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[EN] Mesenchymal stem cells represent an important resource, for bone regenerative medicine and therapeutic applications. This review focuses on new advancements and biophysical tools which exploit different physical and chemical markers of mesenchymal stem cell populations, to finely characterize phenotype changes along their osteogenic differentiation process. Special attention is paid to recently developed label-free methods, which allow monitoring cell populations with minimal invasiveness. Among them, quantitative phase imaging, suitable for single-cell morphometric analysis, and nanoindentation, functional to cellular biomechanics investigation. Moreover, the pool of ion channels expressed in cells during differentiation is discussed, with particular interest for calcium homoeostasis.Altogether, a biophysical perspective of osteogenesis is proposed, offering a valuable tool for the assessment of the cell stage, but also suggesting potential physiological links between apparently independent phenomena.

Over the last decade, toxic events along the Mediterranean coast associated with exceptional harmful blooms of the dinoflagellate Ostreopsis cf. ovata have increased in frequency and distribution, causing not only the death of marine organisms and human health problems, but also economic loss on the tourism and aquaculture industries. In order to reduce the burden of routine algal counting, an innovative automated, low-cost, opto-electronic system called OvMeter was developed. It is able to speed up the monitoring process and therefore it enables early warning of incipient harmful algal blooms. An ad-hoc software tool provides automated cell recognition, counting and real-time calculation of the final algal concentration. The core of dinoflagellate recognition relies on a localization step which takes advantage of the synergistic exploitation of 2D bright-field and quantitative phase microscopy images, and a classification phase performed by a machine learning algorithm based on Boosted Trees approach. The architectural design of the OvMeter device is presented here, together with a performance evaluation on sea samples.

25 p.-5 fig.-1 tab. + 6 fig. supl.+ 1 tab. supl., αβ-Tubulin is a validated target for anticancer drug discovery, and molecules binding to this protein are used to treat several types of tumors. Here, we report on a combined X-ray crystallography and molecular dynamics approach to study drug binding within the colchicine site of αβ-tubulin, focusing on plinabulin, an agent currently in phase 3 clinical testing for the treatment of cancer and chemotherapy-induced neutropenia. We found that plinabulin is more persistently bound to the colchicine site of βII- compared to βIII-tubulin, allowing for a prediction of isotype-expression-dependent drug sensitivity. Additionally, computational residence time and exit paths from the βII-tubulin were compared between plinabulin and two other compounds, colchicine and combretastatin-A4. The former displayed the highest residence time, followed by plinabulin and then distantly by combretastatin-A4. Our combined experimental and computational protocol could help to investigate anti-tubulin drugs, improving our understanding of their mechanism of action, residence time, and tubulin isotype selectivity., This work was financially supported by BeyondSpring Pharmaceuticals Inc. (to M.O.S. and A.C.) and by grants from the Ministerio de Ciencia Innovacion y Universidades (BFU2016-75319-R; to J.F.D.), from the Swiss National Science Foundation (31003A_166608, to M.O.S.) and from the Regione Lombardia (Accordo per la Ricerca e l’Innovazione).

The ability to sense mechanical stimuli and elaborate a response to them is a fundamental process in all organisms, driving crucial mechanisms ranging from cell volume regulation up to organ development or regeneration. Nevertheless, only in few cases the underlying molecular players are known. In particular, mammals possess a large variety of mechanoreceptors, providing highly specialized functions in sensory cells, but also several housekeeping molecular systems are involved in the complex mechanism of mechanotransduction. Recently, a new class of almost ubiquitous membrane channels has been identified in mammalians, namely piezo1 and piezo2, that is thought to play a crucial role in the mechanobiology of mammals. This review focuses on recent findings on these novel channels, and highlights open biophysical questions that largely remain to be addressed.

The stroma of human bone marrow contains a population of skeletal stem cells (hBM-MSC) which are common ancestors, among the others, of osteoblasts and adipocytes. It has been proposed that the imbalance between hBM-MSC osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. The possibility to reproduce this mechanism in vitro has been demonstrated, providing a good model to disclose the details of the complex bone-fat generation homeostasis. Nevertheless, the lack of a simple approach to quantitatively assess the actual stage of a cellular population hindered the adoption of this in vitro model. In this work, the direct differentiation of hBM-MSCs towards a single (osteo or adipo) lineage was characterized using quantitative biophysical and biological approaches, together with the parallel process of trans-differentiation from one lineage to the other. The results confirm that the original plasticity of hBM-MSCs is maintained along the initial stages of the differentiation, showing that in vitro conversion of pre-osteoblasts into adipocytes and, vice versa, of pre-adipocytes into osteoblasts is extremely efficient, comparable with the direct differentiation. Moreover, a method based on digital holography is proposed, providing a quantitative indication of the phenotype stage along differentiation.

The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium- based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo. Moreover, there is evidence that Ca-P substrate triggers osteogenic differentiation through genes (SMAD and RAS family) that are typically regulated during dexamethasone (DEX) induced differentiation.

Meta-analysis of microarray data is increasingly important, considering both the availability of multiple platforms using disparate technologies and the accumulation in public repositories of data sets from different laboratories. We addressed the issue of comparing Gene expression profiles from two microarray platforms by devising a standardized investigative strategy. We tested this procedure by studying MDA-MB-231 cells, which undergo apoptosis on treatment with resveratrol. Gene expression profiles were obtained using high-density. short-oligonucleotide, single-color microarray platforms: GeneChip (Affymetrix) and CodeLink (Amersham). Interplatform analyses were carried out on 8414 common transcripts represented on both platforms, as identified by LocusLink ID, representing 70.8% and 88.6% of annotated GeneChip and CodeLink features, respectively. We identified 105 differentially expressed genes (DEGs) on CodeLink and 42 DEGs on GeneChip. Among them, only 9 DEGs were commonly identified by both platforms. Multiple analyses (BLAST alignment of probes with target sequences. gene ontology, literature mining, and quantitative real-time PCR) permitted LIS to investigate the factors contributing to the generation of platform-dependent results in single-color microarray experiments. Ail effective approach to cross-platform comparison involves microarrays of similar technologies, samples prepared by identical methods, and a standardized battery of bioinformatic and statistical analyses. (c) 2006 Elsevier Inc. All rights reserved.

BACKGROUND: Inflammatory bowel diseases (IBD) refer to a group of inflammatory conditions concerning colon and small intestine, which cause socially uncomfortable symptoms and often are associated with an increased risk of colon cancer. IBD are complex disorders, which rely on genetic susceptibility, environmental factors, deregulation of the immune system, and host relationship with commensal microbiota. The complexity of these pathologies makes difficult to clearly understand the mechanisms of their onset. Therefore, the study of IBD must be faced exploiting an integrated and multilevel approach, ranging from genes, transcripts and proteins to pathways altered in affected tissues, and carefully considering their regulatory mechanisms, which may intervene in the pathology onset. It is also crucial to have a knowledge base about the symbiotic bacteria that are hosted in the human gut. To date, much data exist regarding IBD and human commensal bacteria, but this information is sparse in literature and no free resource provides a homogeneously and rationally integrated view of biomolecular data related to these pathologies. METHODS: Human genes altered in IBD have been collected from literature, paying particular interest for the immune system alterations prompted by the interaction with the gut microbiome. This process has been performed manually to assure the reliability of collected data. Heterogeneous metadata from different sources have been automatically formatted and integrated in order to enrich information about these altered genes. A user-friendly web interface has been created for easy access to structured data. Tools such as gene clustering coefficients, all-pairs shortest paths and pathway lengths calculation have been developed to provide data analysis support. Moreover, the implemented resource is compliant to the Galaxy framework, allowing the collected data to be exploited in the context of high throughput bioinformatics analysis. RESULTS: To fill the lack of a reference resource for 'omics' science analysis in the context of IBD, we developed the IBDsite (available at http://www.itb.cnr.it/ibd), a disease-oriented platform, which collects data related to biomolecular mechanisms involved in the IBD onset. The resource provides a section devoted to human genes identified as altered in IBD, which can be queried at different biomolecular levels and visualised in gene-centred report pages. Furthermore, the system presents information related to the gut microbiota involved in IBD affected patients. The IBDsite is compliant with all Galaxy installations (in particular, it can be accessed from our custom version of Galaxy, http://www.itb.cnr.it/galaxy), in order to facilitate high-throughput data integration and to enable evaluations of the genomic basis of these diseases, complementing the tools embedded in the IBDsite. CONCLUSIONS: Lots of sparse data exist concerning IBD studies, but no on-line resource homogeneously and rationally integrate and collect them. The IBDsite is an attempt to group available information regarding human genes and microbial aspects related to IBD, by means of a multilevel mining tool. Moreover, it constitutes a knowledge base to filter, annotate and understand new experimental data in order to formulate new scientific hypotheses, thanks to the possibility of integrating genomics aspects by employing the Galaxy framework. Discussed use-cases demonstrate that the developed system is useful to infer not trivial knowledge from the existing widespread data or from novel experiments.

miRNA target genes prediction represents a crucial step in miRNAs functional characterization. In this context, the challenging issue remains predictions accuracy and recognition of false positive results. In this article myMIR, a web based system for increasing reliability of miRNAs predicted targets lists, is presented. myMIR implements an integrated pipeline for computing ranked miRNA::target lists and provides annotations for narrowing them down. The system relies on knowledge base data, suitably integrated in order to extend the functional characterization of targeted genes to miRNAs, by highlighting the search on over-represented annotation terms. Validation results show a dramatic reduction in the quantity of predictions and an increase in the sensitivity, when compared to other methods. This improves the predictions accuracy and allows the formulation of novel hypotheses on miRNAs functional involvement.

Recent advances in life sciences suggest that human and rodent cell responses to stimuli might differ significantly. In this context, the results achieved in neurotoxicology and biomedical research practices using neural networks obtained from mouse or rat primary culture of neurons would benefit of the parallel evaluation of the same parameters using fully differentiated neurons with a human genetic background, thus emphasizing the current need of neuronal cells with human origin. In this work, we developed a human functionally active neural network derived by human neuroblastoma cancer cells genetically engineered to overexpress NDM29, a non-coding RNA whose increased synthesis causes the differentiation toward a neuronal phenotype. These cells are here analyzed accurately showing functional and morphological traits of neurons such as the expression of neuron-specific proteins and the possibility to generate the expected neuronal current traces and action potentials. Their morphometrical analysis is carried out by quantitative phase microscopy showing soma and axon sizes compatible with those of functional neurons. The ability of these cells to connect autonomously forming physical junctions recapitulates that of hippocampal neurons, as resulting by connect-ability test. Lastly, these cells self-organize in neural networks able to produce spontaneous firing, in which spikes can be clustered in bursts. Altogether, these results show that the neural network obtained by NDM29-dependent differentiation of neuroblastoma cells is a suitable tool for biomedical research practices.