ExMove: An open-source toolkit for processing and exploring animal-tracking data in R.

Ongoing technological advances have led to a rapid increase in the number, type and scope of animal-tracking studies. In response, many software tools have been developed to analyse animal movement data. These tools generally focus on movement modelling, but the steps required to clean raw data files from different tracking devices have been largely ignored. Such pre-processing steps are often time-consuming and involve a steep learning curve but are crucial for the creation of high-quality, standardised and shareable data. Moreover, decisions made at this early stage can substantially influence subsequent analyses, and in the current age of reproducibility crisis, the transparency of this process is vital. Here we present an open-access, reproducible toolkit written in the programming language R for processing raw data files into a single cleaned data set for analyses and upload to online tracking databases (found here: https://github.com/ExMove/ExMove). The toolkit comprises well-documented and flexible code to facilitate data processing and user understanding, both of which can increase user confidence and improve the uptake of sharing open and reproducible code. Additionally, we provide an overview website (found here: https://exmove.github.io/) and a Shiny app to help users visualise tracking data and assist with parameter determination during data cleaning. The toolkit is generalisable to different data formats and device types, uses modern 'tidy coding' practices, and relies on a few well-maintained packages. Among these, we perform spatial manipulations using the package sf. Overall, by collating all required steps from data collection to archiving on open access databases into a single, robust pipeline, our toolkit provides a valuable resource for anyone conducting animal movement analyses and represents an important step towards increased standardisation and reproducibility in animal movement ecology.

Bioaccessibility of Organic Compounds Associated with Tire Particles Using a Fish In Vitro Digestive Model: Solubilization Kinetics and Effects of Food Coingestion.

Tire and road wear particles (TRWP) account for an important part of the polymer particles released into the environment. There are scientific knowledge gaps as to the potential bioaccessibility of chemicals associated with TRWP to aquatic organisms. This study investigated the solubilization and bioaccessibility of seven of the most widely used tire-associated organic chemicals and four of their degradation products from cryogenically milled tire tread (CMTT) into fish digestive fluids using an in vitro digestion model based on Oncorhynchus mykiss. Our results showed that 0.06-44.1% of the selected compounds were rapidly solubilized into simulated gastric and intestinal fluids within a typical gut transit time for fish (3 h in gastric and 24 h in intestinal fluids). The environmentally realistic scenario of coingestion of CMTT and fish prey was explored using ground Gammarus pulex. Coingestion caused compound-specific changes in solubilization, either increasing or decreasing the compounds' bioaccessibility in simulated gut fluids compared to CMTT alone. Our results emphasize that tire-associated compounds become accessible in a digestive milieu and should be studied further with respect to their bioaccumulation and toxicological effects upon passage of intestinal epithelial cells.

Antibody-Drug Conjugates with Indolinobenzodiazepine Dimer Payloads: DNA-Binding Mechanism of Indolinobenzodiazepine Dimer Catabolites in Target Cancer Cells.

DNA-targeting indolinobenzodiazepine dimer (IGN) payloads are used in several clinical-stage antibody-drug conjugates. IGN drugs alkylate DNA through the single imine moiety present in the dimer in contrast to the pyrrolobenzodiazepine dimer drugs, such as talirine and tesirine, which contain two imine moieties per dimer and cross-link DNA. This study explored the mechanism of binding of IGN to DNA in cells and to synthetic duplex and hairpin oligonucleotides. New, highly sensitive IGN-DNA binding enzyme-linked immunosorbent assay methods were developed using biotinylated IGN analogues (monoimine, diimine, and diamine IGNs) and digoxigenin-labeled duplex oligonucleotides, which allowed the measurement of drug-DNA adducts in viable cells at concentrations below IC50. Furthermore, the release of free drug from the IGN-DNA adduct upon treatment with nuclease ex vivo was tested under physiological conditions. The monoimine IGN drug formed a highly stable adduct with DNA in cells, with stability similar to that of the diimine drug analogue. Both monoimine and diimine IGN-DNA adducts released free drugs upon DNA cleavage by nuclease at 37 °C, although more free drug was released from the monoimine compared to the diimine adduct, which presumably was partly cross-linked. The strong binding of the monoimine IGN drug to duplex DNA results from both the noncovalent IGN-DNA interaction and the covalent bond formation between the 2-amino group of a guanine residue and the imine moiety in IGN.

Optimizing Lysosomal Activation of Antibody-Drug Conjugates (ADCs) by Incorporation of Novel Cleavable Dipeptide Linkers.

Although peptide linkers are used in multiple clinical-stage ADCs, there are only few reports on optimizing peptide linkers for efficient lysosomal proteolysis and for stability in circulation. We screened multiple dipeptide linkers for efficiency of proteolysis and compared them to the dipeptide linkers currently being evaluated in the clinic: Val-Cit, Val-Ala, and Ala-Ala. Lead dipeptide linkers selected from the initial screen were incorporated into ADCs with indolinobenzodiazepine dimer (IGN) payloads to evaluate cellular processing, in vitro cytotoxic activity, plasma stability, and in vivo efficacy. ADCs with several dipeptide linkers bearing l-amino acids showed faster lysosomal processing in target cancer cells compared to the l-Ala-l-Ala linked ADC. These variances in linker processing rates did not result in different in vitro and in vivo activities among peptide linker ADCs, presumably due to accumulation of threshold cytotoxic catabolite levels for ADCs of several peptide linkers in the cell lines and xenografts tested. ADCs with l-amino acid dipeptide linkers exhibited superior in vitro cytotoxic potencies in multiple cell lines compared to an ADC with a d-Ala-d-Ala dipeptide linker and an ADC with a noncleavable linker. This work adds to the toolbox of stable, lysosomally cleavable peptide linkers for ADCs.

LC/MS/MS Bioanalysis of Protein-Drug Conjugates-The Importance of Incorporating Succinimide Hydrolysis Products.

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow-therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy.

Compensatory axon sprouting for very slow axonal die-back in a transgenic model of spinal muscular atrophy type III.

KEY POINTS: Smn+/- transgenic mouse is a model of the mildest form of spinal muscular atrophy. Although there is a loss of spinal motoneurons in 11-month-old animals, muscular force is maintained. This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons. The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity. We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die-back. ABSTRACT: Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn+/- transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die-back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die-back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast-twitch and one slow-twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn+/- transgenic mouse increases their susceptibility to cell death demonstrated that all the motoneurons survived and they sustained their capacity to regenerate their nerve fibres. It is concluded the systematic die-back of motoneurons that innervate both fast- and slow-twitch muscle fibres is not related to immaturity of the neuromuscular system in SMA.

Minor salivary gland carcinoma: a review of 35 cases.

Minor salivary gland carcinomas represent a heterogeneous group of tumors with broad variation in clinical appearance and histopathology. Clinical data of patients with small salivary gland malignancies were collected from the medical records. Tissue microarray was constructed to determine the expression pattern of 24 proteins in 35 patients with minor salivary gland carcinomas. The choice of markers was based on involvement in neoangiogenesis, cell-to-cell contact, cell-cycle regulation and carcinogenesis. Protein expression data were correlated to patients' clinical data. Overexpression of patched (p = 0.046) and Smo (p = 0.032) was linked to a better overall survival and Glutathione S-transferase π overexpression was linked to prolonged disease-free survival (p = 0.005). Cox-1 (p = 0.035) and VEGFR2 (p = 0.009) were significantly linked to decreased survival for recurrent disease. Bcl-x (84 %), ß-catenin (87 %) and Cox-2 (87 %) were significantly overexpressed in minor salivary gland carcinomas. We have shown that Smo resulted in a better overall survival, whereas Gstπ in improved disease-free survival. VEGFR2 was a prognostic factor for survival after recurrence in patients with minor salivary gland carcinomas. Cyclooxygenase inhibitors and anti-Wnt-1 antibodies might be a potential therapeutic option in an adjuvant setting or for patients with unresectable tumors of the minor salivary glands.

Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing.

Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.

Supraorbital keyhole approaches in the first 3 years of practice: outcomes and lessons learned. Patient series.

BACKGROUND: Supraorbital keyhole approaches (SKAs) have garnered criticism for a limited surgical exposure, restrictive surgical freedom, blind spots, and the learning curve. This retrospective study of patients who underwent SKA aims to explore the outcomes, technical nuances, and the learning curve reflected in a single surgeon's experience in the initial 3 years of practice. OBSERVATIONS: A total of 20 SKA operations were performed in 19 patients. Gross- or near-total resection was achieved in 14 of 17 tumor cases. The mean blood loss was 80.5 mL, the mean duration of surgery was 5 hours, and the median stay was 3 days. Endoscopic augmentation was used in 11 cases in which additional tumor removal occurred in 8 of the 11 cases. There were no cases of cerebrospinal fluid leakage or wound infection. A 30-day readmission and typical narcotics after discharge were seen in one patient each. When comparing two halves of a neurosurgery practice over 3 years, the duration of surgery was significantly longer in the later year, which is likely due to operating on a larger tumor size as the years progressed. No cases required static retractors or conversion to larger craniotomies. LESSONS: Careful case selection and respecting the learning curve allows the safe incorporation of SKA in the early stages of neurosurgical practice.

Differing drivers of decline within a migratory metapopulation has implications for future conservation.

Researchers generally ascribe demographic drivers in a single sub-population and presume they are representative. With this information, practitioners implement blanket conservation measures across metapopulations to reverse declines. However, such approaches may not be appropriate in circumstances where sub-populations are spatiotemporally segregated and exposed to different environmental variation. The Greenland White-fronted Goose, Anser albifrons flavirostris, is an Arctic-nesting migrant that largely comprises two sub-populations (delineated by northerly and southerly breeding areas in west Greenland). The metapopulation has declined since 1999 but this trend is only mirrored in one sub-population and the causes of this disparity are unclear. Here we compare the drivers and trends of productivity in both sub-populations using population- and individual-level analysis. We examined how temperature and precipitation influenced population-level reproductive success over 37 years and whether there was a change in the relationship when metapopulation decline commenced. In addition, we used biologging devices to remotely classify incubation events for 86 bird-years and modelled how phenology and environmental conditions influenced individual-level nest survival. Correlations between reproductive success and temperature/precipitation on the breeding grounds have weakened for both sub-populations. This has resulted in lower reproductive success for the northerly, but not southerly breeding sub-population, which at the individual-level appears to be driven by lower nest survival. Earlier breeding ground arrival and less precipitation during incubation increased nest survival in the northerly breeding population, while no factors examined were important for the southerly breeding sub-population. This suggests reproductive success is driven by different factor(s) in the two sub-populations. Demographic rates and their environmental drivers differ between the sub-populations examined here and consequently we encourage further decomposition of demography within metapopulations. This is important for conservation practitioners to consider as bespoke conservation strategies, targeting different limiting factors, may be required for different sub-populations.

Differential regulation of the Tor gene homolog drives the red/green pigmentation phenotype in the aphid Myzuspersicae.

In some aphid species, intraspecific variation in body colour is caused by differential carotenoid content: whilst green aphids contain only yellow carotenoids (ß-, γ-, and ß,γ-carotenes), red aphids additionally possess red carotenoids (torulene and 3,4-didehydrolycopene). Unusually, within animals who typically obtain carotenoids from their diet, ancestral horizontal gene transfer of carotenoid biosynthetic genes from fungi (followed by gene duplication), have imbued aphids with the intrinsic gene repertoire necessary to biosynthesise carotenoids. In the pea aphid, Acyrthosiphon pisum a lycopene (phytoene) desaturase gene (Tor) underpins the red/green phenotype, with this locus present in heterozygous form in red individuals but absent in green aphids, resulting in them being unable to convert lycopene into the red compounds 3,4-didehydrolycopene and torulene. The green peach aphid, Myzus persicae, separated from the pea aphid for ≈45MY also exists as distinct colour variable morphs, with both red and green individuals present. Here, we examined genomic data for both red and green morphs of M. persicae and identified an enlarged (compared to A. pisum) repertoire of 16 carotenoid biosynthetic genes (11 carotenoid desaturases and five carotenoid cyclase/synthase genes). From these, we identify the homolog of A. pisum Tor (here called carotene desaturase 2 or CDE-2) and show through 3D modelling that this homolog can accommodate the torulene precursor lycopene and, through RNA knockdown feeding experiments, demonstrate that disabling CDE-2 expression in red M. persicae clones results in green-coloured offspring. Unlike in A. pisum, we show that functional CDE-2 is present in the genomes of both red and green aphids. However, expression differences between the two colour morphs (350-700 fold CDE-2 overexpression in red clones), potentially driven by variants identified in upstream putative regulatory elements, underpin this phenotype. Thus, whilst aphids have a common origin of their carotenoid biosynthetic pathway, two aphid species separated for over 40MY have evolved very different drivers of intraspecific colour variation.

Impact of vasopressors on outcomes in head and neck free tissue transfer.

OBJECTIVES/HYPOTHESIS: The primary objective of the study was to determine the frequency of intraoperative vasopressor administration among patients undergoing free tissue transfer for head and neck reconstruction, and the secondary objective was to determine the impact of intraoperative vasopressor on free tissue transfer outcomes, including the impact of cumulative vasopressor dose and timing of intraoperative vasopressor administration. STUDY DESIGN/METHODS: A retrospective review was performed of all patients undergoing free tissue transfer for head and neck reconstruction at the University Health Network between 2004 to 2008. RESULTS: From 2004 to 2008 inclusive, 485 patients underwent 496 free tissue transfers for head and neck reconstruction. The complete failure rate was 2.2% (11 of 485 patients). The partial failure rate was 1.4%, and the operative take-back rate for venous congestion or arterial thrombosis was 1.6%. This gave a total major flap complication rate of 5.2%, which was used as the primary free tissue transfer outcome measure. Of the 485 patients who underwent free tissue transfer, 320 (66.0%) received intraoperative vasopressor. Of these patients, the majority (97.5%) received phenylephrine and/or ephedrine. There was no significant relationship between receiving intraoperative vasopressor and major free flap complications, which were defined as complete failure, partial failure, or operative take-back for venous congestion or arterial thrombosis. CONCLUSION: Intraoperative vasopressors are used routinely in free tissue transfer for the reconstruction of head and neck defects. The use of intraoperative vasopressors does not appear to adversely affect free tissue transfer outcomes.

Improving Pneumococcal Vaccination Rates in High-risk Children in Specialty Clinics.

BACKGROUND AND OBJECTIVES: Pediatric patients with immunocompromising or certain chronic medical conditions have an increased risk of acquiring invasive pneumococcal disease (IPD). The 23-valent pneumococcal polysaccharide vaccine (PPSV23) is recommended for patients ≥2 years at high risk for IPDs. The aim of this project was to improve PPSV23 vaccination rates for children at high risk for IPD who were seen in 3 specialty clinics from ∼20% to 50% over a 12-month period. METHODS: The project team included quality improvement champions from the divisions of rheumatology, infectious diseases, and pulmonology in addition to leaders from our population health management subsidiary. Several initiatives were implemented, starting with review of patient inclusion criteria per the vaccination recommendations, that led to the design and deployment of an automated weekly previsit planning report. Additionally, we implemented a process to stock pneumococcal vaccines and shared best practices among the divisions. We monitored improvement through times series and run charts of PPSV23 vaccination rates. RESULTS: The initial PPSV23 vaccination rate for applicable high-risk patients was ∼20%. There was an increase in vaccination rate to ∼60%. All 3 divisions showed improvements in their individual PPSV23 vaccination rates. CONCLUSIONS: Using quality improvement methodology, we increased PPSV23 vaccination rates in 3 pediatric specialty clinics, and this improvement was sustained. We will continue to identify best practices and actively recruit additional divisions because we have the opportunity to reach >9000 high-risk patients.

A pragmatic parallel group implementation study of a prehospital-activated ECPR protocol for refractory out-of-hospital cardiac arrest.

OBJECTIVES: Extracorporeal membrane oxygenation within CPR (ECPR) may improve survival among patients with refractory out-of-hospital cardiac arrest (OHCA). We evaluated outcomes after incorporating ECPR into a conventional resuscitation system. METHODS: We introduced a prehospital-activated ECPR protocol for select refractory OHCAs into one of four metropolitan regions in British Columbia. We prospectively identified ECPR-eligible patients in both the ECPR region and the three other regions to serve as the control group. We compared the proportion with favorable neurological outcomes at hospital discharge (cerebral performance category ≤2) and used logistic regression to estimate the association with treatment region. RESULTS: The study was terminated prematurely due to changes in hospital protocols and COVID-19. In the ECPR region, 15/58 (25.9%) patients had favourable neurological outcomes owing to conventional resuscitation and 2/58 (3.4%) owing to ECPR, for a total of 17/58 (29.3%). In the control regions, 67/250 (26.8%) patients had a favourable outcome owing to conventional resuscitation, for a between-group difference of 2.5% (95% CI -10 to 15%). We did not detect a statistically significant association between treatment region and outcomes. CONCLUSION: In this prematurely-terminated study of ECPR for refractory OHCA, we did not detect an association between a regional ECPR protocol and neurologically favorable outcomes. However, our data suggests that outcomes owing to conventional resuscitation were similar, with the potential for additional survivors due to ECPR therapies.

Medial gastrocnemius muscles fatigue but do not atrophy in paralyzed cat hindlimb after long-term spinal cord hemisection and unilateral deafferentation.

This study of medial gastrocnemius (MG) muscle and motor units (MUs) after spinal cord hemisection and deafferentation (HSDA) in adult cats, asked 1) whether the absence of muscle atrophy and unaltered contractile speed demonstrated previously in HSDA-paralyzed peroneus longus (PerL) muscles, was apparent in the unloaded HSDA-paralyzed MG muscle, and 2) how ankle unloading impacts MG muscle and MUs after dorsal root sparing (HSDA-SP) with foot placement during standing and locomotion. Chronic isometric contractile forces and speeds were maintained for up to 12 months in all conditions, but fatigability increased exponentially. MU recordings at 8-11½ months corroborated the unchanged muscle force and speed with significantly increased fatigability; normal weights of MG muscle confirmed the lack of disuse atrophy. Fast MUs transitioned from fatigue resistant and intermediate to fatigable accompanied by corresponding fiber type conversion to fast oxidative (FOG) and fast glycolytic (FG) accompanied by increased GAPDH enzyme activity in absolute terms and relative to oxidative citrate synthase enzyme activity. Myosin heavy chain composition, however, was unaffected. MG muscle behaved like the PerL muscle after HSDA with maintained muscle and MU contractile force and speed but with a dramatic increase in fatigability, irrespective of whether all the dorsal roots were transected. We conclude that reduced neuromuscular activity accounts for increased fatigability but is not, in of itself, sufficient to promote atrophy and slow to fast conversion. Position and relative movements of hindlimb muscles are likely contributors to sustained MG muscle and MU contractile force and speed after HSDA and HSDA-SP surgeries.

Synthesis of Highly Potent N-10 Amino-Linked DNA-Alkylating Indolinobenzodiazepine Antibody-Drug Conjugates (ADCs).

Indolinobenzodiazepine DNA alkylators (IGNs) are the cytotoxic payloads in antibody-drug conjugates (ADCs) currently undergoing Phase I clinical evaluation (IMGN779, IMGN632, and TAK164). These ADCs possess linkers that have been incorporated into a central substituted phenyl spacer. Here, we present an alternative strategy for the IGNs, linking through a carbamate at the readily available N-10 amine present in the monoimine containing dimer. As a result, we have designed a series of N-10 linked IGN ADCs with a wide range of in vitro potency and tolerability, which may allow us to better match an IGN with a particular target based on the potential dosing needs.

Effect of Linker Stereochemistry on the Activity of Indolinobenzodiazepine Containing Antibody-Drug Conjugates (ADCs).

Antibody-drug conjugates (ADCs) that incorporate potent indolinobenzodiazepine DNA alkylators as the payload component are currently undergoing clinical evaluation. In one ADC design, the payload molecules are linked to the antibody through a peptidase-labile l-Ala-l-Ala linker. In order to determine the role of amino acid stereochemistry on antitumor activity and tolerability, we incorporated l- and d-alanyl groups in the dipeptide, synthesized all four diastereomers, and prepared and tested the corresponding ADCs. Results of our preclinical evaluation showed that the l-Ala-l-Ala configuration provided the ADC with the highest therapeutic index (antitumor activity vs toxicity).

Bioanalytical workflow for novel scaffold protein-drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin-drug conjugate in plasma and tissue samples using liquid chromatography-tandem mass spectrometry.

AIM: Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates. METHODOLOGY: Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively. CONCLUSION: The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.

A DNA-Interacting Payload Designed to Eliminate Cross-Linking Improves the Therapeutic Index of Antibody-Drug Conjugates (ADCs).

Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA-cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here, we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared with those of the cross-linker. Thus, the improved in vivo tolerability and antitumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment. Mol Cancer Ther; 17(3); 650-60. ©2018 AACR.

A comprehensive regional clinical and educational ECPR protocol decreases time to ECMO in patients with refractory out-of-hospital cardiac arrest.

OBJECTIVE: Extracorporeal membrane oxygenation within CPR (ECPR) may improve survival for refractory out-of-hospital cardiac arrest (OHCA). We developed a prehospital, emergency department (ED), and hospital-based clinical and educational protocol to improve the key variable of time-to-ECPR (TTE). METHODS: In a single urban health region we involved key prehospital, clinical, and administrative stakeholders over a 2-year period, to develop a regional ECPR program with destination to a single urban tertiary care hospital. We developed clear and reproducible inclusion criteria and processes, including measures of program efficiency. We conducted seminars and teaching modules to paramedics and hospital-based clinicians including monthly simulator sessions, and performed detailed reviews of each treated case in the form of report cards. In this before-and-after study we compared patients with ECPR attempted prior to, and after, protocol implementation. The primary outcome was TTE, defined as the time of initial professional CPR to establishment of extracorporeal circulation. We compared the median TTE for patients in the two groups using the Wilcoxon signed rank test. RESULTS: Four patients were identified prior to the protocol and managed in an ad hoc basis; for nine patients the protocol was utilized. Overall favourable neurological outcomes among ECPR-treated patients were 27%. The median TTE was 136 minutes (IQR 98 - 196) in the pre-protocol group, and 60 minutes (IQR 49 - 81) minutes in the protocol group (p=0.0165). CONCLUSION: An organized clinical and educational protocol to initiate ECPR for patients with OHCA is feasible and significantly reduces the key benchmark of time-to-ECPR flows.