Caterina Millino

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

BackgroundSessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.ResultsWe have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.ConclusionsThe Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.

Combinatorial cis-Acting Elements Control Tissue-specific Activation of the Cardiac Troponin I Gene in Vitro and in Vivo

The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (−230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the −230/+126 base pair promoter express β-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.

Combinatorial cis-acting elements regulate tissue-specific expression of the cardiac troponin I gene in vitro and in vivo

The cardiac troponin I gene is one of the few sarcomeric protein genes exclusively expressed in cardiac muscle. We show here that this specificity is controlled by a proximal promoter (−230/+16) in transfected cardiac cells in culture, in the adult hearts, and in transgenic animals. Functional analysis indicates that MEF2/Oct-1, Sp1, and GATA regulatory elements are required for optimal gene activation because selective mutations produce weak or inactive promoters. MEF2 and Oct-1 transcription factors bind to the same A/T-rich element. A mutation that blocks this binding markedly reduces gene activation in vivo and in vitro, and overexpression of MEF2A, MEF2C, and MEF2D in noncardiac cells transactivates the cardiac troponin I promoter. Disruption of these elements inactivates the cardiac troponin I promoter in cultured cardiac cells but has a less important role in transfected adult heart. Moreover, nuclear extracts from an almost pure population of adult cardiac cells contain much lower levels of GATA binding activity compared with fetal cardiac cells. These findings point to a differential role of GATA factors in the maintenance of gene expression in the adult heart as compared with the activation of cardiac genes in fetal cardiomyocytes. Overexpression of GATA family members transactivates the cardiac troponin I promoter, and GATA-5 and GATA-6 are stronger transactivators than GATA-4, a property apparently unique to the cardiac troponin I promoter. Transgenic mice carrying the −230/+126 base pair promoter express β-galactosidase reporter gene in the heart both at early stages of cardiogenesis and in the adult animals. These results indicate that the ability of the cardiac troponin I proximal promoter to target expression of a downstream gene in the heart is also maintained when the transgene is integrated into the genome.

Cardiac and smooth muscle cell contribution to the formation of the murine pulmonary veins

Previous studies have demonstrated that the primordial pulmonary veins originate as an outgrowth of the atrial cells and anastomosis with the pulmonary venous plexus. As a consequence of this embryologic origin the tunica media of these vessels is composed of cardiac cells that express atrial specific markers (Lyons et al. [ 1990 ] J Cell Biol 111:2427–2436; Jones et al. [ 1994 ] Dev Dyn 200:117–128). We used transgenic mice for the cardiac troponin I (cTNI) gene and smooth muscle (SM) myosin heavy chain as differentiation markers, to analyze how cardiac and SM cells contribute to the formation and structural remodeling of the pulmonary veins during development. We show here that the tunica media of the adult mouse pulmonary veins contains an outer layer of cardiac cells and an intermediate SM cell compartment lining down on the inner endothelium. This structural organization is well expressed in the intrapulmonary veins from the beginning of vasculogenesis, with cardiac cells accumulating over preexisting roots of endothelial and SM cells and extending to the third bifurcation of the pulmonary branches without reaching the more distal tips of the vessels. On the other hand, SM cells, which are widely distributed in the intrapulmonary veins from the embryonic stage E16, accumulate also in the extrapulmonary branches and reach the posterior wall of the left atrium, including the orifices of the pulmonary veins. This event takes place around birth when the pulmonary blood flow starts to function properly. A model for the development of the pulmonary veins is presented, based upon our analysis. Dev Dyn 2000;218:414–425.

Different atrophy-hypertrophy transcription pathways in muscles affected by severe and mild spinal muscular atrophy

BackgroundSpinal muscular atrophy (SMA) is a neurodegenerative disorder associated with mutations of the survival motor neuron gene SMN and is characterized by muscle weakness and atrophy caused by degeneration of spinal motor neurons. SMN has a role in neurons but its deficiency may have a direct effect on muscle tissue.MethodsWe applied microarray and quantitative real-time PCR to study at transcriptional level the effects of a defective SMN gene in skeletal muscles affected by the two forms of SMA: the most severe type I and the mild type III.ResultsThe two forms of SMA generated distinct expression signatures: the SMA III muscle transcriptome is close to that found under normal conditions, whereas in SMA I there is strong alteration of gene expression. Genes implicated in signal transduction were up-regulated in SMA III whereas those of energy metabolism and muscle contraction were consistently down-regulated in SMA I. The expression pattern of gene networks involved in atrophy signaling was completed by qRT-PCR, showing that specific pathways are involved, namely IGF/PI3K/Akt, TNF-α/p38 MAPK and Ras/ERK pathways.ConclusionOur study suggests a different picture of atrophy pathways in each of the two forms of SMA. In particular, p38 may be the regulator of protein synthesis in SMA I. The SMA III profile appears as the result of the concurrent presence of atrophic and hypertrophic fibers. This more favorable condition might be due to the over-expression of MTOR that, given its role in the activation of protein synthesis, could lead to compensatory hypertrophy in SMA III muscle fibers.

Human MYO18B, a novel unconventional myosin heavy chain expressed in striated muscles moves into the myonuclei upon differentiation

We have characterized a novel unconventional myosin heavy chain, named MYO18B, that appears to be expressed mainly in human cardiac and skeletal muscles and, at lower levels, in testis. MYO18B transcript is detected in all types of striated muscles but at much lower levels compared to class II sarcomeric myosins, and it is up regulated after in vitro differentiation of myoblasts into myotubes. Phylogenetic analysis shows that this myosin belongs to the recently identified class XVIII, however, unlike the other member of this class, it seems to be unique to Vertebrate since it contains two large amino acid domains of unknown function at the N and C-termini. Immunolocalization of MYO18B protein in skeletal muscle cells shows that this myosin heavy chain is located in the cytoplasm of undifferentiated myoblasts. After in vitro differentiation into myotubes, a fraction of this protein is accumulated in a subset of myonuclei. This nuclear localization was confirmed by immunofluorescence experiments on primary cardiomyocytes and adult muscle sections. In the cytoplasm MYO18B shows a punctate staining, both in cardiac and skeletal fibers. In some cases, cardiomyocytes show a partial sarcomeric pattern of MYO18B alternating that of alpha-actinin-2. In skeletal muscle the cytoplasmic MYO18B results much more evident in the fast type fibers.

Altered glucose metabolism and proteolysis in pancreatic cancer cell conditioned myoblasts: searching for a gene expression pattern with a microarray analysis of 5000 skeletal muscle genes

Background and aims: We verified whether conditioned media (CM) from pancreatic cancer cell lines (MIAPaCa2, CAPAN-1, PANC-1, BxPC3) alter glucose metabolism and gene expression profiles (microarray experiment with a platform of 5000 skeletal muscle cDNA) in mice myoblasts. Methods: Myoblasts were incubated with control or pancreatic cancer CM for 24 and 48 hours. Results: Lactate significantly increased in CM compared with non-conditioned myoblasts. No variations in expression levels of the main genes involved in glycolysis were found in CM myoblasts. Propionyl coenzyme A carboxylase and isocitrate dehydrogenase 3 beta genes, which encode enzymes of the tricarboxylic acid cycle, were overexpressed, while IGFIIR and VAMP5 genes were underexpressed in CM myoblasts. PAFAH1B1 and BCL-2 genes (intracellular signal transduction) and the serine protease cathepsin G (proteolysis), were overexpressed in CM myoblasts. Tyrosine accumulation in CM myoblasts suggested that proteolysis overcomes protein synthesis. Sorcin, actin alpha, troponin T1, and filamin A were underexpressed in CM myoblasts. Conclusions: Our findings demonstrate that pancreatic cancer cell conditioned media enhanced lactate production and induced proteolysis, possibly by altering expression levels of a large number of genes, not only those involved in protein biosynthesis and degradation or glucose metabolism, but also those involved in the tricarboxylic acid cycle and in vesicle traffic.

The Antarctic krill Euphausia superba shows diurnal cycles of transcription under natural conditions.

Background Polar environments are characterized by extreme seasonal changes in day length, light intensity and spectrum, the extent of sea ice during the winter, and food availability. A key species of the Southern Ocean ecosystem, the Antarctic krill (Euphausia superba) has evolved rhythmic physiological and behavioral mechanisms to adapt to daily and seasonal changes. The molecular organization of the clockwork underlying these biological rhythms is, nevertheless, still only partially understood. Methodology/Principal Findings The genome sequence of the Antarctic krill is not yet available. A normalized cDNA library was produced and pyrosequenced in the attempt to identify large numbers of transcripts. All available E. superba sequences were then assembled to create the most complete existing oligonucleotide microarray platform with a total of 32,217 probes. Gene expression signatures of specimens collected in the Ross Sea at five different time points over a 24-hour cycle were defined, and 1,308 genes differentially expressed were identified. Of the corresponding transcripts, 609 showed a significant sinusoidal expression pattern; about 40% of these exibithed a 24-hour periodicity while the other 60% was characterized by a shorter (about 12-hour) rhythm. We assigned the differentially expressed genes to functional categories and noticed that those concerning translation, proteolysis, energy and metabolic process, redox regulation, visual transduction and stress response, which are most likely related to daily environmental changes, were significantly enriched. Two transcripts of peroxiredoxin, thought to represent the ancestral timekeeping system that evolved about 2.5 billion years ago, were also identified as were two isoforms of the EsRh1 opsin and two novel arrestin1 sequences involved in the visual transduction cascade. Conclusions Our work represents the first characterization of the krill diurnal transcriptome under natural conditions and provides a first insight into the genetic regulation of physiological changes, which occur around the clock during an Antarctic summer day.

Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new ‘STressREsponse Microarray’ (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 μg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota.

Gene and microRNA Expression Are Predictive of Tumor Response in Rectal Adenocarcinoma Patients Treated with Preoperative Chemoradiotherapy

Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT. Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one‐color microarray technique that compares signatures between responders (R) and non‐responders (NR), as measured based on tumor regression grade. Two groups composed of 38 “exploration cohort” and 21 “validation cohort” LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti‐correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR‐630 appeared only with the NR patients and was anti‐correlated with a single transcript: RAB5B. After PAM, the following eight transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7, and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity, and 82% specificity. All three parameters reached 100% when both cohorts were considered together. In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. J. Cell. Physiol. 232: 426–435, 2017.