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

Transcriptional upregulation of c-MET is associated with invasion and tumor budding in colorectal cancer

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis

Ultra-High Carrier Mobilities in Ferroelectric Domain Wall Corbino Cones at Room Temperature

Recently, electrically conducting heterointerfaces between dissimilar band-insulators (such as lanthanum aluminate and strontium titanate) have attracted considerable research interest. Charge transport has been thoroughly explored and fundamental aspects of conduction firmly established. Perhaps surprisingly, similar insights into conceptually much simpler conducting homointerfaces, such as the domain walls that separate regions of different orientations of electrical polarisation within the same ferroelectric band-insulator, are not nearly so well-developed. Addressing this disparity, we herein report magnetoresistance in approximately conical 180° charged domain walls, which occur in partially switched ferroelectric thin film single crystal lithium niobate. This system is ideal for such measurements: firstly, the conductivity difference between domains and domain walls is extremely and unusually large (a factor of at least 1013) and hence currents driven through the thin film, between planar top and bottom electrodes, are overwhelmingly channelled along the walls; secondly, when electrical contact is made to the top and bottom of the domain walls and a magnetic field is applied along their cone axes (perpendicular to the thin film surface), then the test geometry mirrors that of a Corbino disc, which is a textbook arrangement for geometric magnetoresistance measurement. Our data imply carriers at the domain walls with extremely high room temperature Hall mobilities of up to ~ 3,700cm2V-1s-1. This is an unparalleled value for oxide interfaces (and for bulk oxides too) and is most comparable to mobilities in other systems typically seen at cryogenic, rather than at room, temperature

Microglia protect against age-associated brain pathologies

Microglia are brain-resident macrophages that contribute to central nervous system (CNS) development, maturation, and preservation. Here, we examine the consequences of permanent microglial deficiencies on brain aging using the Csf1r^ΔFIRE/ΔFIRE mouse model. In juvenile Csf1r^ΔFIRE/ΔFIRE mice, we show that microglia are dispensable for the transcriptomic maturation of other brain cell types. By contrast, with advancing age, pathologies accumulate in Csf1r^ΔFIRE/ΔFIRE brains, macroglia become increasingly dysregulated, and white matter integrity declines, mimicking many pathological features of human CSF1R-related leukoencephalopathy. The thalamus is particularly vulnerable to neuropathological changes in the absence of microglia, with atrophy, neuron loss, vascular alterations, macroglial dysregulation, and severe tissue calcification. We show that populating Csf1r^ΔFIRE/ΔFIRE brains with wild-type microglia protects against many of these pathological changes. Together with the accompanying study by Chadarevian and colleagues, our results indicate that the lifelong absence of microglia results in an age-related neurodegenerative condition that can be counteracted via transplantation of healthy microglia.ISSN:0896-6273ISSN:1097-419

Development of Gold Nanocarriers to Deliver Proteins and Peptides to the CNS

Treatment of many CNS disorders, specifically neurodegenerative disorders are severely limited at present, and with an increase in the aging population, research into an effective treatment is necessary. A major issue is that many therapeutic agents cannot access the brain, due to a structure termed the blood brain barrier (BBB), which excludes many potential therapeutic drugs from the CNS of the drugs that could possibly treat these disorders. Cellular delivery of bioactive molecules e.g. antibodies, peptides and cytokines are a growing area of research due to their possible therapeutic potential. There have been several cytokines which have been successfully used in experimental models of many neurodegenerative diseases but have had difficulty in being translated into clinical trials. These cytokines do not cross the BBB by themselves but if attached to an effective nanocarrier that is able to cross this barrier, could be translated into a much-needed treatment. Gold Nanoparticles (AuNP) have been selected due to their numerous useful characteristics, such as easy production, biological compatibility and chemical stability. Previous research has shown that glucose derived-coated gold nanoparticles can bind to, and cross human brain endothelial cells in vitro and rat brain endothelial cells in vivo. 15 formulations of ligand coated AuNPs were investigated using a simple model of the BBB, to determine which formulation could cross the brain endothelium most efficiently. PEGamine/GalactoseC2 and GlucoseC2 ligand coated AuNPs were able to cross most effectively and were taken forward for further experimentation; GalactoseC2 coated AuNPs were used as a control ligand coating. BDNF, the chosen cytokine of interest for attachment to AuNP, was mutagenically altered (Histidine→Cysteine) in the His tag region of a plasmid used to produce recombinant human BDNF to produce a free thiol group on the Cysteine allowing a place-exchange reaction onto the surface of the AuNP. We optimized the production of our modified BDNF (BDNF-H9C) and developed effective techniques to measure the quantity and biological activity of the protein produced (ELISA and TrK-B Assay). However insufficient quantity of BDNF-H9C could be produced from transfected cells and purified to perform an exchange reaction. A series of peptides was analysed for their ability to bind the transferrin receptor expressed on brain endothelium in vitro (hCMEC/D3 cells). One strongly-binding peptide was selected for attachment to AuNPs and this was found to increase the amount of AuNP that crossed a BBB model

Gold nanoparticles for imaging and drug transport to the CNS

Gold nanoparticles with a core size of 2 nm covalently coated with glycans to maintain solubility, targeting molecules for brain endothelium, and cargo molecules hold great potential for delivery of therapies into the CNS. They have low toxicity, pass through brain endothelium in vitro and in vivo, and move rapidly through the brain parenchyma. Within minutes of infusion the nanoparticles can be detected in neurons and glia. These nanoparticles are relatively easy to synthesize in association with their surface ligands. They can be detected by electron microscopy, ICP-mass spectrometry, and spectroscopy. However, modification of the basic gold nanoparticle is required for in vivo imaging by MR or radioactive methods. Depending on their surface coat, the nanoparticles cross the brain endothelium by the plasma membrane/cytosolic route (passive transport) or by vesicular transcytosis (active transport). A primary aim of current research is to improve the biodistribution of the nanoparticles for CNS drug delivery. Smaller gold nanoparticles are removed rapidly via the kidney, while larger nanoparticles are taken up by mononuclear phagocytes in various tissues. Receptors selectively located on brain endothelium can act as targets for the nanoparticles, to increase their delivery to the brain