Biology subjects

Smith, I.

Publications and source records attributed to Smith, I..

24 records · Page 2Linked to original sources

bioRxiv2020

The Hitchhiker's Guide to the Periplasm: Unexpected Molecular Interactions of Antibiotics Revealed by Considering Crowding Effects in E. coli

The periplasm of Gram-negative bacteria is a highly crowded environment comprised of many different molecular species. Antibacterial agents that causes lysis of Gram-negative bacteria by their action against the inner membrane must cross the periplasm to arrive at their target membrane. Very little is currently known about their route through the periplasm, and the interactions they experience. To this end, here atomistic molecular dynamics simulations are used to study the path taken by the antibiotic polymyxin B1 through a number of models of the periplasm which are crowded with proteins and osmolytes to different extents. The simulations reveal that PMB1 forms transient and long-lived interactions with proteins and osmolytes that are free in solution as well as lipoproteins anchored to the outer membrane and bound to the cell wall. We show that PMB1 may be able to hitchhike within the periplasm by binding to lipoprotein carriers. Overall our results show that PMB1 is rarely uncomplexed within the periplasm; an important consideration for interpretations of its therapeutic mechanism of action. It is likely that this observation can be extended to other antibiotics that rely on diffusion to cross the periplasm.

biochemistry↗

bioRxiv2020

Differential DNA methylation in the Vinv promoter region controls Cold Induced Sweetening in potato

Control of potato sprouting is important to ensure constant supply of high-quality potato to the industry. Efficient control of sprouting can be achieved by chemical treatment or cold temperature. Recent bans on anti-sprouting molecules are prompting the use of cold storage in the potato value chain. Unfortunately, storage of potato at low temperatures is associated with cold induced sweetening (CIS) due to the induction of the vacuolar invertase gene under low temperatures. Because CIS is associated with the production of the potentially carcinogenic acrylamide in processed potatoes, concise knowledge on the regulatory mechanisms controlling the CIS-phenotype in potatoes is expected to help pave the way for the production of CIS-resistant potato varieties. Here, we dissect the promoters of the Vacuolar invertase (Vinv) genes from CIS-susceptible and CIS-resistant varieties to investigate their implication in CIS-phenotype determination. Using bisulfite sequencing and CRISPR-dCas9-DRM2-mediated de novo DNA methylation, we show that the CIS-resistant phenotype of Verdi, is in part due to hypermethylation of its Vinv promoter, more specifically in the 1.0-1.7kb region. Those findings open new perspectives to engineer CIS-resistant potatoes by genome and epigenome modifications.

plant biology↗

bioRxiv2019

Differential serological diagnostics of different Rabbit Haemorrhagic Disease Viruses co-circulating in Australia

The lagovirus Rabbit Haemorrhagic Disease Virus (RHDV) has been circulating in Australia since the mid-1990s when it was deliberately released to control overabundant rabbit populations. In recent years, the viral diversity of different RHDVs in Australia has increased, and currently four different types of RHDV are known to be circulating. To allow for ongoing epidemiological studies and impact assessments of these viruses on Australian wild rabbit populations, it is essential that serological tools are updated. To this end, Reference sera were produced against all four virulent RHDVs (including RHDV2) known to be present in Australia and tested in a series of available immunological assays originally developed for the prototype RHDV, to assess patterns of cross reactivity and the usefulness of these assays to detect lagovirus antibodies, either in a generic or specific manner. Enzyme Linked Immuno Sorbent Assays (ELISAs) developed to detect antibody isotypes IgM, IgA and IgG were sufficiently cross reactive to detect antibodies raised against all four virulent lagoviruses. For the more specific detection of antibodies to the antigenically more different RHDV2, a competition ELISA was adapted using RHDV2 specific monoclonal antibodies in combination with Australian viral antigen. Archival serum banks from a long term rabbit monitoring site where rabbits were sampled quarterly over a period of six years were re-screened using this assay, and revealed serological evidence for the arrival of RHDV2 in this population at least six months prior to its initial detection in Australia in a deceased rabbit in May 2015. The serological methods and reference reagents described here will provide valuable tools to study presence, prevalence and impact of RHDV2 on Australian rabbit populations; however the discrimination of different antigenic variants of RHDVs as well as mixed infections at the serological level remains challenging.

epidemiology↗

bioRxiv2019

Pan-filovirus one-step reverse transcription-polymerase chain reaction screening assay

Five species within the genera Ebolavirus and Marburgvirus of the family Filoviridae are known to cause severe hemorrhagic fever with high mortality rates in humans and non-human primates. Recent large outbreaks of Ebola virus disease in West Africa (2014 - 2016) and the Democratic Republic of the Congo (2018 - ongoing) have demonstrated the epidemic potential with devastating public health consequences. Several known and novel filovirus species have been found in bats in recent years. However, the role of each virus species in the disease ecology of human disease is still unclear. In particular, the transmission mechanism from potential animal hosts to humans is not known. Therefore, a simple, flexible, cost-effective screening tool for detecting the presence of any (putative) member of the filovirus family in animal samples is needed. In this study, a one-step conventional pan-filovirus RT-PCR assay was developed. The designed universal consensus primers of this screening test target two highly conserved regions of the nucleoprotein (NP) of all currently known filoviruses. The assay was capable of specific amplification of viral RNA of all six primate-pathogenic (human and non-human) filovirus species and resulted in 317 bp long RT-PCR products. This amplicon length renders the assay suitable for flexible application as conventional reverse transcription polymerase chain reaction (RT-PCR) as well as for future use as rapid real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).

molecular biology↗

bioRxiv2017

Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map

The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss of function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that miRNA-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a Consensus Gene Signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 sgRNAs in 6 cells lines, and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.

bioinformatics↗

bioRxiv2017

A Next Generation Connectivity Map: L1000 Platform And The First 1,000,000 Profiles

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.\n\nHIGHLIGHTSO_LIA new gene expression profiling method, L1000, dramatically lowers cost\nC_LIO_LIThe Connectivity Map database now includes 1.3 million publicly accessible L1000 perturbational profiles\nC_LIO_LIThis expanded Connectivity Map facilitates discovery of small molecule mechanism of action and functional annotation of genetic variants\nC_LIO_LIThe work establishes feasibility and utility of a truly comprehensive Connectivity Map\nC_LI

genomics↗