Plastic Fly: What Drosophila melanogaster Can Tell Us about the Biological Effects and the Carcinogenic Potential of Nanopolystyrene.

Today, plastic pollution is one of the biggest threats to the environment and public health. In the tissues of exposed species, micro- and nano-fragments accumulate, leading to genotoxicity, altered metabolism, and decreased lifespan. A model to investigate the genotoxic and tumor-promoting potential of nanoplastics (NPs) is Drosophila melanogaster. Here we tested polystyrene, which is commonly used in food packaging, is not well recycled, and makes up at least 30% of landfills. In order to investigate the biological effects and carcinogenic potential of 100 µm polystyrene nanoparticles (PSNPs), we raised Oregon [R] wild-type flies on contaminated food. After prolonged exposure, fluorescent PSNPs accumulated in the gut and fat bodies. Furthermore, PSNP-fed flies showed considerable alterations in weight, developmental time, and lifespan, as well as a compromised ability to recover from starvation. Additionally, we noticed a decrease in motor activity in DNAlig4 mutants fed with PSNPs, which are known to be susceptible to dietary stressors. A qPCR molecular investigation of the larval intestines revealed a markedly elevated expression of the genes drice and p53, suggesting a response to cell damage. Lastly, we used warts-defective mutants to assess the carcinogenic potential of PSNPs and discovered that exposed flies had more aberrant masses than untreated ones. In summary, our findings support the notion that ingested nanopolystyrene triggers metabolic and genetic modifications in the exposed organisms, eventually delaying development and accelerating death and disease.

Repeated Rounds of Gonadotropin Stimulation Induce Imbalance in the Antioxidant Machinery and Activation of Pro-Survival Proteins in Mouse Oviducts.

Controlled ovarian stimulation (COS) through gonadotropin administration has become a common procedure in assisted reproductive technologies. COS's drawback is the formation of an unbalanced hormonal and molecular environment that could alter several cellular mechanisms. On this basis, we detected the presence of mitochondrial DNA (mtDNA) fragmentation, antioxidant enzymes (catalase; superoxide dismutases 1 and 2, SOD-1 and -2; glutathione peroxidase 1, GPx1) and apoptotic (Bcl-2-associated X protein, Bax; cleaved caspases 3 and 7; phosphorylated (p)-heat shock protein 27, p-HSP27) and cell-cycle-related proteins (p-p38 mitogen-activated protein kinase, p-p38 MAPK; p-MAPK activated protein kinase 2, p-MAPKAPK2; p-stress-activated protein kinase/Jun amino-terminal kinase, p-SAPK/JNK; p-c-Jun) in the oviducts of unstimulated (Ctr) and repeatedly hyperstimulated (eight rounds, 8R) mice. While all the antioxidant enzymes were overexpressed after 8R of stimulation, mtDNA fragmentation decreased in the 8R group, denoting a present yet controlled imbalance in the antioxidant machinery. Apoptotic proteins were not overexpressed, except for a sharp increase in the inflammatory-related cleaved caspase 7, accompanied by a significant decrease in p-HSP27 content. On the other hand, the number of proteins involved in pro-survival mechanisms, such as p-p38 MAPK, p-SAPK/JNK and p-c-Jun, increased almost 50% in the 8R group. Altogether, the present results demonstrate that repeated stimulations cause the activation of the antioxidant machinery in mouse oviducts; however, this is not sufficient to induce apoptosis, and is efficiently counterbalanced by activation of pro-survival proteins.

The Endocannabinoid System in the Mediterranean Mussel Mytilus galloprovincialis: Possible Mediators of the Immune Activity?

Anandamide (AEA) is one of the best characterized members of the endocannabinoid family and its involvement in many pathophysiological processes has been well documented in vertebrates and invertebrates. Here, we report the biochemical and functional characterization of key elements of the endocannabinoid system in hemocytes isolated from the Mediterranean mussel Mytilus galloprovincialis. We also show the effects of exogenous AEA, as well as of capsaicin, on the cell ability to migrate and to activate the respiratory burst, upon in vitro stimulation of phagocytosis. Interestingly, our findings show that both AEA and capsaicin suppress the hemocyte response and that the use of selective antagonists of CB2 and TRPV1 receptors revert their inhibitory effects. Overall, present data support previous evidence on the presence of endocannabinoid signaling in mollusks and advance our knowledge about the evolutionary origins of this endogenous system and its role in the innate response of mollusks.

Early genotoxic damage through micronucleus test in exfoliated buccal cells and occupational dust exposure in construction workers: a cross-sectional study in L'Aquila, Italy.

AIM: The city of L'Aquila (central Italy) was hit by a strong earthquake in 2009 that caused the collapse of several buildings, deaths and injured people. In the following years, a great number of building sites were activated, building workers resulted intensely exposed and represent a relevant target for research on environmental mutagenesis and epidemiological surveillance. Cells of buccal mucosa are considered an appropriate site for early detecting of cytogenetic damage, since it represents the first barrier in inhalation or ingestion and can metabolize carcinogenic agents into reactive chemicals. Our study is aimed 1) at comparing the early genotoxic damage as measured by the buccal mucosa micronucleus test in two subgroups of workers defined by different occupational exposure and 2) at evaluating possible confounding variables such as lifestyle factors. METHODS AND RESULTS: A cross-sectional study was conducted in L'Aquila, on 24 outdoor workers (OWs) highly exposed on the construction sites and 26 indoor workers (IWs), all subjected to the compulsory occupational surveillance system, in the period 2017-2018. Buccal cells samples were collected and, based on the Micronucleus test, the exfoliated cells were classified in respect of nuclear changes observed. Moreover, a self-report questionnaire composed of 84 items, was administered to the workers. RESULTS: Significant differences were observed between Exp+ (OWs) and Exp- (IWs) in the number of the analyzed cells (expressed as mean value out of 1000 cells): respectively 954.46 vs 990.06 normal cells, (p < 0.001); 19.79 vs 4.95 micronucleated cells, as marker of chromosomal damage (p < 0.001); 13.93 vs 8.96 binucleated cells, as marker of failed cytokinesis (p < 0.001); 2.09 vs 1.18 karyolytic cells, as marker of cell death and damaged DNA (p < 0.05). According with a multivariate regression analysis, in addition to the job exposure (OW vs IW, beta = 12.221, p < 0.001), the only variable independently associated with an increase in Micronuclei (MNs) is the smoking habit (OWs vs IWs, beta = 6.683, p < 0.001) which, even if not associated with dust exposure, worsens cell integrity. Moreover, this worsening effect is weaker in workers not exposed to the site dust (moderation effect). Within social demographic factors, the high educational level only apparently seems to affect MNs number: even if unbalanced in favor of IWs vs OWs, this variable resulted a confounder, since its effect disappears when the interaction between these two factors is considered, because it is a covariate of smoking habit as well as of the job condition. CONCLUSION: Despite some limitation, our findings clearly confirm the role of occupational exposure as a marker of cytogenetic damage associated with MNs number in construction workers. Moreover, smoking status appears as the only other investigated factor independently associated to the outcome. The statistical model, in addition, highlights possible moderation and confounding effects, such as interaction between smoking and occupational exposure and the unbalanced school education level in workers. Micronucleus test in exfoliated buccal cells would be considered a suitable method for studying the early genotoxic damage in the construction occupational setting as well as in evaluating the efficacy of preventive practices.

Particle Debris Generated from Passenger Tires Induces Morphological and Gene Expression Alterations in the Macrophages Cell Line RAW 264.7.

Air pollution in the urban environment is a topical subject. Aero-suspended particles can cause respiratory diseases in humans, ranging from inflammation to asthma and cancer. One of the components that is most prevalent in particulate matter (PM) in urban areas is the set of tire microparticles (1-20 µm) and nanoparticles (<1 µm) that are formed due to the friction of wheels with asphalt and are increased in slow-moving areas that involve a lot of braking actions. In this work, we studied the effect that microparticles generated from passenger tires (PTWP, passenger tire wear particles) have in vitro on murine macrophages cells RAW 264.7 at two concentrations of 25 and 100 µg/mL, for 24 and 48 h. In addition to the chemical characterization of the material and morphological characterization of the treated cells by transmission electron microscopy, gene expression analysis with RT-PCR and active protein analysis with Western blotting were performed. Growth curves were obtained, and the genotoxic effect was evaluated with a comet assay. The results indicate that initially, an induction of the apoptotic process is observable, but this is subsequently reversed by Bcl2. No genotoxic damage is present, but mild cellular abnormalities were observed in the treated cells.

Estimation of DNA Degradation in Archaeological Human Remains.

The evaluation of the integrity and quantity of DNA extracted from archaeological human remains is a fundamental step before using the latest generation sequencing techniques in the study of evolutionary processes. Ancient DNA is highly fragmented and chemically modified; therefore, the present study aims to identify indices that can allow the identification of potentially amplifiable and sequenceable DNA samples, reducing failures and research costs. Ancient DNA was extracted from five human bone remains from the archaeological site of Amiternum L'Aquila, Italy dating back to the 9th-12th century and was compared with standard DNA fragmented by sonication. Given the different degradation kinetics of mitochondrial DNA compared to nuclear DNA, the mitochondrially encoded 12s RNA and 18s ribosomal RNA genes were taken into consideration; fragments of various sizes were amplified in qPCR and the size distribution was thoroughly investigated. DNA damage degree was evaluated by calculating damage frequency (λ) and the ratio between the amount of the different fragments and that of the smallest fragment (Q). The results demonstrate that both indices were found to be suitable for identifying, among the samples tested, those less damaged and suitable for post-extraction analysis; mitochondrial DNA is more damaged than nuclear, in fact, amplicons up to 152 bp and 253 bp, respectively are obtained.

Genotoxicity Response of Fibroblast Cells and Human Epithelial Adenocarcinoma In Vitro Model Exposed to Bare and Ozone-Treated Silica Microparticles.

Indoor air pollutants (IAP), which can pose a serious risk to human health, include biological pollutants, nitric oxide (NO), nitrogen dioxide (NO2), volatile organic compounds (VOC), sulfur dioxide (SO2), carbon monoxide (CO), carbon dioxide (CO2), silica, metals, radon, and particulate matter (PM). The aim of our work is to conduct a multidisciplinary study of fine silica particles (<2.5 µm) in the presence or absence of ozone (O3), and evaluate their potential cytotoxicity using MTS, micronucleus, and the comet test in two cell lines. We analyzed A549 (human basal alveolar epithelial cell adenocarcinoma) and Hs27 (human normal fibroblasts) exposed to dynamic conditions by an IRC simulator under ozone flow (120 ppb) and in the presence of silica particles (40 µg/h). The viability of A549 and Hs27 cells at 48 and 72 h of exposure to silica or silica/ozone decreases, except at 72 h in Hs27 treated with silica/ozone. The micronucleus and comet tests showed a significant increase in the number of micronuclei and the % of DNA in the queue, compared to the control, in both lines in all treatments, even if in different cell times/types. We found that silica alone or with more O3 causes more pronounced genotoxic effects in A549 tumor cells than in normal Hs27 fibroblasts.

A qPCR-duplex assay for sex determination in ancient DNA.

Molecular biology techniques are increasingly being used in sex identification of skeletal remains when traditional anthropometric analyzes are not successful in identifying sex of remains that are incomplete, fragmented and /or of immature individuals. In the present work, we investigated the possibility of determining sex by using the qPCR-duplex method for both ancient and modern DNA samples. This method involves the co-amplification of two genes in a single reaction system and the subsequent analysis of the fusion curves; the gene sequences used for the construction of suitable primers are those of steroid sulfatase (STS) and testis specific protein Y-linked 1 (TSPY) genes which turned out to be two sensitive markers as they have a detection limit of 60 pg and 20 pg respectively on modern DNA. The validity of the method was verified on modern DNA in which gender was identified in all the samples with 100% accuracy; thus, allowing for the same results as the classic method with amelogenin, but in a faster and more immediate way, as it allows for sex determination solely by analyzing the denaturation curves without having to perform an electrophoretic run. The proposed molecular technique proves to be sensitive and precise even on degraded DNA, in fact on 9 archaeological finds dating from the VII-XII century in which sex had been identified through anthropometric analysis, it confirmed the sex of 8 out of 9 finds correctly.

Genotoxicity and oxidative stress induction by polystyrene nanoparticles in the colorectal cancer cell line HCT116.

The potential risks of environmental nanoparticles (NPs), in particular Polystyrene Nanoparticles (PNPs), is an emerging problem; specifically, the interaction of PNPs with intestinal cells has not been characterized so far. The mechanism by which polystyrene particles are transferred to humans has not yet been clarified, whether directly through ingestion from contaminated food. We evaluated the interaction between PNPs and colorectal adenocarcinoma cells (HCT116). Cells were exposed to different concentrations of PNPs, metabolic activity and the consequent cytotoxic potential were assessed through viability test; we evaluated the PNP genotoxic potential through the Cytokinesis-Block Micronucleus cytome (CBMN cyt) assay. Finally, we detected Reactive Oxygen Species (ROS) production after NPs exposure and performed Western Blot analysis to analyze the enzymes (SOD1, SOD2, Catalase, Glutathione Peroxidase) involved in the cell detoxification process that comes into play during the cell-PNPs interaction. This work analyzes the cyto and genotoxicity of PNPs in the colorectal HCT116 cell line, in particular the potential damage from oxidative stress produced by PNPs inside the cells related to the consequent nuclear damage. Our results show moderate toxicity of PNPs both in terms of ROS production and DNA damage. Further studies will be needed on different cell lines to have a more complete picture of the impact of environmental pollution on human health in terms of PNPs cytotoxicity and genotoxicity.

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development.

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17-18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development.

Exposure to particle debris generated from passenger and truck tires induces different genotoxicity and inflammatory responses in the RAW 264.7 cell line.

A number of studies have shown variable grades of cytotoxicity and genotoxicity in in vitro cell cultures, laboratory animals and humans when directly exposed to particle debris generated from tires. However, no study has compared the effects of particles generated from passenger tires with the effects of particles from truck tires. The aim of this study was to investigate and relate the cyto- and genotoxic effects of different types of particles (PP, passenger tire particles vs. TP, truck tire particles) in vitro using the phagocytic cell line RAW 264.7 (mouse leukaemic monocyte macrophage cell line). The viability of RAW 264.7 cells was determined by the 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay following exposure for 4, 24 and 48 hours to different particle concentrations (10 µg / ml, 25 µg / ml, 50 µg / ml, 100 µg / ml). The effects of particles of passenger and truck tires on cell proliferation and genotoxicity were evaluated by means of the cytokinesis-block micronucleus (CBMN) assay following exposure for 24 hours to different particle concentrations (10 µg / ml, 25 µg / ml, 50 µg / ml, 100 µg / ml). In MTS assay, after 24 hours, it was found that PP induced a 30% decrease in metabolic activity at a concentration of 10 µg/ml, while TP caused reductions of 20% and 10% at concentrations of 10 µg/ml and 50 µg/ml, respectively. At 48 hours after the treatments, we observed increased metabolic activity at 50 µg/ml and 100 µg/ml for the PP while only at 50 µg/ml for the TP. The CBMN assay showed a significant increase in the number of micronuclei in the cells incubated with PP in all experimental conditions, while the cells treated with TP showed a meaningful increase only at 10 µg /ml. We utilized the TNF-α ELISA mouse test to detect the production of tumour necrosis factor-alpha (TNF-α) in RAW 264.7 cells. The effect of passenger and truck particles on TNF-α release was evaluated following exposure for 4 and 24 hours. After 4 hours of incubation, the cells treated with PP and TP at 100 µg / ml showed a slight but significant increase in TNF-α release, while there was a significant increase in the release of TNF-α after 24 hours of incubation with both tire samples in the cells treated with 50 and 100 µg / ml PP. The data obtained show a higher cytotoxic, clastogenic/genotoxic and inflammatory effects of passenger compared to the truck tire particles.

In Vitro Genotoxicity of Polystyrene Nanoparticles on the Human Fibroblast Hs27 Cell Line.

Several studies have provided information on environmental nanoplastic particles/debris, but the in vitro cyto-genotoxicity is still insufficiently characterized. The aim of this study is to analyze the effects of polystyrene nanoparticles (PNPs) in the Hs27 cell line. The viability of Hs27 cells was determined following exposure at different time windows and PNP concentrations. The genotoxic effects of the PNPs were evaluated by the cytokinesis-block micronucleus (CBMN) assay after exposure to PNPs. We performed ROS analysis on HS27 cells to detect reactive oxygen species at different times and treatments in the presence of PNPs alone and PNPs added to the Crocus sativus L. extract. The different parameters of the CBMN test showed DNA damage, resulting in the increased formation of micronuclei and nuclear buds. We noted a greater increase in ROS production in the short treatment times, in contrast, PNPs added to Crocus sativus showed the ability to extract, thus reducing ROS production. Finally, the SEM-EDX analysis showed a three-dimensional structure of the PNPs with an elemental composition given by C and O. This work defines PNP toxicity resulting in DNA damage and underlines the emerging problem of polystyrene nanoparticles, which extends transversely from the environment to humans; further studies are needed to clarify the internalization process.

Analysis of ancient mtDNA from the medieval archeological site of Amiternum (L'Aquila), central Italy.

Study of ancient DNA makes it possible to analyze genetic relationships between individuals and populations of past and present. In this paper we have analyzed remains of human bones, dating back to the 8th-10th century AD, from the burials found in the Cathedral of Santa Maria in Civitate, archaeological site of Amiternum, L'Aquila, Italy. As a genetic marker, the hypervariable region 1 of mitochondrial DNA (HVR1) was selected. To obtain reliable sequences from the hypervariable region 1 of mtDNA (HVR1) were performed: multiple extractions, template quantification and cloning of PCR products. The sequences obtained were compared with Anderson's sequence for the identification of polymorphisms (SNP) and haplogroups. The data obtained were analyzed with various software and phylogenetic methods. For the comparison between populations, ancient and modern sequences found in databases and literature have been used. This work provides preliminary information on the correlation between the population of Amiternum, the migrant populations transited and/or established in the territory of Amiternum such as Byzantines, Longobards (Lombards), which dominated the Italian peninsula between 568 and 774 AD, and the current populations of Italy. The study of haplogroups, the analysis of genetic variability and phylogenesis studies on the sequences considered show a genetic closeness between the individuals of Amiternum, the current population of central-northern Italy and the Germanic tribe of Longobards, however, also highlights genetic traits of Byzantines in some samples of Amiternum. Using the analysis of amelogenin gene fragments, we successfully determined the sex of the bone remains on all samples.

The genomic tool-kit of the truffle Tuber melanosporum programmed cell death.

A survey of the truffle Tuber melanosporum genome has shown the presence of 67 programmed cell death (PCD)-related genes. The 67 genes are all expressed during fruit body (FB) development of T. melanosporum development; their expression has been detected by DNA microarrays and qPCR. A set of 14 PCD-related genes have been chosen, those with the highest identities to the homologs of other species, for a deeper investigation. That PCD occurs during T. melanosporum development has been demonstrated by the TUNEL reaction and transmission electron microscopy. The findings of this work, in addition to the discovery of PCD-related genes in the T. melanosporum genome and their expression during the differentiation and development of the FB, would suggest that one of the PCD subroutines, maybe autophagy, is involved in the FB ripening, i.e., sporogenesis.

Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells.

Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

Amphibian transition to the oxidant terrestrial environment affects the expression of glutathione S-transferases isoenzymatic pattern.

It has been postulated that glutathione S-transferases (GST; EC 2.5.1.18) may play a role in protecting against oxidative stress. In previous studies, we have purified and characterised from Bufo bufo embryos a GST isoenzyme (BbGSTP1-1), which falls at very low level in the adult liver, where a novel isoform (BbGSTP2-2), starts to be highly expressed. During transition to adult life, B. bufo leaves the aquatic environment to live predominantly in the terrestrial environment, characterised by higher oxygen concentration. It has been found that BbGSTP2-2 is more efficient in scavenging from organic hydroperoxides. Therefore, the appearance of BbGSTP2-2 may respond to the necessity of providing the adult toad with a more suitable protection against oxygen toxic by-products. In this work, we performed experiments aimed at verifying if oxidative stress (hyperoxic and H(2)O(2) treatments) could act as a modulator of BbGSTP2-2 expression. Results show that: (a) BbGSTP2 mRNA starts to be expressed in the late embryonic period, while protein appears during metamorphosis; (b) oxidative stress induces anticipation of BbGSTP2 gene expression at both transcriptional and translational levels. These findings seem to indicate that the appearance of BbGSTP2-2 is aimed at endowing the adult toad with more efficient antioxidant defence in the terrestrial atmosphere.

Truffles contain endocannabinoid metabolic enzymes and anandamide.

Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

Validation of reference genes for quantitative real-time PCR in Périgord black truffle (Tuber melanosporum) developmental stages.

The symbiotic fungus Tuber melanosporum Vittad. (Périgord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; ß-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; ß-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable genes for fruiting body developmental stages.

Truffle thio-flavours reversibly inhibit truffle tyrosinase.

Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms. In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to harden cell walls. Some of us have previously shown that tyrosinase is involved in truffle development and differentiation. Here we present the purification, the molecular properties and the reversible inhibition of Tuber melanosporum tyrosinase by dimethyl-sulfide and bis[methylthio]methane, the main flavour compounds of black and whitish truffles. The MW(r) is 39000. L-3,4-dihydroxyphenylalanine and L-tyrosine stain corresponding bands as expected for a true tyrosinase. Phenylthiourea, diethyldithiocarbamate and mimosine inhibit L-tyrosine and L-3,4-dihydroxyphenylalanine oxidation.

Transcriptional, biochemical and histochemical investigation on laccase expression during Tuber melanosporum Vittad. development.

The cDNAs of Tuber melanosporum laccases (Tmellcc1 and Tmellcc2) have been cloned. From the cloned cDNAs probes were prepared to investigate the expression levels of the Tmellcc1 and Tmellcc2 genes in the free living mycelium (FLM), ectomycorrhizae (ECM) and different developmental stages of fruit body (FB) by quantitative PCR (qPCR). The mRNA expression levels agree with the changes of laccase activities. The histochemical data agree with the qPCR and biochemical results. The highest laccase expression occurs in the ECM, when the host plant roots are invaded by the fungal mycelium.