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Patients with breast cancer show altered expression of genes within the pectoralis major skeletal muscle cells of the breast. Through analyses of The Cancer Genome Atlas (TCGA)-breast cancer (BRCA), we identified three previously uncharacterized putative novel tumor suppressor genes expressed in normal muscle cells, whose expression was downregulated in breast tumors. We found that NEDD4 binding protein 2-like 1 ( N4BP2L1 ), pleckstrin homology domain-containing family A member 4 ( PLEKHA4 ), and brain-enriched guanylate kinase-associated protein ( BEGAIN ) that are normally highly expressed in breast myoepithelial cells and smooth muscle cells were significantly downregulated in breast tumor tissues of a cohort of 50 patients with this cancer. Our data revealed that the low expression of PLEKHA4 in patients with menopause below 50 years correlated with a higher risk of breast cancer. Moreover, we identified N4BP2L1 and BEGAIN as potential biomarkers of HER2-positive breast cancer. Furthermore, low BEGAIN expression in breast cancer patients with blood fat, heart problems, and diabetes correlated with a higher risk of this cancer. In addition, protein and RNA expression analysis of TCGA-BRCA revealed N4BP2L1 as a promising diagnostic protein biomarker in breast cancer. In addition, the in silico data of scRNA-seq showed high expression of these genes in several cell types of normal breast tissue, including breast myoepithelial cells and smooth muscle cells. Thus, our results suggest their possible tumor-suppressive function in breast cancer and muscle development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dastsooz, Anselmi, Lauria, Cicconetti, Proserpio, Mohammadisoleimani, Firoozi, Mansoori, Haghi-Aminjan, Caizzi and Oliviero.)

The epigenome coordinates spatial-temporal specific gene expression during development and in adulthood, for the maintenance of homeostasis and upon tissue repair. The upheaval of the epigenetic landscape is a key event in the onset of many pathologies including tumours, where epigenetic changes cooperate with genetic aberrations to establish the neoplastic phenotype and to drive cell plasticity during its evolution. DNA methylation, histone modifiers and readers or other chromatin components are indeed often altered in cancers, such as carcinomas that develop in epithelia. Lining the surfaces and the cavities of our body and acting as a barrier from the environment, epithelia are frequently subjected to acute or chronic tissue damages, such as mechanical injuries or inflammatory episodes. These events can activate plasticity mechanisms, with a deep impact on cells' epigenome. Despite being very effective, tissue repair mechanisms are closely associated with tumour onset. Here we review the similarities between tissue repair and carcinogenesis, with a special focus on the epigenetic mechanisms activated by cells during repair and opted by carcinoma cells in multiple epithelia. Moreover, we discuss the recent findings on inflammatory and wound memory in epithelia and describe the epigenetic modifications that characterise them. Finally, as wound memory in epithelial cells promotes carcinogenesis, we highlight how it represents an early step for the establishment of field cancerization., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Long non-coding RNAs (lncRNAs) regulate gene expression through different molecular mechanisms, including DNA binding via the formation of RNA:DNA:DNA triple helices (TPXs). Despite the increasing amount of experimental evidence, TPXs investigation remains challenging. Here we present 3plex , a software able to predict TPX interactions in silico . Given an RNA sequence and a set of DNA sequences, 3plex integrates 1) Hoogsteen pairing rules that describe the biochemical interactions between RNA and DNA nucleotides, 2) RNA secondary structure prediction and 3) determination of the TPX thermal stability derived from a collection of TPX experimental evidences. We systematically collected and uniformly re-analysed published experimental lncRNA binding sites on human and mouse genomes. We used these data to evaluate 3plex performance and showed that its specific features allow a reliable identification of TPX interactions. We compared 3plex with the other available software and obtained comparable or even better accuracy at a fraction of the computation time. Interestingly, by inspecting collected data with 3plex we found that TPXs tend to be shorter and more degenerated than previously expected and that the majority of analysed lncRNAs can directly bind to the genome by TPX formation. Those results suggest that an important fraction of lncRNAs can exert its biological function through this mechanism. The software is available at https://github.com/molinerisLab/3plex., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset., (© 2023. The Author(s).)

The correct establishment of DNA methylation patterns during mouse early development is essential for cell fate specification. However, the molecular targets as well as the mechanisms that determine the specificity of the de novo methylation machinery during differentiation are not completely elucidated. Here we show that the DNMT3B-dependent DNA methylation of key developmental regulatory regions at epiblast-like cells (EpiLCs) provides an epigenetic priming that ensures flawless commitment at later stages. Using in vitro stem cell differentiation and loss of function experiments combined with high-throughput genome-wide bisulfite-, bulk-, and single cell RNA-sequencing we dissected the specific role of DNMT3B in cell fate. We identify DNMT3B-dependent regulatory elements on the genome which, in Dnmt3b knockout (3BKO), impair the differentiation into meso-endodermal (ME) progenitors and redirect EpiLCs towards the neuro-ectodermal lineages. Moreover, ectopic expression of DNMT3B in 3BKO re-establishes the DNA methylation of the master regulator Sox2 super-enhancer, downmodulates its expression, and restores the expression of ME markers. Taken together, our data reveal that DNMT3B-dependent methylation at the epiblast stage is essential for the priming of the meso-endodermal lineages and provide functional characterization of the de novo DNMTs during EpiLCs lineage determination., (© 2023. The Author(s).)

While many single-cell proteomics techniques have been rapidly developed over the past decade, flow cytometry still remains the pillar of single-cell protein analysis, as it allows to rapidly analyze and characterize protein expression in millions of cells.In this chapter, we will describe the main steps to prepare and acquire samples for flow cytometry, with particular focus on the setup of the right controls that are instrumental in analyzing and interpreting the results., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Over the past 7 years, single-cell sequencing has become very popular. For this reason, many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique. For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population, the choice of the best protocol can be difficult, due to the high abundance of available protocols, instruments, and options. For this reason, here we describe the Smart-seq2 protocol for full-length mRNA sequencing of single cell. This protocol can be easily optimized in every molecular biology laboratory provided with standard laboratory equipment. The protocol is suitable for many different cell types, and the cost per cell is relatively small, allowing a good balance between costs and transcript coverage., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases., (© 2021 The Authors.)

There is increasing evidence that long noncoding RNAs (lncRNAs) are among the main regulatory factors of stem cell maintenance and differentiation. They act through various mechanisms and interactions with proteins, DNA, and RNA. This heterogeneity in function increases the capabilities of the lncRNome toolkit but also makes it difficult to predict the function of novel lncRNAs or even rely on biological information produced in animal models. As lncRNAs are species- and tissue-specific, the recent technical advances in self-renewal and differentiation of human embryonic stem cells (ESCs) make these cells the ideal system to identify key regulatory lncRNAs and study their molecular functions. Here we provide an overview of the functional versatility of lncRNA mechanistic heterogeneity in regulating pluripotency maintenance and human differentiation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment., Competing Interests: The authors have read the journal’s policy and have the following competing interests: JF was an employee of Microsoft Research Cambridge at the time the study was conducted, but is no longer an employee of the company. EN is an employee of Aleph Labs, but was not employed by the company at the time the study was conducted. GK is an employee of AstraZeneca, but was not employed by the company at the time the study was conducted. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Objectives: The purpose of this study was to compare data obtained from the reticulocyte channel (RET channel) heated to 41°C with those obtained from impedance channel (I-Channel) at room temperature in the samples with the mean corpuscular hemoglobin concentration (MCHC)<370g/L and in samples with the MCHC>370g/L, in the presence of cold agglutinins., Methods: In this study, 60 blood samples (group 1) with the MCHC<370g/L (without cold agglutinins) and 78 blood samples (group 2) with the MCHC>370g/L (with cold agglutinins) were used to compare the two analytical channels of the XN-9000 analyzer in different preanalytical conditions. The parameters evaluated in both groups were the following: red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), RBC-most frequent volume (R-MFV), mean hemoglobin concentration (MCH) and mean cellular hemoglobin concentration (MCHC)., Results: The results of this study showed an excellent correlation with both channels of the XN-9000 analyzer in samples with and without cold agglutinins, except for the MCHC. The bias between the values obtained in the I-channel and those obtained in the RET channel of both groups was insignificant and remained within the limits of acceptability, as reported by Ricos et al. for all considered parameters, except for MCHC., Conclusions: The presence of cold agglutinins in blood samples can be detected by a spurious lowering of the RBC count and by a spurious increase in the MCHC. The RET channel represents a great opportunity to correct the RBC count in a rapid manner without preheating. However, neither methodology can completely solve the residual presence of cold agglutinins in all samples, despite the MCHC values being < 370g/L., (Copyright © 2020 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier España, S.L.U. All rights reserved.)

Background: Although the clinical assessment of iron status is usually based on iron stores, a rapid and accurate diagnosis of iron deficiency is challenging since ferritin is often unavailable as an urgent test and its value is frequently increased in acute phase conditions. This study was therefore aimed at evaluating the diagnostic performance of the new Sysmex XN "Iron Deficiency?" (Iron-Def) parameter for identifying patients with iron deficiency., Materials and Methods: The study population consisted of 688 consecutive patients (median age: 71 years; 341 women and 347 men), referred for routine diagnostics to the Laboratory of Clinical Pathology of Lecco Hospital, Italy. A complete clinical chemistry profile and haematological testing were performed for identifying iron deficiency anaemia., Results: A significant negative correlation was found between Sysmex XN Iron-Def and ferritin, serum iron, mean cell haemoglobin concentration, mean cell haemoglobin, mean corpuscular volume and age, while a positive correlation was noted with transferrin, percentage of microcytic red cell, red blood cell count and red blood cell distribution width. The diagnostic accuracy of Iron-Def for identifying patients with a percentage of saturation of transferrin <15% (n=104) was 84%, with a sensitivity of 0.952 and specificity of 0.538. A sub-analysis of 71 patients with ferritin <20 ng/dL yielded an even better diagnostic performance (86%, with a sensitivity of 0.935 and specificity of 0.620)., Discussion: Although additional confirmatory investigations would be needed, the preliminary findings of our study attest that Iron-Def may be an easy, inexpensive, rapid and reliable parameter for screening iron deficiency anaemia.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.

Memory of a trauma and how to cope with it is useful for acting rapidly in the event of a second traumatic incident. Recently, Naik et al. (2017) reported in Nature that skin epithelial stem cells have this ability by maintaining long-term chromatin features acquired during the first assault., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells., Results: We perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing., Conclusion: The link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity.

We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4(+) T cells as well as mature effector and memory cells.

In the last lustrum single-cell techniques such as single-cell quantitative PCR, RNA and DNA sequencing, and the state-of-the-art cytometry by time of flight (CyTOF) mass cytometer have allowed a detailed analysis of the sub-composition of different organs from the bone marrow hematopoietic compartment to the brain. These fine-grained analyses have highlighted the great heterogeneity within each cell compartment revealing previously unknown subpopulations of cells. In this review, we analyze how this fast technological evolution has improved our understanding of the biological processes with a particular focus on rare cells of the immune system.

The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system., (© 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.)

The transcriptome of single cells can reveal important information about cellular states and heterogeneity within populations of cells. Recently, single-cell RNA-sequencing has facilitated expression profiling of large numbers of single cells in parallel. To fully exploit these data, it is critical that suitable computational approaches are developed. One key challenge, especially pertinent when considering dividing populations of cells, is to understand the cell-cycle stage of each captured cell. Here we describe and compare five established supervised machine learning methods and a custom-built predictor for allocating cells to their cell-cycle stage on the basis of their transcriptome. In particular, we assess the impact of different normalisation strategies and the usage of prior knowledge on the predictive power of the classifiers. We tested the methods on previously published datasets and found that a PCA-based approach and the custom predictor performed best. Moreover, our analysis shows that the performance depends strongly on normalisation and the usage of prior knowledge. Only by leveraging prior knowledge in form of cell-cycle annotated genes and by preprocessing the data using a rank-based normalisation, is it possible to robustly capture the transcriptional cell-cycle signature across different cell types, organisms and experimental protocols., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent technical developments have enabled the transcriptomes of hundreds of cells to be assayed in an unbiased manner, opening up the possibility that new subpopulations of cells can be found. However, the effects of potential confounding factors, such as the cell cycle, on the heterogeneity of gene expression and therefore on the ability to robustly identify subpopulations remain unclear. We present and validate a computational approach that uses latent variable models to account for such hidden factors. We show that our single-cell latent variable model (scLVM) allows the identification of otherwise undetectable subpopulations of cells that correspond to different stages during the differentiation of naive T cells into T helper 2 cells. Our approach can be used not only to identify cellular subpopulations but also to tease apart different sources of gene expression heterogeneity in single-cell transcriptomes.

T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a "lymphosteroid," a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

It has long been recognized that the hair follicle growth cycle and oscillation in the thickness of the underlying adipocyte layer are synchronized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether the epidermis can regulate adipogenesis. We show that inhibition of epidermal Wnt/β-catenin signaling reduced adipocyte differentiation in developing and adult mouse dermis. Conversely, ectopic activation of epidermal Wnt signaling promoted adipocyte differentiation and hair growth. When the Wnt pathway was activated in the embryonic epidermis, there was a dramatic and premature increase in adipocytes in the absence of hair follicle formation, demonstrating that Wnt activation, rather than mature hair follicles, is required for adipocyte generation. Epidermal and dermal gene expression profiling identified keratinocyte-derived adipogenic factors that are induced by β-catenin activation. Wnt/β-catenin signaling-dependent secreted factors from keratinocytes promoted adipocyte differentiation in vitro, and we identified ligands for the bone morphogenetic protein and insulin pathways as proadipogenic factors. Our results indicate epidermal Wnt/β-catenin as a critical initiator of a signaling cascade that induces adipogenesis and highlight the role of epidermal Wnt signaling in synchronizing adipocyte differentiation with the hair growth cycle.

Single-cell RNA-seq can yield valuable insights about the variability within a population of seemingly homogeneous cells. We developed a quantitative statistical method to distinguish true biological variability from the high levels of technical noise in single-cell experiments. Our approach quantifies the statistical significance of observed cell-to-cell variability in expression strength on a gene-by-gene basis. We validate our approach using two independent data sets from Arabidopsis thaliana and Mus musculus.

Elucidating the epigenetic mechanisms underlying muscle mass determination and skeletal muscle wasting holds the potential of identifying molecular pathways that constitute possible drug targets. Here, we report that the methyltransferase SMYD3 modulates myostatin and c-Met transcription in primary skeletal muscle cells and C2C12 myogenic cells. SMYD3 targets the myostatin and c-Met genes and participates in the recruitment of the bromodomain protein BRD4 to their regulatory regions through protein-protein interaction. By recruiting BRD4, SMYD3 favors chromatin engagement of the pause-release factor p-TEFb (positive transcription elongation factor) and elongation of Ser2-phosphorylated RNA polymerase II (PolIISer2P). Reducing SMYD3 decreases myostatin and c-Met transcription, thus protecting from glucocorticoid-induced myotube atrophy. Supporting functional relevance of the SMYD3/BRD4 interaction, BRD4 pharmacological blockade by the small molecule JQ1 prevents dexamethasone-induced myostatin and atrogene up-regulation and spares myotube atrophy. Importantly, in a mouse model of dexamethasone-induced skeletal muscle atrophy, SMYD3 depletion prevents muscle loss and fiber size decrease. These findings reveal a mechanistic link between SMYD3/BRD4-dependent transcriptional regulation, muscle mass determination, and skeletal muscle atrophy and further encourage testing of small molecules targeting specific epigenetic regulators in animal models of muscle wasting.

How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified an inflammation-activated signaling in muscle stem (satellite) cells, by which the polycomb repressive complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38α kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EZH2, the enzymatic subunit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. TNF-α antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signaling--p38α and EZH2--invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signaling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signaling to Pax7 and satellite cell decision to proliferate or differentiate., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We verified the feasibility of plasma bound method for detecting renal cell carcinoma (RCC) combining the study of plasma DNA concentration and microsatellite alterations (LOH). Plasma DNA concentration was evaluated with real-time PCR in 54 patients with renal neoplasm before surgery and in 20 of these patients during a 26-64 month follow-up. Microsatellite study was performed on tumour tissue DNA of 33 RCC clear cell (RCCcc) and on plasma DNA of 14 RCCcc patients during preoperative and/or follow-up period. Patients had a significantly high (26.4+/-48.3 ng/ml versus controls 3.2+/-1.5 ng/ml; p=0.003) preoperative plasma DNA concentration that decreased after nephrectomy. During follow-up, plasma DNA increased in 12 patients without evidence of neoplasia; 3 patients successively relapsed. Tumour tissue DNA of 25 RCCcc patients (75.8%) displayed microsatellite LOH. Preoperative plasma DNA of 9 patients harboured LOH in 5 cases (55.6%). Augmented plasma DNA of 7 patients displayed LOH in 3 cases (42.9%) at follow-up, and in 1 case preceded the recurrence of disease. Plasma DNA concentration combined with microsatellite LOH in plasma DNA may predict disease recurrence in RCC patients.

Background: Clear cell renal carcinoma (RCC) is the most common and invasive adult renal cancer. For the purpose of identifying RCC biomarkers, we investigated chromosomal regions and individual genes modulated in RCC pathology. We applied the dual strategy of assessing and integrating genomic and transcriptomic data, today considered the most effective approach for understanding genetic mechanisms of cancer and the most sensitive for identifying cancer-related genes., Results: We performed the first integrated analysis of DNA and RNA profiles of RCC samples using Affymetrix technology. Using 100K SNP mapping arrays, we assembled a genome-wide map of DNA copy number alterations and LOH areas. We thus confirmed the typical genetic signature of RCC but also identified other amplified regions (e.g. on chr. 4, 11, 12), deleted regions (chr. 1, 9, 22) and LOH areas (chr. 1, 2, 9, 13). Simultaneously, using HG-U133 Plus 2.0 arrays, we identified differentially expressed genes (DEGs) in tumor vs. normal samples. Combining genomic and transcriptomic data, we identified 71 DEGs in aberrant chromosomal regions and observed, in amplified regions, a predominance of up-regulated genes (27 of 37 DEGs) and a trend to clustering. Functional annotation of these genes revealed some already implicated in RCC pathology and other cancers, as well as others that may be novel tumor biomarkers., Conclusion: By combining genomic and transcriptomic profiles from a collection of RCC samples, we identified specific genomic regions with concordant alterations in DNA and RNA profiles and focused on regions with increased DNA copy number. Since the transcriptional modulation of up-regulated genes in amplified regions may be attributed to the genomic alterations characteristic of RCC, these genes may encode novel RCC biomarkers actively involved in tumor initiation and progression and useful in clinical applications.

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.