The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair.

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.

A Feshbach resonance in collisions between triplet ground-state molecules.

Collisional resonances are important tools that have been used to modify interactions in ultracold gases, for realizing previously unknown Hamiltonians in quantum simulations1, for creating molecules from atomic gases2 and for controlling chemical reactions. So far, such resonances have been observed for atom-atom collisions, atom-molecule collisions3-7 and collisions between Feshbach molecules, which are very weakly bound8-10. Whether such resonances exist for ultracold ground-state molecules has been debated owing to the possibly high density of states and/or rapid decay of the resonant complex11-15. Here we report a very pronounced and narrow (25 mG) Feshbach resonance in collisions between two triplet ground-state NaLi molecules. This molecular Feshbach resonance has two special characteristics. First, the collisional loss rate is enhanced by more than two orders of magnitude above the background loss rate, which is saturated at the p-wave universal value, owing to strong chemical reactivity. Second, the resonance is located at a magnetic field where two open channels become nearly degenerate. This implies that the intermediate complex predominantly decays to the second open channel. We describe the resonant loss feature using a model with coupled modes that is analogous to a Fabry-Pérot cavity. Our observations provide strong evidence for the existence of long-lived coherent intermediate complexes even in systems without reaction barriers and open up the possibility of coherent control of chemical reactions.

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts.

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

Collisional cooling of ultracold molecules.

Since the original work on Bose-Einstein condensation1,2, the use of quantum degenerate gases of atoms has enabled the quantum emulation of important systems in condensed matter and nuclear physics, as well as the study of many-body states that have no analogue in other fields of physics3. Ultracold molecules in the micro- and nanokelvin regimes are expected to bring powerful capabilities to quantum emulation4 and quantum computing5, owing to their rich internal degrees of freedom compared to atoms, and to facilitate precision measurement and the study of quantum chemistry6. Quantum gases of ultracold atoms can be created using collision-based cooling schemes such as evaporative cooling, but thermalization and collisional cooling have not yet been realized for ultracold molecules. Other techniques, such as the use of supersonic jets and cryogenic buffer gases, have reached temperatures limited to above 10 millikelvin7,8. Here we show cooling of NaLi molecules to micro- and nanokelvin temperatures through collisions with ultracold Na atoms, with both molecules and atoms prepared in their stretched hyperfine spin states. We find a lower bound on the ratio of elastic to inelastic molecule-atom collisions that is greater than 50-large enough to support sustained collisional cooling. By employing two stages of evaporation, we increase the phase-space density of the molecules by a factor of 20, achieving temperatures as low as 220 nanokelvin. The favourable collisional properties of the Na-NaLi system could enable the creation of deeply quantum degenerate dipolar molecules and raises the possibility of using stretched spin states in the cooling of other molecules.

An enhancer trap system to track developmental dynamics in Marchantia polymorpha.

A combination of streamlined genetics, experimental tractability and relative morphological simplicity compared to vascular plants makes the liverwort Marchantia polymorpha an ideal model system for studying many aspects of plant biology. Here we describe a transformation vector combining a constitutive fluorescent membrane marker with a nuclear marker that is regulated by nearby enhancer elements and use this to produce a library of enhancer trap lines for Marchantia. Screening gemmae from these lines allowed the identification and characterization of novel marker lines, including markers for rhizoids and oil cells. The library allowed the identification of a margin tissue running around the thallus edge, highlighted during thallus development. The expression of this marker is correlated with auxin levels. We generated multiple markers for the meristematic apical notch region, which have different spatial expression patterns, reappear at different times during meristem regeneration following apical notch excision and have varying responses to auxin supplementation or inhibition. This reveals that there are proximodistal substructures within the apical notch that could not be observed otherwise. We employed our markers to study Marchantia sporeling development, observing meristem emergence as defining the protonema-to-prothallus stage transition, and subsequent production of margin tissue during the prothallus stage. Exogenous auxin treatment stalls meristem emergence at the protonema stage but does not inhibit cell division, resulting in callus-like sporelings with many rhizoids, whereas pharmacologically inhibiting auxin synthesis and transport does not prevent meristem emergence. This enhancer trap system presents a useful resource for the community and will contribute to future Marchantia research.

An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology.

Fgf8 promotes survival of nephron progenitors by regulating BAX/BAK-mediated apoptosis.

Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK/BAX and for subsequent steps involving, as yet, undefined roles in kidney development.

The International Society of Differentiation: Past, present, and future.

The International Society of Differentiation was born from the First International Conference on Cell Differentiation conceived by D.V. and held in Nice, France in 1971. The conference also resulted in the creation of the journal of the Society named Differentiation. The Society advocates for the field of differentiation through the journal Differentiation, organizing and supporting international scientific conferences, honoring scientific achievements, and supporting trainees.

Genetic Variation in Reproductive Investment Across an Ephemerality Gradient in Daphnia pulex.

Species across the tree of life can switch between asexual and sexual reproduction. In facultatively sexual species, the ability to switch between reproductive modes is often environmentally dependent and subject to local adaptation. However, the ecological and evolutionary factors that influence the maintenance and turnover of polymorphism associated with facultative sex remain unclear. We studied the ecological and evolutionary dynamics of reproductive investment in the facultatively sexual model species, Daphnia pulex. We found that patterns of clonal diversity, but not genetic diversity varied among ponds consistent with the predicted relationship between ephemerality and clonal structure. Reconstruction of a multi-year pedigree demonstrated the coexistence of clones that differ in their investment into male production. Mapping of quantitative variation in male production using lab-generated and field-collected individuals identified multiple putative quantitative trait loci (QTL) underlying this trait, and we identified a plausible candidate gene. The evolutionary history of these QTL suggests that they are relatively young, and male limitation in this system is a rapidly evolving trait. Our work highlights the dynamic nature of the genetic structure and composition of facultative sex across space and time and suggests that quantitative genetic variation in reproductive strategy can undergo rapid evolutionary turnover.

A stripe phase with supersolid properties in spin-orbit-coupled Bose-Einstein condensates.

Supersolidity combines superfluid flow with long-range spatial periodicity of solids, two properties that are often mutually exclusive. The original discussion of quantum crystals and supersolidity focused on solid 4He and triggered extensive experimental efforts that, instead of supersolidity, revealed exotic phenomena including quantum plasticity and mass supertransport. The concept of supersolidity was then generalized from quantum crystals to other superfluid systems that break continuous translational symmetry. Bose-Einstein condensates with spin-orbit coupling are predicted to possess a stripe phase with supersolid properties. Despite several recent studies of the miscibility of the spin components of such a condensate, the presence of stripes has not been detected. Here we observe the predicted density modulation of this stripe phase using Bragg reflection (which provides evidence for spontaneous long-range order in one direction) while maintaining a sharp momentum distribution (the hallmark of superfluid Bose-Einstein condensates). Our work thus establishes a system with continuous symmetry-breaking properties, associated collective excitations and superfluid behaviour.

Unique genetic signatures of local adaptation over space and time for diapause, an ecologically relevant complex trait, in Drosophila melanogaster.

Organisms living in seasonally variable environments utilize cues such as light and temperature to induce plastic responses, enabling them to exploit favorable seasons and avoid unfavorable ones. Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, we examined the genetic basis and evolutionary history of ovarian diapause. We exposed outbred females to different temperatures and day lengths, characterized ovarian development for over 2800 flies, and reconstructed their complete, phased genomes. We found that diapause, scored at two different developmental cutoffs, has modest heritability, and we identified hundreds of SNPs associated with each of the two phenotypes. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, we utilized outdoor mesocosms to track diapause under natural conditions. We found that hybrid swarms reared outdoors evolved increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. Our results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space.

A small survey of introduced Zaprionus indianus (Diptera: Drosophilidae) in orchards of the eastern United States.

The African fig fly, Zaprionus indianus (Gupta), is a generalist fruit fly that typically breeds in decaying fruits from over 70 plant species. The species has spread globally from its native range in tropical Africa, becoming an invasive pest on ripening figs in Brazil. First reported in the United States in 2005 in Florida, Z. indianus has since been documented as far north as Canada and is hypothesized to recolonize northwards from southern refugia each year. We sampled drosophilid communities over the growing season at 2 orchards in Virginia from 2020 to 2022 and 11 orchards along the East Coast during the fall of 2022 to quantify the abundance of Z. indianus relative to other drosophilids across locations, seasons, and fruit crops. Massachusetts had the northernmost population, with no Z. indianus detected in Maine and no correlation between latitude and relative abundance. Variation in Z. indianus relative abundance was high between nearby orchards and abundance was higher on peaches relative to apples within orchards. Comparisons of seasonal abundance curves between 2 Virginia orchards showed similar dynamics across years with individuals first detected around July and becoming absent around December, with peaks in late summer and mid-fall. The variation in seasonal and latitudinal abundance shown here highlights a need for broader sampling to accurately characterize the range, spread, and environmental tolerances of Z. indianus in North America.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Effect of Positioning of the Region of Interest on Bone Density of the Hip.

BACKGROUND: Large changes in positioning of the global region of interest (ROI) influence the measurement of bone mineral density (BMD) in the hip and forearm regions. However, it is unknown whether minor shifts in the positioning of the bottom of the global hip ROI affect the measurement of total hip BMD. METHODS: The hip BMDs of 40 clinical densitometry patients were analyzed at baseline with the bottom of the global hip ROI positioned as usual, 10 mm distal to the base of the lesser trochanter (position 0). Then the hip was reanalyzed by shifting the bottom of the global hip ROI 1 mm proximally 10 times (positions +1 through +10) and then by shifting the bottom of the global hip ROI 1 mm distally 10 times (positions -1 through -10). The significance of the differences between mean values at the various distances from baseline was assessed using a Wilcoxon signed-rank test. RESULTS: The mean total hip area, bone mineral content and BMD decreased as the bottom of the global hip ROI was shifted proximally; the decrease was significant when shifted by even 1 mm (p < 0.001). The mean total hip area, bone mineral content and BMD increased as the bottom of the global hip ROI was shifted distally; the increase was significant when shifted by even 1 mm (p < 0.001). The change in BMD with each 1 mm shift was uniform across the range studied from positions +10 through -10, and was approx 0.54%/mm. When the least significant change was based on 40 pairs of measurements, where each pair was comprised of the baseline scan and the same scan at -1 position, the least significant change was 0.01 g/cm2. CONCLUSIONS: The BMD of the total hip is sensitive to even minor changes in the positioning of the bottom of the global hip ROI. Although a 1 mm change in the bottom of the global hip ROI positioning would make little difference in the reported T-score, it could easily affect the determination of significance in changes in BMD over time.

The role of healthcare professionals in HPV communication with head and neck cancer patients: A narrative synthesis of qualitative studies.

INTRODUCTION: Prevalence rates of human papillomavirus positive (HPV+) head and neck cancers (HNC) have increased over the last decades. Communicating about HPV is an increasingly relevant part of HNC patient care. This systematic review was conducted to explore healthcare professionals' (HCP) views and experiences of discussing HPV with HNC patients. It also examined perceptions among different HCP groups of their professional roles in HPV discussions. METHODS: A narrative synthesis of qualitative research was conducted. Three databases-Embase, PsycINFO and CINAHL+-were searched from January 2007 to August 2018. Relevant data were extracted and synthesised thematically. RESULTS: Five studies were identified: four were qualitative and one used mixed methods. HCPs varied in their experience and views of discussing HPV. HCPs who engaged in these discussions believed they were beneficial for patients. All HCPs described the need to address their HPV knowledge deficits in order to provide clear HPV information. Changes in professional roles which were linked to HPV communication for HCPs involved in HNC patient care were also evident. CONCLUSIONS: Effective HPV discussions are an important part of patient-provider interactions. Evidence-based interventions and professional development activities which support HCPs in their HPV discussions with patients would be valuable.

A study of medication errors during the prescription stage in the pediatric critical care services of a secondary-tertiary level public hospital.

BACKGROUND: Medication Errors (MEs) are considered the most common type of error in pediatric critical care services. Moreover, the ME rate in pediatric patients is up to three times higher than the rate for adults. Nevertheless, information in pediatric population is still limited, particularly in emergency/critical care practice. The purpose of this study was to describe and analyze MEs in the pediatric critical care services during the prescription stage in a Mexican secondary-tertiary level public hospital. METHODS: A cross-sectional study to detect MEs was performed in all pediatric critical care services [pediatric emergency care (PEC), pediatric intensive care unit (PICU), neonatal intensive care unit (NICU), and neonatal intermediate care unit (NIMCU)] of a public teaching hospital. A pharmacist identified MEs by direct observation as the error detection method and MEs were classified according to the updated classification for medication errors by the Ruíz-Jarabo 2000 working group. Thereafter, these were subclassified in clinically relevant MEs. RESULTS: In 2347 prescriptions from 301 patients from all critical care services, a total of 1252 potential MEs (72%) were identified, and of these 379 were considered as clinically relevant due to their potential harm. The area with the highest number of MEs was PICU (n = 867). The ME rate was > 50% in all pediatric critical care services and PICU had the highest ME/patient index (13.1). The most frequent MEs were use of abbreviations (50.9%) and wrong speed rate of administration (11.4%), and only 11.7% of the total drugs were considered as ideal medication orders. CONCLUSION: Clinically relevant medication errors can range from mild skin reactions to severe conditions that place the patient's life at risk. The role of pharmacists through the detection and timely intervention during the prescription and other stages of the medication use process can improve drug safety in pediatric critical care services.

Mesoderm-specific Stat3 deletion affects expression of Sox9 yielding Sox9-dependent phenotypes.

To date, mutations within the coding region and translocations around the SOX9 gene both constitute the majority of genetic lesions underpinning human campomelic dysplasia (CD). While pathological coding-region mutations typically result in a non-functional SOX9 protein, little is known about what mechanism(s) controls normal SOX9 expression, and subsequently, which signaling pathways may be interrupted by alterations occurring around the SOX9 gene. Here, we report the identification of Stat3 as a key modulator of Sox9 expression in nascent cartilage and developing chondrocytes. Stat3 expression is predominant in tissues of mesodermal origin, and its conditional ablation using mesoderm-specific TCre, in vivo, causes dwarfism and skeletal defects characteristic of CD. Specifically, Stat3 loss results in the expansion of growth plate hypertrophic chondrocytes and deregulation of normal endochondral ossification in all bones examined. Conditional deletion of Stat3 with a Sox9Cre driver produces palate and tracheal irregularities similar to those described in Sox9+/- mice. Furthermore, mesodermal deletion of Stat3 causes global embryonic down regulation of Sox9 expression and function in vivo. Mechanistic experiments ex vivo suggest Stat3 can directly activate the expression of Sox9 by binding to its proximal promoter following activation. These findings illuminate a novel role for Stat3 in chondrocytes during skeletal development through modulation of a critical factor, Sox9. Importantly, they further provide the first evidence for the modulation of a gene product other than Sox9 itself which is capable of modeling pathological aspects of CD and underscore a potentially valuable therapeutic target for patients with the disorder.

Seasonally fluctuating selection can maintain polymorphism at many loci via segregation lift.

Most natural populations are affected by seasonal changes in temperature, rainfall, or resource availability. Seasonally fluctuating selection could potentially make a large contribution to maintaining genetic polymorphism in populations. However, previous theory suggests that the conditions for multilocus polymorphism are restrictive. Here, we explore a more general class of models with multilocus seasonally fluctuating selection in diploids. In these models, the multilocus genotype is mapped to fitness in two steps. The first mapping is additive across loci and accounts for the relative contributions of heterozygous and homozygous loci-that is, dominance. The second step uses a nonlinear fitness function to account for the strength of selection and epistasis. Using mathematical analysis and individual-based simulations, we show that stable polymorphism at many loci is possible if currently favored alleles are sufficiently dominant. This general mechanism, which we call "segregation lift," requires seasonal changes in dominance, a phenomenon that may arise naturally in situations with antagonistic pleiotropy and seasonal changes in the relative importance of traits for fitness. Segregation lift works best under diminishing-returns epistasis, is not affected by problems of genetic load, and is robust to differences in parameters across loci and seasons. Under segregation lift, loci can exhibit conspicuous seasonal allele-frequency fluctuations, but often fluctuations may be small and hard to detect. An important direction for future work is to formally test for segregation lift in empirical data and to quantify its contribution to maintaining genetic variation in natural populations.

How to Dress Radio-Frequency Photons with Tunable Momentum.

We demonstrate how the combination of oscillating magnetic forces and radio-frequency (rf) pulses endows rf photons with tunable momentum. We observe velocity-selective spin-flip transitions and the associated Doppler shift. Recoil-dressed photons are a promising tool for measurements and quantum simulations, including the realization of gauge potentials and spin-orbit coupling schemes which do not involve optical transitions.