Population density and group size effects on reproductive behavior in a simultaneous hermaphrodite

<p>Abstract</p> <p>Background</p> <p>Despite growing evidence that population dynamic processes can have substantial effects on mating system evolution, little is known about their effect on mating rates in simultaneous hermaphrodites. According to theory, mating rate is expected to increase with mate availability because mating activity is primarily controlled by the male sexual function. A different scenario appears plausible in the hermaphroditic opisthobranch <it>Chelidonura sandrana</it>. Here, field mating rates are close to the female fitness optimum, suggesting that mating activity remains unresponsive to variation in mate availability.</p> <p>Results</p> <p>Applying an experimental design that aims at independent experimental manipulation of density and social group size, we find substantial increases in mate encounter rate with both factors, but no statistically detectable effects on mating rate in <it>C. sandrana</it>. Instead, mating rate remained close to the earlier determined female fitness optimum.</p> <p>Conclusions</p> <p>We demonstrate that mating rate in <it>C. sandrana </it>is largely unresponsive to variation in mate availability and is maintained close to the female fitness optimum. These findings challenge the prevailing notion of male driven mating rates in simultaneous hermaphrodites and call for complementary investigations of mating rate effects on fitness through the male sexual function.</p

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP–causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1

Coordination of dual incision and repair synthesis in human nucleotide excision repair

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1-XPF and 3′ to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5′ incision by ERCC1-XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a ‘cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity

Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research

Correction of <i>XPF</i> mutant cell NER defect by microinjection of XPF-ERCC1.

<p>Primary fibroblasts from XFE progeroid patient XP51RO were fused to create homopolykaryons by treatment with inactivated Sendai virus then plated on glass coverslips. Only homopolykaryons were injected with recombinant XPF-ERCC1 protein complex (A) wild-type (B) XPF^R799W-ERCC1 (C) XPF^R153P-ERCC1. The cultures were irradiated with 10 J/m² UV-C and ³H-thymidine was added to the culture. UV-induced unscheduled DNA synthesis was detected by autoradiography. Homopolykaryons are indicated with arrows. (D) Histogram indicating the average number of radiographic grains in nuclei injected with each of the recombinant protein complexes and uninjected cells in the same sample. Error bars indicate the standard deviation. N indicates the number of nuclei analyzed in each population. P values for the comparison between injected and uninjected cells were calculated using an unpaired two-tailed Student's t-test.</p

Characteristics of <i>XPF</i> mutant cell lines.

<p>UDS unscheduled DNA synthesis</p><p>*The patient had normal levels of <i>XPF</i> transcript, suggesting one allele encodes a full-length mRNA.</p>♦<p>Mutation could not be confirmed on genomic DNA.</p><p>° Siblings.</p><p>n.d.  =  not determined</p

Characterization of XPF-YFP and XPF¹⁵³-YFP in CHO cells.

<p>(A) Western blot analysis of XPF-YFP expressed in <i>Xpf</i> mutant cells. XPF-deficient hamster cell line, UV41, was transiently transfected with wild type <i>XPF-YFP</i> or <i>XPF¹⁵³-YFP</i> and the fusion proteins were detected using an antibody against XPF or GFP. C5RO was used as positive control for the XPF blot and as a negative control for the GFP blot. UV41 cells transfected with YFP alone was used as a negative control for XPF blot and as a positive control for GFP blot. (B) Clonogenic survival of wild-type (wt), XPF-deficient CHO cell line UV41, and UV41 transfected with wild type XPF-YFP and XPF¹⁵³-YFP after UV and MMC treatment. Colonies were counted 7–10 days after treatment and results are plotted as mean 3 independent experiments. (C) Subcellular localization of wild type XPF-YFP and XPF¹⁵³-YFP after transient transfection in XPF-deficient the CHO cell line UV41 detected by fluorescence microscopy.</p

Ischaemic stroke despite antiplatelet therapy: Causes and outcomes.

BACKGROUND Ischaemic stroke may occur despite antiplatelet therapy (APT). We aimed to investigate frequency, potential causes and outcomes in patients with ischaemic stroke despite APT. METHODS In this cohort study, we enrolled patients with imaging-confirmed ischaemic stroke from the Swiss Stroke Registry (01/2014-07/2022). We determined the frequency of prior APT, assessed stroke aetiology (modified TOAST classification) and determined the association of prior APT with unfavourable functional outcome (modified Rankin Scale score 3-6) and recurrent ischaemic stroke at 3 months using regression models. RESULTS Among 53,352 patients, 27,484 (51.5%) had no prior antithrombotic treatment, 17,760 (33.3%) were on APT, 7039 (13.2%) on anticoagulation and 1069 (2.0%) were on APT + anticoagulation. In patients with a history of ischaemic stroke/TIA (n = 11,948; 22.4%), 2401 (20.1%) had no prior antithrombotic therapy, 6594 (55.2%) were on APT, 2489 (20.8%) on anticoagulation and 464 (3.9%) on APT + anticoagulation. Amongst patients with ischaemic stroke despite APT, aetiology was large artery atherosclerosis in 19.8% (n = 3416), cardiac embolism in 23.6% (n = 4059), small vessel disease in 11.7% (n = 2011), other causes in 7.4% (n = 1267), more than one cause in 6.3% (n = 1078) and unknown cause in 31.3% (n = 5388). Prior APT was not independently associated with unfavourable outcome (aOR = 1.06; 95% CI: 0.98-1.14; p = 0.135) or death (aOR = 1.10; 95% CI: 0.99-1.21; p = 0.059) at 3-months but with increased odds of recurrent stroke (6.0% vs 4.3%; aOR 1.26; 95% CI: 1.11-1.44; p < 0.001). CONCLUSIONS One-third of ischaemic strokes occurred despite APT and 20% of patients with a history of ischaemic stroke had no antithrombotic therapy when having stroke recurrence. Aetiology of breakthrough strokes despite APT is heterogeneous and these patients are at increased risk of recurrent stroke