liver- and gene-specific role of Txnip and its upstream/downstream signaling in the pathogenesis of NASH. Methods and Results: Using four independent NASH mouse models, we found that TXNIP protein abnormally accumulated in NASH mouse livers. A decrease in E3 ubiquitin ligase NEDD4L resulted in impaired TXNIP ubiquitination and its accumulation in the liver. TXNIP protein levels were positively correlated with that of CHOP, a major regulator of ER stress-mediated apoptosis, in NASH mouse liver. Moreover, gain- and loss-of-function studies showed that TXNIP increased protein not mRNA levels of Chop both in vitro and in vivo. Mechanistically, the C-terminus of TXNIP associated with the N-terminus of the α-helix domain of CHOP and decreased CHOP ubiquitination, thus increasing the stability of CHOP protein. Lastly, selective knockdown of Txnip by adenovirus-mediated shRNA (not targets Txnip antisense lncRNA) delivery in the livers of both young and aged NASH mice suppressed the expression of CHOP and its downstream apoptotic pathway, and ameliorated NASH by reducing hepatic apoptosis, inflammation, and fibrosis. Conclusions: Our study revealed a pathogenic role of hepatic TXNIP in NASH and identified a novel NEDD4L-TXNIP-CHOP axis in the pathogenesis of NASH.

Using cultured canine cardiac slices to model the autophagic flux with doxorubicin.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 µm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.

Prospective study of dilated cardiomyopathy in dogs eating nontraditional or traditional diets and in dogs with subclinical cardiac abnormalities.

BACKGROUND: Recent studies have investigated dogs with presumed diet-associated dilated cardiomyopathy (daDCM), but prospective studies of multiple breeds are needed. HYPOTHESIS/OBJECTIVES: To evaluate baseline features and serial changes in echocardiography and cardiac biomarkers in dogs with DCM eating nontraditional diets (NTDs) or traditional diets (TDs), and in dogs with subclinical cardiac abnormalities (SCA) eating NTD. ANIMALS: Sixty dogs with DCM (NTD, n = 51; TDs, n = 9) and 16 dogs with SCA eating NTDs. METHODS: Echocardiography, electrocardiography, and measurement of taurine, cardiac troponin I, and N-terminal pro-B-type natriuretic peptide were performed in dogs with DCM or SCA. Diets were changed for all dogs, taurine was supplemented in most, and echocardiography and cardiac biomarkers were reassessed (3, 6, and 9 months). RESULTS: At enrollment, there were few differences between dogs with DCM eating NTDs or TDs; none had low plasma or whole blood taurine concentrations. Improvement in fractional shortening over time was significantly associated with previous consumption of a NTD, even after adjustment for other variables (P = .005). Median survival time for dogs with DCM was 611 days (range, 2-940 days) for the NTD group and 161 days (range, 12-669 days) for the TD group (P = .21). Sudden death was the most common cause of death in both diet groups. Dogs with SCA also had significant echocardiographic improvements over time. CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with DCM or SCA previously eating NTDs had small, yet significant improvements in echocardiographic parameters after diet changes.

VEGF Receptor Inhibitor-Induced Hypertension: Emerging Mechanisms and Clinical Implications.

PURPOSE OF REVIEW: While vascular endothelial growth factor receptor inhibitors (VEGFRis) have dramatically improved cancer survival, these drugs cause hypertension in a majority of patients. This side effect is often dose limiting and increases cardiovascular mortality in cancer survivors. This review summarizes recent advances in our understanding of the molecular mechanisms and clinical findings that impact management of VEGFRi-induced hypertension. RECENT FINDINGS: Recent studies define new connections between endothelial dysfunction and VEGFRi-induced hypertension, including the balance between nitric oxide, oxidative stress, endothelin signaling, and prostaglandins and the potential role of microparticles, vascular smooth muscle cells, vascular stiffness, and microvessel rarefaction. Data implicating genetic polymorphisms that might identify patients at risk for VEGFRi-induced hypertension and the growing body of literature associating VEGFRi-induced hypertension with antitumor efficacy are reviewed. These recent advances have implications for the future of cardio-oncology clinics and the management of VEGFRi-induced hypertension.

Dynamical system model predicts when social learners impair collective performance.

A key question concerning collective decisions is whether a social system can settle on the best available option when some members learn from others instead of evaluating the options on their own. This question is challenging to study, and previous research has reached mixed conclusions, because collective decision outcomes depend on the insufficiently understood complex system of cognitive strategies, task properties, and social influence processes. This study integrates these complex interactions together in one general yet partially analytically tractable mathematical framework using a dynamical system model. In particular, it investigates how the interplay of the proportion of social learners, the relative merit of options, and the type of conformity response affect collective decision outcomes in a binary choice. The model predicts that, when the proportion of social learners exceeds a critical threshold, a bistable state appears in which the majority can end up favoring either the higher- or lower-merit option, depending on fluctuations and initial conditions. Below this threshold, the high-merit option is chosen by the majority. The critical threshold is determined by the conformity response function and the relative merits of the two options. The study helps reconcile disagreements about the effect of social learners on collective performance and proposes a mathematical framework that can be readily adapted to extensions investigating a wider variety of dynamics.

Scaling of urban income inequality in the USA.

Urban scaling analysis, the study of how aggregated urban features vary with the population of an urban area, provides a promising framework for discovering commonalities across cities and uncovering dynamics shared by cities across time and space. Here, we use the urban scaling framework to study an important, but under-explored feature in this community-income inequality. We propose a new method to study the scaling of income distributions by analysing total income scaling in population percentiles. We show that income in the least wealthy decile (10%) scales close to linearly with city population, while income in the most wealthy decile scale with a significantly superlinear exponent. In contrast to the superlinear scaling of total income with city population, this decile scaling illustrates that the benefits of larger cities are increasingly unequally distributed. For the poorest income deciles, cities have no positive effect over the null expectation of a linear increase. We repeat our analysis after adjusting income by housing cost, and find similar results. We then further analyse the shapes of income distributions. First, we find that mean, variance, skewness and kurtosis of income distributions all increase with city size. Second, the Kullback-Leibler divergence between a city's income distribution and that of the largest city decreases with city population, suggesting the overall shape of income distribution shifts with city population. As most urban scaling theories consider densifying interactions within cities as the fundamental process leading to the superlinear increase of many features, our results suggest this effect is only seen in the upper deciles of the cities. Our finding encourages future work to consider heterogeneous models of interactions to form a more coherent understanding of urban scaling.

Intravenous administration of allogeneic Wharton jelly-derived mesenchymal stem cells for treatment of dogs with congestive heart failure secondary to myxomatous mitral valve disease.

OBJECTIVE: To evaluate whether mesenchymal stem cells (MSCs) can be safely administered IV to dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve disease (MMVD) to improve cardiac function and prolong survival time. ANIMALS: 10 client-owned dogs with CHF secondary to MMVD. PROCEDURES: Dogs with an initial episode of CHF secondary to MMVD were enrolled in a double-blind, placebo-controlled clinical trial. Five dogs in the MSC group received allogeneic Wharton jelly-derived MSCs (2 × 106 cells/kg, IV), and 5 dogs in the placebo group received a 1% solution of autologous serum (IV) for 3 injections 3 weeks apart. Cell-release criteria included trilineage differentiation, expression of CD44 and CD90 and not CD34 and major histocompatability complex class II, normal karyotype, and absence of contamination by pathogenic microorganisms. Patients were followed for 6 months or until death or euthanasia. Echocardiographic data, ECG findings, serum cardiac biomarker concentrations, CBC, and serum biochemical analysis results were obtained prior to and 4 hours after the first injection and every 3 months after the final injection. RESULTS: Lymphocyte and eosinophil counts decreased significantly 4 hours after injection, and monocytes decreased significantly only in dogs that received an MSC injection. No significant differences were seen in the echocardiographic variables, ECG results, serum cardiac biomarker concentrations, survival time, and time to first diuretic drug dosage escalation between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: This study showed that MSCs can be easily collected from canine Wharton jelly as an allogeneic source of MSCs and can be safely delivered IV to dogs with CHF secondary to MMVD.

The effects of two intramuscular sedation protocols on echocardiographic variables in cats following sedation and blood donation.

OBJECTIVE: To compare effects of 2 IM sedation protocols, alfaxalone-butorphanol (AB) versus dexmedetomidine-butorphanol (DB), on echocardiographic (ECHO) variables in cats following sedation and blood donation. DESIGN: Experimental randomized, blinded crossover study. SETTING: University teaching hospital. ANIMALS: Eleven client-owned healthy cats. INTERVENTIONS: Cats received a baseline ECHO without sedation prior to their first donation. Cats were sedated intramuscularly with AB (alfaxalone, 2 mg/kg, and butorphanol, 0.2 mg/kg) for 1 donation and DB (dexmedetomidine, 10 µg/kg, and butorphanol 0.2, mg/kg) for another, with a minimum 6 weeks between donations. A post-sedation, post-donation ECHO was performed after each blood donation. MEASUREMENTS AND MAIN RESULTS: Eight cats completed the study. Compared to baseline, DB combined with blood donation decreased heart rate (-84/min; P < 0.0001), fractional shortening (-16.5%; P < 0.0001), ejection fraction (-21.0%; P = 0.0002), and cardiac output (-292 mL/min, P = 0.0001); AB combined with blood donation increased heart rate (+45/min; P = 0.0003) and decreased left ventricular end diastolic volume (-1.57 mL; P < 0.0001). Compared to AB, DB decreased heart rate (-129/min; P < 0.0001) and fractional shortening (-21.6%; P < 0.0001) and increased left ventricular end-systolic (+1.14 mL; P = 0.0004) and diastolic volumes (+1.93 mL; P < 0.0002). Cats administered DB had a significant increase in regurgitant flow across mitral, aortic, and pulmonic valves following blood donation (P < 0.05). One cat administered DB developed spontaneous echo contrast in the left ventricle following donation. CONCLUSIONS AND CLINICAL RELEVANCE: Compared to AB, DB had more pronounced effects on ECHO variables in cats following IM sedation and blood donation. Due to its minimal impact on ECHO variables, AB may be a more desirable sedation protocol in this population of cats.

Falling through the cracks: Modeling the formation of social category boundaries.

Social categorizations divide people into "us" and "them", often along continuous attributes such as political ideology or skin color. This division results in both positive consequences, such as a sense of community, and negative ones, such as group conflict. Further, individuals in the middle of the spectrum can fall through the cracks of this categorization process and are seen as out-group by individuals on either side of the spectrum, becoming inbetweeners. Here, we propose a quantitative, dynamical-system model that studies the joint influence of cognitive and social processes. We model where two social groups draw the boundaries between "us" and 'them" on a continuous attribute. Our model predicts that both groups tend to draw a more restrictive boundary than the middle of the spectrum. As a result, each group sees the individuals in the middle of the attribute space as an out-group. We test this prediction using U.S. political survey data on how political independents are perceived by registered party members as well as existing experiments on the perception of racially ambiguous faces, and find support.

Retrospective study of dilated cardiomyopathy in dogs.

BACKGROUND: The United States Food and Drug Administration is investigating possible diet-associated dilated cardiomyopathy (DCM) in dogs and cats. OBJECTIVES: To retrospectively review DCM cases for signalment, diet information, echocardiographic changes, and survival. ANIMALS: Client-owned dogs (n = 71). METHODS: Medical records of dogs diagnosed with DCM between January 1, 2014 and September 30, 2018 were reviewed. Dogs were grouped into "traditional" or "nontraditional" diet categories and whether or not diet was changed after diagnosis. RESULTS: For dogs eating nontraditional diets, those that had their diets changed had a larger percentage decrease in normalized systolic left ventricular internal dimension (P = .03) and left atrial:aorta ratio (P < .001) compared to those that did not have their diets changed. Survival time was significantly longer for dogs with DCM eating nontraditional diets that had their diets changed (median survival, 337 days; range, 9-1307 days) compared to dogs eating nontraditional diets that did not have their diets changed (median survival, 215 days; range, 1-852 days; P = .002). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with DCM eating nontraditional diets can experience improvement in cardiac function after diet change but additional research is needed to examine possible associations between diet and DCM.

Extracellular vesicular microRNAs as potential biomarker for early detection of doxorubicin-induced cardiotoxicity.

BACKGROUND: Long-term use of doxorubicin (DOX) is limited by cumulative dose-dependent cardiotoxicity. OBJECTIVES: Identify plasma extracellular vesicle (EV)-associated microRNAs (miRNAs) as a biomarker for cardiotoxicity in dogs by correlating changes with cardiac troponin I (cTnI) concentrations and, echocardiographic and histologic findings. ANIMALS: Prospective study of 9 client-owned dogs diagnosed with sarcoma and receiving DOX single-agent chemotherapy (total of 5 DOX treatments). Dogs with clinically relevant metastatic disease, preexisting heart disease, or breeds predisposed to cardiomyopathy were excluded. METHODS: Serum concentration of cTnI was monitored before each treatment and 1 month after the treatment completion. Echocardiography was performed before treatments 1, 3, 5, and 1 month after completion. The EV-miRNA was isolated and sequenced before treatments 1 and 3, and 1 month after completion. RESULTS: Linear mixed model analysis for repeated measurements was used to evaluate the effect of DOX. The miR-107 (P = .03) and miR-146a (P = .02) were significantly downregulated whereas miR-502 (P = .02) was upregulated. Changes in miR-502 were significant before administration of the third chemotherapeutic dose. When stratifying miRNA expression for change in left ventricular ejection fraction, upregulation of miR-181d was noted (P = .01). Serum concentration of cTnI changed significantly but only 1 month after treatment completion, and concentrations correlated with left ventricular ejection fraction and left ventricular internal dimension in diastole. CONCLUSION AND CLINICAL SIGNIFICANCE: Downregulation of miR-502 was detected before significant changes in cTnI concentrations or echocardiographic parameters. Further validation using a larger sample size will be required.

The interpretation of urban scaling analysis in time.

Scaling is a general analytical framework used by many disciplines-from physics to biology and the social sciences-to characterize how population-averaged properties of a collective vary with its size. The observation of scale invariance over some range identifies general system types, be they ideal gases, ecosystems or cities. The use of scaling in the analysis of cities quantifies many of their arguably fundamental general characteristics, especially their capacity to create interrelated economies of scale in infrastructure and increasing returns to scale in socio-economic activities. However, the measurement of these effects, and the relationship of observable parameters to theory, hinge on how scaling analysis is used empirically. Here, we show how two equivalent approaches to urban scaling-cross-sectional and temporal-lead to the measurement of different mixtures of the same fundamental parameters describing pure scale and pure temporal phenomena. Specifically, temporal exponents are sensitive to the intensive growth of urban quantities and to circumstances when population growth vanishes, leading to instabilities and infinite divergences. These spurious effects are avoided in cross-sectional scaling, which is more common and closer to theory in terms of quantitative testable expectations for its parameters.

Extracellular Vesicles from Wharton's Jelly Mesenchymal Stem Cells Suppress CD4 Expressing T Cells Through Transforming Growth Factor Beta and Adenosine Signaling in a Canine Model.

Mesenchymal stem cells (MSCs) are widely investigated as potential therapeutic agents due to their potent immunomodulatory capacity. Although specific mechanisms by which MSC acts on immune cells are emerging, many questions remain, including the potential of extracellular vesicles (EVs) to mediate biological activities. Canine MSCs are of interest for both veterinary and comparative models of disease and have been shown to suppress CD4pos T cell proliferation. The aim of this study was to determine whether EV isolated from canine Wharton's jelly-derived MSC (WJ-MSC EV) suppresses CD4pos T cell proliferation using biochemical mechanisms previously ascribed to soluble mediators [transforming growth factor beta (TGF-ß) and adenosine]. WJ-MSC EV exhibited mode of 125 nm diameter, low buoyant density (1.1 g/mL), and expression of EV proteins Alix and TSG101. Functionally, EVs inhibited CD4pos T cell proliferation in a dose-dependent manner, which was absent in EV-depleted samples and EVs from non-MSC fibroblasts. EV suppression of CD4pos T cell proliferation was inhibited by a TGF-ßRI antagonist, neutralizing antibodies to TGF-ß, or A2A adenosine receptor blockade. TGF-ß was present on EVs as latent complexes most likely tethered to EV membrane by betaglycan. These data demonstrate that canine WJ-MSC EV utilizes TGF-ß and adenosine signaling to suppress proliferation of CD4pos T cell and will enable further investigation into mechanisms of immune cell modulation, as well as refinement of WJ-MSC and their EVs for therapeutic application.

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.

Clinical findings and survival time in dogs with advanced heart failure.

BACKGROUND: Dogs with advanced heart failure are a clinical challenge for veterinarians but there are no studies reporting clinical features and outcome of this population. HYPOTHESIS/OBJECTIVES: To describe clinical findings and outcome of dogs with advanced heart failure caused by degenerative mitral valve disease (DMVD). ANIMALS: Fifty-four dogs with advanced heart failure because of DMVD. METHODS: For study purposes, advanced heart failure was defined as recurrence of congestive heart failure signs despite receiving the initially prescribed dose of pimobendan, angiotensin-converting-enzyme inhibitor (ACEI), and furosemide >4 mg/kg/day. Data were collected for the time of diagnosis of Stage C heart failure and time of diagnosis of advanced heart failure. Date of death was recorded. RESULTS: At the diagnosis of advanced heart failure, doses of pimobendan (n = 30), furosemide (n = 28), ACEI (n = 13), and spironolactone (n = 4) were increased, with ≥1 new medications added in most dogs. After initial diagnosis of advanced heart failure, 38 (70%) dogs had additional medications adjustments (median = 2 [range, 0-27]), with the final total medication number ranging from 2-10 (median = 5). Median survival time after diagnosis of advanced heart failure was 281 days (range, 3-885 days). Dogs receiving a furosemide dose >6.70 mg/kg/day had significantly longer median survival times (402 days [range, 3-885 days] versus 129 days [range 9-853 days]; P = .017). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with advanced heart failure can have relatively long survival times. Higher furosemide dose and non-hospitalization were associated with longer survival.