Talks by SRUK/CERU members – session II

Javier Pardo Díaz, University of Oxford

Construction and analysis of gene expression networks for Rhizobium leguminosarum

Nitrogen is one of the limiting factors during plant growth. nitrogen uptake in legumes is facilitated by bacteria such as Rhizobium leguminosarum. For this bacterium, gene expression data are available, but functional gene annotation is less well developed. More annotations could lead to a better understanding of the pathways for bacterial growth, plant colonisation and nitrogen fixation in R. leguminosarum. We present and evaluate a computational pipeline based on the use of signed distance correlation to construct gene co-expression networks from R. leguminosarum gene expression data. Our method results in more robust and informative networks than state-of-the art methods. We analyse such networks in a principled way, combining novel scores from gene co-expression network analysis, to identify genes which are associated with certain growth conditions or highly co-expressed with a predefined set of genes of interest. We assigned a putative function to those genes. We complement and assess the information from the gene co-expression network with experimental results.

Miguel Alena Rodríguez, University of Birmingham

Dynamic combinatorial chemistry for the detection of cancer

Abnormal glycosylation –and more specifically, sialylation- patterns are known to occur in cancerous cells, becoming those glycans a biomarker for cancer detection. However, the complexity and vast array of glycoforms structurally similar to each other that can be present in cells, makes a challenge the identification of the ones actually involved in tumour growth. Rates of 50% and 25% of false positive and negative results -respectively- in early detection of Prostate Cancer (as an example) are a good proof for the need of an enhanced detection technology.

This project aims to develop a methodology to accurately, efficiently, and selectively find receptors for such glycans.

We employ Dynamic Combinatorial Chemistry (DCC). DCC allows you, not only to synthesise a number of receptors, but also to identify the best one among them. This methodology already gave us some preliminary promising results that will be further confirmed by analytical techniques such as ITC, SPR, and NMR. We are already working on the development of a peptide-based second-generation library, incorporating more complex building blocks and larger library sizes, that will bring even better receptors to be employed for the effective early detection of cancer.

Alicia L. Bruzos, The Francis Crick Institute

Beyond the limits of metastasis: marine contagious cancers

Clonally transmissible cancers, also known as contagious cancers, are somatic cell lineages that are transmitted between individuals via the physical transfer of living cancer cells. No virus, bacteria or parasite infects the new host, it is the cancer cell itself that will be established in the new individual and divide to form a new tumour. In other words, these cancer cells acquire the ability of contagion. Contagious cancers are particularly frequent in marine bivalves, in which the disease is presented in the form of a leukaemia called hemic neoplasia (HN). Tumour cells behave like metastatic cells, leaving their hosts to dive in the marine environment until they reach a new host and propagate inside it. For this reason, they represent an interesting and unique model to illuminate insights into the general mechanisms of cancer development and metastasis.

Since 2016, we have collected 6,700 cockles from 33 locations covering all the Atlantic Coast of Europe, from Morocco to Russia. 342 cockles (5.4%) from 5 different countries were diagnosed with cancer. However, we observe prevalence disparity across cockle populations, with countries where disease reaches high prevalence rates, and others with no disease at all. The contagious nature of these cancer samples was assessed with sequencing data. Phylogenetic analyses of mtDNA revealed nine paraphyletic clonal cancer lineages, some with a distribution extending over thousands of miles. Analysis of nuclear structural variants, however, does not reveal nine cancer lineages, seeming to suggest a maximum of two independent origins of the disease. Multiple captures of the mitochondria from the host cells by the cancer lineages, throughout the long-term evolution of these two cancer lineages, may explain these contradicting observations.

This work reveals that mitochondrial and nuclear DNA of cockle transmissible cancers tell slightly different evolutionary stories that are part of the same book.

Asier R. Muguruza, University of Birmingham

Development of aminocarboxylate coated hybrid silica nanocarriers to overcome antibiotic impermeability in Gram-negative bacteria

Antimicrobial resistance is the cause of an estimate of five million deaths worldwide, and along with its economic cost and the lack of new antibiotics in the pipeline is an increasing threat for current society.  Among the described resistance mechanisms, impermeability of Gram-negative bacterial species is a major barrier for successful treatment and novel drug design. Inclusion of therapeutic agents within nanostructured materials for their delivery and release is a potential approach to explore. Such nanomaterials have been proven to overcome different bacteria resistance mechanisms through surface chemical and/or physical modification. Silica nanoparticles (SiO2) have been highlighted as promising nanocarriers for drug delivery and tracking due to their chemical stability, biocompatibility and turnability regarding nanoparticle size, surface chemistry and porosity.  

In this work we show the design of a novel hybrid nanoparticles with therapeutic and tracking abilities through simultaneous encapsulation of an antibiotic (Vancomycin) and a luminescent agent ([Ru(phen)3]2+). Vancomycin is an FDA approved antibiotic, active only against gram-positive due the inability to cross of gram-negative bacteria. The ruthenium complex [Ru(phen)3]2+, where phen is 1,10-phenantronile, is a popular luminescent agent with photostable red-luminescence upon excitation in the visible range with long lifetimes. Moreover, [Ru(phen)3]2+ and other metal complexes have been previously included in SiO2 to visualisze the particles in tissues and in flows. Furthermore, we covalently modified the surface of the SiO2 nanocarriers with aminocarboxylate moieties to overcome described impermeability taking advantage of this molecule’s ability to bind metal ions and destabilisze cell membranes. Confocal fluorescence imaging of live bacterial cells incubated with designed nanoparticles revealed uptake in Gram negative bacteria (Escherichia coli). Absence of coating, however, leads to no cell uptake in E. coli, highlighting the importance of surface coating for the nanoparticles to overcome outer membrane and release included cargos. Determination of MIC values for six different Gram-negative bacterial species reveals high efficiency of coated hybrid nanoparticles compared with control samples. The presence of Ru(II) complex allows tracking of the particles in cells

Marta Domínguez Prieto, De Montfort University

Endocytosis and transcytosis of amyloid-β peptides by astrocytes: a possible mechanism for amyloid-β clearance in Alzheimer’s disease

Alzheimer’s Disease is characterised by the accumulation and deposition of amyloid-beta (Aβ) within the brain. Aβ accumulation depends not only on the rate of its synthesis but also on the rate of clearance. Since astrocytes have been proposed to participate in Aβ clearance from the brain, we decided to study the effects of different Aβ peptides (Aβ 25-35, Aβ 40, Aβ 42) on rat astrocytes in primary culture. Our results showed that all the peptides assayed significantly decreased astrocyte viability while increasing the production of reactive oxygen species (ROS). In order to examine the localization of Aβ within the cells, we carried out immunocytochemistry against Aβ. We observed that all the Aβ assayed were avidly internalised by astrocytes. To get inside the molecular mechanisms involved in Aβ internalisation, we challenged the astrocytes with inhibitors of endocytosis and transfected them with siRNAs to silence the expression of caveolin1 and clathrin. Our results clearly show that Aβ is internalised by a process of clathrin-mediated endocytosis. Furthermore, once we had checked that astrocytes were able to internalise amyloid-beta we wanted to know if they could also release it. In order to study this possible transcytosis, astrocytes cultured on transwell inserts were treated with amyloid-beta for a short period and then, the inserts were incubated with non-treated astrocytes, in such a way that there was no physical contact among them. Non-treated astrocytes internalised amyloid-beta released to the media by treated-astrocytes resulting in non-treated astrocytes death. In short, astrocytes transcytosed amyloid-beta by a process that includes clathrin-mediated endocytosis. This process could play a role in the clearance of Aβ peptides from the brain to cerebrospinal fluid.

Virginia Ruiz Asenjo, University of Hertfordshire & Universidad Francisco de Vitoria

Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of Aspergillus species

Section Flavi is one of the most significant sections in the genus Aspergillus. Phylogenetically, section Flavi is split in eight clases and the section currently contains 33 species, presenting similar morphological and phylogenetic characteristics.

Five main groups have a significant impact in the agrifood sector: Aspergillus flavus, A. nomius, A. oryzae, A. parasiticus, A. sojae and A. tamarii. Despite the shared common features, A. oryzae, A. sojae, and A. tamarii are non-aflatoxigenic, widely used in fermenting processes, and provide significant value to the industry. On the other hand, A. flavus, A. nomius and A. parasiticus are considered a significant problem for animal and human health. They are toxigenic, widely distributed, and grow in almost any crop or food. The taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. Currently, there are differential mediums such as AFPA (Aspergillus flavus and parasiticus agar) available for distinguishing toxigenic and non-toxigenic Aspergillus. However, results are normally contrasted with molecular techniques to accurately identify each strain.

Matrix-assisted laser desorption ionisation–time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and low-cost technique for microbial identification that can potentially reduce the resources required for proper identification between the different members of the family Aspergillus. However, leading companies in the area of Mass Spectrometry (MS) have focused their efforts on developing libraries mainly for clinical purposes, and there is a lack of consolidated commercial repositories for this purpose. Thus, there is a gap in developing standardised protocols and bespoke libraries for fungal identification by MALDI TOF. The development of both will pave the way for a cost-effective pathogen identification while providing a suitable approach for a diverse array of purposes, such as screening potential biocontrol agents or the rapid identification of environmental microbes.T

his research aimed to assess the effect of the media and incubation time in identifying Aspergillus Section Flavi species and the potential development of a superspectral library for more accurate identification of closely related species belonging to this group