Quantitative Detection of Biological Nanovesicles in Drops of Saliva Using Microcantilevers

Extracellular nanovesicles (EVs) are lipid-based vesicles secreted by cells and are present in all bodily fluids. They play a central role in communication between distant cells and have been proposed as potential indicators for the early detection of a wide range of diseases, including different types of cancer. However, reliable quantification of a specific subpopulation of EVs remains challenging. The process is typically lengthy and costly and requires purification of relatively large quantities of biopsy samples. Here, we show that microcantilevers operated with sufficiently small vibration amplitudes can successfully quantify a specific subpopulation of EVs directly from a drop (0.1 mL) of unprocessed saliva in less than 20 min. Being a complex fluid, saliva is highly non-Newtonian, normally precluding mechanical sensing. With a combination of standard rheology and microrheology, we demonstrate that the non-Newtonian properties are scale-dependent, enabling microcantilever measurements with a sensitivity identical to that in pure water when operating at the nanoscale. We also address the problem of unwanted sensor biofouling by using a zwitterionic coating, allowing efficient quantification of EVs at concentrations down to 0.1 μg/mL, based on immunorecognition of the EVs’ surface proteins. We benchmark the technique on model EVs and illustrate its potential by quantifying populations of natural EVs commonly present in human saliva. The method effectively bypasses the difficulty of targeted detection in non-Newtonian fluids and could be used for various applications, from the detection of EVs and viruses in bodily fluids to the detection of molecular clusters or nanoparticles in other complex fluids

Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea–Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours

Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses

Influence of the scanning temperature on the classification of whisky samples analysed by UV-VIS spectroscopy

The definition of the optimal temperature and its effects (either increasing or variations) during analysis of alcoholic beverages are of importance to develop protocols based in spectroscopy. Although several reports have been published on the use of spectroscopy combined with chemometrics to classify and authenticate alcoholic beverages (e.g., wine, tequila, whisky), few reports deal with issues related with the spectra collection (e.g., temperature, path length) and its effect on the classification performances. The objective of this study was to evaluate the effect of increasing temperature on both the UV-VIS spectra of whisky and on the classification results of the samples according to country of origin. Whisky samples from different commercial labels were analysed at different temperatures (25, 35, 45, 55 ◦C) using a UV-VIS instrument (Agilent, Cary 3500). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models based in cross validation were used to classify whisky samples according to scanning temperature and origin. The results of this study indicated that temperature did not affect the classification of whisky samples according to country of origin. Overall, well defined protocols need to be defined for routine use of these methods in research and by the industr

Bacterial-nanostructure interactions: The role of cell elasticity and adhesion forces

The attachment of single-celled organisms, namely bacteria and fungi, to abiotic surfaces is of great interest to both the scientific and medical communities. This is because the interaction of such cells has important implications in a range of areas, including biofilm formation, biofouling, antimicrobial surface technologies, and bio-nanotechnologies, as well as infection development, control, and mitigation. While central to many biological phenomena, the factors which govern microbial surface attachment are still not fully understood. This lack of understanding is a direct consequence of the complex nature of cell-surface interactions, which can involve both specific and non-specific interactions. For applications involving micro- and nano-structured surfaces, developing an understanding of such phenomenon is further complicated by the diverse nature of surface architectures, surface chemistry, variation in cellular physiology, and the intended technological output. These factors are extremely important to understand in the emerging field of antibacterial nanostructured surfaces. The aim of this perspective is to re-frame the discussion surrounding the mechanism of nanostructured-microbial surface interactions. Broadly, the article reviews our current understanding of these phenomena, while highlighting the knowledge gaps surrounding the adhesive forces which govern bacterial-nanostructure interactions and the role of cell membrane rigidity in modulating surface activity. The roles of surface charge, cell rigidity, and cell-surface adhesion force in bacterial-surface adsorption are discussed in detail. Presently, most studies have overlooked these areas, which has left many questions unanswered. Further, this perspective article highlights the numerous experimental issues and misinterpretations which surround current studies of antibacterial nanostructured surfaces

Chemometrics for environmental monitoring: a review

Environmental monitoring is necessary to ensure the overall health and conservation of an ecosystem. However, ecosystems (e.g.\ua0air, water, soil), are complex, involving numerous processes (both native and external), inputs, contaminants, and living organisms. As such, monitoring an environmental system is not a trivial task. The data obtained from natural systems is often multifaceted and convoluted, as a multitude of inputs can be intertwined within the matrix of the information obtained as part of a study. This means that trends and important results can be easily overlooked by conventional and single dimensional data analysis protocols. Recently, chemometric methods have emerged as a powerful method for maximizing the details contained within a chemical data set. Specifically, chemometrics refers to the use of mathematical and statistical analysis methods to evaluate chemical data, beyond univariant analysis. This type of analysis can provide a quantitative description of environmental measurements, while also having the capacity to reveal previously overlooked trends in data sets. Applying chemometrics to environmental data allows us to identify and describe the inter-relationship of environmental drivers, sources of contamination, and their potential impact upon the environment. This review aims to provide a detailed understanding of chemometric techniques, how they are currently used in environmental monitoring, and how these techniques can be used to improve current practices. An enhanced ability to monitor environmental conditions and to predict trends would be greatly beneficial to government and research agencies in their ability to develop environmental policies and analytical procedures

From Academia to Reality Check: A Theoretical Framework on the Use of Chemometric in Food Sciences

There is no doubt that the current knowledge in chemistry, biochemistry, biology, and mathematics have led to advances in our understanding about food and food systems. However, the so-called reductionist approach has dominated food research, hindering new developments and innovation in the field. In the last three decades, food science has moved into the digital and technological era, inducing several challenges resulting from the use of modern instrumental techniques, computing and algorithms incorporated to the exploration, mining, and description of data derived from this complexity. In this environment, food scientists need to be mindful of the issues (advantages and disadvantages) involved in the routine applications of chemometrics. The objective of this opinion paper is to give an overview of the key issues associated with the implementation of chemometrics in food research and development. Please note that specifics about the different methodologies and techniques are beyond the scope of this review

Adsorbed and near-surface structure of ionic liquids determines nanoscale friction

Surface-adsorbed and near-surface ion layer structure controls nanotribology in the silica–propylammonium nitrate (PAN)–mica system. Atomic Force Microscopy (AFM) imaging and normal force curves reveal that the normal load dictates the number of interfacial ion layers and the lateral layer structure. Shear force measurements show the lubricity of the interface changes with the number, and lateral structure, of the confined ion layer(s)