Absence of known feline MYH7 and MYBPC3 variants in a diverse cohort of cats with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common cause of heart disease in the domestic cat with a genetic predisposition in a few breeds. In the Maine Coon and Ragdoll breeds, two variants associated with the HCM phenotype have been identified in the cardiac myosin binding protein C gene (MYBPC3; p.Ala31Pro and p.Arg820Trp respectively), and a single variant has been identified in the myosin heavy chain gene (MYH7; p.Glu1883Lys) in one domestic cat with HCM. It is not known if these variants influence the development of HCM in other cohorts of the feline population. The objective of this study was to evaluate the presence of the known MYBPC3 and MYH7 variants in a population of cats with HCM. DNA was isolated from samples collected from non-Ragdoll and non-Maine Coon domestic cats diagnosed with HCM through the North Carolina State University College of Veterinary Medicine and genotyped for the three variants. One-hundred and three DNA samples from cats with HCM were evaluated from domestic shorthair, domestic longhair and purebred cats. All samples were wt for the MYBPC3 and MYH7 variants. Although this study was limited by its inclusion of cats from one tertiary hospital, the lack of these MYBPC3 and MYH7 variants in this feline HCM population indicates that the clinical utility of genetic testing for these variants may be isolated to the two cat breeds in which these variants have been identified. Further studies to identify the causative variants for the feline HCM population are warranted.

Raman spectroscopy of stored red blood cell concentrate within sealed transfusion blood bags.

The standard practice in blood banks worldwide involves storage of red blood cells (RBCs) in plastic bags until they are needed for transfusion. During storage, the cells gradually degrade in functionality, a condition described as RBC storage lesion. Standard analytical techniques cannot assess the blood quality without breaching the sterility of the transfusion bag. In this study, we employed a commercially available spatially offset Raman spectroscopy (SORS) system using a custom designed protocol to non-invasively explore the biochemical changes in RBC concentrate of healthy donors over a storage period of approximately 42 days in standard transfusion bags, under standard storage conditions. The results reveal an increase in the oxygenation state of haemoglobin over the storage period for all donors, but different profiles for each donor. This study demonstrates the feasibility of acquiring consistent biochemical information relevant to the quality of stored blood, in situ through sealed blood transfusion bags using a commercially available instrument.

Non-invasive spectroscopy of transfusable red blood cells stored inside sealed plastic blood-bags.

After being separated from (donated) whole blood, red blood cells are suspended in specially formulated additive solutions and stored (at 4 °C) in polyvinyl chloride (PVC) blood-bags until they are needed for transfusion. With time, the prepared red cell concentrate (RCC) is known to undergo biochemical changes that lower effectiveness of the transfusion, and thus regulations are in place that limit the storage period to 42 days. At present, RCC is not subjected to analytical testing prior to transfusion. In this study, we use Spatially Offset Raman Spectroscopy (SORS) to probe, non-invasively, the biochemistry of RCC inside sealed blood-bags. The retrieved spectra compare well with conventional Raman spectra (of sampled aliquots) and are dominated by features associated with hemoglobin. In addition to the analytical demonstration that SORS can be used to retrieve RCC spectra from standard clinical blood-bags without breaking the sterility of the system, the data reveal interesting detail about the oxygenation-state of the stored cells themselves, namely that some blood-bags unexpectedly contain measurable amounts of deoxygenated hemoglobin after weeks of storage. The demonstration that chemical information can be obtained non-invasively using spectroscopy will enable new studies of RCC degeneration, and points the way to a Raman-based instrument for quality-control in a blood-bank or hospital setting.

The effect of enalapril on furosemide-activated renin-angiotensin-aldosterone system in healthy dogs.

Studies in our laboratory have revealed that furosemide-induced RAAS activation, evaluated via the urine aldosterone-to-creatinine ratio (UAldo:C), was not attenuated by the coadministration of benazepril, while enalapril successfully suppressed amlodipine-induced urinary aldosterone excretion. This study was designed to evaluate the efficacy of enalapril in suppressing ACE activity and furosemide-induced circulating RAAS activation. Failure to do so would suggest that this failure may be a drug class effect. We hypothesized that enalapril would suppress ACE activity and furosemide-induced circulating RAAS activation. Sixteen healthy hound dogs. The effect of furosemide (2 mg/kg PO, q12 h; Group F) and furosemide plus enalapril (0.5 mg/kg PO, q12 h; Group FE) on circulating RAAS was determined by plasma ACE activity, 4-6 h post-treatment, and urinary A:C on days -1, -2, 1, 4, and 7. There was a significant increase in the average urine aldosterone-to-creatinine ratio (UAldo:C) after administration of furosemide (P < 0.05). Enalapril inhibited ACE activity (P < 0.0001) but did not significantly reduce aldosterone excretion. A significant (P < 0.05) increase in the UAldo:C was maintained for the 7 days of the study in both groups. Enalapril decreased plasma ACE activity; however, it did not suppress furosemide-induced RAAS activation, as determined by the UAldo:C. While enalapril blunts ACE activity, the absence of circulating RAAS suppression may be due to angiotensin II reactivation, alternative RAAS pathways, and furosemide overriding concurrent ACE inhibition, all indicating the existence of aldosterone breakthrough (ABT). Along with similar findings with benazepril, it appears that failure to suppress aldosterone suppression with furosemide stimulation may be a drug class effect. The discrepancy between the current data and the documented benefits of enalapril likely reflects the efficacy of this ACE inhibitor in suppressing tissue RAAS, variable population responsiveness to ACE-inhibition, and/or providing additional survival benefits, possibly through as yet unknown mechanisms.

Aldosterone breakthrough with benazepril in furosemide-activated renin-angiotensin-aldosterone system in normal dogs.

Pilot studies in our laboratory revealed that furosemide-induced renin-angiotensin-aldosterone system (RAAS) activation was not attenuated by the subsequent co-administration of benazepril. This study was designed to evaluate the effect of benazepril on angiotensin-converting enzyme (ACE) activity and furosemide-induced circulating RAAS activation. Our hypothesis was that benazepril suppression of ACE activity would not suppress furosemide-induced circulating RAAS activation, indicated by urinary aldosterone concentration. Ten healthy hound dogs were used in this study. The effect of furosemide (2 mg/kg p.o., q12h; Group F; n = 5) and furosemide plus benazepril (1 mg/kg p.o., q24h; Group FB; n = 5) on circulating RAAS was determined by plasma ACE activity, 4-6 h posttreatment, and urinary aldosterone to creatinine ratio (UAldo:C) on days -1, -2, 1, 3, and 7. There was a significant increase in the average UAldo:C (µg/g) after the administration of furosemide (Group F baseline [average of days -1 and -2] UAldo:C = 0.41, SD 0.15; day 1 UAldo:C = 1.1, SD 0.56; day 3 UAldo:C = 0.85, SD 0.50; day 7 UAldo:C = 1.1, SD 0.80, P < 0.05). Benazepril suppressed ACE activity (U/L) in Group FB (Group FB baseline ACE = 16.4, SD 4.2; day 1 ACE = 3.5, SD 1.4; day 3 ACE = 1.6, SD 1.3; day 7 ACE = 1.4, SD 1.4, P < 0.05) but did not significantly reduce aldosterone excretion (Group FB baseline UAldo:C = 0.35, SD 0.16; day 1 UAldo:C = 0.79, SD 0.39; day 3 UAldo:C 0.92, SD 0.48, day 7 UAldo:C = 0.99, SD 0.48, P < 0.05). Benazepril decreased plasma ACE activity but did not prevent furosemide-induced RAAS activation, indicating aldosterone breakthrough (escape). This is particularly noteworthy in that breakthrough is observed at the time of initiation of RAAS suppression, as opposed to developing after months of therapy.

Postinjury treatment with rolipram increases hemorrhage after traumatic brain injury.

The pathology caused by traumatic brain injury (TBI) is exacerbated by the inflammatory response of the injured brain. Two proinflammatory cytokines that contribute to inflammation after TBI are tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). From previous studies using the parasagittal fluid-percussion brain injury model, we reported that the anti-inflammatory drug rolipram, a phosphodiesterase 4 inhibitor, reduced TNF-α and IL-1ß levels and improved histopathological outcome when administered 30 min prior to injury. We now report that treatment with (±)-rolipram given 30 min after injury significantly reduced TNF-α levels in the cortex and hippocampus. However, postinjury administration of (±)-rolipram significantly increased cortical contusion volume and increased atrophy of the cortex compared with vehicle-treated animals at 10 days postinjury. Thus, despite the reduction in proinflammatory cytokine levels, histopathological outcome was worsened with post-TBI (±)-rolipram treatment. Further histological analysis of (±)-rolipram-treated TBI animals revealed significant hemorrhage in the contused brain. Given the well-known role of (±)-rolipram of increasing vasodilation, it is likely that (±)-rolipram worsened outcome after fluid-percussion brain injury by causing increased bleeding.

Prevalence of an angiotensin-converting enzyme gene variant in dogs.

BACKGROUND: Genetic heterogeneity of the canine angiotensin converting enzyme (ACE) gene is functionally important because the degree of aldosterone breakthrough with ACE-inhibitor therapy is greater in variant positive dogs compared to variant negative dogs, but the prevalence of the variant is not known. The purpose of this study was to determine ACE gene variant-positive prevalence in a population of 497 dogs of different breeds. RESULTS: Overall variant-positive prevalence was 31%, with 20% of dogs heterozygous and 11% of dogs homozygous. The variant was overrepresented in Irish Wolfhounds (prevalence 95%; P < .001), Dachshunds (prevalence 90%; P < .001), Cavalier King Charles Spaniels (prevalence 85%; P < .001), Great Danes (prevalence 84%; P < .001), and Bull Mastiffs (prevalence 58%; P = .02). Irish Wolfhounds were more likely to be homozygous than heterozygous (P < .001). CONCLUSIONS: Nearly one-third of dogs in this study were positive for a functionally important ACE gene variant, with wide prevalence variability between breeds. The clinical importance of high ACE gene variant-positive prevalence in some breeds requires further study because the highest prevalences were found in breeds that are predisposed to heart disease and therefore may be treated with ACE-inhibitors.

Postural orthostatic tachycardia syndrome: a case report of palpitations and dizziness following prophylactic mefloquine use.

OBJECTIVE: The antimalarial agent mefloquine has been reported to cause a number of possible side effects. Here we describe a case demonstrating a previously unreported complication, the postural orthostatic tachycardia syndrome (POTS). CASE HISTORY: A 44-year-old woman presented with symptoms of severe orthostatic intolerance (palpitations and dizziness) in combination with postural tachycardia (but no fall in blood pressure) following the use of mefloquine prophylaxis. Investigations revealed evidence of autonomic dysfunction (with loss of ECG R-R interval variation) consistent with POTS. Her symptoms responded well to beta-blockade with propranolol. CONCLUSION: The possibility of POTS should be considered in patients presenting with symptoms of palpitations, dizziness, presyncope or other features suggestive of autonomic dysfunction following the use of mefloquine.

Atenolol in cats with subclinical hypertrophic cardiomyopathy: a double-blind, placebo-controlled, randomized clinical trial of effect on quality of life, activity, and cardiac biomarkers.

OBJECTIVE: To compare quality of life (QOL) and activity measures between healthy control cats and cats with subclinical hypertrophic cardiomyopathy (HCM), and to evaluate the effect of oral atenolol therapy on QOL, activity, and circulating biomarkers in cats with subclinical HCM. ANIMALS: Thirty-two client-owned cats with subclinical HCM and 27 healthy control cats. METHODS: Owner responses to a QOL questionnaire, circulating cardiac biomarker concentrations, and accelerometer-based activity measures were compared prospectively in cats with and without HCM, and in cats with HCM before and after treatment with oral atenolol (6.25 mg/cat q 12 h) for 6 months. RESULTS: Owner-assessed activity of daily living score was lower in cats with HCM than in cats in controls (p=0.0420). No differences were identified between control cats and cats with HCM for any activity variable. Compared with placebo, treatment with atenolol was associated with a lower baseline-adjusted mean ± SD heart rate (157 ± 30 vs. 195 ± 20 bpm; p=0.0001) and rate-pressure product (22,446 ± 6,237 vs. 26,615 ± 4,623 mmHg/min; p=0.0146). A treatment effect of atenolol on QOL or activity was not demonstrated. CONCLUSIONS: This study failed to identify an effect of subclinical HCM on owner-assessed QOL or activity or a treatment effect of atenolol on these variables at the dosage evaluated. These findings do not support a treatment benefit of atenolol for the goal of symptom reduction in cats with subclinical HCM.

Acute effect of pimobendan and furosemide on the circulating renin-angiotensin-aldosterone system in healthy dogs.

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) is activated in states of decreased cardiac output and by certain cardiovascular therapeutic agents, such as loop diuretics and vasodilators. HYPOTHESIS: Short-term treatment with the inodilator, pimobendan, will not activate the circulating RAAS because its vasodilatory action will be offset by its positive inotropic property, thereby ameliorating RAAS stimulation at the juxtaglomerular apparatus. Furthermore, pimobendan will suppress RAAS activation produced by furosemide. ANIMALS: Nine healthy laboratory dogs were used in this study. METHODS: Experimental, cross-over study. Dogs were administered pimobendan (0.5 mg/kg q12h) for 4 days followed by furosemide (2 mg/kg q12h) and then, after a wash-out period, a combination of the drugs. Aldosterone : creatinine (A : Cr) was measured at the end of each treatment cycle. RESULTS: There was no significant increase in the average urinary A : Cr with the administration of pimobendan (control urinary A : Cr = 0.46, standard deviation (SD) 0.33; pimobendan A : Cr = 0.48, SD 0.28). There was a significant increase in the average urinary A : Cr after administration of furosemide (urinary A : Cr = 1.3, SD 0.70) and with the combination of furosemide and pimobendan (urinary A : Cr = 2.9, SD 1.6). CONCLUSIONS AND CLINICAL RELEVANCE: Short-term administration of high-dose pimobendan, does not activate the RAAS in healthy dogs. Pimobendan did not prevent RAAS activation associated with furosemide therapy. These results in healthy dogs suggest that furosemide therapy, with or without pimobendan, should be accompanied by RAAS suppressive therapy.

Myxomatous mitral valve disease in the miniature poodle: A retrospective study.

Myxomatous mitral valve disease (MMVD) is the most common cardiovascular disease in the dog. The natural history of the disease is wide ranging and includes patients without clinical signs as well as those with significant clinical consequences from cardiac arrhythmias, pulmonary hypertension and/or congestive heart failure. The factors that determine which dogs remain asymptomatic and which develop clinical disease are not known. Disease characteristics could be breed or family related; some breeds of dogs, particularly the Cavalier King Charles spaniels, develop MMVD at an early age. The purpose of this study was to retrospectively characterize MMVD in the miniature poodle, a commonly affected breed in which MMVD has not been well characterized. Thirty-two miniature poodles met the inclusion criteria. Mean age was 11±three years. Clinical signs included exercise intolerance, syncope and coughing. Eighteen dogs were classified as ACVIM Stage B1, 12 as stage B2, and two as stage C. Mean vertebral heart scale (VHS) was 10.2 (±standard deviation of 0.9); 15 of 28 dogs had a VHS <10.3. One dog had radiographic evidence of congestive heart failure. Mean diastolic left ventricle dimension normalized to body weight was 1.6 (±0.4) and mean systolic was 0.8 (±0.3). Mitral valve prolapse was subjectively classified as mild or moderate in 19 dogs and severe in two. In the miniature poodles reported here, MMVD appears to be a fairly late onset disease and often is a mild phenotype.

Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.

The E6-AP ubiquitin ligase (human/mouse gene UBE3A/Ube3a) promotes the degradation of p53 in association with papilloma E6 protein, and maternal deficiency causes human Angelman syndrome (AS). Ube3a is imprinted with silencing of the paternal allele in hippocampus and cerebellum in mice. We found that the phenotype of mice with maternal deficiency (m-/p+) for Ube3a resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. Long-term potentiation (LTP) was severely impaired in m-/p+ mice despite normal baseline synaptic transmission and neuroanatomy, indicating that ubiquitination may play a role in mammalian LTP and that LTP may be abnormal in AS. The cytoplasmic abundance of p53 was increased in postmitotic neurons in m-/p+ mice and in AS, providing a potential biochemical basis for the phenotype through failure to ubiquitinate and degrade various effectors.

Echocardiographic quantification of Dirofilaria immitis in experimentally infected cats.

The safety of heartworm preventives in heartworm-positive cats has traditionally been evaluated using adult Dirofilaria immitis removed from infected dogs and surgically implanted into the cats. An alternate study model uses infective larvae to establish adult infections in cats. Unfortunately, the number of adult worms resulting from the latter method varies widely from none to more than 30, both unacceptable for studies of natural heartworm infection and for studies evaluating product safety in heartworm-infected cats. We sought to determine infection severity in experimental infections via echocardiography to reduce the chances of enrolling uninfected and heavily infected cats into a study. Eighty adult cats were each inoculated with 60 infective D. immitis larvae and maintained for 8 months to allow for the development of adult worms. Antigen and antibody testing, as well as echocardiographic imaging, were performed to confirm and estimate adult worm burdens. Approximately 8 and 12 months post-infection, echocardiographic examination was performed to confirm and enumerate adult D. immitis populations in the cardiovascular system. Worm burdens were stratified as 0, 1-3, 4-11, and > 11 adults, with 0 being considered uninfected and more than 11 considered too heavily infected to be relevant for anthelmintic studies. Cats with clinically relevant infections (1-10 adults) subsequently received multiple treatments with the investigational drug, and worm burdens were confirmed by necropsy 30 days following the final treatment. Worm burden estimated with echocardiography correlated well, but not precisely, with post-mortem counts (p < 0.001, r2 = 0.67). Echocardiography under-, over-, and exactly estimated heartworm burden 53%, 27%, and 22% of the time, respectively. Although the correct category (0-4) was determined by echocardiography in only 54% of cats, positive cats were distinguished from negative cats 88% of the time and the heaviest infections (> 11) were correctly categorized 95% of the time. Both false negative and false positive results were observed. We conclude that echocardiography is useful for detecting mature experimental heartworm infections, identifying cats that have rejected mature infection, and detecting very heavy heartworm burdens, but it is only moderately accurate in classifying lesser burdens. While echocardiography cannot be relied upon to consistently determine the exact heartworm burden in experimentally infected cats, it is useful in stratifying worm burdens for anthelmintic safety studies.

The MAPK cascade is required for mammalian associative learning.

Mitogen-activated protein kinase (MAPK) is an integral component of cellular signaling during mitogenesis and differentiation of mitotic cells. Recently MAPK activation in post-mitotic cells has been implicated in hippocampal long-term potentiation (LTP), a potential cellular mechanism of learning and memory. Here we investigate the involvement of MAPK in learning and memory in behaving animals. MAPK activation increased in the rat hippocampus after an associative learning task, contextual fear conditioning. Two other protein kinases known to be activated during hippocampal LTP, protein kinase C and alpha-calcium/calmodulin protein kinase II, also were activated in the hippocampus after learning. Inhibition of the specific upstream activator of MAPK, MAPK kinase (MEK), blocked fear conditioning. Thus, classical conditioning in mammals activates MAPK, which is necessary for consolidation of the resultant learning.

Pharmacodynamic assessment of diuretic efficacy and braking in a furosemide continuous infusion model.

INTRODUCTION: Diuretic failure is a potential life-ending event but is unpredictable and poorly understood. The objectives of this study were to evaluate pharmacodynamic markers of furosemide-induced diuresis and to investigate mechanisms of diuretic braking in dogs receiving constant rate infusion (CRI) of furosemide. ANIMALS: Six healthy male dogs. METHODS: Raw data and stored samples from one arm of a previously published study were further analyzed to mechanistically investigate causes of diuretic braking in these dogs. Urine volume was recorded hourly during a 5-h furosemide CRI. Urine and blood samples were collected hourly to measure serum and urine electrolytes, urine aldosterone, and plasma and urine furosemide. Serum electrolyte fractional excretion was calculated. Urine sodium concentration was indexed to urine potassium (uNa:uK) and urine furosemide (uNa:uFur) concentrations, plasma furosemide concentration was indexed to urine furosemide concentration (pFur:uFur), and urine aldosterone was indexed to urine creatinine (UAldo:C). Temporal change and the relationship to urine volume were evaluated for these measured and calculated variables. RESULTS: Urine volume was significantly correlated with urine electrolyte amounts and with uNa:uK. The ratio of pFur:uFur decreased during the infusion, whereas furosemide excretion was unchanged. CONCLUSIONS: There was a strong relationship between urine volume and absolute urine electrolyte excretion. Urine volume was strongly correlated to uNa:uK, giving it potential as a spot indicator of urine production during diuresis. The decrease in uNa:uK over time during the infusion is consistent with mineralocorticoid modification of urinary electrolyte excretion, supporting renin-angiotensin-aldosterone activation as a cause of diuretic braking in this model.

Evaluation of genes associated with human myxomatous mitral valve disease in dogs with familial myxomatous mitral valve degeneration.

Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is believed to be heritable in Cavalier King Charles spaniels (CKCS) and Dachshunds. Myxomatous mitral valve disease is a familial disease in human beings as well and genetic mutations have been associated with its development. We hypothesized that a genetic mutation associated with the development of the human form of MMVD was associated with the development of canine MMVD. DNA was isolated from blood samples from 10 CKCS and 10 Dachshunds diagnosed with MMVD, and whole genome sequences from each animal were obtained. Variant calling from whole genome sequencing data was performed using a standardized bioinformatics pipeline for all samples. After filtering, the canine genes orthologous to the human genes known to be associated with MMVD were identified and variants were assessed for likely pathogenic implications. No variant was found in any of the genes evaluated that was present in least eight of 10 affected CKCS or Dachshunds. Although mitral valve disease in the CKCS and Dachshund is a familial disease, we did not identify genetic cause in the genes responsible for the human disease in the dogs studied here.

Aldosterone breakthrough in dogs with naturally occurring myxomatous mitral valve disease.

INTRODUCTION: Aldosterone breakthrough (ABT) is the condition in which angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers fail to effectively suppress the activity of the renin angiotensin aldosterone system. The objective of this study was to determine if ABT occurs in dogs with naturally occurring myxomatous mitral valve disease receiving an ACEI, using the urine aldosterone to creatinine ratio (UAldo:C) as a measure of renin angiotensin aldosterone system activation. ANIMALS, MATERIALS AND METHODS: This study includes 39 dogs with myxomatous mitral valve disease. A UAldo:C cut-off definition (derived from a normal population of healthy, adult, and client-owned dogs) was used to determine the prevalence of ABT in this population. Spearman analysis and univariate logistic regression were used to evaluate the relationship between UAldo:C and ABT (yes/no) and eight variables (age, serum K+ concentration, serum creatinine concentration, ACEI therapy duration and ACEI dosage, furosemide therapy duration and furosemide dosage, and urine sample storage time). Finally, the UAldo:C in dogs receiving spironolactone, as part congestive heart failure (CHF) therapy, was compared to dogs with CHF that were not receiving spironolactone. RESULTS: The prevalence of ABT was 32% in dogs with CHF and 30% in dogs without CHF. There was no relationship between either the UAldo:C or the likelihood of ABT and the eight variables. Therapy with spironolactone lead to a significant elevation of the UAldo:C. DISCUSSION: Using the UAldo:C and a relatively stringent definition of ABT, it appears that incomplete RAAS blockade is common in dogs with MMVD receiving an ACEI. The prevalence of ABT in this canine population mirrors that reported in humans. While the mechanism of ABT is likely multifactorial and still poorly understood, the proven existence of ABT in dogs offers the potential to improve the prognosis for MMVD with the addition of a mineralocorticoid receptor blocker to current therapeutic regimens. CONCLUSIONS: Approximately 30% of dogs being treated for heart disease and CHF satisfied the definition of ABT. Identifying patient subpopulations experiencing ABT may help guide future study design and clinical decision-making.

Furosemide continuous rate infusion diluted with 5% dextrose in water or hypertonic saline in normal adult dogs: a pilot study.

OBJECTIVE: The goal of this study was to investigate the short-term safety and diuretic efficacy of furosemide constant rate infusion (CRI) diluted with 5% dextrose in water (D5W) compared to dilution with 2.4% hypertonic saline in healthy dogs. ANIMALS: Six healthy dogs. METHODS: Dogs were studied in a randomized, blinded, crossover manner. Furosemide 3.3mg/kg was diluted to 2.2mg/mL with either 1.5mL/kg D5W for the DEX method or with 1.0mL/kg D5W and 0.5mL/kg of 7.2% hypertonic saline for the H-SAL method. After a 0.66mg/kg furosemide IV bolus, the infusion rate was 0.3 mL/kg/hr for 5 h such that both methods delivered 0.66 mg/kg/hr (total 3.3mg/kg) furosemide in equal volume for the study duration. Urine output, water intake, central venous pressure (CVP), physical parameters, furosemide concentrations, blood and urine electrolytes, and urine aldosterone to creatinine ratio (UAldo:C) were evaluated. RESULTS: Measured variables were not different between methods but showed changes over time consistent with diuresis. Mean CVP decreased over time similarly for both methods. Plasma furosemide and urine concentrations were stable and not different between methods. Both furosemide CRI methods showed an increase in the UAldo:C, however, the rise was greater for DEX than for H-SAL. CONCLUSIONS: Diuresis was similar for both furosemide CRI methods; however, the H-SAL method induced less renin-angiotensin-aldosterone system activation than the DEX method. The absence of intravascular volume expansion based on CVP suggests that dilution of a furosemide CRI with 2.4% hypertonic saline may be well tolerated in heart failure.

Long-distance movement of phosphate starvation-responsive microRNAs in Arabidopsis.

Plant microRNAs are small RNAs that are important for genetic regulation of processes such as plant development or environmental responses. Specific microRNAs accumulate in the phloem during phosphate starvation, and may act as long-distance signalling molecules. We performed quantitative PCR on Arabidopsis hypocotyl micrograft tissues of wild-type and hen1-6 mutants to assess the mobility of several phosphate starvation-responsive microRNA species. In addition to the previously confirmed mobile species miR399d, the corresponding microRNA* (miR399d*) was identified for the first time as mobile between shoots and roots. Translocation by phosphate-responsive microRNAs miR827 and miR2111a between shoots and roots during phosphate starvation was evident, while their respective microRNA*s were not mobile. The results suggest that long-distance mobility of microRNA species is selective and can occur without the corresponding duplex strand. Movement of miR399d* and root-localised accumulation of miR2111a* opens the potential for persisting microRNA*s to be mobile and functional in novel pathways during phosphate starvation responses.