The analysis of the joint effect of substances on reversion systems and the assessment of antimutagenicity.

The statistical methods for the analysis of mutagenicity and carcinogenicity underwent considerable theoretical-practical development following the need for assessing the mutagenic and carcinogenic potential of substances. Antimutagenicity is investigated through the analysis of respondents in dose-response assays, when two different molecules are administered separately and as a mixture to a respondent system. When the number of respondent units is high, and doses are orthogonal, it is possible to apply simple models such as analysis of variance. This is not always possible or common, and alternative approaches have been developed, based on multiple regression and on tables of proportions. In this work, some of the most frequently used methods for the assessment of joint responses are reviewed, particularly those based on multiple regression, such as the method of Shaeffer et al. and the method of Hass et al. In order to illustrate these methods, joint responses of perylene and cyclopentapyrene, of N-acetylcysteine and dinitropyrene, and of N-acetylcysteine and extracts from diesel exhausts were analyzed. An antagonistic effect of perylene on the action of CPP was detected by the algorithm of Shaeffer et al. The effect is not multiplicative, i.e., it is not proportional to the product of doses. The antimutagenic effect of N-acetylcysteine on dinitropyrene is multiplicative, as detected by the method of Hass et al. The latter reveals that the inhibition by N-acetylcysteine on the mutagenic effect of extracts from diesel exhausts is also multiplicative.     

The use of GMOs (genetically modified organisms): agricultural biotechnology or agricultural biopolitics?

Agricultural biotechnologies embrace a large array of conventional and modern technologies, spanning from composting organic by-products of agriculture to innovative improvement of quality traits of about twenty out of the mostly cultivated plants. In EU a rather restrictive legislative framework has been installed for GMOs, requiring a risk assessment disproportionate with respect to conventional agriculture and organic farming products. The latter are far from being proved safe for human and animal health, and for the environment.Biotechnology of GMOs has been overtaken by biopolitics. On one side there are biotechnological challenges to be tackled, on another side there is plenty of ground for biopolitical decisions about GMOs. Perhaps the era of harsh confrontation could be fruitfully replaced by sensible cooperation, in order to get a sustainable agricultural development.     

In Vivo and in Vitro Experimental Analysis of Lens Regeneration in Larval Xenopus laevis

After lentectomy of larval Xenopus laevis , the outer cornea undergoes tissue transformation resulting in formation of a new lens. This lens regeneration is triggered and sustained by neural retina. In the present study, lens-forming transformation of the outer cornea was completed in vitro when the outer cornea was cultured within the lentectomized eye-cup. Well-differentiated lens fiber cells, which showed positive immunofluorescence for total crystallins, were also formed when the outer cornea was cultivated with the retina. No lens tissue was formed when the cornea was cultured alone. Lens-forming transformation, originating from the cornea three and five days after lentectomy, completely regressed when the tissue was isolated in vitro . Fom the present and previous findings, we concluded that, the interaction of corneal cells with the retina plays a decisive role in lens regeneration in situ .     

Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a ‘locus a’ myc− mutant of Pisum sativum L.

Pisum sativum L. myc^– mutants which fail to form arbuscular mycorrhiza have recently been identified amongst nod^– mutants (Duc et al., 1989, Plant Sci. 60, 215–222). The reason for this resistance to symbiotic fungi has been investigated in the case of a locus a mutant (P2) inoculated with Glomus mosseae (Nicol. and Gerd.) Gerd, and Trappe. The fungal symbiont formed viable appressoria in contact with the root surface but its development was stopped at the root epidermis. Abundant material was deposited on the inner face of root cell walls adjacent to the appressoria in the P2 mutant, but not in the wild-genotype parent cultivar (Frisson) forming a symbiotic mycorrhizal infection. Fluorescence, histochemical, cytochemical and immunocytological approaches were used to characterize the paramural deposits in epidermal and hypodermal cells of the mutant. Strong fluorescence under blue light indicated the accumulation of phenolic compounds although polymers like lignin or suberin were not localized. Proteins and glycoproteins were homogeneously distributed within the paramural deposits. In the latter, the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for 1,4-polysaccharide detection showed a heterogeneous composition with electron-dense points surrounded by non-reactive material, but cytological tests for cellulose and pectin gave weak responses as compared to epidermal and hypodermal walls of the wild genotype. -1,3-Glucans indicative of callose were detected by in-situ immunolocalization in the paramural deposits below appressoria on mutant roots, but not in walls of the wild genotype. Thus, appressorium formation by G. mosseae on roots of the locus a P. sativum mutant elicits wall modifications usually associated with activation of defence responses to pathogens. It is proposed that this locus must be involved in a key event in symbiotic infection processes in P. sativum, and the possible role of complex regulatory interactions between symbiosis and defence genes in endomycorrhiza development is discussed.Abbreviations DAPI 4,6-diamino-2-phenylindole - FDA fluo-rescein diacetate - PATAg periodic acid-thiocarbohydrazide-silver proteinate The authors are grateful to C. Arnould for technical assistance, K. Niehaus for the purified Sirofluor, K. Roberts for the AFRC JIM5 antibody and J. Lherminier (INRA, Dijon, France), for useful discussion. This collaborative research programme was financially supported by MRT, INRA, EPR-Bourgogne (grant to A.G., Contrat de Plan project 3060A), EEC COST ACTION 8.10 (Endomycorrhizas) and the National Research Council of Italy, Special Project RAISA, Sub-project N.2, Paper N. 801     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Compatible and Incompatible Interactions Between Callus Tissue and Mycorrhizal or Pathogenic Fungi

Tissue cultures of Nicotiana tabacum were utilized to investigate the mechanisms associated with host specificity and non-host incompatibility in mycorrhizal and pathogenic fungi. They were tested for expression of resistance to different species of mycorrhizal fungi and to a fungal pathogen of tobacco, Thielaviopsis basicola , by monitoring the production of callose, phenolic compounds and peroxidases in dual cultures. Tobacco cells reacted to the presence of all the mycorrhizal fungi with callose deposits, whereas callose was nearly always absent in tobacco cells inoculated with their pathogen T. basicola. The broad-host range ectomycorrhizal fungi Hebeloma crustuliniforme, Lac-caria laccata and Suilhis granulatus elicited less intense responses than did Hymenoscyphus ericae. The results obtained for phenolic production and peroxidase activity were consistently similar to those obtained for callose deposition. They showed that H. ericae , an endomycorrhizal symbiont of Ericaceae, was highly incompatible with tobacco cells and that the tobacco pathogen T. basicola did not elicit strong reactions in the cells of its host. In this paper, the possibility of utilizing callus cultures as a simple model system to study both the different degrees of compatibility and the early events of recognition between mycorrhizal fungi and their host or non-host plants is discussed.     

COVID-19 plasma proteome reveals novel temporal and cell-specific signatures for disease severity and high-precision disease management

Coronavirus disease 2019 (COVID-19) is a systemic inflammatory condition with high mortality that may benefit from personalized medicine and high-precision approaches. COVID-19 patient plasma was analysed with targeted proteomics of 1161 proteins. Patients were monitored from Days 1 to 10 of their intensive care unit (ICU) stay. Age- and gender-matched COVID-19-negative sepsis ICU patients and healthy subjects were examined as controls. Proteomic data were resolved using both cell-specific annotation and deep-analysis for functional enrichment. COVID-19 caused extensive remodelling of the plasma microenvironment associated with a relative immunosuppressive milieu between ICU Days 3–7, and characterized by extensive organ damage. COVID-19 resulted in (1) reduced antigen presentation and B/T-cell function, (2) increased repurposed neutrophils and M1-type macrophages, (3) relatively immature or disrupted endothelia and fibroblasts with a defined secretome, and (4) reactive myeloid lines. Extracellular matrix changes identified in COVID-19 plasma could represent impaired immune cell homing and programmed cell death. The major functional modules disrupted in COVID-19 were exaggerated in patients with fatal outcome. Taken together, these findings provide systems-level insight into the mechanisms of COVID-19 inflammation and identify potential prognostic biomarkers. Therapeutic strategies could be tailored to the immune response of severely ill patients.     

Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

IL28B Gene Polymorphisms and US Liver Fatty Changes in Patients Who Spontaneously Cleared Hepatitis C Virus Infection

Background

Recent clinical studies have shown that the presence of CC genotype in the rs12979860 region of IL28B gene is associated with an increase in the probability of spontaneous clearance of hepatitis C virus (HCV). Moreover, IL28B polymorphism seems to influence the probability of developing liver steatosis in chronic HCV patients.

Aims

The aims of our clinical study were 1) to verify the distribution of IL28B genotypes (CC, CT or TT) among subjects with spontaneous clearance of HCV infection and 2) to examine the correlation between IL28B polymorphism and hepatic steatosis among these subjects.

Methods and patients

We enrolled 41 subjects with spontaneous resolution of HCV infection (detectable serum anti-HCV but undetectable HCV-RNA) and 134 healthy controls from the same geographical area. The IL28B single-nucleotide polymorphism (SNP) rs12979860 was genotyped by using a Pyrosequencing™ technique. The presence of steatosis was assessed by liver biopsy or ultrasound examination in the 41 study subjects.

Results

CC, CT and TT-genotypes of the SNP rs1979860 were found in 66%, 24% and 10% of the subjects who spontaneously cleared HCV and in 31%, 54% and 15% of controls, respectively (p = 0.0003). Among the study subjects, females with CC-genotype were significantly more represented (p = 0.02). Hepatic steatosis did not correlate with IL28B genotype (p = 0,14) but only with a high body mass index (BMI) value (p = 0.03).

Conclusions

Female subjects carrying IL28B CC-genotype are significantly more represented among Italian patients who spontaneously cleared HCV infection. In addition, among these subjects, the presence of liver steatosis does not correlate with IL28B genotype but is solely related to the occurrence of high BMI. Thus, the association between IL28B polymorphism and steatosis in chronic HCV patients requires the presence of active HCV replication to occur, while in subjects who have cleared the infection, the mechanism(s) inducing liver steatosis are independent from IL28B profile.     

Lipase production by Aspergillus ibericus using olive mill wastewater

Olive mill wastewater (OMW) characteristics make it a suitable resource to be used as a microbial culture media to produce value-added compounds, such as enzymes. In this work, the ability of the novel species Aspergillus ibericus to discolor OMW and produce lipase was studied. An initial screening on plates containing an OMW-based agar medium and an emulsified olive oil/rhodamine-B agar medium was employed to select the strain A. ibericus MUM 03.49. Then, experiments in conical flasks with liquid OMW-based media showed that the fungus could growth on undiluted OMW, with a chemical oxygen demand (COD) of 97 ± 2 g/L, and to produce up to 2,927 ± 54 U/L of lipase. When pure OMW was used in the media, the maximum COD and color reduction achieved were 45 and 97 %, respectively. When OMW diluted to 10 % was used, A. ibericus was able to reduce phenolic and aromatic compounds by 37 and 39 %, respectively. Additionally, lipase production was found to be promoted by the addition of mineral nutrients. When the fermentations were scaled up to a 2-L bioreactor, A. ibericus produced up to 8,319 ± 33 U/L of lipase, and the maximum COD and color reduction were 57 and 24 %, respectively.