Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 µm and report voltage correlations in pairs of neurons.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

SnapShot: Cellular Senescence in Pathophysiology.

Cellular senescence is a fundamental cell fate, important both in physiological and pathophysiological processes. This SnapShot focuses on the role of cellular senescence in health, disease, and aging.

SnapShot: Cellular Senescence Pathways.

Cellular senescence is a fundamental cell fate, playing important physiological and pathophysiological roles. This SnapShot focuses on major signaling pathways and transcriptional control mechanisms that consolidate the senescence phenotype.

Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.

A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

Cell-Type-Specific Optical Recording of Membrane Voltage Dynamics in Freely Moving Mice.

Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO's capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.

Ultrasound Blood-Brain Barrier Opening and Aducanumab in Alzheimer's Disease.

Antiamyloid antibodies have been used to reduce cerebral amyloid-beta (Aß) load in patients with Alzheimer's disease. We applied focused ultrasound with each of six monthly aducanumab infusions to temporarily open the blood-brain barrier with the goal of enhancing amyloid removal in selected brain regions in three participants over a period of 6 months. The reduction in the level of Aß was numerically greater in regions treated with focused ultrasound than in the homologous regions in the contralateral hemisphere that were not treated with focused ultrasound, as measured by fluorine-18 florbetaben positron-emission tomography. Cognitive tests and safety evaluations were conducted over a period of 30 to 180 days after treatment. (Funded by the Harry T. Mangurian, Jr. Foundation and the West Virginia University Rockefeller Neuroscience Institute.).

The zonula adherens matura redefines the apical junction of intestinal epithelia.

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

Convalescent Plasma for Covid-19-Induced ARDS in Mechanically Ventilated Patients.

BACKGROUND: Passive immunization with plasma collected from convalescent patients has been regularly used to treat coronavirus disease 2019 (Covid-19). Minimal data are available regarding the use of convalescent plasma in patients with Covid-19-induced acute respiratory distress syndrome (ARDS). METHODS: In this open-label trial, we randomly assigned adult patients with Covid-19-induced ARDS who had been receiving invasive mechanical ventilation for less than 5 days in a 1:1 ratio to receive either convalescent plasma with a neutralizing antibody titer of at least 1:320 or standard care alone. Randomization was stratified according to the time from tracheal intubation to inclusion. The primary outcome was death by day 28. RESULTS: A total of 475 patients underwent randomization from September 2020 through March 2022. Overall, 237 patients were assigned to receive convalescent plasma and 238 to receive standard care. Owing to a shortage of convalescent plasma, a neutralizing antibody titer of 1:160 was administered to 17.7% of the patients in the convalescent-plasma group. Glucocorticoids were administered to 466 patients (98.1%). At day 28, mortality was 35.4% in the convalescent-plasma group and 45.0% in the standard-care group (P = 0.03). In a prespecified analysis, this effect was observed mainly in patients who underwent randomization 48 hours or less after the initiation of invasive mechanical ventilation. Serious adverse events did not differ substantially between the two groups. CONCLUSIONS: The administration of plasma collected from convalescent donors with a neutralizing antibody titer of at least 1:160 to patients with Covid-19-induced ARDS within 5 days after the initiation of invasive mechanical ventilation significantly reduced mortality at day 28. This effect was mainly observed in patients who underwent randomization 48 hours or less after ventilation initiation. (Funded by the Belgian Health Care Knowledge Center; ClinicalTrials.gov number, NCT04558476.).

Hydrocortisone in Severe Community-Acquired Pneumonia.

BACKGROUND: Whether the antiinflammatory and immunomodulatory effects of glucocorticoids may decrease mortality among patients with severe community-acquired pneumonia is unclear. METHODS: In this phase 3, multicenter, double-blind, randomized, controlled trial, we assigned adults who had been admitted to the intensive care unit (ICU) for severe community-acquired pneumonia to receive intravenous hydrocortisone (200 mg daily for either 4 or 7 days as determined by clinical improvement, followed by tapering for a total of 8 or 14 days) or to receive placebo. All the patients received standard therapy, including antibiotics and supportive care. The primary outcome was death at 28 days. RESULTS: A total of 800 patients had undergone randomization when the trial was stopped after the second planned interim analysis. Data from 795 patients were analyzed. By day 28, death had occurred in 25 of 400 patients (6.2%; 95% confidence interval [CI], 3.9 to 8.6) in the hydrocortisone group and in 47 of 395 patients (11.9%; 95% CI, 8.7 to 15.1) in the placebo group (absolute difference, -5.6 percentage points; 95% CI, -9.6 to -1.7; P = 0.006). Among the patients who were not undergoing mechanical ventilation at baseline, endotracheal intubation was performed in 40 of 222 (18.0%) in the hydrocortisone group and in 65 of 220 (29.5%) in the placebo group (hazard ratio, 0.59; 95% CI, 0.40 to 0.86). Among the patients who were not receiving vasopressors at baseline, such therapy was initiated by day 28 in 55 of 359 (15.3%) of the hydrocortisone group and in 86 of 344 (25.0%) in the placebo group (hazard ratio, 0.59; 95% CI, 0.43 to 0.82). The frequencies of hospital-acquired infections and gastrointestinal bleeding were similar in the two groups; patients in the hydrocortisone group received higher daily doses of insulin during the first week of treatment. CONCLUSIONS: Among patients with severe community-acquired pneumonia being treated in the ICU, those who received hydrocortisone had a lower risk of death by day 28 than those who received placebo. (Funded by the French Ministry of Health; CAPE COD ClinicalTrials.gov number, NCT02517489.).

Two-point optical manipulation reveals mechanosensitive remodeling of cell-cell contacts in vivo.

Biological tissues acquire reproducible shapes during development through dynamic cell behaviors. Most of these behaviors involve the remodeling of cell-cell contacts. During epithelial morphogenesis, contractile actomyosin networks remodel cell-cell contacts by shrinking and extending junctions between lateral cell surfaces. However, actomyosin networks not only generate mechanical stresses but also respond to them, confounding our understanding of how mechanical stresses remodel cell-cell contacts. Here, we develop a two-point optical manipulation method to impose different stress patterns on cell-cell contacts in the early epithelium of the Drosophila embryo. The technique allows us to produce junction extension and shrinkage through different push and pull manipulations at the edges of junctions. We use these observations to expand classical vertex-based models of tissue mechanics, incorporating negative and positive mechanosensitive feedback depending on the type of remodeling. In particular, we show that Myosin-II activity responds to junction strain rate and facilitates full junction shrinkage. Altogether our work provides insight into how stress produces efficient deformation of cell-cell contacts in vivo and identifies unanticipated mechanosensitive features of their remodeling.

A DELAY OF GERMINATION 1 (DOG1)-like protein regulates spore germination in the moss Physcomitrium patens.

DELAY OF GERMINATION 1 is a key regulator of dormancy in flowering plants before seed germination. Bryophytes develop haploid spores with an analogous function to seeds. Here, we investigate whether DOG1 function during germination is conserved between bryophytes and flowering plants and analyse the underlying mechanism of DOG1 action in the moss Physcomitrium patens. Phylogenetic and in silico expression analyses were performed to identify and characterise DOG1 domain-containing genes in P. patens. Germination assays were performed to characterise a Ppdog1-like1 mutant, and replacement with AtDOG1 was carried out. Yeast two-hybrid assays were used to test the interaction of the PpDOG1-like protein with DELLA proteins from P. patens and A. thaliana. P. patens possesses nine DOG1 domain-containing genes. The DOG1-like protein PpDOG1-L1 (Pp3c3_9650) interacts with PpDELLAa and PpDELLAb and the A. thaliana DELLA protein AtRGA in yeast. Protein truncations revealed the DOG1 domain as necessary and sufficient for interaction with PpDELLA proteins. Spores of Ppdog1-l1 mutant germinate faster than wild type, but replacement with AtDOG1 reverses this effect. Our data demonstrate a role for the PpDOG1-LIKE1 protein in moss spore germination, possibly alongside PpDELLAs. This suggests a conserved DOG1 domain function in germination, albeit with differential adaptation of regulatory networks in seed and spore germination.

Genetic induction and mechanochemical propagation of a morphogenetic wave.

Tissue morphogenesis arises from coordinated changes in cell shape driven by actomyosin contractions. Patterns of gene expression regionalize cell behaviours by controlling actomyosin contractility. Here we report two modes of control over Rho1 and myosin II (MyoII) activation in the Drosophila endoderm. First, Rho1-MyoII are induced in a spatially restricted primordium via localized transcription of the G-protein-coupled receptor ligand Fog. Second, a tissue-scale wave of Rho1-MyoII activation and cell invagination progresses anteriorly away from the primordium. The wave does not require sustained gene transcription, and is not governed by regulated Fog delivery. Instead, MyoII inhibition blocks Rho1 activation and propagation, revealing a mechanical feedback driven by MyoII. We find that MyoII activation and invagination in each row of cells drives adhesion to the vitelline membrane mediated by integrins, apical spreading, MyoII activation and invagination in the next row. Endoderm morphogenesis thus emerges from local transcriptional initiation and a mechanically driven cycle of cell deformation.

Force generation, transmission, and integration during cell and tissue morphogenesis.

Cell shape changes underlie a large set of biological processes ranging from cell division to cell motility. Stereotyped patterns of cell shape changes also determine tissue remodeling events such as extension or invagination. In vitro and cell culture systems have been essential to understanding the fundamental physical principles of subcellular mechanics. These are now complemented by studies in developing organisms that emphasize how cell and tissue morphogenesis emerge from the interplay between force-generating machines, such as actomyosin networks, and adhesive clusters that transmit tensile forces at the cell cortex and stabilize cell-cell and cell-substrate interfaces. Both force production and transmission are self-organizing phenomena whose adaptive features are essential during tissue morphogenesis. A new era is opening that emphasizes the similarities of and allows comparisons between distant dynamic biological phenomena because they rely on core machineries that control universal features of cytomechanics.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

Controlled Second Harmonic Generation with Optically Trapped Lithium Niobate Nanoparticles.

We report the second harmonic generation (SHG) response from a single 34 nm diameter lithium niobate nanoparticle. The experimental setup involves a first beam devoted to the optical trapping of single nanoparticles, whereas a second arm involves a femtosecond laser source leading to the SHG emission from the trapped nanoparticles. SHG operation where one to three nanoparticles are present in the optical trap is first demonstrated, highlighting the transition between coherent and incoherent SHG, the latter known as hyper-Rayleigh scattering (HRS). With a spatial light modulator moving the optical trap in and out of the focus of the femtosecond beam, the SHG intensity is switched back and forth between a low and a high level. This controlled operation opens new avenues for nanoparticle characterization and applications in sensing or communication and information technologies and constitutes the first step in the design of active substrateless metasurfaces.

Endogenous ether lipids differentially promote tumor aggressiveness by regulating the SK3 channel.

SK3 channels are potassium channels found to promote tumor aggressiveness. We have previously demonstrated that SK3 is regulated by synthetic ether lipids, but the role of endogenous ether lipids is unknown. Here, we have studied the role of endogenous alkyl- and alkenyl-ether lipids on SK3 channels and on the biology of cancer cells. Experiments revealed that the suppression of alkylglycerone phosphate synthase or plasmanylethanolamine desaturase 1, which are key enzymes for alkyl- and alkenyl-ether-lipid synthesis, respectively, decreased SK3 expression by increasing micro RNA (miR)-499 and miR-208 expression, leading to a decrease in SK3-dependent calcium entry, cell migration, and matrix metalloproteinase 9-dependent cell adhesion and invasion. We identified several ether lipids that promoted SK3 expression and found a differential role of alkyl- and alkenyl-ether lipids on SK3 activity. The expressions of alkylglycerone phosphate synthase, SK3, and miR were associated in clinical samples emphasizing the clinical consistency of our observations. To our knowledge, this is the first report showing that ether lipids differentially control tumor aggressiveness by regulating an ion channel. This insight provides new possibilities for therapeutic interventions, offering clinicians an opportunity to manipulate ion channel dysfunction by adjusting the composition of ether lipids.

Sculpting with stem cells: how models of embryo development take shape.

During embryogenesis, organisms acquire their shape given boundary conditions that impose geometrical, mechanical and biochemical constraints. A detailed integrative understanding how these morphogenetic information modules pattern and shape the mammalian embryo is still lacking, mostly owing to the inaccessibility of the embryo in vivo for direct observation and manipulation. These impediments are circumvented by the developmental engineering of embryo-like structures (stembryos) from pluripotent stem cells that are easy to access, track, manipulate and scale. Here, we explain how unlocking distinct levels of embryo-like architecture through controlled modulations of the cellular environment enables the identification of minimal sets of mechanical and biochemical inputs necessary to pattern and shape the mammalian embryo. We detail how this can be complemented with precise measurements and manipulations of tissue biochemistry, mechanics and geometry across spatial and temporal scales to provide insights into the mechanochemical feedback loops governing embryo morphogenesis. Finally, we discuss how, even in the absence of active manipulations, stembryos display intrinsic phenotypic variability that can be leveraged to define the constraints that ensure reproducible morphogenesis in vivo.

A mountaineering strategy to excited states: Accurate vertical transition energies and benchmarks for substituted benzenes.

In an effort to expand the existing QUEST database of accurate vertical transition energies [Véril et al. WIREs Comput. Mol. Sci. 2021, 11, e1517], we have modeled more than 100 electronic excited states of different natures (local, charge-transfer, Rydberg, singlet, and triplet) in a dozen of mono- and di-substituted benzenes, including aniline, benzonitrile, chlorobenzene, fluorobenzene, nitrobenzene, among others. To establish theoretical best estimates for these vertical excitation energies, we have employed advanced coupled-cluster methods including iterative triples (CC3 and CCSDT) and, when technically possible, iterative quadruples (CC4). These high-level computational approaches provide a robust foundation for benchmarking a series of popular wave function methods. The evaluated methods all include contributions from double excitations (ADC(2), CC2, CCSD, CIS(D), EOM-MP2, STEOM-CCSD), along with schemes that also incorporate perturbative or iterative triples (ADC(3), CCSDR(3), CCSD(T)(a) ⋆ $$ {}^{\star } $$ , and CCSDT-3). This systematic exploration not only broadens the scope of the QUEST database but also facilitates a rigorous assessment of different theoretical approaches in the framework of a homologous chemical series, offering valuable insights into the accuracy and reliability of these methods in such cases. We found that both ADC(2.5) and CCSDT-3 can provide very consistent estimates, whereas among less expensive methods SCS-CC2 is likely the most effective approach. Importantly, we show that some lower order methods may offer reasonable trends in the homologous series while providing quite large average errors, and vice versa. Consequently, benchmarking the accuracy of a model based solely on absolute transition energies may not be meaningful for applications involving a series of similar compounds.

Population pharmacokinetics and dosing simulations of total and unbound temocillin in the plasma and CSF of neurocritically ill patients with external ventricular drain-related cerebral ventriculitis.

BACKGROUND: Cerebral ventriculitis might be caused by Gram-negative bacteria, including ESBL producers. Temocillin may be a useful treatment option in this scenario; however, no consistent data are available regarding its penetration into the CSF. OBJECTIVES: To describe the population pharmacokinetics of temocillin in plasma and CSF and to determine the probability for different simulated dosing regimens to achieve pharmacokinetic/pharmacodynamic (PK/PD) targets in the CSF. METHODS: Ten post-neurosurgical critically ill adult patients requiring continuous drainage of CSF were included in this monocentric, prospective, open-label, non-randomized study. They received 2 g loading dose temocillin over 30 min IV infusion, followed by a 6 g continuous infusion over 24 h. Total and unbound concentrations were measured in plasma (n = 88 and 86) and CSF (n = 88 and 88) samples and used to build a population PK model. Monte Carlo simulations were performed to estimate the PTA at 100% Css>MIC (steady state concentration above the MIC) in CSF. RESULTS: All patients were infected with Enterobacterales with temocillin MICs ≤8 mg/L. The median (min-max) temocillin penetration in CSF was 12.1% (4.3-25.5) at steady state. Temocillin unbound plasma pharmacokinetics were best described by a one-compartment model. PTA for the applied dosing regimen was >90% for bacteria with MIC ≤ 4 mg/L. CONCLUSIONS: The currently approved dose of 6 g by continuous infusion may be adequate for the treatment of ventriculitis by Enterobacterales with MIC ≤ 4 mg/L if considering 100% Css>MIC as the PK/PD target to reach. Higher maintenance doses could help covering higher MICs, but their safety would need to be assessed.