Methods and computational tools to study eukaryotic cell migration in vitro.

Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion.

Inherited rare variants in homologous recombination and neurodevelopmental genes are associated with increased risk of neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common solid extracranial paediatric tumour. Genome-wide association studies have driven the discovery of common risk variants, but no large study has investigated the contribution of rare variants to NB susceptibility. Here, we conducted a whole-exome sequencing (WES) of 664 NB cases and 822 controls and used independent validation datasets to identify genes with rare risk variants and involved pathways. METHODS: WES was performed at 50× depth and variants were jointly called in cases and controls. We developed two models to identify mutations with high clinical impact (P/LP model) and to discover less penetrant risk mutations affecting non-canonical cancer pathways (RPV model). We performed a gene-level collapsing test using Firth's logistic regression in 242 selected cancer predisposition genes (CPGs) and a gene-sets burden analysis of biologically-informed pathways. FINDINGS: Twelve percent of patients carried P/LP variants in CPGs and showed a significant enrichment (P = 2.3 × 10-4) compared to controls (6%). We identified P/LP variants in 45 CPGs enriched in homologous recombination (HR) pathway. The most P/LP enriched genes in NB were BRCA1, ALK and RAD51C. Additionally, we found higher RPV burden in gene-sets of neuron differentiation, neural tube development and synapse assembly, and in gene-sets associated with neurodevelopmental disorders (NDD). INTERPRETATION: The high fraction of NB patients with P/LP variants indicates the need of genetic counselling. Furthermore, inherited rare variants predispose to NB development by affecting mechanisms related to HR and neurodevelopmental processes, and demonstrate that NDD genes are altered in NB at the germline level. FUNDING: Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la Lotta al Neuroblastoma, Associazione Oncologia Pediatrica e Neuroblastoma, Regione Campania, Associazione Giulio Adelfio onlus, and Italian Health Ministry.

Biological relevance of ZNF224 expression in chronic lymphocytic leukemia and its implication IN NF-kB pathway regulation.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, whose presentation and clinical course are highly variable. Identification of novel prognostic factors may contribute to improving the CLL classification and providing indications for treatment options. The zinc finger protein ZNF224 plays a key role in cell transformation, through the control of apoptotic and survival pathways. In this study, we evaluated the potential application of ZNF224 as a novel marker of CLL progression and therapy responsiveness. To this aim, we analyzed ZNF224 expression levels in B lymphocytes from CLL patients at different stages of the disease and in patients showing different treatment outcomes. The expression of ZNF224 was significantly increased in disease progression and dramatically decreased in patients in complete remission after chemotherapy. Gene expression correlation analysis performed on datasets of CLL patients revealed that ZNF224 expression was well correlated with that of some prognostic and predictive markers. Moreover, bioinformatic analysis coupled ZNF224 to NF-κB pathway, and experimental data demonstrated that RNA interference of ZNF224 reduced the activity of the NF-κB survival pathway in CLL cells. Consistently with a pro-survival role, ZNF224 knockdown raised spontaneous and drug-induced apoptosis and inhibited the proliferation of peripheral blood mononuclear cells from CLL patients. Our findings provide evidence for the involvement of ZNF224 in the survival of CLL cells via NF-κB pathway modulation, and also suggest ZNF224 as a prognostic and predictive molecular marker of CLL disease.

A three component model for superdiffusive motion effectively describes migration of eukaryotic cells moving freely or under a directional stimulus.

Although the simple diffusion model can effectively describe the movement of eukaryotic cells on a culture surface observed at relatively low sampling frequency, at higher sampling rates more complex models are often necessary to better fit the experimental data. Currently available models can describe motion paths by involving additional parameters, such as linearity or directional persistence in time. However sometimes difficulties arise as it is not easy to effectively evaluate persistence in presence of a directional bias. Here we present a procedure which helps solve this problem, based on a model which describes displacement as the vectorial sum of three components: diffusion, persistence and directional bias. The described model has been tested by analysing the migratory behaviour of simulated cell populations and used to analyse a collection of experimental datasets, obtained by observing cell cultures in time lapse microscopy. Overall, the method produces a good description of migration behaviour as it appears to capture the expected increase in the directional bias in presence of wound without a large concomitant increase in the persistence module, allowing it to remain as a physically meaningful quantity in the presence of a directional stimulus.

Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals.

PURPOSE: Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants - referred as qualifying variants (QVs) - of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. METHODS: Exploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. RESULTS: We found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. CONCLUSION: This study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/.

SARS-CoV-2 Pandemic Tracing in Italy Highlights Lineages with Mutational Burden in Growing Subsets.

Tracing the appearance and evolution of virus variants is essential in the management of the COVID-19 pandemic. Here, we focus on SARS-CoV-2 spread in Italian patients by using viral sequences deposited in public databases and a tracing procedure which is used to monitor the evolution of the pandemic and detect the spreading, within the infected population of emergent sub-clades with a potential positive selection. Analyses of a collection of monthly samples focused on Italy highlighted the appearance and evolution of all the main viral sub-trees emerging at the end of the first year of the pandemic. It also identified additional expanding subpopulations which spread during the second year (i.e., 2021). Three-dimensional (3D) modelling of the main amino acid changes in mutated viral proteins, including ORF1ab (nsp3, nsp4, 2'-o-ribose methyltransferase, nsp6, helicase, nsp12 [RdRp]), N, ORF3a, ORF8, and spike proteins, shows the potential of the analysed structural variations to result in epistatic modulation and positive/negative selection pressure. These analyzes will be of importance to the early identification of emerging clades, which can develop into new "variants of concern" (i.e., VOC). These analyses and settings will also help SARS-CoV-2 coronet genomic centers in other countries to trace emerging worldwide variants.

Loss of Detection of sgN Precedes Viral Abridged Replication in COVID-19-Affected Patients-A Target for SARS-CoV-2 Propagation.

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication.

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

Pediatric Celiac Disease Patients Show Alterations of Dendritic Cell Shape and Actin Rearrangement.

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls' DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer.

M2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC). Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-ß enhancement, IL-17F secretion, and extracellular vesicle protein content modulation. We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

The complete 12 Mb genome and transcriptome of Nonomuraea gerenzanensis with new insights into its duplicated "magic" RNA polymerase.

In contrast to the widely accepted consensus of the existence of a single RNA polymerase in bacteria, several actinomycetes have been recently shown to possess two forms of RNA polymerases due the to co-existence of two rpoB paralogs in their genome. However, the biological significance of the rpoB duplication is obscure. In this study we have determined the genome sequence of the lipoglycopeptide antibiotic A40926 producer Nonomuraea gerenzanensis ATCC 39727, an actinomycete with a large genome and two rpoB genes, i.e. rpoB(S) (the wild-type gene) and rpoB(R) (the mutant-type gene). We next analyzed the transcriptional and metabolite profiles in the wild-type gene and in two derivative strains over-expressing either rpoB(R) or a mutated form of this gene to explore the physiological role and biotechnological potential of the "mutant-type" RNA polymerase. We show that rpoB(R) controls antibiotic production and a wide range of metabolic adaptive behaviors in response to environmental pH. This may give interesting perspectives also with regard to biotechnological applications.

TRAP1 controls cell migration of cancer cells in metabolic stress conditions: Correlations with AKT/p70S6K pathways.

Cell motility is a highly dynamic phenomenon that is essential to physiological processes such as morphogenesis, wound healing and immune response, but also involved in pathological conditions such as metastatic dissemination of cancers. The involvement of the molecular chaperone TRAP1 in the regulation of cell motility, although still controversial, has been recently investigated along with some well-characterized roles in cancer cell survival and drug resistance in several tumour types. Among different functions, TRAP1-dependent regulation of protein synthesis seems to be involved in the migratory behaviour of cancer cells and, interestingly, the expression of p70S6K, a kinase responsible for translation initiation, playing a role in cell motility, is regulated by TRAP1. In this study, we demonstrate that TRAP1 silencing enhances cell motility in vitro but compromises the ability of cells to overcome stress conditions, and that this effect is mediated by the AKT/p70S6K pathway. In fact: i) inhibition of p70S6K activity specifically reduces migration in TRAP1 knock-down cells; ii) nutrient deprivation affects p70S6K activity thereby impairing cell migration only in TRAP1-deficient cells; iii) TRAP1 regulates the expression of both AKT and p70S6K at post-transcriptional level; and iii) TRAP1 silencing modulates the expression of genes involved in cell motility and epithelial-mesenchymal transition. Notably, a correlation between TRAP1 and AKT expression is found in vivo in human colorectal tumours. These results provide new insights into TRAP1 role in the regulation of cell migration in cancer cells, tumour progression and metastatic mechanisms.

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern.

BACKGROUND: Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome. RESULTS: PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y's phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism. CONCLUSIONS: In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

TRAP1-dependent regulation of p70S6K is involved in the attenuation of protein synthesis and cell migration: relevance in human colorectal tumors.

TNF receptor-associated protein 1 (TRAP1) is an HSP90 chaperone involved in stress protection and apoptosis in mitochondrial and extramitochondrial compartments. Remarkably, aberrant deregulation of TRAP1 function has been observed in several cancer types with potential new opportunities for therapeutic intervention in humans. Although previous studies by our group identified novel roles of TRAP1 in quality control of mitochondria-destined proteins through the attenuation of protein synthesis, molecular mechanisms are still largely unknown. To shed further light on the signaling pathways regulated by TRAP1 in the attenuation of protein synthesis, this study demonstrates that the entire pathway of cap-mediated translation is activated in cells following TRAP1 interference: consistently, expression and consequent phosphorylation of p70S6K and RSK1, two translation activating kinases, are increased upon TRAP1 silencing. Furthermore, we show that these regulatory functions affect the response to translational stress and cell migration in wound healing assays, processes involving both kinases. Notably, the regulatory mechanisms controlled by TRAP1 are conserved in colorectal cancer tissues, since an inverse correlation between TRAP1 and p70S6K expression is found in tumor tissues, thereby supporting the relevant role of TRAP1 translational regulation in vivo. Taken as a whole, these new findings candidate TRAP1 network for new anti-cancer strategies aimed at targeting the translational/quality control machinery of tumor cells.

DNA sequence capture and next-generation sequencing for the molecular diagnosis of genetic cardiomyopathies.

Hypertrophic cardiomyopathy is a relatively frequent disease with a prevalence of 0.2% worldwide and a remarkable genetic heterogeneity, with more than 30 causative genes reported to date. Current PCR-based strategies are inadequate for genomic investigations involving many candidate genes. Here, we report a next-generation sequencing procedure associated with DNA sequence capture that is able to sequence 202 cardiomyopathy-related genes simultaneously. We developed a complementary data analysis pipeline to select and prioritize genetic variants. The overall procedure can screen a large number of target genes simultaneously, thereby potentially revealing new disease-causing and modifier genes. By using this procedure, we analyzed hypertrophic cardiomyopathy patients in a shorter time and at a lower cost than with current procedures. The specificity of the next-generation sequencing-based procedure is at least as good as other techniques routinely used for mutation searching, and the sensitivity is much better. Analysis of the results showed some novel variants potentially involved in the pathogenesis of hypertrophic cardiomyopathy: a missense mutation in MYH7 and a nonsense variant in INS-IGF2 (patient 1), a splicing variant in MYBPC3 and an indel/frameshift variant in KCNQ1 (patient 2), and two concomitant variations in CACNA1C (patient 3). Sequencing of DNA from the three patients within a pool allowed detection of most variants identified in each individual patient, indicating that this approach is a feasible and cost-effective procedure.

Ras activated ERK and PI3K pathways differentially affect directional movement of cultured fibroblasts.

BACKGROUND: Cell migration is essential in physiological and pathological processes, such as wound healing and metastasis formation. Ras involvement in these processes has been extensively demonstrated. This work attempts to characterize Ras regulation of the phenomena determining directional cell migration by separately analyzing the role of its principal effector pathways, MAPK and PI3K. METHODS: NIH3T3 and NIHRasV12 fibroblasts were followed in wound healing assays to study, in time and under a directional stimulus, cell migration both under standard conditions and in presence of MAPK and PI3K inhibitors. Several parameters, descriptive of specific aspects of cell motion, were evaluated by coupling dynamic microscopy with quantitative and statistical methods. Quantitative Western Blots coupled with immunofluorescence stainings, were used to evaluate ERK activation. RESULTS: Constitutive RasV12 activation confers to NIH3T3 the ability to close the wound faster. Neither increased cell proliferation nor higher speed explains the accelerated healing, but the increased directional migration drives the wound closure. Inhibition of ERK activation, which occurs immediately after wound, greatly blocks the directional migration, while inhibition of PI3K pathway reduces cell speed but does not prevent wound closure. CONCLUSION: Ras is greatly involved in determining and regulating directionality, ERK is its key effector for starting, driving and regulating directional movement.

Androgen-induced cell migration: role of androgen receptor/filamin A association.

BACKGROUND: Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. CONCLUSIONS/SIGNIFICANCE: The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis.

Gliadin peptide P31-43 localises to endocytic vesicles and interferes with their maturation.

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.

Colon OCTN2 gene expression is up-regulated by peroxisome proliferator-activated receptor gamma in humans and mice and contributes to local and systemic carnitine homeostasis.

In the large intestine organic cation transporter type-2 (OCTN2) is recognized as a transporter of compounds such as carnitine and colony sporulation factor, promoting health of the colon intestinal epithelium. Recent reports suggest that OCTN2 expression in small intestine is under control of peroxisome proliferator-activated receptor-alpha (PPARalpha). However, PPARalpha contribution to colonic OCTN2 expression remains controversial. Here we examined the transcriptional regulation of colon OCTN2 gene by PPARgamma. To exclude any additional modulation of other PPAR to OCTN2 expression, we used both in vivo and in vitro PPAR-null models and specific PPAR inhibitors. The PPARgamma agonists thiazolidinediones increased both OCTN2 mRNA and protein expression in colonic epithelial cell lines independently by PPARalpha expression. The induction was blocked only by PPARgamma antagonists or by gammaORF4, a PPARgamma isoform with dominant negative activity, suggesting a PPARgamma-dependent mechanism. A conserved noncanonical PPAR-responsive element was found by computational analysis in the first intron of human OCTN2 gene and validated by EMSA assay. Promoter-reporter assays further confirmed transcriptional functionality of the putative PPAR response element, whereas selective mutation caused complete loss of responsiveness to PPARgamma activation. Finally, adenovirus-mediated overexpression of constitutively active PPARgamma mutant increased colon OCTN2 expression in PPARalpha(-/-) mice. Interestingly, animals overexpressing colon PPARgamma showed a significant increase in plasma carnitine, thus demonstrating the functional contribution of large intestine to systemic carnitine homeostasis. This study reveals a PPARgamma-dependent absorption machinery in colon that is likely involved in the health of colon epithelium, in the microbiota-host interactions and in the absorption of nutraceuticals and drugs.

Analysis and modelling of motility of cell populations with MotoCell.

BACKGROUND: Cell motility plays a central role in development, wound-healing and tumour invasion. Cultures of eukaryotic cells are a complex system where most cells move according to 'random' patterns, but may also be induced to a more coordinate migration by means of specific stimuli, such as the presence of chemical attractants or the introduction of a mechanical stimulus. Various tools have been developed that work by keeping track of the paths followed by specific objects and by performing statistical analysis on the recorded path data. The available tools include desktop applications or macros running within a commercial package, which address specific aspects of the process. RESULTS: An online application, MotoCell, was developed to evaluate the motility of cell populations maintained in various experimental conditions. Statistical analysis of cell behaviour consists of the evaluation of descriptive parameters such as average speed and angle, directional persistence, path vector length, calculated for the whole population as well as for each cell and for each step of the migration; in this way the behaviour of a whole cell population may be assessed as a whole or as a sum of individual entities. The directional movement of objects may be studied by eliminating the modulo effect in circular statistics analysis, able to evaluate linear dispersion coefficient (R) and angular dispersion (S) values together with average angles. A case study is provided where the system is used to characterize motility of RasV12 transformed NIH3T3 fibroblasts. CONCLUSION: Here we describe a comprehensive tool which takes care of all steps in cell motility analysis, including interactive cell tracking, path editing and statistical analysis of cell movement, all within a freely available online service. Although based on a standard web interface, the program is very fast and interactive and is immediately available to a large number of users, while exploiting the web approach in a very effective way. The ability to evaluate the behaviour of single cells allows to draw the attention on specific correlations, such as linearity of movement and deviation from the expected direction. In addition to population statistics, the analysis of single cells allows to group the cells into subpopulations, or even to evaluate the behaviour of each cell with respect to a variable reference, such as the direction of a wound or the position of the closest cell.