UK veterinary professionals' perceptions and experiences of adverse drug reaction reporting.

BACKGROUND: Spontaneous reporting of suspected adverse drug reactions (ADRs) is the cornerstone of pharmacovigilance. Despite this, it is believed that there is significant under-reporting in the veterinary setting. Low reporting rates delay marketing authorisation holders (MAHs) and regulators taking mitigating action in the case of safety concerns. METHOD: We designed a survey to explore the perceptions, attitudes and experiences of UK veterinary professionals towards ADR reporting. The survey was advertised widely through conventional and social media and at several conferences. RESULTS: In total, 260 respondents completed the survey, including 210 veterinary surgeons, 49 veterinary nurses and one suitably qualified person. Respondents generally understood the need to report ADRs. The main barrier to reporting was the suspected ADR being well known, and the most popular potential facilitator identified was the ability to report via the practice management system. Facilitation via education in the form of a pharmacovigilance themed continuing professional development event was particularly popular among veterinary nurses, who reported time as being less of a barrier to reporting than their veterinary surgeon counterparts. CONCLUSIONS: Our findings suggest that technological interventions to facilitate reporting and empowerment of veterinary nurses to report through a tailored training event should be explored further.

Antibody charge variant modulation by in vitro enzymatic treatment in different Chinese hamster ovary cell cultures.

Charge variants represent a critical quality attribute that must be controlled during the development and manufacturing of monoclonal antibodies (mAb). Previously, we reported the development of a cost-effective enzymatic treatment capable of removing the C-terminal lysine from a mAb produced by a Chinese hamster ovary (CHO) GS cell line. This treatment resulted in a significant decrease in basic charge variants and a corresponding improvement in the main peak, enabling a longer cell culture production duration for titer improvement. Here, we describe this enzymatic treatment protocol in detail and demonstrate its applicability to two additional mAbs produced by distinct industrial cell lines. The simple addition of carboxypeptidase B (CpB) at a ratio of 1:10,000 (w/w) to whole cell cultures significantly improved the main peaks for both mAbs without affecting other critical quality attributes, including size exclusion chromatography impurities and N-glycans. Our results demonstrate that this in vitro CpB treatment protocol can be used as a platform strategy to improve main peak for mAbs that exhibit high levels of basic variants attributable to C-terminal lysines. An in vitro enzymatic treatment in general may be another good addition to existing in vivo CHO cell culture strategies for titer improvement and control of critical quality attributes.

LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria.

The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.

Folding the Mitochondrial UPR into the Integrated Stress Response.

Eukaryotic cells must accurately monitor the integrity of the mitochondrial network to overcome environmental insults and respond to physiological cues. The mitochondrial unfolded protein response (UPRmt) is a mitochondrial-to-nuclear signaling pathway that maintains mitochondrial proteostasis, mediates signaling between tissues, and regulates organismal aging. Aberrant UPRmt signaling is associated with a wide spectrum of disorders, including congenital diseases as well as cancers and neurodegenerative diseases. Here, we review recent research into the mechanisms underlying UPRmt signaling in Caenorhabditis elegans and discuss emerging connections between the UPRmt signaling and a translational regulation program called the 'integrated stress response'. Further study of the UPRmt will potentially enable development of new therapeutic strategies for inherited metabolic disorders and diseases of aging.

Conserved juxtamembrane domains in the yeast golgin Coy1 drive assembly of a megadalton-sized complex and mediate binding to tethering and SNARE proteins.

The architecture and organization of the Golgi complex depend on a family of coiled-coil proteins called golgins. Golgins are thought to form extended homodimers that are C-terminally anchored to Golgi membranes, whereas their N termini extend into the cytoplasm to initiate vesicle capture. Previously, we reported that the Saccharomyces cerevisiae golgin Coy1 contributes to intra-Golgi retrograde transport and binds to the conserved oligomeric Golgi (COG) complex and multiple retrograde Golgi Q-SNAREs (where SNARE is soluble NSF-attachment protein receptor). Here, using various engineered yeast strains, membrane protein extraction and fractionation methods, and in vitro binding assays, we mapped the Coy1 regions responsible for these activities. We also report that Coy1 assembles into a megadalton-size complex and that assembly of this complex depends on the most C-terminal coiled-coil and a conserved region between this coiled-coil and the transmembrane domain of Coy1. We found that this conserved region is necessary and sufficient for binding the SNARE protein Sed5 and the COG complex. Mutagenesis of conserved arginine residues within the C-terminal coiled-coil disrupted oligomerization, binding, and function of Coy1. Our findings indicate that the stable incorporation of Coy1 into a higher-order oligomer is required for its interactions and role in maintaining Golgi homeostasis. We propose that Coy1 assembles into a docking platform that directs COG-bound vesicles toward cognate SNAREs on the Golgi membrane.

The Golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs.

Extended coiled-coil proteins of the Golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the Golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde Golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the Conserved Oligomeric Golgi complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, co-immunoprecipitation experiments revealed an association with the Conserved Oliogmeric Golgi (COG) complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5 and Sft1. Thus, our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles.

Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.

Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.