Technical and health governance aspects of the External Quality Assessment Scheme for the SARS-CoV-2 molecular tests: institutional experience performed in all clinical laboratories of a Regional Health Service.

OBJECTIVES: Since December 2019, the worldwide public health has been threatened by a severe acute respiratory syndrome caused by Coronavirus-2. From the beginning, a turning point has been the identification of new cases of infection, in order to minimize the virus spreading among the population. For this reason, it was necessary introducing a panel of tests able to identify positive cases, which became crucial for all countries. METHODS: As a Regional Reference Centre, the CRQ Laboratory (Regional Laboratory for the Quality Control) developed and conducted an External Quality Assessment (EQA) panel of assay, so as to evaluate the quality of real-time reverse transcription polymerase chain reaction (PCR), which were used by 62 Sicilian laboratories, previously authorized to issue certificates for the COVID-19 diagnosis, on behalf of the Public Health Service. RESULTS: The qualitative performance test was based on pooled samples with different viral loads of SARS-CoV-2 or human Coronavirus OC43. 75% of the participating laboratories tested all core samples correctly, while the remaining 25% interpreted incorrectly the EQA exercise samples matching negatively the standards required. CONCLUSIONS: Subsequent inspection visits confirmed the issue of incorrect positive and negative certifications for COVID-19 by private and public laboratories, despite the possession of the authorization requirements currently provided for by current regulations, with a significant impact on the SSR.

Changes in the Transcriptome Profiles of Human Amnion-Derived Mesenchymal Stromal/Stem Cells Induced by Three-Dimensional Culture: A Potential Priming Strategy to Improve Their Properties.

Mesenchymal stromal/stem cells (MSCs) are believed to function in vivo as a homeostatic tool that shows therapeutic properties for tissue repair/regeneration. Conventionally, these cells are expanded in two-dimensional (2D) cultures, and, in that case, MSCs undergo genotypic/phenotypic changes resulting in a loss of their therapeutic capabilities. Moreover, several clinical trials using MSCs have shown controversial results with moderate/insufficient therapeutic responses. Different priming methods were tested to improve MSC effects, and three-dimensional (3D) culturing techniques were also examined. MSC spheroids display increased therapeutic properties, and, in this context, it is crucial to understand molecular changes underlying spheroid generation. To address these limitations, we performed RNA-seq on human amnion-derived MSCs (hAMSCs) cultured in both 2D and 3D conditions and examined the transcriptome changes associated with hAMSC spheroid formation. We found a large number of 3D culture-sensitive genes and identified selected genes related to 3D hAMSC therapeutic effects. In particular, we observed that these genes can regulate proliferation/differentiation, as well as immunomodulatory and angiogenic processes. We validated RNA-seq results by qRT-PCR and methylome analysis and investigation of secreted factors. Overall, our results showed that hAMSC spheroid culture represents a promising approach to cell-based therapy that could significantly impact hAMSC application in the field of regenerative medicine.

Aneuploid IMR90 cells induced by depletion of pRB, DNMT1 and MAD2 show a common gene expression signature.

Chromosome segregation defects lead to aneuploidy which is a major feature of solid tumors. How diploid cells face chromosome mis-segregation and how aneuploidy is tolerated in tumor cells are not completely defined yet. Thus, an important goal of cancer genetics is to identify gene networks that underlie aneuploidy and are involved in its tolerance. To this aim, we induced aneuploidy in IMR90 human primary cells by depleting pRB, DNMT1 and MAD2 and analyzed their gene expression profiles by microarray analysis. Bioinformatic analysis revealed a common gene expression profile of IMR90 cells that became aneuploid. Gene Set Enrichment Analysis (GSEA) also revealed gene-sets/pathways that are shared by aneuploid IMR90 cells that may be exploited for novel therapeutic approaches in cancer. Furthermore, Protein-Protein Interaction (PPI) network analysis identified TOP2A and KIF4A as hub genes that may be important for aneuploidy establishment.

RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. METHODS: We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipitated fraction and the unbound sample resulting from the RIP experiment. We used the expression profile of the input sample to compute several variables, using formulae capable of integrating the information on miRNA binding sites, both in the 3'UTR and coding regions, with miRNA and mRNA expression level profiles. We compared immunoprecipitated vs unbound samples to determine the enriched or underrepresented genes in the immunoprecipitated fractions, independently for AGO2 and GW182 related samples. RESULTS: For each of the two proteins, we trained and tested several support vector machine algorithms capable of distinguishing the enriched from the underrepresented genes that were experimentally detected. The most efficient algorithm for distinguishing the enriched genes in AGO2 immunoprecipitated samples was trained by using variables involving the number of binding sites in both the 3'UTR and coding region, integrated with the miRNA expression profile, as expected for miRNA targets. On the other hand, we found that the best variable for distinguishing the enriched genes in the GW182 immunoprecipitated samples was the length of the coding region. CONCLUSIONS: Due to the major role of GW182 in GW/P-bodies, our data suggests that the AGO2-GW182 RISC recruits genes based on miRNA binding sites in the 3'UTR and coding region, but only the longer mRNAs probably remain sequestered in GW/P-bodies, functioning as a repository for translationally silenced RNAs.

Negative transcriptional control of ERBB2 gene by MBP-1 and HDAC1: diagnostic implications in breast cancer.

BACKGROUND: The human ERBB2 gene is frequently amplified in breast tumors, and its high expression is associated with poor prognosis. We previously reported a significant inverse correlation between Myc promoter-binding protein-1 (MBP-1) and ERBB2 expression in primary breast invasive ductal carcinoma (IDC). MBP-1 is a transcriptional repressor of the c-MYC gene that acts by binding to the P2 promoter; only one other direct target of MBP-1, the COX2 gene, has been identified so far. METHODS: To gain new insights into the functional relationship linking MBP-1 and ERBB2 in breast cancer, we have investigated the effects of MBP-1 expression on endogenous ERBB2 transcript and protein levels, as well as on transcription promoter activity, by transient-transfection of SKBr3 cells. Reporter gene and chromatin immunoprecipitation assays were used to dissect the ERBB2 promoter and identify functional MBP-1 target sequences. We also investigated the relative expression of MBP-1 and HDAC1 in IDC and normal breast tissues by immunoblot analysis and immunohistochemistry. RESULTS: Transfection experiments and chromatin immunoprecipitation assays in SKBr3 cells indicated that MBP-1 negatively regulates the ERBB2 gene by binding to a genomic region between nucleotide -514 and -262 of the proximal promoter; consistent with this, a concomitant recruitment of HDAC1 and loss of acetylated histone H4 was observed. In addition, we found high expression of MBP-1 and HDAC1 in normal tissues and a statistically significant inverse correlation with ErbB2 expression in the paired tumor samples. CONCLUSIONS: Altogether, our in vitro and in vivo data indicate that the ERBB2 gene is a novel MBP-1 target, and immunohistochemistry analysis of primary tumors suggests that the concomitant high expression of MBP-1 and HDAC1 may be considered a diagnostic marker of cancer progression for breast IDC.

The Suitability of RNA from Positive SARS-CoV-2 Rapid Antigen Tests for Whole Virus Genome Sequencing and Variant Identification to Maintain Genomic Surveillance.

The COVID-19 pandemic has transformed laboratory management, with a surge in demand for diagnostic tests prompting the adoption of new diagnostic assays and the spread of variant surveillance tools. Rapid antigen tests (RATs) were initially used only for screening and later as suitable infection assessment tools. This study explores the feasibility of sequencing the SARS-CoV-2 genome from the residue of the nasopharyngeal swab extraction buffers of rapid antigen tests (RATs) to identify different COVID-19 lineages and sub-lineages. METHODS: Viral RNA was extracted from the residue of the nasopharyngeal swab extraction buffers of RATs and, after a confirmation of positivity through a reaction of RT-PCR, viral genome sequencing was performed. RESULTS: Overall, the quality of the sequences obtained from the RNA extracted from the residue of the nasopharyngeal swab extraction buffers of RATs was adequate and allowed us to identify the SARS-CoV-2 variants' circulation and distribution in a period when the use of molecular swabs had been drastically reduced. CONCLUSIONS: This study demonstrates the potential for genomic surveillance by sequencing SARS-CoV-2 from the residue of the nasopharyngeal swab extraction buffers of RATs, highlighting alternative possibilities for tracking variants.

Transcriptomic Changes Following Partial Depletion of CENP-E in Normal Human Fibroblasts.

The centromere is a fundamental chromosome structure in which the macro-molecular kinetochore assembles and is bound by spindle microtubules, allowing the segregation of sister chromatids during mitosis. Any alterations in kinetochore assembly or functioning or kinetochore-microtubule attachments jeopardize chromosome stability, leading to aneuploidy, a common feature of cancer cells. The spindle assembly checkpoint (SAC) supervises this process, ensuring a faithful segregation of chromosomes. CENP-E is both a protein of the kinetochore and a crucial component of the SAC required for kinetochore-microtubule capture and stable attachment, as well as congression of chromosomes to the metaphase plate. As the function of CENP-E is restricted to mitosis, its haploinsufficiency has been used to study the induced cell aneuploidy; however, the gene expression profile triggered by CENP-E reduction in normal cells has never been explored. To fill this gap, here we investigated whether a gene network exists that is associated with an siRNA-induced 50% reduction in CENP-E and consequent aneuploidy. Gene expression microarray analyses were performed at early and late timepoints after transfection. Initially, cell cycle regulation and stress response pathways were downregulated, while afterwards pathways involved in epithelial-mesenchymal transition, hypoxia and xenobiotic metabolism were altered. Collectively, our results suggest that CENP-E reduction triggers a gene expression program that recapitulates some features of tumor cells.

The Secreted Protein C10orf118 Is a New Regulator of Hyaluronan Synthesis Involved in Tumour-Stroma Cross-Talk.

Interaction between cancer cells and their microenvironment is central in defining the fate of cancer development. Tumour cells secrete signals (cytokines, chemokines, growth factors) that modify the surrounding area, while the niche supplies structures and activities necessary for tumour maintenance and growth. Hyaluronan (HA) is a glycosaminoglycan that constitute cancer cell niche and is known to influence tumour functions such as proliferation, migration and neoangiogenesis. The knowledge of the factors regulating HA synthesis and size is crucial in understanding the mechanisms sustaining tumour development. Here we show that a yet uncharacterized protein secreted by breast tumour cell lines, named c10orf118 (accession number NM_018017 in NCBI/BLAST, and Q7z3E2 according to the Uniprot identifier), with a predicted length of 898 amino acids, can induce the secretion of HA by stromal fibroblasts through the up-regulation of the hyaluronan synthase 2 gene (HAS2). Intracellularly, this protein is localized in the Golgi apparatus with a possible role in vesicle maturation and transport. The expression of c10orf118 was verified in breast cancer patient specimens and was found to be associated with the presence of estrogen receptor that characterizes a good patient survival. We suggest c10orf118 as a new player that influences the HA amount in breast cancer microenvironment and is associated with low aggressiveness of cancer.

Viral miRNAs as Active Players and Participants in Tumorigenesis.

The theory that viruses play a role in human cancers is now supported by scientific evidence. In fact, around 12% of human cancers, a leading cause of morbidity and mortality in some regions, are attributed to viral infections. However, the molecular mechanism remains complex to decipher. In recent decades, the uncovering of cellular miRNAs, with their invaluable potential as diagnostic and prognostic biomarkers, has increased the number of studies being conducted regarding human cancer diagnosis. Viruses develop clever mechanisms to succeed in the maintenance of the viral life cycle, and some viruses, especially herpesviruses, encode for miRNA, v-miRNAs. Through this viral miRNA, the viruses are able to manipulate cellular and viral gene expression, driving carcinogenesis and escaping the host innate or adaptive immune system. In this review, we have discussed the main viral miRNAs and virally influenced cellular pathways, and their capability to drive carcinogenesis.

Cellular stress induces cap-independent alpha-enolase/MBP-1 translation.

Myc promoter-binding protein-1 (MBP-1) is a shorter protein variant of the glycolytic enzyme alpha-enolase. Although several lines of evidence indicate that MBP-1 acts as a tumor suppressor, the cellular mechanisms and signaling pathways underlying MBP-1 expression still remain largely elusive. To dissect these pathways, we used the SkBr3 breast cancer cell line and non-tumorigenic HEK293T cells ectopically overexpressing alpha-enolase/MBP-1. Here, we demonstrate that induced cell stresses promote MBP-1 expression through the AKT/PERK/eIF2α signaling axis. Our results contribute to shedding light on the molecular mechanisms underlying MBP-1 expression in non-tumorigenic and cancer cells.

Analysis of the thymidylate synthase gene structure in colorectal cancer patients and its possible relation with the 5-Fluorouracil drug response.

Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and it is the target for the 5-Fluorouracil (5-FU) activity. Barbour et al. showed that variant structural forms of TS in tumour cell lines confer resistance to fluoropyrimidines. We planned to perform the whole TS gene structure by means of sequencing techniques in human colorectal cancer (CRC) samples to try to identify the presence of any possible TS variant form that could be responsible of fluoropyrimidines drug resistance and of the worse prognosis. We performed the TS-DNA gene sequence in 68 CRC from patients of A, B, and C Dukes' stages and different histological grade, but we did not find any mutation in the TS-DNA structure. In the future we intend to widen the TS structure analysis to the metastatic CRCs, because due to their higher genomic instability, they could present a TS variant form responsible of the fluoropyrimidines drug resistance and the worse prognosis.

Myc promoter-binding protein-1 (MBP-1) is a novel potential prognostic marker in invasive ductal breast carcinoma.

BACKGROUND: Alpha-enolase is a glycolytic enzyme that catalyses the formation of phosphoenolpyruvate in the cell cytoplasm. α-Enolase and the predominantly nuclear Myc promoter-binding protein-1 (MBP-1) originate from a single gene through the alternative use of translational starting sites. MBP-1 binds to the P2 c-myc promoter and competes with TATA-box binding protein (TBP) to suppress gene transcription. Although several studies have shown an antiproliferative effect of MBP-1 overexpression on several human cancer cells, to date detailed observations of α-enolase and MBP-1 relative expression in primary tumors versus normal tissues and their correlation with clinicopathological features have not been undertaken. METHODOLOGY AND FINDINGS: We analyzed α-enolase and MBP-1 expression in normal breast epithelium and primary invasive ductal breast carcinoma (IDC) from 177 patients by Western blot and immunohistochemical analyses, using highly specific anti-α-enolase monoclonal antibodies. A significant increase in the expression of cytoplasmic α-enolase was observed in 98% of the tumors analysed, compared to normal tissues. Nuclear MBP-1 was found in almost all the normal tissues while its expression was retained in only 35% of the tumors. Statistically significant associations were observed among the nuclear expression of MBP-1 and ErbB2 status, Ki-67 expression, node status and tumor grade. Furthermore MBP-1 expression was associated with good survival of patients with IDC. CONCLUSIONS: MBP-1 functions in repressing c-myc gene expression and the results presented indicate that the loss of nuclear MBP-1 expression in a large number of IDC may be a critical step in the development and progression of breast cancer and a predictor of adverse outcome. Nuclear MBP-1 appears to be a novel and valuable histochemical marker with potential prognostic value in breast cancer.

The PVT-1 oncogene is a Myc protein target that is overexpressed in transformed cells.

The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations occurring in variant Burkitt's lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastoma cells that do not express c-Myc. Reporter gene assays were used to dissect the PVT-1 promoter and to identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites for Myc proteins. The results of transfection experiments in RAT1-MycER cells and chromatin immunoprecipitation (ChIP) assays in proliferating and differentiated neuroblastoma cells indicate that PVT-1 is a downstream target of Myc proteins.