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This research demonstrates the value of laser scanning confocal microscopy (LSCM) as a research tool in osteological studies, and diagenetic studies in particular. LSCM combines properties of light and scanning electron microscopy using laser light to excite fluorophores throughout the z-axis, developing a 3-D image. Using differential staining and selecting for specific wavelengths of light, one can image targeted materials. This research is divided into two parts: visualizing bone structures such as proteins and their decompositional products and visualizing diagenesis. Part one of this study utilized pig bones as a means of testing the overall ability of LSCM to fluoresce bone. Twenty-three samples were imaged, including 13 samples from a decompositional study conducted 5 years previous, and 10 "fresh" samples collected from a commercial butcher. This part of the study determined that protein and organic components of the bone could be fluoresced and diagenetic alteration could be imaged. The second part of the study used human samples as a means of imaging and mapping diagenetic alterations. The second part of the study used 13 samples, including 4 clinical, 7 ancient, and 2 modern controls. The pig study used Basic Fuchsin and SlowFade Gold stains, while the human study used toluidine blue. Images were also taken with unstained elements. The results of the non-human study found that a fresh bone fluoresced differently than that of a 5-year subset, while the results of the human study confirmed these findings and determined that the bone diagenesis can be mapped using LSCM.

BACKGROUND To determine if racial disparities exist between African Americans (AA) and Non-Hispanic Whites (NHW) for patients undergoing repair of acute type A aortic dissection (ATAAD) at a rural tertiary academic medical center. METHODS There were 215 consecutive AA and NHW patients who underwent ATAAD repair at our institution from 1999 to 2019 included in a retrospective analysis of our Society of Thoracic Surgeons Adult Cardiac Surgery Database. Statistical analysis was performed with a p value of less than .05 considered statistically significant. RESULTS Patients undergoing ATAAD repair were 47% AA despite comprising only 27% of the total population in our region. AAs were significantly younger (54.0 vs. 61.2 years), were more likely to be hypertensive (94.1% vs. 79.7%), had higher creatinine levels (1.7 vs. 1.1 mg/dL), and higher body mass index (30.8 vs. 28.1 kg/m2 ) (all p values

The NEXT collaboration: et al., The Neutrino Experiment with a Xenon TPC (NEXT) searches for the neutrinoless double-beta (0νββ) decay of 136Xe using high-pressure xenon gas TPCs with electroluminescent amplification. A scaled-up version of this technology with about 1 tonne of enriched xenon could reach in less than 5 years of operation a sensitivity to the half-life of 0νββ decay better than 1027 years, improving the current limits by at least one order of magnitude. This prediction is based on a well-understood background model dominated by radiogenic sources. The detector concept presented here represents a first step on a compelling path towards sensitivity to the parameter space defined by the inverted ordering of neutrino masses, and beyond., The NEXT Collaboration acknowledges support from the following agencies and institutions: the European Research Council (ERC) under the Advanced Grant 339787-NEXT; the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014–2020) under the Grant Agreements No. 674896, 690575 and 740055; the Ministerio de Economía y Competitividad and the Ministerio de Ciencia, Innovación y Universidades of Spain under grants FIS2014-53371-C04, RTI2018-095979, the Severo Ochoa Program grants SEV-2014-0398 and CEX2018-000867-S, and the María de Maeztu Program MDM2016-0692; the Generalitat Valenciana of Spain under grants PROMETEO/2016/120 and SEJI/2017/011; the Portuguese FCT under project PTDC/FIS-NUC/2525/2014 and under projects UID/FIS/04559/2020 to fund the activities of LIBPhys-UC; the Pazy Foundation (Israel) under grants 877040 and 877041; the US Department of Energy under contracts number DE-AC02-06CH11357 (Argonne National Laboratory), DE-AC02-07CH11359 (Fermi National Accelerator Laboratory), DE-FG02-13ER42020 (Texas A&M) and DE-SC0019223 / DE-SC0019054 (University of Texas at Arlington); and the University of Texas at Arlington. DGD acknowledges support from the Ramón y Cajal program (Spain) under contract number RYC-2015-18820. JM-A acknowledges support from Fundación Bancaria la Caixa (ID 100010434), grant code LCF/BQ/PI19/11690012, and from the Plan GenT program of the Generalitat Valenciana, grant code CIDEGENT/2019/049.

Wnt signaling regulates cell proliferation and cell differentiation as well as migration and polarity during development. However, it is still unclear how the Wnt ligand distribution is precisely controlled to fulfil these functions. Here, we show that the planar cell polarity protein Vangl2 regulates the distribution of Wnt by cytonemes. In zebrafish epiblast cells, mouse intestinal telocytes and human gastric cancer cells, Vangl2 activation generates extremely long cytonemes, which branch and deliver Wnt protein to multiple cells. The Vangl2-activated cytonemes increase Wnt/β-catenin signaling in the surrounding cells. Concordantly, Vangl2 inhibition causes fewer and shorter cytonemes to be formed and reduces paracrine Wnt/β-catenin signaling. A mathematical model simulating these Vangl2 functions on cytonemes in zebrafish gastrulation predicts a shift of the signaling gradient, altered tissue patterning, and a loss of tissue domain sharpness. We confirmed these predictions during anteroposterior patterning in the zebrafish neural plate. In summary, we demonstrate that Vangl2 is fundamental to paracrine Wnt/β-catenin signaling by controlling cytoneme behaviour., Cytonemes are cellular projections known to transfer Wnt ligands between cells, but their regulation remains unclear. Here, the authors show that activation of the planar cell polarity protein Vangl2 generates long and branched cytonemes increasing paracrine Wnt/β-catenin signaling.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

The α-hydroxytropolones (αHTs) are troponoid inhibitors of hepatitis B virus (HBV) replication that can target HBV RNase H with submicromolar efficacies. αHTs and related troponoids (tropones and tropolones) can be cytotoxic in cell lines as measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays that assess mitochondrial function. Previous studies suggest that tropolones induce cytotoxicity through inhibition of mitochondrial respiration. Therefore, we screened 35 diverse troponoids for effects on mitochondrial function, mitochondrial/nuclear genome ratios, cytotoxicity, and reactive oxygen species (ROS) production. Troponoids as a class did not inhibit respiration or glycolysis, although the α-ketotropolone subclass interfered with these processes. The troponoids had no impact on the mitochondrial DNA/nuclear DNA ratio after 3 days of compound exposure. The patterns of troponoid-induced cytotoxicity among three hepatic cell lines were similar for all compounds, but three potent HBV RNase H inhibitors were not cytotoxic in primary human hepatocytes. Tropolones and αHTs increased ROS production in cells at cytotoxic concentrations but had no effect at lower concentrations that efficiently inhibit HBV replication. Troponoid-mediated cytotoxicity was significantly decreased upon the addition of the ROS scavenger N-acetylcysteine. These studies show that troponoids can increase ROS production at high concentrations within cell lines, leading to cytotoxicity, but are not cytotoxic in primary hepatocytes. Future development of αHTs as potential therapeutics against HBV may need to mitigate ROS production by altering compound design and/or by coadministering ROS antagonists to ameliorate increased ROS levels.

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies., Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. Here the authors show that MLKL trafficking and plasma membrane accumulation are crucial necroptosis checkpoints, and that accumulation of phosphorylated MLKL at intercellular junctions promotes necroptosis.

Fatty acids have been widely used as trophic biomarkers in marine mammals. However, for the South American sea lion, the most abundant otariid in the eastern South Pacific, there is no information about blubber fatty acids and their link to diet. Here, we compare fatty acid profiles of sea lions from two distinct oceanographic regions in northern and southern Chile. Their fatty acids vary greatly between regions, suggesting dietary differences at a spatial scale. The fatty acid C22:6ω3 was more abundant in sea lions from the northern region, likely associated with consumption of anchovy, cephalopods, and crustaceans, which are rich in that fatty acid, and have been reported as their main prey items. Sea lions from the southern region were richer in C22:1 and C20:1, characteristic of teleost fish, suggesting a piscivorous diet. Males displayed a more diverse fatty acid composition than females, suggesting a wider trophic niche. Few individual sea lions within the southern region had unusually high levels of C18:2ω6, commonly found in terrestrial environments. This suggests consumption of farmed salmon, whose diet is usually based on terrestrial sources. This demonstrates how human intervention is being reflected in the tissues of a top predator in a natural environment.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

Individual longitudinal records of diet, movement, and physiological state of endangered Tasmanian devils (Sarcophilus harrisii) are needed for effective management of wild populations, yet most traditional techniques are expensive or labor‐intensive. Stable isotope analysis of inert tissue, such as vibrissae (whiskers), provides a viable and minimally invasive solution to chronologically record the foraging ecology and habitat use of individuals. Species‐specific information on whisker growth (i.e., time‐position growth of isotopic signatures), retention time, and arrangement on the face is required before the implementation of stable isotope analysis in wild populations. Here, whiskers of six captive Tasmanian devils were internally marked with 13C‐ and 15N‐labeled glycine at three‐month intervals followed by isotopic analysis of the longest whisker to provide a time stamp for whisker growth and estimate retention time. Intradermal and extradermal lengths of wild Tasmanian devil whiskers were used to assess the arrangement and relative length of whiskers on the face. We found that whiskers can record at least nine months of an animal's ecological history and that whisker growth is not linear, the growth gradually slows down as the whisker lengthens. Our findings demonstrate that sequentially sampled whiskers have the potential to track monthly and seasonal isotopic changes of an individual animal in the wild, both within its historical range and in areas to which it has recently been introduced. Such information can be used to identify temporal shifts in habitat and prey preferences within populations and help select suitable individuals for translocations. We recommend that the longest mystacial whiskers, positioned posteriorly at the third and fourth row, should be preferentially used for stable isotope studies in this species. The timeframe represented by the root of the whisker (˜3–63 d) can be used to adjust the base of cut whiskers to the correct time period.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here we show that the nuclear-localized LINC complex protein SUN1 regulates vascular sprouting and barrier function via effects on endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted barrier function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and barrier function.SUMMARYThe nuclear membrane protein SUN1 promotes blood vessel formation and barrier function by stabilizing endothelial cell-cell junctions. Communication between SUN1 and endothelial cell junctions relies upon proper microtubule dynamics and Rho signaling far from the nucleus, revealing long-range cellular communication from the nucleus to the cell periphery that is important for vascular development and function.

Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp. induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles. To understand this process and address the debate regarding how the parasitophorous vacuole membrane (PVM) is formed, we developed an approach using lattice light-sheet microscopy, which enables the parasite interaction with the host cell membrane to be tracked and characterized during invasion. Our results show that the PVM is predominantly formed from the erythrocyte membrane, which undergoes biophysical changes as it is remodeled across all stages of invasion, from pre-invasion through to PVM sealing. This approach enables a functional interrogation of parasite-derived lipids and proteins in PVM biogenesis and echinocytosis during Plasmodium falciparum invasion and promises to yield mechanistic insights regarding how this is more generally orchestrated by other intracellular pathogens., Here, Geoghegan, Evelyn et al. provide a lattice light-sheet microscopy based 4D imaging pipeline to quantitatively investigate Plasmodium spp. invasion and show that the nascent parasitophorous vacuole is predominantly formed from host’s erythrocyte membrane and undergoes continuous remodeling throughout invasion.

High temperatures (HT) and high hydrostatic pressures (HHP) are characteristic of deep-sea hydrothermal vents and other deep crustal settings. These environments host vast and diverse microbial populations, yet only a small fraction of those populations have been successfully cultured. This is due, in part, to the difficulty of sampling while maintaining these in situ conditions and also replicating those high-temperature and high-pressure conditions in the laboratory. In an effort to facilitate more HT-HHP cultivation, we present two HT-HHP batch culture incubation systems for cultivating deep-sea vent and subsurface (hyper)thermophilic microorganisms. One HT-HHP system can be used for batch cultivation up to 110 MPa and 121°C, and requires sample decompression during subsampling. The second HT-HHP system can be used to culture microorganisms up to 100 MPa and 160°C with variable-volume, pressure-retaining vessels that negate whole-sample decompression during subsampling. Here, we describe how to build cost effective heating systems for these two types of high-pressure vessels, as well as the protocols for HT-HHP microbial batch cultivation in both systems. Additionally, we demonstrate HHP transfer between the variable-volume vessels, which has utility in sampling and enrichment without decompression, laboratory isolation experiments, as well as HHP filtration.

A novel family of four 1-bromo-2,6-bis{[(λ5-phosphanylidene)imino]methyl}benzene ligands has been synthesized and characterized. The phosphiniminomethyl substituents are decorated with either three phenyl groups, two phenyl and one cyclohexyl group, one phenyl and two cyclohexyl groups, or three cyclohexyl groups. Each ligand was metallated using zero-valent nickel through an oxidative addition to form a family of organonickel(II) complexes, namely (2,6-bis{[(triphenyl-λ5-phosphanylidene)imino]methyl}phenyl-κ3 N,C 1,N′)bromidonickel(II) dichloromethane hemisolvate, [NiBr(C44H37N2P2)]·0.5CH2Cl2, (2,6-bis{[(cyclohexyldiphenyl-λ5-phosphanylidene)imino]methyl}phenyl-κ3 N,C 1,N′)bromidonickel(II) diethyl ether hemisolvate, [NiBr(C44H49N2P2)]·0.5C4H10O, (2,6-bis{[(dicyclohexylphenyl-λ5-phosphanylidene)imino]methyl}phenyl-κ3 N,C 1,N′)bromidonickel(II), [NiBr(C44H61N2P2)], and (2,6-bis{[(tricyclohexyl-λ5-phosphanylidene)imino]methyl}phenyl-κ3 N,C 1,N′)bromidonickel(II), [NiBr(C44H73N2P2)]. This family of complexes represents a useful opportunity to investigate the impact of incrementally changing the steric characteristics of a complex on its structure and reactivity.

Summary: We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation. : Jansz et al. report that the chromatin protein Smchd1 depends on polycomb repressive complex 1-mediated ubiquitylation of histone H2A for its recruitment to the inactive X chromosome and for its protein stability. These data have implications for Smchd1 targeting genome-wide. Keywords: Smchd1, X inactivation, Xist, PRC1, Hnrnpk, Ring1B

Within the framework of xenon-based double beta decay experiments, we propose the possibility to improve the background rejection of an electroluminescent Time Projection Chamber (EL TPC) by reducing the diffusion of the drifting electrons while keeping nearly intact the energy resolution of a pure xenon EL TPC. Based on state-of-the-art microscopic simulations, a substantial addition of helium, around 10 or 15~\%, may reduce drastically the transverse diffusion down to 2.5~mm/$\sqrt{\mathrm{m}}$ from the 10.5~mm/$\sqrt{\mathrm{m}}$ of pure xenon. The longitudinal diffusion remains around 4~mm/$\sqrt{\mathrm{m}}$. Light production studies have been performed as well. They show that the relative variation in energy resolution introduced by such a change does not exceed a few percent, which leaves the energy resolution practically unchanged. The technical caveats of using photomultipliers close to an helium atmosphere are also discussed in detail., 8 pages, 7 figures

Background Firefighters are at increased risk for both problematic alcohol use and suicidality. Research has found that problematic alcohol use is related to suicidality among this population; however, limited data exist regarding what might account for this association. The present two-study investigation (1) examined the association between suicidality and problematic alcohol use among two large samples of firefighters and (2) tested whether interpersonal theory of suicide constructs—perceived burdensomeness (PB) and thwarted belongingness (TB)—serve as indirect indicators of this relationship. Methods Participants in Study 1 were 944 U.S. firefighters (12.5% female); participants in Study 2 were 241 U.S. women firefighters. Participants completed the Interpersonal Needs Questionnaire, Alcohol Use Disorders Identification Test, and the Depressive Symptom Inventory-Suicidality Subscale (Study 1) or the Self-Injurious Thoughts and Behaviors Interview-Short Form (Study 2). Bias-corrected bootstrap indirect effects path analyses were utilized. Results In Study 1, more problematic alcohol use was significantly associated with more severe career suicidal ideation via PB but not TB. In Study 2, problematic alcohol use was associated with career suicidal ideation via both PB and TB. PB seems to account for the relationship between problematic alcohol use and career suicidal ideation among male and female firefighters. Limitations Limitations include use of a cross-sectional design, use of retrospective measures of suicidal ideation, and our findings derived from subsamples of two existing datasets. Conclusions Findings suggest that PB and TB may explain the relationship between problematic alcohol use and suicidal ideation, but that this effect is discrepant based on gender.

Summary: At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation. : Yan et al. identify the major microtubule acetylase in Drosophila and show that the enzyme and microtubule acetylation broadly control mechanosensation, but not other sensory modalities. Acetylation is required for mechanosensation by the TRP channel NOMPC, and possibly other channels, by virtue of its effects on microtubule mechanical stability and/or dynamics. Keywords: Drosophila, mechanosensation, microtubule acetylation, TRP channel, somatosensory neuron

[EN] Double electron capture by proton-rich nuclei is a second-order nuclear process analogous to double beta decay. Despite their similarities, the decay signature is quite di erent, potentially providing a new channel to measure the hypothesized neutrinoless mode of these decays. The Standard-Model-allowed two-neutrino double electron capture has been predicted for a number of isotopes, but only observed in 78Kr, 130Ba and, recently, 124Xe. The sensitivity to this decay establishes a benchmark for the ultimate experimental goal, namely the potential to discover also the lepton-number-violating neutrinoless version of this process. Here we report on the current sensitivity of the NEXT-White detector to 124Xe 2 ECEC and on the extrapolation to NEXT-100. Using simulated data for the 2 ECEC signal and real data from NEXT-White operated with 124Xe-depleted gas as background, we de ne an optimal event selection that maximizes the NEXT-White sensitivity. We estimate that, for NEXT-100 operated with xenon gas isotopically enriched with 1 kg of 124Xe and for a 5-year run, a sensitivity to the two-neutroni double electron capture half-life of 6x10exp22 years (at 90% con dence level) or better can be reached., The NEXT collaboration acknowledges support from the following agencies and institutions: the European Research Council (ERC) under the Advanced Grant 339787-NEXT; the European Union's Framework Programme for Research and Innovation Horizon 2020 (2014-2020) under the Marie Sklodowska-Curie Grant Agreements No. 674896, 690575 and 740055; the Ministerio de Economia y Competitividad and the Ministerio de Ciencia, Innovacion y Universidades of Spain under grants FIS2014-53371-C04, RTI2018-095979, the Severo Ochoa Program grants SEV-2014-0398 and CEX2018-000867-S, and the Maria de Maeztu Program MDM-2016-0692; the GVA of Spain under grants PROMETEO/2016/120 and SEJI/2017/011; the Portuguese FCT under project PTDC/FIS-NUC/2525/2014, under project UID/FIS/04559/2013 to fund the activities of LIBPhys, and under grants PD/BD/105921/2014, SFRH/BPD/109180/2015 and SFRH/BPD/76842/2011; the U.S. Department of Energy under contracts number DE-AC02-06CH11357 (Argonne National Laboratory), DE-AC02-07CH11359 (Fermi National Accelerator Laboratory), DE-FG02-13ER42020 (Texas A&M) and DE-SC0019223/DE-SC0019054 (University of Texas at Arlington); and the University of Texas at Arlington. DGD acknowledges Ramon y Cajal program (Spain) under contract number RYC-2015-18820. We also warmly acknowledge the Laboratori Nazionali del Gran Sasso (LNGS) and the Dark Side collaboration for their help with TPB coating of various parts of the NEXT-White TPC. Finally, we are grateful to the Laboratorio Subterraneo de Canfranc for hosting and supporting the NEXT experiment

From an analytical chemistry standpoint, determining the chemical attribution signatures (CAS) of synthetic reaction mixtures is an impurity profiling exercise. Identifying and understanding the impurity profile and CAS of these chemical agents would allow them to be exploited for chemical forensic information, such as how a particular chemical agent was synthesised. Being able to determine the synthetic route used to make a chemical agent allows for the possibility of batches of the agent, and individual incidents using that agent, to be forensically linked. This information is of particular benefit to agencies investigating the nefarious and illicit use of chemical agents. One such chemical agent of interest to law enforcement and national security agencies is fentanyl. In this study two acylation methods for the final step of fentanyl production, herein termed the Janssen and Siegfried methods, were investigated by liquid chromatography- high resolution mass spectrometry (LC-HRMS) and multivariate statistical analysis (MVA). From these data, fifty-five chemical impurities were identified. Of these, ten were specific CAS for the Janssen method, and five for the Siegfried method. Additionally, analytical data from four different literature methods for production of the fentanyl precursor 4-anilino-N-phenethylpiperidine (ANPP), were compared to the results obtained from the method of production (Valdez) used in this study. Comparison of the LC-HRMS data for these five methods allowed for four Valdez specific impurities to be identified. These may be useful CAS for the Valdez method of ANPP production.

DNA and RNA nucleases are wide-ranging enzymes, taking part in broad cellular processes from DNA repair to immune response control. Growing interest in the mechanisms and activities of newly discovered nucleases inspired us to share the detailed protocol of our nuclease assay ( Sheppard et al., 2019 ). This easy and inexpensive method can provide data that enables understanding of the molecular mechanism for novel or tested nucleases, from substrate preference and cofactors involved to catalytic rate of reaction.

How do firing patterns in a cortical circuit change when inhibitory neurons are excited? We virally expressed an excitatory designer receptor exclusively activated by a designer drug (Gq-DREADD) in all inhibitory interneuron types of the CA1 region of the hippocampus in the rat. While clozapine N-oxide (CNO) activation of interneurons suppressed firing of pyramidal cells, unexpectedly the majority of interneurons also decreased their activity. CNO-induced inhibition decreased over repeated sessions, which we attribute to long-term synaptic plasticity between interneurons and pyramidal cells. Individual interneurons did not display sustained firing but instead transiently enhanced their activity, interleaved with suppression of others. The power of the local fields in the theta band was unaffected, while power at higher frequencies was attenuated, likely reflecting reduced pyramidal neuron spiking. The incidence of sharp wave ripples decreased but the surviving ripples were associated with stronger population firing compared with the control condition. These findings demonstrate that DREADD activation of interneurons brings about both short-term and long-term circuit reorganization, which should be taken into account in the interpretation of chemogenic effects on behavior.

We test the performance of the Bayesian mixing model, MixSIAR, to quantitatively predict diets of consumers based on their fatty acids (FAs). The known diets of six species, undergoing controlled-feeding experiments, were compared with dietary predictions modelled from their FAs. Test subjects included fish, birds and mammals, and represent consumers with disparate FA compositions. We show that MixSIAR with FA data accurately identifies a consumer’s diet, the contribution of major prey items, when they change their diet (diet switching) and can detect an absent prey. Results were impacted if the consumer had a low-fat diet due to physiological constraints. Incorporating prior information on the potential prey species into the model improves model performance. Dietary predictions were reasonable even when using trophic modification values (calibration coefficients, CCs) derived from different prey. Models performed well when using CCs derived from consumers fed a varied diet or when using CC values averaged across diets. We demonstrate that MixSIAR with FAs is a powerful approach to correctly estimate diet, in particular if used to complement other methods.

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3 and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol-is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3 and MLKL signals.

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages ‘dwells’ within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

The majority of the Earth’s microbial biomass exists in the Deep Biosphere, in the deep ocean and within the Earth’s crust. While other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high-pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold pressure on the Earth’s surface. The mechanisms of pressure adaptation are also important to in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here we describe implementation of a high-pressure sample chamber with a 2-photon scanning microscope system allowing for the first time, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for Fluorescence Lifetime Imaging Microscopy with Phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon,Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning Number and Brightness (sN&B) and Raster scanning Imaging Correlation Spectroscopy (RICS)) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein inE. coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation.SIGNIFICANCEThe majority of the Earth’s microbial biomass exists in high-pressure environments where pressures can reach over 100 MPa. The molecular mechanisms that allow microbes to flourish under such extreme conditions remain to be discovered. The high pressure, high resolution imaging system presented here revealed pressure dependent changes in metabolism and protein interactions in live microbial cells, demonstrating great promise for understanding deep life.

International audience; High hydrostatic pressure (HHP) batch cultivation of a model extremophile, Archaeoglobus fulgidus type strain VC-16, was performed to explore how elevated pressures might affect microbial growth and physiology in the deep marine biosphere. Though commonly identified in high-temperature and high-pressure marine environments (up to 2–5 km below sea level, 20–50 MPa pressures), A. fulgidus growth at elevated pressure has not been characterized previously. Here, exponential growth of A. fulgidus was observed up to 60 MPa when supported by the heterotrophic metabolism of lactate oxidation coupled to sulfate reduction, and up to 40 MPa for autotrophic CO2 fixation coupled to thiosulfate reduction via H2. Maximum growth rates for this heterotrophic metabolism were observed at 20 MPa, suggesting that A. fulgidus is a moderate piezophile under these conditions. However, only piezotolerance was observed for autotrophy, as growth rates remained nearly constant from 0.3 to 40 MPa. Experiments described below show that A. fulgidus continues both heterotrophic sulfate reduction and autotrophic thiosulfate reduction nearly unaffected by increasing pressure up to 30 MPa and 40 MPa, respectively. As these pressures encompass a variety of subsurface marine environments, A. fulgidus serves as a model extremophile for exploring the effects of elevated pressure on microbial metabolisms in the deep subsurface. Further, these results exemplify the need for high-pressure cultivation of deep-sea and subsurface microorganisms to better reflect in situ physiological conditions.

Importance Intravitreous bevacizumab (0.25 mg to 0.625 mg) is commonly used to treat type 1 retinopathy of prematurity (ROP), but there are concerns about systemic toxicity, particularly the risk of neurodevelopmental delay. A much lower dose may be effective for ROP while reducing systemic risk. Previously, after testing doses of 0.25 mg to 0.031 mg, doses as low as 0.031 mg were found to be effective in small cohorts of infants. Objective To find the lowest dose of intravitreous bevacizumab effective for severe ROP. Design, Setting, and Participants Between April 2017 and May 2019, 59 premature infants with type 1 ROP in 1 or both eyes were enrolled in a masked, multicenter, dose de-escalation study. In cohorts of 10 to 14 infants, 1 eye per infant received 0.016 mg, 0.008 mg, 0.004 mg, or 0.002 mg of intravitreous bevacizumab. Diluted bevacizumab was prepared by individual research pharmacies and delivered using 300-µL syringes with 5/16-inch, 30-guage fixed needles. Analysis began July 2019. Interventions Bevacizumab intravitreous injections at 0.016 mg, 0.008 mg, 0.004 mg, or 0.002 mg. Main Outcomes and Measures Success was defined as improvement by 4 days postinjection and no recurrence of type 1 ROP or severe neovascularization requiring additional treatment within 4 weeks. Results Fifty-five of 59 enrolled infants had 4-week outcomes completed; the mean (SD) birth weight was 664 (258) g, and the mean (SD) gestational age was 24.8 (1.6) weeks. A successful 4-week outcome was achieved for 13 of 13 eyes (100%) receiving 0.016 mg, 9 of 9 eyes (100%) receiving 0.008 mg, 9 of 10 eyes (90%) receiving 0.004 mg, but only 17 of 23 eyes (74%) receiving 0.002 mg. Conclusions and Relevance These data suggest that 0.004 mg may be the lowest dose of bevacizumab effective for ROP. Further investigation is warranted to confirm effectiveness of very low-dose intravitreous bevacizumab and its effect on plasma vascular endothelial growth factor levels and peripheral retinal vascularization.

Cytonemes are specialized signalling filopodia that have a role in development and cellular differentiation. However, they are not well preserved by standard fixation techniques to study protein localization and interactions. A recent methodological advance has yielded improvements in cytoneme preservation using glutaraldehyde fixation and sodium borohydride treatment to reduce background. We herein describe a safer method for effective blocking using glycine following glutaraldehyde fixation of cytonemes on cultured adherent cells and demonstrate its effectiveness in immunocytochemistry.

Motivated by the possibility of guiding daughter ions from double beta decay events to single-ion sensors for barium tagging, the NEXT collaboration is developing a program of R&D to test radio frequency (RF) carpets for ion transport in high pressure xenon gas. This would require carpet functionality in regimes at higher pressures than have been previously reported, implying correspondingly larger electrode voltages than in existing systems. This mode of operation appears plausible for contemporary RF-carpet geometries due to the higher predicted breakdown strength of high pressure xenon relative to low pressure helium, the working medium in most existing RF carpet devices. In this paper we present the first measurements of the high voltage dielectric strength of xenon gas at high pressure and at the relevant RF frequencies for ion transport (in the 10 MHz range), as well as new DC and RF measurements of the dielectric strengths of high pressure argon and helium gases at small gap sizes. We find breakdown voltages that are compatible with stable RF carpet operation given the gas, pressure, voltage, materials and geometry of interest.

Suspended particulate organic carbon (POCsusp) in the Gulf of Mexico is unique compared to other seas and oceans. In addition to surface primary production, isotopic analysis indicates that microbial cycling of oil and riverine inputs are primary sources of carbon to POCsusp in the Gulf. To characterize POCsusp from seep sites and non-seep north central Gulf (NCG) sites potentially affected by the Deepwater Horizon (DWH) spill, we analyzed 277 and 123 samples for δ13C and Δ14C signatures, respectively. Depth, partitioned into euphotic (300 m), was the main driver of spatial δ13C differences, with deep depths exhibiting 13C depletion. Both deep depths and proximity to sources of natural seepage resulted in 14C depletion. A two-endmember mixing model based on Δ14C indicated that sources to POCsusp were 14–29% fossil carbon at NCG sites and 19–57% at seep sites, with the balance being modern surface production. A six-component Bayesian mixing model MixSIAR, using both 13C and 14C, suggested that riverine inputs were an important carbon source to POCsusp contributing 34–46%. The influence of seeps was localized. Below the euphotic zone at seep sites, 46 ± 5% (n = 9) of the carbon in POCsusp was derived from environmentally degraded, transformed oil; away from seeps, transformed oil contributed 15 ± 4% (n = 39). We hypothesized that, at NCG sites removed from hydrocarbon seep sources, isotopic signatures would be depleted following the spill and then shift towards background-like enriched values over time. At deep depths we observed decreasing Δ14C signatures in POCsusp from 2010 to 2012, followed by isotopic enrichment from 2012 to 2014 and a subsequent recovery rate of 159‰ per year, consistent with this hypothesis and with biodegraded material from DWH hydrocarbons contributing to POCsusp.

This work describes a fully-integrated portable microfluidic analysis system for real-time monitoring of dynamic changes in glucose and lactate occurring in the brain as a result of cardiac arrest and resuscitation. Brain metabolites are sampled using FDA-approved microdialysis probes and coupled to a high-temporal resolution 3D printed microfluidic chip housing glucose and lactate biosensors. The microfluidic biosensors are integrated with a wireless 2-channel potentiostat forming a compact analysis system that is ideal for use in a crowded operating theatre. Data are transmitted to a custom-written app running on a tablet for real-time visualisation of metabolic trends. In a proof-of-concept porcine model of cardiac arrest, the integrated analysis system proved reliable in a challenging environment resembling a clinical setting; noise levels were found to be comparable with those seen in the lab and were not affected by major clinical interventions such as defibrillation of the heart. Using this system, we were able, for the first time, to measure changes in brain glucose and lactate levels caused by cardiac arrest and resuscitation; the system was sensitive to clinical interventions such as infusion of adrenaline. Trends suggest that cardiopulmonary resuscitation alone does not meet the high energy demands of the brain as metabolite levels only return to their values preceding cardiac arrest upon return of spontaneous circulation.

Excellent energy resolution is one of the primary advantages of electroluminescent high pressure xenon TPCs, and searches for rare physics events such as neutrinoless double-beta decay ($\beta\beta0\nu$) require precise energy measurements. Using the NEXT-White detector, developed by the NEXT (Neutrino Experiment with a Xenon TPC) collaboration, we show for the first time that an energy resolution of 1% FWHM can be achieved at 2.6 MeV, establishing the present technology as the one with the best energy resolution of all xenon detectors for $\beta\beta0\nu$ searches., Comment: 13 pages, 7 figures; accepted to JHEP

Purpose: Mounds from the Pennsylvanian aged San Emiliano Formation (Cantabrian Mountains, Spain) are commonly well exposed. These mounds range from 2 to 50 m in height and are observed to be primary geological features. The mounds are described and classified and the factors and controls of mound nucleation, growth and demise have been established. Methods: Microfacies analysis of 177 thin sections has revealed the composition of the mounds and surrounding carbonates. Results: Composite mounds, exhibiting characteristic components of both Cluster mounds and Agglutinated Microbial mounds are described. The mounds are skeletal-microbial/pack-wackestones. Peloidal, homogenous and clotted micrites are the main sedimentological constituents of the mounds. Microfossils are dominant with Donezella, Claracrusta, Rothpletzella and Girvanella being common. Small foraminifera, bryozoans, corals and algae are all present within the mounds, but are more common within off-mound carbonates. Conclusions: The formation of the mounds was controlled by a dynamic relationship between Donezellacean algae, and microscopic encrusters, a bio-mechanism not observed in mud mounds elsewhere. Fluctuating environmental conditions lead to the alternate dominance between the two groups, resulting in accretion and stabilisation of carbonate muds. These mounds are compositionally different to their Pennsylvanian counterparts.

Industrial dimerization of ethylene to 1-butene is achieved with catalysts bearing ligand structures and activated by a co-catalyst. Here, we report that a Cr-promoted cobalt oxide on carbon catalyst was able to produce 1-butene with 53.5–82.4% total selectivity at ethylene conversions between 8.9 and 31.5% without the use of a co-catalyst. The Cr-promoted catalyst showed enhanced activity and stability compared to the nonpromoted catalyst. The characterization results revealed that the incorporation of Cr into the cobalt oxide on carbon catalyst was able to decrease the cobalt oxide particle size. Charge transfer from Cr to Co was also observed in the Cr-promoted catalyst compared to monometallic Co and Cr on carbon. The kinetic model built to rationalize the experimental observations predicted a 50% increase in active site dispersion in the Cr-promoted catalyst, which explained the 1.6× apparent reaction rate increase compared to the nonpromoted catalyst.

Cobalt oxide supported on N-doped carbon catalysts were investigated for 1-butene oligomerization. The materials were synthesized by treating activated carbon with nitric acid and subsequently with NH3 at 200, 400, 600, and 800 °C, followed by impregnation with cobalt. The 1-butene oligomerization selectivity increased with ammonia treatment temperature of the carbon support. The oligomerization selectivity of cobalt oxide on N-doped carbon synthesized at 800 °C (800A-CoOx/N-C) is 2.6 times higher than previously reported cobalt oxide on N-doped carbon synthesized with NH4OH (2A-CoOx/N-C). Over 70% of the butene dimers were linear C8 olefins for all catalysts. The oligomerization selectivity increased with 1-butene conversion. The catalysts were characterized by elemental analysis, N2 adsorption, X-ray diffraction (XRD), X-ray absorption spectroscopy (XAS), and X-ray photoelectron spectroscopy (XPS). The nitrogen content of the catalysts increases with ammonia treatment temperature as confirmed by element...

A nitrogen-doped carbon-supported cobalt oxide catalyst is able to oligomerize ethylene, propylene, 1-butene and 1-hexene into mixtures of oligomers with above 94.1% dimers. Higher than 72.5% of the dimers produced from 1-butene and 1-hexene are internal linear olefins, while the dimer products from propylene oligomerization are 47.0% linear including 5.9% 1-hexene. Ethylene had the highest oligomerization activity with 56.1–87.0% 1-butene selectivity. The selectivity to linear alpha olefins decreases with an increasing oligomer chain length during ethylene oligomerization. The oligomers formed from ethylene conversion follow a Schulz-Flory distribution. Cossee type mechanism rationalizes the product selectivity from the four olefin feeds, suggesting that a 1,2-2,1 insertion sequence is critical to obtaining linear olefin products. The catalyst was inactive in oligomerizing internal olefins.

We explored the cross-national association between population-level alcohol consumption and homicide victimization rates. The very few prior studies of this association had small homogeneous sample...

Purpose To determine the accuracy of identifying referral-warranted retinopathy of prematurity (RW-ROP, defined as any zone I ROP, stage 3 or worse, or plus disease) from retinal image sets using three grading protocols: a single optic disk–centered image, a set of 3 horizontal images, and a 5-image set. Methods In this secondary analysis of images from the e-ROP study, a weighted sample of 250 image sets from 250 infants (125 with RW-ROP and 125 without RW-ROP) was randomly selected. The sensitivities and specificities for detecting RW-ROP and its components from a single disk center image, along with nasal and temporal retinal images, were calculated and compared with the e-ROP grading of RW-ROP of all 5 retinal images (disk center and nasal, temporal, superior, and inferior retinal images). Results RW-ROP was identified with a sensitivity of 11.2% (95% CI, 6.79%-17.9%) using a single disk center image, with a sensitivity of 70.4% (95% CI, 61.9%-77.9%) using 3 horizontal images, and a statistically higher sensitivity of 82.4% (95% CI, 75.0%-89.0%) using all 5 images ( P = 0.002). The specificities were 100%, 86.4%, and 90.4%, respectively. For grading using 3 horizontal images, sensitivity was 14.3% for plus disease, 25% for zone I ROP, and 71.2% for stage 3 or worse compared to 40.8%, 50%, and 79.8% for grading using 5-image sets, respectively. Conclusions Both a single, disk-centered, posterior pole image and 3 horizontal images were less effective than a 5-image set in determining the presence of RW-ROP on qualitative grading by trained readers.

The Deep Water Horizon (DWH) incident caused the release of an unprecedented amount of 13C- and 14C-depleted oil and gas to the Gulf of Mexico (GoM), which formed surface slicks and deep oil/gas plumes that extended laterally at 1000–1200 m. We conducted three research cruises between 2010 and 2012 to study the potential assimilation of petrocarbon (C derived from oil and/or gas) into the GoM microbial food web. In 2010, we found low δ13C (−30 to −25‰) and Δ14C (−603 to −55‰) values for suspended particles at 1000–1200 m depth as far as 289 km SW of the wellhead, providing direct evidence of the spatial extent of the subsurface plumes. At those depths in 2010, methane and oil carbon accounted for up to 28% and 62% of total particulate carbon (Csp), respectively. In the total area affected by the DWH, 80 ± 56 to 104 ± 91 tonnes (t) of methane-derived and 216 ± 174 to 292 ± 165 t of oil-derived carbon were incorporated into Csp. In 2011 and 2012, the δ13C values were distributed throughout the water column indicating that petrocarbon was still present and recycling, especially in the section closest to the DWH, where oil supplied up to 53% and 75% of Csp, respectively. Relatively low δ15N (< 4‰) values in suspended particles at 1000–1200 m in 2010 indicate stimulation of nitrogen fixation linked to methane oxidation in the months after the spill, which accounted for up to 40% of the particulate nitrogen in the water column at those depths.

Thermophiles are challenging to be studied for ethanol production using agricultural waste containing lignocellulosic materials rich in hexose and pentose. These bacteria have many advantages such as utilizing a wide range of substrates, including pentose (C5) and hexose (C6). In ethanol production, it is important to use ethanol tolerant strain capable in converting lignocellulosic hydrolysate. This study was aimed to investigate the growth profile of ethanol-tolerant thermophile Geobacillus thermoglucosidasius M10EXG using a defined growth medium consisted of single carbon glucose (TGTV), xylose (TXTV), and a mixture of glucose and xylose (TGXTV), together with the effect of yeast extract additionto the media. The experiments were conducted at the School of Biotechnology and Biomolecular Sciences of The University of New South Wales, Australia on a shake flask fermentation at 60°C in duplicate experiment. Cultures were sampled every two hours and analised for their kinetic parameters including the maximum specific growth rate (µmax), biomass yield (Yx/s), ethanol and by-product yields (acetate and L-lactate) (Yp/s), and the doubling time (Td). Results showed that this strain was capable of growing on minimal medium containing glucose or xylose as a single carbon source. This strain utilized glucose and xylose simultaneously (co-fermentation), although there was glucose repression of xylose at relatively low glucose concentration (0.5% w/v), particularly when yeast extract (0.2% w/v) was added to the medium. The highest biomass yield was obtained at 0.5 g l-1 on glucose medium; the yield increased when yeast extract was added (at 0.59 g l-1). The highest specific growth rate of 0.25 was obtained in the phase I growth when the strain was grown on a mixture of glucose and xylose (0.5% : 0.5% w/v) medium. Diauxic growth was shown on the mixture of glucose, xylose, and yeast extract. The strain produced low level of ethanol (0.1 g l-1), as well as low level (0.2 g l-1) of by-products (L-lactate and acetate) after 15 hours. The results suggests its potential application for fermenting lignocellulosic agricultural wastes for ethanol production.

Adeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

We report new measurements of the drift velocity and longitudinal diffusion coefficients of electrons in pure xenon gas and in xenon-helium gas mixtures at 1-9 bar and electric field strengths of 50-300 V/cm. In pure xenon we find excellent agreement with world data at all $E/P$, for both drift velocity and diffusion coefficients. However, a larger value of the longitudinal diffusion coefficient than theoretical predictions is found at low $E/P$ in pure xenon, below the range of reduced fields usually probed by TPC experiments. A similar effect is observed in xenon-helium gas mixtures at somewhat larger $E/P$. Drift velocities in xenon-helium mixtures are found to be theoretically well predicted. Although longitudinal diffusion in xenon-helium mixtures is found to be larger than anticipated, extrapolation based on the measured longitudinal diffusion coefficients suggest that the use of helium additives to reduce transverse diffusion in xenon gas remains a promising prospect.

The Deepwater Horizon oil spill released petroleum hydrocarbons that were depleted in δ13C and Δ14C at depth into the Gulf of Mexico. Stable-carbon and radiocarbon isotopic values and high-resolution mass spectrometry were used to follow the distributions of this petroleum and to track its transformation into petrocarbon, a term used to describe crude oil or transformed crude oil following biodegradation, weathering, oxygenation, or loss of lighter components. The term petrocarbon includes oil- or methane-derived carbon assimilated or incorporated into microbial biomass or into the food web as well as degraded and undegraded petroleum constituents. Here we report (1) the increase in the relative abundance of oxygen-containing carbon compounds making up the dissolved organic matter (DOM) with increasing depth through the water column, indicating the biodegradation of DOM as it was transported to depth in the water column, (2) the finding of 14C depletion in DOM indicating petrocarbon inputs, and (3) the decrease and subsequent increase of 14C in the isotopic composition of sinking particles indicating the capture of petrocarbon in sediment traps. In addition, we discuss the 14C depletion of this material once it is sedimented to the seafloor and the implications for oil spill budgets of seafloor petrocarbon deposition.

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and regulatory components can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we have developed a quick, safe, reproducible, cost-effective, and real-time nuclease assay toolkit that could be used for small- and large- scale experimental assays. Additionally, these data can be analysed to determine each reaction's unique enzyme kinetics. We have designed a library of substrates that can be used to study catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended this assay to consider analysing the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, and RNA:DNA nucleases, which are also involved in DNA repair and immune regulation, and have been associated with various disease conditions, including cancer and immune disorders.