Biology subjects

Lin, R. C.

Publications and source records attributed to Lin, R. C..

3 recordsLinked to original sources

bioRxiv2023

Mammalian cells internalize bacteriophages and utilize them as a food source to enhance cellular growth and survival

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following eight hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage internalization led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a food source to promote cellular growth and metabolism.

microbiology↗

bioRxiv2022

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy: A new mechanism for motor neuron degeneration.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and 4 disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild type full length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic C. elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

neuroscience↗

bioRxiv2019

Safety and Tolerability of Bacteriophage Therapy in Severe Staphylococcus aureus Infection

ImportanceThe effect of IV administration of a bacteriophage cocktail produced under GMP conditions on patients with severe S. aureus infection, including complicated bacteraemia, endocarditis and septic shock, is unknown.\n\nObjectiveTo assess safety and tolerability of adjunctive bacteriophage therapy in patients with severe S. aureus infections.\n\nDesign, Setting, ParticipantsObservational, open-label clinical trial of thirteen critically-ill patients admitted to a tertiary-referral hospital with S. aureus bacteraemia (including infective endocarditis, n=6) were assessed by the treating clinician and two consulting infectious diseases physicians to independently verify that routine medical and surgical therapy was optimal and that a poor outcome remained likely. Compassionate access to therapy was approved by both US and Australian regulators and by the Westmead Hospital Human Research Ethics Committee.\n\nInterventionA GMP-quality preparation of three combined Myoviridae bacteriophages with specific activity against S. aureus (AB-SA01), was administered intravenously in conjunction with optimal antibiotic therapy.\n\nMain Outcome and MeasurementsPhysiological, haematological and biochemical markers of infection, bacterial and bacteriophage kinetics in blood, development of resistance to bacteriophages, and mortality at 28 (D28) and 90 (D90) days were measured. Main outcomes were safety and tolerability.\n\nResultsBacteriophage therapy was initiated 4-10 days after antibiotic commencement, at 109 plaque-forming units (PFU) twice daily. Infecting staphylococci were typical of common local subtypes. Initial input ratio of phages to bacteria in the bloodstream (MOIinput) was >100. Five of the thirteen patients died by D28 and a sixth patient suffered sudden cardiac death on D90. Bacteriophage therapy coincided with a marked reduction in staphylococcal bacterial DNA in the blood and in sepsis-associated inflammatory responses in almost all cases. No bacteriophage-attributable adverse events were identified. Development of bacteriophage resistance was not observed. Population analysis revealed no significant effect of bacteriophage therapy on the gut microflora.\n\nConclusions and RelevanceAdjunctive bacteriophage therapy appears to be safe and well-tolerated in critically ill patients with severe S. aureus infection. Two weeks of twice daily intravenous administration may be a suitable protocol. Controlled trials are needed.\n\nTrial RegistrationWestmead Hospital Human Research Ethics Committee approval July 11, 2017; ClinicalTrials.gov Identifier: NCT03395769, AB-SA01-EAP01 (January 10, 2018); Clinical Trials Notification (Australian Therapeutic Goods Association): CT-2018-CTN-02372-1 (July 23, 2018).\n\nKey Points\n\nQuestionIs intravenous (IV) administration of investigational bacteriophage (phage) therapy safe and well-tolerated in patients with severe Staphylococcus aureus infection?\n\nFindingsThirteen patients with severe S. aureus infections received AB-SA01, a bacteriophage product prepared according to Good Manufacturing Practices (GMP), as adjunctive therapy to antibiotics. AB-SA01 was well-tolerated with no adverse events identified. Bacterial burden and inflammatory responses were reduced and no phage-resistant staphylococci were isolated during or after therapy.\n\nMeaningOur results will inform future randomised controlled trials assessing the antibacterial and anti-inflammatory potential of bacteriophages in the treatment of severe S. aureus infection.

clinical trials↗