Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.

We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo -JetPEI ® . Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR- mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6 ± 2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo . Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.

Nasal continuous positive airway pressure practices in preterm infants: A survey of neonatal providers.

BACKGROUND: The standard of care for respiratory support of preterm infants is nasal continuous positive airway pressure (CPAP), yet practices are not standardized. Our aim was to survey CPAP practices in infants < 32 weeks gestation among the American Academy of Pediatrics Neonatal-Perinatal section. METHODS: A US, web-based survey inquired about the initiation, management, and discontinuation of CPAP, and chinstrap use and oral feedings on CPAP. RESULTS: 857 providers consented. Regarding criteria to discontinue/wean CPAP: 69% use specific respiratory stability criteria; 22% a specific post-menstrual age; 8% responded other. 64% did not have guidelines for CPAP discontinuation; 54% did not have guidelines for CPAP initiation. 66% believe chinstraps improve CPAP efficacy; however, 11% routinely apply a chinstrap. 22% allow oral feeds on CPAP in certain circumstances. CONCLUSION: There are meaningful variabilities in CPAP practices among neonatal providers across the US. Given the potential long-term implications this can have on the growth and development of the preterm lung, further evidence-based research is needed in relation to respiratory outcomes to optimize and standardize CPAP strategies.

Inhaled mRNA therapy for treatment of cystic fibrosis: Interim results of a randomized, double-blind, placebo-controlled phase 1/2 clinical study.

BACKGROUND: MRT5005, a codon-optimized CFTR mRNA, delivered by aerosol in lipid nanoparticles, was designed as a genotype-agnostic treatment for CF lung disease. METHODS: This was a randomized, double-blind, placebo-controlled Phase 1/2 study performed in the US. Adults with 2 severe class I and/or II CFTR mutations and baseline ppFEV1 values between 50 and 90% were randomized 3:1 (MRT5005: placebo). Six dose levels of MRT5005 (4, 8, 12, 16, 20, and 24 mg) or placebo (0.9% Sodium Chloride) were administered by nebulization. The single ascending dose cohort was treated over a range from 8 to 24 mg; the multiple ascending dose cohort received five weekly doses (range 8-20 mg); and the daily dosing cohort received five daily doses (4 mg). RESULTS: A total of 42 subjects were assigned to MRT5005 [31] or placebo [11]. A total of 14 febrile reactions were observed in 10 MRT5005-treated participants, which were mild [3] or moderate [11] in severity; two subjects discontinued related to these events. Additionally, two MRT5005-treated patients experienced hypersensitivity reactions, which were managed conservatively. The most common treatment emergent adverse events were cough and headache. No consistent effects on FEV1 were noted. CONCLUSIONS: MRT5005 was generally safe and well tolerated through 28 days of follow-up after the last dose, though febrile and hypersensitivity reactions were noted. The majority of these reactions resolved within 1-2 days with supportive care allowing continued treatment with MRT5005 and careful monitoring. In this small first-in-human study, FEV1 remained stable after treatment, but no beneficial effects on FEV1 were observed.

Short communication: Evaluation of an eating time sensor for use in pasture-based dairy systems.

The assessment of grazing behavior is important for research and practice in pasture-grazed dairy farm systems. However, few devices are available that enable assessment of cow grazing behavior at an individual animal level. This study investigated whether commercially available Smarttag "eating time" sensors (Nedap Livestock Management, Groenlo, the Netherlands) were suitable for recording the grazing time of cows. Smarttag sensors were mounted on the neck collars of multiparous Holstein-Friesian cows in a herd in Taranaki, New Zealand. Cows were randomly selected each observation day from the milking herd for 8 separate days across a 1-mo period. Trained observers conducted 90-min observation periods to evaluate the relationship between the sensor eating time measure and grazing time. A set of 5 defined cow behaviors (2 "head up" and 3 "head down" behaviors) were assessed. In total, observations of 37 cows were recorded in 14 sessions over 8 d in the study period, providing 55.5 total hours of observations. Observation data were aligned with sensor data according to the sensor time stamps and grouped into matching 15-min intervals. Interobserver reliability was assessed both before and after the main trial period, and the mean percentage eating time per observer had a coefficient of variation of 0.46% [mean 93.2, standard deviation (SD) 0.425] before and 0.07% (mean 96.3, SD 0.074) after. In the main trial, the relationship between observed (mean 70.8%) and sensor-derived (mean 69.3%) percentage eating time over the observation period gave a Pearson correlation coefficient of 0.971, concordance correlation coefficient 0.968, mean difference 1.50% points, and SD 5.8% points. Therefore, sensor-identified percentage "eating time" and observed percentage active grazing time were shown to be both very well correlated and concordant (in agreement, with high correlation and little bias). Therefore, the relationship between observed and sensor-derived data had a high degree of agreement for identifying cow grazing activity. In conclusion, Smarttag sensors are a valid and useful tool for estimating grazing activity at time periods of 1 h or more.

Imaging of cerebral venous thrombosis.

The incidence of cerebral venous thrombosis (CVT) is increasing due to the widespread use of computed tomography (CT) and magnetic resonance imaging (MRI) for investigating patients with acute headaches and new onset of seizures. Alternatively referred to as cerebral venous sinus thrombosis (CVST) or dural venous sinus thrombosis (DVST), the terms encompass a broad spectrum of neurological pathologies. These include dural venous sinus thrombosis, cortical vein thrombosis, cavernous sinus thrombosis, venous infarction/haemorrhage, and the rare sequelae of intracranial hypertension or dural arteriovenous fistula. Timely and accurate diagnosis is critical; most patients are young adults and up to 15% will die in the acute phase of the condition. Imaging diagnosis using unenhanced CT or CT venography (CTV) can be readily achieved by the general radiologist. MRI or MRI venography (MRV) are powerful techniques, provided the radiologist is aware of critical diagnostic pitfalls. In selected cases, cerebral digital subtraction angiography (DSA) can facilitate both diagnosis and anticoagulant/transcatheter thrombolytic therapy improving clinical outcome. This article will outline the condition, highlighting cerebral venous anatomy, diagnostic techniques, and pitfalls pertinent to all practising radiologists.

A quantitative case study assessment of biophysical and economic effects from altering season of calving in temperate pasture-based dairy systems.

In theory, a late winter-early spring calving date in temperate grazing systems best matches pasture supply and herd demand, thereby minimizing the need for nonpasture feeds and maximizing profitability. We used a quantitative case study approach to define the effects of season of calving on biophysical and financial performance in a grazing system without the confounding effects of imported feeds (i.e., milk production directly from grazed pasture). A 2-yr production system experiment was established to quantify the effects of changing onset of seasonal calving (i.e., planned start of calving; PSC) from winter (July in the Southern Hemisphere) to spring (October), summer, (January), or autumn (April) on pasture and animal production and profitability. Eighty Holstein-Friesian cows were randomly allocated to 1 of 4 PSC treatments, each of which had a different PSC [mean calving date of January 10 (JAN), April 10 (APR), July 10 (JUL), or October 10 (OCT)]. Data were analyzed for consistency of treatment response over years using ANOVA procedures with year, PSC treatment, and year × PSC treatment interactions as fixed effects. Collated biological data and financial data extracted from a national economic database were used as fixed variables to model the financial performance for the different treatments. A stochastic risk analysis was undertaken, where historical pasture growth and milk price data were used to estimate the probability distributions for stochastic input variables. Gross farm revenue and operating profit per hectare were modeled under 2 scenarios: (A) milk price did not include a premium for milk supplied during the winter, and (B) milk price included a realistic premium for milk supplied in winter. Annual and seasonal pasture growth did not differ between treatments, but the pasture growth (kg of dry matter/ha) and profile of the JUL treatment best matched the lactation nutrient demand profile. In comparison, profiles for JAN, APR, and OCT calving treatments had periods of greater surplus and deficit due to the time of calving and herd demand relative to the pasture growth profile. As a result, the JAN and OCT treatments conserved more pasture as silage and cows consumed a larger proportion of their annual diet as silage. Although the amount of silage conserved and consumed did not differ between the JUL and APR calving treatments, the timing of the silage consumption was different, with silage making up a greater proportion of the diets in the APR treatment 1 to 90 and 91 to 180 d postcalving and being offered to the JUL calving treatment only 271 to 365 d postcalving. As a result of differences in the quantity and proportion of pasture and pasture silage in the lactating diet, the JUL treatment herd tended to produce greater milk, 4% fat-corrected milk, fat, protein, and lactose yields (kg/cow) than the other PSC treatments, which did not differ from each other. Operating expenses per hectare did not differ materially between calving date scenarios, but operating expenses per kilogram of fat-corrected milk and kilogram of fat and protein were 15 to 20% less in the JUL treatment. With or without a realistic winter milk premium, gross farm revenue and operating profit per hectare were greater in the JUL treatment than in the APR treatment, which had greater revenue and profitability than the remaining 2 calving date treatments. In summary, our results indicate that a PSC in late winter is most profitable in a grazing system not importing feed, with or without a realistic price incentive scheme.

Short communication: Milk fat payment affects the relative profitability of Jersey and Holstein-Friesian cows at optimal comparative stocking rate.

Choice of stocking rate and breed of cow are 2 strategic decisions that affect the profitability of pasture-based dairy farm businesses. This study sought to analyze the effects of a range of fat and protein prices on the profitability of the Jersey (J) and Holstein-Friesian (HF) breeds at 2 comparative stocking rates (CSR): 80 kg of body weight (BW) per tonne of dry matter (DM) of feed (CSR80), and 100 kg of BW per tonne of DM of feed (CSR100). Data were obtained from a recently published study, and equations constructed to determine the values for fat and protein at which each breed broke even (profit = NZ$0/ha; at time of writing, NZ$1 = US$0.69 or €0.60), returned equal profit, and exceeded the other breed by 1% or 5%. At CSR100 there were few combinations of fat and protein prices for which HF were more profitable than J. At CSR80, J and HF were equally profitable at a fat price of NZ$5.67 ± NZ$0.20 per kilogram, depending on protein price. The study also highlighted the importance of including volume adjustments in milk price calculations when differences in milk composition exist, as the fat price at which the profitability of HF and J were equal was NZ$1.23/kg lower when volume adjustments were included. The recent increase in the value of fat relative to protein favors J. Farmers should consider the medium- to long-term outlook of fat price when evaluating breed choice for their farm system.

Methods to manufacture regulatory T cells for cell therapy.

Regulatory T cell (Treg ) therapy has shown promise in early clinical trials for treating graft-versus-host disease, transplant rejection and autoimmune disorders. A challenge has been to isolate sufficiently pure Tregs and expand them to a clinical dose. However, there has been considerable progress in the development and optimization of these methods, resulting in a variety of manufacturing protocols being tested in clinical trials. In this review, we summarize methods that have been used to manufacture Tregs for clinical trials, including the choice of cell source and protocols for cell isolation and expansion. We also discuss alternative culture or genome editing methods for modulating Treg specificity, function or stability that could be applied to future clinical manufacturing protocols to increase the efficacy of Treg therapy.

Hot topic: Selecting cattle for low residual feed intake did not affect daily methane production but increased methane yield.

Reducing enteric methane (CH4) production and improving feed conversion efficiency of dairy cows is of high importance. Residual feed intake (RFI) is one measure of feed efficiency, with low RFI animals being more efficient in feed conversion. Enteric CH4 is an important source of digestible energy loss in ruminants and, because research in beef cattle has reported a positive relationship between RFI and daily CH4 production, we hypothesized that low RFI dairy heifers, which are more feed efficient, would produce less CH4/d. We measured the daily methane production (g of CH4/d), methane yield [g of CH4/kg of dry matter intake (DMI)], and CH4 per kilogram of body weight (BW) gain for 56 heifers (20-22 mo old) in a 2 × 2 factorial arrangement: factors included 2 breeds (Holstein-Friesian and Jersey; n = 28/breed), with equal numbers of animals previously determined as being either high [+2.0 kg of dry matter (DM)/d] or low RFI (-2.1 kg of DM/d; n = 28/RFI category). All heifers were commingled and offered unrestricted access to the same diet of dried alfalfa cubes. Between RFI categories, heifers did not differ in BW or BW gain but low RFI heifers had 9.3 and 10.6% lower DMI and DMI/kg of BW, respectively, than high RFI heifers. Similarly, RFI category did not affect CH4/d or CH4/kg of BWg, but CH4/kg of DMI was higher in low RFI heifers because of their lower DMI. These results might reflect more complete digestion of ingested feed in the more efficient, low RFI heifers, consistent with previous reports of greater apparent digestibility of organic matter. Holstein-Friesian heifers were heavier and consumed more total DM than Jersey heifers, but breed did not affect DMI/kg of BW or BWg. Jersey heifers produced less CH4/d, but not CH4/kg of DMI or CH4/kg of BWg. We detected no interaction between breed and RFI category in any of the variables measured. In conclusion, differences in RFI in dairy heifers did not affect daily CH4 production (g/d); however, low RFI heifers had a greater CH4 yield (g/kg of DMI) on a high forage diet.

Tailoring the homing capacity of human Tregs for directed migration to sites of Th1-inflammation or intestinal regions.

Cell-based therapy with CD4+ FOXP3+ regulatory T cells (Tregs) is a promising strategy to limit organ rejection and graft-vs-host disease. Ongoing clinical applications have yet to consider how human Tregs could be modified to direct their migration to specific inflammation sites and/or tissues for more targeted immunosuppression. We show here that stable, homing-receptor-tailored human Tregs can be generated from thymic Tregs isolated from pediatric thymus or adult blood. To direct migration to Th1-inflammatory sites, addition of interferon-γ and IL-12 during Treg expansion produced suppressive, epigenetically stable CXCR3+ TBET+ FOXP3+ T helper (Th)1-Tregs. CXCR3 remained expressed after injection in vivo and Th1-Tregs migrated efficiently towards CXCL10 in vitro. To induce tissue-specific migration, addition of retinoic acid (RA) during Treg expansion induced expression of the gut-homing receptors α4ß7-integrin and CCR9. FOXP3+ RA-Tregs had elevated expression of the functional markers latency-associated peptide and glycoprotein A repetitions predominant, increased suppressive capacity in vitro and migrated efficiently to healthy and inflamed intestine after injection into mice. Homing-receptor-tailored Tregs were epigenetically stable even after long-term exposure to inflammatory conditions, suppressive in vivo and characterized by Th1- or gut-homing-specific transcriptomes. Tailoring human thymic Treg homing during in vitro expansion offers a new and clinically applicable approach to improving the potency and specificity of Treg therapy.

Review: New considerations to refine breeding objectives of dairy cows for increasing robustness and sustainability of grass-based milk production systems.

Although food from grazed animals is increasingly sought by consumers because of perceived animal welfare advantages, grazing systems provide the farmer and the animal with unique challenges. The system is dependent almost daily on the climate for feed supply, with the importation of large amounts of feed from off farm, and associated labour and mechanisation costs, sometimes reducing economic viability. Furthermore, the cow may have to walk long distances and be able to harvest feed efficiently in a highly competitive environment because of the need for high levels of pasture utilisation. She must, also, be: (1) highly fertile, with a requirement for pregnancy within ~80 days post-calving; (2) 'easy care', because of the need for the management of large herds with limited labour; (3) able to walk long distances; and (4) robust to changes in feed supply and quality, so that short-term nutritional insults do not unduly influence her production and reproduction cycles. These are very different and are in addition to demands placed on cows in housed systems offered pre-made mixed rations. Furthermore, additional demands in environmental sustainability and animal welfare, in conjunction with the need for greater system-level biological efficiency (i.e. 'sustainable intensification'), will add to the 'robustness' requirements of cows in the future. Increasingly, there is evidence that certain genotypes of cows perform better or worse in grazing systems, indicating a genotype×environment interaction. This has led to the development of tailored breeding objectives within countries for important heritable traits to maximise the profitability and sustainability of their production system. To date, these breeding objectives have focussed on the more easily measured traits and those of highest relative economic importance. In the future, there will be greater emphasis on more difficult to measure traits that are important to the quality of life of the animal in each production system and to reduce the system's environmental footprint.

Dairy cow breed interacts with stocking rate in temperate pasture-based dairy production systems.

Economic optimum stocking rates for grazing dairy systems have been defined by accounting for the pasture production potential of the farm [t of dry matter (DM)/ha], the amount of feed imported from outside the farm (t of DM/ha), and the size of the cow (kg). These variables were combined into the comparative stocking rate [CSR; kg of body weight (BW)/t of feed DM available] measure. However, CSR assumes no effect of cow genetics beyond BW, and there is increasing evidence of within-breed differences in residual feed intake and between-breed differences in the gross efficiency with which cows use metabolizable energy for milk production. A multiyear production system experiment was established to determine whether Jersey (J) and Holstein-Friesian (HF) breeds performed similarly at the same CSR. Fifty-nine J cows and 51 HF cows were randomly allocated to 1 of 2 CSR in a 2 × 2 factorial arrangement; systems were designed to have a CSR of either 80 or 100 kg of BW/t of feed DM (J-CSR80, J-CSR100, HF-CSR80, and HF-CSR100 treatment groups). Data were analyzed for consistency of farmlet response over years using ANOVA procedures, with year and farmlet as fixed effects and the interaction of farmlet with year as a random effect. The collated biological data and financial data extracted from a national economic database were used to model the financial performance for the different breed and CSR treatments. On average, annual and individual season pasture DM production was greater for the J farmlets and was less in the CSR100 treatment; however, the effect of CSR was primarily driven by a large decline in pasture DM production in the HF-CSR100 treatment (breed × CSR interaction). This interaction in feed availability resulted in a breed × CSR interaction for the per-cow and per-hectare milk production variables, with HF cows producing more milk and milk components per cow in the CSR80 treatment but the same amount as the J cows in the CSR100 treatment. On a per-hectare basis, HF cows produced the same amount of 4% fat-corrected milk and lactose as J cows in the CSR80 treatment, but less fat; at CSR100, J cows produced more 4% fat-corrected milk, fat, and protein per hectare than HF cows. Our results support a greater gross efficiency for use of metabolizable energy by the J cow; 11% less total metabolizable energy was required to produce 1 kg of fat and protein at a system level. Economic modeling indicated that profitability of both breeds was less at CSR100, but the decline in profitability with increasing stocking rate was much greater in the HF breed. Holstein-Friesian cows were more profitable at CSR80 but were less profitable at CSR100.

How do patients trade-off surgeon choice and waiting times for total joint replacement: a discrete choice experiment.

OBJECTIVE: Patients face significant waiting times for hip and knee total joint replacement (TJR) in publicly funded healthcare systems. We aimed to assess how surgeon selection and reputation affect patients' willingness to wait for TJR. DESIGN: We assessed patient preferences using a discrete choice experiment questionnaire with 12 choice scenarios administered to patients referred for TJR. Based on qualitative research, pre- and pilot-testing, we characterized each scenario by five attributes: surgeon reputation, surgeon selection, waiting time to surgeon visit (initial consultation), waiting time to surgery, and travel time to hospital. Preferences were assessed using hierarchical Bayes (HB) analysis and evaluated for goodness-of-fit. We conducted simulation analyses to understand how patients value surgeon reputation and surgeon selection in terms of willingness to wait for surgeon visit and surgery. RESULTS: Of 422 participants, 68% were referred for knee TJR. The most important attribute was surgeon reputation followed by waiting times, surgeon selection process and travel time. Patients appear willing to wait 10 months for a visit with an excellent reputation surgeon before switching to a good reputation surgeon. Patients in the highest pain category were willing to wait 7.3 months before accepting the next available surgeon, compared to 12 months for patients experiencing the least pain. CONCLUSIONS: Our findings confirm that patients value surgeon reputation in the context of wait times and surgeon selection. We suggest opportunities to reduce wait times by explicitly offering the next available surgeon to increase patient choice, and by reporting surgeon performance to reduce potential misinformation about reputation.

Vitamin D status and risk for sarcopenia in youth with inflammatory bowel diseases.

Suboptimal vitamin D (vitD) status and reduced lean body mass are highly prevalent in pediatric inflammatory bowel diseases (IBD). The study objective was to determine sarcopenia prevalence and associations with vitD status in newly diagnosed pediatric IBD. Children with Crohn's disease (CD; n = 58) and ulcerative colitis (UC; n = 27) were included. Primary outcomes included body composition (total/regional/percent fat mass (FM), fat-free mass (FFM), skeletal muscle mass (SMM)), and vitD status (serum 25(OH)D). Sarcopenia was defined as SMM-z < -2. Additional variables measured included serum CRP, ESR, anthropometric, Pediatric Crohn's Disease Activity Index (PCDAI), and the Pediatric Ulcerative Colitis Disease Activity index (PUCAI). Sarcopenia and suboptimal 25(OH)D levels (< 50 nmol/l) were found in 23.5% (n = 20) and 52% (n = 44) of children, respectively. Younger children (< 13 years) with CD with suboptimal 25(OH)vitD (< 50 nmol/l) had the greatest frequency of sarcopenia (57.1%) (p = 0.004). Sarcopenia was prevalent in newly diagnosed, young children with CD with vitD deficiency.

Pharmacokinetics of the anticonvulsant levetiracetam in neonatal foals.

BACKGROUND: Seizures are a common manifestation of neurological disease in the neonatal foal and are an important cause of morbidity and mortality in this population. Current antiepileptic options are effective, but often have undesirable adverse effects, short duration of action and high cost. Levetiracetam has an ideal safety and pharmacokinetic profile in multiple species, including the adult horse, and may be a safe and cost-effective alternative anticonvulsant in neonatal foals. Due to differences in drug disposition and clearance dosages in neonates, dosing recommendations in other species or adult horses cannot be extrapolated to foals. OBJECTIVE: To establish the pharmacokinetic profile of single-dose i.v. and intragastric administration of levetiracetam in healthy neonatal foals. STUDY DESIGN: Randomised crossover experimental study. METHODS: Levetiracetam was administered as a single dose to six healthy foals (ages 1-10 days) at a dose of 32 mg/kg bwt i.v. or intragastrically. Plasma levetiracetam concentrations were measured using a validated HPLC protocol. RESULTS: After i.v. administration to healthy foals, levetiracetam had a mean (±s.d.) elimination half-life of 7.76 ± 0.51 h, a mean systemic clearance of 61.67 ± 10.96 (mL/h/kg) and a mean apparent volume of distribution at steady state of 0.670 ± 0.124 (L/kg). Following intragastric administration, levetiracetam had a peak concentration of 38.34 ± 7.42 mg/L and time to achieve peak concentration was 0.875 (0.5-1.5) h. Mean bioavailability for IG administration was excellent (103.04 ± 14.51%). No significant differences in pharmacokinetic variables between routes and order of administration were observed. MAIN LIMITATIONS: Small sample size and single-dose administration. CONCLUSIONS: Levetiracetam has excellent intragastric bioavailability in foals and is predicted to maintain plasma concentrations at or above the proposed target concentration with twice daily i.v. or oral administration. Once-daily administration may be possible in some foals based on the therapeutic range recommended in other species.

Longitudinal Analysis of Quality of Life, Clinical, Radiographic, Echocardiographic, and Laboratory Variables in Dogs with Preclinical Myxomatous Mitral Valve Disease Receiving Pimobendan or Placebo: The EPIC Study.

BACKGROUND: Changes in clinical variables associated with the administration of pimobendan to dogs with preclinical myxomatous mitral valve disease (MMVD) and cardiomegaly have not been described. OBJECTIVES: To investigate the effect of pimobendan on clinical variables and the relationship between a change in heart size and the time to congestive heart failure (CHF) or cardiac-related death (CRD) in dogs with MMVD and cardiomegaly. To determine whether pimobendan-treated dogs differ from dogs receiving placebo at onset of CHF. ANIMALS: Three hundred and fifty-four dogs with MMVD and cardiomegaly. MATERIALS AND METHODS: Prospective, blinded study with dogs randomized (ratio 1:1) to pimobendan (0.4-0.6 mg/kg/d) or placebo. Clinical, laboratory, and heart-size variables in both groups were measured and compared at different time points (day 35 and onset of CHF) and over the study duration. Relationships between short-term changes in echocardiographic variables and time to CHF or CRD were explored. RESULTS: At day 35, heart size had reduced in the pimobendan group: median change in (Δ) LVIDDN -0.06 (IQR: -0.15 to +0.02), P < 0.0001, and LA:Ao -0.08 (IQR: -0.23 to +0.03), P < 0.0001. Reduction in heart size was associated with increased time to CHF or CRD. Hazard ratio for a 0.1 increase in ΔLVIDDN was 1.26, P = 0.0003. Hazard ratio for a 0.1 increase in ΔLA:Ao was 1.14, P = 0.0002. At onset of CHF, groups were similar. CONCLUSIONS AND CLINICAL IMPORTANCE: Pimobendan treatment reduces heart size. Reduced heart size is associated with improved outcome. At the onset of CHF, dogs treated with pimobendan were indistinguishable from those receiving placebo.

Sex hormones play a role in vulnerability to sleep loss on emotion processing tasks.

The central aim of this study was to investigate hormones as a predictor of individual vulnerability or resiliency on emotion processing tasks following one night of sleep restriction. The restriction group was instructed to sleep 3 a.m.-7 a.m. (13 men, 13 women in follicular phase, 10 women in luteal phase of menstrual cycle), and a control group slept 11 p.m.-7 a.m. (12 men, 12 follicular women, 12 luteal women). Sleep from home was verified with actigraphy. Saliva samples were collected on the evening prior to restriction, and in the morning and afternoon following restriction, to measure testosterone, estradiol, and progesterone. In the laboratory, event-related potentials (ERPs) were recorded during presentation of images and faces to index neural processing of emotional stimuli. Compared to controls, sleep-restricted participants had a larger amplitude Late Positive Potential (LPP) ERP to positive vs neutral images, reflecting greater motivated attention towards positive stimuli. Sleep-restricted participants were also less accurate categorizing sad faces and exhibited a larger N170 to sad faces, reflecting greater neural reactivity. Sleep-restricted luteal women were less accurate categorizing all images compared to control luteal women, and progesterone was related to several outcomes. Morning testosterone in men was lower in the sleep-restricted group compared to controls; lower testosterone was associated with lower accuracy to positive images, a greater difference between positive vs neutral LPP amplitude, and lower accuracy to sad and fearful faces. In summary, women higher in progesterone and men lower in testosterone were more vulnerable to the effects of sleep restriction on emotion processing tasks. This study highlights a role for sex and sex hormones in understanding individual differences in vulnerability to sleep loss.

A 100-Year Review: A century of change in temperate grazing dairy systems.

From 1917 to 2017, dairy grazing systems have evolved from uncontrolled grazing of unimproved pastures by dual-purpose dairy-beef breeds to an intensive system with a high output per unit of land from a fit-for-purpose cow. The end of World War I signaled significant government investments in agricultural research institutes around the world, which coincided with technological breakthroughs in milk harvesting and a recognition that important traits in both plants and animals could be improved upon relatively rapidly through genetic selection. Uptake of milk recording and herd testing increased rapidly through the 1920s, as did the recognition that pastures that were rested in between grazing events yielded more in a year than those continuously grazed. This, and the invention and refinement of the electric fence, led to the development of "controlled" rotational grazing. This, in itself, facilitated greater stocking rates and a 5 to 10% increase in milk output per hectare but, perhaps more importantly, it allowed a more efficient use of nitrogen fertilizer, further increasing milk output/land area by 20%. Farmer inventions led to the development of the herringbone and rotary milking parlors, which, along with the "unshortable" electric fence and technological breakthroughs in sperm dilution rates, allowed further dairy farm expansion. Simple but effective technological breakthroughs in reproduction ensured that cows were identified in estrus early (a key factor in maintaining the seasonality of milk production) and enabled researchers to quantify the anestrus problem in grazing herds. Genetic improvement of pasture species has lagged its bovine counterpart, but recent developments in multi-trait indices as well as investment in genetic technologies should significantly increase potential milk production per hectare. Decades of research on the use of feeds other than pasture (i.e., supplementary feeds) have provided consistent milk production responses when the reduction in pasture intake associated with the provision of supplementary feed (i.e., substitution rate) is accounted for. A unique feature of grazing systems research over the last 70 yr has been the use of multi-year farm systems experimentation. These studies have allowed the evaluation of strategic changes to a component of the system on all the interacting features of the system. This technique has allowed excellent component research to be "systemized" and is an essential part of the development of the intensive grazing production system that exists today. Future challenges include the provision of skilled labor or specifically designed automation to optimize farm management and both environmental sustainability and animal welfare concerns, particularly relating to the concentration of nitrogen in each urine patch and the associated risk of nitrate leaching, as well as concerns regarding exposure of animals to harsh climatic conditions. These combined challenges could affect farmers' "social license" to farm in the future.

Characterization of exosomes from body fluids of dairy cows.

Exosomes are a specific subpopulation of extracellular vesicles that are widely released by cells of different origins with divergent functions that make their way into body fluids that can be conveniently sampled. In the current study, we isolated and evaluated exosomes from concurrently collected samples of milk, plasma, saliva, and urine from a group of 6 pregnant Holstein-Friesian dairy cows (aged 7 mo, 174 to 203 d of gestation). The cows had BCS of 3.5 to 5.25 (on a scale of 1 to 10), and the milk production for the season to the time of sampling ranged between 5,118 and 6,959 kg. The low levels of extracellular vesicles in saliva and urine (more than 86% fewer compared to the extracellular vesicles in milk and plasma) precluded further detailed evaluation since utility for diagnostics was deemed unlikely. In exosomes isolated from milk and plasma, size distribution, morphology, and the presence of exosome markers was confirmed by nanoparticle tracking analysis, electron microscopy, and Western blot. In addition, a targeted proteomic approach using the quadrupole ion trap mass spectrometer was also used in the study to screen for the exosome marker (e.g., Tumor susceptibility gene 101). Following confirmation of the presence of exosomes, the proteomic profiles of milk and plasma exosomes were evaluated using information-dependent acquisition-mediated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The milk exosomes contain proteins that differed greatly from the plasma exosomes, with only 8 similar proteins harbored in both the milk and plasma exosomes. The milk and plasma exosomes were found to contain proteins (e.g., immunoglobulin J chain and α2 macroglobulin) associated with specific biological processes and molecular functions. Hence, the fluid of origin required for exosome analysis will be dependent on the specific information needed. In conclusion, isolated exosomes from milk and plasma samples collected at the same time point from the same dairy cows encapsulated different profiles of proteins associated with different biological processes and molecular functions.

Generating an image that affords slant perception from stereo, without pictorial cues.

This paper describes an algorithm for generating a planar image that when tilted provides stereo cues to slant, without contamination from pictorial gradients. As the stimuli derived from this image are ultimately intended for use in studies of slant perception under magnification, a further requirement is that the generated image be suitable for high-definition printing or display on a monitor. A first stage generates an image consisting of overlapping edges with sufficient density that when zoomed, edges that nearly span the original scale are replaced with newly emergent content that leaves the visible edge statistics unchanged. A second stage reduces intensity clumping while preserving edges by enforcing a broad dynamic range across the image. Spectral analyses demonstrate that the low-frequency content of the resulting image, which would correspond to the pictorial cue of texture gradient changes under slant, (a) has a power fall-off deviating from 1/f noise (to which the visual system is particularly sensitive), and (b) does not offer systematic cues under changes in scale or slant. Two behavioral experiments tested whether the algorithm generates stimuli that offer cues to slant under stereo viewing only, and not when disparities are eliminated. With a particular adjustment of dynamic range (and nearly so with the other version that was tested), participants viewing without stereo cues were essentially unable to discriminate slanted from flat (frontal) stimuli, and when slant was reported, they failed to discriminate its direction. In contrast, non-stereo viewing of a control stimulus with pictorial cues, as well as stereoscopic observation, consistently allowed participants to perceive slant correctly. Experiment 2 further showed that these results generalized across a population of different stimuli from the same generation process and demonstrated that the process did not substitute biased slant cues.