Biomolecular condensates regulate cellular electrochemical equilibria.

Control of the electrochemical environment in living cells is typically attributed to ion channels. Here, we show that the formation of biomolecular condensates can modulate the electrochemical environment in bacterial cells, which affects cellular processes globally. Condensate formation generates an electric potential gradient, which directly affects the electrochemical properties of a cell, including cytoplasmic pH and membrane potential. Condensate formation also amplifies cell-cell variability of their electrochemical properties due to passive environmental effect. The modulation of the electrochemical equilibria further controls cell-environment interactions, thus directly influencing bacterial survival under antibiotic stress. The condensate-mediated shift in intracellular electrochemical equilibria drives a change of the global gene expression profile. Our work reveals the biochemical functions of condensates, which extend beyond the functions of biomolecules driving and participating in condensate formation, and uncovers a role of condensates in regulating global cellular physiology.

Magnesium Flux Modulates Ribosomes to Increase Bacterial Survival.

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

Acetate Production from Glucose and Coupling to Mitochondrial Metabolism in Mammals.

Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.

Lactate Metabolism in Human Lung Tumors.

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Lactate is an epigenetic metabolite that drives survival in model systems of glioblastoma.

Lactate accumulates to a significant amount in glioblastomas (GBMs), the most common primary malignant brain tumor with an unfavorable prognosis. However, it remains unclear whether lactate is metabolized by GBMs. Here, we demonstrated that lactate rescued patient-derived xenograft (PDX) GBM cells from nutrient-deprivation-mediated cell death. Transcriptome analysis, ATAC-seq, and ChIP-seq showed that lactate entertained a signature of oxidative energy metabolism. LC/MS analysis demonstrated that U-13C-lactate elicited substantial labeling of TCA-cycle metabolites, acetyl-CoA, and histone protein acetyl-residues in GBM cells. Lactate enhanced chromatin accessibility and histone acetylation in a manner dependent on oxidative energy metabolism and the ATP-citrate lyase (ACLY). Utilizing orthotopic PDX models of GBM, a combined tracer experiment unraveled that lactate carbons were substantially labeling the TCA-cycle metabolites. Finally, pharmacological blockage of oxidative energy metabolism extended overall survival in two orthotopic PDX models in mice. These results establish lactate metabolism as a novel druggable pathway for GBM.

Saturation of the mitochondrial NADH shuttles drives aerobic glycolysis in proliferating cells.

Proliferating cells exhibit a metabolic phenotype known as "aerobic glycolysis," which is characterized by an elevated rate of glucose fermentation to lactate irrespective of oxygen availability. Although several theories have been proposed, a rationalization for why proliferating cells seemingly waste glucose carbon by excreting it as lactate remains elusive. Using the NCI-60 cell lines, we determined that lactate excretion is strongly correlated with the activity of mitochondrial NADH shuttles, but not proliferation. Quantifying the fluxes of the malate-aspartate shuttle (MAS), the glycerol 3-phosphate shuttle (G3PS), and lactate dehydrogenase under various conditions demonstrated that proliferating cells primarily transform glucose to lactate when glycolysis outpaces the mitochondrial NADH shuttles. Increasing mitochondrial NADH shuttle fluxes decreased glucose fermentation but did not reduce the proliferation rate. Our results reveal that glucose fermentation, a hallmark of cancer, is a secondary consequence of MAS and G3PS saturation rather than a unique metabolic driver of cellular proliferation.

Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function.

Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+.

Estrogen-related receptors are targetable ROS sensors.

Excessive reactive oxygen species (ROS) can cause oxidative stress and consequently cell injury contributing to a wide range of diseases. Addressing the critical gaps in our understanding of the adaptive molecular events downstream ROS provocation holds promise for the identification of druggable metabolic vulnerabilities. Here, we unveil a direct molecular link between the activity of two estrogen-related receptor (ERR) isoforms and the control of glutamine utilization and glutathione antioxidant production. ERRα down-regulation restricts glutamine entry into the TCA cycle, while ERRγ up-regulation promotes glutamine-driven glutathione production. Notably, we identify increased ERRγ expression/activation as a hallmark of oxidative stress triggered by mitochondrial disruption or chemotherapy. Enhanced tumor antioxidant capacity is an underlying feature of human breast cancer (BCa) patients that respond poorly to treatment. We demonstrate that pharmacological inhibition of ERRγ with the selective inverse agonist GSK5182 increases antitumor efficacy of the chemotherapeutic paclitaxel on poor outcome BCa tumor organoids. Our findings thus underscore the ERRs as novel redox sensors and effectors of a ROS defense program and highlight the potential therapeutic advantage of exploiting ERRγ inhibitors for the treatment of BCa and other diseases where oxidative stress plays a central role.

Uptake-independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates.

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX-1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface-exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX-1-mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake-independent killing of macrophages and that the 50-kDa isoform of the ESX-1-secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX-1 inhibitor reduces uptake-independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti-phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.

Massive seasonal high-altitude migrations of nocturnal insects above the agricultural plains of East China.

Long-distance migrations of insects contribute to ecosystem functioning but also have important economic impacts when the migrants are pests or provide ecosystem services. We combined radar monitoring, aerial sampling, and searchlight trapping, to quantify the annual pattern of nocturnal insect migration above the densely populated agricultural lands of East China. A total of ~9.3 trillion nocturnal insect migrants (15,000 t of biomass), predominantly Lepidoptera, Hemiptera, and Diptera, including many crop pests and disease vectors, fly at heights up to 1 km above this 600 km-wide region every year. Larger migrants (>10 mg) exhibited seasonal reversal of movement directions, comprising northward expansion during spring and summer, followed by southward movements during fall. This north-south transfer was not balanced, however, with southward movement in fall 0.66× that of northward movement in spring and summer. Spring and summer migrations were strongest when the wind had a northward component, while in fall, stronger movements occurred on winds that allowed movement with a southward component; heading directions of larger insects were generally close to the track direction. These findings indicate adaptations leading to movement in seasonally favorable directions. We compare our results from China with similar studies in Europe and North America and conclude that ecological patterns and behavioral adaptations are similar across the Northern Hemisphere. The predominance of pests among these nocturnal migrants has severe implications for food security and grower prosperity throughout this heavily populated region, and knowledge of their migrations is potentially valuable for forecasting pest impacts and planning timely management actions.

Uncovering underlying physical principles and driving forces of cell differentiation and reprogramming from single-cell transcriptomics.

Recent advances in single-cell sequencing technology have revolutionized our ability to acquire whole transcriptome data. However, uncovering the underlying transcriptional drivers and nonequilibrium driving forces of cell function directly from these data remains challenging. We address this by learning cell state vector fields from discrete single-cell RNA velocity to quantify the single-cell global nonequilibrium driving forces as landscape and flux. From single-cell data, we quantified the Waddington landscape, showing that optimal paths for differentiation and reprogramming deviate from the naively expected landscape gradient paths and may not pass through landscape saddles at finite fluctuations, challenging conventional transition state estimation of kinetic rate for cell fate decisions due to the presence of the flux. A key insight from our study is that stem/progenitor cells necessitate greater energy dissipation for rapid cell cycles and self-renewal, maintaining pluripotency. We predict optimal developmental pathways and elucidate the nucleation mechanism of cell fate decisions, with transition states as nucleation sites and pioneer genes as nucleation seeds. The concept of loop flux quantifies the contributions of each cycle flux to cell state transitions, facilitating the understanding of cell dynamics and thermodynamic cost, and providing insights into optimizing biological functions. We also infer cell-cell interactions and cell-type-specific gene regulatory networks, encompassing feedback mechanisms and interaction intensities, predicting genetic perturbation effects on cell fate decisions from single-cell omics data. Essentially, our methodology validates the landscape and flux theory, along with its associated quantifications, offering a framework for exploring the physical principles underlying cellular differentiation and reprogramming and broader biological processes through high-throughput single-cell sequencing experiments.

A microtubule stability switch alters isolated vascular smooth muscle calcium flux in response to matrix rigidity.

During ageing, the extracellular matrix of the aortic wall becomes more rigid. In response, VSMCs generate enhanced contractile forces. Our previous findings demonstrate that VSMC volume is enhanced in response to increased matrix rigidity, but our understanding of mechanisms regulating this process remain incomplete. In this current study, we show that microtubule stability in VSMCs is reduced in response to enhanced matrix rigidity via piezo1-mediated Ca2+ influx. Moreover, VSMC volume and Ca2+ flux was regulated by microtubule dynamics; microtubule stabilising agents reduced both VSMC volume and Ca2+ flux on rigid hydrogels, whereas microtubule destabilising agents increased VSMC volume and Ca2+ flux on pliable hydrogels. Finally, we show that disruption of the microtubule deacetylase HDAC6 uncoupled these processes and increased K40 alpha tubulin acetylation, VSMC volume and Ca2+ flux on pliable hydrogels, but did not alter VSMC microtubule stability. These findings uncover a microtubule stability switch that controls VSMC volume by regulating Ca2+ flux. Together, these data demonstrate that manipulation of microtubule stability can modify VSMC response to matrix stiffness.

Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis.

The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using 13C and 2H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.

Interactome Analysis Identifies the Role of BZW2 in Promoting Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Metabolism.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

Aquatic biomass is a major source to particulate organic matter export in large Arctic rivers.

Arctic rivers provide an integrated signature of the changing landscape and transmit signals of change to the ocean. Here, we use a decade of particulate organic matter (POM) compositional data to deconvolute multiple allochthonous and autochthonous pan-Arctic and watershed-specific sources. Constraints from carbon-to-nitrogen ratios (C:N), δ13C, and Δ14C signatures reveal a large, hitherto overlooked contribution from aquatic biomass. Separation in Δ14C age is enhanced by splitting soil sources into shallow and deep pools (mean ± SD: -228 ± 211 vs. -492 ± 173‰) rather than traditional active layer and permafrost pools (-300 ± 236 vs. -441 ± 215‰) that do not represent permafrost-free Arctic regions. We estimate that 39 to 60% (5 to 95% credible interval) of the annual pan-Arctic POM flux (averaging 4,391 Gg/y particulate organic carbon from 2012 to 2019) comes from aquatic biomass. The remainder is sourced from yedoma, deep soils, shallow soils, petrogenic inputs, and fresh terrestrial production. Climate change-induced warming and increasing CO2 concentrations may enhance both soil destabilization and Arctic river aquatic biomass production, increasing fluxes of POM to the ocean. Younger, autochthonous, and older soil-derived POM likely have different destinies (preferential microbial uptake and processing vs. significant sediment burial, respectively). A small (~7%) increase in aquatic biomass POM flux with warming would be equivalent to a ~30% increase in deep soil POM flux. There is a clear need to better quantify how the balance of endmember fluxes may shift with different ramifications for different endmembers and how this will impact the Arctic system.

Assessing the costs of ozone pollution in India for wheat producers, consumers, and government food welfare policies.

We assess wheat yield losses occurring due to ozone pollution in India and its economic burden on producers, consumers, and the government. Applying an ozone flux-based risk assessment, we show that ambient ozone levels caused a mean 14.18% reduction in wheat yields during 2008 to 2012. Furthermore, irrigated wheat was particularly sensitive to ozone-induced yield losses, indicating that ozone pollution could undermine climate-change adaptation efforts through irrigation expansion. Applying an economic model, we examine the effects of a counterfactual, "pollution-free" scenario on yield losses, wheat prices, consumer and producer welfare, and government costs. We explore three policy scenarios in which the government support farmers at observed levels of either procurement prices (fixed-price), procurement quantities (fixed-procurement), or procurement expenditure (fixed-expenditure). In pollution-free conditions, the fixed-price scenario absorbs the fall in prices, thus increasing producer welfare by USD 2.7 billion, but total welfare decreases by USD 0.24 billion as government costs increase (USD 2.9 billion). In the fixed-procurement and fixed-expenditure scenarios, ozone mitigation allows wheat prices to fall by 38.19 to 42.96%. The producers lose by USD 5.10 to 6.01 billion, but the gains to consumers and governments (USD 8.7 to 10.2 billion) outweigh these losses. These findings show that the government and consumers primarily bear the costs of ozone pollution. For pollution mitigation to optimally benefit wheat production and maximize social welfare, new approaches to support producers other than fixed-price grain procurement may be required. We also emphasize the need to consider air pollution in programs to improve agricultural resilience to climate change.

Beyond spreading rate: Controls on the thermal regime of mid-ocean ridges.

The thermal state of mid-ocean ridges exerts a crucial modulation on seafloor spreading processes that shape ~2/3 of our planet's surface. Standard thermal models treat the ridge axis as a steady-state boundary layer between the hydrosphere and asthenosphere, whose thermal structure primarily reflects the local spreading rate. This framework explains the deepening of axial melt lenses (AMLs)-a proxy for the basaltic solidus isotherm-from ~1 to ~3 km from fast- to intermediate-spreading ridges but fails to account for shallow crustal AMLs documented at slow-ultraslow spreading ridges. Here, we show that these can be explained by a numerical model that decouples the potentially transient ridge magma supply from spreading rate, captures the essential physics of hydrothermal convection, and considers multiple modes of melt emplacement. Our simulations show that melt flux is a better thermal predictor than spreading rate. While multiple combinations of melt/dike emplacement modes, permeability structure, and temporal fluctuations of melt supply can explain shallow crustal AMLs at slow-ultraslow ridges, they all require elevated melt fluxes compared to most ridge sections of comparable spreading rates. This highlights the importance of along-axis melt focusing at slow-ultraslow ridges and sheds light on the natural variability of their thermal regimes.

Engineering a universal and efficient platform for terpenoid synthesis in yeast.

Engineering microbes for the production of valuable natural products is often hindered by the regulation of native competing metabolic networks in host. This is particularly evident in the case of terpenoid synthesis in yeast, where the canonical terpenoid precursors are tightly coupled to the biosynthesis of sterols essential for yeast viability. One way to circumvent this limitation is by engineering product pathways less connected to the host native metabolism. Here, we introduce a two-step isopentenol utilization pathway (IUP) in Saccharomyces cerevisiae to augment the native mevalonate pathway by providing a shortcut to the synthesis of the common terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). As such, the IUP was capable of elevating the IPP/DMAPP pool by 147-fold compared with the native pathway. We further demonstrate that cofeeding isoprenol and prenol enhances geranyl diphosphate (GPP) content for monoterpene biosynthesis. More importantly, we established a synthetic three-step route for efficient synthesis of di-and tetraterpene precursor geranylgeranyl diphosphate (GGPP), circumventing the competition with farnesyl diphosphate (FPP) for sterol biosynthesis and elevating the GGPP level by 374-fold. We combine these IUP-supported precursor-forming platforms with downstream terpene synthases to harness their potential and improve the production of industrially relevant terpenoids by several fold. Our exploration provides a universal and effective platform for supporting terpenoid synthesis in yeast.

Non-equilibrium early-warning signals for critical transitions in ecological systems.

Complex systems can exhibit sudden transitions or regime shifts from one stable state to another, typically referred to as critical transitions. It becomes a great challenge to identify a robust warning sufficiently early that action can be taken to avert a regime shift. We employ landscape-flux theory from nonequilibrium statistical mechanics as a general framework to quantify the global stability of ecological systems and provide warning signals for critical transitions. We quantify the average flux as the nonequilibrium driving force and the dynamical origin of the nonequilibrium transition while the entropy production rate as the nonequilibrium thermodynamic cost and thermodynamic origin of the nonequilibrium transition. Average flux, entropy production, nonequilibrium free energy, and time irreversibility quantified by the difference in cross-correlation functions forward and backward in time can serve as early warning signals for critical transitions much earlier than other conventional predictors. We utilize a classical shallow lake model as an exemplar for our early warning prediction. Our proposed method is general and can be readily applied to assess the resilience of many other ecological systems. The early warning signals proposed here can potentially predict critical transitions earlier than established methods and perhaps even sufficiently early to avert catastrophic shifts.

Nitrate alleviates ammonium toxicity in Brassica napus by coordinating rhizosphere and cell pH and ammonium assimilation.

In natural and agricultural situations, ammonium ( NH 4 + ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as NH 4 + toxicity. Nitrate ( NO 3 - ) has long been recognized to reduce NH 4 + toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high NH 4 + . Here, we found that NO 3 - can mitigate NH 4 + toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. NO 3 - increased the uptake of NO 3 - and NH 4 + under high NH 4 + circumstances by triggering the expression of NO 3 - and NH 4 + transporters, while NO 3 - and H+ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of NO 3 - efflux transporters and genes encoding plasma membrane H+ -ATPase. In addition, NO 3 - increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by NO 3 - under high NH 4 + conditions to enhance the assimilation of NH 4 + into amino acids, thereby reducing NH 4 + accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. NO 3 - might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate NH 4 + toxicity. Collectively, our results indicate that NO 3 - balances both rhizosphere and intracellular pH via effective NO 3 - transmembrane cycling, accelerates NH 4 + assimilation, and up-regulates malate metabolism to mitigate NH 4 + toxicity in oilseed rape.