Suppressed basal melting in the eastern Thwaites Glacier grounding zone.

Thwaites Glacier is one of the fastest-changing ice-ocean systems in Antarctica1-3. Much of the ice sheet within the catchment of Thwaites Glacier is grounded below sea level on bedrock that deepens inland4, making it susceptible to rapid and irreversible ice loss that could raise the global sea level by more than half a metre2,3,5. The rate and extent of ice loss, and whether it proceeds irreversibly, are set by the ocean conditions and basal melting within the grounding-zone region where Thwaites Glacier first goes afloat3,6, both of which are largely unknown. Here we show-using observations from a hot-water-drilled access hole-that the grounding zone of Thwaites Eastern Ice Shelf (TEIS) is characterized by a warm and highly stable water column with temperatures substantially higher than the in situ freezing point. Despite these warm conditions, low current speeds and strong density stratification in the ice-ocean boundary layer actively restrict the vertical mixing of heat towards the ice base7,8, resulting in strongly suppressed basal melting. Our results demonstrate that the canonical model of ice-shelf basal melting used to generate sea-level projections cannot reproduce observed melt rates beneath this critically important glacier, and that rapid and possibly unstable grounding-line retreat may be associated with relatively modest basal melt rates.

High-capacity sample multiplexing for single cell chromatin accessibility profiling.

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

Protected fish spawning aggregations as self-replenishing reservoirs for regional recovery.

Dispersal of eggs and larvae from spawning sites is critical to the population dynamics and conservation of marine fishes. For overfished species like critically endangered Nassau grouper (Epinephelus striatus), recovery depends on the fate of eggs spawned at the few remaining aggregation sites. Biophysical models can predict larval dispersal, yet these rely on assumed values of key parameters, such as diffusion and mortality rates, which have historically been difficult or impossible to estimate. We used in situ imaging to record three-dimensional positions of individual eggs and larvae in proximity to oceanographic drifters released into egg plumes from the largest known Nassau grouper spawning aggregation. We then estimated a diffusion-mortality model and applied it to previous years' drifter tracks to evaluate the possibility of retention versus export to nearby sites within 5 days of spawning. Results indicate that larvae were retained locally in 2011 and 2017, with 2011 recruitment being a substantial driver of population recovery on Little Cayman. Export to a nearby island with a depleted population occurred in 2016. After two decades of protection, the population appears to be self-replenishing but also capable of seeding recruitment in the region, supporting calls to incorporate spawning aggregation protections into fisheries management.

The proto-oncometabolite fumarate binds glutathione to amplify ROS-dependent signaling.

The tricarboxylic acid cycle enzyme fumarate hydratase (FH) has been identified as a tumor suppressor in a subset of human renal cell carcinomas. Human FH-deficient cancer cells display high fumarate concentration and ROS levels along with activation of HIF-1. The underlying mechanisms by which FH loss increases ROS and HIF-1 are not fully understood. Here, we report that glutamine-dependent oxidative citric acid cycle metabolism is required to generate fumarate and increase ROS and HIF-1 levels. Accumulated fumarate directly bonds the antioxidant glutathione in vitro and in vivo to produce the metabolite succinated glutathione (GSF). GSF acts as an alternative substrate to glutathione reductase to decrease NADPH levels and enhance mitochondrial ROS and HIF-1 activation. Increased ROS also correlates with hypermethylation of histones in these cells. Thus, fumarate serves as a proto-oncometabolite by binding to glutathione which results in the accumulation of ROS.

Reductive carboxylation supports growth in tumour cells with defective mitochondria.

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.

Pyruvate carboxylase is required for glutamine-independent growth of tumor cells.

Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how "glutamine-addicted" cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis.

Direct observations of melting, freezing, and ocean circulation in an ice shelf basal crevasse.

Ocean conditions near the grounding zones of Antarctica's ice shelves play a key role in controlling the outflow and mass balance of the ice sheet. However, ocean observations in these regions are largely absent. Here, we present a detailed spatial survey collected with an underwater vehicle in a basal crevasse located in the ocean cavity at the Ross Ice Shelf grounding zone. The observations depict fine-scale variability in ocean forcing that drives asymmetric melting along the lower crevasse sidewalls and freezing in the upper reaches of the crevasse. Freshwater release from melting at depth and salt rejection from freezing above drives an overturning circulation. This vertical circulation pattern overlays a dominant throughflow jet, which funnels water parallel to the coastline, orthogonal to the direction of tidal currents. Importantly, these data reveal that basal crevasses influence ocean circulation and mixing at ice shelf grounding zones to an extent previously unknown.

An electroencephalogram biomarker of fentanyl drug effects.

Opioid drugs influence multiple brain circuits in parallel to produce analgesia as well as side effects, including respiratory depression. At present, we do not have real-time clinical biomarkers of these brain effects. Here, we describe the results of an experiment to characterize the electroencephalographic signatures of fentanyl in humans. We find that increasing concentrations of fentanyl induce a frontal theta band (4 to 8 Hz) signature distinct from slow-delta oscillations related to sleep and sedation. We also report that respiratory depression, quantified by decline in an index of instantaneous minute ventilation, occurs at ≈1700-fold lower concentrations than those that produce sedation as measured by reaction time. The electroencephalogram biomarker we describe could facilitate real-time monitoring of opioid drug effects and enable more precise and personalized opioid administration.

Adaptive Manufacturing for Healthcare During the COVID-19 Emergency and Beyond.

During the COVID-19 pandemic, global health services have faced unprecedented demands. Many key workers in health and social care have experienced crippling shortages of personal protective equipment, and clinical engineers in hospitals have been severely stretched due to insufficient supplies of medical devices and equipment. Many engineers who normally work in other sectors have been redeployed to address the crisis, and they have rapidly improvised solutions to some of the challenges that emerged, using a combination of low-tech and cutting-edge methods. Much publicity has been given to efforts to design new ventilator systems and the production of 3D-printed face shields, but many other devices and systems have been developed or explored. This paper presents a description of efforts to reverse engineer or redesign critical parts, specifically a manifold for an anaesthesia station, a leak port, plasticware for COVID-19 testing, and a syringe pump lock box. The insights obtained from these projects were used to develop a product lifecycle management system based on Aras Innovator, which could with further work be deployed to facilitate future rapid response manufacturing of bespoke hardware for healthcare. The lessons learned could inform plans to exploit distributed manufacturing to secure back-up supply chains for future emergency situations. If applied generally, the concept of distributed manufacturing could give rise to "21st century cottage industries" or "nanofactories," where high-tech goods are produced locally in small batches.

N-Acetyl cysteine abrogates silver-induced reactive oxygen species in human cells without altering silver-based antimicrobial activity.

Silver-based antimicrobials are widely used topically to treat infections associated with multi-drug resistant (MDR) pathogens. Expanding this topical use to aerosols to treat lung infections requires understanding and preventing silver toxicity in the respiratory tract. A key mechanism resulting in silver-induced toxicity is the production of reactive oxygen species (ROS). In this study, we have verified ROS generation in silver-treated bronchial epithelial cells prompting evaluation of three antioxidants, N-acetyl cysteine (NAC), ascorbic acid, and melatonin, to identify potential prophylactic agents. Among them, NAC was the only candidate that abrogated the ROS generation in response to silver acetate exposure resulting in the rescue of these cells from silver-associated toxicity. Further, this protective effect directly translated to preservation of metabolic activity, as demonstrated by the normal levels of citric acid cycle metabolites in NAC-pretreated silver acetate-exposed cells. Because the citric acid cycle remained functional, silver-exposed cells pre-incubated with NAC demonstrated significantly higher levels of adenosine triphosphate levels compared with NAC-free controls. Moreover, we found that this prodigious capacity of NAC to rescue silver acetate-exposed cells was due not only to its antioxidant activity, but also to its ability to directly bind silver. Despite binding to silver, NAC did not alter the antimicrobial activity of silver acetate.

Altering alpha-frequency brain oscillations with rapid analog feedback-driven neurostimulation.

Oscillations of the brain's local field potential (LFP) may coordinate neural ensembles and brain networks. It has been difficult to causally test this model or to translate its implications into treatments, because there are few reliable ways to alter LFP oscillations. We developed a closed-loop analog circuit to enhance brain oscillations by feeding them back into cortex through phase-locked transcranial electrical stimulation. We tested the system in a rhesus macaque with chronically implanted electrode arrays, targeting 8-15 Hz (alpha) oscillations. Ten seconds of stimulation increased alpha oscillatory power for up to 1 second after stimulation offset. In contrast, open-loop stimulation decreased alpha power. There was no effect in the neighboring 15-30 Hz (beta) LFP rhythm or on a neighboring array that did not participate in closed-loop feedback. Analog closed-loop neurostimulation might thus be a useful strategy for altering brain oscillations, both for basic research and the treatment of neuro-psychiatric disease.

Underwater microscopy for in situ studies of benthic ecosystems.

Microscopic-scale processes significantly influence benthic marine ecosystems such as coral reefs and kelp forests. Due to the ocean's complex and dynamic nature, it is most informative to study these processes in the natural environment yet it is inherently difficult. Here we present a system capable of non-invasively imaging seafloor environments and organisms in situ at nearly micrometre resolution. We overcome the challenges of underwater microscopy through the use of a long working distance microscopic objective, an electrically tunable lens and focused reflectance illumination. The diver-deployed instrument permits studies of both spatial and temporal processes such as the algal colonization and overgrowth of bleaching corals, as well as coral polyp behaviour and interspecific competition. By enabling in situ observations at previously unattainable scales, this instrument can provide important new insights into micro-scale processes in benthic ecosystems that shape observed patterns at much larger scales.

Pleural Hypercarbia After Lung Surgery Is Associated With Persistent Alveolopleural Fistulae.

BACKGROUND: Persistent air leak (PAL) > 5 days due to alveolopleural fistulae is a leading cause of morbidity following surgical resection. Elevated CO2 levels reportedly inhibit alveolar epithelial cell proliferation and impair wound healing in vitro. Because the injured lung surface is in direct communication with the pleural cavity, we investigated whether the pleural gaseous milieu affected lung healing. METHODS: Oxygen and CO2 levels in pleural gas were determined prospectively in consecutive patients (N = 116) undergoing lung resection by using an infrared spectroscopy-based analyzer. Poisson and logistic regression analyses were used to determine the relationship between time to resolution of air leaks and pleural oxygen and CO2. In addition, patients with pleural CO2 concentrations ? 6% on postoperative day 1 (n = 20) were alternatively treated with supplemental oxygen and extrapleural suction to reduce the pleural CO2 levels. RESULTS: Poisson analyses revealed that every 1% increase in CO2 was associated with a delay in resolution of air leak by 9 h (95% CI, 7.1 to 10.8; P < .001). Linear regression showed that every 1% increase in CO2 increased the odds of PAL by 10-fold (95% CI, 2.2 to 47.8; P = .003). In patients with pleural CO2 ? 6%, a reduction in CO2 promoted resolution of air leak (6.0 ± 1.2 vs 3.4 ± 1.1 days; P < .001). CONCLUSIONS: Pleural hypercarbia seems to be associated with persistent alveolopleural fistulae following lung resection. Analysis of pleural gases could allow for better chest tube management following lung resection. Patients with intrapleural hypercarbia seem to benefit from supplemental oxygen and suction, whereas patients who do not have hypercarbia can be maintained on water seal drainage.

Oxidation of alpha-ketoglutarate is required for reductive carboxylation in cancer cells with mitochondrial defects.

Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse.

Metabolic changes in cancer cells upon suppression of MYC.

BACKGROUND: Cancer cells engage in aerobic glycolysis and glutaminolysis to fulfill their biosynthetic and energetic demands in part by activating MYC. Previous reports have characterized metabolic changes in proliferating cells upon MYC loss or gain of function. However, metabolic differences between MYC-dependent cancer cells and their isogenic differentiated counterparts have not been characterized upon MYC suppression in vitro. RESULTS: Here we report metabolic changes between MYC-dependent mouse osteogenic sarcomas and differentiated osteoid cells induced upon MYC suppression. While osteogenic sarcoma cells increased oxygen consumption and spare respiratory capacity upon MYC suppression, they displayed minimal changes in glucose and glutamine consumption as well as their respective contribution to the citrate pool. However, glutamine significantly induced oxygen consumption in the presence of MYC which was dependent on aminotransferases. Furthermore, inhibition of aminotransferases selectively diminished cell proliferation and survival of osteogenic sarcoma MYC-expressing cells. There were minimal changes in ROS levels and cell death sensitivity to reactive oxygen species (ROS)-inducing agents between osteoid cells and osteogenic sarcoma cells. Nevertheless, the mitochondrial-targeted antioxidant Mito-Vitamin E still diminished proliferation of MYC-dependent osteogenic sarcoma cells. CONCLUSION: These data highlight that aminotransferases and mitochondrial ROS might be attractive targets for cancer therapy in MYC-driven tumors.

Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development.

Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and ß-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.

Genetically-defined metabolic reprogramming in cancer.

Oncogenes and tumor suppressors regulate cell metabolism. Evidence demonstrates that tumorigenic mutations in these genes tend to orchestrate metabolic activity into a platform that promotes cell survival, growth, and proliferation. Recent work has shown that some metabolic enzymes are also mutated in cancer, and that these mutations may influence malignancy directly. Thus, these enzymes seem to function as oncogenes and tumor suppressors, and would appear to be compelling targets for therapeutic intervention. Here, we review several enzymes mutated in cancer - phosphoglycerate dehydrogenase, isocitrate dehydrogenases 1 and 2, succinate dehydrogenase, and fumarate hydratase - and discuss exciting new work that has begun to pull back the curtain on how mutations in these enzymes influence tumorigenesis.

Glutamine: pleiotropic roles in tumor growth and stress resistance.

Tumors and tumor cell lines rapidly consume the amino acid glutamine (Gln) and use it to supply metabolic pathways that support cell growth and proliferation. Much of the research regarding the relationship between glutamine metabolism and cancer is based on the premise that this abundant nutrient represents an important driver of tumor cell anabolism. However, Gln's influence in cell biology and cancer extends far beyond its use as a carbon and nitrogen source for the structural components of dividing cells. Gln is truly a multipurpose nutrient, feeding many additional pathways that boost the ability of cells to communicate with each other and to cope with stress by oncogenic signaling and by the tumor microenvironment. A number of recent reports have highlighted these "non-anabolic" functions of Gln metabolism in regulating cell survival, oxidative stress resistance, signal transduction, and autophagy. Here, we review some of these findings and discuss their relevance to tumor biology and the potential for cancer therapy.

SERPINE1 (PAI-1) is deposited into keratinocyte migration "trails" and required for optimal monolayer wound repair.

Cutaneous tissue injury, both in vivo and in vitro, initiates activation of a "wound repair" transcriptional program. One such highly induced gene encodes plasminogen activator inhibitor type-1 (PAI-1, SERPINE1). PAI-1-GFP, expressed as a fusion protein under inducible control of +800 bp of the wound-activated PAI-1 promoter, prominently "marked" keratinocyte migration trails during the real-time of monolayer scrape-injury repair. Addition of active recombinant PAI-1 to wounded wild-type keratinocyte monolayers as well as to PAI-1(-/-) MEFs and PAI-1(-/-) keratinocytes significantly stimulated directional motility above basal levels in all cell types. PAI-1 expression knockdown or antibody-mediated functional inhibition, in contrast, effectively attenuated injury repair. The defect in wound-associated migratory activity as a consequence of antisense-mediated PAI-1 down-regulation was effectively reversed by addition of recombinant PAI-1 immediately after scrape injury. One possible mechanism underlying the PAI-1-dependent motile response may involve fine control of the keratinocyte substrate detachment/re-attachment process. Exogenous PAI-1 significantly enhanced keratinocyte spread cell "footprint" area while PAI-1 neutralizing antibodies, but not control non-immune IgG, effectively inhibited spreading with apoptotic hallmarks evident within 24 h. Importantly, PAI-1 not only stimulated keratinocyte adhesion and wound-initiated planar migration but also rescued keratinocytes from plasminogen-induced substrate detachment/anoikis. The early transcriptional response of the PAI-1 gene to monolayer trauma and its prominence in the injury repair genetic signature are consistent with its function as both a survival factor and regulator of the time course of epithelial migration as part of the cutaneous injury response program.