Macroautophagic cargo sequestration assays

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.

TAP Reactor Development and Application in the Kinetic Characterization of Catalysts for Heterogeneously Catalyzed Gas Phase Reactions

This manuscript describes the application and further development of the TAP technique in kinetic characterization of heterogeneous catalysis. The major application of TAP systems is to study mechanisms, kinetics and transport phenomena in heterogeneous catalysis, all of which is made possible by the sub-millisecond time resolution. Furthermore, the kinetic information obtained can be used to gain an insight into the mechanism occurring over the catalyst system. This is advantageous as heterogeneous catalysts with an improved efficiency can be developed as a result. TAP kinetic studies are carried out at low pressure (~1x10-7 mbar) and TAP pulses are sufficiently small (1013-1015 molecules) so as to maintain this low pressure. The use of a small number of molecules in comparison to the total number of active sites means the state of the catalyst remains relatively unchanged. The use of the low intensity pulses also makes the pressure gradient negligible and so allows the TAP reactor system to operate in the Knudsen Diffusion regime, where gas-gas reactions are eliminated. Hence only gas-catalyst reactions are investigated and, by the use of moment analysis of observed exit flow, rate constants of elementary steps of the reaction can be obtained.

In this manuscript, two attempts to further the TAP technique are reported. Firstly, the work undertaken at QUB to attempt to control the number of molecules of condensable reagents that can be pulsed during a TAP pulse experiment is disclosed. Secondly, a collaborative project with SAI Ltd Manchester is discussed in a separate chapter, where technical details and validation of a customised time of flight mass spectrometer (ToF MS) for the QUB TAP-1 system are reported. A collaborative project with Cardiff Catalysis Institute focusing on the study of CO oxidation over hopcalite catalysts is also reported. The analysis of the experimental results has provided an insight into the possible mechanism of the oxidation of CO over these catalysts. A correction function has also been derived which accounts for the adsorption of reactant molecules over inert materials that are used for the reactor packing in TAP experiments. This function was then applied to the selective reduction of O2 in a H2 rich ethene feed, so that more accurate TAP moment based analysis could be conducted.

Development of a Novel Assay for the Detection of Active Neutrophil Elastase in Patients with Chronic Obstructive Pulmonary Disease

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including chronic obstructive pulmonary disease (COPD). However, it’s detection and quantification in biological samples is confounded by a lack of reliable and robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex clinical samples containing multiple proteolytic and hydrolytic enzymes which have the ability to hydrolyse the substrate, thereby resulting in an over-estimation of the target protease. Furthermore, ELISA systems measure total protease levels which can be a mixture of latent, active and protease-inhibitor complexes. Therefore, we have developed a novel immunoassay (ProteaseTag™ Active NE Immunoassay) which is selective and specific for the capture of active NE in sputum and Bronchoalveolar Lavage (BAL) in patients with COPD. The objective of this study was to clinically validate ProteaseTag™ Active NE Ultra Immunoassay for the detection of NE in sputum from COPD patients. 20 matched sputum sol samples were collected from 10 COPD patients (M=6, F=4; 73 ± 6 years) during stable and exacerbation phases. Samples were assayed for NE activity utilising both ProteaseTag™ Active NE Ultra Immunoassay and a fluorogenic substrate-based kinetic activity assay. Both assays detected elevated levels of NE in the majority of patients (n=7) during an exacerbation (mean=217.2 μg/ml ±296.6) compared to their stable phase (mean=92.37 μg/ml ±259.8). However, statistical analysis did not show this difference to be significant (p=0.07, ProteaseTag™ Active NE Ultra Immunoassay; p=0.06 kinetic assay), most likely due to the low study number. A highly significant correlation was found between the 2 assay types (p≤0.0001, r=0.996). NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE Immunoassay specifically measures only active NE in clinical samples, is quick and easy to use (< 3 hours) and has no dependency on a kinetic readout. ProteaseTag™ technology is currently being transferred to a lateral flow device for use at Point of Care.

Considerations for optimization of microRNA PCR assays for molecular diagnosis

The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.