Tracer Experiments Research Articles

Nitrogen form differently modulates nitrogen uptake and utilization and related gene expression between two tea cultivars

Nitrogen (N) is essential for the growth and development of tea plants, and ammonium (NH4+) and nitrate (NO3-) are crucial N sources for tea yield and amino acid contents. However, the uptake and utilization of different N forms in tea plants are different. Reasonable N form is an important means to enhance the growth and development of tea plants. Therefore, supplying suitable N forms may be effective way to optimize N use efficiency. A hydroponic trial was conducted with 'Chuancha No.2′ (CC) and 'Emeiwenchun' (EW) tea cultivars with N form treatments: NH4+ and NO3-. The results showed that there were significant difference in the NH4+/NO3- uptake kinetics, dynamic changes in 15N abundance, enzyme activities, and related gene expression in N metabolism between CC and EW after NH4+/NO3- treatment. In CC and EW, NH4+ and NO3- uptake followed Michaelis-Menten kinetics at low N concentrations (< 1 mmol L-1), but CC exhibited a slightly higher uptake rate than EW when supplied with 2 mmol L-1 NH4+. In a 0-24h 15N tracing experiment, CC roots accumulated 15N faster than EW under NH4+. NH4+-fed CC exhibited higher GS activities and higher expression levels of CsAMTs and genes involved in N utilization, such as CsNR, CsGS2, CsGDH1, and CsTS1, compared to EW. When NO3- was provided, EW roots accumulated more 15N than CC roots from 8-24 h, which could be attributed to the higher NR activities and higher expression levels of CsNRT1.5, CsNR, CsGS1.1 and CsGS1.2 than those in CC. In summary, the two tea varieties exhibited distinct characteristics of N uptake and utilization, as well as gene expression patterns under NH4+/NO3- treatments. CC demonstrated an advantage in NH4+ uptake and assimilation, while EW exhibited an advantage in NO3-.

Quantifying protein kinetics in vivo: Influence of precursor dynamics on product labeling.

Protein kinetics can be quantified by coupling stable isotope tracer methods with mass spectrometry readouts; however, inter-connected decision points in the experimental design affect the complexity of the workflow and impact data interpretations. For example, choosing between a single bolus (pulse-chase) or a continuous exposure protocol influences subsequent decisions regarding when to measure and how to model the temporal labeling of a target protein. Herein, we examine the merits of in vivo tracer protocols, we direct attention towards stable isotope tracer experiments that rely on administering a single bolus since these are generally more practical to use as compared to continuous administration protocols. We demonstrate how the interplay between precursor and product kinetics impacts downstream analytics and calculations by contrasting fast vs slow turnover precursors (e.g. 13C-leucine vs 2H-water, respectively). Although the data collected here underscore certain advantages of using longer lived precursors (e.g. 2H- or 18O-water) the results also highlight the influence of tracer recycling on measures of protein turnover. We discuss the impact of tracer recycling and consider how the sampling interval is critical for interpreting studies. Finally, we demonstrate that tracer recycling does not limit the ability to perform back-to-back studies of protein kinetics. It is possible to run experiments in which subjects are used as their own controls even though the precursor and product remain labeled following an initial tracer dosing.

Snowmelt-mediated isotopic homogenization of shallow till soil

Abstract. The hydrological cycle of sub-arctic areas is dominated by the snowmelt event. An understanding of the mechanisms that control water fluxes during high-volume infiltration events in sub-arctic till soils is needed to assess how future changes in the timing and magnitude of snowmelt can affect soil water storage dynamics. We conducted a tracer experiment in which deuterated water was used to irrigate a plot on a forested hilltop in Lapland, tracked water fluxes of different mobility and monitored how the later snowmelt modifies the labelled soil water storage. We used lysimeters and destructive soil coring for soil water sampling and monitored and sampled the groundwater. Large spatiotemporal variability between the waters of different mobility was observed in the subsurface, while surface water flow during the tracer experiment was largely controlled by a fill-and-spill mechanism. Extensive soil saturation induced the flow of labelled water into the roots of nearby trees. We found that labelled water remained in deeper soil layers over the winter, but the snowmelt event gradually displaced all deuterated water and fully homogenized all water fluxes at the soil–vegetation interface. The conditions required for the full displacement of the old soil water occur only during a snowmelt with a persistently high groundwater table. We propose a conceptual model where infiltration into the soil and eventual soil water replenishment occur in three stages. First, unsaturated macropore flow is initiated via the surface microtopography and is directed towards the groundwater storage. The second stage is characterized by groundwater rise through the macropore network, subsequent pore water saturation and increased horizontal connectivity of macropores. Shallow subsurface lateral fluxes develop in more permeable shallow soil layers. In the third stage, which materializes during a long period with a high groundwater table and high hydrological connectivity within the soil, the soil water is replenished via enhanced matrix flow and pore water exchange with the macropore network.

Exploring hydrogen isotope fractionation in lipid biomolecules of freshwater algae: implications for ecological and paleoenvironmental studies

ABSTRACT Understanding the stable hydrogen isotope (δ 2H) composition and fractionation in lipid biomolecules of primary producers, such as terrestrial and aquatic plants, is crucial for deciphering past environmental conditions, as well as applying compound-specific stable isotope analysis for the study of metabolic and ecological processes. We conducted a new tracer experiment to explore the δ 2H composition of algal fatty acid biomarkers, focusing on freshwater algae, which form the base of aquatic food webs. We selected a range of algal species widely found in freshwater ecosystems and cultivated them under controlled conditions. First, we added 2H2O to ambient water as a tracer to investigate the net hydrogen isotope fractionation during algal lipid synthesis at isotopic equilibrium, which is particularly informative for paleo-geochemical studies. Then, we conducted kinetic experiments to quantify the time needed for algal fatty acids to achieve isotopic steady-state conditions in response to the change in ambient water δ 2H values. Our findings revealed substantial variability in hydrogen isotope fractionation among different algal taxa and various fatty acids. Based on taxa, different fatty acids exhibited faster integration of water hydrogen than others, but they were not necessarily in the order of the biosynthetic pathway. This experiment underscores the complexity of hydrogen isotope fractionation and the requirement for controlled laboratory studies to properly apply compound-specific stable H isotope analysis techniques in ecological and paleo-environmental studies.

Effects of mono- and multicomponent nonaqueous-phase liquid on the migration and retention of pollutant-degrading bacteria in porous media

The successful implementation of in-situ bioremediation of nonaqueous-phase liquid (NAPL) contamination in soil-groundwater systems is greatly influenced by the migration performance of NAPL-degrading bacteria. However, the impact and mechanisms of NAPL on the migration/retention of pollutant-degrading bacteria remain unclear. This study investigated the migration/retention performance of A. lwoffii U1091, a strain capable of degrading diesel while producing surfactants, in porous media without and with the presence of mono- and multicomponent NAPL (n-dodecane and diesel) under environmentally relevant conditions. The results showed that under all examined conditions (5 and 50 mM NaCl solution at flow rates of 4 and 8 m/d), the presence of n-dodecane/diesel in porous media could reduce the migration and enhance retention of A. lwoffii in quartz sand columns. Moreover, comparing with mutlicomponent NAPLs of n-dodecane, the monocomponent NAPLs (diesel) exhibited a greater reduction effect on the retention of A. lwoffii in porous media. Through systemically investigating the potential mechanisms via tracer experiment, visible chamber experiment, and theoretical calculation, we found that the reduction in porosity, repulsive forces and movement speeds, the presence of stagnant flow zones in porous media, particularly the biosurfactants generated by A. lwoffii contributed to the enhanced retention of bacteria in NAPL-contaminated porous media. Moreover, owing to presence of the greater amount of hydrophilic components in diesel than in n-dodecane, the available binding sites for the adsorption of bacteria were lower in diesel, resulting in the slightly decreased retention of A. lwoffii in porous media containing diesel than n-dodecane. This study demonstrated that comparing with porous media without NAPL contamination, the retention of strain capable of degrading NAPL in porous media with NAPL contamination was enhanced, beneficial for the subsequent biodegradation of NAPL.

Impact of freeze-thaw cycles on the remobilization behaviors of microplastics in natural soils

Freeze-thaw (FT) cycle would greatly influence the fate of plastic particles (one of emerging contaminants with great concerns) in soils, yet its impacts and mechanisms remain unclear. The vertical migration/release behaviors of plastic particles (with diameters of 0.2 μm and 1 μm) in two natural soils and one model soil (i.e. quartz sand) without/with FT treatments (1 and 3 cycles) were examined. Owing to the presence of Fe/Al oxide minerals, finer pore structure, and uneven surfaces of natural soils, the breakthrough ratio of plastics in two natural soils was over 25% lower than in quartz sand. However, regardless of porous media type, FT processes (increasing cycles) significantly promoted the remobilization of plastics initially retained in three media during the subsequent water flushing processes. Via theoretical calculation, tracer experiments, and visible chamber experiments, the mechanisms driving plastics release from natural soils subjected to FT treatments during the water elution processes were determined to be different from those from pure quartz sand. The change of sand local configuration (the rearrangement of local sand pore spaces) during FT process mainly drove to plastics released from quartz sand columns. While the alteration in local soil configuration, the formation of preferential pathways, and increased release of soil particles contributed to plastics remobilization from soil columns subjected to FT. Clearly, FT processes significantly increased the vertical migration of plastics in soils potentially to groundwater, enhancing environmental risks of plastics.

Hyperspectral Image Transects during Transient Events in Rivers (HITTER): Framework Development and Application to a Tracer Experiment on the Missouri River, USA

Rivers convey a broad range of materials, such as sediment, nutrients, and contaminants. Much of this transport can occur during or immediately after an episodic, pulsed event like a flood or an oil spill. Understanding the flow processes that influence the motion of these substances is important for managing water resources and conserving aquatic ecosystems. This study introduces a new remote sensing framework for characterizing dynamic phenomena at the scale of a channel cross-section: Hyperspectral Image Transects during Transient Events in Rivers (HITTER). We present a workflow that uses repeated hyperspectral scan lines acquired from a hovering uncrewed aircraft system (UAS) to quantify how a water attribute of interest varies laterally across the river and evolves over time. Data from a tracer experiment on the Missouri River are used to illustrate the components of the end-to-end processing chain we used to quantify the passage of a visible dye. The framework is intended to be flexible and could be applied in a number of different contexts. The results of this initial proof-of-concept investigation suggest that HITTER could potentially provide insight regarding the dispersion of a range of materials in rivers, which would facilitate ecological and geomorphic studies and help inform management.

Structural diversity inside the mouse subiculum revealed by a new marker protein fibronectin 1.

The subiculum is one of the major output structures of the hippocampal formation and is an important brain region for memory. We have previously reported that the subiculum of rodents can be morphologically divided into its temporal (ventral) two-thirds and the septal (dorsal) third and that the former can be further subdivided into the distal (Sub1) and proximal (Sub2) regions, on a basis of immunohistochemical localizations of several Sub2-specific proteins. However, it remains unclear whether detailed structural organization found in the temporal subiculum is applicable to the septal subiculum. In this study, we found that the distribution of fibronectin (FN1)-positive non-GABAergic, presumptive pyramidal cells exactly coincided with the extent of the Sub1 region of male mice. Using FN1 immunohistochemistry, the Sub1 was found to keep relatively constant size throughout the septotemporal axis of the subiculum. In contrast, the size of the Sub2 became smaller as it approached the septal side, and the Sub2 finally disappeared at the most septal level of the subiculum. Retrograde tracer experiments confirmed that FN1-positive Sub1 neurons projected to the retrosplenial cortex, which is thought to be associated with spatial memory, whereas FN1-negative Sub2 neurons projected to the nucleus accumbens associated with emotional memory. Considering both the functional segregation of these two subicular targets and the relative abundance of the Sub2 on the temporal side, the subiculum can be one of the neural substrates for functional differences between the septal and temporal hippocampal formation associated with the spatial and emotional memory, respectively.

NMR-Based Stable Isotope Tracing of Cancer Metabolism.

NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.e., isotopomer distribution analysis).In this protocol, we give an overview with specific protocols for NMR-based stable isotope-resolved metabolomics, or SIRM, with a workflow from administration of isotope-enriched precursors, via sample preparation through to NMR data collection and reduction. We focus on indirect detection of common NMR-active stable isotopes including 13C, 15N, 31P, and 2H, using a variety of 1H-based two-dimensional experiments. We also include the application and analyses of multiplex tracer experiments.

The effect of Southern Ocean topography on the global MOC and abyssal water mass distribution

Abstract We investigate the role of Southern Ocean topography and wind stress in the deep and abyssal ocean overturning and water mass composition using a suite of idealized global ocean circulation models. Specifically, we address how the presence of a meridional ridge in the vicinity of Drake Passage and the formation of an associated Southern Ocean gyre influences the water mass composition of the abyssal cell. Our experiments are carried out using a numerical representation of the global ocean circulation in an idealized two-basin geometry under varying wind-stress and Drake Passage ridge height. In the presence of a low Drake Passage ridge the overall strength of the meridional overturning circulation is primarily influenced by wind stress, with a topographically-induced weakening of the mid-depth cell and concurrent strengthening of the abyssal cell occurring only after ridge height passes 2500m. Passive tracer experiments show that a strengthening mid-depth cell leads to increased abyssal ventilation by North Atlantic water masses, as more North Atlantic Deep Water (NADW) enters the Southern Ocean and then spreads into the Indo-Pacific. We repeat our tracer experiments without restoring in the high-latitude Southern Ocean in order to identify the origin of water masses that circulate through the Southern Ocean before sinking into the abyss as Antarctic Bottom Water. Our results from these “exchange” tracer experiments show that an increasing ridge height in Drake Passage and the concurrent gyre spin-up lead to substantially decreased NADW-origin waters in the abyssal ocean, as more surface waters from north of the ACC are transferred into the Antarctic Bottom Water formation region.

Modelling the recovery time from peak loads in a full-scale horizontal flow wetland in Sicily

The aim of this study was to simulate the water flow and reactive transport of pollutants in a horizontal flow (HF) wetland to better understand the recovery time of the treatment performance for peak load events. For the simulation, the processes-based model HYDRUS and its Wetland Module is used. The system under investigation is the first stage of the 9-years old hybrid treatment wetland of a large retail store, located in Catania, Italy. For the calibration of the hydraulic model, the data of a tracer test was used. The data set of the systems is available for a seven year period including organic matter and ammonia nitrogen. The data was split into a standard event representing low loading conditions and determined peak load events with high loadings. The results show that the response time of the model correlates with the hydraulic retention time from the tracer experiment and indicates that higher peak load concentrations at the inlet of the system lead to a longer recovery time of the wetland.

Model-driven design and validation of glucose supply control in a tubular photobioreactor operated under oxygen-balanced mixotrophy

Scaling up bioprocesses remains challenging due to the partially unpredictable and suboptimal behavior of microbes during scale transition. Mathematical modelling serves as a valuable tool in navigating these challenges. In this study, we developed and validated a kinetic model based on the tank-in-series approach for a novel bioprocess known as oxygen-balanced mixotrophy with the microalga Galdieria sulphuraria. This model aims to facilitate the design of an effective glucose feeding control strategy tailored for two-phase tubular photobioreactors (TPBRs). The residence time distribution of our reference reactor was investigated by means of a tracer experiment. Qualitative analysis of the results indicated that our system was optimally represented by simulations with a 100 stirred-tank reactors (STRs).The controlled variable, oxygen concentration in the gas phase, showed a downward trend in simulations with simplified conditions: fixed oxygen production and balanced glucose supply. The decrease was caused by dominant net heterotrophic activity and accumulation of CO2 in the gas phase. Different configurations of a proportional-integral-derivative (PID) controller were designed by means of the ultimate gain method and compared under these simplified conditions. The most effective PID controller was implemented and refined in simulations with outdoor weather conditions. The model was validated successfully by simulating over time in a lab-scale STR the spatial fluctuations happening in a TPBR. The empirical oxygen consumption and production were faster and steeper than in the model, probably due to inaccuracies in parameter estimation or biomass adaptation.

Tailor-made polymer tracers reveal the role of clay minerals on colloidal transport in carbonate media

HypothesisHost rock weathering and incipient pedogenesis result in the exposition of minerals, e.g., clay minerals in sedimentary limestones. Once exposed, these minerals provide the surfaces for fluid–solid interactions that control the fate of dissolved or suspended compounds such as organic matter and colloids. However, the functional and compositional diversity of organic matter and colloids limits the assessment of reactivity and availability of clay mineral interfaces. Such assessment demands a mobile compound with strong affinity to clay surfaces that is alien to the subsurface. ExperimentWe approached this challenge by using poly(ethylene glycol) (PEG) as interfacial tracer in limestone weathering experiments. FindingsPEG adsorption and transport was dependent on the availability of clay mineral surfaces and carbonate dissolution dynamics. In addition, PEG adsorption featured adsorption–desorption hysteresis which retained PEG mass on clay mineral surfaces. This resulted in different PEG transport for experiments conducted consecutively in the same porous medium. As such, PEG transport was reconstructed with a continuum-scale model parametrized by a Langmuir-type isotherm including hysteresis. Thus, we quantified the influence of exposed clay mineral surfaces on the transport of organic colloids in carbonate media. This renders PEG a suitable model colloid tracer for the assessment of clay surface exposition in porous media.

Preparation of PBAT microplastics and their potential toxicity to zebrafish embryos and juveniles

The extensive use of traditional non-biodegradable plastics results in the generation of microplastics (MPs), forming a new pollutant that can pose significant environmental risks. Biodegradable plastics (BP) possess degradation properties and can partially replace conventional plastics, thereby reducing pollution. However, further investigation is needed into the toxicity of biodegradable microplastics (BMPs) on aquatic organisms. This study explores the toxic effects of PBAT microplastics (PBAT-BMPs) and microplastics produced from degradable PBAT/TPS (thermoplastic starch) composite film (PBAT/TPS-BMPs) on zebrafish embryos. Our findings indicate that the presence of microplastics on the embryo's surface increases with higher BMPs concentration. Nonetheless, PBAT-BMPs tend to aggregate and are blocked by the embryonic membrane, thus diminishing their toxic effects on the embryo. Acute toxicity experiments revealed that 30 mg/L of PBAT-BMPs significantly reduced the survival rate of zebrafish embryos, whereas PBAT/TPS-BMPs had a lesser effect on survival. Both types of BMPs influenced the hatching rate of the embryos, leading to prolonged incubation periods. Additionally, both types of BMPs impacted the locomotor behavior of zebrafish larvae, causing an increase in larval locomotor speed. However, these BMPs had little impact on larval body development and heartbeat behavior. Fluorescent microplastic tracer experiments demonstrated that PBAT-BMPs persisted in juvenile fish for at least 144 h and were difficult to metabolize and excrete. Our study aims to gain a better understanding of the potential effects of BMPs on aquatic ecosystems and biological health, as well as to propose effective strategies for reducing environmental pollution and protecting organisms.

Enhancing soil gross nitrogen transformation through regulation of microbial nitrogen-cycling genes by biodegradable microplastics

Microplastics (MPs) in agricultural plastic film mulching system changes microbial functions and nutrient dynamics in soils. However, how biodegradable MPs impact the soil gross nitrogen (N) transformations and crop N uptake remain significantly unknown. In this study, we conducted a paired labeling 15N tracer experiment and microbial N-cycling gene analysis to investigate the dynamics and mechanisms of soil gross N transformation processes in soils amended with conventional (polyethylene, PE) and biodegradable (polybutylene adipate co-terephthalate, PBAT) MPs at concentrations of 0 %, 0.5 %, and 2 % (w/w). The biodegradable MPs-amended soils showed higher gross N mineralization rates (0.5–16 times) and plant N uptake rates (16–32 %) than soils without MPs (CK) and with conventional MPs. The MPs (both PE and PBAT) with high concentration (2 %) increased gross N mineralization rates compared to low concentration (0.5 %). Compare to CK, MPs decreased the soil gross nitrification rates, except for PBAT with 2 % concentration; while PE with 0.5 % concentration and PBAT with 2 % concentration increased but PBAT with 0.5 % concentration decreased the gross N immobilization rates significantly. The results indicated that there were both a concentration effect and a material effect of MPs on soil gross N transformations. Biodegradable MPs increased N-cycling gene abundance by 60–103 %; while there was no difference in the abundance of total N-cycling genes between soils without MPs and with conventional MPs. In summary, biodegradable MPs increased N cycling gene abundance by providing enriched nutrient substrates and enhancing microbial biomass, thereby promoting gross N transformation processes and maize N uptake in short-term. These findings provide insights into the potential consequences associated with the exposure of biodegradable MPs, particularly their impact on soil N cycling processes.

Microbial life in preferential flow paths in subsurface clayey till revealed by metataxonomy and metagenomics

BackgroundSubsurface microorganisms contribute to important ecosystem services, yet little is known about how the composition of these communities is affected by small scale heterogeneity such as in preferential flow paths including biopores and fractures. This study aimed to provide a more complete characterization of microbial communities from preferential flow paths and matrix sediments of a clayey till to a depth of 400 cm by using 16S rRNA gene and fungal ITS2 amplicon sequencing of environmental DNA. Moreover, shotgun metagenomics was applied to samples from fractures located 150 cm below ground surface (bgs) to investigate the bacterial genomic adaptations resulting from fluctuating exposure to nutrients, oxygen and water.ResultsThe microbial communities changed significantly with depth. In addition, the bacterial/archaeal communities in preferential flow paths were significantly different from those in the adjacent matrix sediments, which was not the case for fungal communities. Preferential flow paths contained higher abundances of 16S rRNA and ITS gene copies than the corresponding matrix sediments and more aerobic bacterial taxa than adjacent matrix sediments at 75 and 150 cm bgs. These findings were linked to higher organic carbon and the connectivity of the flow paths to the topsoil as demonstrated by previous dye tracer experiments. Moreover, bacteria, which were differentially more abundant in the fractures than in the matrix sediment at 150 cm bgs, had higher abundances of carbohydrate active enzymes, and a greater potential for mixotrophic growth.ConclusionsOur results demonstrate that the preferential flow paths in the subsurface are unique niches that are closely connected to water flow and the fluctuating ground water table. Although no difference in fungal communities were observed between these two niches, hydraulically active flow paths contained a significantly higher abundance in fungal, archaeal and bacterial taxa. Metagenomic analysis suggests that bacteria in tectonic fractures have the genetic potential to respond to fluctuating oxygen levels and can degrade organic carbon, which should result in their increased participation in subsurface carbon cycling. This increased microbial abundance and activity needs to be considered in future research and modelling efforts of the soil subsurface.

Enhancement of nitrogen removal in constructed wetlands through boron mud and elemental sulfur addition: Regulation of sulfur and oxygen cycling

Constructed wetlands (CWs) utilizing elemental sulfur (S0) as an electron donor are effective for nitrate (NO3–-N) removal from low C/N wastewater. However, the limited bioavailability of chemical S0 (chem-S0) due to low water solubility and inadequate mass transfer impedes complete denitrification. In this study, boron mud waste and S0 (S0&BM) were processed and combined to convert chem-S0 to biological S0 (bio-S0) in CWs. The employment of S0&BM improved NO3–-N and total nitrogen (TN) removal rates by 15.36 % and 10.55 % compared to S0 alone. The 15N isotope tracing experiment unveiled the pathways of N2O production and demonstrated that S0&BM facilitated full denitrification, resulting in a higher percentage of nitrogen being degraded in a non-polluting form (54.70 %). Releasing trace amounts of boron and magnesium in S0&BM mitigated the excessive acidification caused by the sulfur autotrophic process and promoted biofilm formation, thereby pronouncing vertical oxygen fluctuations. Meanwhile, oxygen vacancies generated in the S0&BM promoted the closed-loop sulfur cycle, starting from the oxidation of chem-S0 to sulfate (SO42-), the important process of SO42--reducing bacteria using partial organic compounds as electron donors to reduce SO42- to sulfide (S2-) was realized, and finally the S2- was oxidized to bio-S0 with better properties. S0&BM enriched functional microbial communities in the biofilm related to nitrogen and sulfur transformation. Functional gene analysis showed an increase in the narG, nirS, norB, and nosZ genes supporting denitrification. The dsrA and sqr genes responsible for the sulfur cycle exhibited the highest abundance. This realization allowed for the utilization of waste materials for treatment, enhancing the energy efficiency of carbon reduction and pollution reduction in CWs.

Research on CO Migration Patterns in Shallowly Buried Coal Seam Composite Goafs.

To address the issue of CO exceedance in the close-range composite goaf, this paper focuses on the composite goaf of the Shaping Mine as the research subject and investigated the migration dynamics of CO within shallowly buried composite goaf areas. Sulfur hexafluoride gas (SF6) tracer experiments were conducted at the 13103 working face in Shaping Mine to assess surface fissures and air leakage, yielding insights into the distribution patterns of air leakage channels and facilitating the identification of critical areas for channel sealing. Programmed heating and oxidation experiments were conducted on coal seams 8# and 13# to determine the CO generation patterns during coal oxidation. The results show that higher concentrations of O2 corresponded to elevated CO production at equivalent temperatures. Subsequent data analysis unveiled exponential relationships between the O2 consumption rate and CO production rate within the goaf area, offering a theoretical framework for understanding CO migration patterns. Through numerical simulations, the study analyzed the CO migration patterns in the composite goaf area, observing downward diffusion of CO emanating from coal oxidation in the overlying goaf areas followed by dispersion toward the working face and roadways. Driven by airflow dynamics, CO accrued in the return air corner of the working face. Building upon these insights, comprehensive CO management strategies were implemented, resulting in sustained reductions of temperatures and CO concentrations to safe levels within the original high-temperature, high-concentration CO zones. Notably, CO concentrations at the return air corner of the working face continued to decline over the management period, reaching below 24 ppm within 10 to 15 days, highlighting the effectiveness of the management measures in ensuring safe underground production.

A 3D-printed tumor-on-chip: user-friendly platform for the culture of breast cancer spheroids and the evaluation of anti-cancer drugs

Tumor-on-chips (ToCs) are useful platforms for studying the physiology of tumors and evaluating the efficacy and toxicity of anti-cancer drugs. However, the design and fabrication of a ToC system is not a trivial venture. We introduce a user-friendly, flexible, 3D-printed microfluidic device that can be used to culture cancer cells or cancer-derived spheroids embedded in hydrogels under well-controlled environments. The system consists of two lateral flow compartments (left and right sides), each with two inlets and two outlets to deliver cell culture media as continuous liquid streams. The central compartment was designed to host a hydrogel in which cells and microtissues can be confined and cultured. We performed tracer experiments with colored inks and 40 kDa fluorescein isothiocyanate dextran to characterize the transport/mixing performances of the system. We also cultured homotypic (MCF7) and heterotypic (MCF7-BJ) spheroids embedded in gelatin methacryloyl hydrogels to illustrate the use of this microfluidic device in sustaining long-term micro-tissue culture experiments. We further demonstrated the use of this platform in anticancer drug testing by continuous perfusion of doxorubicin, a commonly used anti-cancer drug for breast cancer. In these experiments, we evaluated drug transport, viability, glucose consumption, cell death (apoptosis), and cytotoxicity. In summary, we introduce a robust and friendly ToC system capable of recapitulating relevant aspects of the tumor microenvironment for the study of cancer physiology, anti-cancer drug transport, efficacy, and safety. We anticipate that this flexible 3D-printed microfluidic device may facilitate cancer research and the development and screening of strategies for personalized medicine.

Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.