The Arabidopsis MEI1 gene encodes a protein with five BRCT domains that is involved in meiosis-specific DNA repair events independent of SPO11-induced DSBs

Arabidopsis thaliana MEI1 was first described as a gene involved in male meiosis, encoding a short protein showing homology with a human acrosin-trypsin inhibitor. We have isolated a new allele of mei1, and shown that in both mutants male and female meiosis are affected. In both reproductive pathways, meiosis proceeds while chromosomes become fragmented, resulting in aberrant meiotic products and in a strongly reduced fertility. We have shown that the gene mutated in mei1 mutants actually encodes a protein of 972 amino acids that contains five BRCA1 C-terminus (BRCT) domains and is similar to proteins involved in the response to DNA damage and replication blocks in eukaryotes. During meiosis, recombination is initiated by the formation of DNA double strand breaks (DSBs) induced by the protein SPO11. We analysed meiotic chromosome behaviour of the mei1 mutant in a spo11 mutant background and proved that the meiotic fragmentation observed in mei1 mutants was not the consequence of defects in the repair of meiotic DSBs induced by SPO11. We also analysed the effect of mei1 on the mitotic cell cycle but could not detect any sensitivity of mei1 seedlings to DNA-damaging agents like gamma-rays or UV. Therefore, MEI1 is a BRCT-domain-containing protein that could be specific to the meiotic cell cycle and that plays a crucial role in some DNA repair events independent of SPO11 DSB recombination repair.     

Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a sphingomonas strain that degrades various polycyclic aromatic hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

Tip60 acetyltransferase activity is controlled by phosphorylation

Here we show that the phosphorylation of histone acetyltransferase Tip60, a target of human immunodeficiency virus, type 1-encoded transactivator Tat, plays a crucial role in the control of its catalytic activity. Baculovirus-based expression and purification of Tip60 combined with mass spectrometry allowed the identification of serines 86 and 90 as two major sites of phosphorylation in vivo. The phosphorylation of Tip60 was found to modulate its histone acetyltransferase activity. One of the identified phosphorylated serines, Ser-90, was within a consensus cyclin B/Cdc2 site. Ser-90 was specifically phosphorylated in vitro by the cyclin B/Cdc2 complex. Accordingly, the phosphorylation of Tip60 was enhanced after drug-induced arrest of cells in G(2)/M. This G(2)/M-dependent phosphorylation of Tip60 was abolished by treating cells with a specific inhibitor of the cyclin-dependent kinase, roscovitin. All together, these results strongly suggest a G(2)/M-dependent control of Tip60 activity.     

Dynamic instability of microtubules is regulated by force

Microtubules are long filamentous protein structures that randomly alternate between periods of elongation and shortening in a process termed dynamic instability. The average time a microtubule spends in an elongation phase, known as the catastrophe time, is regulated by the biochemical machinery of the cell throughout the cell cycle. In this light, observed changes in the catastrophe time near cellular boundaries (Brunner, D., and P. Nurse. 2000. Cell. 102:695-704; Komarova, Y.A., I.A. Vorobjev, and G.G. Borisy. 2002. J. Cell Sci. 115:3527-3539) may be attributed to regulatory effects of localized proteins. Here, we argue that the pushing force generated by a microtubule when growing against a cellular object may itself provide a regulatory mechanism of the catastrophe time. We observed an up to 20-fold, force-dependent decrease in the catastrophe time when microtubules grown from purified tubulin were polymerizing against microfabricated barriers. Comparison with catastrophe times for microtubules growing freely at different tubulin concentrations leads us to conclude that force reduces the catastrophe time only by limiting the rate of tubulin addition.     

Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation

Loss of plasma membrane asymmetry, resulting in the exposure of phosphatidylserine (PS), is considered to be an early event in apoptosis. It is generally accepted to precede nuclear condensation, independent of the apoptosis inductive agent.In the present study we focus on 2 apoptotic parameters: PS exposure in comparison with morphological alterations. Peripheral blood lymphocytes were irradiated in vitro (5 Gy Co--rays) or incubated with staurosporine (1 M, 6 hours). PS exposure was measured flow cytometrically using FITC-labelled annexin V, combined with PI. Morphological alterations were evaluated by electron microscopy (EM). Results are based on 3 independent experiments.For the irradiated lymphocytes the amount of viable cells (annexin V-/PI-) as scored by flow cytometry was comparable or slightly lower than the number of viable cells as scored by EM (75% compared to 79%). However, for the staurosporine treated lymphocytes only about 24% of the cells were designated as viable by EM, whereas by flow cytometry about 65% of the cells were annexin V-/PI-. Examination by EM showed about 40% cells with a morphology distinct from that of a normal viable cell, but without the clear-cut characteristics of apoptotic cells. Time studies revealed that these cells went into apoptosis after prolonged incubation times up to 18 hours.Application of biotinilated annexin V for EM detection with gold-conjugated anti-biotin, showed that only clear-cut apoptotic, apoptotic necrotic and oncotic cells showed the gold-label at their membranes. Cells that could be detected under the EM as non-viable but without the clear-cut characteristics of apoptotic cells, were not labelled. Data indicate that, dependent on the apoptosis inductive mechanism, morphological alterations can occur before PS exposure.     

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.     

Artificial sweeteners and cancer risk: Results from the NutriNet-Santé population-based cohort study

BackgroundThe food industry uses artificial sweeteners in a wide range of foods and beverages as alternatives to added sugars, for which deleterious effects on several chronic diseases are now well established. The safety of these food additives is debated, with conflicting findings regarding their role in the aetiology of various diseases. In particular, their carcinogenicity has been suggested by several experimental studies, but robust epidemiological evidence is lacking. Thus, our objective was to investigate the associations between artificial sweetener intakes (total from all dietary sources, and most frequently consumed ones: aspartame [E951], acesulfame-K [E950], and sucralose [E955]) and cancer risk (overall and by site).Methods and findingsOverall, 102,865 adults from the French population-based cohort NutriNet-Santé (2009–2021) were included (median follow-up time = 7.8 years). Dietary intakes and consumption of sweeteners were obtained by repeated 24-hour dietary records including brand names of industrial products. Associations between sweeteners and cancer incidence were assessed by Cox proportional hazards models, adjusted for age, sex, education, physical activity, smoking, body mass index, height, weight gain during follow-up, diabetes, family history of cancer, number of 24-hour dietary records, and baseline intakes of energy, alcohol, sodium, saturated fatty acids, fibre, sugar, fruit and vegetables, whole-grain foods, and dairy products. Compared to non-consumers, higher consumers of total artificial sweeteners (i.e., above the median exposure in consumers) had higher risk of overall cancer (n = 3,358 cases, hazard ratio [HR] = 1.13 [95% CI 1.03 to 1.25], P-trend = 0.002). In particular, aspartame (HR = 1.15 [95% CI 1.03 to 1.28], P = 0.002) and acesulfame-K (HR = 1.13 [95% CI 1.01 to 1.26], P = 0.007) were associated with increased cancer risk. Higher risks were also observed for breast cancer (n = 979 cases, HR = 1.22 [95% CI 1.01 to 1.48], P = 0.036, for aspartame) and obesity-related cancers (n = 2,023 cases, HR = 1.13 [95% CI 1.00 to 1.28], P = 0.036, for total artificial sweeteners, and HR = 1.15 [95% CI 1.01 to 1.32], P = 0.026, for aspartame). Limitations of this study include potential selection bias, residual confounding, and reverse causality, though sensitivity analyses were performed to address these concerns.ConclusionsIn this large cohort study, artificial sweeteners (especially aspartame and acesulfame-K), which are used in many food and beverage brands worldwide, were associated with increased cancer risk. These findings provide important and novel insights for the ongoing re-evaluation of food additive sweeteners by the European Food Safety Authority and other health agencies globally.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03335644","term_id":"NCT03335644"}}NCT03335644.

Charlotte Debras and colleagues investigate investigate associations between artificial sweetener intakes and cancer risk in adults from a French population-based cohort.     

Marker-assisted introgression of five QTLs controlling fruit quality traits into three tomato lines revealed interactions between QTLs and genetic backgrounds

The evaluation of organoleptic quality of tomato fruit requires physical, chemical and sensory analyses, which are expensive and difficult to assess. Therefore, their practical use in phenotypic selection is difficult. In a previous study, the genetic control of several traits related to organoleptic quality of fresh-market tomato fruit was investigated. Five chromosome regions strongly involved in organoleptic quality attributes were then chosen to be introgressed into three different recipient lines through marker-assisted selection. A marker-assisted backcross (MABC) strategy was performed, as all the favorable alleles for quality traits were provided by the same parental tomato line, whose fruit weight (FW) and firmness were much lower than those of the lines commonly used to develop fresh market varieties. Three improved lines were obtained after three backcrossing and two selfing generations. The implementation of the MABC scheme is described. The three improved lines were crossed together and with the recipient lines in a half-diallel mating scheme, and the simultaneous effect of the five quantitative trait locus (QTL) regions was compared in different genetic backgrounds. Significant effects of the introgressed regions and of the genetic backgrounds were shown. Additive effects were detected for soluble solid and reducing sugar content in two genetic backgrounds. A partially dominant effect on titratable acidity was detected in only one genetic background. In contrast, additive to dominant unfavorable effects of the donor alleles were detected for FW and locule number in the three genetic backgrounds. Recessive QTL effects on firmness were only detected in the two firmest genetic backgrounds. Comparison of the hybrids in the half-diallel gave complementary information on the effects of: (1) the alleles at the selected regions, (2) the genetic backgrounds and (3) their interaction. Breeding efficiency strongly varied according to the recipient parent, and significant interactions between QTLs and genetic backgrounds were shown for all of the traits studied.     

Pap smear quality parameters of 3 sampling devices used by auxiliary nurse midwives on symptomatic rural Indian women

OBJECTIVE: To determine whether auxiliary nurse midwives (ANMs) could make good-quality Pap smears after focused training and to determine which sampling device is most effective in their hands in field practice areas. STUDY DESIGN: In a downstaging cervical cancer screening program, 394 symptomatic rural Indian women between the ages of 35 and 60 were identified by the ANMs in 2 villages, with a total population of 14,747, and were invited to have a smear taken. Two hundred of these symptomatic women responded. The ANMs were educated to render information on screening for cervical cancer and to take smears with 3 sampling devices--Ayre spatula, modified spatula (with extended tip) and Cytobrush (Medscand AB, Malm?, Sweden). The smears were evaluated for 6 adequacy parameters. Smears made by gynecologists were used as controls. To establish the superiority of a method, chi2 tests were used. RESULTS: All smears made by the ANMs could be used to render a cytologic diagnosis. The adequacy parameters of all the smears made by ANMs at least matched those of the gynecologists. The best results were obtained with modified spatula (with extended tip) and combination of Cytobrush with modified spatula. CONCLUSION: Since only 50% of symptomatic rural women came for Pap testing, we conclude it is not easy to motivate women for such testing, even if a large-scale educational effort is made. ANMs can be taught to take reasonably good qualisuperior sampling device for ANMs is ty Pap smears. The superior sampling device for ANMs is the modified spatula (with extended tip).