Interference with lysosomal biogenesis by Salmonella Typhimurium

Salmonella enterica is an intracellular bacterial pathogen that replicates within membrane-bound vacuoles by delivering virulence (effector) proteins across the vacuolar membrane by the SPI-2 type III secretion system (T3SS). In this thesis I show that through the action of this T3SS, Salmonella selectively interferes with a subset of endosome-to-trans-Golgi network (TGN) traffic that is involved in mannose-6-phosphate receptor (MPR) recycling. Two pathways of endosome-to-TGN traffic have been described: one involves the Q-SNARE syntaxin 6; the other is defined by the Q-SNARE syntaxin 10 and involves retrograde transport of the MPRs. Salmonella specifically disrupted syntaxin 10-dependent endosome-to- TGN traffic and this was accompanied by redistribution of MPR from the TGN, misrouting of newly synthesized lysosomal enzymes and attenuation of lysosome function. The SPI-2 T3SS effector SifA is an important virulence determinant that is known to interact with the endolysosomal system. SifA was shown to be required and sufficient to mediate the effects on lysosome biogenesis during infection or following transfection of host cells. By contrast, a variant of SifA with a single amino acid substitution that prevents interaction with its host protein target, SKIP, failed to redistribute MPR or to reduce lysosomal function. SKIP was found to contribute to lysosomal biogenesis in uninfected cells. Inhibition of lysosomal enzyme function with a lysosomal protease inhibitor or by depleting cells of syntaxin 10 enhanced intracellular bacterial replication. I conclude that SifA attenuates lysosomal activity through its interaction with SKIP and that this facilitates intracellular bacterial replication

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the “insulin paradox”). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders

Inhibition of apoptosis and NF-κB activation by vaccinia protein N1 occur via distinct binding surfaces and make different contributions to virulence.

Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections

Regional mechanical and biochemical properties of the porcine cortical meninges

peer-reviewedThe meninges are pivotal in protecting the brain against traumatic brain injury (TBI), an ongoing issue in most mainstream sports. Improved understanding of TBI biomechanics and pathophysiology is desirable to improve preventative measures, such as protective helmets, and advance our TBI diagnostic/prognostic capabilities. This study mechanically characterised the porcine meninges by performing uniaxial tensile testing on the dura mater (DM) tissue adjacent to the frontal, parietal, temporal, and occipital lobes of the cerebellum and superior sagittal sinus region of the DM. Mechanical characterisation revealed a significantly higher elastic modulus for the superior sagittal sinus region when compared to other regions in the DM. The superior sagittal sinus and parietal regions of the DM also displayed local mechanical anisotropy. Further, fatigue was noted in the DM following ten preconditioning cycles, which could have important implications in the context of repetitive TBI. To further understand differences in regional mechanical properties, regional variations in protein content (collagen I, collagen III, fibronectin and elastin) were examined by immunoblot analysis. The superior sagittal sinus was found to have significantly higher collagen I, elastin, and fibronectin content. The frontal region was also identified to have significantly higher collagen I and fibronectin content while the temporal region had increased elastin and fibronectin content. Regional differences in the mechanical and biochemical properties along with regional tissue thickness differences within the DM reveal that the tissue is a non-homogeneous structure. In particular, the potentially influential role of the superior sagittal sinus in TBI biomechanics warrants further investigation

Enzymatic biofuel cells for self-powered, controlled drug release.

Self-powered drug delivery systems based on conductive polymers (CPs) that eliminate the need for external power sources, are of significant interest for use in clinical applications. Osmium redox polymer mediated glucose/O2 enzymatic biofuel cells (EBFCs) were prepared with an addi-tional CP-drug layer on the cathode. On discharging the EBFCs in the presence of glucose and dioxygen, model drug compounds incorporated in the CP layer were rapidly released with negligible amounts released when the EBFCs were held at open circuit. Controlled and ex situ release of three model compounds, ibuprofen (IBU), fluorescein (FLU) and 4',6-diamidino-2-phenylindole (DAPI), was achieved with this self-powered drug release system. DAPI released in situ in cell culture media was incorporated into retinal pigment epithelium (RPE) cells. This work demonstrates a proof-of-concept responsive drug release system that may be used in implantable devices

The mechanical responses of advecting cells in confined flow

Fluid dynamics have long influenced cells in suspension. Red blood cells and white blood cells are advected through biological microchannels in both the cardiovascular and lymphatic systems and, as a result, are subject to a wide variety of complex fluidic forces as they pass through. In vivo, microfluidic forces influence different biological processes such as the spreading of infection, cancer metastasis, and cell viability, highlighting the importance of fluid dynamics in the blood and lymphatic vessels. This suggests that in vitro devices carrying cell suspensions may influence the viability and functionality of cells. Lab-on-a-chip, flow cytometry, and cell therapies involve cell suspensions flowing through microchannels of approximately 100–800 μm. This review begins by examining the current fundamental theories and techniques behind the fluidic forces and inertial focusing acting on cells in suspension, before exploring studies that have investigated how these fluidic forces affect the reactions of suspended cells. In light of these studies’ findings, both in vivo and in vitro fluidic cell microenvironments shall also be discussed before concluding with recommendations for the fiel

Cell specific variation in viability in suspension in in vitro poiseuille flow conditions

The influence of Poiseuille flow on cell viability has applications in the areas of cancer metastasis, lab-on-a-chip devices and flow cytometry. Indeed, retaining cell viability is important in the emerging field of adoptive cell therapy, as cells need to be returned to patients’ bodies, while the viability of other cells, which are perhaps less accustomed to suspension in a fluidic environment, is important to retain in flow cytometers and other such devices. Despite this, it is unclear how Poiseuille flow affects cell viability. Following on from previous studies which investigated the viability and inertial positions of circulating breast cancer cells in identical flow conditions, this study investigated the influence that varying flow rate, and the corresponding Reynolds number has on the viability of a range of different circulating cells in laminar pipe flow including primary T cells, primary fibroblasts and neuroblastoma cells. It was found that Reynolds numbers as high as 9.13 had no effect on T-cells while the viabilities of neuroblastoma cells and intestinal fibroblasts were significantly reduced in comparison. This indicates that in vitro flow devices need to be tailored to cell-specific flow regime

The in vitro inertial positions and viability of cells in suspension under different in vivo flow conditions

The influence of Poiseuille flow on cell viability has applications in the areas of cancer metastasis, lab-on-a-chip devices and flow cytometry. Indeed, retaining cell viability is important in the emerging field of cell therapy as cells need to be returned to patients’ bodies. Despite this, it is unclear how this fundamental fluid regime affects cell viability. This study investigated the influence that varying flow rate, and the corresponding wall shear stress (τw) has on the viability and inertial positions of circulating cells in laminar pipe flow. The viability of two representative cell lines under different shear stresses in two different systems were investigated while particle streak imaging was used to determine their inertial positions. It was found that peristaltic pumps have a negative effect on cell viability in comparison to syringe pumps. Increasing shear stress in a cone and plate above 3 Pa caused an increase in cell death, however, τw as high as 10 Pa in circulation has little to no effect on cell viability. Inertial lift forces that move cells towards the centre of the channel protect them from experiencing detrimental levels of τw, indicating that τw in Poiseuille flow is not a good predictor of cell viability during advectio

Synthesis of poly(acrylic acid)-cysteine-based hydrogels with highly customizable mechanical properties for advanced cell culture applications

The fabrication of highly customizable scaffolds is a key enabling technology in the development of predictive in vitro cell models for applications in drug discovery, cancer research, and regenerative medicine. Naturally derived and synthetic hydrogels are good candidates for in vitro cell growth studies, owing to their soft and biocompatible nature; however, they are often hindered by limited ranges of stiffness and the requirement to modify the gel with additional extracellular matrix (ECM) proteins for cell adherence. Here, we report on the synthesis of a printable synthetic hydrogel based on cysteine-modified poly(acrylic acid) (PAA-Cys) with tuneable mechanical and swelling properties by incorporating acrylic acid into the PAA-Cys network and subsequent photoinitiated thiol-acrylate cross-linking. Control of the acrylic acid concentration and UV curing time produces a series of hydrogels with swelling ratios in excess of 100% and Young’s modulus values ranging from ∼2 to ∼35 kPa, of which most soft tissues fall within. Biocompatibility studies with RPE1 cells showed excellent cell adhesion and cell viability without the need for further modification with ECM proteins, but still can be modified as needed. The versatility of the hydrogel tuneable properties is demonstrated by culturing with RPE1 cells, which in vivo perform an important function in the visual process and the dysfunction of which may lead to various retinal abnormalities, such as glaucoma

The Metallome as a link between the “Omes” in autism spectrum disorders

Metal dyshomeostasis plays a significant role in various neurological diseases such as Alzheimer’s disease, Parkinson’s disease, Autism Spectrum Disorders (ASD), and many more. Like studies investigating the proteome, transcriptome, epigenome, microbiome, etc., for years, metallomics studies have focused on data from their domain, i.e., trace metal composition, only. Still, few have considered the links between other “omes,” which may together result in an individual’s specific pathologies. In particular, ASD have been reported to have multitudes of possible causal effects. Metallomics data focusing on metal deficiencies and dyshomeostasis can be linked to functions of metalloenzymes, metal transporters, and transcription factors, thus affecting the proteome and transcriptome. Furthermore, recent studies in ASD have emphasized the gut-brain axis, with alterations in the microbiome being linked to changes in the metabolome and inflammatory processes. However, the microbiome and other “omes” are heavily influenced by the metallome. Thus, here, we will summarize the known implications of a changed metallome for other “omes” in the body in the context of “omics” studies in ASD. We will highlight possible connections and propose a model that may explain the so far independently reported pathologies in ASD