Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.

Immunopeptidomic Analysis of the Phosphopeptidome Displayed by HLA Class I Molecules.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a powerful tool that allows the characterization of MHC-associated peptidomes. In addition to conventional nonmodified peptides, these complex mixtures also contain phosphorylated species, which may be of great interest for personalized cancer immunotherapy. Here, we provide a detailed protocol to identify phosphopeptides displayed by human HLA class I molecules consisting of four main steps: (1) immunopurification of MHC-I molecules, (2) phosphopeptide enrichment, (3) LC-MS/MS analysis, and (4) MS/MS ion search.

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself.

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry.

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

Data Collection for the Fourth Multicentre Confidential Enquiry into Perioperative Equine Fatalities (CEPEF4) Study: New Technology and Preliminary Results.

It is almost 20 years since the largest observational, multicentre study evaluating the risks of mortality associated with general anaesthesia in horses. We proposed an internet-based method to collect data (cleaned and analysed with R) in a multicentre, cohort, observational, analytical, longitudinal and prospective study to evaluate peri-operative equine mortality. The objective was to report the usefulness of the method, illustrated with the preliminary data, including outcomes for horses seven days after undergoing general anaesthesia and certain procedures using standing sedation. Within six months, data from 6701 procedures under general anaesthesia and 1955 standing sedations from 69 centres were collected. The results showed (i) the utility of the method; also, that (ii) the overall mortality rate for general anaesthesia within the seven-day outcome period was 1.0%. In horses undergoing procedures other than exploratory laparotomy for colic ("noncolics"), the rate was lower, 0.6%, and in "colics" it was higher, at 3.4%. For standing sedations, the overall mortality rate was 0.2%. Finally, (iii) we present some descriptive data that demonstrate new developments since the previous CEPEF2. In conclusion, horses clearly still die unexpectedly when undergoing procedures under general anaesthesia or standing sedation. Our method is suitable for case collection for future studies.

Genetic architecture and major genes for backfat thickness in pig lines of diverse genetic backgrounds.

BACKGROUND: Backfat thickness is an important carcass composition trait for pork production and is commonly included in swine breeding programmes. In this paper, we report the results of a large genome-wide association study for backfat thickness using data from eight lines of diverse genetic backgrounds. METHODS: Data comprised 275,590 pigs from eight lines with diverse genetic backgrounds (breeds included Large White, Landrace, Pietrain, Hampshire, Duroc, and synthetic lines) genotyped and imputed for 71,324 single-nucleotide polymorphisms (SNPs). For each line, we estimated SNP associations using a univariate linear mixed model that accounted for genomic relationships. SNPs with significant associations were identified using a threshold of p < 10-6 and used to define genomic regions of interest. The proportion of genetic variance explained by a genomic region was estimated using a ridge regression model. RESULTS: We found significant associations with backfat thickness for 264 SNPs across 27 genomic regions. Six genomic regions were detected in three or more lines. The average estimate of the SNP-based heritability was 0.48, with estimates by line ranging from 0.30 to 0.58. The genomic regions jointly explained from 3.2 to 19.5% of the additive genetic variance of backfat thickness within a line. Individual genomic regions explained up to 8.0% of the additive genetic variance of backfat thickness within a line. Some of these 27 genomic regions also explained up to 1.6% of the additive genetic variance in lines for which the genomic region was not statistically significant. We identified 64 candidate genes with annotated functions that can be related to fat metabolism, including well-studied genes such as MC4R, IGF2, and LEPR, and more novel candidate genes such as DHCR7, FGF23, MEDAG, DGKI, and PTN. CONCLUSIONS: Our results confirm the polygenic architecture of backfat thickness and the role of genes involved in energy homeostasis, adipogenesis, fatty acid metabolism, and insulin signalling pathways for fat deposition in pigs. The results also suggest that several less well-understood metabolic pathways contribute to backfat development, such as those of phosphate, calcium, and vitamin D homeostasis.

A critical evaluation for validation of composite and unidimensional postoperative pain scales in horses.

Proper pain therapy requires adequate pain assessment. This study evaluated the reliability and validity of the Unesp-Botucatu horse acute pain scale (UHAPS), the Orthopedic Composite Pain Scale (CPS) and unidimensional scales in horses admitted for orthopedic and soft tissue surgery. Forty-two horses were assessed and videotaped before surgery, up to 4 hours postoperatively, up to 3 hours after analgesic treatment, and 24 hours postoperatively (168 video clips). After six evaluators viewing each edited video clip twice in random order at a 20-day interval, they chose whether analgesia would be indicated and applied the Simple Descriptive, Numeric and Visual Analog scales, CPS, and UHAPS. For all evaluators, intra-observer reliability of UHAPS and CPS ranged from 0.70 to 0.97. Reproducibility was variable among the evaluators and ranged from poor to very good for all scales. Principal component analysis showed a weak association among 50% and 62% of the UHAPS and CPS items, respectively. Criterion validity based on Spearman correlation among all scales was above 0.67. Internal consistency was minimally acceptable (0.51-0.64). Item-total correlation was acceptable (0.3-0.7) for 50% and 38% of UHAPS and CPS items, respectively. UHAPS and CPS were specific (90% and 79% respectively), but both were not sensitive (43 and 38%, respectively). Construct validity (responsiveness) was confirmed for all scales because pain scores increased after surgery. The cut-off point for rescue analgesia was ≥ 5 and ≥ 7 for the UHAPS and CPS, respectively. All scales presented adequate repeatability, criterion validity, and partial responsiveness. Both composite scales showed poor association among items, minimally acceptable internal consistency, and weak sensitivity, indicating that they are suboptimal instruments for assessing postoperative pain. Both composite scales require further refinement with the exclusion of redundant or needless items and reduction of their maximum score applied to each item or should be replaced by other tools.

Recovery after General Anaesthesia in Adult Horses: A Structured Summary of the Literature.

Recovery remains the most dangerous phase of general anaesthesia in horses. The objective of this publication was to perform a structured literature review including levels of evidence (LoE) of each study with the keywords "recovery anaesthesia horse", entered at once, in the search browsers PubMed and Web of Science. The two authors independently evaluated each candidate article. A final list with 444 articles was obtained on 5 April 2021, classified as: 41 "narrative reviews/expert opinions", 16 "retrospective outcome studies", 5 "surveys", 59 "premedication/sedation and induction drugs", 27 "maintenance with inhalant agents", 55 "maintenance with total intravenous anaesthesia (TIVA)", 3 "TIVA versus inhalants", 56 "maintenance with partial intravenous anaesthesia (PIVA)", 27 "other drugs used during maintenance", 18 "drugs before/during recovery", 18 "recovery systems", 21 "respiratory system in recovery", 41 "other factors", 51 "case series/reports" and 6 "systems to score recoveries". Of them, 167 were LoE 1, 36 LoE 2, 33 LoE 3, 110 LoE 4, 90 LoE 5 and 8 could not be classified based on the available abstract. This review can be used as an up-to-date compilation of the literature about recovery after general anaesthesia in adult horses that tried to minimise the bias inherent to narrative reviews.

Development and validation of the facial scale (FaceSed) to evaluate sedation in horses.

Although facial characteristics are used to estimate horse sedation, there are no studies measuring their reliability and validity. This randomised controlled, prospective, horizontal study aimed to validate a facial sedation scale for horses (FaceSed). Seven horses received detomidine infusion i.v. in low or high doses/rates alone (DL 2.5 µg/kg+6.25 µg/kg/h; DH 5 µg/kg+12.5 µg/kg/h) or combined with methadone (DLM and DHM, 0.2 mg/kg+0.05 mg/kg/h) for 120 min, or acepromazine boli i.v. in low (ACPL 0.02 mg/kg) or high doses (ACPH 0.09 mg/kg). Horses' faces were photographed at i) baseline, ii) peak, iii) intermediate, and iv) end of sedation. After randomisation of moments and treatments, photos were sent to four evaluators to assess the FaceSed items (ear position, orbital opening, relaxation of the lower and upper lip) twice, within a one-month interval. The intraclass correlation coefficient of intra- and interobserver reliability of FaceSed scores were good to very good (0.74-0.94) and moderate to very good (0.57-0.87), respectively. Criterion validity based on Spearman correlation between the FaceSed versus the numerical rating scale and head height above the ground were 0.92 and -0.75, respectively. All items and the FaceSed total score showed responsiveness (construct validity). According to the principal component analysis all FaceSed items had load factors >0.50 at the first dimension. The high internal consistency (Cronbach´s α = 0.83) indicated good intercorrelation among items. Item-total Spearman correlation was adequate (rho 0.3-0.73), indicating homogeneity of the scale. All items showed sensitivity (0.82-0.97) to detect sedation, however only orbital opening (0.79) and upper lip relaxation (0.82) were specific to detect absence of sedation. The limitations were that the facial expression was performed using photos, which do not represent the facial movement and the horses were docile, which may have reduced specificity. The FaceSed is a valid and reliable tool to assess tranquilisation and sedation in horses.

Description of a novel ultrasound guided peribulbar block in horses: a cadaveric study.

BACKGROUND: Standing surgery in horses combining intravenous sedatives, analgesics and local anaesthesia is becoming more popular. Ultrasound guided (USG) peribulbar nerve block (PB) has been described in dogs and humans for facial and ocular surgery, reducing the risk of complications versus retrobulbar nerve block (RB). OBJECTIVE: To describe a technique for USG PB in horse cadavers. METHODS: Landmarks and PB technique were described in two equine cadaver heads (Phase 1), with computed tomography (CT) imaging confirming contrast location and spread. In Phase 2, ten equine cadaver heads were randomised to two operators naïve to the USG PB, with moderate experience with ultrasonography and conventional "blind" RB. Both techniques were demonstrated once. Subsequently, operators performed five USG PB and five RB each, unassisted. Contrast location and spread were evaluated by CT. Injection site success was defined for USG PB as extraconal contrast, and for RB intraconal contrast. RESULTS: Success was 10/10 for USG PB and 0/10 for RB (p < 0.001). Of the RB injections, eight resulted in extraconal contrast and two in the masseter muscle (p = 0.47). CONCLUSIONS: The USG PB had a high injection site success rate compared with the RB technique; however, we cannot comment on clinical effect. The USG technique was easily learnt, and no potential complications were seen. The USG PB nerve block could have a wide application for use in horses for ocular surgeries (enucleations, eyelid, corneal, cataract surgeries, and ocular analgesia) due to reduced risk of iatrogenic damage. Further clinical studies are needed.

Development, Validation, and Reliability of a Sedation Scale in Horses (EquiSed).

The lack of standardization of sedation scales in horses limits the reproducibility between different studies. This prospective, randomized, blinded, horizontal and controlled trial aimed to validate a scale for sedation in horses (EquiSed). Seven horses were treated with intravenous detomidine in low/high doses alone (DL 2.5 µg/kg + 6.25 µg/kg/h; DH 5 µg/kg +12.5 µg/kg/h) or associated with methadone (DLM and DHM, 0.2 mg/kg + 0.05 mg/kg/h) and with low (ACPL 0.02 mg/kg) or high (ACPH 0.09 mg/kg) doses of acepromazine alone. Horses were filmed at (i) baseline (ii) peak, (iii) intermediate, and (iv) end of sedation immediately before auditory, visual and pressure stimuli were applied and postural instability evaluated for another study. Videos were randomized and blindly evaluated by four evaluators in two phases with 1-month interval. Intra- and interobserver reliability of the sum of EquiSed (Intraclass correlation coefficient) ranged between 0.84-0.94 and 0.45-0.88, respectively. The criterion validity was endorsed by the high Spearman correlation between the EquiSed and visual analog (0.77), numerical rating (0.76), and simple descriptive scales (0.70), and average correlation with head height above the ground (HHAG) (-0.52). The Friedman test confirmed the EquiSed responsiveness over time. The principal component analysis showed that all items of the scale had a load factor ≥ 0.50. The item-total Spearman correlation for all items ranged from 0.3 to 0.5, and the internal consistency was good (Cronbach's α = 0.73). The area under the curve of EquiSed HHAG as a predictive diagnostic measure was 0.88. The sensitivity of the EquiSed calculated according to the cut-off point (score 7 of the sum of the EquiSed) determined by the receiver operating characteristic curve, was 96% and specificity was 83%. EquiSed has good intra- and interobserver reliabilities and is valid to evaluate tranquilization and sedation in horses.

Thermal, mechanical and electrical stimuli in antinociceptive studies in standing horses: an update.

OBJECTIVE: To perform a literature review of the thermal and mechanical antinociceptive devices used in pharmacological studies in standing horses published after 2011 (2012-2019). To complete a full literature review about electrical stimulation used for evaluation in similar studies. DATABASES USED: PubMed, Google Scholar and Web of Science. CONCLUSIONS: A high level of standardization has been reached in antinociceptive studies in standing horses using thermal and mechanical stimuli in most recent years. Commercially available testing devices to deliver thermal, mechanical and electrical stimuli, with observation of aversive responses to these stimuli, are reliable, sensitive and specific. For electrical stimulus testing, there is evidence that the resistance between the electrodes should be measured and should not exceed 3 kΩ to guarantee consistent and reproducible stimuli. The specific analysis of electromyographic activity after an electrical stimulus provides more detailed information about the neurons stimulated.

A possible solution to model nonlinearity in elimination and distributional clearances with α2 -adrenergic receptor agonists: Example of the intravenous detomidine and methadone combination in sedated horses.

The alpha(α)2 -agonist detomidine is used for equine sedation with opioids such as methadone. We retrieved the data from two randomized, crossover studies where detomidine and methadone were given intravenously alone or combined as boli (STUDY 1) (Gozalo-Marcilla et al., 2017, Veterinary Anaesthesia and Analgesia, 2017, 44, 1116) or as 2-hr constant rate infusions (STUDY 2) (Gozalo-Marcilla et al., 2019, Equine Veterinary Journal, 51, 530). Plasma drug concentrations were measured with a validated tandem Mass Spectrometry assay. We used nonlinear mixed effect modelling and took pharmacokinetic (PK) data from both studies to fit simultaneously both drugs and explore their nonlinear kinetics. Two significant improvements over the classical mammillary two-compartment model were identified. First, the inclusion of an effect of detomidine plasma concentration on the elimination clearances (Cls) of both drugs improved the fit of detomidine (Objective Function Value [OFV]: -160) and methadone (OFV: -132) submodels. Second, a detomidine concentration-dependent reduction of distributional Cls of each drug further improved detomidine (OFV: -60) and methadone (OFV: -52) submodel fits. Using the PK data from both studies (a) helped exploring hypotheses on the nonlinearity of the elimination and distributional Cls and (b) allowed inclusion of dynamic effects of detomidine plasma concentration in the model which are compatible with the pharmacology of detomidine (vasoconstriction and reduction in cardiac output).

Clinical applicability of detomidine and methadone constant rate infusions for surgery in standing horses.

OBJECTIVE: To determine the required rate of a detomidine infusion (loading dose 5 µg kg-1; initial rate 12.5 µg kg-1 hour-1) added to a constant infusion of methadone (0.2 mg kg-1; 0.05 mg kg-1 hour-1) for sedation in standing horses and ponies undergoing elective surgeries with appropriate local anaesthetic techniques. STUDY DESIGN: Prospective, clinical study. ANIMALS: Adult, healthy, client-owned, non-food-producing horses or ponies sedated for elective standing surgeries longer than 45 minutes. METHODS: At baseline (in the stables before administration of sedative agents), at 10 minutes after sedation and every 5 minutes thereafter, ataxia, sedation and surgical condition were evaluated; each scored 0-3. These scores were used to adjust the detomidine administration rate using the Ghent Sedation Algorithm. A 10 cm visual analogue scale (VAS) was used by the main surgeon at the end of the procedure to evaluate the surgical conditions. Heart rate, systolic arterial pressure and respiratory frequency were also recorded at each time point. For statistical analysis, anova for normal, Kruskal-Wallis H-test for non-normal variables, and Mann-Whitney U test for VAS were used. RESULTS: From the 42 horses/ponies included in this study, 28 underwent dental procedures and 14 other types of procedures. Overall, dental procedures required higher mean detomidine rates compared with other types of surgeries (16.9 ± 4.5 versus 9.0 ± 1.9 µg kg-1 hour-1) (p < 0.001). Dental procedures were assigned similar VAS scores, median (range), of 7.8 (5.8-10) with other procedures, 8.7 (2.8-10). Cardiovascular changes were not clinically significant. No signs or behavioural changes of abdominal pain were observed postoperatively. CONCLUSIONS AND CLINICAL RELEVANCE: Satisfactory surgical conditions were achieved using a combination of detomidine and methadone infusions with locoregional anaesthesia, with no adverse effects. Dental procedures required higher detomidine dose rates compared with other surgeries.

The cochaperone CHIP marks Hsp70- and Hsp90-bound substrates for degradation through a very flexible mechanism.

Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). Both ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinated the substrate at a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, showed the substrate located between the chaperone and the cochaperone, suggesting a ubiquitination mechanism in which the chaperone-bound substrate is presented to CHIP. These complexes are inherently flexible, which is important for the ubiquitination process.

How to score sedation and adjust the administration rate of sedatives in horses: a literature review and introduction of the Ghent Sedation Algorithm.

OBJECTIVE: To summarize the different methods used to assess sedation and/or adjust the dose or administration rate of alpha-2 agonists in horses and to propose an algorithm to adjust the administration rate of a constant rate infusion of an alpha-2 agonist in horses. DATABASES USED: PubMed and Web of Science; search terms: horse, sedation and score. CONCLUSIONS: Most authors distinguish between sedation depth, sedation quality and degree of ataxia. These three features are evaluated using scoring systems similar to those classically used to assess pain, i.e. simple descriptive scales, numerical rating scales (NRS), visual analogue scales and/or multifactorial sedation scales. In addition, head height above the ground is often used as a measure of the depth of sedation. Very few authors have described how to adjust the administration rate or dose of alpha-2 agonists. Based on the available literature, the Ghent Sedation Algorithm was developed, which assigns scores (NRS) for degree of ataxia, sedation depth and surgical conditions, and uses these to prescribe changes in the administration rate of constant rate infusions of alpha-2 agonists. Studies are needed to validate this algorithm.

Identification of the Missing Protein Hyaluronan Synthase 1 in Human Mesenchymal Stem Cells Derived from Adipose Tissue or Umbilical Cord.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.

Minimal Information About an Immuno-Peptidomics Experiment (MIAIPE).

Minimal information about an immuno-peptidomics experiment (MIAIPE) is an initiative of the members of the Human Immuno-Peptidome Project (HIPP), an international program organized by the Human Proteome Organization (HUPO). The aim of the MIAIPE guidelines is to deliver technical guidelines representing the minimal information required to sufficiently support the evaluation and interpretation of immunopeptidomics experiments. The MIAIPE document has been designed to report essential information about sample preparation, mass spectrometric measurement, and associated mass spectrometry (MS)-related bioinformatics aspects that are unique to immunopeptidomics and may not be covered by the general proteomics MIAPE (minimal information about a proteomics experiment) guidelines.