Genome and transcriptome guided gene discovery in plant secondary metabolism

Plants produce a wide range of complex secondary metabolites that have many applications, for example as pharmaceutical agents. Gene discovery and the elucidation of these unique biosynthetic pathways is challenging since many of the enzymatic transformations are unprecedented. In Catharanthus roseus, the sole producer of the valuable anti-cancer compounds vinblastine and vincristine, the biosynthetic pathway for these alkaloids is highly complex and crucial steps are still unknown. Recently, the tight transcriptional co-regulation of the early part of this pathway enabled discovery of some of the central enzymatic steps by analysing the gene co-expression patterns and testing potential candidates using virus induced gene silencing. Additionally, it has become apparent that some plant secondary metabolite pathways exhibit physical clustering of pathway related genes in the genome. This thesis highlights how both strategies of gene discovery can be applied for the targeted discovery of genes for missing steps in biosynthesis of non-model plants. Co-expression analysis to identify candidates and subsequent testing of these candidates using virus induced gene silencing has led to the discovery and subsequent characterisation of the enzyme tabersonine 3-oxygenase (T3O), a key oxidation step in vindoline biosynthesis. This thesis furthermore reports the first C. roseus whole genome sequence. Additionally a BAC library was obtained and selected BACs sequenced. Analysis of the combined sequencing data established that gene clustering does indeed occur for alkaloid biosynthesis in C. roseus and yielded a new set of candidates for so far unknown pathway enzymes. Selected candidates have been tested by silencing or expression and results are discussed. The sequence information provides a valuable resource for the wider community, available as a searchable, publically available database (http://medicinalplantgenomics.msu.edu/). The work presented in this thesis highlights how next generation sequence data can be exploited to elucidate complex secondary metabolic pathways

Community-Based Outbreaks in Vulnerable Populations of Invasive Infections Caused by Streptococcus pneumoniae Serotypes 5 and 8 in Calgary, Canada

BACKGROUND: Outbreaks of invasive pneumococcal disease (IPD) typically occur within institutions. Beginning in 2005, we detected an increase in serotype (ST) 5 and ST8 IPD cases, predominantly in homeless persons living in an open community. METHODOLOGY/PRINCIPAL FINDINGS: CASPER (Calgary Area S. pneumoniae Epidemiology Research) surveillance study of all IPD (sterile site isolates) in our region (pop ~1,100,000). Interviews and chart reviews of all cases and all isolates phenotypically analyzed and selected isolated tested by multi-locus sequence typing (MLST). CONCLUSIONS/SIGNIFICANCE: During 2005-2007, 162 cases of ST5 IPD and 45 cases of ST8 IPD were identified. The isolates demonstrated phenotypic and genotypic clonality. The ST5 isolates were sequence type (ST) 289 and demonstrated intermediate susceptibility to TMP-SMX. The ST8 isolates were predominantly ST1268, with a susceptible antimicrobial susceptibility profile. Individuals with ST5 IPD were more likely to be middle aged (OR 2.6), homeless (OR 4.4), using illicit drugs(OR 4.8), and asthmatic(OR 2.6). Those with ST8 were more likely to be male (OR 4.4), homeless (OR 2.6), aboriginal (OR7.3), and a current smoker (OR 2.5). Overlapping outbreaks of ST5 and ST8 IPD occurred in an open community in Calgary, Canada and homelessness was a predominant risk factor. Homelessness represents a unique community in which pneumococcal outbreaks can occur

Interseasonal RSV infections in Switzerland - rapid establishment of a clinician-led national reporting system (RSV EpiCH).

In anticipation of an interseasonal respiratory syncytial virus (RSV) epidemic, a clinician-led reporting system was rapidly established to capture RSV infections in Swiss hospitals, starting in January 2021. Here, we present details of the reporting system and first results to June 2021. An unusual epidemiology was observed with an interseasonal surge of RSV infections associated with COVID-19-related non-pharmacological interventions. These data allowed real-time adjustment of RSV prophylaxis guidelines and consequently underscore the need for and continuation of systematic nationwide RSV surveillance

嶺南通訊 Lingnan Newsletter (第63期)

https://commons.ln.edu.hk/lingnan_newsletter/1062/thumbnail.jp

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia

A single-cell screening approach identifies targets for CAR-T cells in acute myeloid leukemia. Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development

Stimuli-Responsive Thiomorpholine Oxide-Derived Polymers with Tailored Hydrophilicity and Hemocompatible Properties

International audienceThermo-responsive hydrophilic polymers, including those showing tuneable lower critical solution temperature (LCST), represent a continuous subject of exploration for a variety of applications, but particularly in nanomedicine. Since biological pH changes can inform the organism about the presence of disequilibrium or diseases, the development of dual LCST/pH-responsive hydrophilic polymers with biological potential is an attractive subject in polymer science. Here, we present a novel polymer featuring LCST/pH double responsiveness. The monomer ethylthiomorpholine oxide methacrylate (THOXMA) can be polymerised via the RAFT process to obtain well-defined polymers. Copolymers with hydroxyethyl methacrylate (HEMA) were prepared, which allowed the tuning of the LCST behaviour of the polymers. Both, the LCST behaviour and pH responsiveness of hydrophilic PTHOXMA were tested by following the evolution of particle size by dynamic light scattering (DLS). In weak and strong alkaline conditions, cloud points ranged between 40–60 °C, while in acidic medium no LCST was found due to the protonation of the amine of the THOX moieties. Additional cytotoxicity assays confirmed a high biocompatibility of PTHOXMA and haemolysis and aggregation assays proved that the thiomorpholine oxide-derived polymers did not cause aggregation or lysis of red blood cells. These preliminary results bode well for the use of PTHOXMA as smart material in biological application

Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism

International audienc

Discovery of a P450-catalyzed step in vindoline biosynthesis:a link between the aspidosperma and eburnamine alkaloids

Here we report the discovery of a cytochrome P450 that is required for the biosynthesis of vindoline, a plant-derived natural product used for semi-synthesis of several anti-cancer drugs. This enzyme catalyzes the formation of an epoxide that can undergo rearrangement to yield the vincamine-eburnamine backbone, thereby providing evidence for the long-standing hypothesis that the aspidosperma- and eburnamine-type alkaloids are biosynthetically related