The Cellular Localisation and Role of S100P in the Motility and Invasion of Trophoblasts

The S100 family of proteins are calcium-binding proteins expressed in a wide variety of tissues that participate in both intracellular and extracellular activities. One family member, S100P, has gained attention primarily in the context of promotion of carcinogenesis. Containing no enzymatic activity of its own, it interacts with multiple target proteins to regulate motility and invasion of cancer cells. Little is known about the role of S100P in trophoblast cells of the placenta, but our initial work has shown that it plays a crucial role in regulating both cellular motility and invasion. In this work, we sought to first establish the cellular localisation of S100P using different fractionation analysis. We first demonstrated the strictly cytoplasmic and membrane-associated localisation of S100P in different trophoblast cells including JEG-3, BeWo and HTR8, as well as cancer cells, regardless of the levels of S100P or any other changes such as excess calcium, addition of a non-ionic detergent, or the use of nuclear export inhibitors (Leptomycin B). Interestingly, tagging S100P with the fluorescent marker YFP led to a relocalisation of S100P from the cytoplasm/membrane fractions to nuclear fractions, suggesting the fusion protein had differential properties and cellular localisation. Further cellular compartmentalisation allowed us to determine that some of the S100P pools were also found in the plasma membrane fraction in both trophoblasts and in cancer cells. S100P was detectable at the extracellular surface using plasma membrane purification and biotinylation experiments. Analysis of the S100P structure suggests the presence of possible membrane-interacting residues. The C-terminal polybasic domain, in combination with potentially lipid-modified residues, may promote the association of S100P with negatively-charged membrane structures. Blockade of extracellular S100P through use of an S100P antibody or with cromolyn, a molecule shown to interact with helix 4 of S100P, results in decreases in both migration and invasion of choriocarcinoma cell line JEG-3, EVT-like HTR8/SVneo cells and EVT cells isolated from first trimester placenta, but did not lead to any changes in the formation of focal adhesions. This is in contrast to changes in total cellular levels of S100P in JEG-3 and HTR8 cells where a decrease in paxillin-containing focal adhesions can be seen. These studies therefore show that different pools of S100P, either intracellular or membrane-associated, promote trophoblast motility and invasion through two independent pathways. Further analysis in order to fully characterise the S100P interactome demonstrated the presence of S100P in high molecular weight complexes in trophoblast cells, and mass spectrometry analysis presented a number of proteins with increased abundance in S100P-expressing cells, although characterisation of specific S100P interactors remains to be fully explored

The influence of customer loyalty on small island economies: an empirical and exploratory study

There is growing consensus that companies' long-term success is reliant on building and sustaining strong customer relationships. This study explores the antecedents of loyalty in business to business (B2Bs) using Guernsey's telecommunication industry as a case study. It examines how these influence customer loyalty orientation and factors that help service providers improve loyalty rates. Extant literature pays little attention to the antecedents of loyalty in small island economies. Prior research focuses on cultural, environmental and macro-economic issues. Drawing on Dick and Basu's (Customer loyalty: Toward an integrated conceptual framework. Journal of the Academy of Marketing Science, 22(Spring), 99–113, 1994) loyalty model, this research explores loyalty antecedents that are cognisant of distinct market conditions that can impact customer loyalty within the telecommunications sector of a small island economy. It seeks to advance understanding of loyalty in B2B relationships in this context and identify factors that contribute towards converting passively loyal customers to being actively loyal customers

The role of the C-terminal lysine of S100P in S100P-induced cell migration and metastasis

S100P protein is a potent inducer of metastasis in a model system, and its presence in cancer cells of patients is strongly associated with their reduced survival times. A well-established Furth Wistar rat metastasis model system, methods for measuring cell migration, and specific inhibitors were used to study pathways of motility-driven metastasis. Cells expressing C-terminal mutant S100P proteins display markedly-reduced S100P-driven metastasis in vivo and cell migration in vitro. These cells fail to display the low focal adhesion numbers observed in cells expressing wild-type S100P, and the mutant S100P proteins exhibit reduced biochemical interaction with non-muscle myosin heavy chain isoform IIA in vitro. Extracellular inhibitors of the S100P-dependent plasminogen activation pathway reduce, but only in part, wild-type S100P-dependent cell migration; they are without effect on S100P-negative cells or cells expressing C-terminal mutant S100P proteins and have no effect on the numbers of focal adhesions. Recombinant wild-type S100P protein, added extracellularly to S100P-negative cells, stimulates cell migration, which is abolished by these inhibitors. The results identify at least two S100P-dependent pathways of migration, one cell surface and the other intracellularly-linked, and identify its C-terminal lysine as a target for inhibiting multiple migration-promoting activities of S100P protein and S100P-driven metastasis

An extracellular/membrane bound S100P pool regulates motility and invasion of human extravillous trophoblast lines and primary cells

Whilst S100P has been shown to be a marker for carcinogenesis, we have shown, in non physio-pathological states, that its expression promotes trophoblast motility and invasion but the mechanisms explaining these cellular processes are unknown. Here we identify the presence of S100P in the plasma membrane/cell surface of all trophoblast cells tested, whether lines, primary extravillous (EVT) cells or section tissue samples using either biochemical purification ofplasma membrane material, cell surface protein isolation through biotinylation or microscopy analysis. Using extracellular loss of function studies, through addition of a specific S100P antibody, our work shows that inhibiting the cell surface/membrane-bound or extracellular S100P pools significantly reduces, but importantly only in part, both cell motility and cellular invasion in different trophoblastic cell lines, as well as primary EVTs. Interestingly, this loss in cellular motility/invasion did not result in changes to the overall actin organisation and focal adhesion complexes. These findings shed new light on at least two newly characterized pathways by which S100P promotes trophoblast cellular motility and invasion. One where cellular S100P levels involve the remodelling of focal adhesions whilst another, an extracellular pathway, appears to be focal adhesion independent. Both pathways could lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation

SARS-CoV-2 anti-spike antibody levels following second dose of ChAdOx1 nCov-19 or BNT162b2 in residents of long-term care facilities in England (VIVALDI)

General population studies have shown strong humoral response following SARS-CoV-2 vaccination with subsequent waning of anti-spike antibody levels. Vaccine-induced immune responses are often attenuated in frail and older populations, but published data are scarce. We measured SARS-CoV-2 anti-spike antibody levels in Long-Term Care Facility residents and staff following second vaccination dose with Oxford-AstraZeneca or Pfizer-BioNTech. Vaccination elicited robust antibody responses in older residents, suggesting comparable levels of vaccine-induced immunity to that in the general population. Antibody levels are higher after Pfizer-BioNTech vaccination but fall more rapidly compared to Oxford-AstraZeneca recipients and are enhanced by prior infection in both groups

Robust SARS-CoV-2-specific and heterologous immune responses in vaccine-naïve residents of long-term care facilities who survive natural infection

We studied humoral and cellular immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 152 long-term care facility staff and 124 residents over a prospective 4-month period shortly after the first wave of infection in England. We show that residents of long-term care facilities developed high and stable levels of antibodies against spike protein and receptor-binding domain. Nucleocapsid-specific responses were also elevated but waned over time. Antibodies showed stable and equivalent levels of functional inhibition against spike-angiotensin-converting enzyme 2 binding in all age groups with comparable activity against viral variants of concern. SARS-CoV-2 seropositive donors showed high levels of antibodies to other beta-coronaviruses but serostatus did not impact humoral immunity to influenza or other respiratory syncytial viruses. SARS-CoV-2-specific cellular responses were similar across all ages but virus-specific populations showed elevated levels of activation in older donors. Thus, survivors of SARS-CoV-2 infection show a robust and stable immunity against the virus that does not negatively impact responses to other seasonal viruses

Surgical Standards for Management of the Axilla in Breast Cancer Clinical Trials with Pathological Complete Response Endpoint.

Advances in the surgical management of the axilla in patients treated with neoadjuvant chemotherapy, especially those with node positive disease at diagnosis, have led to changes in practice and more judicious use of axillary lymph node dissection that may minimize morbidity from surgery. However, there is still significant confusion about how to optimally manage the axilla, resulting in variation among practices. From the viewpoint of drug development, assessment of response to neoadjuvant chemotherapy remains paramount and appropriate assessment of residual disease-the primary endpoint of many drug therapy trials in the neoadjuvant setting-is critical. Therefore decreasing the variability, especially in a multicenter clinical trial setting, and establishing a minimum standard to ensure consistency in clinical trial data, without mandating axillary lymph node dissection, for all patients is necessary. The key elements which include proper staging and identification of nodal involvement at diagnosis, and appropriately targeted management of the axilla at the time of surgical resection are presented. The following protocols have been adopted as standard procedure by the I-SPY2 trial for management of axilla in patients with node positive disease, and present a framework for prospective clinical trials and practice

Erratum: Author Correction: Surgical Standards for Management of the Axilla in Breast Cancer Clinical Trials with Pathological Complete Response Endpoint.

[This corrects the article DOI: 10.1038/s41523-018-0074-6.]