Search

The use of lipid nanoparticles (LNPs) for therapeutic RNA delivery has gained significant interest, particularly highlighted by recent milestones such as the approval of Onpattro and two mRNA-based SARS-CoV-2 vaccines. However, despite substantial advancements in this field, our understanding of the structure and internal organization of RNA-LNPs -and their relationship to efficacy, both in vitro and in vivo- remains limited. In this study, we present a coarse-grained molecular dynamics (MD) approach that allows for the simulations of full-size LNPs. By analyzing MD-derived structural characteristics in conjunction with cellular experiments, we investigate the effect of critical parameters, such as pH and composition, on LNP structure and potency. Additionally, we examine the mobility and chemical environment within LNPs at a molecular level. Our findings highlight the significant impact that LNP composition and internal molecular mobility can have on key stages of LNP-based intracellular RNA delivery., Competing Interests: Competing interests statement:All authors are current employees of Bayer AG or Nuvisan ICB GmbH.

The clinical application of heart valve scaffolds is hindered by complications associated with the activation of valvular interstitial cell-like (VIC-like) cells and their transdifferentiation into myofibroblasts. This study aimed to examine several molecular pathway(s) that may trigger the overactive myofibroblast phenotypes in the implanted scaffolds. So, we investigated the influence of three molecular pathways - macrophage-induced inflammation, the TGF-β1-SMAD2, and WNT/β-catenin β on VIC-like cells during tissue engineering of heart valve scaffolds. We implanted electrospun heart valve scaffolds in adult sheep for up to 6 months in the right ventricular outflow tract (RVOT) and analyzed biomolecular (gene and protein) expression associated with the above three pathways by the scaffold infiltrating cells. The results showed a gradual increase in gene and protein expression of markers related to the activation of VIC-like cells and the myofibroblast phenotypes over 6 months of scaffold implantation. Conversely, there was a gradual increase in macrophage activity for the first three months after scaffold implantation. However, a decrease in macrophage activity from three to six months of scaffold tissue engineering suggested that immunological signal factors were not the primary cause of myofibroblast phenotype. Similarly, the gene and protein expression of factors associated with the TGF-β1-SMAD2 pathway in the cells increased in the first three months but declined in the next three months. Contrastingly, the gene and protein expression of factors associated with the WNT/β-catenin pathway increased significantly over the six-month study. Thus, the WNT/β-catenin pathway could be the predominant mechanism in activating VIC-like cells and subsequent myofibroblast phenotype.

Objective: To describe the detailed surgical procedure for open-chest CPR (OC-CPR) through a transdiaphragmatic (TD) approach during planned laparotomy and to evaluate the procedure time and damage to organs., Animals: 7 mixed-breed canine cadavers., Methods: The procedure was divided into 3 stages. Durations for each of the 3 stages of the procedure and total time from diaphragmatic incision to the end of Rumel tourniquet application were recorded. Subjective assessment of ease of procedures and postprocedural physical evaluation of thoracoabdominal organs were also performed., Results: Mean time from diaphragmatic incision to pericardiotomy was 15.1 seconds (SD, 4.0). Performing 10 cardiac compressions took 12.0 seconds (SD, 1.8). Dissection of the aorta and application of a Rumel tourniquet took 130.4 seconds (SD, 52.2). The mean total time from start of first procedure to end of last procedure was 157.6 seconds (SD, 21.5). The mean length of diaphragmatic incision was 11.5 cm (SD, 2.2). Lung laceration was identified in one dog, and liver laceration was identified in another dog. The mean ease of pericardiotomy was 10, and application of a Rumel tourniquet was 4 (SD, 1.9). There was no instance of abdominal organs moving into the thoracic cavity during the procedure in any of the dogs., Clinical Relevance: Resuscitation techniques during TD OC-CPR can be performed with acceptable timing and effort, except for aortic Rumel tourniquet application, which was difficult and time consuming. Avoidable damage to thoracoabdominal organs can occur.

Objective: To compare the acute strength (failure load and work to failure) of standard incisional gastropexy (SIG) and modified incisional gastropexy (MIG)., Animals: 37 pig cadavers., Procedures: Stomachs and right abdominal walls were harvested from pigs euthanized for reasons unrelated to this study. The tissues were stored in lactated Ringer's solution overnight in a 5 °C cooler. Matching body wall and stomach tissue pairs were randomized and divided into 2 groups, on which either SIG or MIG was performed the following day. The MIG technique was identical to SIG except 2 additional simple interrupted sutures, 1 cranial and 1 caudal to the continuous suture line, were placed full thickness into the stomach to ensure engagement of the submucosa. After gastropexy, the samples underwent biomechanical testing. Information regarding change in position and load was generated by the MTESTQuattro software. Mode of failure was examined after the procedure was complete., Results: The MIG had higher failure load and work to failure compared to SIG. All failures were caused by gastric tissue tearing., Clinical Relevance: The MIG is biomechanically superior to SIG and may provide more security than SIG during healing. However, clinical study is needed to ascertain if there is a difference in gastropexy failure and complications between these 2 techniques.

Objective: To compare complications between a modified incisional gastropexy (MIG) technique and standard incisional gastropexy (SIG)., Animals: 347 client-owned dogs., Procedures: Dogs that had undergone SIG or MIG from March 2005 through April 2019 were identified through a medical record search of the University of Missouri Veterinary Health Center. The MIG technique is identical to SIG except 2 additional simple interrupted sutures are added, 1 cranial and 1 caudal to the continuous suture line, going full thickness into the stomach to ensure engagement of submucosa. Medical record information was used to identify intraoperative, postoperative, and short-term complications, and telephone or email communication to pet owners and/or referring veterinarians was used to identify complications (short-term and long-term) after discontinuance of care at the University of Missouri Veterinary Health Center. Intraoperative, postoperative, short-term, and long-term complications were analyzed in aggregate within 6 matched groupings: (1) gastropexy for gastric dilatation-volvulus, (2) prophylactic gastropexy without other procedures, (3) gastropexy with ovariohysterectomy, (4) gastropexy with castration, (5) gastropexy with splenectomy, and (6) gastropexy with celiotomy other than splenectomy. Overall rates of complications potentially attributed to gastropexy were compared between SIG and MIG using the Fisher exact test. Overall rates of complications not attributed to gastropexy were compared between SIG and MIG using the χ2 test., Results: There were no significant differences in overall complication rates between SIG and MIG., Clinical Relevance: Surgeons who feel that engagement of gastric submucosa is important for gastropexy success may use the MIG technique with minimal fear of complications. However, superiority of one technique over the other cannot be determined on the basis of this study.

Single-wall carbon nanotubes (SWCNTs) are used in diverse applications that require chemical tailoring of the SWCNT surface, including optical sensing, imaging, targeted drug delivery and single-photon generation. SWCNTs have been noncovalently modified with (bio)polymers to preserve their intrinsic near-infrared fluorescence. However, demanding applications (e.g., requiring stability in biological fluids) would benefit from a stable covalent linkage between the SWCNT and the functional unit (e.g., antibody, fluorophore, drug). Here we present how to use diazonium salt chemistry to introduce sp 3 quantum defects in the SWCNT carbon lattice to serve as handles for conjugation while preserving near-infrared fluorescence. In this protocol, we describe the straightforward, stable (covalent), highly versatile and scalable functionalization of SWCNTs with biomolecules such as peptides and proteins to yield near-infrared fluorescent SWCNT bioconjugates. We provide a step-by-step procedure covering SWCNT dispersion, quantum defect incorporation, bioconjugation, in situ peptide synthesis on SWCNTs, and characterization, which can be completed in 5-7 d., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The molecular determinants of atypical clinical variants of Alzheimer's disease, including the recently discovered rapidly progressive Alzheimer's disease (rpAD), are unknown to date. Fibrilization of the amyloid-β (Aβ) peptide is the most frequently studied candidate in this context. The Aβ peptide can exist as multiple proteoforms that vary in their post-translational processing, amyloidogenesis, and toxicity. The current study was designed to identify these variations in Alzheimer's disease patients exhibiting classical (sAD) and rapid progression, with the primary aim of establishing if these variants may constitute strains that underlie the phenotypic variability of Alzheimer's disease. We employed two-dimensional polyacrylamide gel electrophoresis and MALDI-ToF mass spectrometry to validate and identify the Aβ proteoforms extracted from targeted brain tissues. The biophysical analysis was conducted using RT-QuIC assay, confocal microscopy, and atomic force microscopy. Interactome analysis was performed by co-immunoprecipitation. We present a signature of 33 distinct pathophysiological proteoforms, including the commonly targeted Aβ 40 , Aβ 42 , Aβ 4-42 , Aβ 11-42 , and provide insight into their synthesis and quantities. Furthermore, we have validated the presence of highly hydrophobic Aβ seeds in rpAD brains that seeded reactions at a slower pace in comparison to typical Alzheimer's disease. In vitro and in vivo analyses also verified variations in the molecular pathways modulated by brain-derived Aβ. These variations in the presence, synthesis, folding, and interactions of Aβ among sAD and rpAD brains constitute important points of intervention. Further validation of reported targets and mechanisms will aid in the diagnosis of and therapy for Alzheimer's disease., (© 2021. The Author(s).)

Objective: To determine whether ease of access to thoracic structures for performing open-chest cardiopulmonary resuscitation (OC-CPR) differed between fourth and fifth intercostal space (ICS) left lateral thoracotomies in dogs, and to determine if "shingling" improved access for OC-CPR manipulations., Design: Prospective single-blinded study., Setting: Laboratory., Animals: Twelve mixed breed canine cadavers weighing approximately 20 kg., Interventions: Left lateral thoracotomies were performed at the 4th ICS (n = 6) or 5th ICS (n = 6). Shingling at the 4th or 5th ICS, as applicable, was performed after initial data collection and outcomes were reassessed., Measurements and Main Results: Three evaluators blinded to the surgical approach scored the following parameters on a 0 to 10 scale (0 = easiest, 10 = most difficult): ease of access of the phrenicopericardial ligament, ease of pericardial incision, ease of appropriate hand position, ease of aortic access, ease of Rumel tourniquet application, and ease of proper placement of defibrillation paddles. Objective measurements (time to completion or number of attempts) were made for all but ease of pericardial incision and ease of appropriate hand position. Outcomes were reassessed after shingling. The 5th ICS was superior for ease of aortic access (P = 0.042), time to visualization of aorta (P = 0.009), and ease of application of a Rumel tourniquet (P = 0.019). When comparing scores pre- and post-shingling, shingling improved time to visualization of the aorta (P < 0.001), time to placement of Rumel tourniquet (P < 0.001), ease of paddle placement (P = 0.017), and time to paddle placement (P < 0.001)., Conclusions: Either 4th or 5th ICS thoracotomy may provide adequate access to intrathoracic structures pertinent to performing OC-CPR in dogs weighing approximately 20 kg, but 5th ICS was preferred for most manipulations, and shingling improved access for most of the measured parameters., (© 2021 The Authors. Journal of Veterinary Emergency and Critical Care published by Wiley Periodicals LLC on behalf of Veterinary Emergency and Critical Care Society.)

Single-walled carbon nanotubes (SWCNTs) are a 1D nanomaterial that shows fluorescence in the near-infrared (NIR, >800 nm). In the past, covalent chemistry was less explored to functionalize SWCNTs as it impairs NIR emission. However, certain sp 3 defects (quantum defects) in the carbon lattice have emerged that preserve NIR fluorescence and even introduce a new, red-shifted emission peak. Here, we report on quantum defects, introduced using light-driven diazonium chemistry, that serve as anchor points for peptides and proteins. We show that maleimide anchors allow conjugation of cysteine-containing proteins such as a GFP-binding nanobody. In addition, an Fmoc-protected phenylalanine defect serves as a starting point for conjugation of visible fluorophores to create multicolor SWCNTs and in situ peptide synthesis directly on the nanotube. Therefore, these quantum defects are a versatile platform to tailor both the nanotube's photophysical properties as well as their surface chemistry., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Objective: To compare the durations of surgery and anesthesia and the likelihoods of short- and long-term postoperative complications between cats positioned in sternal recumbency versus dorsal recumbency for perineal urethrostomy (PU)., Animals: 247 client-owned cats that underwent PU between January 2004 and December 2015 at 6 veterinary teaching hospitals and 1 private veterinary referral hospital., Procedures: Medical records were reviewed, and signalment, presenting complaints, previous history of urethral obstruction or PU, diet fed, medications administered, indication for PU, durations of surgery and anesthesia for PU, suture type and size, suture pattern for skin closure, and short- and long-term postoperative complications were recorded. Univariable and multivariable analyses were performed to identify differences in durations of surgery and anesthesia and the likelihoods of short- and long-term complications between cats positioned in sternal recumbency and those positioned in dorsal recumbency., Results: Patient position was not associated with durations of surgery and anesthesia for PU, even if a concurrent cystotomy was necessary or the patient required repositioning from sternal to dorsal recumbency. Likewise, patient position was not associated with the likelihood of short- and long-term complications., Conclusions and Clinical Relevance: The observed lack of differences in outcomes between sternal and dorsal recumbency suggested that logistic considerations and personal preference can continue to guide veterinarians when positioning cats for PU.

Case Description: A 10-year-old 7-kg (15.4-lb) neutered male Shih Tzu was referred for evaluation because of a sudden onset of dullness and intermittent vomiting of 1 to 2 weeks' duration. Two days prior to evaluation, clinical signs had worsened and 1 seizure was reported., Clinical Findings: A 3.5 × 2.5-cm soft, dome-shaped mass of the right occipital region of the head was noted on physical examination. Radiography and CT confirmed the presence of the mass and indicated its intra- and extracranial expansion. The MRI images showed compression of the cerebellum by the mass, with distinct margins of hypointensity on both T1- and T2-weighted images., Treatment and Outcome: Surgery was performed, and the mass was completely excised. The dog had mild ataxia, hypermetria, and head tremors after surgery. Histologic examination of the mass yielded a diagnosis of intradiploic epidermoid cyst. On examination 3 weeks after surgery, the previous neurologic signs had resolved. On examination 25 months after surgery, the dog remained free of clinical signs., Clinical Relevance: The present report described the clinical signs, diagnostic imaging results, and successful surgical removal of an intradiploic epidermoid cyst in a dog. Long-term prognosis may be good with complete removal of intradiploic epidermoid cysts in dogs.

An abrupt balance impairment, including leaning, falling, and rolling, occurred after IV administration of 0.2 mg/kg midazolam as a preanesthetic medication in two geriatric dogs with a history of nystagmus and head tilt. In the second case, leaning, falling, and rolling recurred after recovery from general anesthesia but gradually ceased after IV administration of 0.01 mg/kg flumazenil. These two cases suggest that the IV administration of midazolam was responsible for the balance impairment in dogs who were suspected to have idiopathic peripheral vestibular disease.

Objective: To evaluate the time and number of laser beam passes required to make full-thickness skin incisions and extent of laser-induced tissue artifacts following use of a CO 2 laser at various settings., Sample: 24 skin specimens from six 5-month-old porcine carcasses., Procedures: 4 full-thickness skin specimens were harvested from the flank regions of each carcass within 30 minutes after euthanasia and randomly assigned to 4 treatment groups. Three 5-cm-long incisions were made in each specimen with a CO 2 laser (beam diameter, 0.4 mm) set to deliver a continuous wave of energy alone (groups 1 and 2) or in superpulse mode (groups 3 and 4) at 10 (groups 1 and 3) or 20 (groups 2 and 4) W of power. The time and number of passes required to achieve a full-thickness incision were recorded, and extent of laser-induced tissue artifact (as determined by histologic evaluation) was compared among the 4 groups., Results: Mean time required to make a full-thickness skin incision for groups 2 and 4 (power, 20 W) was significantly less than that for groups 1 and 3 (power, 10 W). Mean number of passes was lowest for group 2 (continuous wave at 20 W). Extent of laser-induced tissue artifact was greatest for group 4 (superpulse mode at 20 W)., Conclusions and Clinical Relevance: Results provided preliminary information regarding use of CO 2 lasers to make skin incisions in veterinary patients. In vivo studies are necessary to evaluate the effect of various CO 2 laser settings on tissue healing and patient outcome.

Objective: To determine if there is a difference in intra-abdominal pressure (IAP) among 3 anatomic body positions (right lateral, left lateral, and sternal recumbence) in apparently healthy dogs., Design: Prospective, observational study., Setting: University Veterinary Medical Teaching Hospital., Animals: Fourteen apparently healthy male dogs., Measurements and Main Results: After mild sedation with dexmedetomidine, a water manometer attached to a Foley urinary catheter was used to measure IAP in 3 different body positions in each dog. There was no significant difference in IAP between right lateral (3.87± 3.16 cm H 2 O), left lateral (4.45 ± 3.22 cm H 2 O), and sternal recumbence (4.04 ± 3.57 cm H 2 O)., Conclusions: Based on data from these 14 apparently healthy dogs, these 3 body positions can be used interchangeably for monitoring an individual dog. However, more research in dogs is needed to see if this conclusion holds true when IAP is abnormal., (© Veterinary Emergency and Critical Care Society 2020.)

Cells can take up nanoscale materials, which has important implications for understanding cellular functions, biocompatibility as well as biomedical applications. Controlled uptake, transport and triggered release of nanoscale cargo is one of the great challenges in biomedical applications of nanomaterials. Here, we study how human immune cells (neutrophilic granulocytes, neutrophils) take up nanomaterials and program them to release this cargo after a certain time period. For this purpose, we let neutrophils phagocytose DNA-functionalized single-walled carbon nanotubes (SWCNTs) in vitro that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling, subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis, we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation, cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release the SWCNTs. The transported and released SWCNT sensors are still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils transport and release functional nanomaterials.

Imaging of complex (biological) samples in the near-infrared (NIR) is beneficial due to reduced light scattering, absorption, phototoxicity, and autofluorescence. However, there are few NIR fluorescent materials known and suitable for biomedical applications. Here we exfoliate the layered pigment CaCuSi 4 O 10 (Egyptian Blue, EB) via ball milling and facile tip sonication into NIR fluorescent nanosheets (EB-NS). The size of EB-NS can be tailored to diameters <20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the fluorescence intensity correlates with the number of Cu 2+ ions. Furthermore, EB-NS display no bleaching and high brightness compared with other NIR fluorophores. The versatility of EB-NS is demonstrated by in-vivo single-particle tracking and microrheology measurements in Drosophila melanogaster embryos. EB-NS can be uptaken by plants and remotely detected in a low-cost stand-off detection setup. In summary, EB-NS have the potential for a wide range of bioimaging applications.

A 3yr old spayed female Brittany was referred with a cylindrical mass on the right side of her face. Three months earlier, a small stone embedded in the right parotid duct was noted and removed through a ductal incision by the referring veterinarian. At referral, the dog's general physical condition was normal except for a cylindrical mass on the right cheek. Skull radiographs showed a possibly retained sialolith or dystrophic mineralization within the previous surgical site. Aspiration of the mass yielded a thick, yellow/tan, mucopurulent fluid. Cytology of the fluid demonstrated degenerative neutrophils without bacteria. The cylindrical mass was excised with a carbon dioxide laser. The caudal end of the mass was connected to the parotid salivary gland and the rostral one-third of the mass tapered to a point. The caudal end of the mass was ligated with 3-0 polydioxanone and excised immediately rostral to the ligation. Histopathology revealed that the mass was parotid duct ectasia. The dog completely recovered with some mild temporary facial paresis and had no recurrence of parotid duct ectasia at 4 mo follow-up. The case report describes a dog with an unusual parotid salivary duct ectasia caused by parotid duct sialolith removal.

Serotonin is an important neurotransmitter involved in various functions of the nervous, blood, and immune system. In general, detection of small biomolecules such as serotonin in real time with high spatial and temporal resolution remains challenging with conventional sensors and methods. In this work, we designed a near-infrared (nIR) fluorescent nanosensor (NIRSer) based on fluorescent single-walled carbon nanotubes (SWCNTs) to image the release of serotonin from human blood platelets in real time. The nanosensor consists of a nonbleaching SWCNT backbone, which is fluorescent in the beneficial nIR tissue transparency window (800-1700 nm) and a serotonin binding DNA aptamer. The fluorescence of the NIRSer sensor (995 nm emission wavelength for (6,5)-SWCNTs) increases in response to serotonin by a factor up to 1.8. It detects serotonin reversibly with a dissociation constant of 301 nM ± 138 nM and a dynamic linear range in the physiologically relevant region from 100 nM to 1 μM. As a proof of principle, we detected serotonin release patterns from activated platelets on the single-cell level. Imaging of the nanosensors around and under the platelets enabled us to locate hot spots of serotonin release and quantify the time delay (≈ 21-30 s) between stimulation and release in a population of platelets, highlighting the spatiotemporal resolution of this nanosensor approach. In summary, we report a nIR fluorescent nanosensor for the neurotransmitter serotonin and show its potential for imaging of chemical communication between cells.

Fluorescent nanomaterials such as single-walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP-binding nanobodies to DNA-wrapped SWCNTs. This approach combines the targeting capabilities of GFP-binding nanobodies and the nonbleaching near-infrared fluorescence (850-1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin-5-GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Single-walled carbon nanotubes (SWCNTs) have unique photophysical properties and serve as building blocks for biosensors, functional materials and devices. For many applications it is crucial to use chirality-pure SWCNTs, which requires sophisticated processes. Purification procedures such as wrapping by certain polymers, phase separation, density gradient centrifugation or gel chromatography have been developed and yield distinct SWCNT species wrapped by a specific polymer or surfactant. However, many applications require a different organic functionalization (corona) around the SWCNTs instead of the one used for the purification process. Here, we present a novel efficient and straightforward process to gain chirality pure SWCNTs with tunable functionalization. Our approach uses polyfluorene (PFO) polymers to enrich certain chiralities but the polymer is removed again and finally exchanged to any desired organic phase. We demonstrate this concept by dispersing SWCNTs in poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(6,6'-{2,2'-bipyridine})] (PFO-BPy), which is known to preferentially solubilize (6,5)-SWCNTs. Then PFO-BPy is removed and recycled, while letting the SWCNTs adsorb/agglomerate on sodium chloride (NaCl) crystals, which act as a toluene-stable but water-soluble filler material. In the last step these purified SWCNTs are redispersed in different polymers, surfactants and ssDNA. This corona phase exchange purification (CPEP) approach was also extended to other PFO variants to enrich and functionalize (7,5)-SWCNTs. CPEP purified and functionalized SWCNTs display monodisperse nIR spectra, which are important for fundamental studies and applications that rely on spectral changes. We show this advantage for SWCNT-based nIR fluorescent sensors for the neurotransmitter dopamine and red-shifted sp 3 defect peaks . In summary, CPEP makes use of PFO polymers for chirality enrichment but provides access to chirality enriched SWCNTs functionalized in any desired polymer, surfactant or biopolymer.

Background: Megaesophagus (ME) carries a poor long-term prognosis in dogs. In people, lower esophageal sphincter (LES) achalasia is a rare cause of ME that may respond to targeted intervention. Dogs with lower esophageal sphincter achalasia-like syndrome (LES-AS) have been described recently, warranting investigation of analogous targeted treatment., Hypothesis/objectives: Evaluate response of dogs with LES-AS to LES mechanical dilation and botulinum toxin A (BTA) injections, with or without surgical myotomy and fundoplication. We hypothesized that clinical and videofluoroscopic swallow study (VFSS) features of LES-AS would improve after treatment targeting functional LES obstruction., Animals: Fourteen client-owned dogs with LES-AS diagnosed by VFSS., Methods: Retrospective study. Dogs diagnosed with LES-AS underwent treatment between April 2015 and December 2017. Outcome measures included client perception of clinical severity, body weight (BW), body condition score (BCS), regurgitation frequency, and VFSS parameters (ME, esophageal motility, gastric filling). Dogs with positive responses were considered candidates for LES myotomy with fundoplication., Results: By a median IQR of 21 (IQR, 14-25) days after mechanical dilation and BTA, clients reported clinical improvement in 100% of dogs, BW increased 20.4% (IQR, 12.7%-25%), pre- and post-treatment BCS was 3 (IQR, 3-4) and 5 (IQR, 4-5), respectively, and frequency of regurgitation decreased by 80% (IQR, 50%-85%). Duration of effect was 40 (IQR, 17-53) days. Despite clinical improvement, ME and abnormal esophageal motility persisted in 14 dogs. Six dogs subsequently underwent myotomy and fundoplication and maintained improvement observed after mechanical dilation and BTA., Conclusions and Clinical Importance: Dogs with LES-AS experienced significant, temporary, clinical improvement after mechanical dilation and BTA. Preliminary results suggest myotomy with fundoplication provide lasting clinical benefit despite persistence of ME., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Specific functionalization of 1D nanomaterials such as near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) is essential for colloidal stability and tailoring of their interactions with the environment. Here, we show that de novo designed alpha-helical coiled-coil peptide barrels (αHBs) with appropriate pores encapsulate and solubilize SWCNTs. In contrast, barrels without or with narrow pores showed a much smaller efficiency. Absorption/fluorescence spectroscopy and atomic force microscopy indicate that the SWCNTs are incorporated into the αHB's pore. The resulting hybrid SWCNT@αHBs display periodic surface coverage with a 40 nm pitch and remain fluorescent in the nIR. This approach presents a novel concept to encapsulate, discriminate and functionalize SWCNTs non-covalently with peptides and holds great promise for future applications in bioimaging or drug delivery., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

OBJECTIVE To compare the leak pressure and amount of time required to perform closure for 2 double-layer esophagotomy closure techniques. SAMPLE 28 intrathoracic esophageal segments harvested from 38 porcine cadavers. PROCEDURES Longitudinal 3-cm esophagotomy incisions made in porcine cadaveric esophagi were closed with 2 double-layer closure techniques. Fifteen incisions were closed with a simple interrupted pattern, and 13 incisions were closed with a simple continuous pattern. Leak pressure, bursting wall tension, and closure time were compared between suture patterns by use of a t test or Mann-Whitney rank sum test. RESULTS Median leak pressures differed significantly between segments closed with the simple interrupted pattern (16.0 mm Hg; range, 5.4 to 54.9 mm Hg) and the simple continuous pattern (38.7 mm Hg; range, 11.3 to 81.9 mm Hg). Median bursting wall tension differed significantly between the simple interrupted pattern (0.63 × 10 5 dynes/cm; range, 0.16 × 10 5 dynes/cm to 2.89 × 10 5 dynes/cm) and the simple continuous pattern (1.79 × 10 5 dynes/cm; range, 0.44 × 10 5 dynes/cm to 4.70 × 10 5 dynes/cm). Mean ± SD closure time differed significantly between the simple interrupted pattern (19.2 ± 2.0 minutes) and the simple continuous pattern (14.7 ± 1.5 minutes). CONCLUSIONS AND CLINICAL RELEVANCE In the study reported here, double-layer simple continuous closure resulted in a higher median postoperative leak pressure and higher median postoperative bursting wall tension and could be performed more rapidly than the double-layer simple interrupted closure on these porcine cadaveric specimens.

OBJECTIVE To determine whether stored (cooled or frozen-thawed) jejunal segments can be used to obtain dependable leak pressure data after enterotomy closure. SAMPLE 36 jejunal segments from 3 juvenile pigs. PROCEDURES Jejunal segments were harvested from euthanized pigs and assigned to 1 of 3 treatment groups (n = 12 segments/group) as follows: fresh (used within 4 hours after collection), cooled (stored overnight at 5°C before use), and frozen-thawed (frozen at -12°C for 8 days and thawed at room temperature [23°C] for 1 hour before use). Jejunal segments were suspended and 2-cm enterotomy incisions were made on the antimesenteric border. Enterotomies were closed with a simple continuous suture pattern. Lactated Ringer solution was infused into each segment until failure at the suture line was detected. Leak pressure was measured by use of a digital transducer. RESULTS Mean ± SD leak pressure for fresh, cooled, and frozen-thawed segments was 68.3 ± 23.7 mm Hg, 55.3 ± 28.1 mm Hg, and 14.4 ± 14.8 mm Hg, respectively. Overall, there were no significant differences in mean leak pressure among pigs, but a significant difference in mean leak pressure was detected among treatment groups. Mean leak pressure was significantly lower for frozen-thawed segments than for fresh or cooled segments, but mean leak pressure did not differ significantly between fresh and cooled segments. CONCLUSIONS AND CLINICAL RELEVANCE Fresh porcine jejunal segments or segments cooled overnight may be used for determining intestinal leak pressure, but frozen-thawed segments should not be used.

Objective: To evaluate the effects of intravenous (IV) infusion of fish oil (FO) emulsion following ovariohysterectomy (OVH) on inflammatory mediators and plasma omega-3 nonesterified fatty acids (NEFA) concentrations in dogs., Design: Prospective clinical study., Setting: University teaching hospital., Animals: Twenty-nine privately owned dogs undergoing routine OVH., Interventions: Postoperative 3-hour IV infusion of saline (n = 9), FO (Omegaven, n = 10), or soybean oil (SO, intralipid, n = 10) emulsion and blood collected before, 5 and 24 hours following OVH for plasma NEFA and RBC membrane fatty acids (FAs) concentrations, leukocyte cytokine production capacity, and C-reactive protein (CRP) measurement., Measurements and Main Results: Plasma omega-3 NEFA, eicosapentaenoic acid, docosahexaenoic acid, and total long-chain omega-3 FA significantly increased shortly after FO infusion (8.8 ± 3.3 μM, 13.6 ± 5.6 μM, and 25.1 ± 9.6 μM, respectively) compared to SO (0.7 ± 0.9, 2.3 ± 1.8, and 4.2 ± 3.0 μM, respectively) and saline infusion (1.6 ± 2.5, 2.6 ± 3.1, and 5.9 ± 6.4 μM, respectively). Plasma CRP concentration significantly increased after OVH, but with no significant group differences. A weak negative correlation occurred between post-OVH CRP and postinfusion total long-chain omega-3 FA concentrations (r 2 = 0.21, P = 0.014). Stimulated leukocyte interleukin (IL) 6 production capacity increased (P = 0.001) after OVH in all groups; SO infusion resulted in reduced leukocyte IL-6 production capacity (1048.1 ± 277.7 pg/mL) compared to FO (1299.9 ± 302.1 pg/mL, P = 0.048) and saline infusions (1499.0 ± 363.1 pg/mL, P = 0.01). No significant group difference was observed in leukocyte IL-10 and tumor necrosis factor α production capacities., Conclusions: Postoperative administration of FO emulsion increases plasma omega-3 NEFA concentrations promptly, but does not significantly attenuate CRP production or leukocyte cytokine production capacity. FO infusion at the dosage used in the present study can be safely used in dogs, but it was not clearly beneficial in decreasing post-OVH indices of inflammation., (© Veterinary Emergency and Critical Care Society 2017.)

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Detection of neurotransmitters is an analytical challenge and essential to understand neuronal networks in the brain and associated diseases. However, most methods do not provide sufficient spatial, temporal, or chemical resolution. Near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have been used as building blocks for sensors/probes that detect catecholamine neurotransmitters, including dopamine. This approach provides a high spatial and temporal resolution, but it is not understood if these sensors are able to distinguish dopamine from similar catecholamine neurotransmitters, such as epinephrine or norepinephrine. In this work, the organic phase (DNA sequence) around SWCNTs was varied to create sensors with different selectivity and sensitivity for catecholamine neurotransmitters. Most DNA-functionalized SWCNTs responded to catecholamine neurotransmitters, but both dissociation constants ( K d ) and limits of detection were highly dependent on functionalization (sequence). K d values span a range of 2.3 nM (SWCNT-(GC) 15 + norepinephrine) to 9.4 μM (SWCNT-(AT) 15 + dopamine) and limits of detection are mostly in the single-digit nM regime. Additionally, sensors of different SWCNT chirality show different fluorescence increases. Moreover, certain sensors (e.g., SWCNT-(GT) 10 ) distinguish between different catecholamines, such as dopamine and norepinephrine at low concentrations (50 nM). These results show that SWCNTs functionalized with certain DNA sequences are able to discriminate between catecholamine neurotransmitters or to detect them in the presence of interfering substances of similar structure. Such sensors will be useful to measure and study neurotransmitter signaling in complex biological settings., Competing Interests: The authors declare no conflict of interest.

Objective: To describe the use of postoperative intrajejunal feeding and to evaluate the association of preoperative plasma albumin concentrations with intrajejunal feeding-related complications and clinical outcome., Design: Prospective, observational study., Setting: University veterinary teaching hospital., Animals: Sixty-four dogs., Interventions: Jejunostomy tube placement during abdominal surgery., Measurements and Main Results: Most dogs (81%) survived. The median intrajejunal feeding period was 2.1 days (range: 1-16 days; n = 64). Only 3 (5%) dogs received their estimated resting energy requirement by intrajejunal feeding. Of dogs that were fed intrajejunally (58 out of 64), most (55 out of 58) received intrajejunal feeding within 24 hours after surgery. Energy provision via the jejunal feeding tube did not differ between dogs with and without complications (P = 0.592), or between nonsurvivors and survivors (P = 0.298). Thirty-five dogs ate voluntarily concurrently with intrajejunal feeding. Of dogs that ate voluntarily concurrently with intrajejunal feeding for ≤50% of the postoperative period, most (74%) survived to discharge. Complications were seen in 22% of dogs, and none were life-threatening; gastrointestinal signs were most common. There was no difference in preoperative plasma albumin concentration between dogs with and without complications (P = 0.432) and between nonsurvivors and survivors (P = 0.727). Fecal score was not significantly different between the 2 liquid diets studied (FormulaV Enteral Care HLP and CliniCare Canine/Feline; P = 0.927)., Conclusions: A jejunostomy tube placed during abdominal surgery was likely to be used at the study institution. Few complications were seen and none were life-threatening. Intrajejunal feeding was initiated early after surgery and did not interfere with the initiation of voluntary oral intake. Fecal scores were high and were useful for an objective assessment of fecal consistency in dogs with intrajejunal feeding., (© Veterinary Emergency and Critical Care Society 2016.)

This study was conducted to investigate the clinical data of two cases of canine sialolithiasis and to analyze 29 cases identified in the Veterinary Medical Database by year of admission, breed, gender, and age. Medical records from the University of Missouri Veterinary Medical Teaching Hospital were reviewed and two dogs diagnosed with sialolithiasis (calcium oxalate) were identified between 1990 and 2010. The two dogs had cervical or pharyngeal sialocele and were successfully treated by sialolith removal and concurrent sialoadenectomy. Signalments of dogs with sialolithiasis between 1964 and 2010 were collected from the Veterinary Medical Database and evaluated. Several breeds of dogs were represented and the 10 to <15 yr old age group was shown to have significant association with sialolithiasis. Sialolithiasis is a rare veterinary condition. In this study, older dogs were at higher risk. In dogs, concurrent sialocele was common and good outcome could be expected after surgical removal of sialoliths with concurrent sialoadenectomy.

A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To compare perioperative RBC transfusion among dogs undergoing liver lobectomy, splenectomy, partial gastrectomy, rhinotomy, thyroidectomy, perineal herniorrhaphy, and intrathoracic surgery., Design: Retrospective case series., Animals: 207 client-owned dogs that underwent various surgeries., Procedures: Medical records were reviewed for dogs that had undergone liver lobectomy, splenectomy, partial gastrectomy, rhinotomy, neoplastic thyroidectomy, perineal herniorrhaphy, or intrathoracic surgery. Transfusion requirement (packed RBC, whole blood, and bovine hemoglobin-based oxygen carrier) and survival rate at 2 weeks after surgery were compared among dogs undergoing the various surgeries., Results: Patients undergoing splenectomy and liver lobectomy were significantly more likely to receive RBC transfusion when each was compared with patients undergoing all other procedures. A significant association was found between body weight and perioperative RBC transfusion, with greater odds of transfusion as body weight increased. Dogs receiving perioperative RBC transfusions were significantly less likely to survive to 2 weeks after surgery., Conclusions and Clinical Relevance: Results indicated that dogs undergoing splenectomy and liver lobectomy may require RBC transfusion perioperatively. Veterinarians who perform these procedures should plan accordingly and have packed RBCs or whole blood donors readily available.

Objective: To establish a reference interval for plasma lactate in a population of healthy adult cats on a laboratory analyzer (Nova Biomedical Critical Care Xpress [CCX]) and 2 commercially available point-of-care (POC) analyzers (Abbott i-STAT [i-STAT] and Nova Biomedical Lactate Plus [LP]), and to compare the level of agreement of lactate measurement between the laboratory analyzer and POC analyzers., Design: Prospective observational study., Setting: University veterinary teaching hospital., Animals: Forty-seven healthy adult cats., Interventions: Jugular phlebotomy., Measurements and Main Results: In this population, plasma lactate reference interval was 0.67-5.44 mmol/L for the CCX, 0.65-5.16 mmol/L for the i-STAT, and 0.68-4.39 mmol/L for the LP. Comparisons were made between lactate measurements on 2 point-of-care analyzers and the laboratory analyzer using the Bland-Altman method. For the comparison of CCX and i-STAT, the bias was -0.10 mmol/L; for the CCX and LP, the bias was -0.24 mmol/L., Conclusions: Measurements of plasma lactate in cats using the i-STAT showed acceptable agreement with the CCX. The LP showed weaker agreement. However, both POC analyzers are suitable for measurement of lactate in cats, provided results from different POC analyzers are not directly compared. This study identified a larger reference interval for plasma lactate concentration in cats than what has been previously reported., (© Veterinary Emergency and Critical Care Society 2015.)

Opioids have immunomodulatory properties in many species, but there is little information pertaining to these properties in dogs. Our objective was to compare the in vivo effects of morphine, buprenorphine, and control solution on innate immune system function and apoptosis in healthy dogs. Six adult dogs received a 24-hour infusion of morphine, buprenorphine, or control solution (saline) in a randomized, controlled, crossover block design. Leukocyte apoptosis, phagocytosis, and oxidative burst were evaluated using flow cytometry. Lipopolysaccharide, lipoteichoic acid, and peptidoglycan-stimulated leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined using canine specific multiplex assays. No significant treatment effects were detected among groups. These data suggest that healthy dogs could be less sensitive to the immunomodulatory effects of acute opioid administration compared with other species. Larger investigations in healthy and immunologically challenged dogs are recommended prior to application of these results in clinical patients., (Copyright © 2015. Published by Elsevier Ltd.)

This is the 11th installment of a series that will highlight one case per publication issue from the bank of cases available online as part of the American Society of Emergency Radiology (ASER) educational resources. Our goal is to generate more interest in the use of our online materials. To view more cases online, please visit the ASER Core Curriculum and Recommendations for Study online at http://www.aseronline.org/curriculum/toc.htm .

Objective: To evaluate the effects of topical zinc gluconate on healing, analgesia, and bacterial growth of full-thickness rat skin wounds., Study Design: Experimental study., Animals: Male Wistar rats (n = 98) METHODS: One full-thickness round 2 cm × 2 cm surgically created wound on the dorsum of each rat received 1 of the following randomized treatments: zinc gluconate in carrier solution, carrier solution, saline (0.9% NaCl) solution, chlorhexidine, chlorhexidine + zinc gluconate, corticosteroid, and no treatment. At 4 and 21 days, biopsy samples were taken for histopathology. Bacterial cultures were performed on day 4. Subjective pain scores and weight were recorded daily., Results: No significant differences were noted among the groups except the corticosteroid group, in which delayed healing was noted at 4 and 21 days. Bacterial isolates were similar and no differences in pain scores or weight gain were noted among groups. The wounds treated with corticosteroid at day 4 had decreased inflammatory cells and serocellular crust formation, and minimal neovascularization and granulation tissue formation. At day 21, that group had prolonged neutrophilic inflammatory cells, immature fibrosis, less re-epithelialization, and more prominent serocellular crust., Conclusion: Wounds treated with zinc gluconate healed similarly and contained similar bacterial load as wounds with other topical treatments except for corticosteroids. No treatment demonstrated the negative effects seen with topical corticosteroid nor did any demonstrate analgesic advantage., (© Copyright 2014 by The American College of Veterinary Surgeons.)

This is the ninth installment of a series that will highlight one case per publication issue from the bank of cases available online as part of the American Society of Emergency Radiology (ASER) educational resources. Our goal is to generate more interest in and use of our online materials. To view more cases online, please visit the ASER Core Curriculum and Recommendations for Study online at http://www.aseronline.org/curriculum/toc.htm .

Objective: To establish reference intervals for whole blood and plasma colloid osmotic pressure (COP) in healthy cats between the ages of 1 and 10 years using a cage-side colloid osmometer., Design: Prospective, observational study., Settings: University veterinary teaching hospital., Animals: Sixty-three healthy cats., Interventions: Phlebotomy., Measurements and Main Results: Whole blood COP mean was 24.4 (±2.78) mmHg and plasma COP mean was 24.3 (±2.59) mmHg. Reference interval for our study population of feline whole blood COP was 18.9 to 30.4 mmHg, and for our study population of feline plasma COP was 18.3 to 30.8 mmHg. Difference of paired whole blood COP and plasma COP was +0.23 ± 1.68 mmHg (P = 0.32). There was no significant difference when comparing COP from neutered male and neutered female cats. Total protein and albumin were significantly correlated with whole blood COP (total protein to whole blood COP P < 0.0001, r = 0.53; albumin to whole blood COP P <0.0001, r = 0.68) and plasma COP (total protein to plasma COP P = 0.0025, r = 0.41; albumin to plasma COP P < 0.0001, r = 0.66)., Conclusions: No significant difference was found between mean whole blood and plasma COP in this study population of cats. Even though not statistically significant, evaluation of paired whole blood COP and plasma COP did reveal a slight difference; therefore, it seems prudent to maintain sample consistency for serial evaluations in cats., (© Veterinary Emergency and Critical Care Society 2014.)

Multiple organ dysfunction syndrome (MODS), defined as the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention, is a cause of high morbidity and mortality in humans and animals. Many advances have been made in understanding the pathophysiology and treatment of this syndrome in human medicine, but much still is unknown. This comparative review will provide information regarding the history and pathophysiology of MODS in humans and discuss how MODS affects each major organ system in animals., (Copyright © 2014 by the American College of Veterinary Internal Medicine.)