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BackgroundCurrent minimally invasive fat reduction modalities use equipment that can cost thousands of U.S. dollars. Electrochemical lipolysis (ECLL), using low-cost battery and electrodes (approximately $10), creates acid/base within fat (width, approximately 3 mm), damaging adipocytes. Longitudinal effects of ECLL have not been studied. In this pilot study, the authors hypothesize that in vivo ECLL induces fat necrosis, decreases adipocyte number/viability, and forms lipid droplets.MethodsTwo female Yorkshire pigs (50 to 60 kg) received ECLL. In pig 1, 10 sites received ECLL, and 10 sites were untreated. In pig 2, 12 sites received ECLL and 12 sites were untreated. For ECLL, two electrodes were inserted into dorsal subcutaneous fat and direct current was applied for 5 minutes. Adverse effects of excessive pain, bleeding, infection, and agitation were monitored. Histology, live-dead (calcein, Hoechst, ethidium homodimer-1), and morphology (Bodipy and Hoechst) assays were performed on day 0 and postprocedure days 1, 2, 7, 14 (pig 1 and pig 2), and 28 (pig 2). Average particle area, fluorescence signal areas, and adipocytes and lipid droplet numbers were compared.ResultsNo adverse effects occurred. Live-dead assays showed adipocyte death on the anode on days 0 to 7 and the cathode on days 1 to 2 (not significant). Bodipy showed significant adipocyte loss at all sites ( P < 0.001) and lipid droplet formation at the cathode site on day 2 ( P = 0.0046). Histology revealed fat necrosis with significant increases in average particle area at the anode and cathode sites by day 14 (+277.3% change compared with untreated, P < 0.0001; +143.4%, P < 0.0001) and day 28 (+498.6%, P < 0.0001; +354.5%, P < 0.0001).ConclusionsIn vivo ECLL induces fat necrosis in pigs. Further studies are needed to evaluate volumetric fat reduction.Clinical relevance statementIn vivo ECLL induces adipocyte death and fat necrosis. ECLL has the potential to be utilized in body fat contouring.

A single locus on the X chromosome codes for androgen receptor (AR) although this gene is subject to alternative splicing. AR is expressed in multiple tissues in males and females and is essential for reproductive success in the male. Since male and female mice are viable following naturally occurring and engineered loss of function with male mice infertile as anticipated, functional deletion of AR in pigs was hypothesized to provide a genetic containment strategy for males with edited genomes. In addition, deletion of AR might be a method to manage boar taint, hence contributing to a perceived improvement in animal welfare. The CRISPR/Cas9 technology was used to edit either exon 2 or exon 5 of the pig AR gene. Although pregnancies were established following embryo transfer of edited embryos, they were not maintained beyond day 25. Furthermore, normal M:F sex ratios were present in edited blastocysts and 19-day fetuses, but all fetuses recovered on day 21 or later were female. The pig AR gene differs from the mouse in having a U2 spliceosome component encoded in the intronic region. Hence, the absence of fetal survival beyond day 25 may be due to interference with the U2 component rather than AR.

Introduction: The creation of synthetic reservoirs for bladder replacement has been limited by challenges of interfacing synthetic materials and native tissue. We sought to overcome this challenge by utilizing a novel bilayer silk fibroin scaffold (BLSF) as an intermediary toward the development of an acellular prosthetic reservoir. Methods: Under institutionally approved protocols, 3D-printed reservoirs were implanted in six juvenile female pigs after cystectomy. BLSF was attached to the in situ prosthetic reservoir serving as an intermediary to native ureteral and urethral tissue anastomoses. Our first protocol allowed four pigs to be survived up to 7 days, and the second protocol allowed two pigs to be survived for up to 1 year. At the first sign of functional decline or the end of the study period, the animals were euthanized, and kidneys, ureters, prosthetic bladder, and urethra were harvested en bloc for histopathology analysis. Results: The first two pigs had anastomotic urine leaks because of design flaws resulting in early termination. The third pig had acute renal failure resulting in early termination. The artificial bladder design was modified in subsequent iterations. The fourth pig survived for 7 days and, upon autopsy, had intact urethral and ureteral anastomoses. The fifth and sixth pigs survived for 11 and 12 weeks, respectively, before they were sacrificed because of failure to thrive. One animal developed an enteric fistula. The other animal had an intact anastomosis, and the BLFS was identified at the ureteral and urethral anastomoses on histopathologic analysis. Conclusions: Replacing the porcine bladder with a prosthetic bladder was achieved for up to 3 months, the second longest survival period for a nonbiologic bladder alternative. BLSF was used for the first time to create an interface between synthetic material and biologic tissue by allowing ingrowth of urothelium onto the acellular alloplastic bladder.

The goal of this project was to determine if standardized ileal digestible (SID) lysine provided at 40% above estimated requirements, with the concomitant increase in protein intake, from days 90 to 110 of gestation would stimulate mammary development in gilts. From day 90 of gestation, Yorkshire × Landrace gilts were fed 2.65 kg of either a conventional diet (CTL, control, n = 19) providing 18.6 g/d of SID Lys or a diet providing 26.0 g/d of SID Lys via additional soybean meal (HILYS, n = 19). Both diets were isoenergetic. Jugular blood samples obtained on days 90 and 110 of gestation were used to measure concentrations of insulin-like growth factor-1 (IGF-1), metabolites, and amino acids (AA). Gilts were necropsied on day 110 ± 1 of gestation to obtain mammary glands for compositional analyses, immunohistochemistry, and analysis of mRNA abundance for AA transporters and markers of cell proliferation and differentiation. The HILYS gilts gained more body weight (P < 0.01) during the experimental period compared with CTL gilts, and had greater fetal weights (1.29 vs. 1.21 ± 0.03 kg, P < 0.05). There was no difference in circulating IGF-1, glucose, or albumin (P > 0.10) between HILYS and CTL gilts on day 110 of gestation, whereas concentrations of urea and free fatty acids were greater (P < 0.01), and those of Trp and Ala were lower (P < 0.05), in HILYS than CTL gilts. The provision of lysine at 40% above estimated requirements increased total mammary parenchymal mass by 44%, as well as total parenchymal fat, protein, DNA, and RNA (P < 0.01). The mRNA abundance of ACACA was greater (P < 0.05) in HILYS than CTL gilts, while only the AA transporter SLC6A14 tended (P < 0.10) to be greater. Results demonstrate that providing dietary Lys above current National Research Council recommendations in late gestation increases mammary development in gilts. Results also indicate that Lys may have been limiting for protein retention. These data suggest that the use of a two-phase feeding strategy during gestation, whereby dietary Lys is increased from day 90, could benefit potential sow milk yield in the subsequent lactation.

Judgement bias testing has emerged as a potential tool for assessing affective states in animals. Researchers infer an animal's affective state based on an animal's response to an ambiguous stimulus that is intermediate to both the rewarded and punished conditioned stimuli. Animals can be classified as "optimistic" or having a positive affective state if the animal displays behaviors that suggest an increased expectation of reward in the face of ambiguous stimuli. Alternatively, animals can be classified "pessimistic" or having a negative affective state if the animal displays behaviors that suggest an increased expectation of punishment in the face of ambiguous stimuli. Recent reports in multiple species question what factors influence performance in judgement bias testing, and which may allow for erroneous conclusions regarding individual affective state. In order to better understand this concern, 25 female swine were subjected to behavioral assessments at critical rearing stages to determine response variability. These same individuals were then assessed for physical measures of welfare and judgement bias using the "go/no-go" task as breeding adults. Sows which were more aggressive approached the ambiguous, but not the positive, stimulus significantly faster than others. Both optimistic and pessimistic biases were observed despite all sows living in enriched housing, and, sows with more positive physical welfare measures (fewer skin lesions and healthy body condition) did not exhibit more optimistic judgement biases. Our data demonstrate that behavior traits, such as aggressiveness, can affect a sow's performance in a judgement bias test, while measures of physical health did not. We suggest that individual differences in behavior (e.g., bold-aggressive behavioral syndrome, or, proactive coping style) generate different emotional responses and can contribute to the animal's overall affective state more so than physical ailment. Our findings highlight the complexity of how different factors impact an animal's overall affective state and support the need for complementary measures in future JBT studies, including personality assessment.

ObjectivesMinimally invasive fat sculpting techniques are becoming more widespread with the development of office-based devices and therapies. Electrochemical lipolysis (ECLL) is a needle-based technology that uses direct current (DC) to electrolyze tissue water creating acid and base in situ. In turn, fat is saponified and adipocyte cell membrane lysis occurs. The electrolysis of water can be accomplished using a simple open-loop circuit (V-ECLL) or by incorporating a feedback control circuit using a potentiostat (P-ECLL). A potentiostat utilizes an operational amplifier with negative feedback to allow users to precisely control voltage at specific electrodes. To date, the variation between the two approaches has not been studied. The aim of this study was to assess current and charge transfer variation and lipolytic effect created by the two approaches in an in vivo porcine model.MethodsCharge transfer measurements from ex vivo V-ECLL and P-ECLL treated porcine skin and fat were recorded at -1 V P-ECLL, -2 V P-ECLL, -3 V P-ECLL, and -5 V V-ECLL each for 5 min to guide dosimetry parameters for in vivo studies. In follow-up in vivo studies, a sedated female Yorkshire pig was treated with both V-ECLL and P-ECLL across the dorsal surface over a range of dosimetry parameters, including -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL, and 5 V V-ECLL each treated for 5 min. Serial biopsies were performed at baseline before treatment, 1, 2, 7, 14, and 28 days after treatment. Tissue was examined using fluorescence microscopy and histology to compare the effects of the two ECLL approaches.ResultsBoth V-ECLL and P-ECLL treatments induced in-vivo fat necrosis evident by adipocyte membrane lysis, adipocyte denuclearization, and an acute inflammatory response across a 28-day longitudinal study. However, -1.5 V P-ECLL produced a smaller spatial necrotic effect compared to 5 V V-ECLL. In addition, 5 V V-ECLL produced a comparable necrotic effect to that of -2.5 V and -3.5 V P-ECLL.ConclusionsV-ECLL and P-ECLL at the aforementioned dosimetry parameters both achieved fat necrosis by adipocyte membrane lysis and denuclearization. The -2.5 V and -3.5 V P-ECLL treatments created spatially similar fat necrotic effects when compared to the 5 V V-ECLL treatment. Quantitatively, total charge transfer between dosimetry parameters suggests that -2.5 V P-ECLL and 5 V V-ECLL produce comparable electrochemical reactions. Such findings suggest that a low-voltage closed-loop potentiostat-based system is capable of inducing fat necrosis to a similar extent compared to that of a higher voltage direct current system.

Porcine reproductive and respiratory syndrome (PRRS) is an extremely contagious disease that causes great damage to the U.S. pork industry. PRRS is not subject to official control in the U.S., but most producers adopt control strategies, including vaccination. However, the PRRS virus mutates frequently, facilitating its ability to infect even vaccinated animals. In this paper we analyze how increased vaccination on sow farms reduces PRRS losses and when vaccination is profitable. We develop a SIR model to simulate the spread of an outbreak between and within swine farms located in a region of Minnesota. Then, we estimate economic losses due to PRRS and calculate the benefits of vaccination. We find that increased vaccination of sow farms increases the private profitability of vaccination, and also transmits positive externalities to farms that do not vaccinate. Although vaccination reduces industry losses, a low to moderate vaccine efficacy implies that large PRRS losses remain, even on vaccinated farms. Our approach provides useful insight into the dynamics of an endemic animal disease and the benefits of different vaccination regimens.

This study investigated activity, preferred pen location and social interactions in female piglets (0–10 weeks of age, N = 98) intended for breeding. Piglets were housed in pens where the sow and the piglets were loose-housed without (CP) or with access to the neighbouring pen week 2–5 (AP). Female piglets of two genetic lines (Dutch and Swedish Yorkshire (DY, SY)) from 26 litters were selected within 24 h after birth. DY piglets in the AP treatment spent more time in the neighbouring pen than SY (24.0% vs 19.0%), while AP piglets of both genetic lines spent less time lying down before weaning than CP. At weaning, CP piglets increased their time in the piglet corner and spent less time lying. SY piglets were less responsive to social interactions. The results confirm previous findings on favourable effects of early social mixing on piglets' behavioural responses to weaning also when sows are individually loose-housed. [ABSTRACT FROM AUTHOR]

Acute ischemic stroke patients benefit from reperfusion in a short time-window after debut. Later treatment may be indicated if viable brain tissue is demonstrated and this outweighs the inherent risks of late reperfusion. Magnetic resonance imaging (MRI) with hyperpolarized [1-13C]pyruvate is an emerging technology that directly images metabolism. Here, we investigated its potential to detect viable tissue in ischemic stroke. Stroke was induced in pigs by intracerebral injection of endothelin 1. During ischemia, the rate constant of pyruvate-to-lactate conversion, kPL, was 52% larger in penumbra and 85% larger in the infarct compared to the contralateral hemisphere (P = 0.0001). Within the penumbra, the kPL was 50% higher in the regions that later infarcted compared to non-progressing regions (P = 0.026). After reperfusion, measures of pyruvate-to-lactate conversion were slightly decreased in the infarct compared to contralateral. In addition to metabolic imaging, we used hyperpolarized pyruvate for perfusion-weighted imaging. This was consistent with conventional imaging for assessment of infarct size and blood flow. Lastly, we confirmed the translatability of simultaneous assessment of metabolism and perfusion with hyperpolarized MRI in healthy volunteers. In conclusion, hyperpolarized [1-13C]pyruvate may aid penumbral characterization and increase access to reperfusion therapy for late presenting patients.

Catheters used for endovascular navigation in interventional procedures lack dexterity at the distal tip. Neurointerventionists, in particular, encounter challenges in up to 25% of aneurysm cases largely due to the inability to steer and navigate the tip of the microcatheters through tortuous vasculature to access aneurysms. We overcome this problem with submillimeter diameter, hydraulically actuated hyperelastic polymer devices at the distal tip of microcatheters to enable active steerability. Controlled by hand, the devices offer complete 3D orientation of the tip. Using saline as a working fluid, we demonstrate guidewire-free navigation, access, and coil deployment in vivo, offering safety, ease of use, and design flexibility absent in other approaches to endovascular intervention. We demonstrate the ability of our device to navigate through vessels and to deliver embolization coils to the cerebral vessels in a live porcine model. This indicates the potential for microhydraulic soft robotics to solve difficult access and treatment problems in endovascular intervention.

Majority of drugs are administered orally, yet their efficient absorption is often difficult to achieve, with a low dose fraction reaching the blood compartment. Here, a microstirring pill technology is reported with built-in mixing capability for oral drug delivery that greatly enhances bioavailability of its therapeutic payload. Embedding microscopic stirrers into a pill matrix enables faster disintegration and dissolution, leading to improved release profiles of three widely used model drugs, aspirin, levodopa, and acetaminophen, without compromising their loading. Unlike recently developed drug-carrying nanomotors, drug molecules are not associated with the microstirrers, and hence there is no limitation on the loading capacity. These embedded microstirrers are fabricated through the asymmetric coating of titanium dioxide thin film onto magnesium microparticles. In vitro tests illustrate that the embedded microstirrers lead to substantial enhancement of local fluid transport. In vivo studies using murine and porcine models demonstrate that the localized stirring capability of microstirrers leads to enhanced bioavailability of drug payloads. Such improvements are of considerable importance in clinical scenarios where fast absorption and high bioavailability of therapeutics are critical. The encouraging results obtained in porcine model suggest that the microstirring pill technology has translational potential and can be developed toward practical biomedical applications.

Individuals with heart failure (HF) frequently present with comorbidities, including obesity, insulin resistance, hypertension, and dyslipidemia. Many patients with HF experience cardiogenic dementia, yet the pathophysiology of this disease remains poorly understood. Using a swine model of cardiometabolic HF (Western diet+aortic banding; WD-AB), we tested the hypothesis that WD-AB would promote a multidementia phenotype involving cerebrovascular dysfunction alongside evidence of Alzheimer's disease (AD) pathology. The results provide evidence of cerebrovascular insufficiency coupled with neuroinflammation and amyloidosis in swine with experimental cardiometabolic HF. Although cardiac ejection fraction was normal, indices of arterial compliance and cerebral blood flow were reduced, and cerebrovascular regulation was impaired in the WD-AB group. Cerebrovascular dysfunction occurred concomitantly with increased MAPK signaling and amyloidogenic processing (i.e., increased APP, BACE1, CTF, and Aβ40 in the prefrontal cortex and hippocampus) in the WD-AB group. Transcriptomic profiles of the stellate ganglia revealed the WD-AB group displayed an enrichment of gene networks associated with MAPK/ERK signaling, AD, frontotemporal dementia, and a number of behavioral phenotypes implicated in cognitive impairment. These provide potentially novel evidence from a swine model that cerebrovascular and neuronal pathologies likely both contribute to the dementia profile in a setting of cardiometabolic HF.

[Figure: see text].

Interneurons contribute to the complexity of neural circuits and maintenance of normal brain function. Rodent interneurons originate in embryonic ganglionic eminences, but developmental origins in other species are less understood. Here, we show that transcription factor expression patterns in porcine embryonic subpallium are similar to rodents, delineating a distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of Nkx2.1, Lhx6, and Dlx2 expression, in vitro differentiation into neurons expressing GABA, and robust migratory capacity in explant assays, we propose that cortical and hippocampal interneurons originate from a porcine MGE region. Following xenotransplantation into adult male and female rat hippocampus, we further demonstrate that porcine MGE progenitors, like those from rodents, migrate and differentiate into morphologically distinct interneurons expressing GABA. Our findings reveal that basic rules for interneuron development are conserved across species, and that porcine embryonic MGE progenitors could serve as a valuable source for interneuron-based xenotransplantation therapies.SIGNIFICANCE STATEMENT Here we demonstrate that porcine medial ganglionic eminence, like rodents, exhibit a distinct transcriptional and interneuron-specific antibody profile, in vitro migratory capacity and are amenable to xenotransplantation. This is the first comprehensive examination of embryonic interneuron origins in the pig; and because a rich neurodevelopmental literature on embryonic mouse medial ganglionic eminence exists (with some additional characterizations in other species, e.g., monkey and human), our work allows direct neurodevelopmental comparisons with this literature.

Atrioventricular heart valves (AHVs) regulate the unidirectional flow of blood through the heart by opening and closing of the leaflets, which are supported in their functions by the chordae tendineae (CT). The leaflets and CT are primarily composed of collagen fibers that act as the load-bearing component of the tissue microstructures. At the CT-leaflet insertion, the collagen fiber architecture is complex, and has been of increasing focus in the previous literature. However, these previous studies have not been able to quantify the load-dependent changes in the tissues collagen fiber orientations and alignments. In the present study, we address this gap in knowledge by quantifying the changes in the collagen fiber architecture of the mitral and tricuspid valves strut CT-leaflet insertions in response to the applied loads by using a unique approach, which combines polarized spatial frequency domain imaging with uniaxial mechanical testing. Additionally, we characterized these microstructural changes across the same specimen without the need for tissue fixatives. We observed increases in the collagen fiber alignments in the CT-leaflet insertion with increased loading, as described through the degree of optical anisotropy. Furthermore, we used a leaflet-CT-papillary muscle entity method during uniaxial testing to quantify the chordae tendineae mechanics, including the derivation of the Ogden-type constitutive modeling parameters. The results from this study provide a valuable insight into the load-dependent behaviors of the strut CT-leaflet insertion, offering a research avenue to better understand the relationship between tissue mechanics and the microstructure, which will contribute to a deeper understanding of AHV biomechanics.

BackgroundMethyl mercaptan occurs naturally in the environment and is found in a variety of occupational settings, including the oil, paper, plastics, and pesticides industries. It is a toxic gas and deaths from methyl mercaptan exposure have occurred. The Department of Homeland Security considers it a high threat chemical agent that could be used by terrorists. Unfortunately, no specific treatment exists for methyl mercaptan poisoning.MethodsWe conducted a randomized trial in 12 swine comparing no treatment to intramuscular injection of the vitamin B12 analog cobinamide (2.0 mL, 12.5 mg/kg) following acute inhalation of methyl mercaptan gas. Physiological and laboratory parameters were similar in the control and cobinamide-treated groups at baseline and at the time of treatment.ResultsAll six cobinamide-treated animals survived, whereas only one of six control animals lived (17% survival) (p = 0.0043). The cobinamide-treated animals returned to a normal breathing pattern by 3.8 ± 1.1 min after treatment (mean ± SD), while all but one animal in the control group had intermittent gasping, never regaining a normal breathing pattern. Blood pressure and arterial oxygen saturation returned to baseline values within 15 minutes of cobinamide-treatment. Plasma lactate concentration increased progressively until death (10.93 ± 6.02 mmol [mean ± SD]) in control animals, and decreased toward baseline (3.79 ± 2.93 mmol [mean ± SD]) by the end of the experiment in cobinamide-treated animals.ConclusionWe conclude that intramuscular administration of cobinamide improves survival and clinical outcomes in a large animal model of acute, high dose methyl mercaptan poisoning.

The use of renal biopsy through laparoscopy is increasingly present both in human and veterinary medicine. However, both techniques require skill and training to make the operator capable to do it. The learning curve allows the quantitative and qualitative assessment of the number of attempts and minimum time for the surgical procedure. The objective included establish the learning curve for laparoscopy-guided kidney biopsy procedures in dog and pig corpses. Six dogs and six pigs corpses weighing less than 10 kg were used for this study. All corpses underwent kidney biopsy performed through laparoscopy. Twenty-four operators, two per animal, performed 20 renal biopsies each (10 for each kidney), with 480 collection-procedures in total. Duration and difficulty of the procedure and the biopsy sample quality were evaluated and statistical analysis was performed using a mixed regression model with a random effect of individuals and multivariate analysis of data. There were 91.5% of the samples that were adequate for evaluation. There was no significant difference in the number of glomeruli or cortex percentage considering the attempts in either species, demonstrating the operators ability since first collection. Swine samples showed higher amounts of renal cortex than canine samples. The procedure duration was shorter as more attempts were performed in dogs and pigs. From the fourth repetition, the professional reached a plateau for the variable related to collection, and from the second, the professional presented uniform duration for sample storage. Operators of the swine model acquired more agility than the dog ones. The variable difficulty decreased as more repetitions were performed, reaching a plateau in the sixth attempt. Seven renal biopsies laparoscopy-guided are required for an operator to be considered capable to perform the procedure in the referred species included. The learning curve for image-guided kidney biopsy procedures improves the implementation of this technique and benefits patients that undergo this procedure.

BackgroundBoth iron deficiency and overload may adversely affect neurodevelopment.ObjectivesThe study assessed how changes in early-life iron status affect iron homeostasis and cytoarchitecture of hippocampal neurons in a piglet model.MethodsOn postnatal day (PD) 1, 30 Hampshire × Yorkshire crossbreed piglets (n = 15/sex) were stratified by sex and litter and randomly assigned to experimental groups receiving low (L-Fe), adequate (A-Fe), or high (H-Fe) levels of iron supplement during the pre- (PD1-21) and postweaning periods (PD22-35). Pigs in the L-Fe, A-Fe, and H-Fe groups orally received 0, 1, and 30 mg Fe · kg weight-1 · d-1 preweaning and were fed a diet containing 30, 125, and 1000 mg Fe/kg postweaning, respectively. Heme indexes were analyzed weekly, and gene and protein expressions of iron regulatory proteins in duodenal mucosa, liver, and hippocampus were analyzed through qRT-PCR and western blot, respectively, on PD35. Hippocampal neurons stained using the Golgi-Cox method were traced and their dendritic arbors reconstructed in 3-D using Neurolucida. Dendritic complexity was quantified using Sholl and branch order analyses.ResultsPigs in the L-Fe group developed iron deficiency anemia (hemoglobin = 8.2 g/dL, hematocrit = 20.1%) on PD35 and became stunted during week 5 with lower final body weight than H-Fe group pigs (6.6 compared with 9.6 kg, P

The functional annotation of livestock genomes is crucial for understanding the molecular mechanisms that underpin complex traits of economic importance, adaptive evolution and comparative genomics. Here, we provide the most comprehensive catalogue to date of regulatory elements in the pig (Sus scrofa) by integrating 223 epigenomic and transcriptomic data sets, representing 14 biologically important tissues. We systematically describe the dynamic epigenetic landscape across tissues by functionally annotating 15 different chromatin states and defining their tissue-specific regulatory activities. We demonstrate that genomic variants associated with complex traits and adaptive evolution in pig are significantly enriched in active promoters and enhancers. Furthermore, we reveal distinct tissue-specific regulatory selection between Asian and European pig domestication processes. Compared with human and mouse epigenomes, we show that porcine regulatory elements are more conserved in DNA sequence, under both rapid and slow evolution, than those under neutral evolution across pig, mouse, and human. Finally, we provide biological insights on tissue-specific regulatory conservation, and by integrating 47 human genome-wide association studies, we demonstrate that, depending on the traits, mouse or pig might be more appropriate biomedical models for different complex traits and diseases.

Proper, tissue-specific regulation of CYP19, the gene encoding aromatase, the key enzyme of estrogen synthesis, is essential for reproductive processes. Here, we analyzed transcriptional regulation of the porcine CYP19 in female and male gonads and brain by 5'RACE and RT-PCR and comprehensively mapped the pig CYP19 locus by in silico analysis. Our data revealed that the complete locus, including three paralogous copies, CYP19A1, CYP19A2 and CYP19A3, spans approximately 330 kb of the porcine chromosome 1. The locus also harbors the first exon of the Gliomedin gene (GLDN) in reverse orientation. Only transcripts of the CYP19A3 paralog were substantially expressed in gonads and hypothalamus. We identified CYP19A3-associated untranslated exons approximately 160 kb and 50 kb distal from the first codon. The 5´ untranslated regions of transcripts were derived from either a proximal or from one of these distal untranslated exons. Transcripts including only untranslated exons could be amplified from testis, thus suggesting long non-coding transcripts. The data revealed an additional layer of complexity in the regulation of the porcine CYP19 locus. Tissue-specific expression is not only achieved by tissue- and stage-specific expression of the three different CYP19 paralogs, but also by directing the expression of CYP19A3 from different, proximal and distal promoter regions.

PurposeTo study the feasibility and efficacy of a new remote wet lab for microsurgical education using a corneal suturing task.SettingDepartment of Ophthalmology, University of California San Francisco, San Francisco, California, USA.DesignProspective randomized controlled study.MethodsTen ophthalmology residents were stratified by postgraduate year and randomized to perform a corneal suturing task consisting of placing the 4 cardinal sutures for a penetrating keratoplasty in porcine eyes with or without remote ophthalmology attending feedback. Subsequently, both groups repeated the same task without remote feedback to test whether initial remote feedback affected subsequent performance. Finally, the group without feedback was crossed over to repeat the same corneal suturing task with remote feedback. The effectiveness of the remote wet lab was assessed subjectively by survey and objectively by grading each suture pass.ResultsResident-reported comfort with corneal suturing improved significantly after the remote wet lab for all residents. Residents and attendings rated the remote wet lab as equally or more effective compared with previous in-person wet labs and overall effective in corneal suturing. Attendings rated the remote wet lab as effective in multiple domains of microsurgical education using a modified microsurgical global rating scale. Objective corneal suturing performance was similar for both groups.ConclusionsThe remote wet lab was feasible and effective for training ophthalmology residents in corneal suturing. This represents a new social distancing compliant platform for microsurgical education during the COVID-19 pandemic.

A study determined the effects of supplementing corn-based diets for weaned pigs with multi-enzymes on growth performance, apparent total tract digestibility (ATTD) of nutrients, fecal score, and fecal microbial composition. A total of 132 pigs (initial body weight = 7.23 kg) that had been weaned at 21 d of age and fed a drug-free nursery diet for 7 d were housed in 33 pens of 4 barrows or gilts, blocked by body weight and gender, and fed 3 experimental diets at 11 pens per diet. The diets were corn-based diet without or with multi-enzyme A or B. Multi-enzyme A supplied 4,000 U of xylanase, 150 U of β-glucanase, 3,500 U of protease, and 1,500 U of amylase per kilogram of diet. Multi-enzyme B was the same as multi-enzyme A except that it supplied amylase at 150 U/kg, and that its source of amylase was different from that of multi-enzyme A. All diets contained phytase at 1,000 U/kg. The diets were fed for 35 d in 2 phases; phase 1 for the first 14 d and phase 2 for the last 21 d of the trial. Fecal score was determined daily during the first 7 d of the trial. Fecal samples were collected from rectum of 1 pig per pen on days 2, 7, 14, and 35 of the trial for determining bacterial composition. Also, fresh fecal samples were collected from each pen on days 41 and 42 to determine ATTD of nutrients. Multi-enzyme B increased (P < 0.05) average daily gain (ADG) for phases 1 and 2. For the overall study period, multi-enzyme B increased (P < 0.05) ADG from 262 to 313 g, and average daily feed intake (ADFI) from 419 to 504 g. Multi-enzyme A increased (P < 0.05) overall ADG from 262 to 290 g, but did not affect ADFI. Multi-enzyme A or B did not affect ATTD of gross energy, but increased (P < 0.05) the ATTD of ether extract from 30% to 36% or 37%, respectively. Multi-enzyme A did not affect fecal score; however, multi-enzyme B tended to decrease (P = 0.09) fecal score, implying that it tended to decrease diarrhea. Firmicutes were the most abundant phylum of fecal bacteria (its relative abundance ranged from 58% to 72%). Bacteroidetes and Actinobacteria were the 2nd and 3rd most abundant phyla of fecal bacteria. Neither multi-enzyme affected fecal bacterial composition. In conclusion, the addition of multi-enzyme A or B to phytase-supplemented corn-based diet for weaned pigs can improve their growth performance and fat digestibility. However, multi-enzyme B was more effective than multi-enzyme A in terms of improving the growth performance of weaned pigs fed corn-based diet.

Two experiments were conducted to determine the effects of protease supplementation on degradation of soybean meal (SBM) allergenic proteins (glycinin and β-conglycinin) and gut health of weaned pigs fed soybean meal-based diets. In experiment 1, 2 SBM samples from 2 different sources were subjected to porcine in vitro gastric degradation to determine the effects of protease (at 15,000 U/kg of feedstuff) on degradation of the soybean allergenic proteins. In experiment 2, 48 weaned pigs (body weight = 6.66 kg) were obtained in 2 batches of 24 pigs each. Pigs were individually housed in metabolic crates and fed 4 diets (12 pigs/diet). The diets were corn-based diet with SBM 1 or SBM 2 without or with protease at 15,000 U/kg of diet in 2 × 2 factorial arrangement. Diets were fed for 10 d and pigs were sacrificed on day 10 for measurement of small intestinal histomorphology, permeability of small intestine mounted in Ussing chambers, and serum concentration of pro-inflammatory cytokines. Two SBM sources (SBM 1 and SBM 2) contained 46.9% or 47.7% CP, 14.0% or 14.6% glycinin, and 9.90% or 10.3% β-conglycinin, respectively. Protease and SBM source did not interact on any of the response criteria measured in the current study. Protease supplementation tended to increase (P = 0.069) the in vitro gastric degradation of glycinin. Protease supplementation tended to reduce (P = 0.099) fluorescein isothiocyanate dextran 4,000 Da (which is a marker probe for intestinal permeability) flow in jejunum, and reduced (P = 0.037) serum TNF-α concentration. Protease did not affect small intestinal histomorphology. In conclusion, protease tended to increase gastric degradation of glycinin and reduce gut permeability, and serum concentration of pro-inflammatory cytokines, indicating that the protease used in the current study can be added to SBM-based diets for weanling pigs to improve gut health.

The study was conducted to investigate the efficacy of a probiotic Bacillus subtilis strain on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli and to compare the efficacy of B. subtilis with that of carbadox. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of four dietary treatments: negative control (NC, control diet without E. coli challenge), positive control (PC, control diet with E. coli challenge), and supplementation of 50 mg/kg of carbadox (antibiotic growth promotor [AGP]) or 2.56 × 109 CFU/kg of B. subtilis probiotics (PRO). The experiment lasted for 28 d with 7 d before and 21 d after the first E. coli inoculation. Fecal and blood samples were collected on days 0, 3, 7, 14, and 21 post inoculation (PI) to analyze β-hemolytic coliforms and complete blood cell count, respectively. Diarrhea score was recorded daily for each pig to calculate the frequency of diarrhea. All pigs were euthanized at day 21 PI to collect jejunal and ileal mucosa for gene expression analysis. Pigs in AGP had greater (P < 0.05) BW on days 7, 14, and 21 PI than pigs in PC and PRO groups. Supplementation of PRO enhanced pigs' BW on day 21 PI compared with the PC. Escherichia coli F18 challenge reduced (P < 0.05) average daily gain (ADG) and feed efficiency from day 0 to 21 PI, while supplementation of carbadox or PRO enhanced ADG and feed efficiency in E. coli F18-challenged pigs from day 0 to 21 PI. Pigs in AGP and PRO groups had reduced (P < 0.05) frequency of diarrhea throughout the experiment and fecal β-hemolytic coliforms on day 7 PI than pigs in the PC. Pigs in PRO had greater (P < 0.05) gene expression of CLDN1 in jejunal mucosa than pigs in the PC. Supplementation of carbadox or PRO reduced (P < 0.05) the gene expression of IL6 and PTGS2 in ileal mucosa of E. coli-infected pigs compared with pigs in the PC. Pigs in the PRO group had lower (P < 0.05) white blood cell number and neutrophil count, and serum haptoglobin concentration on day 7 PI, and less (P < 0.05) monocyte count on day 14 PI, compared with PC. In conclusion, supplementation of probiotic B. subtilis could enhance disease resistance and promote the growth performance of weaned pigs under disease challenge conditions. The potential mechanisms include but not limited to enhanced gut barrier integrity and local and systemic immune responses of weaned pigs.

BackgroundEndothelial dysfunction in the nitric oxide-cyclic guanosine monophosphate pathway is a potential contributor to perioperative myocardial ischemia. The nitric oxide precursor, L-arginine, and the cyclic guanosine monophosphate degradation blocker, sildenafil, have vasodilatory effects under high dosage.ObjectiveThis study examined the hemodynamic safety and effect profiles of the combined administration of L-arginine and sildenafil using an in-vivo pig model.MethodsHemodynamic safety including mean arterial pressure, central venous pressure, heart rate, coronary vascular resistance, and systemic vascular resistance, as well as effect profiles including cardiac output and left anterior descending blood flow were measured in ten female swine after administrations of L-arginine, sildenafil, as well as combined L-arginine and sildenafil. Measurements were compared using repeated-measures analysis of variance and linear mixed models.ResultsThe combination of L-arginine and sildenafil produced a significant dose-dependent increase in left anterior descending flow and cardiac output. In contrast, mean arterial pressure, heart rate, central venous pressure, coronary vascular resistance, and systemic vascular resistance did not show any significant changes. No significant change in serum osmolality was observed after administrations of L-arginine.ConclusionsThe combined intravenous administration of sildenafil and L-arginine in a porcine animal model was safe, well tolerated, and had at least additive effects on left anterior descending artery blood flow. Simultaneous application of both drugs might have dose-sparing effects leading to desired coronary effects at lower and safer sildenafil and L-arginine plasma concentrations. Hyperosmolality was only a minor factor in L-arginine hemodynamic effects.

The microbiome represents a vast resource for drug discovery, as its members engage in constant conflict to outcompete one another by deploying diverse strategies for survival. Cutibacterium acnes is one of the most common bacterial species on human skin and can promote the common disease acne vulgaris. By employing a combined strategy of functional screening, genetics, and proteomics we discovered a strain of Staphylococcus capitis (S. capitis E12) that selectively inhibited growth of C. acnes with potency greater than antibiotics commonly used in the treatment of acne. Antimicrobial peptides secreted from S. capitis E12 were identified as four distinct phenol-soluble modulins acting synergistically. These peptides were not toxic to human keratinocytes and the S. capitis extract did not kill other commensal skin bacteria but was effective against C. acnes on pig skin and on mice. Overall, these data show how a member of the human skin microbiome can be useful as a biotherapy for acne vulgaris.

BackgroundDispersion in ventricular repolarization is relevant for arrhythmogenesis.ObjectiveThe purpose of this study was to determine the spatiotemporal effects of sympathetic stimulation on ventricular repolarization.MethodsIn 5 anesthetized female open-chest pigs, ventricular repolarization was measured from the anterior, lateral, and posterior walls of the left ventricle (LV) and right ventricle using up to 40 transmural plunge needles (4 electrodes each) before and after left stellate ganglion stimulation (LSGS) and right stellate ganglion stimulation. In addition, LSGS was performed in 3 pigs (2 male, 1 female) before and after verapamil (5-10 mg/h) administration.ResultsLSGS yielded a biphasic response in repolarization in the lateral and posterior walls of the LV, with prolongation at ∼5 seconds (10 ± 1.5 ms) and shortening at 20-30 seconds of stimulation (-28.9 ± 4.4 ms) during a monotonic pressure increase. While the initial prolongation was abolished by verapamil, late shortening was augmented. Sequential transections of the vagal nerve and stellate ganglia augmented repolarization dispersion responses to LSGS in 2 of 5 hearts. An equal pressure increase by aortic occlusion resulted in a homogeneous shortening of repolarization in the LV, and the effects were smaller than those during LSGS. Right stellate stimulation shortened repolarization mainly in the anterior LV wall, but the effects were smaller than those of LSGS.ConclusionLSGS first prolongs (through the L-type calcium current) and then shortens repolarization. The effect of LSGS was prominent in the posterior and lateral, not the anterior, LV walls.

A number of oxylipins have been described as endogenous PPAR ligands. The very short biological half-lives of oxylipins suggest roles as autocrine or paracrine signaling molecules. While coronary arterial atherosclerosis is the root of myocardial infarction, aortic atherosclerotic plaque formation is a common readout of in vivo atherosclerosis studies in mice. Improved understanding of the compartmentalized sources of oxylipin PPAR ligands will increase our knowledge of the roles of PPAR signaling in diverse vascular tissues. Here, we performed a targeted lipidomic analysis of ex vivo-generated oxylipins from porcine aorta, coronary artery, pulmonary artery and perivascular adipose. Cyclooxygenase (COX)-derived prostanoids were the most abundant detectable oxylipin from all tissues. By contrast, the coronary artery produced significantly higher levels of oxylipins from CYP450 pathways than other tissues. The TLR4 ligand LPS induced prostanoid formation in all vascular tissue tested. The 11-HETE, 15-HETE, and 9-HODE were also induced by LPS from the aorta and pulmonary artery but not coronary artery. Epoxy fatty acid (EpFA) formation was largely unaffected by LPS. The pig CYP2J homologue CYP2J34 was expressed in porcine vascular tissue and primary coronary artery smooth muscle cells (pCASMCs) in culture. Treatment of pCASMCs with LPS induced a robust profile of pro-inflammatory target genes: TNFα, ICAM-1, VCAM-1, MCP-1 and CD40L. The soluble epoxide hydrolase inhibitor TPPU, which prevents the breakdown of endogenous CYP-derived EpFAs, significantly suppressed LPS-induced inflammatory target genes. In conclusion, PPAR-activating oxylipins are produced and regulated in a vascular site-specific manner. The CYP450 pathway is highly active in the coronary artery and capable of providing anti-inflammatory oxylipins that prevent processes of inflammatory vascular disease progression.

Interventional magnetic resonance imaging (MRI) could allow for diagnosis and immediate treatment of ischemic stroke; however, such endovascular catheter-based procedures under MRI guidance are inherently difficult. One major challenge is tracking the tip of the catheter, as standard fabrication methods for building inductively coupled coil markers are rigid and bulky. Here, we report a new approach that uses aerosol jet deposition to three-dimensional (3-D) print an inductively coupled RF coil marker on a polymer catheter. Our approach enables lightweight conforming markers on polymer catheters and these low-profile markers allow the catheter to be more safely navigated in small caliber vessels. Prototype markers with an inductor with the geometry of a double helix are incorporated on catheters for in vitro studies, and we show that these markers exhibit good signal amplification. We report temperature measurements and, finally, demonstrate feasibility in a preliminary in vivo experiment. We provide material properties and electromagnetic simulation performance analysis. This paper presents fully aerosol jet-deposited and functional wireless resonant markers on polymer catheters for use in 3T clinical scanners.

Background: Cyanide is a metabolic poison used in multiple industries and is a high threat chemical agent. Current antidotes require intravenous administration, limiting their usefulness in a mass casualty scenario. Sodium tetrathionate reacts directly with cyanide yielding thiosulfate and the non-toxic compound thiocyanate. Thiosulfate, in turn, neutralizes a second molecule of cyanide, thus, per mole, sodium tetrathionate neutralizes two moles of cyanide. Historical studies examined its efficacy as a cyanide antidote, but it has not been evaluated in a clinically relevant, large animal model, nor has it previously been administered by intramuscular injection.Objective: The objective of this study is to evaluate the efficacy of intramuscular sodium tetrathionate on survival and clinical outcomes in a large, swine model of severe cyanide toxicity.Methods: Anesthetized swine were instrumented for continuous monitoring of hemodynamics, then acclimated and breathing spontaneously prior to potassium cyanide infusion (0.17 mg/kg/min). At 6-min post-apnea (no breaths for 20 s), the cyanide infusion was terminated, and animals were treated with sodium tetrathionate (∼18 mg/kg) or normal saline control. Clinical parameters and laboratory values were evaluated at various time points until death or termination of the experiment (90 min post-treatment).Results: Laboratory values, vital signs, and time to apnea were similar in both groups at baseline and treatment. Survival in the sodium tetrathionate treated group was 100% and 17% in controls (p = 0.0043). All animals treated with sodium tetrathionate returned to breathing at a mean time of 10.85 min after antidote, and all but one control remained apneic through end of the experiment. Animals treated with tetrathionate showed improvement in blood lactate (p ≤ 0.002) starting at 30 min post-treatment. The average time to death in the control group is 63.3 ± 23.2 min. No systemic or localized adverse effects of intramuscular administration of sodium tetrathionate were observed.Conclusion: Sodium tetrathionate significantly improves survival and clinical outcomes in a large, swine model of acute cyanide poisoning.

In 2018, a 15-year-old female adolescent in Australia was infected with swine influenza A(H3N2) variant virus. The virus contained hemagglutinin and neuraminidase genes derived from 1990s-like human seasonal viruses and internal protein genes from influenza A(H1N1)pdm09 virus, highlighting the potential risk that swine influenza A virus poses to human health in Australia.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

The current study was designed to evaluate the pathogenesis, pathology and immune response of female genital tract infection with Chlamydia trachomatis L2c, the most recently discovered lymphogranuloma venereum strain, using a porcine model of sexually transmitted infections. Pigs were mock infected, infected once or infected and re-infected intravaginally, and samples were obtained for chlamydial culture, gross and microscopic pathology, and humoral and cell-mediated immunity. Intravaginal inoculation of pigs with this bacterium resulted in an infection that was confined to the urogenital tract, where inflammation and pathology were caused that resembled what is seen in human infection. Re-infection resulted in more severe gross pathology than primary infection, and chlamydial colonization of the urogenital tract was similar for primary infected and re-infected pigs. This indicates that primary infection failed to induce protective immune responses against re-infection. Indeed, the proliferative responses of mononuclear cells from blood and lymphoid tissues to C. trachomatis strain L2c were never statistically different among groups, suggesting that C. trachomatis-specific lymphocytes were not generated following infection or re-infection. Nevertheless, anti-chlamydial antibodies were elicited in sera and vaginal secretions after primary infection and re-infection, clearly resulting in a secondary systemic and mucosal antibody response. While primary infection did not protect against reinfection, the porcine model is relevant for evaluating immune and pathogenic responses for emerging and known C. trachomatis strains to advance drug and/or vaccine development in humans.

About 30% of patients with impaired cardiac function have ventricular dyssynchrony and seek cardiac resynchronization therapy (CRT). In this study, we demonstrate synchronized biventricular (BiV) pacing in a leadless fashion by implementing miniaturized and wirelessly powered pacemakers. With their flexible form factors, two pacemakers were implanted epicardially on the right and left ventricles of a porcine model and were inductively powered at 13.56 MHz and 40.68 MHz industrial, scientific, and medical (ISM) bands, respectively. The power consumption of these pacemakers is reduced to µW-level by a novel integrated circuit design, which considerably extends the maximum operating distance. Leadless BiV pacing is demonstrated for the first time in both open-chest and closed-chest porcine settings. The clinical outcomes associated with different interventricular delays are verified through electrophysiologic and hemodynamic responses. The closed-chest pacing only requires the external source power of 0.3 W and 0.8 W at 13.56 MHz and 40.68 MHz, respectively, which leads to specific absorption rates (SARs) 2-3 orders of magnitude lower than the safety regulation limit. This work serves as a basis for future wirelessly powered leadless pacemakers that address various cardiac resynchronization challenges.

BackgroundCell based therapies, such as bone marrow derived mesenchymal stem cells (BM-MSCs; also known as mesenchymal stromal cells), are currently under investigation for a number of disease applications. The current challenge facing the field is maintaining the consistency and quality of cells especially for cell dose production for pre-clinical testing and clinical trials. Here we determine how BM-donor variability and thus the derived MSCs factor into selection of the optimal primary cell lineage for cell production and testing in a pre-clinical swine model of trauma induced acute respiratory distress syndrome.MethodsWe harvested bone marrow and generated three different primary BM-MSCs from Yorkshire swine. Cells from these three donors were characterized based on (a) phenotype (morphology, differentiation capacity and flow cytometry), (b) in vitro growth kinetics and metabolic activity, and (c) functional analysis based on inhibition of lung endothelial cell permeability.ResultsCells from each swine donor exhibited varied morphology, growth rate, and doubling times. All expressed the same magnitude of standard MSC cell surface markers by flow cytometry and had similar differentiation potential. Metabolic activity and growth potential at each of the passages varied between the three primary cell cultures. More importantly, the functional potency of the MSCs on inhibition of endothelial permeability was also cell donor dependent.ConclusionThis study suggests that for production of MSCs for cell-based therapy, it is imperative to examine donor variability and characterize derived MSCs for marker expression, growth and differentiation characteristics and testing potency in application dependent assays prior to selection of the optimal cell lineage for large scale expansion and dose production.

BackgroundVascular grafts are widely used as a treatment in coronary artery bypass surgery, hemodialysis, peripheral arterial bypass and congenital heart disease. Various types of synthetic and natural materials were experimented to produce tissue engineering vascular grafts. In this study, we investigated in vivo tissue engineering technology in miniature pigs to prepare decellularized autologous extracellular matrix-based grafts that could be used as vascular grafts for small-diameter vascular bypass surgery.MethodsAutologous tissue conduits (3.9 mm in diameter) were fabricated by embedding Teflon tubings in the subcutaneous pocket of female miniature pigs (n = 8, body weight 25-30 kg) for 4 weeks. They were then decellularized by CHAPS decellularization solution. Heparin was covalently-linked to decellularized tissue conduits by Sulfo-NHS/EDC. We implanted these decellularized, completely autologous extracellular matrix-based grafts into the carotid arteries of miniature pigs, then sacrificed the pigs at 1 or 2 months after implantation and evaluated the patency rate and explants histologically.ResultsAfter 1 month, the patency rate was 100% (5/5) while the inner diameter of the grafts was 3.43 ± 0.05 mm (n = 5). After 2 months, the patency rate was 67% (2/3) while the inner diameter of the grafts was 2.32 ± 0.14 mm (n = 3). Histological staining confirmed successful cell infiltration, and collagen and elastin deposition in 2-month samples. A monolayer of endothelial cells was observed along the inner lumen while smooth muscle cells were dominant in the graft wall.ConclusionA completely autologous acellular conduit with excellent performance in mechanical properties can be remodeled into a neoartery in a minipig model. This proof-of-concept study in the large animal model is very encouraging and indicates that this is a highly feasible idea worthy of further study in non-human primates before clinical translation.

TEA domain family transcription factor 4 (Tead4) is known to be important for the trophectoderm (TE) segregation in murine embryos. However, the role of TEAD4 in early development of porcine embryos is still unknown. We examined TEAD4 expression patterns and attempted to determine the functions of TEAD4 during porcine preimplantation development using RNA interference. TEAD4 mRNA was upregulated from the 2-4-cell to 8-16-cell stages and then decreased to the blastocyst stage. Nuclear localization of TEAD4 protein was detected at the 16-cell stage, as well as at subsequent developmental stages. In porcine embryos injected with TEAD4 siRNA, transformation from morula to blastocyst was inhibited. Although TEAD4 downregulation did not affect the expression levels of POU class 5 homeobox 1 (OCT-4), transcription of SRY-related HMG-box gene 2 (SOX2) was detected at high level in TEAD4-downregulated embryos. It is possible that TEAD4 contributes to blastocyst formation in porcine embryos through downregulation of SOX2 expression. Collectively, our results indicate that TEAD4 is an important factor for the preimplantation development of porcine embryos.

BACKGROUND: Although damage control resuscitation (DCR) is routinely performed for short durations, prolonged DCR may be required in military conflicts as a component of prolonged field care. Valproic acid (VPA) has been shown to have beneficial properties in lethal hemorrhage/trauma models. We sought to investigate whether the addition of a single dose of VPA to a 72-hour prolonged DCR protocol would improve clinical outcomes. METHODS: Fifteen Yorkshire swine (40-45 kg) were subjected to lethal (50% estimated total blood volume) hemorrhagic shock (HS) and randomized to three groups: (1) HS, (2) HS-DCR, (3) HS-DCR-VPA (150 mg/kg over 3 hours) (n = 5/cohort). In groups assigned to receive DCR, Tactical Combat Casualty Care guidelines were applied (1 hour into the shock period), targeting a systolic blood pressure of 80 mm Hg. At 72 hours, surviving animals were given transfusion of packed red blood cells, simulating evacuation to higher echelons of care. Survival rates, physiologic parameters, resuscitative fluid requirements, and laboratory profiles were used to compare the clinical outcomes. RESULTS: This model was 100% lethal in the untreated animals. DCR improved survival to 20%, although this was not statistically significant. The addition of VPA to DCR significantly improved survival to 80% (p < 0.01). The VPA-treated animals also had significantly (p < 0.05) higher systolic blood pressures, lower fluid resuscitation requirements, higher hemoglobin levels, and lower creatinine and potassium levels. CONCLUSION: VPA administration improves survival, decreases resuscitation requirements, and improves hemodynamic and laboratory parameters when added to prolonged DCR in a lethal hemorrhage model.

There is a need for noninvasive, quantitative methods to characterize wound healing in the context of longitudinal investigations related to regenerative medicine. Such tools have the potential to inform the assessment of wound status and healing progression and aid the development of new treatments. We employed spatial frequency domain imaging (SFDI) to characterize the changes in optical properties of tissue during wound healing progression in a porcine model of split-thickness skin grafts and also in a model of burn wound healing with no graft intervention. Changes in the reduced scattering coefficient measured using SFDI correlated with structural changes reported by histology of biopsies taken concurrently. SFDI was able to measure spatial inhomogeneity in the wounds and predicted heterogeneous healing. In addition, we were able to visualize differences in healing rate, depending on whether a wound was debrided and grafted, versus not debrided and left to heal without intervention apart from topical burn wound care. Changes in the concentration of oxy- and deoxyhemoglobin were also quantified, giving insight into hemodynamic changes during healing.

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/β-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Impact statementThe search for an ideal "off-the-shelf" biomaterial for augmentation cystoplasty remains elusive and current scaffold configurations are hampered by mechanical and biocompatibility restrictions. In addition, preclinical evaluations of potential scaffold designs for bladder repair are limited by the lack of tractable large animal models of obstructive bladder disease that can mimic clinical pathology. The results of this study describe a novel, minimally invasive, porcine model of partial bladder outlet obstruction that simulates clinically relevant phenotypes. Utilizing this model, we demonstrate that acellular, bi-layer silk fibroin grafts can support the formation of vascularized, innervated bladder tissues with functional properties.

The aim of this study was to determine the effects of dietary grape seed polyphenols (GSP) supplementation during the late gestation and lactation period on reproductive performance, antioxidative status in serum, nutrient composition, and Ig content in colostrum of multiparous sows. On day 80 of gestation, a total of 64 sows with similar body condition were allocated to a completely randomized block design with 4 dietary treatments (n = 16 sows per treatment): 1) basal diet (CON, control group); 2) basal diet supplemented with 200 IU/kg vitamin E (200VE, positive control group); 3) basal diet supplemented with 200 mg/kg GSP (200GSP); and 4) basal diet supplemented with 300 mg/kg GSP (300GSP). The trial lasted 56 d until the piglets were weaned on day 21 of lactation. Reproductive performance, parameters of antioxidative status, and levels of progesterone (P4) and estradiol (E2) in serum, nutrient composition, and Ig content in colostrum of sows were determined. The number of dead fetuses was reduced, and farrowing survival was significantly improved in the litters from 300GSP-fed (P < 0.05). Preweaning survivability significantly increased in the litters from sows fed 200GSP and 200VE (P < 0.05). The activity of superoxide dismutase and glutathione peroxidase (GSH-Px) in the serum was significantly increased in sows fed 200GSP and 300GSP (P < 0.05). The activity of GSH-Px in the serum also significantly increased in sows fed 200VE (P < 0.05). Sows fed 300GSP had the greatest levels of P4 and E2 in the serum, which was significantly greater than sows fed 200VE and CON (P < 0.05). No significant differences were found among treatments for the content of solids-not-fat, fat, protein, and lactose in colostrum (P > 0.05). However, sows fed GSP had greater IgM and IgG content in colostrum compared with sows fed 200VE and CON (P < 0.05). In conclusion, dietary GSP supplementation during late gestation and lactation improved the farrowing survival and preweaning survivability, enhanced the antioxidant status and hormone levels in serum, and increased the IgM and IgG content in colostrum of sows.

A primary goal in the management of burn wounds is early wound closure. The use of skin allografts represents a lifesaving strategy for severe burn patients, but their ultimate rejection limits their potential efficacy and utility. IL-6 is a major pleiotropic cytokine which critically links innate and adaptive immune responses. Here, we devised anti-IL-6 receptor eluting gelatin methacryloyl (GelMA) biomaterials (GelMA/anti-IL-6), which were implanted at the interface between the wound beds and skin allografts. Our visible light crosslinked GelMA/anti-IL-6 immunomodulatory biomaterial (IMB) demonstrated a stable kinetic release profile of anti-IL-6. In addition, the incorporation of anti-IL-6 within the GelMA hydrogel had no effect on the mechanical properties of the hydrogels. Using a highly stringent skin transplant model, the GelMA/anti-IL-6 IMB almost doubled the survival of skin allografts. The use of GelMA/anti-IL-6 IMB was far superior to systemic anti-IL-6 receptor treatment in prolonging skin allograft survival. As compared to the untreated control group, skin from the GelMA/anti-IL-6 IMB group contained significantly fewer alloreactive T cells and macrophages. Interestingly, the environmental milieu of the draining lymph nodes (DLNs) of the mice implanted with the GelMA/anti-IL-6 IMB was also considerably less pro-inflammatory. The percentage of CD4+ IFNγ+ cells was much lower in the DLNs of the GelMA/anti-IL-6 IMB group in comparison to the GelMA group. These data highlight the importance of localized immune delivery in prolonging skin allograft survival and its potential utility in treating patients with severe burns.

Neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Huntington's disease manifest with the neuronal accumulation of toxic proteins. Since autophagy upregulation enhances the clearance of such proteins and ameliorates their toxicities in animal models, we and others have sought to re-position/re-profile existing compounds used in humans to identify those that may induce autophagy in the brain. A key challenge with this approach is to assess if any hits identified can induce neuronal autophagy at concentrations that would be seen in humans taking the drug for its conventional indication. Here we report that felodipine, an L-type calcium channel blocker and anti-hypertensive drug, induces autophagy and clears diverse aggregate-prone, neurodegenerative disease-associated proteins. Felodipine can clear mutant α-synuclein in mouse brains at plasma concentrations similar to those that would be seen in humans taking the drug. This is associated with neuroprotection in mice, suggesting the promise of this compound for use in neurodegeneration.

BackgroundCyanide is a deadly compound used as a terrorist agent. Current FDA approved antidotes require intravenous administration, limiting their utility in a mass casualty scenario. Dimethyl trisulfide (DMTS), a sulfur-based molecule, binds cyanide converting it to the less toxic by-product thiocyanate. Studies evaluating efficacy in rodents have been performed, but a large, clinically relevant animal model has not been reported.ObjectiveThis study evaluates the efficacy of intramuscular DMTS on survival and clinical outcomes in a swine model of acute, severe cyanide toxicity.MethodsAnesthetized swine were instrumented for continuous monitoring of hemodynamics. Prior to potassium cyanide infusion animals were acclimated and breathing spontaneously. At 5-minutes post-apnea animals were treated with DMTS or saline. Vital signs, hemodynamics, and laboratory values were evaluated at various time points.ResultsBaseline values and time to apnea were similar in both groups. Survival in the DMTS treated group was 83.3% and 0% in saline controls (p = .005). The DMTS group returned to breathing at a mean time of 19.3 ± 10 min after antidote, control animals did not return to breathing (CI difference 8.8, 29.8). At the end of the experiment or time of death, mean lactate was 9.41 mmol/L vs. 4.35 mmol/L (CI difference -10.94,0.82) in the saline and DMTS groups, respectively and pH was 7.20 vs. 7.37 (CI difference -0.04, 0.38). No adverse effects were observed at the injection site.ConclusionIntramuscular administration of DMTS improves survival and clinical outcomes in our large animal swine model of acute cyanide toxicity.

Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes >540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

IntroductionHydrogen sulfide (H2S) is found in petroleum, natural gas, and decaying organic matter. Terrorist groups have attempted to use it in enclosed spaces as a chemical weapon. Mass casualty scenarios have occurred from industrial accidents and release from oil field sites. There is no FDA approved antidote for sulfide poisoning. We have previously reported that intravenous cobinamide is effective for sulfide poisoning. A rapid-acting antidote that is easy to administer intramuscularly (IM) would be ideal for use in a prehospital setting. In this study, we assessed survival in sulfide-poisoned swine treated with IM cobinamide.MethodsEleven swine (45-55 kg) were anesthetized, intubated, and instrumented with continuous femoral and pulmonary artery pressure monitoring. After stabilization, anesthesia was adjusted such that animals ventilated spontaneously with a FiO2 of 0.21. Sodium hydrosulfide (NaHS, 8 mg/mL) was infused intravenously at 0.9 mg/kg.min until apnea or severe hypotension. Animals were randomly assigned to receive cobinamide (4 mg/kg), or no treatment at the apnea/hypotension trigger. The NaHS infusion rate was sustained for 1.5 min post trigger, decreased to 0.2 mg/kg.min for 10 min, and then discontinued.ResultsThe amount of NaHS required to produce apnea or hypotension was not statistically different in both groups (cobinamide: 9.0 mg/kg ±6.1; saline: 5.9 mg/kg ±5.5; mean difference: -3.1, 95% CI: -11.3, 5.0). All of the cobinamide treated animals survived (5/5), none of the control (0/6) animals survived (p

Magnetic resonance-guided focused ultrasound (MRgFUS) is a novel non-invasive ablation technique that uses focused sound energy to destroy focal tumors, primarily via heat deposition. It is widely used for palliation of pain from bone metastases and has also recently gained popularity as a technique for ablation of benign bone tumors and facet degenerative joint disease (rhizotomy). Clinically, in a subset of patients who have undergone MRgFUS of bone, a variety of treatment responses have been noted on follow-up imaging, including focal sclerosis within the target lesion or more exuberant proliferative changes associated with the periosteum. In this study, high resolution peripheral quantitative CT (HR-pQCT) was used to evaluate remodeling of bone following ablation in a swine model of MRgFUS and compared to samples from a control, non-treated femur. Within each treated femur, two lesions were created: a higher energy focused ultrasound dose was used for one lesion compared to a lower energy dose for the second lesion. Exuberant, extra-cortical bone formation was detected at the higher energy ablation zones, with volumes ranging from 340 mm3 to 1040 mm3. More subtle endosteal and cortical changes were detected in the lower energy ablation zones, however cortical thickness was significantly increased at these sites compared to control bone. For both high and low energy lesions, lower bone mineral density and tissue mineral density was noted in treated regions compared to control regions, consistent with the formation of newly mineralized tissue. Following HR-pQCT analysis, Fourier transform infrared (FTIR) spectroscopy was subsequently used to detect biochemical changes associated with remodeling of bone following MRgFUS, and compared to samples from the control, non-treated femur. Findings were compared with histopathologic examination following hematoxylin-eosin staining. FTIR analysis demonstrated lower mineral/phosphate ratio and increased crystallinity compared to the control samples (p = 0.013). Histopathologic review demonstrated associated areas of endosteal inflammation, scarring, fat necrosis, and new extra-cortical bone formation associated with the ablations. Overall, these findings provide novel characterization of new bone formation following MRgFUS ablation.

Due to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols.

Animal studies have demonstrated beneficial effects of therapeutic hypothermia on myocardial function, yet exact mechanisms remain unclear. Impaired autophagy leads to heart failure and mitophagy is important for mitigating ischemia/reperfusion injury. This study aims to investigate whether the beneficial effects of therapeutic hypothermia are due to preserved autophagy and mitophagy. Under general anesthesia, the left anterior descending coronary artery of 19 female farm pigs was occluded for 90 minutes with consecutive reperfusion. 30 minutes after reperfusion, we performed pericardial irrigation with warm or cold saline for 60 minutes. Myocardial tissue analysis was performed one and four weeks after infarction. Therapeutic hypothermia induced a significant increase in autophagic flux, mitophagy, mitochondrial mass and function in the myocardium after infarction. Cell stress, apoptosis, inflammation as well as fibrosis were reduced, with significant preservation of systolic and diastolic function four weeks post infarction. We found similar biochemical changes in human samples undergoing open chest surgery under hypothermic conditions when compared to the warm. These results suggest that autophagic flux and mitophagy are important mechanisms implicated in cardiomyocyte recovery after myocardial infarction under hypothermic conditions. New therapeutic strategies targeting these pathways directly could lead to improvements in prevention of heart failure.