Exploring uses for an algorithmically generated Animal Welfare Indicator for welfare assessment of dairy herds.

On-farm welfare assessment is time-consuming and costly. Assessing welfare using routinely collected herd data has been proposed as a more economical alternative. The online Animal Welfare Indicator (AWI), developed by a Norwegian dairy cooperative, applies an algorithm to routinely collected health, production, and management data to "indicate" aspects of animal welfare at herd level. The overall AWI score is based on 10 AWI subindicator scores, representative of elements of animal welfare such as claw health, udder health, and mortality. Our cross-sectional study explored 2 ways in which the AWI may enable more efficient welfare assessment of Norwegian dairy herds. First, we investigated using the AWI to reduce the duration of on-farm assessments by replacing on-farm measures. Second, we examined reducing the number of on-farm welfare assessments by using the AWI to predict which herds have poorer welfare with respect to specific on-farm measures. Using Spearman rank analyses, we investigated if the AWI scores for 157 herds were associated with 24 on-farm welfare variables measured contemporaneously by Welfare Quality assessment. The mortality AWI subindicator score and the percentage mortality in the previous 12 mo were moderately correlated, as were the udder health AWI subindicator score and the percentage high somatic cell count (SCC) in the previous 3 recordings. Only negligible or weak correlations were found between the other AWI scores and the on-farm assessment variables. We built Generalized Linear Models using AWI scores as independent variables to predict herds with poorer welfare. Herds were classified as having poorer welfare based on their results in specific on-farm welfare measures. We evaluated the models' predictive ability and accuracy. Moderately accurate models were built for predicting poorer herds with respect to high SCC, mortality, and moderate or severe lameness. The other models were less accurate. The AWI scores were generally unsuitable as replacements of on-farm welfare measures. The AWI subindicators for udder health and mortality could replace the on-farm welfare measures related to those 2 topics, but there was some overlap in the data used to calculate them. Despite a lack of independence, the use of those 2 AWI subindicators may marginally reduce the duration of on-farm assessments. A prediction model based on AWI scores showed potential for identifying herds with poorer welfare in terms of moderate or severe lameness, facilitating more efficient use of resources for on-farm lameness assessment. As a consequence of the data used in the AWI, it was only reflective of health-related welfare outcomes.

Improved Physical Stability of an Antibody-Drug Conjugate Using Host-Guest Chemistry.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceutical products for oncology, with the cytotoxic pyrrolobenzodiazepine (PBD) family of "warheads" well-established in the clinic. While PBDs offer high potency, they are also characterized by their hydrophobicity, which can make formulation of the ADC challenging. Several approaches have been investigated to improve the physicochemical properties of PBD-containing ADCs, and herein a supramolecular approach was explored using cucurbit[8]uril (CB[8]). The ability of CB[8] to simultaneously encapsulate two guests was exploited to incorporate a 12-mer polyethylene glycol harboring a methyl viologen moiety at one terminus (MV-PEG12), together with a PBD harboring an indole moiety at the C2' position (SG3811). This formulation approach successfully introduced a hydrophilic PEG to mask the hydrophobicity of SG3811, improving the physical stability of the ADC while avoiding any loss of potency related to chemical modification.

A new classification for mandibular defects after oncological resection.

No universally accepted classification system exists for mandibular defects after oncological resection. Here, we discuss the scientific literature on classifications for mandibular defects that are sufficiently presented either pictorially or descriptively, and propose a new classification system based on these findings. Of 167 studies included in the data analysis, 49 of these reports sufficiently described the defect for analysis. These reports were analysed for classification, reconstruction, size of defect, number of osteotomies needed, and complications. On the basis of these findings, a new classification is proposed based on the four corners of the mandible (two angles and two canines): class I (lateral), class II (hemimandibulectomy), class III (anterior), and class IV (extensive). Further classes (Ic, IIc, and IVc) include condylectomy. The increasing defect class relates to the size of the defect, osteotomy rate, and functional and aesthetic outcome, and could guide the method of reconstruction.

The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

'Naked' and hydrated conformers of the conserved core pentasaccharide of N-linked glycoproteins and its building blocks.

N-glycosylation of eukaryotic proteins is widespread and vital to survival. The pentasaccharide unit -Man3GlcNAc2- lies at the protein-junction core of all oligosaccharides attached to asparagine side chains during this process. Although its absolute conservation implies an indispensable role, associated perhaps with its structure, its unbiased conformation and the potential modulating role of solvation are unknown; both have now been explored through a combination of synthesis, laser spectroscopy, and computation. The proximal -GlcNAc-GlcNAc- unit acts as a rigid rod, while the central, and unusual, -Man-ß-1,4-GlcNAc- linkage is more flexible and is modulated by the distal Man-α-1,3- and Man-α-1,6- branching units. Solvation stiffens the 'rod' but leaves the distal residues flexible, through a ß-Man pivot, ensuring anchored projection from the protein shell while allowing flexible interaction of the distal portion of N-glycosylation with bulk water and biomolecular assemblies.

Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis.

The detection of tuberculosis currently relies upon insensitive and unspecific techniques; newer diagnostics would ideally co-opt specific bacterial processes to provide real-time readouts. The trehalose mycolyltransesterase enzymes (antigens 85A, 85B and 85C (Ag85A, Ag85B, Ag85C)) serve as essential mediators of cell envelope function and biogenesis in Mycobacterium tuberculosis. Through the construction of a systematically varied sugar library, we show here that Ag85 enzymes have exceptionally broad substrate specificity. This allowed exogenously added synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. Even bulky substituents, such as a fluorescein-containing trehalose probe (FITC-trehalose), were incorporated by growing bacilli, thereby producing fluorescent bacteria; microscopy revealed selective labeling of poles and membrane. Addition of FITC-trehalose to M. tuberculosis-infected macrophages allowed selective, sensitive detection of M. tuberculosis within infected mammalian macrophages. These studies suggest that analogs of trehalose may prove useful as probes of function and for other imaging modalities.

Obtaining an animal welfare status in Norwegian dairy herds-A mountain to climb.

Introduction: Knowing the national status of animal welfare, one can identify welfare problems and set a benchmark against which improvements can be compared. Such a status is potentially invaluable for tangible, sustained animal welfare improvement. The objective of this cross-sectional study was to report the status of animal welfare in Norwegian loose-housed dairy herds as assessed using the Welfare Quality® Assessment Protocol. Additionally, we investigated if the welfare status varied on a regional basis. Methods: In total, 155 herds in eight of Norway's eleven counties were assessed by six trained Welfare Quality® assessors. This article presents the herd prevalences of common welfare issues in dairy production in Norway, as well as integrated welfare scores. To determine whether welfare status varied regionally in Norway, generalized linear modeling was used to estimate the mean welfare score for five regions in the four Welfare Quality® principles: A. Good feeding, B. Good housing, C. Good health, and D. Appropriate behavior. These estimated mean welfare scores and their 95% confidence intervals were subsequently assessed for significant variation. Results: Encouraging findings included the low mean herd prevalence of 'very lean' cows (3.0%) and the high proportion of cows (59.8%) which could be touched during avoidance distance testing, indicating a positive relationship between stockpeople and their cattle. Challenges affecting the welfare of Norwegian dairy cows were also identified. Of particular concern were issues related to the cows' environment such as prolonged times needed to complete lying down movements and integument alterations. No herd was completely free of changes to the integument and, on average, 77.9% of each herd were affected either mildly or severely. Animal welfare did not appear to vary much between the five regions assessed. Our investigation revealed significant regional variation between two regions (Trøndelag and Vestlandet North) in only the Welfare Quality® principle Good housing (p < 0.01). Discussion: The almost complete absence of regional variation demonstrates that animal welfare status generally varies most at herd level. In conclusion, both welfare challenges and encouraging findings were identified in loose-housed Norwegian dairy herds. To improve animal welfare, herd-specific interventions are most likely to be effective in these herds.

Corrigendum: Obtaining an animal welfare status in Norwegian dairy herds-a mountain to climb.

[This corrects the article DOI: 10.3389/fvets.2023.1125860.].

Substrate and metal ion promiscuity in mannosylglycerate synthase.

The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.

Efficacy, Tolerability, and Pharmacokinetic Studies of Antibody-Drug Conjugates Containing a Low-Potency Pyrrolobenzodiazepine Dimer.

Antibody-drug conjugate (ADC) research has typically focused on the release of highly potent cytotoxic agents to achieve antitumor efficacy. However, recently approved ADCs trastuzumab deruxtecan and sacituzumab govitecan release lower-potency topoisomerase inhibitors. This has prompted interest in ADCs that release lower-potency cytotoxic drugs to potentially enhance therapeutic index and reduce unwanted toxicity. Pyrrolobenzodiazepine (PBD) dimer ADCs have been widely investigated in human clinical trials, which have focused on high-potency PBDs. In this study, we evaluated five ADCs that release the low-potency PBD dimer SG3650. The relatively low clogD for this agent facilitated higher drug-to-antibody ratio (DAR) conjugation without the need for antibody engineering or functionalization of the drug. The rank order of potency for DAR 2 site-specific ADCs (conjugated at the C239i position) matched the order for the corresponding free drugs in vitro. Despite free drug SG3650 being inactive in vivo, the DAR 2 ADCs derived from the corresponding drug-linker SG3584 showed antitumor efficacy in solid (anti-HER2) and hematologic (anti-CD22) xenograft models. Antitumor activity could be enhanced by conjugating SG3584 to trastuzumab at higher DARs of 4 and 8 and by adjusting dosing and schedule. Higher-DAR conjugates were stable and displayed good rat pharmacokinetic profiles as measured by ELISA and LC/MS-MS. A single intravenous dose of isotype control SG3584 DAR 2 ADC resulted in no mortality in rats or monkeys at doses of up to 25 and 30 mg/kg, respectively. These findings suggest that further investigations of low-potency PBD dimers in ADCs that target hematologic and solid tumors are warranted.

ESI-MS assay of M. tuberculosis cell wall antigen 85 enzymes permits substrate profiling and design of a mechanism-based inhibitor.

Mycobacterium tuberculosis Antigen 85 enzymes are vital to the integrity of the highly impermeable cell envelope and are potential therapeutic targets. Kinetic analysis using a label-free assay revealed both mechanistic details and a substrate profile that allowed the design and construction of a selective in vitro mechanism-based inhibitor.

Simultaneous monitoring of multiple attributes of pyrrolobenzodiazepine antibody-drug conjugates by size exclusion chromatography - high resolution mass spectrometry.

Antibody-drug conjugates (ADCs) are an emerging class of oncology treatments combining the unique specificity of monoclonal antibodies with the highly cytotoxic properties of small molecule compounds. Pyrrolobenzodiazepines (PBDs) are highly potent agents capable of inhibiting cellular DNA replication which leads to apoptosis. To ensure efficacy and patient safety upon administration of such toxic and heterogeneous molecules, their structure and quality attributes must be closely monitored. Size exclusion chromatography (SEC) is a powerful, fast and robust tool for the separation of compounds varying in molecular weight. When using volatile components in the chromatographic mobile phase, SEC has also been shown to be amenable for interfacing to mass spectrometry, providing potential for reliable identification of protein isoforms across the size variants present. Here, we present a SEC-MS method developed for the characterisation of PBD-based ADCs on the intact molecular level. We demonstrate that information on ADC monomers such as the glycoform distribution and the average drug-antibody ratio (DAR) can be obtained in 15 minutes of analysis time. Qualitative and quantitative information on low and high molecular weight impurities such as aggregates and fragments, fundamental for critical quality attribute analysis of biopharmaceuticals, can be generated simultaneously. SEC-MS enables the characterisation of multiple product quality attributes of complex biotherapeutics at the same time.

Pre-operative testing for SARS-CoV-2 and outcomes in otolaryngology surgery during the pandemic: A multi-center experience.

INTRODUCTION: Preoperative testing for COVID-19 has become widely established to avoid inadvertent surgery on patients with COVID-19 and prevent hospital outbreaks. METHODS: A prospective cross sectional study was carried out in two university hospitals examining the pre-operative protocols for patients undergoing otolaryngology surgery and the incidence of COVID-19 within 30 days of surgery in patients and the otolaryngologists performing surgery. RESULTS: One hundred and seventy-three patients were recruited. One hundred and twenty-three (71%) patients "cocooned" for 14 days prior to surgery. All completed a questionnaire prior to admission. One hundred and fifty-six patients (90%) had reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs, 14 patients (8%) had CT thorax. No cases of COVID-19 were detected among patients followed up at 30 days. Two surgeons developed COVID-19 early during the study period. CONCLUSION: Current pre-operative testing protocols consisting primarily of questionnaires and RT-PCR resulted in zero cases of COVID in this cohort. It is possible that COVID-19 restrictions and high proportion of patients cocooning preoperatively were factors in ensuring a low rate of COVID-19 post-operatively.

The use of 3D printing technology in the creation of patient-specific facial prostheses.

BACKGROUND: Personalised medicine aims to optimise patient outcomes by tailoring treatments and interventions to the individual. While this approach can offer a number of benefits, it can be accompanied by significant overheads in terms of resources. Prostheses exist in order to restore and replicate the normal functions and appearance of the body but if these are not individually tailored to the patient's needs then a true restoration cannot be fully achieved. Traditionally a labour intensive process, the fabrication of craniofacial prostheses, involves taking a plaster cast of the area to be treated, hand carving wax models of the restoration and multiple meetings with the patient to alter this wax restoration before making a final prosthesis in silicone. AIMS: Utilising the patient's pre-existing computed tomography (CT) images and 3D printing technology, a patient-specific prosthesis can be created with improved efficiency and accuracy. METHODS: This study demonstrates methods used to create a patient-specific orbital prosthesis using CT images. These images were manipulated in a way which allowed for the intact orbit to be mirrored and used to develop a 3D printed model which acted as the starting point to create a silicone prosthesis. RESULTS: The benefits of using this method include reduced manufacturing time, decreased outpatient appointments, improved personalised outcomes and a repeatable process allowing multiple prostheses to be made. CONCLUSIONS: 3D printing is a valuable tool which can provide significant savings in time and improve patient outcomes by offering a tailored approach to each individual's treatment.

Recurrent Ameloblastoma Involving Fibula Neo-mandible: Management with Digital Planning and Reconstruction Using a Contralateral Free Fibula Flap.

Ameloblastoma is a locally aggressive tumor that most commonly arises in the mandible. It has a high rate of recurrence if inadequately excised. We report a case of a patient who developed recurrence of his ameloblastoma in his fibula flap mandibular reconstruction despite clear resection margins 23 years after resection. This is the first reported case of recurrent ameloblastoma in a neo-mandible reconstruction in the setting of negative margins. We discuss its surgical management using digital planning and reconstruction using a contralateral free fibula flap. Ameloblastoma is a locally aggressive entity that requires complete excision. Recurrence can even occur in the reconstruction, which can present a challenge to manage. Consideration should be given to repeat excision and second osseous flap reconstruction.

TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.

cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody-drug conjugates (ADCs). However, the clinical benefit from cMet-targeted therapy has been limited. We developed a novel cMet-targeted 'third-generation' ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin-linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin-linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet-expressing cell lines but also in medium-to-low cMet cell lines (40 000-90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801-ADC had excellent efficacy with significant antitumor activity in 90% of tested patient-derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single-dose administration. Altogether, TR1801-ADC is a new generation cMet ADC with best-in-class preclinical efficacy and good tolerability in rats.

Pneumocephalus and Meningitis as Complications of Mastoiditis.

Pneumocephalus in the absence of trauma, tumour, or surgery is a rare entity. We report a case of a 73-year-old lady who presented with sepsis leading to confusion and unresponsiveness. A CT of brain revealed mastoiditis, sinusitis, and associated pneumocephalus. Further investigations led to an eventual diagnosis of pneumococcal meningitis. The combination of pneumocephalus and meningitis as complications of mastoiditis is rare with very few cases published in the literature. We describe one such case.

Synthesis and evaluation of pyrrolobenzodiazepine dimer antibody-drug conjugates with dual ß-glucuronide and dipeptide triggers.

Antibody-drug conjugates (ADCs) containing pyrrolobenzodiazepine (PBD) dimers are currently being evaluated in human oncology clinical trials with encouraging results. To further improve the therapeutic window, next-generation PBD drug-linker design has focused on the inclusion of additional tumor-selective triggers and use of lower-potency PBDs. ß-Glucuronidase is a well-known target for discovery prodrugs due to increased presence in tumor cells and microenvironment. In this study, a ß-glucuronidase cleavable cap was investigated at the PBD N10-position and compared with corresponding free imine ADCs. SG3600 (glucuronide) ADCs showed in vitro and in vivo efficacy/tolerability comparable to SG3400 (imine) ADCs, and good 50% inhibitory concentration differentials were observed in vitro between control non-antigen-targeted ADCs and targeted ADCs. Dependence on ß-glucuronidase for SG3600 activity was demonstrated through CRISPRCas9 knockdown studies and addition of exogenous ß-glucuronidase. SG3600 showed better serum stability, improved conjugation efficiency and was able to reach high drug-to-antibody ratio without aggregation.

A single dose of antibody-drug conjugate cures a stage 1 model of African trypanosomiasis.

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.