Effects of vatinoxan on xylazine-induced pulmonary alterations in sheep.

It was hypothesized that premedication with vatinoxan, a peripheral α2 -adrenoceptor antagonist, would mitigate xylazine-induced pulmonary alterations in sheep. Fourteen adult sheep were allotted into two equal groups and premedicated with either vatinoxan (750 µg/kg IV) or saline and sedated 10 min later with xylazine (500 µg/kg IV). Arterial oxygen saturation (SpO2 ) was measured and respiratory rate (RR) counted at intervals. The sheep were euthanized with IV pentobarbital 10 min after xylazine administration. The severity of pulmonary parenchymal alterations was assessed and graded grossly and histologically and correlations of the morphological changes with SpO2 evaluated. Following xylazine injection, SpO2 was significantly higher and RR significantly lower with vatinoxan than with saline and the sheep administered vatinoxan exhibited significantly smaller quantities of tracheal foam than those receiving saline. No significant differences in macroscopic oedema scores were detected between treatments. In contrast, the vatinoxan-treated animals exhibited significantly graver microscopic interstitial alveolar oedema and haemorrhage than saline-treated animals. The histological severity scores did not correlate with changes in SpO2 . In conclusion, xylazine induced a marked reduction in SpO2 which was abolished by the prior administration of vatinoxan. The histologically detected alterations after pentobarbital euthanasia with vatinoxan premedication need to be studied further.

Concentrations of vatinoxan and xylazine in plasma, cerebrospinal fluid and brain tissue following intravenous administration in sheep.

OBJECTIVES: To investigate the extent of vatinoxan distribution into sheep brain, and whether vatinoxan influences brain concentrations of xylazine; and to examine the utility of cerebrospinal fluid (CSF) as a surrogate of brain tissue concentrations for vatinoxan and xylazine. STUDY DESIGN: Randomised, blinded, experimental study. ANIMALS: A total of 14 adult female sheep. METHODS: Sheep were randomly allocated into two equal groups and premedicated with either intravenous (IV) vatinoxan (750 µg kg-1, VX) or saline (SX) administered 10 minutes before IV xylazine (500 µg kg-1). Sedation was subjectively assessed at selected intervals before and after treatments. At 10 minutes after xylazine administration, a venous blood sample was collected and the sheep were immediately euthanised with IV pentobarbital (100 mg kg-1). Plasma, CSF and brain tissues were harvested, and concentrations of vatinoxan and xylazine were quantified using liquid chromatography-tandem mass spectrometry. Drug ratios were then calculated and the data were analysed as appropriate. RESULTS: The brain-to-plasma and CSF-to-plasma ratios of vatinoxan were 0.06 ± 0.013 and 0.05 ± 0.01 (mean ± standard deviation), respectively. Xylazine brain concentrations were not significantly different (835 ± 262 versus 1029 ± 297 ng g-1 in groups VX and SX, respectively) and were approximately 15-fold higher than those in plasma. The CSF-to-brain ratio of vatinoxan was 0.8 ± 0.2, whereas xylazine concentrations in the brain were approximately 17-fold greater than those in CSF, with and without vatinoxan. CONCLUSIONS AND CLINICAL RELEVANCE: Vatinoxan did not significantly affect sedation with xylazine or the concentrations of xylazine in the brain. CSF is not a good predictor of xylazine concentrations in the brain, whereas vatinoxan concentrations were concordant between the brain and CSF, using the dosages in this study.

Effects of vatinoxan in rats sedated with a combination of medetomidine, midazolam and fentanyl.

BACKGROUND: Alpha2-adrenoceptor agonists (α2-agonists) are widely used in animals as sedatives and for pre-anaesthetic medication. Medetomidine has often been given subcutaneously (SC) to rats, although its absorption rate is slow and the individual variation in serum drug concentrations is high via this route. In addition, α2-agonists have various effects on metabolic and endocrine functions such as hypoinsulinaemia, hyperglycaemia and diuresis. Vatinoxan is a peripherally acting α2-adrenoceptor antagonist that, as a hydrophilic molecule, does not cross the blood-brain barrier in significant quantities and thus alleviates peripheral cardiovascular effects and adverse metabolic effects of α2-agonists. Aim of this study was to evaluate the effects of vatinoxan on sedation, blood glucose concentration, voiding and heart and respiratory rates and arterial oxygen saturation in rats sedated with subcutaneous medetomidine, midazolam and fentanyl. RESULTS: Onset of sedation and loss of righting reflex occurred significantly faster with vatinoxan [5.35 ± 1.08 (mean ± SD) versus 12.97 ± 6.18 min and 6.53 ± 2.18 versus 14.47 ± 7.28 min, respectively]. No significant differences were detected in heart and respiratory rates and arterial oxygen saturation between treatments. Blood glucose concentration (18.3 ± 3.6 versus 11.8 ± 1.2 mmol/L) and spontaneous urinary voiding [35.9 (15.1-41.6), range (median) versus 0.9 (0-8.0) mL /kg/min] were significantly higher without vatinoxan. CONCLUSIONS: Acceleration of induction of sedation, alleviation of hyperglycaemia and prevention of profuse diuresis by vatinoxan may be beneficial when sedating rats for clinical and experimental purposes with subcutaneous medetomidine, midazolam and fentanyl.

CRISPR/Cas9-mediated knockout of the ubiquitin variant UbKEKS reveals a role in regulating nucleolar structures and composition.

Ubiquitination is a post-translational modification responsible for one of the most complex multilayered communication and regulation systems in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Recently discovered ubiquitin variant UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. Depletion of UbKEKS induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. Using data-independent acquisition mass spectrometry and microscopy, we show that despite not impacting protein stability, UbKEKS is required to maintain a normal nucleolar organization. The absence of UbKEKS increases nucleoli's size and accentuate their circularity while disrupting dense fibrillar component and fibrillar centre structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Lack of UbKEKS favours nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis observed by flow cytometry and real-time monitoring. Overall, these results identify the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the ubiquitination system.

Decoupling of mRNA and Protein Expression in Aging Brains Reveals the Age-Dependent Adaptation of Specific Gene Subsets.

During aging, changes in gene expression are associated with a decline in physical and cognitive abilities. Here, we investigate the connection between changes in mRNA and protein expression in the brain by comparing the transcriptome and proteome of the mouse cortex during aging. Our transcriptomic analysis revealed that aging mainly triggers gene activation in the cortex. We showed that an increase in mRNA expression correlates with protein expression, specifically in the anterior cingulate cortex, where we also observed an increase in cortical thickness during aging. Genes exhibiting an aging-dependent increase of mRNA and protein levels are involved in sensory perception and immune functions. Our proteomic analysis also identified changes in protein abundance in the aging cortex and highlighted a subset of proteins that were differentially enriched but exhibited stable mRNA levels during aging, implying the contribution of aging-related post- transcriptional and post-translational mechanisms. These specific genes were associated with general biological processes such as translation, ribosome assembly and protein degradation, and also important brain functions related to neuroplasticity. By decoupling mRNA and protein expression, we have thus characterized distinct subsets of genes that differentially adjust to cellular aging in the cerebral cortex.

Mining Proteomics Datasets to Uncover Functional Pseudogenes.

Analysis of the proteome, combined with the human genome sequence, improved gene annotation and confirmed that some genes are actually expressed as proteins, including pseudogenes, alternative reading frames, and additional protein isoforms. Although sequencing a genome is a challenge in itself, the annotation of all genes encoding proteins is a bigger one. Here, we describe an in silico approach to identify evidence of pseudogene expression, as well as an experimental approach for the validation of the protein encoded by pseudogenes, including the steps necessary to quantify these proteins. This technique enables a comprehensive analysis for the expression of genes into proteins that were not suspected of existing.

Retinal Temperature Determination Based on Photopic Porcine Electroretinogram.

OBJECTIVE: Subthreshold retinal laser therapy (SLT) is a treatment modality where the temperature of the retinal pigment epithelium (RPE) is briefly elevated to trigger the therapeutic benefits of sublethal heat shock. However, the temperature elevation induced by a laser exposure varies between patients due to individual differences in RPE pigmentation and choroidal perfusion. This study describes an electroretinography (ERG)-based method for controlling the temperature elevation during SLT. METHODS: The temperature dependence of the photopic ERG response kinetics were investigated both ex vivo with isolated pig retinas and in vivo with anesthetized pigs by altering the temperature of the subject and recording ERG in different temperatures. A model was created for ERG-based temperature estimation and the feasibility of the model for controlling SLT was assessed through computational simulations. RESULTS: The kinetics of the photopic in vivo flash ERG signaling accelerated between 3.6 and 4.7%/°C, depending on the strength of the stimulus. The temperature dependence was 5.0%/°C in the entire investigated range of 33 to 44°C in ex vivo ERG. The simulations showed that the method is suitable for determining the steady-state temperature elevation in SLT treatments with a sufficiently long laser exposure and large spot size, e.g., during > 30 s laser exposures with > 3 mm stimulus spot diameter. CONCLUSIONS: The described ERG-based temperature estimation model could be used to control SLT treatments such as transpupillary thermotherapy. SIGNIFICANCE: The introduced ERG-based method for controlling SLT could improve the repeatability, safety, and efficacy of the treatment of various retinal disorders.

Comparison Between Arthroscopic and Histological International Cartilage Repair Society Scoring Systems in Porcine Cartilage Repair Model.

OBJECTIVE: The arthroscopic and histological International Cartilage Repair Society (ICRS) scores are designed to evaluate cartilage repair quality. Arthroscopic ICRS score can give a maximum score of 12 and the histological score can give values between 0% and 100% for each of its 14 subscores. This study compares these methods in an animal cartilage repair model. This study hypothesizes that there is a significant correlation between these methods. DESIGN: A chondral defect was made in the medial femoral condyle of 18 pigs. Five weeks later, 9 pigs were treated with a novel recombinant human type III collagen/polylactide scaffold and 9 were left untreated to heal spontaneously. After 4 months, the medial condyles were evaluated with a simulated arthroscopy using the ICRS scoring system followed by a histological ICRS scoring. RESULTS: This porcine cartilage repair model produced repaired cartilage tissue ranging from good to poor repair tissue quality. The mean arthroscopic ICRS total score was 6.8 (SD = 2.2). Histological ICRS overall assessment subscore was 38.2 (SD = 31.1) and histological ICRS average points were 60.5 (SD = 19.5). Arthroscopic ICRS compared with histological ICRS average points or its overall assessment subscore showed moderate correlation (r = 0.49 and r = 0.50, respectively). The interrater reliability with the intraclass correlation coefficients for arthroscopic ICRS total scores, histological ICRS overall assessment subscore, and ICRS average points showed moderate to excellent reliability. CONCLUSIONS: Arthroscopic and histological ICRS scoring methods for repaired articular cartilage show a moderate correlation in the animal cartilage repair model.

Patient-Specific Bioimplants and Reconstruction Plates for Mandibular Defects: Production Workflow and In Vivo Large Animal Model Study.

A major challenge with extensive craniomaxillofacial bone reconstruction is the limited donor-site availability to reconstruct defects predictably and accurately according to the anatomical shape of the patient. Here, patient-specific composite bioimplants, consisting of cross-linked poly(trimethylene carbonate) (PTMC) networks and ß-tricalcium phosphate (ß-TCP), are tested in vivo in twelve Göttingen minipigs in a large mandibular continuity defect model. The 25 mm defects are supported by patient-specific titanium reconstruction plates and receive either osteoconductive composite bioimplants (PTMC+TCP), neat polymer network bioimplants (PTMC), autologous bone segments (positive control), or are left empty (negative control). Postoperatively, defects treated with bioimplants show evident ossification at 24 weeks. Histopathologic evaluation reveals that neat PTMC bioimplant surfaces are largely covered with fibrous tissue, while in the PTMC+TCP bioimplants, bone attached directly to the implant surface shows good osteoconduction and histological signs of osteoinductivity. However, PTMC+TCP bioimplants are associated with high incidence of necrosis and infection, possibly due to rapid resorption and/or particle size of the used ß-TCP. The study highlights the importance of testing bone regeneration implants in a clinically relevant large animal model and at the in situ reconstruction site, since results on small animal models and studies in nonloadbearing areas do not translate directly.

Phage Treatment Trial to Eradicate LA-MRSA from Healthy Carrier Pigs.

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10-100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined.

Arthroscopic International Cartilage Repair Society Classification System Has Only Moderate Reliability in a Porcine Cartilage Repair Model.

BACKGROUND: The International Cartilage Repair Society (ICRS) score was designed for arthroscopic use to evaluate the quality of cartilage repair. PURPOSE: To evaluate the reliability of the ICRS scoring system using an animal cartilage repair model. STUDY DESIGN: Controlled laboratory study. METHODS: A chondral defect with an area of 1.5 cm2 was made in the medial femoral condyle of 18 domestic pigs. Five weeks later, 9 pigs were treated using a novel recombinant human type III collagen/polylactide scaffold, and 9 were left to heal spontaneously. After 4 months, the pigs were sacrificed, then 3 arthroscopic surgeons evaluated the medial femoral condyles via video-recorded simulated arthroscopy using the ICRS scoring system. The surgeons repeated the evaluation twice within a 9-month period using their recorded arthroscopy. RESULTS: The porcine cartilage repair model produced cartilage repair tissue of poor to good quality. The mean ICRS total scores for all observations were 6.6 (SD, 2.6) in arthroscopy, 5.9 (SD, 2.7) in the first reevaluation, and 6.2 (SD, 2.8) in the second reevaluation. The interrater reliability with the intraclass correlation coefficient (ICC) for the ICRS total scores (ICC, 0.46-0.60) and for each individual subscore (ICC, 0.26-0.71) showed poor to moderate reliability. The intrarater reliability with the ICC also showed poor to moderate reliability for ICRS total scores (ICC, 0.52-0.59) and for each individual subscore (ICC, 0.29-0.58). A modified Bland-Altman plot for the initial arthroscopy and for the 2 reevaluations showed an evident disagreement among the observers. CONCLUSION: In an animal cartilage repair model, the ICRS scoring system seems to have poor to moderate reliability. CLINICAL RELEVANCE: Arthroscopic assessment of cartilage repair using the ICRS scoring method has limited reliability. We need more objective methods with acceptable reliability to evaluate cartilage repair outcomes.

UBB pseudogene 4 encodes functional ubiquitin variants.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.

Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study.

The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo-PLA) in the repair of full-thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4-month-old, n = 20) were randomized into three study groups and a circular full-thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo-PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo-PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo-PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo-PLA biomaterial showed promising results in this proof-of-concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745-753, 2016.

The effect of dividing walls, a tunnel, and restricted feeding on cardiovascular responses to cage change and gavage in rats (Rattus norvegicus).

Cage change and gavage are routine procedures in animal facilities, yet little is known about whether housing modifications change responses to these procedures. Telemetric activity and cardiovascular parameters were assessed in this experiment. BN and F344 male rats were housed in open or individually ventilated cages, each containing 3 rats, 1 of which had a transponder. A crossover design was used, in which 2 groups were given dividers made of 2 intersecting boards (1 form contained holes loaded with food pellets; the other did not) and 1 group was given a rectangular tunnel. On day 8 of each 2-wk period, the cages were changed; on day 11, rats were gavaged. The parameters were evaluated for the first hour and for the following 17 h. Baseline values for each rat were subtracted from the corresponding response values. The presence of objects did not affect the responses of F344 rats to cage changing or gavage. In BN rats with IVCs, the presence of the plain divider modified the response to both procedures. Responses to procedures appeared to be dependent on both the strain and the cage object, thus complicating the establishment of valid general recommendations.