Biology subjects

Gray, M.

Publications and source records attributed to Gray, M..

22 records · Page 2Linked to original sources

bioRxiv2019

Rapid, selective and single cell electrical detection of antibiotic resistant bacteria

The rapid increase in antibiotic resistant pathogenic bacteria has become a global threat, which besides the development of new drugs, requires rapid, cheap, scalable, and accurate diagnostics. Label free biosensors relying on electrochemical, mechanical, and mass based detection of whole bacterial cells have attempted to meet these requirements. However, the trade-off between selectivity and sensitivity of such sensors remains a key challenge. In particular, point-of-care diagnostics that are able to reduce and/or prevent unneeded antibiotic prescriptions require highly specific probes with sensitive and accurate transducers that can be miniaturized and multiplexed, and that are easy to operate and cheap. Towards achieving this goal, we present a number of advances in the use of graphene field effect transistors (G-FET) including the first use of peptide probes to electrically detect antibiotic resistant bacteria in a highly specific manner. In addition, we dramatically reduce the needed concentration for detection by employing dielectrophoresis for the first time in a G-FET, allowing us to monitor changes in the Dirac point due to individual bacterial cells. Specifically, we realized rapid binding of bacterial cells to a G-FET by electrical field guiding to the device to realize an overall 3 order of magnitude decrease in cell-concentration enabling a single-cell detection limit, and 9-fold reduction in needed time to 5 minutes. Utilizing our new biosensor and procedures, we demonstrate the first selective, electrical detection of the pathogenic bacterial species Staphylococcus aureus and antibiotic resistant Acinetobacter baumannii on a single platform.

microbiology↗

bioRxiv2019

Rheumatoid arthritis patients express a skewed repertoire of polyclonal, hypomutated B-cell receptors

ObjectivesThe success of B cell depletion therapy in rheumatoid arthritis (RA) therapy testifies to their importance in disease pathogenesis, but the precise B cells mediating this are unclear. For example, it is unknown if RA patients predominantly express a limited number of circulating clonally expanded populations of B cells with highly mutated B cell antigen receptors (BCRs) that would constitute a shared antigen driven response.\n\nMethodsTo address this, we have undertaken the largest study to date utilising next generation sequencing (NGS), to identify the full length of the peripheral blood BCR sequences from the antigen-binding heavy chain. Between 25,000 to 200,000 BCR sequences per patient were analysed from 127 newly diagnosed RA patients, 16 heathy controls, 16 RA patients with established disease and 8 paired blood and synovial samples. This was complemented with B cell subset analysis from an additional 64 RA patients and 22 healthy controls.\n\nResultsRA patients expressed a significantly higher percentage of circulating poorly mutated polyclonal IgG+ve variable heavy (IgG-Vh) BCR sequences, both at the time of diagnosis and following treatment. These sequences resided predominantly within TNF-alpha secreting IgG+veCD27-ve B cells, that were expanded in RA peripheral blood and enriched in the rheumatoid synovium. Surprisingly, peripheral and synovial B cell repertoires of RA patients are quite distinct, sharing very few IgG sequences.\n\nConclusionsThis is the first report to conclusively establish that a substantial component of the peripheral B cell repertoire in RA consists of polyclonal hypomutated IgG+ve BCRs that may play a critical role in driving an autoimmune mediated inflammation.

immunology↗

bioRxiv2018

Changes in excitability and ion channel expression in neurons of the major pelvic ganglion in female type II diabetic mice

Bladder cystopathy is a common urological complication of diabetes, and has been associated with changes in parasympathetic ganglionic transmission and some measures of neuronal excitability in male mice. To determine whether type II diabetes also impacts excitability of parasympathetic ganglionic neurons in females, we investigated neuronal excitability and firing properties, as well as underlying ion channel expression, in major pelvic ganglion (MPG) neurons in control, 10-week, and 21-week db/db mice. Type II diabetes in Leprdb/db animals caused a non-linear change in excitability and firing properties of MPG neurons. At 10 weeks, cells exhibited increased excitability as demonstrated by an increased likelihood of firing multiple spikes upon depolarization, decreased rebound spike latency, and overall narrower action potential half-widths as a result of increased depolarization and repolarization slopes. Conversely, at 21 weeks MPG neurons of db/db mice reversed these changes, with spiking patterns and action-potential properties largely returning to control levels. These changes are associated with numerous time-specific changes in calcium, sodium, and potassium channel subunit mRNA levels. However, Principal Components Analysis of channel expression patterns revealed that the rectification of excitability is not simply a return to control levels, but rather a distinct ion channel expression profile in 21-week db/db neurons. These data indicate that type II diabetes can impact the excitability of post-ganglionic, parasympathetic bladder-innervating neurons of female mice, and suggest that the non-linear progression of these properties with diabetes may be the result of compensatory changes in channel expression that act to rectify disrupted firing patterns of db/db MPG neurons.

neuroscience↗

bioRxiv2018

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen binding affinity

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination, and instead that it would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain super-physiologic numbers of bNAb precursor expressing B cells and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center B cell recrutiment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the germinal center when there are as few as 10 such cells per mouse. However, at low precursor frequencies the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to germinal centers is variable and dependent on re-circulation.\n\nSignificance statementAn essential requirement for an HIV-vaccine is to elicit antibodies to conserved regions of the spike protein (Env) becasue these antibodies can protect against infection. Although broadly neutralizing antibodies develop naturally in rare individuals after prolongued HIV infection, eliciting them by vaccination has only been possible in artificial knock-in mouse models wherein the number of B cells expressing the antibody precursor is super-physiologic. To understand the relationship between precursor frequency, antigen affinity and germinal center recruitment we have performed adoptive transfer experiments in which fixed numbers of precursor cells are engrafted in wild type mice. Our results provide a framework for understanding how precursor frequency and antigen affinity shape humoral immunity to HIV.

immunology↗