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Abstract Objective African American women with breast cancer experience disproportionately poor survival outcomes, primarily due to the high prevalence of the deadliest subtype; triple-negative breast cancer (TNBC). The CRYβB2 gene is upregulated in tumors from African American patients across all breast cancer subtypes, including TNBC, and is associated with worse survival rates. This study investigated the effect of CRYβB2 on the invasion of TNBC cells and the underlying mechanisms contributing to this phenotype. Results We utilized the SUM159 cells with stable CRYβB2 overexpression in a 3D-culture tumor spheroids model in our investigation. A quantitative 3D invasion assay demonstrated that CRYβB2 overexpression significantly enhanced invasion (median invasion %; SUM159 = 0.14 and SUM159 + CRYβB2 = 0.33). RNA sequencing analysis indicated that CRYβB2 overexpression modulated cell-cell adhesion and extracellular matrix organization pathways, which are critical to invasion of cancer cells. Specifically, CRYβB2 suppressed the expression of key cell-cell adhesion genes known as clustered protocadherins and promoted the expression of PCDH7, a nonclustered protocadherin with known oncogenic roles in various cancers. Notably, the knockout of PCDH7 diminished the invasive capacity induced by CRYβB2 (median invasion %; SUM159 = 0.093, SUM159 + CRYβB2 = 0.184 and SUM159 + CRYβB2/PCDH7−/−=0.082). These findings provide a novel link between a previously identified differentially expressed gene, CRYβB2, in driving breast cancer phenotypes by modulating a class of adhesion proteins. more...

Abstract Background European cancer programmes and policies lack a unified health-related quality of life (HRQoL) assessment tool. The European oncology quality of life toolkit (EUonQoL-Kit) is a novel set of HRQoL questionnaires, co-designed with cancer patients and survivors, translated and culturally adapted into 31 European languages, and with both static and dynamic electronic administration modes. The main aim of this study is the psychometric assessment of the static version. Secondary aims include evaluating the EUonQoL-Kit acceptability, cross-validating the administration modes, exploring individual factors potentially affecting HRQoL and HRQoL inequalities between countries. Methods A sample of 4,500 participants, including three groups (active treatment, survivors, and palliative care) from 45 centres in 25 EU Member States and 7 associated countries, will be enrolled in a multicentre observational cross-sectional study. All participants will complete the static EUonQoL-Kit; three subsamples (each 10% of the total sample) will also respectively complete the following: a) dynamic EUonQoL-Kit, based on Item Response Theory (IRT)/Computer Adaptive Testing (CAT), b) FACT-G and EQ-5L-5D, and c) static EUonQoL-Kit (re-test). Psychometric analyses will encompass exploratory and confirmatory factor analyses (measurement model and structural validity), Cronbach's alpha (internal consistency), intraclass correlation coefficient (test–retest reliability), Pearson/Spearman correlation (concurrent validity), comparison of group scores (construct validity), and Differential Item Functioning (cross-country item equivalence). Secondary analyses will evaluate participant response time and rate, and static/dynamic score differences. Regression models will estimate associations between individual factors and HRQoL. Discussion The EUonQoL-Kit will serve to systematically incorporate patient perspectives into European cancer policies and to address HRQoL inequalities across Europe. Trial registration ClinicalTrials.gov Identifier: NCT05947903, 2023–06-28. more...

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are few effective treatments available to treat infection and KSHV oncogenesis. Disrupting the KSHV infectious cycle would diminish the viral spread. The KSHV lytic phase and production of new virions require efficient copying and packaging of the KSHV genome. KSHV encodes its own lytic DNA replication machinery, including the processivity factor (PF-8), which presents itself as an attractive target for antiviral development. We characterized PF-8 at the single molecule level using transmission electron microscopy to identify key molecular interactions that mediate viral DNA replication initiation. Our results indicate that PF-8 forms oligomeric ring structures (tetramer, hexamer, and/or dodecamer) similar to the related Epstein–Barr virus processivity factor (BMRF1). Our DNA positional mapping revealed high-frequency binding locations of PF-8 within the lytic origin of replication (OriLyt). A multi-variable analysis of PF-8 DNA-binding activity with three mutant OriLyts provides new insights into the mechanisms that PF-8 associates with viral DNA and complexes to form multi-ring-like structures. Collectively, these data enhance the mechanistic understanding of the molecular interactions (protein–protein and protein-DNA) of an essential KSHV DNA replication protein. more...

Abstract Background There is widespread agreement about the importance of communication skills training (CST) for healthcare professionals caring for cancer patients. Communication can be effectively learned and improved through specific CST. Existing CSTs have some limitations with regard to transferring the learning to the workplace. The aim of the study is developing, piloting, and preliminarily assessing a CST programme for hospital physicians caring for advanced cancer patients to improve communication competences. Methods This is a Phase 0-I study that follows the Medical Research Council framework; this paper describes the following sections: a literature review on CST, the development of the Teach to Talk training programme (TtT), the development of a procedure for assessing the quality of the implementation process and assessing the feasibility of the implementation process, and the pilot programme. The study was performed at a 900-bed public hospital. The programme was implemented by the Specialized Palliative Care Service. The programme was proposed to 19 physicians from 2 departments. Results The different components of the training course were identified, and a set of quality indicators was developed. The TtT programme was implemented; all the physicians attended the lesson, videos, and role-playing sessions. Only 25% of the physicians participated in the bedside training. It was more challenging to involve Haematology physicians in the programme. Conclusions The programme was completed as established for one of the two departments in which it was piloted. Thus, in spite of the good feedback from the trainees, a re-piloting of a different training program will be developed, considering in particular the bed side component. The program should be tailored on specific communication attitude and believes, probably different between different specialties. more...

Abstract Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single‐particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three‐dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo‐EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated more...

Hypertrophic pyloric stenosis (HPS) is the most important cause of non-bilious vomiting during infancy and it is usually idiopathic. It is very rare in older children and adolescents, in which gastric outlet obstruction (GOO) is more typically secondary to other conditions. Gastrointestinal malformation are frequently associated with Down syndrome and some of them (especially duodenal abnormalities) can be detected quite late. In infants the standard treatment of HPS is represented by a Ramstedt pyloromyotomy, while in adults a distal gastric resection is more indicated and it is usually associated to a Billroth I or II reconstruction. Case report: We present an unusual case of a 12-year-old girl with Down syndrome, diagnosed with late-onset HPS. She came to our attention with periodic non-bilious vomiting and growth delay. She underwent multiple imaging examinations, which highlighted the presence of an HPS. Based on patient's age and the thickness of the pyloric muscle, we decided to perform a partial gastrectomy with a Roux-en-y reconstruction. Patient was discharged on the X day. She is now on follow up, gains weight and presents good conditions. Discussion: HPS is rare but possible during childhood and adolescence. Diagnosis is usually performed through ultrasounds (US) and upper gastrointestinal radiological series (UGI), but sometimes further examinations are required (abdominal MRI, esophagogastroduodenoscopy). In older children since less invasive techniques are not effective, we have to consider partial gastrectomy. Based on our experience, Roux-en-y reconstruction is a valid option, associated with poor complication and good quality of life. more...

This data article is related to the research article entitled “Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression and TGF-beta secretion”, available in the journal Nanomedicine: Nanotechnology, Biology, and Medicine [1]. This Data in Brief consists of data that describe changes in the expression of basement membrane (BM)-associated genes and proteins in three non-transformed epithelial cell lines following acute (6 h) and chronic (24 h plus 7-day chase) exposure to silver nanoparticles (AgNPs). Human BEAS2B (lung), MCF10AI (breast), and CCD-18Co (colon) cultured epithelia were analyzed for protein expression by LC-MS/MS and for gene expression by pathway-focused QRT-PCR arrays of 168 focal adhesion, integrin, and extracellular matrix (ECM) genes known to be localized to the plasma membrane, the BM/ECM, or secreted into the extracellular space. Ingenuity pathway analysis (IPA) of combined gene and protein expression datasets was then used to predict canonical pathways affected by AgNP exposure. Keywords: Silver nanoparticles, Epithelial cells, Basement membrane, Extracellular matrix, TGF-Beta, Intracellular signalling pathway analysis more...

Extracellular signaling is a mechanism that higher eukaryotes have evolved to facilitate organismal homeostasis. Recent years have seen an emerging interest in the role of secreted microvesicles, termed extracellular vesicles (EV) or exosomes in this signaling network. EV contents can be modified by the cell in response to stimuli, allowing them to relay information to neighboring cells, influencing their physiology. Here we show that the tumor virus Kaposi's Sarcoma-associated herpesvirus (KSHV) hijacks this signaling pathway to induce cell proliferation, migration, and transcriptome reprogramming in cells not infected with the virus. KSHV-EV activates the canonical MEK/ERK pathway, while not alerting innate immune regulators, allowing the virus to exert these changes without cellular pathogen recognition. Collectively, we propose that KSHV establishes a niche favorable for viral spread and cell transformation through cell-derived vesicles, all while avoiding detection. more...

Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods. more...

ABSTRACT Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells. IMPORTANCE Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. Nef is dispensable for viral replication but essential for AIDS progression in vivo. Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression. more...

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with the human malignancy Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. KSHV establishes lytic infection of monocytes in vivo, which may represent an important cellular reservoir during KS disease progression. KS tumors consist of latently infected endothelial cells; however, lytic phase gene products are important for KS onset. Early KS lesion progression is driven by proinflammatory cytokines supplied by immune cell infiltrates including T cells and monocytes. KSHV-infected monocytes may supply the lytic viral products and the inflammatory milieu conducive to KS tumor progression. To establish successful infection, KSHV extensively modulates the host immune system. KSHV antigens activate both innate and adaptive immune responses including KSHV-specific T cells, but lifelong infection is still established. Programmed death ligand 1 (PD-L1) is a prosurvival cell surface protein that suppresses T-cell-mediated killing. PD-L1 is variably present on various tumor cells and is a targetable marker for cancer treatment. We show that KSHV infection of human monocytes increases PD-L1 expression and transcription in a dose-dependent manner. We also saw evidence of lytic gene expression in the KSHV-infected monocytes. Intact KSHV is needed for full PD-L1 response in human monocytes. KSHV induces a general proinflammatory cytokine milieu including interleukins 1α, 1β, and 6, which have been implicated in early KS lesion progression. KSHV-mediated PD-L1 increase may represent a novel mechanism of KSHV-mediated immune modulation to allow for virus survival and eventually malignant progression. IMPORTANCE KSHV is the etiologic agent of Kaposi’s sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman’s disease. Programmed death ligand 1 (PD-L1) is an immunosuppressive cell surface marker that inhibits T cell activation. We report that KSHV infection of primary human monocytes upregulates PD-L1 transcription and protein expression. Analysis of the cytokine and chemokine milieu following KSHV infection of monocytes revealed that KSHV induces interleukins 1α, 1β, and 6, all of which have been implicated in KS development. Our work has identified another potential immune evasion strategy for KSHV and a potential target for immunotherapy of KSHV-derived disease. more...