ORCID Profile
0000-0002-2932-1890
Current Organisation
University of Melbourne
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Publisher: Elsevier BV
Date: 08-2009
DOI: 10.1016/J.NEUROSCIENCE.2009.04.063
Abstract: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury-induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freund's adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.
Publisher: Elsevier BV
Date: 09-2011
Publisher: Springer Science and Business Media LLC
Date: 28-04-2012
DOI: 10.1007/S00441-012-1405-9
Abstract: Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2014
DOI: 10.1007/S00441-014-1886-9
Abstract: A sub-group of enteroendocrine cells (L cells) release gastrointestinal hormones, GLP-1 and PYY, which have different but overlapping physiological effects, in response to intraluminal nutrients. Whilst their release profiles are not identical, how the plasma levels of these two hormones are differentially regulated is not well understood. We investigate the possibility that GLP-1 and PYY are in separate storage vesicles. In this study, the subcellular location of GLP-1 and PYY storage organelles is investigated using double-labelling immunohistochemistry, super resolution microscopy and high-resolution confocal microscopy. In all species tested, human, pig, rat and mouse, most cytoplasmic stores that exhibited GLP-1 or PYY immunofluorescence were distinct from each other. The volume occupancy, determined by 3D analysis, overlapped by only about 10∼20 %. At the lower resolution achieved by conventional confocal microscopy, there was also evidence of GLP-1 and PYY being in separate storage compartments but, in subcellular regions where there were many storage vesicles, separate storage could not be resolved. The results indicate that different storage vesicles in L cells contain predominantly GLP-1 or predominantly PYY. Whether GLP-1 and PYY storage vesicles are selectively mobilised and their products are selectively released needs to be determined.
Publisher: Wiley
Date: 16-12-2021
DOI: 10.1002/JBMR.4484
Abstract: Cortical bone develops and changes in response to mechanical load, which is sensed by bone‐embedded osteocytes. The bone formation response to load depends on STAT3 intracellular signals, which are upregulated after loading and are subject to negative feedback from Suppressor of Cytokine Signaling 3 ( Socs3 ). Mice with Dmp1Cre‐ targeted knockout of Socs3 have elevated STAT3 signaling in osteocytes and display delayed cortical bone maturation characterized by impaired accrual of high‐density lamellar bone. This study aimed to determine whether these mice exhibit an altered response to mechanical load. The approach used was to test both treadmill running and tibial compression in female Dmp1Cre.Socs3 f/f mice. Treadmill running for 5 days per week from 6 to 11 weeks of age did not change cortical bone mass in control mice, but further delayed cortical bone maturation in Dmp1Cre.Socs3 f/f mice accrual of high‐density bone was suppressed, and cortical thickness was less than in genetically‐matched sedentary controls. When strain‐matched anabolic tibial loading was tested, both control and Dmp1Cre.Socs3 f/f mice exhibited a significantly greater cortical thickness and periosteal perimeter in loaded tibia compared with the contralateral non‐loaded bone. At the site of greatest compressive strain, the loaded Dmp1Cre.Socs3 f/f tibias showed a significantly greater response than controls, indicated by a greater increase in cortical thickness. This was due to a greater bone formation response on both periosteal and endocortical surfaces, including formation of abundant woven bone on the periosteum. This suggests a greater sensitivity to mechanical load in Dmp1Cre.Socs3 f/f bone. In summary, mice with targeted SOCS3 deletion and immature cortical bone have an exaggerated response to both physiological and experimental mechanical loads. We conclude that there is an optimal level of osteocytic response to mechanical load required for cortical bone maturation and that load‐induced bone formation may be increased by augmenting STAT3 signaling within osteocytes. © 2021 American Society for Bone and Mineral Research (ASBMR).
Publisher: Springer Science and Business Media LLC
Date: 27-11-2019
DOI: 10.1038/S41467-019-13385-X
Abstract: Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack.
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-0897-4_3
Abstract: The digestive system is innervated through its connections with the central nervous system (CNS) and by the enteric nervous system (ENS) within the wall of the gastrointestinal tract. The ENS works in concert with CNS reflex and command centers and with neural pathways that pass through sympathetic ganglia to control digestive function. There is bidirectional information flow between the ENS and CNS and between the ENS and sympathetic prevertebral ganglia.The ENS in human contains 200-600 million neurons, distributed in many thousands of small ganglia, the great majority of which are found in two plexuses, the myenteric and submucosal plexuses. The myenteric plexus forms a continuous network that extends from the upper esophagus to the internal anal sphincter. Submucosal ganglia and connecting fiber bundles form plexuses in the small and large intestines, but not in the stomach and esophagus. The connections between the ENS and CNS are carried by the vagus and pelvic nerves and sympathetic pathways. Neurons also project from the ENS to prevertebral ganglia, the gallbladder, pancreas and trachea.The relative roles of the ENS and CNS differ considerably along the digestive tract. Movements of the striated muscle esophagus are determined by neural pattern generators in the CNS. Likewise the CNS has a major role in monitoring the state of the stomach and, in turn, controlling its contractile activity and acid secretion, through vago-vagal reflexes. In contrast, the ENS in the small intestine and colon contains full reflex circuits, including sensory neurons, interneurons and several classes of motor neuron, through which muscle activity, transmucosal fluid fluxes, local blood flow and other functions are controlled. The CNS has control of defecation, via the defecation centers in the lumbosacral spinal cord. The importance of the ENS is emphasized by the life-threatening effects of some ENS neuropathies. By contrast, removal of vagal or sympathetic connections with the gastrointestinal tract has minor effects on GI function. Voluntary control of defecation is exerted through pelvic connections, but cutting these connections is not life-threatening and other functions are little affected.
Publisher: American Physiological Society
Date: 10-2011
Abstract: Hyperpolarization-activated inward currents ( I h ) contribute to neuronal excitability in sensory neurons. Four subtypes of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate I h , with different activation kinetics and cAMP sensitivities. The aim of the present study was to examine the postnatal development of I h and HCN channel subunits in trigeminal ganglion (TG) neurons. I h was investigated in acutely dissociated TG neurons from rats aged between postnatal day (P)1 and P35 with whole cell patch-cl electrophysiology. In voltage-cl studies, I h was activated by a series of hyperpolarizing voltage steps from −40 mV to −120 mV in −10-mV increments. Tail currents from a common voltage step (−100 mV) were used to determine I h voltage dependence. I h activation was faster in older rats and occurred at more depolarized potentials the half-maximal activation voltage ( V 1/2 ) changed from −89.4 mV (P1) to −81.6 mV (P35). In current-cl studies, blocking I h with ZD7288 caused membrane hyperpolarization and increases in action potential half-duration at all postnatal ages examined. ZD7288 also reduced the action potential firing frequency in multiple-firing neurons. Western blot analysis of the TG detected immunoreactive bands corresponding to all HCN subtypes. HCN1 and HCN2 band density increased with postnatal age, whereas the low-intensity HCN3 and moderate-intensity HCN4 bands were not changed. This study suggests that functional I h are activated in rat trigeminal sensory neurons from P1 during postnatal development, have an increasing role with age, and modify neuronal excitability.
Publisher: eLife Sciences Publications, Ltd
Date: 13-04-2023
Publisher: American Society for Microbiology
Date: 16-10-2023
Publisher: Cold Spring Harbor Laboratory
Date: 02-05-2022
DOI: 10.1101/2022.05.02.490237
Abstract: Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction but its cellular expression in the GI tract remains to be characterised. Localisation of NLGN3 protein is challenging in intestinal tissue due to the lack of target-specific antibodies. Here, we combined RNAScope in situ hybridization for Nlgn3 mRNA and immunofluorescence for markers of all enteric neurons, cholinergic submucosal neurons, non-cholinergic submucosal neurons, nitregic and calretinin-containing myenteric neurons as well as glial cells. We also developed a quantitative 3-dimensional image analysis method to measure Nlgn3 mRNA cellular expression levels in enteric neurons and glia. We show that Nlgn3 mRNA is expressed in most submucosal and myenteric neurons as well as in enteric glia. The R451C mutation reduces Nlgn3 mRNA expression levels in cholinergic, nitrergic and calretinin enteric neuronal subpopulations but does not affect Nlgn3 mRNA expression in VIPergic submucosal neurons. In summary, we show that the autism-associated R451C mutation in Nlgn3 reduces Nlgn3 mRNA expression in the mouse ENS. These findings could shed light on the pathophysiology of GI dysfunction in ASD.
Publisher: MDPI AG
Date: 16-06-2023
DOI: 10.20944/PREPRINTS202306.1181.V1
Abstract: Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization for Nlgn3 mRNA and immunofluorescence for cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells. We measured Nlgn3 mRNA neuronal and glial expression via quantitative 3-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for Neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin and NOS labelled neurons and S100ß labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.
Publisher: Elsevier BV
Date: 11-2022
Publisher: MDPI AG
Date: 02-03-2022
DOI: 10.3390/IJMS23052770
Abstract: Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs’ specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.
Publisher: Wiley
Date: 09-2007
DOI: 10.1002/DDR.20195
Publisher: American Society for Microbiology
Date: 20-02-2019
DOI: 10.1128/IAI.00534-19
Abstract: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella -containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes.
Publisher: Springer Science and Business Media LLC
Date: 27-08-2022
DOI: 10.1038/S41467-022-32579-4
Abstract: The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum , adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite’s nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pf centrin-1 and −4, microtubule-associated protein, End-binding protein-1, kinetochore protein, Pf NDC80 and centromere-associated protein, Pf CENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.
Publisher: Springer Science and Business Media LLC
Date: 18-01-2014
DOI: 10.1007/S00441-013-1780-X
Abstract: TRPA1 is an ion channel that detects specific chemicals in food and also transduces mechanical, cold and chemical stimulation. Its presence in sensory nerve endings is well known and recent evidence indicates that it is expressed by some gastrointestinal enteroendocrine cells (EEC). The purpose of the present work is to identify and quantify EEC that express TRPA1 in the mouse gastrointestinal tract. Combined in situ hybridisation histochemistry for TRPA1 and immunofluorescence for EEC hormones was used. TRPA1 expressing EEC were common in the duodenum and jejunum, were rare in the distal small intestine and were absent from the stomach and large intestine. In the duodenum and jejunum, TRPA1 occurred in EEC that contained both cholecystokinin (CCK) and 5-hydroxytryptamine (5HT) and in a small number of cells expressing 5HT but not CCK. TRPA1 was absent from CCK cells that did not express 5HT and from EEC containing glucagon-like insulinotropic peptide. Thus TRPA1 is contained in very specific EEC populations. It is suggested that foods such as garlic and cinnamon that contain TRPA1 stimulants may aid digestion by facilitating the release of CCK.
Publisher: Wiley
Date: 13-02-2022
DOI: 10.1111/TRA.12831
Abstract: The intracellular trafficking of β‐site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid‐β production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans ‐Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aβ production in non‐neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high‐resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis ‐Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP‐1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.
Publisher: American Physiological Society
Date: 02-2022
DOI: 10.1152/AJPLUNG.00165.2021
Abstract: The mechanisms driving idiopathic pulmonary fibrosis (IPF) remain undefined, however it is postulated that coagulation imbalances may play a role. The impact of blood-derived clotting factors, including factor XII (FXII) has not been investigated in the context of IPF. Plasma levels of FXII were measured by ELISA in patients with IPF and in age-matched healthy donors. Expression of FXII in human lung tissue was quantified using multiplex immunohistochemistry and Western blotting. Mechanistic investigation of FXII activity was assessed in vitro on primary lung fibroblasts using qPCR and specific receptor/FXII inhibition. The functional outcome of FXII on fibroblast migration was examined by high-content image analysis. Compared with 35 healthy donors, plasma levels of FXII were not higher in patients with IPF ( n = 27, P 0.05). Tissue FXII was elevated in IPF ( n = 11) and increased numbers of FXII+ cells were found in IPF ( n = 8) lung tissue compared with nondiseased controls ( n = 6, P 0.0001). Activated FXII induced IL6 mRNA and IL-6 protein in fibroblasts that was blocked by anti-FXII antibody, CSL312. FXII induced IL-6 production via PAR-1 and NF-κB. FXII induced migration of fibroblasts in a concentration-dependent manner. FXII is normally confined to the circulation but it leaks from damaged vessels into the lung interstitium in IPF where it 1) induces IL-6 production and 2) enhances migration of resident fibroblasts, critical events that drive chronic inflammation and therefore, contribute to fibrotic disease progression. Targeting FXII-induced fibroblastic processes in IPF may ameliorate pulmonary fibrosis.
Publisher: Wiley
Date: 03-03-2021
DOI: 10.1002/JBM4.10477
Abstract: The development of the musculoskeletal system and its maintenance depends on the reciprocal relationship between muscle and bone. The size of skeletal muscles and the forces generated during muscle contraction are potent sources of mechanical stress on the developing skeleton, and they shape bone structure during growth. This is particularly evident in hypermuscular global myostatin (Mstn)‐ null mice, where larger muscles during development increase bone mass and alter bone shape. However, whether muscle hypertrophy can similarly influence the shape of bones after the embryonic and prepubertal period is unknown. To address this issue, bone structure was assessed after inducing muscle hypertrophy in the lower hindlimbs of young‐adult C57BL/6J male mice by administering intramuscular injections of recombinant adeno‐associated viral vectors expressing follistatin (FST), a potent antagonist of Mstn. Two FST isoforms were used: the full‐length 315 amino acid isoform (FST‐315) and a truncated 288 amino acid isoform (FST‐288). In both FST‐treated cohorts, muscle hypertrophy was observed, and the anterior crest of the tibia, adjacent to the tibialis anterior muscle, was lengthened. Hypertrophy of the muscles surrounding the tibia caused the adjacent cortical shell to recede inward toward the central axis: an event driven by bone resorption adjacent to the hypertrophic muscle. The findings reveal that inducing muscle hypertrophy in mice can confer changes in bone shape in early adulthood. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
Publisher: Springer Science and Business Media LLC
Date: 07-11-2014
DOI: 10.1007/S00441-014-2033-3
Abstract: This study has investigated the patterns of colocalisation of the conventional K cell marker, glucagon-like insulinotropic peptide (GIP), and the L cell markers, glucagon like peptide-1 (GLP-1) and peptide YY (PYY), in enteroendocrine cells (EEC) of the small intestine and colon of mouse and pig. All combinations of the hormones, 3 in a cell, 2 in a cell and 1 at a time, were encountered. In both species, the three most common EEC types contained (1) both GLP-1 and PYY but not GIP, (2) GLP-1 alone or (3) GIP plus GLP-1 without PYY. Few GIP plus PYY cells and rare cells containing all 3 hormones were encountered. Gradients of cell types occurred along the intestine. For ex le, in mouse, there were no PYY cells in the duodenum and few in the jejunum, but >50% of labelled EEC in the distal ileum and colon were PYY immunoreactive. By contrast, over 40% of EEC in the pig duodenum contained PYY, and most also contained either GLP-1 or GIP. The gradient in pig was less pronounced. It is concluded that the traditional classification of K and L cells requires revision, and that there are major inter-species differences in the patterns of colocalisation of hormones that have been used to characterise K and L cells.
Publisher: Springer US
Date: 14-12-2023
Publisher: Elsevier BV
Date: 04-2020
Publisher: MDPI AG
Date: 12-11-2020
DOI: 10.3390/APP10228017
Abstract: To investigate the effects of microwave (MW) treatment on hays nutritive value, five types of hay (50 g) were treated with MW for 0 (control), 20, 40, 60, and 80 s (0, 440, 880, 1320, and 1760 kJ kg−1 of MW energy, respectively) and analyzed for nutritive value prior to scanning by an electron microscope to observe microstructure changes. The dry matter (DM) % of hays were increased with increasing treatment time (p 0.001). The improved DM and digestible organic matter in the DM were recorded from MW treated lucerne (60 s), wheat (40 s), and canola (20 s) (p 0.001), which might have been due to the MW ruptured cell wall. The MW energy required for maximal DM digestibility improvement was linearly related to control hay crude protein content (r2 = 0.79 p 0.001). Overall, the study showed MW treatment can increase some hays’ digestibility. In addition, the crude protein content from control hay may be a proxy to indicate DM digestibility improvement.
Publisher: Springer Science and Business Media LLC
Date: 15-01-2018
DOI: 10.1007/S10585-018-9873-2
Abstract: Surgery remains the curative treatment modality for colorectal cancer in all stages, including stage IV with resectable liver metastasis. There is emerging evidence that the stress response caused by surgery as well as other perioperative therapies such as anesthesia and analgesia may promote growth of pre-existing micro-metastasis or potentially initiate tumor dissemination. Therapeutically targeting the perioperative period may therefore reduce the effect that surgical treatments have in promoting metastases, for ex le by combining β-adrenergic receptor antagonists and cyclooxygenase-2 (COX-2) inhibitors in the perioperative setting. In this paper, we highlight some of the mechanisms that may underlie surgery-related metastatic development in colorectal cancer. These include direct tumor spillage at the time of surgery, suppression of the anti-tumor immune response, direct stimulatory effects on tumor cells, and activation of the coagulation system. We summarize in more detail results that support a role for catecholamines as major drivers of the pro-metastatic effect induced by the surgical stress response, predominantly through activation of β-adrenergic signaling. Additionally, we argue that an improved understanding of surgical stress-induced dissemination, and more specifically whether it impacts on the level and nature of heterogeneity within residual tumor cells, would contribute to the successful clinical targeting of this process. Finally, we provide a proof-of-concept demonstration that ex-vivo analyses of colorectal cancer patient-derived s les using RGB-labeling technology can provide important insights into the heterogeneous sensitivity of tumor cells to stress signals. This suggests that intra-tumor heterogeneity is likely to influence the efficacy of perioperative β-adrenergic receptor and COX-2 inhibition, and that ex-vivo characterization of heterogeneous stress response in tumor s les can synergize with other models to optimize perioperative treatments and further improve outcome in colorectal and other solid cancers.
Publisher: Elsevier BV
Date: 08-2023
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.JPEDSURG.2019.09.038
Abstract: Undescended Testes (UDT) are prevalent in 2%-5% of male infants and cause malignancy and infertility. During germ cell development, abnormal gonocytes usually undergo apoptosis. This process is believed to involve BAX (Bcl-2 Associated X) protein in clearing abnormal gonocytes which may fail in UDT, resulting in persisting gonocytes causing seminomas later in life. We aim to investigate the role of BAX in gonocyte apoptosis. BAXKO (BAX-knockout) mice were back-crossed to OG2 mice (Oct4-promoter driving enhanced green fluorescent protein-eGFP) to produce BAXOG2 mice. Testes (wildtype-BAX BAX This study demonstrated that apoptosis is important in regulating germ cell migration and differentiation during gonocyte transformation in neonatal mice. In addition, inhibition of apoptosis results in persisting neonatal gonocytes which might become seminomas in patients with UDT.
Publisher: American Physiological Society
Date: 07-2023
DOI: 10.1152/JAPPLPHYSIOL.00651.2022
Abstract: Patients with Duchenne muscular dystrophy (DMD) develop bone fragility because of muscle weakness and immobilization. We investigated whether increasing muscle contractile activity through low-frequency stimulation (LFS) could alter bone architecture in dystrophin-deficient ( mdx) or dystrophin- and utrophin-deficient ( dko) mouse models of DMD. Chronic LFS reduced tibial diaphysis cross sections in mdx and dko mice, without affecting bone shape in healthy mice. LFS affected the distribution of bone mineral density across all phenotypes, with the magnitude of effect being dependent on disease severity.
Publisher: MDPI AG
Date: 24-01-2022
Abstract: Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed “optical barcoding” (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of in idual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.
Publisher: Wiley
Date: 08-2022
Abstract: The small G protein Arl5b is localised on the trans ‐Golgi network (TGN) and regulates endosomes‐to‐TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane‐bound Arl5b(Q70L)‐GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP‐Trap pull down. From MS analysis, 22 Golgi proteins were identified 50% were TGN‐localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.
Publisher: Elsevier BV
Date: 07-2020
Publisher: Wiley
Date: 16-03-2011
DOI: 10.1111/J.1365-2982.2011.01696.X
Abstract: Damage to mucosal epithelial cells, muscle cells and enteric neurons has been extensively studied following intestinal ischemia and reperfusion (I/R). Interestingly, the effects of intestinal I/R on enteric glia remains unexplored, despite knowledge that glia contribute to neuronal maintenance. Here, we describe structural damage to enteric glia and associated changes in distribution and immunoreactivity of the neuronal protein Hu. The mouse small intestine was made ischemic for 3 h and reperfused from 1 to 12 h. Immunohistochemical localisation of glial fibrillary acidic protein (GFAP), Hu and TUNEL were used to evaluate changes. At all time points glial cells became distorted, which was evident by their altered GFAP immunoreactivity, including an unusual appearance of bright perinuclear GFAP staining and the presence of GFAP globules. The numbers of neurons per ganglion area were significantly fewer in ganglia that contained distorted glia when compared with ganglia that contained glia of normal appearance. The distribution of Hu immunoreactivity was altered at all reperfusion time points. The presence of vacuoles and Hu granules in neurons was evident and an increase in nuclear Hu, relative to cytoplasmic Hu, was observed in ganglia that contained both normal and distorted glial cells. A number of neurons appeared to lose their Hu immunoreactivity, most noticeably in ganglia that contained distorted glial cells. TUNEL reaction occurred in a minority of glial cells and neurons. Structural damage to gliofilaments occurs following I/R and may be associated with damage to neighboring neurons.
Publisher: Springer Science and Business Media LLC
Date: 10-10-2009
DOI: 10.1007/S00441-009-0869-8
Abstract: Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels are active at resting membrane potential and thus are likely to contribute to neuronal excitability. Four HCN channel subunits (HCN1-4) have previously been cloned. The aim of the current study was to investigate the immunoreactivity of HCN4 channel protein in rat trigeminal (TG) and dorsal root ganglion (DRG) sensory neurons. HCN4 was present in 9% of TG neurons and 4.7% of DRG neurons, it was distributed in a discrete population of small-diameter neurons in the TG but was located in cells of all sizes in the DRG. Approximately two thirds of HCN4-containing neurons in each ganglia were labelled with antisera raised against the 200-kDa neurofilament (NF200). The remaining HCN4-containing neurons were NF200-negative, were not labelled with antisera raised against calcitonin-gene related peptide (CGRP), and did not bind the isolectin B4 (IB4). HCN4-containing neurons made up more than half of the population of small-diameter primary afferent neurons that did not contain either NF200 or CGRP or bind IB4 in both TG and DRG. This population was not insignificant, comprising 5% of TG neurons and 2% of DRG neurons.
Publisher: American Chemical Society (ACS)
Date: 29-11-2018
Abstract: An effective strategy to inhibit endocytosis in cancer cells is presented where modified net-type graphene oxide (GO) sheets, bound with multiple cell surface receptors, are introduced and synthesized as novel anticancer agents. The results suggest that the binding connects GO sheets with neighboring lipid rafts, neutralizes endocytosis, and causes metabolic deprivation. As a result, tumor cell survival and proliferation are reduced. Live cell confocal microscopy imaging reveals that GO-PEGFA (folate-PEGylated GO) (PEG, polyethylene glycol) is internalized by tumor cells, while GO-PEGRGD (tripeptide Arg-Gly-Asp PEGylated GO) associates with the external cell membrane (not internalized). In vitro exposure of tumor cells to GO-PEGFA or GO-PEGRGD reduces the cell viability by 35%, compared to 50% reduction using methotrexate (100 μM). The combination of modified GO sheets with methotrexate or doxorubicin shows a greater toxicity (80% reduction in cell viability) than the in idual agents. The proposed setup demonstrates a significant synergy in limiting tumor cell growth.
Publisher: American Society for Microbiology
Date: 28-04-2020
Abstract: The trafficking of the virulence antigen Pf EMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer’s clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.
Publisher: Springer Science and Business Media LLC
Date: 29-09-2011
DOI: 10.1007/S00441-011-1240-4
Abstract: Ghrelin, a peptide hormone from the stomach, has been recently discovered to reduce sodium excretion from the kidney. Although the effects on the kidney suggest actions in the distal nephron, the sites of expression of ghrelin receptors have not been localised. In the present work we have used a mouse that expresses green fluorescent protein under the control of the ghrelin receptor promoter to locate sites of receptor expression in the kidney. Receptor expression was confined to the straight parts of the distal tubules and the thin limbs of the loops of Henle. No expression was detected in other structures, including the glomeruli, proximal tubules and collecting ducts. Ghrelin receptors were not found in extra-renal or intra-renal arteries, despite observations that ghrelin is a vasodilator. The distribution revealed by in situ hybridisation histochemistry was the same as that revealed by the reporter. In conclusion, ghrelin receptors have a restricted distribution in the kidney. The location in the straight parts of the distal tubules accords with observations that ghrelin promotes sodium retention.
Publisher: Springer Science and Business Media LLC
Date: 12-06-2012
DOI: 10.1007/S00441-012-1451-3
Abstract: Recent investigation of the intestine following ischemia and reperfusion (I/R) has revealed that nitric oxide synthase (NOS) neurons are more strongly affected than other neuron types. This implies that NO originating from NOS neurons contributes to neuronal damage. However, there is also evidence of the neuroprotective effects of NO. In this study, we compared the effects of I/R on the intestines of neuronal NOS knockout (nNOS(-/-)) mice and wild-type mice. I/R caused histological damage to the mucosa and muscle and infiltration of neutrophils into the external muscle layers. Damage to the mucosa and muscle was more severe and greater infiltration by neutrophils occurred in the first 24 h in nNOS(-/-) mice. Immunohistochemistry for the contractile protein, α-smooth muscle actin, was used to evaluate muscle damage. Smooth muscle actin occurred in the majority of smooth muscle cells in the external musculature of normal mice but was absent from most cells and was reduced in the cytoplasm of other cells following I/R. The loss was greater in nNOS(-/-) mice. Basal contractile activity of the longitudinal muscle and contractile responses to nerve stimulation or a muscarinic agonist were reduced in regions subjected to I/R and the effects were greater in nNOS(-/-) mice. Reductions in responsiveness also occurred in regions of operated mice not subjected to I/R. This is attributed to post-operative ileus that is not significantly affected by knockout of nNOS. The results indicate that deleterious effects are greater in regions subjected to I/R in mice lacking nNOS compared with normal mice, implying that NO produced by nNOS has protective effects that outweigh any damaging effect of this free radical produced by enteric neurons.
Publisher: MDPI AG
Date: 10-10-2016
DOI: 10.3390/NU8100623
Publisher: Wiley
Date: 28-03-2023
Abstract: The coral skeleton harbours a erse community of bacteria and microeukaryotes exposed to light, O 2 and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O 2 and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef‐building corals Porites lutea and Paragoniastrea benhami . The physicochemical environment was more stable in the deep skeleton, and the ersity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O 2 habitat, whereas pH varied from pH 6–9 with depth. Physicochemical gradients of O 2 and pH of the coral skeleton explained the β‐ ersity of the bacterial communities, and skeletal layers that showed O 2 peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.
Publisher: eLife Sciences Publications, Ltd
Date: 08-06-2023
DOI: 10.7554/ELIFE.84778
Abstract: Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic ersity of intracellular bacteria. Using a well-established epithelial cell line model , we have developed a platform called InToxSa ( in tracellular tox icity of S. a ureus ) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S . aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA , encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.
Publisher: European Respiratory Society
Date: 05-09-2021
Publisher: MDPI AG
Date: 24-06-2014
DOI: 10.3390/NU6062478
Publisher: Wiley
Date: 11-09-2023
DOI: 10.1002/LARY.31022
Publisher: Springer Science and Business Media LLC
Date: 09-02-2011
DOI: 10.1007/S00441-010-1126-X
Abstract: Changes in intestinal function, notably impaired transit, following ischemia/reperfusion (I/R) injury are likely to derive, at least in part, from damage to the enteric nervous system. Currently, there is a lack of quantitative data and methods on which to base quantitation of changes that occur in enteric neurons. In the present work, we have investigated quantifiable changes in response to ischemia of the mouse small intestine followed by reperfusion from 1 h to 7 days. I/R caused distortion of nitric oxide synthase (NOS)-containing neurons, the appearance of a TUNEL reaction in neurons, protein nitrosylation and translocation of Hu protein. Protein nitrosylation was detected after 1 h and was detectable in 10% of neurons by 6 h in the ischemic region, indicating that reactive peroxynitrites are rapidly produced and can interact with proteins soon after reperfusion. Apoptosis, revealed by TUNEL staining, was apparent at 6 h. The profile sizes of NOS neurons were increased by 60% at 2 days and neurons were still swollen at 7 days, both in the ischemic region and proximal to the ischemia. The distribution of the enteric neuron marker and oligonucleotide binding protein, Hu, was significantly changed in both regions. Hu protein translocation to the nucleus was apparent by 3 h and persisted for up to 7 days. Particulate Hu immunoreactivity was observed in the ganglia 3 h after I/R but was never observed in control. Our observations indicate that effects of I/R injury can be detected after 1 h and that neuronal changes persist to at least 7 days. Involvement of NO and reactive oxygen species in the changes is indicated by the accumulation of nitrosylated protein aggregates and the swelling and distortion of nitrergic neurons. It is concluded that damage to the enteric nervous system, which is likely to contribute to functional deficits following ischemia and re-oxygenation in the intestine, can be quantified by Hu protein translocation, protein nitrosylation, swelling of nitrergic neurons and apoptosis.
Publisher: MDPI AG
Date: 30-06-2023
DOI: 10.3390/BIOM13071063
Abstract: Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron–glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.
Publisher: Springer Science and Business Media LLC
Date: 07-09-2020
Publisher: Research Square Platform LLC
Date: 03-10-2023
Publisher: Springer Science and Business Media LLC
Date: 24-09-2013
DOI: 10.1038/NRGASTRO.2013.180
Abstract: The gastrointestinal tract presents the largest and most vulnerable surface to the outside world. Simultaneously, it must be accessible and permeable to nutrients and must defend against pathogens and potentially injurious chemicals. Integrated responses to these challenges require the gut to sense its environment, which it does through a range of detection systems for specific chemical entities, pathogenic organisms and their products (including toxins), as well as physicochemical properties of its contents. Sensory information is then communicated to four major effector systems: the enteroendocrine hormonal signalling system the innervation of the gut, both intrinsic and extrinsic the gut immune system and the local tissue defence system. Extensive endocrine-neuro-immune-organ-defence interactions are demonstrable, but under-investigated. A major challenge is to develop a comprehensive understanding of the integrated responses of the gut to the sensory information it receives. A major therapeutic opportunity exists to develop agents that target the receptors facing the gut lumen.
Location: Korea, Republic of
No related grants have been discovered for Hyun-Jung Cho.