ApoA-I Protects Pancreatic ß-Cells From Cholesterol-Induced Mitochondrial Damage and Restores Their Ability to Secrete Insulin.

BACKGROUND: High cholesterol levels in pancreatic ß-cells cause oxidative stress and decrease insulin secretion. ß-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves ß-cell insulin secretion by reducing oxidative stress. METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-ß-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by ß-cells was monitored by flow cytometry. The effects of apoA-I internalization on ß-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the ß-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in ß-cells and isolated islets with MitoSOX and confocal microscopy. RESULTS: An F1-ATPase ß-subunit on the ß-cell surface was identified as the main apoA-I-binding partner. ß-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F1-ATPase ß-subunit-dependent. ß-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase ß-subunit levels than ß-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E ß-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function. CONCLUSIONS: These results establish that ß-cells are functionally heterogeneous, and apoA-I restores insulin secretion in ß-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.

Biosynthesis and differential spatial distribution of the 3-deoxyanthocyanidins apigenidin and luteolinidin at the interface of a plant-cyanobacteria symbiosis exposed to cold.

Aquatic ferns of the genus Azolla (Azolla) form highly productive symbioses with filamentous cyanobacteria fixing N2 in their leaf cavities, Nostoc azollae. Stressed symbioses characteristically turn red due to 3-deoxyanthocyanidin (DA) accumulation, rare in angiosperms and of unknown function. To understand DA accumulation upon cold acclimation and recovery, we integrated laser-desorption-ionization mass-spectrometry-imaging (LDI-MSI), a new Azolla filiculoides genome-assembly and annotation, and dual RNA-sequencing into phenotypic analyses of the symbioses. Azolla sp. Anzali recovered even when cold-induced DA-accumulation was inhibited by abscisic acid. Cyanobacterial filaments generally disappeared upon cold acclimation and Nostoc azollae transcript profiles were unlike those of resting stages formed in cold-resistant sporocarps, yet filaments re-appeared in leaf cavities of newly formed green fronds upon cold-recovery. The high transcript accumulation upon cold acclimation of AfDFR1 encoding a flavanone 4-reductase active in vitro suggested that the enzyme of the first step in the DA-pathway may regulate accumulation of DAs in different tissues. However, LDI-MSI highlighted the necessity to describe metabolite accumulation beyond class assignments as individual DA and caffeoylquinic acid metabolites accumulated differentially. For example, luteolinidin accumulated in epithelial cells, including those lining the leaf cavity, supporting a role for the former in the symbiotic interaction during cold acclimation.

Myeloperoxidase Gene Deletion Causes Drastic Microbiome Shifts in Mice and Does Not Mitigate Dextran Sodium Sulphate-Induced Colitis.

Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.

3M™ Defib-Pads as a Reusable Alternative to Commercial Ultrasound Gel in Resource-Limited Settings.

INTRODUCTION: With point-of-care ultrasound (POCUS) use in austere environments comes the challenge of having an ever-available coupling medium for image generation. Commercial gel has numerous drawbacks that can limit its utility in these settings, and no studies have evaluated the potential for a reusable coupling medium. This study aimed to determine whether 3M™ Defib-Pads could be utilized as a reusable alternative to commercial gel for image generation in resource-limited settings. METHODS: A descriptive, cross-sectional survey of Canadian physicians with POCUS interest was conducted to evaluate the interpretability of various POCUS images in a blinded fashion. Three anatomic regions (cardiac, abdominal, and nerve) were utilized, and image generation from the commercial gel and 7 Defib-Pad conditions were evaluated. These included pads that were 1) newly opened, 2) dirtied then rinsed, 3) air dried, 4) rinsed after being air dried, 5) frozen then thawed, 6) used in double thickness, and 7) used with a probe cover. RESULTS: Compared to commercial gel, 3M™ Defib-Pads performed similarly, with adequate image interpretability of up to 100% in some conditions. The exception was pads that had prolonged air exposure, which produced images that were never interpretable. However, subsequent rinsing of these pads with water resulted in restored image generation. CONCLUSION: 3M™ Defib-Pads were found to produce interpretable POCUS images under multiple environmental stressors and with different modalities of use, suggesting that 3M™ Defib-Pads can perform as a reusable gel alternative in resource-limited settings.

Deep Characterisation of the sn-Isomer Lipidome Using High-Throughput Data-Independent Acquisition and Ozone-Induced Dissociation.

In recent years there has been a significant interest in the development of innovative lipidomics techniques capable of resolving lipid isomers. To date, methods applied to resolving sn-isomers have resolved only a limited number of species. We report a workflow based on ozone-induced dissociation for untargeted characterisation of hundreds of sn-resolved glycerophospholipid isomers from biological extracts in under 20 min, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn-isomer pairs identified as compared to previous reports and reveals that sn-isomer populations are tightly regulated and significantly different between cell lines. The sensitivity of this method and potential for de novo molecular discovery is further demonstrated by the identification of unexpected lipids containing ultra-long monounsaturated acyl chains at the sn-1 position.

State-dependent modulation of activity in distinct layer 6 corticothalamic neurons in barrel cortex of awake mice.

Layer 6 corticothalamic (L6 CT) neurons are in a strategic position to control sensory input to the neocortex, yet we understand very little about their functions. Apart from studying their anatomical, physiological and synaptic properties, most recent efforts have focused on the activity-dependent influences CT cells can exert on thalamic and cortical neurons through causal optogenetic manipulations. However, few studies have attempted to study them during behavior. To address this gap, we performed juxtacellular recordings from optogenetically identified CT neurons in whisker-related primary somatosensory cortex (wS1) of awake, head-fixed mice (either sex) free to rest quietly or self-initiate bouts of whisking and locomotion. We found a rich diversity of response profiles exhibited by CT cells. Their spiking patterns were either modulated by whisking-related behavior (∼28%) or not (∼72%). Whisking-responsive neurons exhibited either increases, activated-type, or decreases in firing rates, suppressed-type, that aligned with whisking onset better than locomotion. We also encountered responsive neurons with preceding modulations in firing rate before whisking onset. Overall, whisking better explained these changes in rates than overall changes in arousal. Whisking-unresponsive CT cells were generally quiet, with many having low spontaneous firing rates, sparse-type, and others being completely silent. Remarkably, the sparse firing CT population preferentially spiked at the state transition point when pupil diameter constricted and the mouse entered quiet wakefulness. Thus, our results demonstrate that L6 CT cells in wS1 show diverse spiking patterns, perhaps subserving distinct functional roles related to precisely timed responses during complex behaviors and transitions between discrete waking states.SIGNIFICANCE STATEMENTLayer 6 corticothalamic neurons provide a massive input to the sensory thalamus and local connectivity within cortex, but their role in thalamocortical processing remains unclear due to difficulty accessing and isolating their activity. Although several recent optogenetic studies reveal that the net influence of corticothalamic actions, suppression versus enhancement, depends critically on the rate these neurons fire, the factors that influence their spiking are poorly understood, particularly during wakefulness. Using the well-established Ntsr1-Cre line to target this elusive population in the whisker somatosensory cortex of awake mice, we found that corticothalamic neurons show diverse state-related responses and modulations in firing rate. These results suggest separate corticothalamic populations can differentially influence thalamocortical excitability during rapid state transitions in awake, behaving animals.

Time-Resolved Imaging of High Mass Proteins and Metastable Fragments Using Matrix-Assisted Laser Desorption/Ionization, Axial Time-of-Flight Mass Spectrometry, and TPX3CAM.

The Timepix (TPX) is a position- and time-sensitive pixelated charge detector that can be coupled with time-of-flight mass spectrometry (TOF MS) in combination with microchannel plates (MCPs) for the spatially and temporally resolved detection of biomolecules. Earlier generation TPX detectors used in previous studies were limited by a moderate time resolution (at best 10 ns) and single-stop detection for each pixel that hampered the detection of ions with high mass-to-charge (m/z) values at high pixel occupancies. In this study, we have coupled an MCP-phosphor screen-TPX3CAM detection assembly that contains a silicon-coated TPX3 chip to a matrix-assisted laser desorption/ionization (MALDI)-axial TOF MS. A time resolution of 1.5625 ns, per-pixel multihit functionality, simultaneous measurement of TOF and time-over-threshold (TOT) values, and kHz readout rates of the TPX3 extended the m/z detection range of the TPX detector family. The detection of singly charged intact Immunoglobulin M ions of m/z value approaching 1 × 106 Da has been demonstrated. We also discuss the utilization of additional information on impact coordinates and TOT provided by the TPX3 compared to conventional MS detectors for the enhancement of the quality of the mass spectrum in terms of signal-to-noise (S/N) ratio. We show how the reduced dead time and event-based readout in TPX3 compared to the TPX improves the sensitivity of high m/z detection in both low and high mass measurements (m/z range: 757-970,000 Da). We further exploit the imaging capabilities of the TPX3 detector for the spatial and temporal separation of neutral fragments generated by metastable decay at different locations along the field-free flight region by simultaneous application of deflection and retarding fields.

Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.

Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an ∼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.

EGFR as a potent CAR T target in triple negative breast cancer brain metastases.

PURPOSE: There is currently no curative treatment for patients diagnosed with triple-negative breast cancer brain metastases (TNBC-BM). CAR T cells hold potential for curative treatment given they retain the cytolytic activity of a T cell combined with the specificity of an antibody. In this proposal we evaluated the potential of EGFR re-directed CAR T cells as a therapeutic treatment against TNBC cells in vitro and in vivo. METHODS: We leveraged a TNBC-BM tissue microarray and a large panel of TNBC cell lines and identified elevated epidermal growth factor receptor (EGFR) expression. Next, we designed a second-generation anti-EGFR CAR T construct incorporating a clinically relevant mAb806 tumor specific single-chain variable fragment (scFv) and intracellular 4-1BB costimulatory domain and CD3ζ using a lentivirus system and evaluated in vitro and in vivo anti-tumor activity. RESULTS: We demonstrate EGFR is enriched in TNBC-BM patient tissue after neurosurgical resection, with six of 13 brain metastases demonstrating both membranous and cytoplasmic EGFR. Eleven of 13 TNBC cell lines have EGFR surface expression ≥ 85% by flow cytometry. EGFR806 CAR T treated mice effectively eradicated TNBC-BM and enhanced mouse survival (log rank p < 0.004). CONCLUSION: Our results demonstrates anti-tumor activity of EGFR806 CAR T cells against TNBC cells in vitro and in vivo. Given EGFR806 CAR T cells are currently undergoing clinical trials in primary brain tumor patients without obvious toxicity, our results are immediately actionable against the TNBC-BM patient population.

DNA Modifications Enabling Proximity Biotinylation.

Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

Temporal Trends of Legacy and Current-Use Halogenated Flame Retardants in Lake Ontario in Relation to Atmospheric Loadings, Sources, and Environmental Fate.

Since the phase-out of polybrominated diphenyl ethers (PBDEs), large amounts of alternative halogenated flame retardants (AHFRs) have been introduced to the market. Due to their persistence and toxicity, halogenated flame retardants (HFRs) have become a concern for the ecosystem and human health. However, there remains limited assessment of the atmospheric loadings, sources, and environmental fate of HFRs in Lake Ontario, which receives urban-related inputs and cumulative chemical inputs from the upstream Great Lakes from Canada and the United States. We combined long-term measurements with a modified multimedia model based on site-specific environmental parameters from Lake Ontario to understand the trends and fate of HFRs. All HFRs were detected in the air, precipitation, lake trout, and herring gull egg samples throughout the sampling periods. General decreasing trends were found for PBDEs, while the temporal trends for AHFRs were not clear. Physical-chemical properties and emissions significantly influence the levels, profiles, and trends. Using the probabilistic modeling, HFR concentrations in lake water and sediment were predicted to be close to the measurement, suggesting a good performance for the modified model. The loadings from tributaries and wastewater effluent were the primary input pathways. Transformations in the water and sedimentation were estimated to be the dominant output pathway for the three HFRs.

Short-Term Facilitation of Long-Range Corticocortical Synapses Revealed by Selective Optical Stimulation.

Short-term plasticity regulates the strength of central synapses as a function of previous activity. In the neocortex, direct synaptic interactions between areas play a central role in cognitive function, but the activity-dependent regulation of these long-range corticocortical connections and their impact on a postsynaptic target neuron is unclear. Here, we use an optogenetic strategy to study the connections between mouse primary somatosensory and motor cortex. We found that short-term facilitation was strong in both corticocortical synapses, resulting in far more sustained responses than local intracortical and thalamocortical connections. A major difference between pathways was that the synaptic strength and magnitude of facilitation were distinct for individual excitatory cells located across all cortical layers and specific subtypes of GABAergic neurons. Facilitation was dependent on the presynaptic calcium sensor synaptotagmin-7 and altered by several optogenetic approaches. Current-clamp recordings revealed that during repetitive activation, the short-term dynamics of corticocortical synapses enhanced the excitability of layer 2/3 pyramidal neurons, increasing the probability of spiking with activity. Furthermore, the properties of the connections linking primary with secondary somatosensory cortex resemble those between somatosensory-motor areas. These short-term changes in transmission properties suggest long-range corticocortical synapses are specialized for conveying information over relatively extended periods.

Controlling a Digital Cursor With a Sterile Gyroscopic Mouse in Digitally Assisted Vitreoretinal Surgery.

PURPOSE: To describe our experience using the gyroscopic mouse in digitally assisted vitreoretinal surgery. METHODS: We used a commercially available gyroscopic mouse to control the on-screen cursor of the NGENUITY System for digitally assisted vitreoretinal surgery. The gyroscopic mouse is sealed in a clear sterile plastic bag to allow for intraoperative use. This allowed both the surgeon and assistant to be fully scrubbed while retaining full control of the NGENUITY system's functions. The mouse also allowed the mentor to provide detailed instructions through the on-screen cursor by highlighting important landmarks. CONCLUSION: Using a sterile gyroscopic mouse improved the teaching utility and surgical workflow of digitally assisted vitreoretinal surgery.

Obscurin regulates ankyrin macromolecular complex formation.

Obscurin is a large scaffolding protein in striated muscle that maintains sarcolemmal integrity and aligns the sarcoplasmic reticulum with the underlying contractile machinery. Ankyrins are a family of adaptor proteins with some isoforms that interact with obscurin. Previous studies have examined obscurin interacting with individual ankyrins. In this study, we demonstrate that two different ankyrins interact with obscurin's carboxyl terminus via independent ankyrin-binding domains (ABDs). Using in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis, we show that obscurin interacts with small ankyrin 1.5 (sAnk1.5) and the muscle-specific ankyrin-G isoform (AnkG107). While there is no direct interaction between sAnk1.5 and AnkG107, obscurin connects the two ankyrins both in vitro and in cells. Moreover, AnkG107 recruits ß-spectrin to this macromolecular protein complex and mutating obscurin's ABDs disrupts complex formation. To further characterize AnkG107 interaction with obscurin, we measure obscurin-binding to different AnkG107 isoforms expressed in the heart and find that the first obscurin-binding domain in AnkG107 principally mediates this interaction. We also find that AnkG107 does not bind to filamin-C and displays minimal binding to plectin-1 compared to obscurin. Finally, both sAnk1.5-GFP and AnkG107-CTD-RFP are targeted to the M-lines of ventricular cardiomyocytes and mutating their obscurin-binding domains disrupts the M-line localization of these ankyrin constructs. Altogether, these findings support a model in which obscurin can interact via independent binding domains with two different ankyrin protein complexes to target them to the sarcomeric M-line of ventricular cardiomyocytes.

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Unraveling the antitrypanosomal mechanism of benznidazole and related 2-nitroimidazoles: From prodrug activation to DNA damage.

Nitroheterocycles represent an important class of compound used to treat trypanosomiasis. They often function as prodrugs and can undergo type I nitroreductase (NTR1)-mediated activation before promoting their antiparasitic activities although the nature of these downstream effects has yet to be determined. Here, we show that in an NTR1-dependent process, benznidazole promotes DNA damage in the nuclear genome of Trypanosoma brucei, providing the first direct link between activation of this prodrug and a downstream trypanocidal mechanism. Phenotypic and protein expression studies revealed that components of the trypanosome's homologous recombination (HR) repair pathway (TbMRE11, γH2A, TbRAD51) cooperate to resolve the benznidazole-induced damage, indicating that the prodrug-induced lesions are most likely double stand DNA breaks, while the sequence/recruitment kinetics of these factors parallels that in other eukaryotes HR systems. When extended to other NTR1-activated 2-nitroimidazoles, some were shown to promote DNA damage. Intriguingly, the lesions induced by these required TbMRE11 and TbCSB activities to fix leading us to postulate that TbCSB may operate in systems other than the transcription-coupled nucleotide excision repair pathway. Understanding how existing trypanosomal drugs work will aid future drug design and help unlock novel reactions/pathways that could be exploited as targets for therapeutic intervention.

Mass Spectrometry Imaging of Lipids Using MALDI Coupled with Plasma-Based Post-Ionization on a Trapped Ion Mobility Mass Spectrometer.

Here we report the development and optimization of a mass spectrometry imaging (MSI) platform that combines an atmospheric-pressure matrix-assisted laser desorption/ionization platform with plasma postionization (AP-MALDI-PPI) and trapped ion mobility spectrometry (TIMS). We discuss optimal parameters for operating the source, characterize the behavior of a variety of lipid classes in positive- and negative-ion modes, and explore the capabilities for lipid imaging using murine brain tissue. The instrument generates high signal-to-noise for numerous lipid species, with mass spectra sharing many similarities to those obtained using laser postionization (MALDI-2). The system is especially well suited for detecting lipids such as phosphatidylethanolamine (PE), as well as numerous sphingolipid classes and glycerolipids. For the first time, the coupling of plasma-based postionization with ion mobility is presented, and we show the value of ion mobility for the resolution and identification of species within rich spectra that contain numerous isobaric/isomeric signals that are not resolved in the m/z dimension alone, including isomeric PE and demethylated phosphatidylcholine lipids produced by in-source fragmentation. The reported instrument provides a powerful and user-friendly approach for MSI of lipids.

Galectin-3 enhances neutrophil motility and extravasation into the airways during Aspergillus fumigatus infection.

Aspergillus fumigatus is an opportunistic mold that infects patients who are immunocompromised or have chronic lung disease, causing significant morbidity and mortality in these populations. While the factors governing the host response to A. fumigatus remain poorly defined, neutrophil recruitment to the site of infection is critical to clear the fungus. Galectin-3 is a mammalian ß-galactose-binding lectin with both antimicrobial and immunomodulatory activities, however the role of galectin-3 in the defense against molds has not been studied. Here we show that galectin-3 expression is markedly up-regulated in mice and humans with pulmonary aspergillosis. Galectin-3 deficient mice displayed increased fungal burden and higher mortality during pulmonary infection. In contrast to previous reports with pathogenic yeast, galectin-3 exhibited no antifungal activity against A. fumigatus in vitro. Galectin-3 deficient mice exhibited fewer neutrophils in their airways during infection, despite normal numbers of total lung neutrophils. Intravital imaging studies confirmed that galectin-3 was required for normal neutrophil migration to the airspaces during fungal infection. Adoptive transfer experiments demonstrated that stromal rather than neutrophil-intrinsic galectin-3 was necessary for normal neutrophil entry into the airspaces. Live cell imaging studies revealed that extracellular galectin-3 directly increases neutrophil motility. Taken together, these data demonstrate that extracellular galectin-3 facilitates recruitment of neutrophils to the site of A. fumigatus infection, and reveals a novel role for galectin-3 in host defense against fungal infections.

Using remote sensing to quantify the additional climate benefits of California forest carbon offset projects.

Nature-based climate solutions are a vital component of many climate mitigation strategies, including California's, which aims to achieve carbon neutrality by 2045. Most carbon offsets in California's cap-and-trade program come from improved forest management (IFM) projects. Since 2012, various landowners have set up IFM projects following the California Air Resources Board's IFM protocol. As many of these projects approach their 10th year, we now have the opportunity to assess their effectiveness, identify best practices, and suggest improvements toward future protocol revisions. In this study, we used remote sensing-based datasets to evaluate the carbon trends and harvest histories of 37 IFM projects in California. Despite some current limitations and biases, these datasets can be used to quantify carbon accumulation and harvest rates in offset project lands relative to nearby similar "control" lands before and after the projects began. Five lines of evidence suggest that the carbon accumulated in offset projects to date has generally not been additional to what might have otherwise occurred: (1) most forests in northwestern California have been accumulating carbon since at least the mid-1980s and continue to accumulate carbon, whether enrolled in offset projects or not; (2) harvest rates were high in large timber company project lands before IFM initiation, suggesting they are earning carbon credits for forests in recovery; (3) projects are often located on lands with higher densities of low-timber-value species; (4) carbon accumulation rates have not yet increased on lands that enroll as offset projects, relative to their pre-enrollment levels; and (5) harvest rates have not decreased on most project lands since offset project initiation. These patterns suggest that the current protocol should be improved to robustly measure and reward additionality. In general, our framework of geospatial analyses offers an important and independent means to evaluate the effectiveness of the carbon offsets program, especially as these data products continue improving and as offsets receive attention as a climate mitigation strategy.