Category: 7. Science

  • Twenty years of tuberculosis-driven selection shaped the evolution of the meerkat major histocompatibility complex

    Twenty years of tuberculosis-driven selection shaped the evolution of the meerkat major histocompatibility complex

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  • Juice team resolves anomaly on approach to Venus

    Juice team resolves anomaly on approach to Venus

    Enabling & Support

    25/08/2025
    2899 views
    39 likes

    The European Space Agency’s Jupiter Icy Moons Explorer (Juice) is on track for its gravity-assist flyby at Venus on 31 August, following the successful resolution of a spacecraft communication anomaly that temporarily severed contact with Earth.

    The issue, which emerged during a routine ground station pass on 16 July, temporarily disrupted Juice’s ability to transmit information about its health and status (telemetry).

    Thanks to swift and coordinated action by the teams at ESA’s European Space Operations Centre (ESOC) in Darmstadt, Germany, and Juice’s manufacturer, Airbus, communication was restored in time to prepare for the upcoming planetary encounter.

    Juice falls silent

    The anomaly began when ESA’s deep space antenna in Cebreros, Spain, failed to establish contact with Juice at the expected time of 04:50 CEST on 16 July. Initial checks ruled out ground station issues, prompting escalation to the Juice control team at ESOC. Attempts to reach the spacecraft via ESA’s New Norcia station also failed, confirming that the problem was on board.

    With no signal and no telemetry, engineers feared Juice might have entered survival mode – a last-resort configuration that is triggered by multiple onboard system failures. In such a state, the spacecraft spins slowly, sweeping its antenna across Earth once per hour. However, no such intermittent signal was detected.

    “Losing contact with a spacecraft is one of the most serious scenarios we can face,” said Angela Dietz, Juice Spacecraft Operations Manager. “With no telemetry, it is much more difficult to diagnose and resolve the root cause of an issue.”

    Attention turned to the communications subsystem. Engineers suspected either a misalignment of Juice’s medium-gain antenna or a failure in the signal transmitter or amplifier. 

    Juice is designed for the cold of the Jupiter system. The spacecraft is currently much closer to the Sun and must point its large, high-gain antenna towards the Sun to act as a heat shield. The steerable medium-gain antenna seen above is used to communicate with Earth during this period.

    Two recovery strategies were considered: waiting for the next automatic spacecraft reset in 14 days’ time, or sending commands ‘blind’ into space in the direction that Juice should be and hoping that they are received by one of the backup low-gain antennas.

    “Waiting was not an option,” explained Angela. “We had to act fast. Waiting two weeks for the reset would have meant delaying important preparations for the Venus flyby.”

    20 hours of troubleshooting

    Blind commanding was a challenge: Juice was around 200 million km from Earth and located on the other side of the Sun. It took each attempted rescue signal 11 minutes to reach the spacecraft, and the team then had to wait another 11 minutes to determine whether they had been successful.

    Six attempts to steer the medium-gain antenna back towards Earth were unsuccessful. Recovery efforts continued overnight, lasting almost 20 hours and focusing on manually powering up Juice’s onboard communication systems.

    Eventually, a command succeeded in reaching Juice and triggering a response. The command activated the signal amplifier that boosts the strength of the signal that Juice sends towards Earth. Contact was re-established, and Juice was found to be in excellent condition. No systems had failed, and all telemetry was nominal.

    The root cause was traced to a software timing bug. The software function that switches the signal amplifier on and off relies on an internal timer. This timer is constantly counting up and restarts from zero once every 16 months. If the function happens to be using the timer at the exact moment it restarts, the amplifier remains switched off, and Juice’s signal is too weak to detect from Earth.

    “It was a subtle bug, but one that we were prepared to investigate and resolve,” said Angela. “We have identified a number of possible ways to ensure that this does not happen again, and we are now deciding which solution would be the best to implement.”

    All clear for the Venus flyby

    Juice’s flyby of Venus (close-up)

    Despite the high stakes and technical complexity, the recovery by ESA’s mission operations team was achieved with minimal disruption.

    “This was a textbook example of teamwork under pressure,” said Angela. “Thanks to the team’s calm and methodical approach, we were able to recover Juice without any lasting impact on the mission.”

    With the anomaly behind them, the Juice team returned their focus to preparations for the Venus flyby. Juice will pass its closest point to Venus at 07:28 CEST on Sunday, 31 August as it completes the second of four planned gravity assists.

    Designed for the cold, dark environment of Jupiter, Juice must adapt to the intense solar heat near Venus. To protect its sensitive components, the spacecraft is using its main, high-gain antenna as a thermal shield. Due to thermal constraints, its remote sensing instruments cannot be active during the flyby, and so no images of Venus will be captured.

    Juice’s journey to Jupiter

    To go directly to Jupiter, Juice would have needed to leave Earth with a velocity of 11 km/s. However, Juice is one of the heaviest interplanetary spacecraft ever launched, at almost 6000 kg. With such a massive payload, its Ariane 5 launcher provided an escape velocity of 2.5 km/s.

    The spacecraft is using gravity-assist manoeuvres to pick up the rest of the required speed. Juice will use the gravity of Venus this week to bend its orbit around the Sun and gain speed relative to Earth without using fuel.

    Juice’s journey to Jupiter

    The Venus flyby will give Juice a significant boost. When it next encounters Earth in September 2026, the spacecraft will have reached the required Jupiter transfer velocity of 11 km/s. However, Jupiter won’t be in the right place to send Juice out towards it just yet.

    Juice will use the Earth flyby in 2026 to further fine-tune its trajectory. After one more orbit around the Sun, the spacecraft will return to Earth for a final flyby in January 2029. This flyby will be used to send Juice on a transfer trajectory that intercepts Jupiter in July 2031.

    About Juice

    ESA’s Jupiter Icy Moons Explorer, ‘Juice’, is humankind’s next bold mission to the outer Solar System. It will make detailed observations of gas giant Jupiter and its three large ocean-bearing moons – Ganymede, Callisto and Europa. This ambitious mission will characterise these moons with a powerful suite of remote sensing, geophysical and in situ instruments to discover more about these compelling destinations as potential habitats for past or present life.

    Juice will monitor Jupiter’s complex magnetic, radiation and plasma environment in depth and its interplay with the moons, studying the Jupiter system as an archetype for gas giant systems across the Universe.

    Juice launched on an Ariane 5 from Europe’s Spaceport in Kourou in April 2023. It has an eight-year cruise with flybys of Earth and Venus to slingshot it to Jupiter. It will make 35 flybys of the three large moons while orbiting Jupiter, before changing orbits to Ganymede and becoming the first spacecraft in history to orbit the moon of a gas giant.

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  • NASA’s Psyche asteroid probe beams home haunting view of distant Earth (photo)

    NASA’s Psyche asteroid probe beams home haunting view of distant Earth (photo)

    NASA’s Psyche spacecraft, which is headed toward a big and bizarre metal asteroid, has delivered a stunning perspective of our home planet from deep space.

    Psyche launched atop a SpaceX Falcon Heavy rocket in October 2023 with the objective of visiting the metallic asteroid 16 Psyche, which is believed to be the exposed core of a demolished planetesimal, or tiny planet.

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  • Malia Bee Pendant: A 3,800-year-old accessory found in a Minoan ‘pit of gold’

    Malia Bee Pendant: A 3,800-year-old accessory found in a Minoan ‘pit of gold’

    QUICK FACTS

    Name: Malia Bee Pendant

    What it is: A gold pendant

    Where it is from: Malia, Crete

    When it was made: Between 1800 and 1700 B.C.

    This gold pendant was discovered in 1930 at the cemetery of Chrysolakkos, which means “pit of gold,” in the ancient Minoan town of Malia in Crete. Although the famed archaeologist Sir Arthur Evans suggested the jewelry depicted bees, the identity of the insects on the pendant and the meaning behind the design have been debated for nearly a century.

    According to the Heraklion Archaeological Museum in Crete, where the pendant is on display, it is 1.8 inches (4.6 centimeters) long and weighs 0.2 ounces (5.5 grams) — about as much as a U.S. quarter. The ancient goldsmith combined several techniques to create the piece — filigree, granulation, repoussé and incised decoration — and the pendant is considered a “masterpiece of Minoan miniature art,” according to the museum.

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  • Nasa rover spots strange hat-shaped rock on Mars

    Nasa rover spots strange hat-shaped rock on Mars

    Nasa’s Perseverance Mars rover has spotted a bizarre helmet-shaped rock, shedding more light on how winds shape the Red Planet’s surface even today.

    The unusual “witch hat” or “helmet-shaped” rock composed entirely of smaller spherules drew attention online.

    “This hat-shaped rock is composed of spherules. This rock’s target name is Horneflya, and it’s distinctive less because of its hat shape and more because it’s made almost entirely of spherules,” David Agle, spokesperson for the Nasa Perseverance team, told Space.com.

    The pioneering Nasa rover is currently studying sand ripples on Mars to better understand how winds currently sculpt the Red Planet’s surface.

    While the rover has so far been focused on unravelling processes in Mars’ distant past that are recorded in ancient rocks, researchers have yet to further explore the science behind the modern Martian environment.

    For instance, the Perseverance rover’s predecessor, Curiosity, captured iconic images of the active sand dune at “Namib Dune” on the floor of the Martian Gale crater.

    But smaller megaripples as well as inactive dusty ones are also common across the surface of Mars.

    Recently, the rover explored a site called “Kerrlaguna,” where the steep slopes give way to a field of megaripples.

    Nasa's Mars Perseverance rover acquired this image of a witch hat or helmet-shaped rock 'Horneflya' (NASA/JPL-Caltech/ASU)

    Nasa’s Mars Perseverance rover acquired this image of a witch hat or helmet-shaped rock ‘Horneflya’ (NASA/JPL-Caltech/ASU)

    These are large windblown sand formations up to 1m (3ft) tall.

    After examining clay and olivine-rich rocks at a site named “Westport”, the rover began moving South.

    But as it attempted to climb toward a rock outcrop called “Midtoya”, the steep hill and its rubble-filled ground made progress nearly impossible, Nasa said.

    The Perseverance team then redirected the rover to smoother terrain where it spotted spherule-rich rocks like Horneflya, which likely rolled down from the Midtoya site.

    All these features recently spotted by Perseverance can help better understand the role played by wind and water on the modern Martian surface, scientists say.

    The Nasa rover uses its SuperCam, Mastcam-Z, and MEDA science instruments to characterise these environments, the size and chemistry of the sand grains and salty crusts that may have developed over time.

    These observations could help document and prepare terrain maps when future astronauts explore the Red Planet and potentially help them find resources within Martian soils to help them survive, researchers say.

    Scientists hope analysis of data collected from “Kerrlaguna” can provide a practice run for a more comprehensive campaign to a larger field of Martian rocks at “Lac de Charmes”, which lies further along the rover’s planned route.

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  • Apoplastic metabolomics reveals sugars as mesophyll messengers regulating guard cell ion transport under red light

    Apoplastic metabolomics reveals sugars as mesophyll messengers regulating guard cell ion transport under red light

    Plant growth

    A. thaliana seeds were plated on plates containing half-strength Murashige and Skoog (MS) basal salt mixture along with 10 g l−1 sucrose and stratified at 4 °C for 10 days to promote a high rate and synchrony of germination. Seedlings were then transplanted to soil (Metro-Mix 360 SC and Sunshine Mix #1, mix at 1:1, Sun Gro) in trays (21″ × 10.5″ × 2.5″, Thunder Acres; 40 seedlings per tray).

    For apoplastic fluid collection, A. thaliana plants were grown in a growth chamber with 8 h/16 h day/night light cycles at a white light intensity of 125 µmol m−2 s−1. Plants were watered once a week. A. thaliana used for stomatal bioassays in response to red light were grown in a growth chamber with an 8 h/16 h day/night light cycle at a white light intensity of 300 µmol m−2 s−1 and 22 °C/20 °C day/night temperature cycles and relative humidity of 50–60%. Plants were watered twice a week. Fully expanded leaves from ~4-week-old plants were used.

    V. faba plants were grown individually in pots (4″ × 4″ × 3.5″; Greenhouse Megastore) filled with soil (Metro-Mix 360, Sun Gro). For apoplastic fluid collection and red-light stomatal assays, V. faba plants were grown in a growth chamber with 8 h/16 h day/night light cycles at a white light intensity of 125 µmol m−2 s−1 and 22 °C/20 °C day/night temperature cycles. Plants were watered once a week. Fully expanded leaves from ~3-week-old plants were used.

    Stomatal bioassays in response to red light

    Leaves were excised before lights turned on in the chamber. Epidermal peels were prepared following the method of ref. 74. Epidermal peels were incubated in 5 mM KCl, 1 mM CaCl2 and 10 mM MES-KOH at pH 6.15 in the dark for 1.5–2 h to ensure stomatal closure. Then peels were transferred to a solution containing 5 mM KCl, 0.1 mM CaCl2 (as mock control) or tested sample (for example, red-light-induced apoplastic fluid or fractions from apoplastic fluid; Fig. 1 and Extended Data Fig. 1a).

    For tests of selected metabolites, epidermal peels were transferred to a solution containing 5 mM KCl, 0.1 mM CaCl2, 10 mM MES-KOH at pH 6.15 as mock opening solution, apoplastic fluid obtained from plants treated with red light as control, or to the solutions containing metabolites being tested. The metabolites were dissoved at various concentrations in a buffer containing 5 mM KCl, 0.1 mM CaCl2 and 10 mM MES-KOH at pH 6.15.

    Peels were placed under red light provided by an ABI LED red light bulb (JacobsParts) at an intensity of 125 μmol m−2 s−1. The emission spectrum of this red-light source is given in Extended Data Fig. 5. Stomata were imaged after 3 h red-light treatment or over the indicated time course (Extended Data Fig. 3c) by light microscopy (Nikon Diaphot 300 or Revolve ECHO Microscope, Model R4 with attached camera (Nikon E990)). Stomatal aperture measurements were performed using Fiji (ImageJ2 v.2.14.0; https://fiji.sc). Image acquisition and analysis were performed blinded. At least three independent biological replicates were performed for each treatment.

    Metabolites that were tested were: fructose (Sigma-Aldrich, 57-48-7), malic acid (Sigma-Aldrich, 97-67-6), sorbose (Sigma-Aldrich, 87-79-6), threonic acid (Sigma-Aldrich, 7306-96-9), methyl indole acetate (Sigma-Aldrich, 1912-48-7), 5′-deoxy-5′methylthio-adenosine (Sigma-Aldrich, 2457-80-9), maleic acid (Sigma-Aldrich, 110-16-7), fructose, glucose and sucrose.

    Gas exchange measurements were conducted using an LI-6800 portable gas-exchange system (LI-COR) on petiole-fed leaves of 5-week-old plants. Petioles were immersed in buffer solutions containing 0, 1 or 100 mM of either sucrose or mannitol, and excised in-solution to prevent embolism formation. Leaves were kept in the buffer with only the petioles submerged for 1 h in the dark to acclimate. Afterwards, they were clamped in the LI-6800 chamber with a 2 cm2 leaf chamber, CO2 concentration of 420 µmol mol−1, leaf temperature of 25 °C for V. faba and 22 °C for Arabidopsis, relative humidity maintained at 60% and a flow rate of 500 μmol s⁻¹. Following 30 min in the dark, leaves were exposed to 400 μmol m⁻² s⁻¹ of 100% red light (λ 620–750 nm) for 1 h. Stomatal conductance (gsw) was recorded every minute using the LI-COR autologging mode.

    Apoplastic fluid preparation from A. thaliana and V. faba

    Apoplastic fluids were prepared as previously described27, with modifications. Plants were placed under red light at an intensity of 125 μmol m−2 s−1 for 3 h. Meanwhile, another set of plants was placed in the dark for 3 h, from which the control apoplastic fluids were collected. Leaves were detached immediately after either dark or red-light treatment and briefly rinsed with deionized water. After immersing leaves in infiltration solution containing 5 mM KCl and 0.1 mM CaCl2, vacuum was applied for 75 s and then slowly released. Excess fluid on the infiltrated leaves was then removed using Kimwipes (Kimberly-Clark). Apoplastic fluids were collected from leaves by centrifugation for 10 min at room temperature using a speed setting of 3 (~1,400 rotations per minute (r.p.m.), corresponding to ~200 g) on an IEC clinical tabletop centrifuge (International Equipment) with #215 4-place central rotor. Previous research utilizing V. faba and other species confirmed that at g forces <1,000, there is negligible cytoplasmic contamination of the apoplastic fluid28. For A. thaliana, 700 g has been used to collect symplast-free extracellular vesicles29, and 1,000 g was recently reported to be appropriate for collection of apoplastic fluid from this species30, confirming the suitability of our protocol. Our yield of apoplastic fluid was ~0.4 ml g−1 A. thaliana leaves (fresh weight) and ~1.0 ml g−1 of V. faba leaves (fresh weight).

    Ion content measurement

    Ion contents of apoplastic fluids from V. faba incubated in darkness or under red light were measured using inductively coupled plasma-atomic emission spectrometry (XSERIES 2ICP-AES, Thermo Fisher) at the Laboratory for Isotopes and Metals in the Environment, Department of Geosciences at Pennsylvania State University.

    Treatment of apoplastic fluid

    To characterize the features of the effective components in apoplastic fluid that promote red-light-induced stomatal opening, heat and pepsin (Sigma-Aldrich, P6887) treatments were performed on red-light-treated apoplastic fluid samples from V. faba. Heat treatment consisted of heating samples at 95 °C in a water bath for 5 min. Pepsin digestion (final pepsin concentration 4 mg ml−1) was performed at 37 °C for 1 h. Treated apoplastic fluids were immediately used for stomatal responses to red light, with untreated apoplastic fluid as control.

    Fractionation of apoplastic fluid using centrifugal filter, solid-phase extraction and HPLC

    To separate apoplastic fluid samples into macromolecular and small-metabolite fractions, centrifugation using an Amicon Ultra filter (4 ml or 15 ml) with a 3-kD-cut-off membrane (EMD Millipore) was performed at 4 °C. Flow-through from the centrifugal filter contains small molecules (<3-kD fraction), whereas sample remaining above the filter membrane (>3 kD) contains macromolecules.

    To further identify hydrophobicity of mesophyll messenger(s), HyperSep Retain PEP cartridge (Thermo Fisher) was used for fractionation. Briefly, the column was conditioned using methanol once, then deionized water twice. Red-light-induced apoplastic fluid (5 ml) from V. faba was then applied onto the column. Flow-through was collected. Methanol (5% methanol, 95% water) was used to wash the column and was collected. Then 25%, 50%, 75% and 100% methanol were applied onto the column and fractions (1 ml for each) were collected. All samples (original, flow-through, wash and all fractions) were lyophilised (Labconco). One ml of 5 mM KCl and 0.1 mM CaCl2 was used to reconstitute each sample for stomatal aperture assays.

    Reverse-phase chromatography (RP-HPLC) uses water and organic solvent (for example, methanol) as the solvent system, and a column embedded with resins containing small hydrophobic groups, which efficiently separates analytes with varying degree of hydrophobicity. An UltiMate 3000 HPLC system (Thermo Fisher) was used to collect fine fractions of V. faba or A. thaliana apoplastic fluid. Lyophilised apoplastic fluid was first reconstituted with solvent A (0.1% trifluoride acid (TFA) in 10% methanol). Then 80 µl was injected and separated on a Gemini C18 column (particle size 5 µm, pore size 110 Å, 250 mm length, 4.6 mm diameter, Phenomenex) using a gradient with solvent B (0.1% TFA in 90% methanol) at a flow rate of 0.5 ml min−1. The HPLC gradient started at 100% A for 5 min, followed by ramping from 0% B to 30% B over 40 min, then ramping to 100% B over 5 min, holding at 100% B for 5 min, and then returning to 0% B and holding at 0% B for 5 min. HPLC fractions were collected, lyophilised and reconsititued using a solution containing 5 mM KCl and 0.1 mM CaCl2. Reconstitued fractions were then tested for enhancement of red-light-induced stomatal opening in V. faba epidermal peels as described above. Eluates between 6.5–9 min for A. thaliana apoplastic fluid and 6.5–10 min for V. faba apoplastic fluid were effective in promoting stomatal opening under red light (Extended Data Fig. 1d) and were thus collected for metabolomics analysis.

    Metabolomics analysis of effective HPLC fractions of A. thaliana and V. faba apoplastic fluid

    Effective HPLC fractions of red-light-induced A. thaliana and V. faba apoplastic fluids were prepared as above. Parallel fractions from dark-treated apoplastic fluids that eluted in the identical time windows were taken as controls. Five biological replicates of each sample (that is, dark-treated A. thaliana (AtD), red-light-treated A. thaliana (AtRL), dark-treated V. faba (VfD) and red-light-treated V. faba (VfRL)) fractions were analysed on three different metabolomics platforms: GC–MS (targeted, GCMS-TQ8040, Shimadzu) at the RIKEN Center for Sustainable Resource Science (Japan), GC–MS (untargeted, Pegasus IV GC–TOFMS, LECO) at the University of California Davis, and LC–MS (targeted, QTRAP4000, Sciex) at the University of Florida. The data are summarized in Supplementary Tables 2–4.

    For targeted analysis on the GCMS-TQ8040, adonitol was first added to each sample as an internal standard, then all samples were lyophilised. Methoxamine reagent (MOX, 100 µl; Thermo Fisher) was then added to each lyophilised sample, followed by overnight incubation at 30 °C on a shaker (1,200 r.p.m.). Then, 50 µl of N-methyl-N-trimethylsilyltrifluoroacetamide plus 1% trimethylchlorosilane (Thermo Fisher) was added to each sample, followed by shaking at 1,200 r.p.m. at 37 °C for 30 min. After spin-down, the supernatant of each sample was transferred to a glass vial. One microlitre of each sample was then injected into a Shimadzu GCMS-TQ8040 with a temperature gradient: 60 °C for 2 min, ramping to 330 °C at 15 °C min−1, followed by 330 °C for 3 min.

    For targeted analysis on the QTRAP4000, effective fractions were lyophilised, redissolved in 10% methanol (with lidocaine as internal standard) and vortexed for 15 min at room temperature. After centrifugation (4 °C, 13,000 g) for 15 min, supernatants were transferred to glass vials for HPLC–MRM–MS analysis. Samples were separated on an Eclipse XDB-C18 column (diameter 4.6 mm, length 250 mm, particle size 5 μm) with 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B. The HPLC gradient started at 1% solvent B for 5 min, followed by ramping from 1% B to 99.5% B over 41.5 min, holding at 99.5% B for 4.5 min, and then returning to 1% B over 0.3 min and holding at 1% B for 8.7 min. The flow rate was 0.5 ml min−1. The mass spectrometer conditions were: 30 psi curtain gas, 50 psi GS1, 55 psi GS2, ion source voltage 4,500 V, with a TurboIon electrospray ionization interface temperature of 350 °C. Multiple reaction monitoring (MRM) transitions were distributed into different periods on the basis of the compounds’ retention time to increase the number of compounds detected in a single run75.

    For untargeted analysis on the Pegasus IV GC–TOFMS, samples were derivatized as previously described76 and then injected (0.5 µl of each sample) into an Agilent 6890 gas chromatograph (Agilent). Separation was performed on an Rtx-5Sil MS column (length 30 m, internal diameter 0.25 mm, 0.25 µm film made of 95% dimethyl/5% diphenylpolysiloxane; Restek) with a 20-min temperature gradient: 50 °C for 1 min, ramping to 330 °C at 20 °C min−1, followed by 330 °C for 5 min. The transfer line temperature between the gas chromatograph and the mass spectrometer (Pegasus IV GC–TOFMS, LECO) was set to 230 °C. Mass spectra were acquired at a mass range of 80–500 Da (17 spectra per second, −70 eV ionization energy and 1,800 V detector voltage) with ion source at 250 °C. Data processing and metabolite identification were performed as previously described76.

    Metabolomics data analysis

    For analyses on targeted metabolomics platforms (QTRAP4000 and GCMS-TQ8040), metabolites were identified by paired mass/charge (m/z) ratios of the precursor ion and a selected product ion along with the chromatographic retention time, as acquired from analysis of authentic compounds (standards). For quantification of QTRAP4000 data, the peak area of each metabolite from each sample was first normalized to the internal standard. For quantification of GCMS-TQ8040 data, the peak area of each metabolite from each sample was first normalized by the LOWESS/Spline normalization algorithm with a pooled QC sample using MRMPROBS77. For analysis on the untargeted metabolomics platform (Pegasus IV GC–TOFMS), peak height was reported because peak heights are more precise for low-abundance metabolites than peak areas, due to the larger influence of baseline determinations on areas compared with peak heights78. Peak heights were log2 transformed for statistical analysis. Fold change was calculated using the average value of each group (dark and red light) and Student’s t-test was performed to assess the statistical significance of the difference between the two groups. Metabolites with a fold change >1.2 or <0.8 are defined as significantly changed metabolites between dark and red-light treatments. According to the Metabolomics Standards Initiative64,65, our targeted LC–MS analyses are at confidence ‘level 1’ identification based on fragmentation spectra from authentic standards. Our GCMS-TQ8040 and GC–TOFMS identifications are based on fragmentation pattern-matching against the Smart Metabolites Database V2 (Shimadzu, https://www.shimadzu.com/an/products/gas-chromatograph-mass-spectrometry/gc-ms-software/smart-metabolites-database/index.html) and in-house mass spectral libraries at the West Coast Metabolomics Center, respectively, and can thus be considered as ‘level 2’ identification.

    Quantification of apoplastic sugar concentrations

    Apoplastic fluid of V. fabia leaves was collected as previously described79 using indigo carmine (Thermo Scientific, 860-22-0) to calculate the dilution factor. Analysis of sugars in the apoplastic fluid was carried out using a GC–MS/MS system that consisted of a gas chromatograph Trace 1300 coupled with a triple-quadrupole mass spectrometer TSQ 9000 (Thermo Scientific). The sample was spiked with a mixture of internal standards (glucose-13C6, fructose-13C6, sorbitol-13C6), followed by evaporation with dry nitrogen and chemical derivatization, namely, conversion to oximes with hydroxylamine and afterwards, trimethylsilylation. The derivatized samples were separated on a BPX5 capillary column (30 m × 0.22 mm, 0.25 µm, SGE) using hydrogen as a carrier gas. The mass spectrometer was operated in positive electron impact ionization and SRM mode. Nitrogen (>99.999% purity) was used as a collision gas. Analytes were quantified against calibration curves of the internal standards of the corresponding carbohydrates. The acquisition was controlled using Xcalibur 4.0, and the data were analysed using TraceFinder software v.5.1 SP1 (Thermo Scientific).

    Immunohistochemical analyses of guard-cell plasma membrane H+-ATPase phosphorylation status

    Immunohistochemical analyses using Arabidopsis epidermal fragments were conducted as described previously with slight modifications47,80. A. thaliana Col-0 plants were grown as previously described49. Plants 4–6-weeks of age were used for the experiments. In brief, the epidermal fragments were kept in the basal buffer (5 mM MES-1,3-bis[tris(hydroxymethyl)methylamino] propane, 50 mM KCl and 0.1 mM CaCl2) and treated with sucrose or mannitol at the indicated concentration in the dark. The buffer pH was adjusted to 5.5 or 6.5 with bis-tris propane. The phosphorylation level of the guard-cell plasma membrane H+-ATPase was estimated as described previously49 using an antibody that recognizes phosphorylated H+-ATPase Thr-948, at a dilution of 1:1,000. Three or more experiments were performed, with at least 30 stomata analysed at each measured timepoint. The specimens were observed under a fluorescence microscope (BX50, Olympus) with a narrow excitation band-pass filter set: BP460–480HQ, BA495–540HQ (U-MGFPHQ, Olympus) for Alexa Fluor 488 using an Hg arc lamp as a source of excitation light. Fluorescence images were collected using a CCD camera system (DP72, Olympus) and processed using DP2-BSW software (Olympus). For estimation of fluorescence intensities, all images were taken at identical exposure times (334.79 ms).

    Preparation of V. faba guard-cell protoplasts

    V. faba guard-cell protoplasts were prepared largely as we previously described74. Briefly, per preparation, 5 healthy, young and fully expanded leaflets were excised into 2 cm × 0.5 cm pieces using a scalpel. Major veins were removed using a razor blade, and leaf pieces were then blended in cold distilled H2O with an Osterizer blender (Sunbeam) for 45 s to isolate epidermal fragments. Epidermal fragments were then collected by filtration on a 100-μm nylon mesh and washed thoroughly with distilled H2O to remove damaged mesophyll cells. Washed epidermal peels were incubated in 10 ml of Enzyme Solution 1 for 40 min under darkness at 29 °C, with shaking at 140 excursions per minute. Next, the osmolality of the solution was adjusted to 510 mOsmol kg−1 by adding 30 ml basic solution, followed by shaking for 5 additional minutes. After collecting the partially digested epidermal tissue on a 100-μm nylon mesh and washing with basic solution, the peels were treated with 10 ml of Enzyme Solution 2 for 1.5–2 h at 20 °C in darkness, with shaking at 40 excursions per minute. Once approximately half of the guard-cell protoplasts had started to round up, the epidermal fragments were collected on a 30-μm nylon mesh and washed gently with basic solution into 50 ml volume. The filtrate was centrifuged at 150 × g for 5 min and the small pellet of guard-cell protoplasts concentrated at the bottom was carefully preserved during removal of the supernatant. The pellet was washed again in 50 ml basic solution and re-centrifuged before collecting the purified guard-cell protoplasts in 3–5 ml final volume of solution. The protoplasts were kept on ice in darkness for 1 h to recover before electrophysiological recording.

    The basic solution consisted of 0.45 M mannitol, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.5 mM ascorbic acid, 10 µM KH2PO4 and 5 mM MES, pH 5.5 (adjusted with KOH). Enzyme Solution 1 was made in basic solution/water 55/45 (v/v) and contained 0.7% (w/v) Cellulysin cellulase Trichoderma viride (Thermo Fisher), 0.1% PVP40, 0.25% BSA (bovine serum albumin) and 0.5 mM ascorbic acid, pH 5.5 (KOH). Enzyme Solution 2 was made in 100% basic solution and contained 1.5% Onozuka Cellulase RS (Yakult), 0.03% Pectolyase Y-23 (Thermo Fisher), 0.25% BSA and 0.5 mM ascorbic acid, pH 5.5 (KOH).

    Electrophysiology of V. faba guard-cell protoplasts and Xenopus oocytes

    We followed a previously established protocol to record anion channel currents of V. faba guard-cell protoplasts81. A 100 μl aliquot of protoplasts was transferred into 1.9 ml of external control solution containing 30 mM CsCl, 1 mM CaCl2, 2 mM MgCl2 and 10 mM MES, pH 5.6 (adjusted with Tris base) at a final osmolality of 490 mOsmol kg−1 (adjusted with d-sorbitol). The pipette solution consisted of 150 mM CsCl, 5.864 mM CaCl2, 2 mM MgCl2, 6.7 mM EGTA, 10 mM HEPES and 2 mM Mg-ATP, pH 7.1 (adjusted with Tris-HCl) at a final osmolality of 510 mOsmol kg−1 (adjusted with d-sorbitol). This combination of divalent cations and EGTA yields 2 μM cytosolic free Ca2+. Recording electrodes made from capillary glass tubes (34500, Kimble) were pulled with two-stage puller PP-83 (Narishige) and fire polished with micro-forge MF-900 (Narishige) before coating with melted wax (Kerr). Such electrodes filled with the abovementioned pipette solution gave an average pipette resistance of 6–8 MOhms in the external control solution. A 1 M KCl agarose bridge was used to connect the bath to an Ag-AgCl ground wire placed in 1 M KCl solution. Liquid junction potential was corrected and the slow/fast capacitance transients were compensated by the functions of Axon pClamp software (Molecular Devices). Recordings were conducted using Multiclamp 700A amplifier (Axon Instruments). Data were sampled at 10 kHz and filtered at 1.4 kHz.

    After the whole-cell configuration was formed, protoplasts were incubated for 5 min to equilibrate the cytosol with the pipette solution. The holding potential for recordings was 40 mV and 6-s voltage sweeps were applied from 80 mV to −160 mV at −40-mV intervals. The first voltage family, applied 5 min after achieving the whole-cell configuration, was designated as the initial condition (T0). Subsequent voltage family recordings (T1, T2.) were initiated 3 min after solution changes. The seal resistance of each protoplast was checked before and after every voltage-step protocol to confirm stability. Average values of steady-state currents for I–V curves were taken within the 10-ms interval just before the end of each voltage step. Data from each protoplast were normalized relative to current amplitude at −160 mV.

    Xenopus oocyte recordings were performed as previously described82,83,84,85,86,87,88,89,90,91, with methodological details provided in Extended Data Fig. 4.

    Statistics and data visualization

    Statistical analyses and data visualization were performed using R statistical software (v.4.4.3; https://cran.r-project.org), with the packages ggplot2, dplyr and ggpubr.

    Reporting summary

    Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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  • LLMs as all-in-one tools to easily generate publication-ready citation diversity reports

    LLMs as all-in-one tools to easily generate publication-ready citation diversity reports

    It has been recognized that women and minority scientific authors are cited less frequently than male and majority authors, a systemic phenomenon that negatively affects the visibility and career success of female and minority scientists and biases the prominent research questions of a field1,2. A citation diversity report (CDR), an optional section immediately preceding the reference section of a manuscript, addresses this by quantifying the demographic distribution of cited authors in manuscripts, enabling analysis and potential revision of the proportion of cited scholars from historically excluded groups as a means to advance diversity and inclusivity in science1,2. Journals can also benefit from authors including CDRs in their papers so that they may track the overall citation diversity of their journal.

    To provide an accurate basis for analysing citation diversity, academic databases such as ORCID have begun to ask authors to voluntarily self-report their gender, race and ethnicity; however, not all scholars choose to disclose this information. Because such data are not widely available at this time, name-based prediction of demographics such as gender, race and ethnicity has become common practice for CDR preparation3. For example, current CDR analysis tools such as cleanBib (https://github.com/dalejn/cleanBib) automate name-based gender and race/ethnicity prediction. However, the databases that cleanBib queries, Gender API and Ethnicolr, have imperfect accuracies of 96.1% and 83%, respectively (ref. 4; https://ethnicolr.readthedocs.io/ethnicolr.html#evaluation).

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  • My moonshot to preserve endangered species

    My moonshot to preserve endangered species

    Mary Hagedorn, who studies cryopreservation of corals, wants to put a biobank on the Moon.Credit: Marco Garcia

    Working scientist profiles

    This article is part of an occasional series in which Nature profiles scientists with unusual career histories or outside interests.

    Mary Hagedorn has spent decades studying coral reproduction as part of an effort to save reefs from being destroyed by rapidly warming oceans. To collect precious fragments of new coral life, she must carefully synchronize her activities to the Moon’s phases.

    Corals breed at or near a full moon, releasing a blizzard of sperm and eggs into shimmering waters, but the unpredictability of which full moon corals choose makes fieldwork a gamble. On one trip “we dove for 60 nights straight” before capturing the magic moment, Hagedorn says.

    The patience and persistence required to collect coral sperm, cryopreserve it, transport it to the laboratory for rearing coral larvae and then releasing them into the ocean could serve her well for another planned mission: a literal moonshot to preserve threatened organisms.

    Working Scientist career profiles

    Hagedorn, a research scientist at the Smithsonian Conservation Biology Institute in Kāne‘ohe, Hawaii, is part of an interdisciplinary team proposing to build a frozen repository in a permanently shadowed polar area on the far side of the Moon. Initially, the effort would focus on cryopreserving a veritable Noah’s ark of Earth’s animal life. It would start by banking tissue samples from endangered and threatened animals, as well as from priority species. These would include pollinators and ecosystem engineers such as beavers, that, through dam-building, create whole systems of aquatic environments for other organisms. Hagedorn and ten collaborators published the lunar biorepository idea in 2024 in the journal BioScience as a backup plan for biodiversity — a way to introduce life back to Earth, or to other planets, in the event of catastrophic loss (M. Hagedorn et al. BioScience 74, 561–566; 2024).

    She likens the planned repository to the Svalbard Global Seed Vault in Arctic Norway, which stores seeds of genetic importance for food, agriculture and biodiversity in a rocky cavern deep under an icy mountain held at −18 °C. It’s an ambitious idea that faces many challenges, including a political climate in which science in the United States and beyond is being undermined and underfunded, especially when related to the impacts of climate change. Nevertheless, the lunar biorepository idea is gaining traction and being given serious consideration.

    “We wanted something that could act like Svalbard,” but there’s no place on Earth that is naturally cold enough, says Hagedorn. Even Svalbard needs refrigeration to keep its samples frozen. In 2016, extraordinarily warm winter temperatures sent a flood of melt water into the vault’s entrance. It was a wake-up call for a facility thought to be a fail-safe because it is surrounded by permafrost.

    Thinking big, and extraterrestrially, Hagedorn reasoned that the lunar south pole is spared the vagaries of climate and temperature (see ‘Quick-fire Q&A’). Being stored under the Moon’s surface, also protects the samples from another damaging factor: radiation. Another advantage is the Moon’s lack (so far) of war, violence, natural disasters, overpopulation and resource depletion. Samples could be stored and retrieved using robots similar to the Mars rovers.

    Addressing criticism that retrieving samples could be challenging, Hagedorn responds that, barring an apocalypse, “we will be travelling into space regularly in the future”.

    Quick-fire Q&A

    If you could do a site visit for a biorepository on the Moon, would you go?

    In a heartbeat. I would love to go into space. I actually applied to be an astronaut with NASA, but my eyesight was not good enough.

    Every author on our 2024 BioScience paper proposing the biorepository is like me — they’re frustrated astronauts, sci-fi buffs or both.

    How did your biorepository team come together?

    Around 2015, I was giving a talk in London about biorepositories in general. I said, “you know, one of the best places we could probably have a biorepository is on the Moon”.

    Then, I brought this concept up at the Smithsonian Institution in Washington DC, and the response of my colleagues there was, “this is really stupid — don’t do this”. So I didn’t pursue it, for about four years. But during the COVID-19 pandemic, I had some time, and I decided to get a group together, meeting on Zoom. It just grew from there.

    What is the biggest challenge to getting this project off the ground?

    Money. This project is going to cover so many different areas, from space engineering to ethics, and there are going to be a lot of scientific changes and breakthroughs.

    It’s all new territory; I think that is going to be very exciting. We just have to be patient and try to get some small grants to keep us going. There will be a way.

    A composite image of the South Pole of the moon.

    Composite image of the lunar south pole.Credit: Stocktrek Images/Getty

    Location, location, location

    One favoured site for a lunar bio-repository lies in a crater some 6 kilometres deep, “way deeper than the Grand Canyon” in Arizona, Hagedorn says. In this permanently shadowed space, the temperature is stable — at or below −196 °C.

    Protecting Earth’s life must be a top priority in the rush to stake out lunar sites for industry and research, argue Hagedorn and her co-authors, whose expertise encompasses cryobiology, medicine, engineering, atmospheric research, coral and fish biology, and law and policy. They issued an open call for others to collaborate on this ambitious, decades-long programme.

    The process would start by banking skin samples that contain fibroblast cells. Those fibroblasts are isolated from a cell-culture process, and then cryopreserved. They can later be thawed and transformed into sperm and egg cells from the specific species. Eventually, whole organisms could be reintroduced into their natural habitat.

    A sit in the sauna can save endangered frogs

    As proof of concept, the team will test one species on the International Space Station. The researchers plan to cryopreserve pelvic fins from a coral-reef dwelling fish aptly named the starry goby (Asterropteryx semipunctata), and test them in space for sensitivity to radiation and microgravity. They will also refine the optimal storage materials for cryopreserved cells and study how frozen storage in space affects DNA and the ability to derive and culture cells from thawed fin samples.

    Once the team works out the kinks for starry gobies, it wants to expand to other species. There are plans to collaborate with continental-scale sampling already being undertaken by entities such as the National Ecological Observatory Network, which is funded by the US National Science Foundation (NSF) and collects 100,000 biological samples annually from freshwater and terrestrial habitats.

    Cryobiology, corals and lunar missions were not always Hagedorn’s focus. After earning a PhD in marine biology at the Scripps Institute of Oceanography in La Jolla, California, in 1983, Hagedorn next studied the physiology of electric fishes and a cichlid fish (Astatotilapia burtoni) as a postdoctoral fellow. That research abruptly ended after a boating accident in the Peruvian Amazon claimed the lives of two colleagues. Hagedorn could not bring herself to go back, but realized she wanted to work on the impacts of warming oceans, which led her to bleached and dying coral — the ocean’s canary in the coal mine.

    An article by Canadian molecular physiologist Ken Storey, on the ability of tree frogs to freeze solid in winter and thaw again in spring (K. B. Storey and J. M. Storey Sci. Am. 263, 92–97; 1990), sparked the idea of using cryobiology for ocean conservation work. “Nothing had been done at the time with cryo-preservation of corals,” says Hagedorn. She received a mid-career fellowship at the Smithsonian Institution in Washington DC in 1996 to start work on fish-embryo cryopreservation. In 2004, she transferred her lab group to Hawaii, expanding the scope of her work to include developing techniques for coral cryopreservation.

    Close up view of coral releasing gametes during a spawning event.

    The lunar biobank could start with tissue samples from corals and other endangered species.Credit: Andrew Heyward, AIMS

    Mehmet Toner, a biomedical engineer at Harvard University in Cambridge, Massachusetts, who has known Hagedorn for more than 30 years, says: “I don’t think there’s anyone else in the world who knows coral biology and cryobiology like her.”

    Toner, Hagedorn and their colleagues are funded by the NSF as part of the ATP-Bio programme, which brings together partners from industry, academia, the non-profit sector and government to investigate how to cryo-preserve and store samples ranging from cells to whole organisms.

    Toner’s cryobiology research includes work to understand how to freeze and thaw cells without damaging them. “When I learned about the southern lunar pole being in cryogenic temperatures, it sparked my interest,” says Toner, a co-author of the lunar biorepository proposal.

    Moonstruck collaborators

    The cryobiology involved in preserving life across a spectrum of biodiversity is extremely complex, he explains. “You’re taking a living thing to −196 °C and bringing it back” to the temperature of its habitat, alive. “I call that a miracle.” At the same time, he notes that cryobiology techniques have advanced significantly in the past 20–30 years. “It’s much more predictable and doable now,” he says.

    Toner notes that their team is not the only one vying for lunar real estate. “That part of the Moon is becoming very popular,” with scientists also proposing polar craters as sites for mines, telescopes and temporary human habitation. Indeed, NASA’s Artemis programme, which aims to land humans on the Moon again, is encouraging the exploration of lunar resources.

    John Bischof, a bioengineer at the University of Minnesota in Minneapolis and the director of ATP-Bio, notes Hagedorn’s talent for identifying scientists to join their team. “She’ll bring you into the collaboration, show you exactly where you can make the contribution, and explain why it’s so important. So, even before you do anything, you’re just so pumped up,” he says, describing her as enthusiastic and empathetic. “It’s fun to be around somebody like Mary,” says Bischof, describing her as “untethered in a good way”.

    This company claimed to ‘de-extinct’ dire wolves. Then the fighting started

    Claire Lager, Hagedorn’s lab manager since 2016, and once her graduate student, notes that “somebody who wants to put things on the Moon has to be optimistic, enthusiastic and very, very charismatic”, and that Hagedorn ticks all the boxes.

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  • Zoonotic microorganisms in native and exotic invasive urban small mammals of bamako, Mali

    Zoonotic microorganisms in native and exotic invasive urban small mammals of bamako, Mali

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  • Advanced integrated strategy for structural and mineralogical exploration of inaccessible regions employing remote sensing and multiscale analysis of aeromagnetic data

    Advanced integrated strategy for structural and mineralogical exploration of inaccessible regions employing remote sensing and multiscale analysis of aeromagnetic data

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