Biology subjects

Tran, J.

Publications and source records attributed to Tran, J..

44 records · Page 3Linked to original sources

bioRxiv2019

Combined use of design of experiments and metabolic engineering for optimization of nogalamycin production

Nogalamycin is an anthracycline antibiotic produced from Streptomyces nogalater that exerts its mechanism of action via inhibition of topoisomerase I. Nogalamycin has a unique tetracyclic structure composed of a 7-O-glycosidically linked L-nogalose sugar and a C2-C5\"-linked L-nogalamine residue that forms an epoxyoxacin ring. Nogalamycin was originally developed as an anticancer agent in the 1970s, however, it exhibited dose-limiting cardiotoxicity. Shortly after the discovery of nogalamycin, the semi-synthetic analogue 7-O-methylnogarol, or menogaril, was synthesized and exhibited a mild improvement in cardiotoxicity and anticancer efficacy. Menogaril lacks the 7-O-L-nogalose moiety and functions as a topoisomerase II inhibitor, like most anthracyclines, however this agent still proved too toxic for clinical use. Our laboratory is developing a production platform for microbial synthesis of novel nogalamycin analogs useful for treatment of human cancers or as antibiotics. Our initial hypothesis is that overexpression of structural genes responsible for synthesis of TDP-deoxysugar and polyketide precursor substrates, respectively, will increase carbon flux towards nogalamycin production. In this study, we have employed metabolic engineering to enhance nogalamycin production in Streptomyces nogalater. In this work, we used an optimized soytone glucose production medium to produce nogalamycin. We also overexpressed copies of structural biosynthetic genes to bolster substrate precursor building blocks for nogalamycin production. First, overexpression of the TDP-glucose synthase and TDP-D-glucose-4,6-dehydratase enzymes (mtmDE) resulted in a 50% increase in nogalamycin production (160 mg/L) as compared to wildtype S. nogalater (100 mg/L). Secondly, overexpression of the minimal polyketide synthase genes (snoa123) resulted in a fourfold production increase in nogalamycin (400 mg/L). This production platform will serve as the fundament for production of nogalamycin analogues for future drug development efforts.

microbiology↗

bioRxiv2019

Persistent Elevation of Electrical Pain Threshold following Continuous Theta Burst Stimulation over Primary Somatosensory Cortex in Humans

BackgroundPrimary somatosensory cortex (S1) is involved in pain processing and thus its suppression using neuromodulatory techniques such as continuous theta burst stimulation (cTBS) might be a potential pain management strategy in patients with neuropathic pain. S1 cTBS is known to elevate pain threshold in young adults. However, the persistence of this effect is unknown.\n\nObjective/HypothesisWe hypothesized persistent elevation of pain threshold following cTBS over S1 in healthy, young adults.\n\nMethodsWe recruited ten subjects in a sham-controlled crossover design and recorded their electrical pain threshold (EPT) for 40 min following cTBS over S1. We assessed corticospinal excitability (CSE) to rule out the involvement of primary motor cortex due to spread of current.\n\nResultscTBS over S1 elevated EPT without modulating CSE compared to sham stimulation. EPT was elevated for 40 min post-cTBS.\n\nConclusionsS1 can be focally targeted using cTBS for a longer lasting pain relief in patients.

neuroscience↗

bioRxiv2019

Alternative Splicing of MR1 regulates antigen presentation to MAIT cells

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are not well characterized.\n\nIn this report, we sought to characterize these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. Despite the widespread expression of MR1A and MR1B, only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A serves to decrease MAIT cell activation following bacterial infection. However, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Relative MR1A/MR1B expression in MR1-expressing thymocytes is associated with their prevalence.\n\nOur results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand.\n\nFundingThis work was supported by NIH T32HL083808 (EK, GAN, EM), VA Merit Award I01CX001562 (MJH), NIH R01AI29976 (MJH), NIH R01AI048090 (DML), NIH R21AI124225-01A1 (FT) and VA Merit Award I01BX000533 (DML). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

immunology↗

bioRxiv2019

Hypoxia-Induced PIM Kinase and Laminin-Activated Integrin α6 Mediate Resistance to PI3K Inhibitors in Bone-Metastatic CRPC

Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin 6{beta}1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin 6{beta}1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.

cancer biology↗

bioRxiv2019

tagPAINT: Stoichiometric and covalent labelling of protein tags for multiplexed and quantitative DNA-PAINT imaging

Recently, DNA-PAINT single molecule localisation microscopy (SMLM) has shown great promise for quantitative imaging. However, labelling strategies so far have relied on approaches that are multivalent or affinity-based. Here, we demonstrate tagPAINT - the covalent labelling of expressed protein tags (SNAP tag and Halo tag) with single DNA docking strands for single molecule localisation microscopy via DNA-PAINT. We utilised tagPAINT for T-cell receptor signalling proteins at the immune synapse as a proof of principle.

biophysics↗

bioRxiv2018

SIS-seq, a molecular ‘time machine’, connects single cell fate with gene programs

Conventional single cell RNA-seq methods are destructive, such that a given cell cannot also then be tested for fate and function, without a time machine. Here, we develop a clonal method SIS-seq, whereby single cells are allowed to divide, and progeny cells are assayed separately in SISter conditions; some for fate, others by RNA-seq. By cross-correlating progenitor gene expression with mature cell fate within a clone, and doing this for many clones, we can identify the earliest gene expression signatures of dendritic cell subset development. SIS-seq could be used to study other populations harboring clonal heterogeneity, including stem, reprogrammed and cancer cells to reveal the transcriptional origins of fate decisions.

systems biology↗

bioRxiv2018

UBE3A-mediated p18/LAMTOR1 ubiquitination and degradation regulate mTORC1 activity and synaptic plasticity

Accumulating evidence indicates that the lysosomal Ragulator complex is essential for full activation of the mechanistic target of rapamycin complex 1 (mTORC1). Abnormal mTORC1 activation has been implicated in several developmental neurological disorders, including Angelman syndrome (AS), which is caused by maternal deficiency of the ubiquitin E3 ligase UBE3A. Here we report that Ube3a regulates mTORC1 signaling by targeting p18, a subunit of the Ragulator. Ube3a ubiquinates p18, resulting in its proteasomal degradation, and Ube3a deficiency in hippocampus of AS mice results in increased lysosomal localization of p18 and other members of the Ragulator-Rag complex, such as RagA, and increased mTORC1 activity. P18 down-regulation by siRNA or shRNA in hippocampal CA1 neurons of AS mice reduces elevated mTORC1 activity and improves long-term potentiation (LTP) and dendritic spine maturation. Our results indicate that Ube3a-mediated regulation of p18 and subsequent mTORC1 signaling is critical for typical synaptic plasticity and dendritic spine development.

neuroscience↗

bioRxiv2017

Naturally-segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster

Identifying the causative sequence polymorphisms underlying complex trait variation is a key goal of evolutionary and biomedical genetics. By knowing the precise molecular events that confer phenotypic changes we can gain insight into the pathways underlying complex traits and the evolutionary forces acting on variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative nucleotide-level polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource, and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNAi of this gene reduced resistance. We discovered a naturally-segregating 22-bp frameshift deletion in Ugt86Dd, and overexpression of the insertion-containing allele in a range of tissues enhanced resistance. Accounting for the InDel event during mapping largely eliminates the QTL, implying the InDel explains the bulk of the effect associated with the mapped locus. Finally, we edited a relatively resistant genetic background to generate lesions in Ugt86Dd that recapitulate the naturally-occurring putative loss-of-function allele, and succeeded in radically reducing resistance. The putatively causative coding InDel in Ugt86Dd can be a launchpad for future mechanistic exploration of xenobiotic detoxification.\n\nARTICLE SUMMARYResolving the mutations that contribute to among-individual trait variation is a major goal of biomedical and evolutionary genetics. In general however, genetic mapping experiments do not allow immediate resolution of the underlying causative variants. Previous mapping work revealed several loci contributing to nicotine resistance in Drosophila melanogaster. We employed a battery of functional tests to demonstrate that the detoxification gene Ugt86Dd has a major phenotypic effect, and that a segregating frameshift mutation is likely causative. Editing the gene to introduce a premature stop codon led to a significant reduction in resistance, validating its role in xenobiotic detoxification.

genetics↗