Search

Background Green feed diet in ruminants exerts a beneficial effect on rumen metabolism and enhances the content of milk nutraceutical quality. At present, a comprehensive analysis focused on the identification of genes, and therefore, biological processes modulated by the green feed in buffalo rumen has never been reported. We performed RNA-sequencing in the rumen of buffaloes fed a total mixed ration (TMR) + the inclusion of 30% of ryegrass green feed (treated) or TMR (control), and identified differentially expressed genes (DEGs) using EdgeR and NOISeq tools. Results We found 155 DEGs using EdgeR (p-values Gene Ontology analysis of DEGs identified using EdgeR revealed that green feed modulates biological processes relevant for the rumen physiology and, then, health and well-being of buffaloes, such as lipid metabolism, response to the oxidative stress, immune response, and muscle structure and function. Accordingly, we found: (i) up-regulation of HSD17B13, LOC102410803 (or PSAT1) and HYKK, and down-regulation of CDO1, SELENBP1 and PEMT, encoding factors involved in energy, lipid and amino acid metabolism; (ii) enhanced expression of SIM2 and TRIM14, whose products are implicated in the immune response and defense against infections, and reduced expression of LOC112585166 (or SAAL1), ROR2, SMOC2, and S100A11, encoding pro-inflammatory factors; (iii) up-regulation of NUDT18, DNAJA4 and HSF4, whose products counteract stressful conditions, and down-regulation of LOC102396388 (or UGT1A9) and LOC102413340 (or MRP4/ABCC4), encoding detoxifying factors; (iv) increased expression of KCNK10, CACNG4, and ATP2B4, encoding proteins modulating Ca2+ homeostasis, and reduced expression of the cytoskeleton-related MYH11 and DES. Conclusion Although statistically unpowered, this study suggests that green feed modulates the expression of genes involved in biological processes relevant for rumen functionality and physiology, and thus, for welfare and quality production in Italian Mediterranean dairy buffaloes. These findings, that need to be further confirmed through the validation of additional DEGs, allow to speculate a role of green feed in the production of nutraceutical molecules, whose levels might be enhanced also in milk.

Improving fruit quality and storability is crucial to alleviate quantitative as well as qualitative post-harvest losses that occur worldwide, and particularly in developing countries. In this study, we investigated the regulatory networks underlying tomato fruit shelf-life in the landrace Lucariello (LUC) belonging to the so-called 'da serbo' tomatoes. LUC fruits have a regular timing of ripening associated with a prolonged shelf-life of more than 150 days. The physiochemical characterization of LUC fruits is consistent with their shelf-life extension. In particular, the changes in fruit firmness and fresh weight confirmed the delay in fruit softening and withering during post-harvest. We compared by multi-omics analysis fruits at 60 and 150 days of post-harvest to those at an advanced stage of ripening, i.e. red ripe fruits at 8 days after breaker coincident with the harvest. Gene expression was quantified by RNA-seq while epigenomic analysis included the profiling of DNA methylation, small non-coding RNAs, and post-translational modifications of histones. The expression profiles of LUC fruits were compared with those from public dataset of fruits during ripening in the short shelf-life cultivar Ailsa Craig (AC). Principal Component Analysis highlighted that LUC samples at harvest do not cluster with the corresponding stage of AC fruits thereby suggesting an overall delay of over-ripening program in LUC fruits. In particular, the delay appears to be highly correlated with the downregulation of RIPENING INHIBITOR (RIN), encoding a ripening-specific MADS-box transcription factor. RIN, as well as its targets such as PECTATE LYASE (PL), a fruit softening-specific gene, and LoxB, involved in the loss of cellular membrane integrity, showed a significant downregulation during ripening in LUC with respect to AC as observed by Real Time PCR. We argue that the decreased expression of these genes in LUC is an important factor in determining its extended shelf-life, since downregulation of these genes has been shown to result in fruits that are firmer for longer than controls. In order to understand the mechanisms underlying the observed transcriptional changes, an integrated analysis is now being carried out to investigate the regulation of gene expression in relation to epigenetic modulations

Tomato (Solanum lycopersicum L.) is the model system for investigating the molecular basis of ripening in fleshy fruits. Ripening is orchestrated by a complex regulative network including multiple transcription factors, ethylene signaling pathway, and epigenome changes. It is still unclear whether fruit ripening has to be regarded as a process of senescence or as a distinct process. We investigated the biological relevance of histone post-translational modifications (HMs) H3K4me3, H3K9/K14ac, and H3K27me3 in fruit post-harvest using ChIP-Seq. H3K4me3 and H3K9/K14ac are histone marks that positively correlate with gene expression while H3K27me3 plays a repressive role. In this study, HMs were mapped in fruit pericarp of Lucariello, a landrace with long shelf-life belonging to "Pomodorino del Piennolo del Vesuvio DOP". Three different time points have been analyzed: red ripe fruit (Breaker+8) coincident with the harvest, 60 and 150 days after harvest. Based on HM profiles, the genome was partitioned into eight chromatin states, which correspond to regions marked by only one HM, co-occurrence of HMs and unmarked regions. H3K27me3-only regions were strongly associated with repetitive sequences. Chromatin states marked by H3K4me3 and/or H3K9/K14ac were found to be associated with gene bodies and promoters. Based on chromatin state changes between time points, we estimated that 12% of chromatin is dynamic during post-harvest. One-third of the changes is related to gain/loss of H3K27me3 mark. We identified differentially enriched peaks of HMs. We focused on those at +/-2kb from predicted transcription start site regions that correlate with differentially expressed genes. Data gathering and interpretation are now being carried out to identify the relative contribution of HMs to transcriptional regulation in tomato fruit post-harvest.

Quantitative and qualitative post-harvest losses occur worldwide up to 50%. It would be of great benefit to control fruit senescence thereby improving fruit quality and storability. Tomato (Solanum lycopersicum L.) is probably the best model for understanding the fleshy climacteric fruit ripening process. However, it is still unclear whether fruit ripening is a process of organ senescence or fruit senescence is a distinct process downstream the ripening. To accomplish a comprehensive investigation on the senescence-related processes, a multi-omics approach including RNA-seq for coding and non-coding RNAs, BS-seq for DNA methylation, and ChIP-seq for histone post-translational modifications (H3K4me3, H3K27me3, H3K9K14ac) was planned in tomato by using the landrace Lucariello (LUC), recognized as 'Pomodorino del Piennolo del Vesuvio', that has a shelf-life higher than 150 days. Fruit pericarps were sampled at the red ripe stage, 60 and 150 days in post-harvest (dph). Dry weight, soluble solids content and firmness decreased in LUC fruits at 150 dph by 13%, 10% and 67%, respectively, in comparison with the earliest time point. Differentially expressed genes (DEGs, n=10285) from RNA-seq included many transcription factor genes involved in fruit ripening-related processes. The occurrence of a genome-wide hypermethylation at CG and CHG sites in 150 dph fruits was concomitant with a progressive silencing of the fruit-specific DNA demethylase, SlDML2, thereby suggesting a role for the epigenome in fruit senescence. We focused on DEGs that were likely regulated by DNA methylation as well as by the abovementioned histone marks or miRNAs. In particular, more than 2000 putative miRNAs were associated with anti-correlated DEG targets in a predictive manner. Among the regions of significant H3K4me3, H3K27me3, H3K9K14ac ChIP enrichment, the peaks of read counts that were different between time points and located around the transcription start site were correlated with DEGs. Similarly, the regions that significantly gained/lost DNA methylation across time points (Differentially Methylated Regions) were analysed for the association with DEGs. Based on the identified DEGs, co-expression analysis is currently underway to find activator and/or repressor candidates in the regulatory pathways underlying tomato fruit senescence. Furthermore, all the data are being reconciled in a web-based platform.

A better understanding of regulatory networks underlying fruit senescence would greatly impact on fruit quality and storability. Tomato (Solanum lycopersicum L.) is currently the best model for studying ripening process in climacteric fleshy fruit. However, the boundary between fruit ripening and senescence is still unclear. The Italian landrace Lucariello (LUC), recognized as 'Pomodorino del Piennolo del Vesuvio', is characterized by a long shelf-life reaching more than 150 days after harvest, thus it represents an excellent model to investigate the relationship between ripening and senescence and a genetic resource to transfer its long shelflife to other cultivars. For a full characterization of Lucariello, a multi-omics approach was followed, in which the genome was resequenced, assembled and annotated. In addition, coding and non-coding RNAs, DNA methylation, and histone post-translational modifications (H3K4me3, H3K27me3, H3K9K14ac) were analysed in fruit pericarps at the red ripe stage, 60 and 150 days in post-harvest (dph). More than 10,000 genes were found to be differentially expressed across the time points and many were related to transcription factors and to fruit ripening. About 2,000 putative miRNAs were predicted as anti-correlated to DEG targets. Differentially enriched regions for H3K4me3, H3K27me3, H3K9K14ac and differentially methylated regions were identified around the transcription start site and correlated with gene expression. An integrated analysis is now being carried out to find activator and/or repressor candidates regulating senescence process in tomato fruits. All the genomic and epigenomic datasets are gathered into a dedicated and user-friendly web-based platform.

It has been a long trip from 1992, the year of the discovery of MECP2, to the present day. What is surprising is that some of the pivotal roles of MeCP2 were already postulated at that time, such as repression of inappropriate expression from repetitive elements and the regulation of pericentric heterochromatin condensation. However, MeCP2 performs many more functions. MeCP2 is a reader of epigenetic information contained in methylated (and hydroxymethylated) DNA, moving from the 'classical' CpG doublet to the more complex view addressed by the non-CpG methylation, which is a feature of the postnatal brain. MECP2 is a transcriptional repressor, although when it forms complexes with the appropriate molecules, it can become a transcriptional activator. For all of these aspects, Rett syndrome, which is caused by MECP2 mutations, is considered a paradigmatic example of a 'chromatin disorder'. Even if the hunt for bona-fide MECP2 target genes is far from concluded today, the role of MeCP2 in the maintenance of chromatin architecture appears to be clearly established. Taking a cue from the non-scientific literature, we can firmly attest that MeCP2 is a player with 'a great future behind it'*.*V. Gassmann 'Un grande avvenire dietro le spalle'.

Epigenetics has been proven to be involved in the regulation of tomato fruit ripening, but a comprehensive investigation on post-harvest related processes is still lacking. By applying next generation sequencing technologies, the EPITOM Consortium is performing integrative analyses including coding and non-coding RNAs, DNA methylation, and histone post-translational modifications in long shelf-life traditional variety Lucariello, recognized as 'Pomodorino del Piennolo del Vesuvio PDOP'. Fruit pericarps have been sampled at the red ripe stage and at two and five months in post-harvest. Currently, the data of high throughput sequencing of mRNA, small RNAs (sRNA), and long non-coding RNAs (lncRNA) are available. RNA-sequencing (RNA-Seq) analysis allowed the identification of several new genes in comparison to the current tomato genome annotation (ITAG2.4, SL2.50). In addition, a large number of alternative transcript variants were identified at the post-harvest stages compared to the red ripe one. Gene ontology (GO) enrichment analysis of all differentially expressed genes highlighted nine over-represented GO terms involved in epigenetic mechanisms, thereby suggesting a pivotal role of the epigenome during tomato post-harvest. Putative miRNAs were also predicted to target differentially expressed mRNAs involved in fruit ripening-related processes. In addition, a number of miRNA precursors showed upstream binding sites for stress related transcription factors. LncRNAs, isolated from RNA-seq experiment selecting ORFs longer than 200 nt are under evaluation to identify novel lncRNAs and to investigate their role in the regulatory mechanisms of tomato fruit ripening during post-harvest. All the data are being reconciled in a web-based platform to allow multilevel integrated analyses of the available collections.

BACKGROUND: Cyclosporine A (CsA) is the first-line immunosuppressant used in transplant patients and in auto- immune diseases. Nephrotoxicity is the major limitation of CsA use. Although the mechanisms of nephrotoxicity have not been completely defined, some evidence suggests that reactive oxygen species (ROS) play a causal role. The present study was designed to investigate in vivo effects of hydroxytyrosol (DOPET), a natural olive oil antioxidant, on oxidative stress, renal histology and haemodynamic alterations induced in rats by CsA treatment. METHODS: Adult Sprague-Dawley rats were treated i.p. with CsA (15 mg/kg) alone or in combination with DOPET (20 mg/kg) for 3 weeks. At the end of the treatment, superoxide concentration within the cells of the abdominal aorta and renal artery was quantified from the oxidation of dihydroethidium (DHE) using fluorescence microscopic imaging analysis. In kidney tissues, lipid peroxidation was measured by thiobarbituric acid-reacting substances (TBARS) assay, glutathione level was assessed enzymatically and the expression of haem oxygenase-1 (HO-1) gene was evaluated by semiquantitative RT-PCR. Renal morphology was studied by classical histological techniques, while the glomerular filtration rate (GFR) was estimated by inulin clearance. Systemic blood pressure was monitored by the tail method and through the catheterization of the carotid artery. RESULTS: CsA administration increased superoxide concentration both in the aorta and in the renal artery, while DOPET completely prevented this effect. Higher levels of TBARS, a significant decrease in GSH and an upregulation of HO-1 mRNA were observed in the kidneys of CsA-treated rats. DOPET treatment reversed quantitatively these effects. However, CsA-dependent changes in renal histology were only partially reversed by DOPET. Finally, CsA induced a severe reduction in GFR and a significant increase in both systolic and diastolic blood pressure; the DOPET treatment had no significant effect on these haemodynamic alterations. CONCLUSION: The reported data indicate that effective DOPET protection from CsA-induced oxidative stress is associated with a mild effect on histological damages and does not affect the altered glomerular function and the hypertension, thus indicating that kidney injury by CsA is only in part dependent on oxidative stress.

Book, page 104

Book, O9.5

Cell cycle regulatory genes are frequently altered in a variety of malignancies. The structure and pattern of expression of eight genes involved in cell division cycle control were studied in leukemic cell samples prepared from bone marrow of patients affected by chronic myelogenous leukemia.Ten cell preparations were obtained from patients in the chronic phase, five from those in myeloid blast crisis and five from those in the lymphoid acute phase. Moreover, bone marrow CD34+ cells, purified from healthy subjects and patients with chronic myelogenous leukemia (both during chronic and acute phases), were analyzed. The investigated genes were RB1, p53 and six cyclin-dependent kinase inhibitor genes (p15INK4B, p16INK4A, p18INK4C, p21WAF1/CIP1, p27Kip1, p57Kip2).We found that none of these genes is structurally altered in either the chronic or acute phases, with the single exception of the p16INK4A gene, which was homozygously deleted in 1 case of lymphoid evolution. p57Kip2 expression is down-regulated during the evolution towards the blast crisis both in malignant and CD34+ cells. In addition, a significant up-regulation of p15INK4B gene expression is observable during the development of the acute phase of malignancy.The transcriptional modulation of some cyclin-dependent kinase inhibitors might contribute to the fatal blast crisis of chronic myelogenous leukemia.

p16(INK4A) and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase activities required for the overcoming of the G1 checkpoint in the eukaryotic cell division cycle. The frequent cytogenetic aberrations occurring in several human neoplasms at the level of their codifying genes along with their molecular function strongly suggest that they might be important tumor suppressor genes. We looked for homozygous deletions of p16(INK4A) and p18 genes in 21 cases of childhood T cell lineage acute lymphoblastic leukemia (ALL). Twenty of 21 patients (95%) had homozygous deletions of p16(INK4A) gene while three out of 21 (14%) showed p18 gene biallelic deletion. Loss of heterozygosity studies were performed in 18 of the T cell ALL investigated by means of two highly polymorphic 9p21 markers. The results obtained demonstrated that genetic deletions of different extension occur on the short arms of the 9 chromosome pair. Karyotypic analyses, performed in 13 cases, failed to demonstrate 9p alterations in 12 samples, (92%) thus demonstrating that p16(INK4A) gene homozygous deletions are not restricted to cases with cytogenetically detectable 9p aberrations. The high incidence of p16(INK4A) gene deletions in pediatric T cell lineage ALL suggests that this genetic alteration could represent an early and key event in the development of such a malignancy but it should not have any prognostic value. Conversely, the inactivation of p18 gene, observed in a lower but significant number of cases, could participate in the progression of acute leukemias towards a more aggressive disease. Finally, our results may suggest that p16(INK4A) protein plays a key role in the control of proliferation and/or differentiation of human T lymphocytes.

The aim of our study is to evaluate in vitro the response of bone marrow stromal cells (BMSCs) to platelet-rich plasma (PRP), in order to clarify the potential role of their combined use in a preclinical phase preceding BMSCs transplantation for bone repair and regeneration procedures. The incubation of BMSCs with PRP promoted a remarkable, dose- and time- dependent, growth stimulation, that was paralleled to a strong increase in the quantity of type I collagen and to a significant decrease in the activity of the early osteoblastic differentiation marker, alkaline phosphatase (AP). Once PRP was removed and osteogenic inducers were added, AP returned to levels comparable to the control, while the late phenotypic markers, osteocalcin and matrix calcification, were enhanced to higher levels than in controls. Our data demonstrate that PRP induces a remarkable ex vivo enrichment of BMSCs maintaining their differentiative potential. Thus PRP represents a valid preclinical tool for obtaining an effective, rapid and safe ex vivo expansion of BMSCs prior to their clinical utilization in bone engineering.

The role and regulation of signal transduction pathways in proliferation and differentiation of intestinal epithelial cells are still poorly understood. However, growing evidences have been recently accumulated demonstrating that mitogen-activated protein kinases (MAPKs) play a pivotal function in the normal development of intestine. We have investigated, in the intestinal cell line HT-29, the regulation (namely activity and phosphorylation degree) of MAP kinases ERK 1 (p44) and ERK 2 (p42) during differentiation. Addition of fetal calf serum to HT-29 undifferentiated resting cells caused a rapid phosphorylation of both ERKs and an increase of their specific kinase activity. Moreover, nuclear translocation of ERK 1 and ERK 2 occurred concurrently to their activation, leading to the conclusion that ERK 1 and ERK 2 are classically regulated when quiescent HT-29 cells are induced to proliferate. Butyrate addition to the intestinal cell line resulted in terminal differentiation and in a selective down-regulation of ERK 2 activity (and phosphorylation degree) without any effect on ERK 1. Conversely, when HT-29 cells were differentiated by repeated passages in a glucose-free medium, we observed a progressive dephosphorylation and inactivation of p42 and p44 kinases along with the failure of serum to activate both the enzymes. Our findings suggest that, during the differentiation of intestinal cells, remarkable changes occur in ERK 1 and ERK 2 control mechanisms leading to an unresponsiveness of MAP kinase pathway.

A series of biodegradable composites with natural hydroxyapatite, designed for possible use in orthopedics application, were preliminarily screened for biocompatibility by employing primary cultures of human osteoblasts in a direct contact method. The cells were seeded at low density onto the materials under investigation and allowed to grow for 2 weeks. They then were analyzed for morphology, proliferation, viability, alkaline phosphatase activity (AP), osteolcalcin (OC) production, and extracellular matrix mineralization. The results showed that all materials have good biocompatibility. Cell viability tests demonstrated that in all cases the values were comparable to the control, and the addition of hydroxyapatite always resulted in an enhancement of performance with respect to the plain polymer. AP and OC analyses confirmed that all composites allowed the expression of phenotype markers. Scanning electron microscopy provided direct evidence of intense cell adhesion and proliferation on the tested materials.A series of biodegradable composites with natural hydroxyapatite, designed for possible use in orthopedics application, were preliminarily screened for biocompatibility by employing primary cultures of human osteoblasts in a direct contact method. The cells were seeded at low density onto the materials under investigation and allowed to grow for 2 weeks. They then were analyzed for morphology, proliferation, viability, alkaline phosphatase activity (AP), osteolcalcin (OC) production, and extracellular matrix mineralization. The results showed that all materials have good biocompatibility. Cell viability tests demonstrated that in all cases the values were comparable to the control, and the addition of hydroxyapatite always resulted in an enhancement of performance with respect to the plain polymer. AP and OC analyses confirmed that all composites allowed the expression of phenotype markers. Scanning electron microscopy provided direct evidence of intense cell adhesion and proliferation on the tested materials.

Pyrrolidine dithiocarbamate (PDTC) is a synthetic antioxidant molecule, which has been recently proposed as an antitumoral agent on the basis of its capability of inducing apoptosis. We investigated the effect of PDTC on the proliferation and survival of the promyelocitic cell line HL-60. Concentration as low as 10 μM of PDTC induces a significant reduction of the growth rate and the contemporaneous activation of the apoptotic process. Programmed cell death was demonstrated by biochemical analyses, including the activation of procaspase 3 and the cleavage of poly(ADP-ribose) polymerase (PARP). PDTC-dependent apoptosis was associated with an early release of cytochrome c from mitochondria, while the involvement of pathways due to cell death receptors engagement was ruled out by detailed time-course analyses of caspases 3 and 8 activation. Moreover, no up-regulation of p21 CIP1 level, a pivotal cyclin-dependent kinase inhibitor, occurred at PDTC concentration able to induce apoptosis. Finally, in vitro incubation of purified mitochondria with PDTC demonstrated that the molecule is directly able to induce cytochrome c release from the intermembrane space, thus confirming that mitochondria are a primary cellular target of the molecule.

The regulation of the D-type cyclin-dependent kinase (CDK4 and CDK6) activity appears to be the key step in the progression of eukaryotic cells through the G 1 cell cycle phase. One of the mechanisms involved in this process is the binding of some small protein inhibitors, with a molecular mass ranging between 14 and 20 kDa, to these CDKs. We have evaluated the amount of two such inhibitors, namely p16(INK4) and p18, in normal and transformed cells, as well as the biochemical features of the macromolecular complexes containing these proteins. The results obtained indicated that (i) p18 gene expression, unlike p16(INK4) gene, is not regulated by pRb status, (ii) no evident relationship exists between the expression of p16(INK4) and p18 genes, (iii) significant amounts of the two proteins are not bound to CDKs but occur as free molecules, (iv) each inhibitor forms a complex with the CDK protein with a 1:1 stoichiometry, and (v) a competition exists between cyclin D and the inhibitor protein toward the CDK protein resulting in the absence of detectable cellular free kinase. Moreover, employing the human native partially purified p16(INK4) or the pure recombinant protein, we have been able to demonstrate in vitro the dissociation of CDK4-cyclin D1 complex and the formation of CDK4-p16(INK4) bimolecular complex. Our findings suggest that during the cell division cycle the members of the p16(INK4) protein family and cyclin Ds compete for binding to CDK4/CDK6 and that their quantitative ratio is essential for G 1 -> S transition.

Malignant transformation is due to mutations that modify the mechanisms regulating normal cellular growth and development. These alterations include the somatic activation of cancer-promoting genes1 (cellular oncogenes) and the germline or somatic inactivation of tumor suppressor genes, also known as antioncogenes or recessive oncogenes2. While the identification of oncogenes is facilitated by their ability to transform appropriate host cells3, the search for tumor suppressor genes is remarkably complicated by the lack of strong selection procedures.