Reproducibility is severely limited if instrument performance is assumed rather than measured. Within optical microscopy, instrument performance is typically measured using sub-resolution fluorescent beads. However, the process is performed infrequently as it is requires time and suitably trained staff to acquire and then process the bead images.
View Article and Find Full Text PDFPersister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells.
View Article and Find Full Text PDFEnvironmental and intracellular stresses can perturb protein homeostasis and trigger the formation and accumulation of protein aggregates. It has been recently suggested that the level of protein aggregates accumulated in bacteria correlates with the frequency of persister and viable but nonculturable cells that transiently survive treatment with multiple antibiotics. However, these findings have often been obtained employing fluorescent reporter strains.
View Article and Find Full Text PDFBackground And Purpose: Functional brain imaging using genetically encoded Ca sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABA antagonism. Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug-induced seizures and testing antiepileptic drug efficacy.
View Article and Find Full Text PDFThe double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to rapidly quantify antibiotic accumulation in hundreds of individual Escherichia coli cells.
View Article and Find Full Text PDFDimethyl sulfoxide (DMSO) is widely used in a number of biological and biotechnological applications, mainly because of its effects on the cell plasma membrane, but the molecular origins of this action are yet to be fully clarified. In this work, we used two- and three-component synthetic membranes (liposomes) and the plasma membrane of human erythrocytes to investigate the effect of DMSO when added to the membrane-solvating environment. Fourier transform infrared spectroscopy and thermal fluctuation spectroscopy revealed significant differences in the response of the two types of liposome systems to DMSO in terms of the bilayer thermotropic behavior, available free volume of the bilayer, its excess surface area, and bending elasticity.
View Article and Find Full Text PDFMitochondria are highly pleomorphic, undergoing rounds of fission and fusion. Mitochondria are essential for energy conversion, with fusion favouring higher energy demand. Unlike fission, the molecular components involved in mitochondrial fusion in plants are unknown.
View Article and Find Full Text PDFUncovering the heterogeneity of cellular populations and multicellular constructs is a long-standing goal in fields ranging from antimicrobial resistance to cancer research. Emerging technology platforms such as droplet microfluidics hold the promise to decipher such heterogeneities at ultra-high-throughput. However, there is a lack of methods able to rapidly identify and isolate single cells or 3D cell cultures.
View Article and Find Full Text PDFMorgana (Mora, also known as CHORD in flies) and its mammalian homologue, called CHORDC1 or CHP1, is a highly conserved cysteine and histidine-rich domain (CHORD)-containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in , loss of causes lethality and a complex mitotic phenotype that is rescued by a human transgene. Here, we show that Mora localises to mitotic spindles and co-purifies with the Hsp90-R2TP-TTT supercomplex and with additional well-known Hsp90 co-chaperones.
View Article and Find Full Text PDFBackground: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity.
View Article and Find Full Text PDFThe dynamic architecture of chromatin, the macromolecular complex comprised primarily of DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to chromatin are important markers of functional and developmental processes in cells. However, chromatin architecture regulation has not yet been fully elucidated.
View Article and Find Full Text PDFLive-cell imaging in microfluidic devices now allows the investigation of cellular heterogeneity within microbial populations. In particular, the mother machine technology developed by Wang et al. has been widely employed to investigate single-cell physiological parameters including gene expression, growth rate, mutagenesis, and response to antibiotics.
View Article and Find Full Text PDFPeroxisomes can be frequently found in proximity to other subcellular organelles such as the endoplasmic reticulum (ER), mitochondria or lysosomes. The tail-anchored protein ACBD5 was recently identified as part of a tethering complex at peroxisome-ER contact sites, interacting with the ER resident protein VAPB. Contact site disruption was found to significantly increase peroxisome motility, apparently interfering with intracellular positioning systems.
View Article and Find Full Text PDFAcute Lymphoblastic Leukemia (ALL) remains the most frequent cause of cancer-related mortality in children and novel therapies are needed for the treatment of relapsed/refractory childhood ALL. One approach is the targeting of ALL blasts with the immunotoxin CAT-8015. Although CAT-8015 has potent anti-leukemia activity, with a 32% objective response rate in a phase 1 study of childhood ALL, haemolytic-uremic syndrome (HUS) and vascular leak syndrome (VLS), major dose-limiting toxicities, have limited the use of this therapeutic approach in children.
View Article and Find Full Text PDFA correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
View Article and Find Full Text PDFBackground: Injections into the tendinous portion of the common extensor origin are a common intervention in the treatment of Lateral Elbow Tendinopathy (LET). Clinical trials report a heterogeneous selection of injectate volumes and delivery techniques, with systematic reviews finding no clear consensus. The aim of this study was to assess the intratendinous distribution and surrounding tissue contamination of ultrasound-guided injections into the Common Extensor Tendon (CET) of the elbow.
View Article and Find Full Text PDFPeroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors.
View Article and Find Full Text PDFBackground: Clonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population's resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health.
View Article and Find Full Text PDFOrganelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors.
View Article and Find Full Text PDFFunctional neuroimaging, using genetically-encoded Ca sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity.
View Article and Find Full Text PDFCellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons.
View Article and Find Full Text PDFThe hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex , the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of Augmin from and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information.
View Article and Find Full Text PDFPeroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells.
View Article and Find Full Text PDFEven distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions.
View Article and Find Full Text PDFRapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish.
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