DNA-binding site II is required for RAD51 recombinogenic activity in Arabidopsis thaliana.

Homologous recombination is a major pathway for the repair of DNA double strand breaks, essential both to maintain genomic integrity and to generate genetic diversity. Mechanistically, homologous recombination involves the use of a homologous DNA molecule as a template to repair the break. In eukaryotes, the search for and invasion of the homologous DNA molecule is carried out by two recombinases, RAD51 in somatic cells and RAD51 and DMC1 in meiotic cells. During recombination, the recombinases bind overhanging single-stranded DNA ends to form a nucleoprotein filament, which is the active species in promoting DNA invasion and strand exchange. RAD51 and DMC1 carry two major DNA-binding sites-essential for nucleofilament formation and DNA strand exchange, respectively. Here, we show that the function of RAD51 DNA-binding site II is conserved in the plant, Arabidopsis. Mutation of three key amino acids in site II does not affect RAD51 nucleofilament formation but inhibits its recombinogenic activity, analogous to results from studies of the yeast and human proteins. We further confirm that recombinogenic function of RAD51 DNA-binding site II is not required for meiotic double-strand break repair when DMC1 is present. The Arabidopsis AtRAD51-II3A separation of function mutant shows a dominant negative phenotype, pointing to distinct biochemical properties of eukaryotic RAD51 proteins.

Acceptability and feasibility of home and hospital follow-up in Burkina Faso and Guinea: A mixed-method study among patients of the COVID-19 Coverage-Africa clinical trial.

Patient experiences and perspectives on trial participation and follow-up may influence their compliance with research procedures or negatively impact their well-being. We aimed to explore the acceptability and feasibility of home-based and hospital-based follow-up modalities among COVID-19 patients enrolled in the ANTICOV ANRS COV33 Coverage-Africa trial in Burkina Faso and Guinea. The trial (2021-2022) evaluated the efficacy of treatments to prevent clinical worsening among COVID-19 patients with mild to moderate symptoms. Patients were either based at home or hospitalized, as per national recommendations, and followed-up through face-to-face visits and phone calls. We conducted a mixed-methods sub-study administering a questionnaire to all consenting participants and individually interviewing purposively selected participants. We performed descriptive analyses of Likert scale questions for the questionnaires and thematic analysis for the interviews. We conducted framework analysis and interpretation. Of the 400 trial patients, 220 completed the questionnaire (n = 182 in Burkina Faso, n = 38 in Guinea) and 24 were interviewed (n = 16 and n = 8, respectively). Participants were mostly followed-up at home in Burkina Faso; all patients from Guinea were first hospitalized, then followed-up at home. Over 90% of participants were satisfied with follow-up. Home follow-up was considered acceptable if (i) participants perceived they were not severely ill, (ii) it was combined with telemedicine, and (iii) the risk of stigma could be avoided. Hospital-based follow-up was viewed as a way to prevent contamination of family members, but could be badly experienced when mandatory and conflicting with family responsibilities and commitments. Phone calls were seen as reassuring and as a way to ensure continuity of care. These overall positive findings support the development of home-based follow-up for mildly ill patients in West-Africa, provided that both emotional and cognitive factors at individual, familial/inter-relational, healthcare and national levels be addressed when planning the implementation of a trial, or developing any public health strategy.

The plant-specific DDR factor SOG1 increases chromatin mobility in response to DNA damage.

Homologous recombination (HR) is a conservative DNA repair pathway in which intact homologous sequences are used as a template for repair. How the homology search happens in the crowded space of the cell nucleus is, however, still poorly understood. Here, we measure chromosome and double-strand break (DSB) site mobility in Arabidopsis thaliana, using lacO/LacI lines and two GFP-tagged HR reporters. We observe an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. This increase in mobility is lost in the sog1-1 mutant, a central transcription factor of the DNA damage response in plants. Also, DSB sites show particularly high mobility levels and their enhanced mobility requires the HR factor RAD54. Our data suggest that repair mechanisms promote chromatin mobility upon DNA damage, implying a role of this process in the early steps of the DNA damage response.

DMC1 attenuates RAD51-mediated recombination in Arabidopsis.

Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.

Rewiring Meiosis for Crop Improvement.

Meiosis is a specialized cell division that contributes to halve the genome content and reshuffle allelic combinations between generations in sexually reproducing eukaryotes. During meiosis, a large number of programmed DNA double-strand breaks (DSBs) are formed throughout the genome. Repair of meiotic DSBs facilitates the pairing of homologs and forms crossovers which are the reciprocal exchange of genetic information between chromosomes. Meiotic recombination also influences centromere organization and is essential for proper chromosome segregation. Accordingly, meiotic recombination drives genome evolution and is a powerful tool for breeders to create new varieties important to food security. Modifying meiotic recombination has the potential to accelerate plant breeding but it can also have detrimental effects on plant performance by breaking beneficial genetic linkages. Therefore, it is essential to gain a better understanding of these processes in order to develop novel strategies to facilitate plant breeding. Recent progress in targeted recombination technologies, chromosome engineering, and an increasing knowledge in the control of meiotic chromosome segregation has significantly increased our ability to manipulate meiosis. In this review, we summarize the latest findings and technologies on meiosis in plants. We also highlight recent attempts and future directions to manipulate crossover events and control the meiotic division process in a breeding perspective.

RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis.

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates the homology search and strand invasion activities of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.

DNA polymerase epsilon is required for heterochromatin maintenance in Arabidopsis.

BACKGROUND: Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. RESULTS: We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. CONCLUSION: These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

SPO11.2 is essential for programmed double-strand break formation during meiosis in bread wheat (Triticum aestivum L.).

Meiotic recombination is initiated by formation of DNA double-strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11-1 and SPO11-2. We analysed the sequences of SPO11-2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11-2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon-2 of the A copy lead to a premature stop codon and suggest that it encodes a non-functional protein. Remarkably, the mutation was absent from the diploid A-relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11-2 in meiotic recombination in wheat. In accordance with its 2-nucleotide deletion in exon-2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.

Bread wheat TaSPO11-1 exhibits evolutionarily conserved function in meiotic recombination across distant plant species.

The manipulation of meiotic recombination in crops is essential to develop new plant varieties rapidly, helping to produce more cultivars in a sustainable manner. One option is to control the formation and repair of the meiosis-specific DNA double-strand breaks (DSBs) that initiate recombination between the homologous chromosomes and ultimately lead to crossovers. These DSBs are introduced by the evolutionarily conserved topoisomerase-like protein SPO11 and associated proteins. Here, we characterized the homoeologous copies of the SPO11-1 protein in hexaploid bread wheat (Triticum aestivum). The genome contains three SPO11-1 gene copies that exhibit 93-95% identity at the nucleotide level, and clearly the A and D copies originated from the diploid ancestors Triticum urartu and Aegilops tauschii, respectively. Furthermore, phylogenetic analysis of 105 plant genomes revealed a clear partitioning between monocots and dicots, with the seven main motifs being almost fully conserved, even between clades. The functional similarity of the proteins among monocots was confirmed through complementation analysis of the Oryza sativa (rice) spo11-1 mutant by the wheat TaSPO11-1-5D coding sequence. Also, remarkably, although the wheat and Arabidopsis SPO11-1 proteins share only 55% identity and the partner proteins also differ, the TaSPO11-1-5D cDNA significantly restored the fertility of the Arabidopsis spo11-1 mutant, indicating a robust functional conservation of the SPO11-1 protein activity across distant plants. These successful heterologous complementation assays, using both Arabidopsis and rice hosts, are good surrogates to validate the functionality of candidate genes and cDNA, as well as variant constructs, when the transformation and mutant production in wheat is much longer and more tedious.

The Linker Histone GH1-HMGA1 Is Involved in Telomere Stability and DNA Damage Repair.

Despite intensive searches, few proteins involved in telomere homeostasis have been identified in plants. Here, we used pull-down assays to identify potential telomeric interactors in the model plant species Arabidopsis (Arabidopsis thaliana). We identified the candidate protein GH1-HMGA1 (also known as HON4), an uncharacterized linker histone protein of the High Mobility Group Protein A (HMGA) family in plants. HMGAs are architectural transcription factors and have been suggested to function in DNA damage repair, but their precise biological roles remain unclear. Here, we show that GH1-HMGA1 is required for efficient DNA damage repair and telomere integrity in Arabidopsis. GH1-HMGA1 mutants exhibit developmental and growth defects, accompanied by ploidy defects, increased telomere dysfunction-induced foci, mitotic anaphase bridges, and degraded telomeres. Furthermore, mutants have a higher sensitivity to genotoxic agents such as mitomycin C and γ-irradiation. Our work also suggests that GH1-HMGA1 is involved directly in the repair process by allowing the completion of homologous recombination.

Analysis of the impact of the absence of RAD51 strand exchange activity in Arabidopsis meiosis.

The ploidy of eukaryote gametes must be halved to avoid doubling of numbers of chromosomes with each generation and this is carried out by meiosis, a specialized cell division in which a single chromosomal replication phase is followed by two successive nuclear divisions. With some exceptions, programmed recombination ensures the proper pairing and distribution of homologous pairs of chromosomes in meiosis and recombination defects thus lead to sterility. Two highly related recombinases are required to catalyse the key strand-invasion step of meiotic recombination and it is the meiosis-specific DMC1 which is generally believed to catalyse the essential non-sister chromatid crossing-over, with RAD51 catalysing sister-chromatid and non-cross-over events. Recent work in yeast and plants has however shown that in the absence of RAD51 strand-exchange activity, DMC1 is able to repair all meiotic DNA breaks and surprisingly, that this does not appear to affect numbers of meiotic cross-overs. In this work we confirm and extend this conclusion. Given that more than 95% of meiotic homologous recombination in Arabidopsis does not result in inter-homologue crossovers, Arabidopsis is a particularly sensitive model for testing the relative importance of the two proteins-even minor effects on the non-crossover event population should produce detectable effects on crossing-over. Although the presence of RAD51 protein provides essential support for the action of DMC1, our results show no significant effect of the absence of RAD51 strand-exchange activity on meiotic crossing-over rates or patterns in different chromosomal regions or across the whole genome of Arabidopsis, strongly supporting the argument that DMC1 catalyses repair of all meiotic DNA breaks, not only non-sister cross-overs.

Augmented reality in gynecologic surgery: evaluation of potential benefits for myomectomy in an experimental uterine model.

BACKGROUND: Augmented Reality (AR) is a technology that can allow a surgeon to see subsurface structures. This works by overlaying information from another modality, such as MRI and fusing it in real time with the endoscopic images. AR has never been developed for a very mobile organ like the uterus and has never been performed for gynecology. Myomas are not always easy to localize in laparoscopic surgery when they do not significantly change the surface of the uterus, or are at multiple locations. OBJECTIVE: To study the accuracy of myoma localization using a new AR system compared to MRI-only localization. METHODS: Ten residents were asked to localize six myomas (on a uterine model into a laparoscopic box) when either using AR or in conditions that simulate a standard method (only the MRI was available). Myomas were randomly divided in two groups: the control group (MRI only, AR not activated) and the AR group (AR activated). Software was used to automatically measure the distance between the point of contact on the uterine surface and the myoma. We compared these distances to the true shortest distance to obtain accuracy measures. The time taken to perform the task was measured, and an assessment of the complexity was performed. RESULTS: The mean accuracy in the control group was 16.80 mm [0.1-52.2] versus 0.64 mm [0.01-4.71] with AR. In the control group, the mean time to perform the task was 18.68 [6.4-47.1] s compared to 19.6 [3.9-77.5] s with AR. The mean score of difficulty (evaluated for each myoma) was 2.36 [1-4] versus 0.87 [0-4], respectively, for the control and the AR group. DISCUSSION: We developed an AR system for a very mobile organ. This is the first user study to quantitatively evaluate an AR system for improving a surgical task. In our model, AR improves localization accuracy.

The Structure-Specific Endonucleases MUS81 and SEND1 Are Essential for Telomere Stability in Arabidopsis.

Structure-specific endonucleases act to repair potentially toxic structures produced by recombination and DNA replication, ensuring proper segregation of the genetic material to daughter cells during mitosis and meiosis. Arabidopsis thaliana has two putative homologs of the resolvase (structure-specific endonuclease): GEN1/Yen1. Knockout of resolvase genes GEN1 and SEND1, individually or together, has no detectable effect on growth, fertility, or sensitivity to DNA damage. However, combined absence of the endonucleases MUS81 and SEND1 results in severe developmental defects, spontaneous cell death, and genome instability. A similar effect is not seen in mus81 gen1 plants, which develop normally and are fertile. Absence of RAD51 does not rescue mus81 send1, pointing to roles of these proteins in DNA replication rather than DNA break repair. The enrichment of S-phase histone γ-H2AX foci and a striking loss of telomeric DNA in mus81 send1 further support this interpretation. SEND1 has at most a minor role in resolution of the Holliday junction but acts as an essential backup to MUS81 for resolution of toxic replication structures to ensure genome stability and to maintain telomere integrity.

Centromere Associations in Meiotic Chromosome Pairing.

Production of gametes of halved ploidy for sexual reproduction requires a specialized cell division called meiosis. The fusion of two gametes restores the original ploidy in the new generation, and meiosis thus stabilizes ploidy across generations. To ensure balanced distribution of chromosomes, pairs of homologous chromosomes (homologs) must recognize each other and pair in the first meiotic division. Recombination plays a key role in this in most studied species, but it is not the only actor and particular chromosomal regions are known to facilitate the meiotic pairing of homologs. In this review, we focus on the roles of centromeres and in particular on the clustering and pairwise associations of nonhomologous centromeres that precede stable pairing between homologs. Although details vary from species to species, it is becoming increasingly clear that these associations play active roles in the meiotic chromosome pairing process, analogous to those of the telomere bouquet.

Fat-containing lesions of the liver: a pictorial essay.

The presence of fat within a hepatic lesion is unusual and can help to direct the radiologist's diagnosis. The aim of this iconographic review is to specify the various hepatic lesions that may contain fat and their appearance particularly on MRI. A histological correlation is also suggested for the most commonly found tumors. The identification of fat within a hepatic tumor, along with other radiological signs and reflection on the clinical and epidemiological context, can lead to a diagnosis being reached or suggested, with confirmation if necessary, by a pathological examination.

Hepatic capsular retraction: spectrum of diagnosis at MRI.

Hepatic capsular retraction is an imaging feature that deserves the attention of the radiologist. Hepatic capsular retraction is associated with a number of hepatic lesions, benign or malignant, treated or untreated. The purpose of this pictorial review is to discuss the most common benign and malignant hepatic lesions associated with this feature with an emphasis on magnetic resonance imaging (MRI).

Resection of pancreatic ductal adenocarcinoma with synchronous distant metastasis: is it worthwhile?

BACKGROUND: The purpose of this study is to report prolonged survival in patients with metastatic pancreatic ductal adenocarcinoma (PDAC) managed by chemotherapy and surgery. METHODS: Between January 2009 and August 2013, 284 patients with metastatic PDAC were managed in our oncologic department. Among them, three (1%) with a single metastasis (liver in two cases and interaorticaval in one case) underwent one- or two-stage surgical resection of the metastasis and the main tumor. Perioperative data were recorded retrospectively, including disease-free and overall survival. RESULTS: The three patients had chemotherapy (FOLFOX or FOLFIRINOX regimen) with objective response or stable disease prior to surgery. Median time between chemotherapy and surgery was 9 (8 to 15) months. Resection consisted in pancreaticoduodenectomy in the three cases. None of the patients had grade III/IV postoperative complications, and median hospital stay was 12 (12 to 22) days. All the patients had postoperative chemotherapy. Only one patient experienced recurrence 11 months after surgery and died after 32.5 months. The two other patients were alive with no recurrence 26.3 and 24.7 months after initial treatment. CONCLUSION: Radical resection of PDAC with single distant metastases can offer prolonged survival with low morbidity after accurate selection by neoadjuvant chemotherapy.

CT imaging of peritoneal carcinomatosis and its mimics.

Invasive peritoneal disease includes more than just peritoneal carcinomatosis. Although this is the most common aetiology, especially when a primary is found, other conditions may be responsible for peritoneal invasion. A rigorous analysis of CT features taken together with the clinical and biological context usually allows the main differential diagnoses, which entail different types of management, to be drawn out. Pseudomyxoma peritonei, peritoneal lymphomatosis, tuberculosis, peritoneal mesothelioma, diffuse peritoneal leiomyomatosis, and benign splenosis are the main differential diagnoses.