Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research.
Serine proteases are extensively known to play key roles in many physiological processes. However, their dysregulation is often associated to several diseases including inflammatory bowel diseases (IBD). Here, we used specific substrates to monitor fecal protease activities in a large cohort of healthy and IBD patients. Of interest, serine protease activity was 10-fold higher in IBD fecal samples compared to healthy controls. Moreover, functional analysis of these fecal proteolytic activities revealed that the most increased activities are trypsin-like, elastase-like and cathepsin G-like. We also show for the first time, an increase of proteinase 3-like activity in these samples compared to controls. Results presented here will guide further investigations to better understand the relevance of these peptidases in IBD.
Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor β2 (RARβ2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and 9 normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RARβ2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RARβ2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RARβ2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RARβ2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis.To assess the functional significance of the epigenetic silencing of RARβ2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RARβ2 through aberrant methylation (p = 0.003) in NPC biopsies.We concluded that the inactivation of RASSF1A, RARβ2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.
About 29 olive (Olea europaea L.) cultivars including oil and table olive cultivars originating from Tunisia and other Mediterranean countries, were genotyped using amplified fragment length polymorphism (AFLP) DNA markers. This technique is a rapid and efficient method for producing DNA fingerprints. Using nine AFLP primer combinations, we produced a total of 410 AFLP markers, among which 172 revealed polymorphism. The results demonstrated a high degree of polymorphism in the olive germplasm we examined with an average of 39%. These AFLP markers were analyzed to estimate genetic distances between pairs of cultivars using Jaccard's similarity coefficient. Furthermore, cluster and principal component analyses were performed in order to identify the genetic variation patterns. Two main groups were obtained: one comprising primarily small-fruited cultivars grown mainly for oil production and the other comprising large fruited cultivars (regardless of their end-use). Our results show no evidence of clustering of olive cultivars according to their geographic origin.
Our preliminary findings suggested that aberrant methylation of RASSF1A and RARbeta2 occurs frequently in Tunisian breast cancer patients compared with others. Furthermore, RASSF1A hypermethylation could be used as a potential marker of poor prognosis.
The TP53 gene, frequently mutated in human cancers, carries several polymorphisms. The one most informative and studied concerns codon 72; a single base changes the CGC (arginine) to CCC (proline). The arginine form was considered to be a significant risk factor in the development of cancer. However, various reports on this polymorphism are controversial. We carried out the same investigation in two groups of patients, a group with bladder cancer and another with breast cancer, and in healthy controls in two regions of our country, using an improved PCR-RFLP method. The number of Arg/Arg, Arg/Pro, and Pro/Pro genotypes was as follows: 21, 23, 3 and 13, 19, 2 for patients (total 47) and controls (34), respectively, in the first group; 18, 9, 3 and 19, 26, 4 for patients (30) and controls (49), respectively, in the second group. Statistical analysis of the genotype and allele frequencies did not reveal any difference between patients and controls in both groups except for a weak difference between the homozygotes to heterozygotes in the second group with a chi square of 4.1 (P = 0.045); the number of breast cancer patients is actually low (30) and should be increased in order to assess such a conclusion. Our overall results are therefore not consistent with a high risk associated with TP53 codon 72 polymorphism in breast and in bladder cancers.
p53 over expression in yeast results in cell death with typical markers of apoptosis such as DNA fragmentation and phosphatidylserine externalization. We aimed to substitute/supplement classical fluorescent techniques (TUNEL, Annexin V, ROS detection) usually used to detect biochemical changes occurring during yeast apoptosis mediated by p53 over expression and the effect of anti-apoptotic purified molecules from Nigel (Nigella sativa) extracts on these same yeasts by the label free technique of FTIR spectroscopy. The comparison of the entire IR spectra highlighted clear modifications between apoptotic p53-expressing yeasts and normal ones. More precisely, DNA damage was detected by the decrease of band intensities at 1079 and 1048 cm-1. While phosphatidylserine exposure was followed by the increase of νsCH2 and νasCH2 bands of unsaturated fatty acids that were exhibited at 2855 and 2926 cm-1, and the appearance of the C = O ester functional group band at 1740 cm-1. In a second step, this FTIR approach was used to estimate the effect of a purified fraction of the Nigel extract. The modulation of band intensities specific to DNA and membrane status was in agreement with apoptosis supression in presence of the Nigel extracts. FTIR spectroscopy is thus proven to be a very reliable technique to monitor the apoptotic cell death in yeast and to be used as a means of evaluating the biomolecules effect on yeast survival.
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