DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

Multisensory mechanisms in temporo-parietal cortex support self-location and first-person perspective

Self-consciousness has mostly been approached by philosophical enquiry and not by empirical neuroscientific study, leading to an overabundance of diverging theories and an absence of data-driven theories. Using robotic technology, we achieved specific bodily conflicts and induced predictable changes in a fundamental aspect of self-consciousness by altering where healthy subjects experienced themselves to be (self-location). Functional magnetic resonance imaging revealed that temporo-parietal junction (TPJ) activity reflected experimental changes in self-location that also depended on the first-person perspective due to visuo-tactile and visuo-vestibular conflicts. Moreover, in a large lesion analysis study of neurological patients with a well-defined state of abnormal self-location, brain damage was also localized at TPJ, providing causal evidence that TPJ encodes self-location. Our findings reveal that multisensory integration at the TPJ reflects one of the most fundamental subjective feelings of humans: the feeling of being an entity localized at a position in space and perceiving the world from this position and perspective.     

T-SPOT.TB: an in vitro diagnostic assay measuring T-cell reaction to Mycobacterium tuberculosis-specific antigens

The overall sensitivity of the RD1 ELISPOT assay (T-SPO.TB), which counts T cells sensitized to specific Mycobacterium tuberculosis peptide sequences, was 81-97% in populations with confirmed tuberculosis, in whom 39-78% had immunosuppressive conditions. In patients with confirmed tuberculosis, the respective RD1 ELISPOT versus tuberculin skin test (TST) sensitivities were 100% versus 89% in adults and 77% versus 35% in children (of whom 39% were HIV-positive). In contrast to that of the TST, the sensitivity of the RD1 ELISPOT assay was not significantly affected by age <36 months, or the immunological or nutritional status of the subjects. Specificity was 100% in two UK-based studies. Isolated false positives have been recorded in patients infected with non-tuberculous M. kansasii. A study investigating latent tuberculosis infection found no significant difference in results between HIV-positive and -negative participants for the RD1 ELISPOT assay, while the TST varied significantly with HIV status. Contact-tracing studies have demonstrated concordance between the RD1 ELISPOT assay and the TST of 65-89%. There is a significant correlation between a positive result and the degree of exposure to the index case for the RD1 ELISPOT assay, but not for the TST, in contact-tracing studies. Unlike the TST, the RD1 ELISPOT assay is not confounded by bacille Calmette-Guérin vaccination.     

Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT‐induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT‐induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1‐lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT. The lack of Mre11 nuclease activity restores wild‐type levels of reversed forks in CPT‐treated tel1Δ cells without affecting fork reversal in wild‐type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild‐type, tel1Δ, and mre11 nuclease‐deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1‐mediated reversal in the presence of CPT.     

Optimizing passive acoustic sampling of bats in forests

Passive acoustic methods are increasingly used in biodiversity research and monitoring programs because they are cost‐effective and permit the collection of large datasets. However, the accuracy of the results depends on the bioacoustic characteristics of the focal taxa and their habitat use. In particular, this applies to bats which exhibit distinct activity patterns in three‐dimensionally structured habitats such as forests. We assessed the performance of 21 acoustic sampling schemes with three temporal sampling patterns and seven sampling designs. Acoustic sampling was performed in 32 forest plots, each containing three microhabitats: forest ground, canopy, and forest gap. We compared bat activity, species richness, and sampling effort using species accumulation curves fitted with the clench equation. In addition, we estimated the sampling costs to undertake the best sampling schemes. We recorded a total of 145,433 echolocation call sequences of 16 bat species. Our results indicated that to generate the best outcome, it was necessary to sample all three microhabitats of a given forest location simultaneously throughout the entire night. Sampling only the forest gaps and the forest ground simultaneously was the second best choice and proved to be a viable alternative when the number of available detectors is limited. When assessing bat species richness at the 1‐km² scale, the implementation of these sampling schemes at three to four forest locations yielded highest labor cost‐benefit ratios but increasing equipment costs. Our study illustrates that multiple passive acoustic sampling schemes require testing based on the target taxa and habitat complexity and should be performed with reference to cost‐benefit ratios. Choosing a standardized and replicated sampling scheme is particularly important to optimize the level of precision in inventories, especially when rare or elusive species are expected.     

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.