Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

The past and future of global river ice.

More than one-third of Earth's landmass is drained by rivers that seasonally freeze over. Ice transforms the hydrologic1,2, ecologic3,4, climatic5 and socio-economic6-8 functions of river corridors. Although river ice extent has been shown to be declining in many regions of the world1, the seasonality, historical change and predicted future changes in river ice extent and duration have not yet been quantified globally. Previous studies of river ice, which suggested that declines in extent and duration could be attributed to warming temperatures9,10, were based on data from sparse locations. Furthermore, existing projections of future ice extent are based solely on the location of the 0-°C isotherm11. Here, using satellite observations, we show that the global extent of river ice is declining, and we project a mean decrease in seasonal ice duration of 6.10 ± 0.08 days per 1-°C increase in global mean surface air temperature. We tracked the extent of river ice using over 400,000 clear-sky Landsat images spanning 1984-2018 and observed a mean decline of 2.5 percentage points globally in the past three decades. To project future changes in river ice extent, we developed an observationally calibrated and validated model, based on temperature and season, which reduced the mean bias by 87 per cent compared with the 0-degree-Celsius isotherm approach. We applied this model to future climate projections for 2080-2100: compared with 2009-2029, the average river ice duration declines by 16.7 days under Representative Concentration Pathway (RCP) 8.5, whereas under RCP 4.5 it declines on average by 7.3 days. Our results show that, globally, river ice is measurably declining and will continue to decline linearly with projected increases in surface air temperature towards the end of this century.

Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.

RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways.

Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

Not4-dependent targeting of MMF1 mRNA to mitochondria limits its expression via ribosome pausing, Egd1 ubiquitination, Caf130, no-go-decay and autophagy.

The Ccr4-Not complex is a conserved multi protein complex with diverse roles in the mRNA life cycle. Recently we determined that the Not1 and Not4 subunits of Ccr4-Not inversely regulate mRNA solubility and thereby impact dynamics of co-translation events. One mRNA whose solubility is limited by Not4 is MMF1 encoding a mitochondrial matrix protein. In this work we uncover a mechanism that limits MMF1 overexpression and depends upon its co-translational targeting to the mitochondria. We have named this mechanism Mito-ENCay. This mechanism relies on Not4 promoting ribosome pausing during MMF1 translation, and hence the co-translational docking of the MMF1 mRNA to mitochondria via the mitochondrial targeting sequence of the Mmf1 nascent chain, the Egd1 chaperone, the Om14 mitochondrial outer membrane protein and the co-translational import machinery. Besides co-translational Mitochondrial targeting, Mito-ENCay depends upon Egd1 ubiquitination by Not4, the Caf130 subunit of the Ccr4-Not complex, the mitochondrial outer membrane protein Cis1, autophagy and no-go-decay.

The importance of hydrology in routing terrestrial carbon to the atmosphere via global streams and rivers.

SignificanceStream/river carbon dioxide (CO2) emission has significant spatial and seasonal variations critical for understanding its macroecosystem controls and plumbing of the terrestrial carbon budget. We relied on direct fluvial CO2 partial pressure measurements and seasonally varying gas transfer velocity and river network surface area estimates to resolve reach-level seasonal variations of the flux at the global scale. The percentage of terrestrial primary production (GPP) shunted into rivers that ultimately contributes to CO2 evasion increases with discharge across regions, due to a stronger response in fluvial CO2 evasion to discharge than GPP. This highlights the importance of hydrology, in particular water throughput, in terrestrial-fluvial carbon transfers and the need to account for this effect in plumbing the terrestrial carbon budget.

eIF4E3 forms an active eIF4F complex during stresses (eIF4FS) targeting mTOR and re-programs the translatome.

The eIF4E are a family of initiation factors that bind the mRNA 5' cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active during stress and that it selects specific mRNA populations based on 5' TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have 'moon-lighting' functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote 'stress resistance' and adaptation.

Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots.

Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.

Specialty pharmacy interventions benefit patients receiving HIV post-exposure prophylaxis.

BACKGROUND: Specialty pharmacies service many different complex disease states that require high-cost medication, including the treatment of patients prescribed HIV post-exposure prophylaxis (PEP). PEP requires time-sensitive initiation and patient counseling for therapeutic efficacy. OBJECTIVE: The objective of this study was to examine all PEP referrals received at a specialty pharmacy and demonstrate how they aided in interventions including assisting in obtaining financial assistance, making clinical interventions, and offering counseling to patients. METHODS: This is an observational retrospective chart review of patients who received PEP from one specialty pharmacy. All patients that filled PEP at the pharmacy between January 1st, 2017-July 1st, 2022, were included. Information was collected from documentation provided in the electronic medication record utilized by the pharmacy. The PEP regimen prescribed were raltegravir (RAL) + emtricitabine/tenofovir disoproxil fumarate (FTC/TDF) and dolutegravir (DTG) + emtricitabine/tenofovir disoproxil fumarate (FTC/TDF). RESULTS: A total of 52 patients were treated with PEP during the measurement period. Patients who received a PEP regimen of RAL + FTC/TDF experienced a total cost-savings of $1,692.60 and $218.40 for those who were fully insured and uninsured, respectively. Patients who received a PEP regimen of DTG + FTC/TDF experienced a total cost-savings of $676.20 and $2,725.50 for those who were fully insured and uninsured, respectively. Counseling by a pharmacist was offered to all patients and 74.5% of patients accepted. Pharmacists made clinical interventions on 29.4% of PEP referrals. CONCLUSION: PEP medications are expensive, time-sensitive, and can require clinical interventions and specific patient counseling. This study indicates that specialty pharmacies can provide and ensure access to care in the areas of financial assistance, patient counseling, and clinical interventions.

Regional trends and drivers of the global methane budget.

The ongoing development of the Global Carbon Project (GCP) global methane (CH4 ) budget shows a continuation of increasing CH4 emissions and CH4 accumulation in the atmosphere during 2000-2017. Here, we decompose the global budget into 19 regions (18 land and 1 oceanic) and five key source sectors to spatially attribute the observed global trends. A comparison of top-down (TD) (atmospheric and transport model-based) and bottom-up (BU) (inventory- and process model-based) CH4 emission estimates demonstrates robust temporal trends with CH4 emissions increasing in 16 of the 19 regions. Five regions-China, Southeast Asia, USA, South Asia, and Brazil-account for >40% of the global total emissions (their anthropogenic and natural sources together totaling >270 Tg CH4  yr-1 in 2008-2017). Two of these regions, China and South Asia, emit predominantly anthropogenic emissions (>75%) and together emit more than 25% of global anthropogenic emissions. China and the Middle East show the largest increases in total emission rates over the 2000 to 2017 period with regional emissions increasing by >20%. In contrast, Europe and Korea and Japan show a steady decline in CH4 emission rates, with total emissions decreasing by ~10% between 2000 and 2017. Coal mining, waste (predominantly solid waste disposal) and livestock (especially enteric fermentation) are dominant drivers of observed emissions increases while declines appear driven by a combination of waste and fossil emission reductions. As such, together these sectors present the greatest risks of further increasing the atmospheric CH4 burden and the greatest opportunities for greenhouse gas abatement.

PRMT5 protects genomic integrity during global DNA demethylation in primordial germ cells and preimplantation embryos.

Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation.

Hierarchical molecular events driven by oocyte-specific factors lead to rapid and extensive reprogramming.

Nuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.

How Have Global River Widths Changed Over Time?

Changes in a river's width reflect natural and anthropogenic impacts on local and upstream/downstream hydraulic and hydrologic processes. Temporal variation of river width also impacts biogeochemical exchange and reflects geomorphologic evolution. However, while global maps of mean river width and dynamic water surface extent exist, there is currently no standardized global assessment of river widths that documents changes over time. Therefore, we made repeated width measurements from Landsat images for all rivers wider than 90 m collected from 1984 to 2020 (named Global LOng-term river Width, GLOW), which consists of ∼1.2 billion cross-sectional river width measurements, with an average of 3,000 width measurements per 10-km reach. With GLOW, we investigated the temporal variations of global river width, quantified by the interquartile range (IQR) and temporal trend. We found that 85% of global rivers have a width IQR <150 m. We also found that 37% of global river segments show significant temporal trends in width over the past 37 years, and this number is higher (46%) for human-regulated rivers. Further, we leveraged machine learning to identify the most important factors explaining river width variations and revealed that these driving factors are significantly different between free-flowing and non-free-flowing rivers. Specifically, the most important factor driving temporal variations in river width is the climate for free-flowing rivers, and is soil condition for human-impacted rivers. Finally, we anticipate that this study and the public release of GLOW will improve the understanding of river dynamics and catalyze additional interdisciplinary studies.

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips.

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.

mTOR activates the VPS34-UVRAG complex to regulate autolysosomal tubulation and cell survival.

Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.

Sperm is epigenetically programmed to regulate gene transcription in embryos.

For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.

Global Reconstruction of Naturalized River Flows at 2.94 Million Reaches.

Spatiotemporally continuous global river discharge estimates across the full spectrum of stream orders are vital to a range of hydrologic applications, yet they remain poorly constrained. Here we present a carefully designed modeling effort (Variable Infiltration Capacity land surface model and Routing Application for Parallel computatIon of Discharge river routing model) to estimate global river discharge at very high resolutions. The precipitation forcing is from a recently published 0.1° global product that optimally merged gauge-, reanalysis-, and satellite-based data. To constrain runoff simulations, we use a set of machine learning-derived, global runoff characteristics maps (i.e., runoff at various exceedance probability percentiles) for grid-by-grid model calibration and bias correction. To support spaceborne discharge studies, the river flowlines are defined at their true geometry and location as much as possible-approximately 2.94 million vector flowlines (median length 6.8 km) and unit catchments are derived from a high-accuracy global digital elevation model at 3-arcsec resolution (~90 m), which serves as the underlying hydrography for river routing. Our 35-year daily and monthly model simulations are evaluated against over 14,000 gauges globally. Among them, 35% (64%) have a percentage bias within ±20% (±50%), and 29% (62%) have a monthly Kling-Gupta Efficiency ≥0.6 (0.2), showing data robustness at the scale the model is assessed. This reconstructed discharge record can be used as a priori information for the Surface Water and Ocean Topography satellite mission's discharge product, thus named "Global Reach-level A priori Discharge Estimates for Surface Water and Ocean Topography". It can also be used in other hydrologic applications requiring spatially explicit estimates of global river flows.