Quantification of proteins in whole blood, plasma and DBS, with element-labelled antibody detection by ICP-MS

Over recent years, quantification of multiple proteins in body fluids has become increasingly prominent, which is beneficial to a number of scientific fields, not least biomedical. Several techniques have been developed based on conventional ELISA; one of these techniques is analysis of proteins labelled with element-tagged antibodies by ICP-MS in serum, allowing quantification of multiple targets within a single sample. This research aimed to quantify albumin and immunoglobulin G (IgG) levels in plasma, whole blood and dried blood spots using NANOGOLD and Europium labelled antibodies analysed by ICP-MS. Before the proteins were quantified simultaneously, albumin and IgG concentrations were measured separately and compared to protein levels obtained by ELISA. It was found that protein concentrations for both albumin and IgG obtained with element-labelled antibody detection correspond to those determined by ELISA. Furthermore, albumin and IgG levels measured simultaneously by ICP-MS correspond to concentrations found when the proteins were analysed separately by ICP-MS. Finally, development of this method has provided a positive indication that it can be extended to quantification of additional proteins, which could be related to a disease or as a minimum provide additional information for a protein profile of an individual. [Abstract copyright: Copyright © 2019. Published by Elsevier Inc.

Adaptation of the kirkstall QV600 LLI microfluidics system for the study of gastrointestinal absorption by mass spectrometry imaging and LC-MS/MS

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 μm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development

Multimodal imaging of metals in a retinal degeneration model to inform on ocular disease

The metallome has been involved in the pathological investigation into ocular tissue for decades; however, as technologies advance, more information can be ascertained from individual tissue sections that were not previously possible. Herein, a demonstration of complementary techniques has been utilized to describe the distribution and concentrations of essential metals in both wildtype (WT) and rhodopsin (Rho−/−) ocular tissues. The multimodal approach described is an example of complementary datasets that can be produced when employing a multifaceted analytical approach. Heterogenous distributions of copper and zinc were observable within both WT and Rho−/− tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), and the distributions of further trace elements notoriously problematic for ICP-MS analysis (phosphorous, Sulfur, chlorine, potassium, calcium, iron, and aluminum) were analysed by particle-induced X-ray emission (PIXE)

Comparison of Osteosarcoma Aggregated Tumour Models with Human Tissue by Multimodal Mass Spectrometry Imaging

Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Carbon budgets for Caribbean mangrove forests of varying structure and with phosphorus enrichment

There are few detailed carbon (C) budgets of mangrove forests, yet these are important for understanding C sequestration in mangrove forests, how they support the productivity of the coast and their vulnerability to environmental change. Here, we develop C budgets for mangroves on the islands of Twin Cays, Belize. We consider seaward fringing forests and interior scrub forests that have been fertilized with phosphorus (P), which severely limits growth of trees in the scrub forests. We found that respiration of the aboveground biomass accounted for 60%–80% of the fixed C and that respiration of the canopy and aboveground roots were important components of respiration. Soil respiration accounted for only 7%–11% of total gross primary production (GPP) while burial of C in soils was ~4% of GPP. Respiration by roots can account for the majority of soil respiration in fringing forests, while microbial processes may account 80% of respiration in scrub forests. Fertilization of scrub forests with P enhanced GPP but the proportion of C buried declined to ~2% of GPP. Net ecosystem production was 17%–27% of GPP similar to that reported for other mangrove forests. Carbon isotope signatures of adjacent seagrass suggest that dissolved C from mangroves is exported into the adjacent ecosystems. Our data indicate that C budgets can vary among mangrove forest types and with nutrient enrichment and that low productivity mangroves provide a disproportionate share of exported C

The acute-aged care interface: Exploring the dynamics of 'bed blocking'

Objective: To understand the dynamics underlying 'bed-blocking' in Australian public hospitals that is frequently blamed on older patients. Methods: Analysis of primary and secondary data of utilisation patterns of hospital and aged care services by older Australians. Results: A model of the dynamics at the acute–aged care interface was developed, in which the pathway into permanent high-care Residential Aged Care (RAC) is conceptualised as competing queues for available places by applicants from the hospital, the community and from within RAC facilities. The hospital effectively becomes a safety net to accommodate people with high-care needs who cannot be admitted into RAC in a timely manner. Conclusion: The model provides a useful tool to explore some of the issues that give rise to access-block within the public hospital system. Access-block cannot be understood by viewing the hospital system in isolation from other sectors that support the health and well-being of older Australians

Platinum catalysed hydrosilylation of propargylic alcohols

A facile and user-friendly protocol has been developed for the selective synthesis of E-vinyl silanes derived from propargylic alcohols using a PtCl2/XPhos catalyst system. The reaction is generally high yielding and provides a single regioisomer at the β-position with E-alkene geometry. The reaction is extremely tolerant of functionality and has a wide scope of reactivity both in terms of alkynes and silanes used. The catalyst loading has been investigated and it is found that good reactivity is observed at extremely low catalyst loadings. This methodology has also been extended to a one-pot hydrosilylation Denmark–Hiyama coupling

Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine

The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II-binding HER3 peptides, which can generate HER3-specific CD4 Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II-binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide-pulsed dendritic cell vaccination resulted in anti-HER3 CD4 Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4 T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4 Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors