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Marine macroalgae are a key primary producer in coastal ecosystems, but are often overlooked in blue carbon inventories. Large quantities of macroalgal detritus deposit on beaches, but the fate of wrack carbon (C) is little understood. If most of the wrack carbon is respired back to CO2, there would be no net carbon sequestration. However, if most of the wrack carbon is converted to bicarbonate (alkalinity) or refractory DOC, wrack deposition would represent net carbon sequestration if at least part of the metabolic products (e.g., reduced Fe and S) are permanently removed (i.e., long-term burial) and the DOC is not remineralised. To investigate the release of macroalgal C via porewater and its potential to contribute to C sequestration (blue carbon), we monitored the degradation of Ecklonia radiata in flow-through mesocosms simulating tidal flushing on sandy beaches. Over 60 days, 81% of added E. radiata organic matter (OM) decomposed. Per 1 mol of detritus C, the degradation produced 0.48 ± 0.34 mol C of dissolved organic carbon (DOC) (59%) and 0.25 ± 0.07 mol C of dissolved inorganic carbon (DIC) (31%) in porewater, and a small amount of CO2 (0.3 ± 0.0 mol C; ca. 3%) which was emitted to the atmosphere. A significant amount of carbonate alkalinity was found in porewater, equating to 33% (0.27 ± 0.05 mol C) of the total degraded C. The degradation occurred in two phases. In the first phase (days 0–3), 27% of the OM degraded, releasing highly reactive DOC. In the second phase (days 4–60), the labile DOC was converted to DIC. The mechanisms underlying E. radiata degradation were sulphate reduction and ammonification. It is likely that the carbonate alkalinity was primarily produced through sulphate reduction. The formation of carbonate alkalinity and semi-labile or refractory DOC from beach wrack has the potential to play an overlooked role in coastal carbon cycling and contribute to marine carbon sequestration. Graphical abstract

Nitrogen that has been recycled in the benthos supports high rates of primary and secondary production in estuaries. However, little is known about the effect of future climate on benthic nitrogen recycling and assimilation. An ex situ core incubation was used to assess the impact of combinations of warming (8°C range) and ocean acidification (OA) (i.e. increased pCO2 and decreased pH) on ammonium (NH4+) and nitrate/nitrite (NOx) fluxes and 15N-nitrate assimilation in shallow unvegetated estuarine sediments. Dissolved inorganic nitrogen (DIN = NH4+ + NOx) fluxes were significantly affected by the interaction of warming and OA, highlighting the importance of considering combined stressor treatments when investigating ecosystem responses to future climates. Warming alone increased DIN efflux from the sediments. At current mean ambient temperatures (23°C) and below (Δ-3°C), OA significantly increased DIN effluxes, but there was little to no effect of OA on DIN fluxes at warmer temperatures (Δ+3°C and Δ+5°C). OA reduced the 15N assimilation/retention of the sediments across all temperatures, suggesting that nitrogen retention in bacterial biomass was reduced, despite OA also increasing primary productivity. As such, under the projected future climate of ~3°C warming and doubling of pCO2 (~1000 µatm), unvegetated estuarine sediments are likely to have a more rapid turnover of DIN driven by greater microphytobenthos production and recycling.

Here, a new class of ester-functionalized polychalcogenophene is reported, the poly(3-alkylesterselenophene). Moreover, a series of thiophene–selenophene copolymers were prepared.

“They call it life, we call it pollution” is an infamous quote which ignores many facts about why carbon dioxide (CO2) poses a significant problem for the ocean. But before we get to this, let’s start at the beginning. All organisms on Earth require a particular set of elements for growth. In the case of plants, these elements are needed to synthesise organic matter in a process called primary production via photosynthesis, and in the case of animals, these elements are directly assimilated by either consuming plant material or by preying on other animals. In this respect, one of the key elements is carbon. Being the molecular backbone for a number of vital organic compounds such as sugars, proteins and nucleic acids (containing genetic information), carbon can be considered as the building block of life.

Climate change is expected to alter the intensity and frequency of upwelling in high productive coastal regions, thus impacting nutrient fluxes, primary productivity and consequently carbon cycling. However, it is unknown how these changes will impact the planktonic (phytoplankton and bacteria) community structure, which affects community respiration (CR) and hence the carbon available for sequestration or transfer to upper trophic levels. Here we present results from a 37-day mesocosm experiment where we examined the response of CR to nutrient additions by simulating upwelling events at different intensities (low, medium, high and extreme) and modes (singular and recurring additions). We also analysed the potential contribution of different plankton size classes and functional groups to CR. The trend in accumulated CR with respect to nutrient fertilisation (total nitrogen added during the experiment) was linear in the two modes. Microplankton (mostly diatoms) and nanoplankton (small flagellates) dominated under extreme upwelling intensities and high CR in both singular and recurring upwelling modes, explaining >65% of the observed variability in CR. In contrast, prokaryotic picoplankton (heterotrophic bacteria and autotrophic cyanobacteria) explained

Certain cellular processes are dose-dependent, requiring a specific quantity of gene products or a defined stoichiometry between them. This is exemplified by haploinsufficiency or by the need for dosage compensation for X-linked genes between the sexes in many species. Understanding dosage-sensitive processes requires the ability to perturb endogenous gene products in a quantitative manner. Here we present CasTuner, a CRISPR-based toolkit that allows analog tuning of endogenous gene expression. In the CasTuner system, activity of Cas-derived repressors is controlled through a FKBP12F36V degron domain and can thereby be quantitatively tuned by titrating the small molecule degrader dTAG-13. The toolkit can be applied at the transcriptional level, using the histone deacetylase hHDAC4 fused to dCas9, or at the post-transcriptional level, using the RNA-targeting CasRx. To optimise efficiency, inducibility and homogeneity of repression we target a fluorescently tagged endogenous gene, Esrrb, in mouse embryonic stem cells. Through flow cytometry, we show that CasTuner allows analog tuning of the target gene in a homogeneous manner across cells, as opposed to the widely used KRAB repressor domain, which exhibits a digital mode of action. We quantify repression and derepression dynamics for CasTuner and use it to measure dose-response curves between the pluripotency factor NANOG and several of its target genes, providing evidence for target-specific dose dependencies. CasTuner thus provides an easy-to-implement tool to perturb gene expression in an inducible, tunable and reversible manner and will be useful to study dose-responsive processes within their physiological context.

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene.

Earth’s Radiation Budget is partly dictated by the fragile and complex balance between biogenic volatile organic compounds (BVOCs) and greenhouse gases (GHGs), which have the potential to impose cooling or warming once emitted to the atmosphere. Whilst methane (CH4) is strictly associated with global warming due to its solar-radiation absorbing properties, dimethyl sulfide (DMS) is generally considered a cooling gas through the light scattering properties of its atmospheric oxidation products. However, DMS may also partially contribute to the Earth’s warming through a small portion of it being degraded to CH4 in the water column. Coral reefs emit both DMS and CH4 but they have not previously been simultaneously measured. Here, we report DMS and CH4 fluxes as well as aerosol particle counts at Heron Island, southern Great Barrier Reef, during the austral summer of 2016. Sea-to-air DMS and CH4 fluxes were on average 24.9 ± 1.81 and 1.36 ± 0.11 µmol m-2 d-1, whilst intermediate (< 0.5-2.5 um) and large (> 2.5 um) particle number concentrations averaged 5.51 x 106 ± 1.73 x 105 m-3 and 1.15 x 106 ± 4.63 x 104 m-3, respectively. Positive correlations were found between DMS emissions and the abundance of intermediate (R2 = 0.1669, p < 0.001, n = 93) and large (R2 = 0.0869, p = 0.004, n = 93) aerosol particles, suggesting that DMS sea-to-air emissions significantly contribute to the growth of existing particles to the measured size ranges at the Heron Island lagoon. Additionally, a strong positive correlation was found between DMS and CH4 fluxes (R2 = 0.7526, p < 0.00001, n = 93), suggesting that the emission of these volatile compounds from coral reefs is closely linked. The slope of the regression between DMS and CH4 suggests that CH4 emissions at the Heron Island lagoon represent 5% of that of DMS, which is consistent with the average sea-to-air fluxes reported in this study (i.e. 24.9 ± 1.81 µmol m-2 d-1 for DMS and 1.36 ± 0.11 for CH4). These findings provide new insights on the complexity of BVOC and GHG emissions in coral reef systems and their potential role in climate regulation.

One snapshot of the peer-review process for "Somatic XIST activation and features of X chromosome inactivation in male human cancers" (Sadagopan et al., 2022).

Pooled CRISPR screens are emerging as a powerful tool to dissect regulatory networks, by assessing how a protein responds to genetic perturbations in a highly multiplexed manner. A large number of genes are perturbed in a cell population through genomic integration of one single-guide RNA (sgRNA) per cell. A subset of cells with the phenotype of interest can then be enriched through fluorescence-activated cell sorting (FACS). SgRNAs with altered abundance after phenotypic enrichment allow identification of genes that either promote or attenuate the investigated phenotype. Here we provide detailed guidelines on how to design and execute a pooled CRISPR screen to investigate molecular phenotypes. We describe how to generate a custom sgRNA library and how to perform a FACS-based screen using readouts such as intracellular antibody staining or Flow-FISH to assess phosphorylation levels or RNA abundance. Through the variety of available perturbation systems and readout options many different molecular and cellular phenotypes can now be tackled with pooled CRISPR screens.

Oxygen minimum zones (OMZs) are characterized by enhanced carbon dioxide (CO2) levels and low pH and are being further acidified by uptake of anthropogenic atmospheric CO2. With ongoing intensification and expansion of OMZs due to global warming, carbonate chemistry conditions may become more variable and extreme, particularly in the eastern boundary upwelling systems. In austral summer (February–April) 2017, a large-scale mesocosm experiment was conducted in the coastal upwelling area off Callao (Peru) to investigate the impacts of ongoing ocean deoxygenation on biogeochemical processes, coinciding with a rare coastal El Niño event. Here we report on the temporal dynamics of carbonate chemistry in the mesocosms and surrounding Pacific waters over a continuous period of 50 d with high-temporal-resolution observations (every second day). The mesocosm experiment simulated an upwelling event in the mesocosms by addition of nitrogen (N)-deficient and CO2-enriched OMZ water. Surface water in the mesocosms was acidified by the OMZ water addition, with pHT lowered by 0.1–0.2 and pCO2 elevated to above 900 µatm. Thereafter, surface pCO2 quickly dropped to near or below the atmospheric level (405.22 µatm in 2017; Dlugokencky and Tans, 2021; NOAA/Global Monitoring Laboratory (GML)) mainly due to enhanced phytoplankton production with rapid CO2 consumption. Further observations revealed that the dominance of the dinoflagellate Akashiwo sanguinea and contamination of bird excrements played important roles in the dynamics of carbonate chemistry in the mesocosms. Compared to the simulated upwelling, natural upwelling events in the surrounding Pacific waters occurred more frequently with sea-to-air CO2 fluxes of 4.2–14.0 mmol C m−2 d−1. The positive CO2 fluxes indicated our site was a local CO2 source during our study, which may have been impacted by the coastal El Niño. However, our observations of dissolved inorganic carbon (DIC) drawdown in the mesocosms suggest that CO2 fluxes to the atmosphere can be largely dampened by biological processes. Overall, our study characterized carbonate chemistry in nearshore Pacific waters that are rarely sampled in such a temporal resolution and hence provided unique insights into the CO2 dynamics during a rare coastal El Niño event.

Leaf oysters (Isognomon ephippium) are large intertidal bivalves that form shellfish reefs. They have a patchy and restricted distribution in estuaries in northern New South Wales, Australia, where the water quality is impacted by a range of anthropogenic stressors from coastal agriculture, urbanisation, industry and recreational activities, along with natural stochastic events such as flooding. Little, however, is currently known about the tolerance of leaf oysters to poor water quality. This study investigated the condition of leaf oyster populations in four estuaries, by assessing the density, size-frequency and condition index. These biological parameters were modelled against habitat water quality parameters including nutrient loads, organic and inorganic carbon, algal biomass, sedimentation and total alkalinity. The water quality assessments were replicated during dry conditions and after rain in two estuaries. Leaf oyster density, size, and body condition significantly differed among the four estuaries. Density ranged from as few as 0.04 oysters per m

Nuclear Overhauser Effect (NOE) methods in NMR are an important tool for 3D structural analysis of small molecules. Quantitative NOE methods conventionally rely on reference distances, known distances that have to be spectrally separated and are not always available. Here we present a new method for evaluation and 3D structure selection that does not require a reference distance, instead utilizing structures optimized by molecular mechanics, enabling NOE evaluation even on molecules without suitable reference groups., A quantitative Nuclear Overhauser Effect (NOE) analysis approach that avoids the use of and internal reference distance to perform molecular configuration selection.

BACKGROUND Numerous studies investigated cell-based therapies for myocardial infarction (MI). The conflicting results of these studies have established the need for developing innovative approaches for applying cell-based therapy for MI. Experimental studies on animal models demonstrated the potential of fresh, uncultured, unmodified, autologous adipose-derived regenerative cells (UA-ADRCs) for treating acute MI. In contrast, studies on the treatment of chronic MI (CMI; > 4 wk post-MI) with UA-ADRCs have not been published so far. Among several methods for delivering cells to the myocardium, retrograde delivery into a temporarily blocked coronary vein has recently been demonstrated as an effective option. AIM To test the hypothesis that in experimentally-induced chronic myocardial infarction (CMI; > 4 wk post-MI) in pigs, retrograde delivery of fresh, uncultured, unmodified, autologous adipose-derived regenerative cells (UA-ADRCs) into a temporarily blocked coronary vein improves cardiac function and structure. METHODS The left anterior descending (LAD) coronary artery of pigs was blocked for 180 min at time point T0. Then, either 18 × 106 UA-ADRCs prepared at "point of care" or saline as control were retrogradely delivered via an over-the-wire balloon catheter placed in the temporarily blocked LAD vein 4 wk after T0 (T1). Effects of cells or saline were assessed by cardiac magnetic resonance (CMR) imaging, late gadolinium enhancement CMR imaging, and post mortem histologic analysis 10 wk after T0 (T2). RESULTS Unlike the delivery of saline, delivery of UA-ADRCs demonstrated statistically significant improvements in cardiac function and structure at T2 compared to T1 (all values given as mean ± SE): Increased mean LVEF (UA-ADRCs group: 34.3% ± 2.9% at T1 vs 40.4 ± 2.6% at T2, P = 0.037; saline group: 37.8% ± 2.6% at T1 vs 36.2% ± 2.4% at T2, P > 0.999), increased mean cardiac output (UA-ADRCs group: 2.7 ± 0.2 L/min at T1 vs 3.8 ± 0.2 L/min at T2, P = 0.002; saline group: 3.4 ± 0.3 L/min at T1 vs 3.6 ± 0.3 L/min at T2, P = 0.798), increased mean mass of the left ventricle (UA-ADRCs group: 55.3 ± 5.0 g at T1 vs 71.3 ± 4.5 g at T2, P < 0.001; saline group: 63.2 ± 3.4 g at T1 vs 68.4 ± 4.0 g at T2, P = 0.321) and reduced mean relative amount of scar volume of the left ventricular wall (UA-ADRCs group: 20.9% ± 2.3% at T1 vs 16.6% ± 1.2% at T2, P = 0.042; saline group: 17.6% ± 1.4% at T1 vs 22.7% ± 1.8% at T2, P = 0.022). CONCLUSION Retrograde cell delivery of UA-ADRCs in a porcine model for the study of CMI significantly improved myocardial function, increased myocardial mass and reduced the formation of scar tissue.

Diatoms, large bloom-forming marine microorganisms, build frustules out of silicate, which ballasts the cells and aids their export to the deep ocean. This unique physiology forges an important link between the marine silicon and carbon cycles. However, the effect of ocean acidification on the silicification of diatoms is unclear. Here we show that diatom silicification strongly diminishes with increased acidity in a natural Antarctic community. Analyses of single cells from within the community reveal that the effect of reduced pH on silicification differs among taxa, with several species having significantly reduced silica incorporation at CO2 levels equivalent to those projected for 2100. These findings suggest that, before the end of this century, ocean acidification may influence the carbon and silicon cycle by both altering the composition of the diatom assemblages and reducing cell ballasting, which will probably alter vertical flux of these elements to the deep ocean. Diatoms have silicate skeletons that affect their buoyancy in the ocean. Ocean acidification reduces silicification, with varying effects between species, and could alter the marine carbon and silica cycles through changes in community composition and sinking rates.

Objective assessment of surgical skill is gaining traction in a number of specialty fields. In robot-assisted surgery in particular, the availability of data from the operating console and patient-side robot offers the potential to derive objective metrics of performance based on tool movement kinematics. While these techniques are becoming established in the laparoscopic domain, current assessment techniques for robotic endovascular surgery are based primarily on observation, checklists, and grading scales. This work presents an objective and quantitative means of measuring technical competence based on analysis of the kinematics of endovascular tool tip motions controlled with a robotic interface. We designed an experiment that recorded catheter tip movement from 21 subjects performing fundamental endovascular robotic navigation tasks on a physical model. Motion-based measures of smoothness (spectral arc length and number of submovements) were computed and tested for correlation with subjective scores from a global rating scale assessment tool that has been validated for use when performing manual catheterization. Results show that the smoothness metrics that produced significant correlations with the global rating scale for manual catheterization show similar correlations for robotic catheterization. This finding is notable, since with the robotic interface, tool tip motion is commanded discretely via a control button interface, while in manual procedures the tools are controlled through continuous movements of the surgeon's hands. Logistic regression analysis using a single motion metric was capable of classifying subjects by expertise with better than 90% accuracy. These objective and quantitative metrics that capture movement quality could be incorporated into future training protocols to provide detailed feedback on trainee performance.

Ocean acidification (OA) can induce shifts in plankton community composition, with coccolithophores being mostly negatively impacted. This is likely to change particulate inorganic and organic carbon (PIC and POC, respectively) production, with impacts on the biological carbon pump. Hence, assessing and, most importantly, understanding species‐specific sensitivities of coccolithophores is paramount. In a multispecies comparison, spanning more than two orders of magnitude in terms of POC and PIC production rates, among Calcidiscus leptoporus, Coccolithus pelagicus subsp. braarudii, Emiliania huxleyi, Gephyrocapsa oceanica, and Scyphosphaera apsteinii, we found that cellular PIC : POC was a good predictor for a species’ OA sensitivity. This is likely related to the need for cellular pH homeostasis, which is challenged by the process of calcification producing protons internally, especially when seawater pH decreases in an OA scenario. With higher PIC : POC, species and strains being more sensitive to OA coccolithophores may shift toward less calcified varieties in the future.

Mangrove soil carbon stocks are known to decrease following forest loss due to respiration and enhanced soil CO2 emissions. However, changes in carbon outwelling to the coastal ocean due to mangrove forest disturbance have not been considered. In December 2015, an extremely large mangrove dieback event (∼7000 hectares, spanning 1000 km of coastline) occurred in the Gulf of Carpentaria, Australia. To assess the effect this dieback event had on carbon outwelling, we used radium isotopes and dissolved carbon measurements (dissolved organic carbon, DOC, dissolved inorganic carbon, DIC, and total alkalinity, TAlk) to estimate cross-shelf carbon transport from living and dead mangrove areas and to calculate the carbon losses from living and dead forest soils via SGD. Radium distributions imply cross shelf eddy diffusivity of 107.5 ± 26.9 and 104.6 ± 23.9 m−2 s−1 from dead and living areas and radium water ages reveal that mangrove carbon reaches 10 km offshore within 7 days. Outwelling rates from living and dead areas were explained by soil carbon losses via SGD. This study suggests a decrease in carbon outwelling to the ocean from dead forest areas compares to living areas by 0–12% for DOC, 50–52% for DIC and by 37–51% for TAlk ∼8 months after the dieback event occurred. Changes to oceanic carbon outwelling rates following mangrove loss are likely driven by a gradual depletion of carbon stocks from the sediment profile.

Bei etwa 10 % aller hospitalisierten Patienten kommt es zu unerwarteten Zwischenfallen, die mit einer erhohten Letalitat einhergehen. Die Etablierung von innerklinischen Notfallteams nimmt deshalb immer hoheren Stellenwert in der interdisziplinaren Notfallversorgung einer Klinik ein. Dabei variiert die Versorgung der Patienten ja nach Krankenhausern und Regionen. Die Versorgung innerklinischer Notfallpatienten des Universitatsklinikums Koln im Vierjahreszeitraum von 2013 bis 2016 soll analysiert und deskriptiv dargestellt werden. Grundlage der vorliegenden Datenanalyse waren innerklinische Notfalleinsatze des Universitatsklinikums Koln („5555-Team“ der Klinik fur Anasthesiologie und Operative Intensivmedizin). Im Untersuchungszeitraum vom 01.01.2013 bis zum 31.12.2016 wurde jeder telefonische Alarm bezuglich des Einsatzstichworts, des Einsatzorts sowie der Einsatzzeiten in einem standardisierten Protokoll dokumentiert. Die Datenanalyse erfolgte retrospektiv und deskriptiv. Ein Ethikvotum war nicht erforderlich. Die Registrierungsnummer auf ClinicalTrials.gov lautet NCT03786445. Insgesamt wurden 1664 Notfallanrufe im Analysezeitraum ausgewertet. Im Studienverlauf von 2013 bis 2016 stiegen die Anzahl der Einsatze insgesamt um 11,4 %, die gesamte Einsatzzeit um 29,5 % und die durchschnittliche Einsatzzeit um 14,3 % an. Das fuhrende Einsatzstichwort war „Kollaps/Synkope“ und machte 29 % des Ausruckegrunds aus. Bei steigenden Pflegetagen innerhalb des Versorgungsgelandes waren 6,5 % der Einsatze Reanimationsbehandlungen. Das Einsatzaufkommen sank zum Wochenende um 50 % im Vergleich zum Einsatzaufkommen an Wochentagen. Uber die letzten 4 Jahre zeigte sich eine deutliche Steigerung der Einsatzzahlen, der gesamten Einsatzzeit und der durchschnittlichen Einsatzdauer. Der fuhrende Alarmierungsgrund war in den Jahren 2013–2016 das Stichwort „Kollaps/Synkope“. Jeder 17. Einsatz war eine Reanimation. Die Inzidenz von Herz-Kreislauf-Stillstanden sank im Verlauf des Analysezeitraums.

SummaryGene-regulatory networks control establishment and maintenance of alternative gene expression states during development. A particular challenge is the acquisition of opposing states by two copies of the same gene, as it is the case in mammals for Xist at the onset of random X-chromosome inactivation (XCI). The regulatory principles that lead to stable mono-allelic expression of Xist remain unknown. Here, we uncovered the minimal Xist regulatory network, by combining mathematical modeling and experimental validation of central model predictions. We identified a symmetric toggle switch as the basis for random mono-allelic Xist up-regulation, which reproduces data from several mutant, aneuploid and polyploid murine cell lines with various Xist expression patterns. Moreover, this toggle switch explains the diversity of strategies employed by different species at the onset of XCI. In addition to providing a unifying conceptual framework to explore X-chromosome inactivation across mammals, our study sets the stage for identifying the molecular mechanisms required to initiate random XCI.

Upwelling of nutrient-rich deep waters make eastern boundary upwelling systems (EBUSs), such as the Humboldt Current system, hot spots of marine productivity. Associated settling of organic matter to depth and consecutive aerobic decomposition results in large subsurface water volumes being oxygen depleted. Under these circumstances, organic matter remineralisation can continue via denitrification, which represents a major loss pathway for bioavailable nitrogen. Additionally, anaerobic ammonium oxidation can remove significant amounts of nitrogen in these areas. Here we assess the interplay of suboxic water upwelling and nitrogen cycling in a manipulative offshore mesocosm experiment. Measured denitrification rates in incubations with water from the oxygen-depleted bottom layer of the mesocosms (via 15N label incubations) mostly ranged between 5.5 and 20 nmol N2 L−1 h−1 (interquartile range), reaching up to 80 nmol N2 L−1 h−1. However, actual in situ rates in the mesocosms, estimated via Michaelis–Menten kinetic scaling, did most likely not exceed 0.2–4.2 nmol N2 L−1 h−1 (interquartile range) due to substrate limitation. In the surrounding Pacific, measured denitrification rates were similar, although indications of substrate limitation were detected only once. In contrast, anammox (anaerobic ammonium oxidation) made only a minor contribution to the overall nitrogen loss when encountered in both the mesocosms and the Pacific Ocean. This was potentially related to organic matter C / N stoichiometry and/or process-specific oxygen and hydrogen sulfide sensitivities. Over the first 38 d of the experiment, total nitrogen loss calculated from in situ rates of denitrification and anammox was comparable to estimates from a full nitrogen budget in the mesocosms and ranged between ∼ 1 and 5.5 µmol N L−1. This represents up to ∼ 20 % of the initially bioavailable inorganic and organic nitrogen standing stocks. Interestingly, this loss is comparable to the total amount of particulate organic nitrogen that was exported into the sediment traps at the bottom of the mesocosms at about 20 m depth. Altogether, this suggests that a significant portion, if not the majority of nitrogen that could be exported to depth, is already lost, i.e. converted to N2 in a relatively shallow layer of the surface ocean, provided that there are oxygen-deficient conditions like those during coastal upwelling in our study. Published data for primary productivity and nitrogen loss in all EBUSs reinforce such conclusion.

Primary production in the Southern Ocean is dominated by diatom-rich phytoplankton assemblages, whose individual physiological characteristics and community composition are strongly shaped by the environment, yet knowledge on how diatoms allocate cellular energy in response to ocean acidification (OA) is limited. Understanding such changes in allocation is integral to determining the nutritional quality of diatoms and the subsequent impacts on the trophic transfer of energy and nutrients. Using synchrotron-based Fourier transform infrared microspectroscopy, we analysed the macromolecular content of selected individual diatom taxa from a natural Antarctic phytoplankton community exposed to a gradient of fCO2 levels (288-1263 µatm). Strong species-specific differences in macromolecular partitioning were observed under OA. Large taxa showed preferential energy allocation towards proteins, while smaller taxa increased both lipid and protein stores at high fCO2 . If these changes are representative of future Antarctic diatom physiology, we may expect a shift away from lipid-rich large diatoms towards a community dominated by smaller taxa, but with higher lipid and protein stores than their present-day contemporaries, a response that could have cascading effects on food web dynamics in the Antarctic marine ecosystem.

Developmental genes such asXist, the master regulator of X-chromosome inactivation (XCI), are controlled by complexcis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns.Xistintegrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements ofXistduring the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of theXistpromoter. The strongest distal element is part of an enhancer cluster ∼200 kb upstream of theXistgene which is associated with a previously unannotatedXist-enhancing regulatory transcript, we namedXert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specificXistupregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.

BackgroundX-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation.ResultsTo identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression.ConclusionsHere, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

Upwelling of nutrient-rich deep waters make Eastern Boundary upwelling systems (EBUS), such as the Humboldt Current System, hotspots of marine productivity. Associated settling of organic matter to depth and consecutive aerobic de composition results in large sub-surface water volumes being oxygen-depleted. Under these circumstances organic matter remineralisation can continue via denitrification which represents a major loss pathway of bioavailable nitrogen. Another process removing significant amounts of nitrogen in these areas is anaerobic ammonium oxidation. Here we assess the interplay of suboxic water upwelling and nitrogen cycling in a manipulative off-shore mesocosm experiment. Measured denitrification rates in the oxygen-depleted bottom layer of the mesocosms mostly ranged between 5.5–20 (interquartile range), reaching up to 80 nmol N2 L−1 h−1. However, realised in-situ rates did most likely not exceed 0.6–1.6 nmol N2 L−1 h−1 (interquartile range), due to substrate limitation in the mesocosms. This was in contrast to realised rates in the surrounding Pacific. Both in the mesocosms and the Pacific Ocean anammox made only a minor contribution to overall nitrogen loss when encountered. Over the first 38 days of the experiment, total nitrogen loss calculated from denitrification and anammox rates was comparable to estimates from a full nitrogen budget in the mesocosms and ranged between ∼ 2–6 μmol N L−1. This represents up to ∼ 20 % of the initially bioavailable inorganic and organic nitrogen standing stocks. Interestingly, this loss is comparable to the total amount of particulate organic nitrogen that was exported into the sediment traps at the bottom of the mesocosms in about 20 metres depth. Altogether, this suggests that a significant portion, if not the majority of nitrogen that could be exported to depth, is already lost, i.e. converted to N2 in a relatively shallow layer of the surface ocean, provided oxygen-deficient conditions like during coastal upwelling in our study.

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibitionin vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenograftsin vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identifiedFOSgene family members as inducers of resistance against ATR inhibitors. Mechanistically,FOSgene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

Quantification of the rate of gas exchange across the air-water interface is essential in understanding the biogeochemical cycling of carbon in mountain streams. However, estimating the gas transfe...

Large-scale dinoflagellate blooms have appeared in recent decades in the Taiwan Strait, Southeast China. To study spatial variability of phytoplankton community composition, physical and chemical environmental drivers in surface seawater of the Taiwan Strait, we conducted cruises in May and July 2019. Cell numbers of dinoflagellates were significantly higher than that of diatoms in most sampling stations during the cruise in May, whereas diatoms were the major contributor to autotrophic biomass in July. Phytoplankton community shifted from a dinoflagellate- and diatom-dominated system in May to diatom dominance in July. The dominant phytoplankton species (genera) were the harmful algal bloom dinoflagellates Prorocentrum donghaiense and Scrippsiella trochoidea and the diatoms Coscinodiscus in May, and Rhizosolenia, Pseudo-nitzschia, and Guinardia in July. Cell densities of dinoflagellates and P. donghaiense reduced exponentially with increasing seawater temperature and salinity and decreasing dissolved inorganic nitrogen (DIN) concentrations. Based on the results of our work and previous studies, it becomes obvious that harmful dinoflagellate blooms are likely to be a major component of the planktonic food web in the Taiwan Strait at a temperature of 17.0&ndash, 23.0 &deg, C, a salinity of 29.0&ndash, 33.0 psu, and a DIN concentration higher than 2.0 &mu, mol L&ndash, 1.