The Biochemical Basis of Congenital Disorders of Glycosylation

The enzyme peptide: N-glycanase (EC 3.5.1.52) is an endoglycosidase which cleaves N-linked glycans from incorrectly folded glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). Clinical mutations in this enzyme (resulting in loss or decrease in function) are responsible for the rare congenital disorder termed N-GLY1. N-GLY1 disorder was first identified by next generation sequencing in 2012, and as such there exists little information as to the cellular effects of N-glycanase deficiency. In this study, N-glycanase deficiency was examined in a well-characterised cell model (HEK-293). Cellular effects were examined following pharmacological inhibition of N-glycanase using carbobenzoxy-valyl-ananyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) and, genetic knockdown by siRNA. Attempts were also made to generate a knockout cell line using CRISPR/Cas9. Using molecular and cellular techniques, this study has confirmed that in HEK-293, pharmacological N-glycanase inhibition or siRNA knockdown does not activate ER stress, oxidative stress or change the glycoprotein profile of the cell. Both treatments were associated with a small increase in ThT fluorescence associated with protein aggregates and identified that autophagy was increased in pharmacological N-glycanase inhibition and siRNA knockdown, and that this likely represents a protective measure. Autophagy dysregulation using ATG13 KO MEF cells found a change in the redox environment of the cell. This effect was further exacerbated in both HEK293, WT MEFs and ATG13 KO MEFs by growth in glucose-free media supplemented with galactose to reduce glycolysis as an energy source. This is suggestive of mitochondrial dysregulation, although no changes in mitochondrial membrane potential or mitochondrial content were identified. Proteomics analysis of immuno-precipitated autophagosomes induced under pharmacological N-glycanase inhibition and siRNA knockdown revealed increased intensity of mitochondrial proteins in these samples indicative of mitophagy which may play a role in the cellular response to impairments of N-glycanase function

Building open lab hardware to tackle antimicrobial resistance

Microbiological imaging can be lengthy and labour-intensive without expensive lab-automation. Using Open Source hardware and software, Dr Al Edwards’ Biomedical Technology Lab designed a robot to take high-resolution images of experiments capturing changes in colour and fluorescence due to bacterial growth. This affordable solution has increased the Lab’s capability to image and screen samples with higher throughput, accuracy, and more kinetic data. The system is fully customisable to suit any imaging experiment and costs only £600 to build from Open Source resources

Data associated with the article ‘Remote videolink observation of model home sampling and home testing devices to simplify usability studies for point-of-care diagnostics’

This dataset contains the video instruction resource used for remote testing of model home testing devices. The questionnaire given to participants along with a summary table of responses to the rating questions. This dataset contains images of model tests returned by participants during the study ‘Remotely evaluating user experience of Covid-19 lateral flow devices’. A spreadsheet summary of the analysis of the returned images is also included

Investigation of gibberellin signalling and the effect of n-acyl homoserine lactones on germination in Physcomitrella patens and an investigation of the interaction between PGE2 and Phospholipase B1 in a Cryptococcus/Macrophage Infection Model

This project consists of two separate research projects each covering 4 months. The first project studies the role of gibberellin signalling and the impact of bacterial quorum sensing molecules, AHLs, in $Physcomitrella$. $Physcomitrella$ patens is a model organism of early land plants and is a useful tool for studying plant evolution. By studying the germination rates of wild type spores and spores that lack the enzyme to produce gibberellins and spores in the presence of AHLs it was found that a gibberellin precursor is important in controlling germination signals in the model moss system. It was also found that long chain AHLs in all substitution states, oxo and hydroxyl and non-substituted, significantly promoted germination in $Physcomitrella$. The second project looked at the role of PGE2 during $Cryptococcus$ infection using a murine macrophage like cell line to study infection. $Cryptococcus$ $neoformans$ is an intracellular opportunistic fungal pathogen that can cause fatal infections in individuals with compromised immune systems. By studying the intracellular proliferation rates and cell viability counts of the wild type fungi with a mutant that did not produce PGE2 it was found that PGE2 produced by the fungi was important for intracellular survival and growth

Dataset associated with the article 'Exploiting Open Source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology'

This dataset is for data analysis associated with the article 'Exploiting Open Source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology'. It contains the raw images and Excel analysis of microtitre plates (MTP) and microcapillary film (MCF) for milk matrix experiments, POLIR quantification, and first experiment testing fluorescence on POLIR

Challenges in microfluidic and point-of-care phenotypic antimicrobial resistance tests

To combat the threat to public health of antimicrobial resistance, there is a need for faster, more portable diagnostic tools to aid in antibiotic selection. Current methods for determining antimicrobial resistance of pathogens in clinical samples take days to result and require high levels of user input. Microfluidics offers many potential benefits, reducing time to result, user input, and allowing point of care testing. This review focuses on the challenges of developing functional or phenotypic microfluidic antimicrobial susceptibility tests; such methods complement other vital tools such as nucleic acid detection. Some of the most important challenges identified here are not unique to microfluidics but apply to most antimicrobial susceptibility testing innovations and relate to the nature of the sample being tested. For many high priority samples, mixtures of bacteria, highly variable target cell density, and the sample matrix can all affect measurements, and miniaturization can create sensitivity problems if target bacteria are dilute. Recent advances including smartphone capability, new sensors, microscopy, and a resurgence in paper microfluidics offer important opportunities for microfluidic engineering to simplify functional and phenotypic antimicrobial susceptibility testing. But the complexity of most clinical samples remains one of the biggest barriers to rapid uptake of microfluidics for antimicrobial resistance testing

Dilution reduces sample matrix effects for rapid, direct, and miniaturised phenotypic antibiotic susceptibility tests for bovine mastitis

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic “dip-and-test” devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 105 CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy

Open hardware for microfluidics: exploiting Raspberry Pi singleboard computer and camera systems for customisable laboratory instrumentation

The integration of Raspberry Pi miniature computer systems with microfluidics has revolu-tionized the development of low-cost and customizable analytical systems in life science labor-atories. This review explores the applications of Raspberry Pi in microfluidics, with a focus on imaging, including microscopy and automated image capture. By leveraging the low-cost, flexi-bility and accessibility of Raspberry Pi components, high-resolution imaging and analysis have been achieved in direct mammalian and bacterial cellular imaging and a plethora of image based biochemical and molecular assays, from immunoassays, through microbial growth, to nucleic acid methods such as real-time-qPCR. The control of image capture permitted by Raspberry Pi hard-ware can also be combined with onboard image analysis. Open-source hardware offers an op-portunity to develop complex laboratory instrumentation systems at a fraction of the cost of commercial equipment and importantly, offer an opportunity to completely customise to meet the users’ needs. However, these benefits come with a trade-off: challenges remain for those wishing to incorporate open-source hardware equipment in their own work, including requirements for construction and operator skill, need for good documentation and the availability of rapid pro-totyping such as 3D printing plus other components. These advances in open-source hardware have the potential to improve efficiency, accessibility, and cost-effectiveness of microfluidic-based experiments and applications

Off‐target inhibition of NGLY1 by the poly‐caspase inhibitor Z‐VAD‐fmk induces cellular autophagy

The pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-inked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER-associated degradation (ERAD). Both pharmacological N-glycanase inhibition by Z-VAD-fmk and siRNA-mediated knockdown (KD) of NGLY1 induce GFP-LC3 positive puncta in HEK 293 cells. Activation of ER stress markers or induction of reactive oxygen species (ROS) are not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry-based proteomics analysis reveal comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation

NGLY1 knockdown or pharmacological inhibition induces cellular autophagy

Pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). Pharmacological N-glycanase inhibition by Z-VAD-fmk or siRNA knockdown (KD) induces GFP-LC3 positive puncta in HEK 293 cells. Activation of ER stress markers or reactive oxygen species (ROS) induction are not observed. In NGLY1 inhibition or KD, upregulation of autophagosome formation without impairment of autophagic flux are observed. Enrichment and proteomics analysis of autophagosomes after Z-VAD-fmk treatment or NGLY1 KD reveals comparable autophagosomal protein content. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation