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Justificación: La mayoría de las infecciones por microsporidios en el ser humano se han reportado en el tracto gastrointestinal, sin embargo, es posible hallarlos parasitando otros sitios anatómicos como el tracto genitourinario, en donde las patologías pueden variar desde uretritis hasta falla renal. Tradicionalmente esas infecciones se deben al género Encephalitozoon, parásito contra el cual el Albendazol ha demostrado ser un medicamento efectivo. Objetivo: Determinar la prevalencia de microsporidios en personas que son referidas al laboratorio para urocultivo, dados los antecedentes clínicos que presentan. Métodos: El procesamiento de las muestras incluyó su toma con técnica aséptica, su cultivo en los medios de Agar Sangre, MacConkey y Manitol - sal para su estudio por presencia de bacterias y/u hongos y la realización del antibiograma, según los parámetros establecidos por la CCSS para los urocultivos. Adicional a esto se centrifugó una alícuota de 9 ml separando 50 microlitos del sedimento urinario, al cual se le añadió igual volumen de KOH al 5%. Se hicieron frotis los cuales se fijaron con alcohol metílico una vez secos, y se tiñeron con la técnica trierómica de Weber, obviando el paso de la decoloración y la deshidratación. Resultados: Se encontró un 25% de orinas positivas por microsporidios. Los pacientes con edades entre 26 y 55 años presentaron mayor positividad. Conclusiones: El porcentaje de prevalencia encontrado (25%) y el hecho de que estas infecciones pueden llegar a causar severos daños al ser humano, hacen imperativa la necesidad de realizar diagnósticos más certeros de esta parasitosis, para lo cual proponemos la tinción de Weber con la modificación presentada en este trabajo para el diagnóstico de las infecciones por microsporidios en el tracto urinario.

Neurological diseases are associated with disruptions in the brain lipidome that are becoming central to disease pathogenesis. Traditionally perceived as static structural support in membranes, lipids are now known to be actively involved in cellular signaling, energy metabolism, and other cellular activities involving membrane curvature, fluidity, fusion or fission. Glia are critical in the development, health, and function of the brain, and glial regulation plays a major role in disease. The major pathways of glial dysregulation related to function are associated with downstream products of metabolism including lipids. Taking advantage of significant innovations and technical advancements in instrumentation, lipidomics has emerged as a popular omics discipline, serving as the prevailing approach to comprehensively define metabolic alterations associated with organismal development, damage or disease. A key technological platform for lipidomics studies is mass spectrometry (MS), as it affords large-scale profiling of complex biological samples. However, as MS-based techniques are often refined and advanced, the relative comfort level among biologists with this instrumentation has not followed suit. In this review, we aim to highlight the importance of the study of glial lipids and to provide a concise record of best practices and steps for MS-based lipidomics. Specifically, we outline procedures for glia lipidomics workflows ranging from sample collection and extraction to mass spectrometric analysis to data interpretation. To ensure these approaches are more accessible, this tutorial aims to familiarize glia biologists with sample handling and analysis techniques for MS-based lipidomics, and to guide non-experts toward generating high quality lipidomics data., (© 2025 The Author(s). GLIA published by Wiley Periodicals LLC.)

Lipids are small molecule immunomodulators that play critical roles in maintaining cellular health and function. Microglia, the resident immune cells of the central nervous system, regulate lipid metabolism both in the extracellular environment and within intracellular compartments through various mechanisms. For instance, glycerophospholipids and fatty acids interact with protein receptors on the microglial surface, such as the Triggering Receptor Expressed on Myeloid Cells 2, influencing cellular functions like phagocytosis and migration. Moreover, cholesterol is essential not only for microglial survival but, along with other lipids such as fatty acids, is crucial for the formation, function, and accumulation of lipid droplets, which modulate microglial activity in inflammatory diseases. Other lipids, including acylcarnitines and ceramides, participate in various signaling pathways within microglia. Despite the complexity of the microglial lipidome, only a few studies have investigated the effects of specific lipid classes on microglial biology. In this review, we focus on major lipid classes and their roles in modulating microglial function. We also discuss novel analytical techniques for characterizing the microglial lipidome and highlight gaps in current knowledge, suggesting new directions for future research on microglial lipid biology., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Background: Sow longevity and reproductivity are essential in the modern swine industry. Although many studies have focused on the genetic and genomic factors for selection, little is known about the associations between the microbiome and sows with longevity in reproduction., Results: In this study, we collected and sequenced rectal and vaginal swabs from 48 sows, nine of which completed up to four parities (U4P group), exhibiting reproductive longevity. We first identified predictors of sow longevity in the rectum (e.g., Akkermansia) and vagina (e.g., Lactobacillus) of the U4P group using RandomForest in the early breeding stage of the first parity. Interestingly, these bacteria in the U4P group showed decreased predicted KEGG gene abundance involved in the biosynthesis of amino acids. Then, we tracked the longitudinal changes of the microbiome over four parities in the U4P sows. LEfSe analysis revealed parity-associated bacteria that existed in both the rectum and vagina (e.g., Streptococcus in Parity 1, Lactobacillus in Parity 2, Veillonella in Parity 4). We also identified patterns of bacterial change between the early breeding stage (d 0) and d 110, such as Streptococcus, which was decreased in all four parties. Furthermore, sows in the U4P group with longevity potential also showed better reproductive performance. Finally, we discovered bacterial predictors (e.g., Prevotellaceae NK3B31 group) for the total number of piglets born throughout the four parities in both the rectum and vagina., Conclusions: This study highlights how the rectal and vaginal microbiome in sows with longevity in reproduction changes within four parities. The identification of parity-associated, pregnancy-related, and reproductive performance-correlated bacteria provides the foundation for targeted microbiome modulation to improve animal production., Competing Interests: Declarations. Ethics approval and consent to participate: Pig experiments were approved and were managed according to the Institutional Animal Care and Use Committee of the University of Arkansas under protocol #21044. Consent for publication: Not applicable. Competing interests: The authors declare no competing financial interest., (© 2025. The Author(s).)

Several microglia-expressed genes have emerged as top risk variants for Alzheimer's disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (Aβ), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent on age and disease progression and is prominent in the hippocampus in mice and humans. Despite differences in microglial LD load between brain regions and sexes in mice, LD-laden microglia exhibited a deficit in Aβ phagocytosis. Unbiased lipidomic analysis identified a decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TGs) as the key metabolic transition underlying LD formation. DGAT2, a key enzyme for converting FFAs to TGs, promotes microglial LD formation and is increased in 5xFAD and human AD brains. Inhibition or degradation of DGAT2 improved microglial uptake of Aβ and drastically reduced plaque load in 5xFAD mice, respectively. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD., Competing Interests: DECLARATION OF INTERESTS G.C. is the Director of the Merck-Purdue Center for Measurement Science funded by Merck Sharp & Dohme, a subsidiary of Merck and a co-founder of Meditati Inc., a startup developing smart drugs for mental health indications. The remaining authors declare no competing interests.

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major public health concern that is exacerbated by the obesity pandemic. Dietary interventions have the potential to alleviate obesity-associated MASLD through variable mechanisms, including optimizing the gut microbiota. Previously, we reported that soy protein concentrate (SPC) with low or high levels of isoflavone (LIF or HIF) protected young obese Zucker rats from developing liver steatosis. The current study was designed to test whether SPC-LIF and SPC-HIF diets would reverse liver steatosis and alter fecal microbial composition in adult obese Zucker rats with existing steatosis., Methods: Six-week-old male obese Zucker rats ( n = 26) were fed a casein control diet (CAS) for 8 weeks and 7 rats were randomly selected and sacrificed to confirm liver steatosis. The remaining rats were randomly assigned to receive CAS, SPC-LIF, or SPC-HIF diet ( n = 6-7/group) for an additional 10 weeks., Results: Compared to CAS diet, feeding SPC-LIF and SPC-HIF diets resulted in significantly lower liver weight, liver steatosis score, and liver microvesicular score ( p < 0.05), but did not lead to difference in body weight, liver macrovesicular score, serum ALT, or serum AST. Isoflavone levels (e.g., LIF vs. HIF) did not affect any of these measurements except in the SPC-HIF group, which had an additional decrease in liver weight ( p < 0.05) compared to the SPC-LIF group. The SPC-HIF group also had significantly higher levels of the aglycone forms of daidzein, genistein, and equol as well as the total levels of daidzein, genistein, and equol compared to SPC-LIF or CAS diet fed rats ( p < 0.05). The distribution of microbial communities based on measures of beta diversity of both SPC-LIF and SPC-HIF groups were significantly different to that of the CAS group ( p ≤ 0.005). Alpha-diversity did not differ between any of the groups., Conclusion: Taken together, dietary soy protein can reverse liver steatosis in adult Zucker rats, and the reversal of steatosis is accompanied by alterations in gut microbial composition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hakkak, Korourian, Li, Spray, Twaddle, Randolph, Børsheim and Robeson.)

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species 1,2 . Acutely, senescent cells promote wound healing 3,4 and prevent tumour formation 5 ; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy 6-11 , why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear 12,13 . Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence 14,15 , we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation., (© 2024. The Author(s).)

In multiple myeloma (MM), increased osteoclast differentiation leads to the formation of osteolytic lesions in most MM patients. Bisphosphonates, such as zoledronic acid (ZA), are used to ameliorate bone resorption, but due to risk of serious side effects as well as the lack of repair of existing lesions, novel anti-bone resorption agents are required. Previously, the absence of osteolytic lesions in MM was strongly associated with elevated levels of cystatin M/E (CST6), a cysteine protease inhibitor, secreted by MM cells. In this study, both MM- and ovariectomy (OVX)-induced osteoporotic mouse models were used to compare the effects of recombinant mouse CST6 (rmCst6) and ZA on preventing bone loss. μCT showed that rmCst6 and ZA had similar effects on improving percent bone volume, and inhibited differentiation of non-adherent bone marrow cells into mature osteoclasts. Single-cell RNA sequencing showed that rmCst6 and not ZA treatment reduced bone marrow macrophage percentage in the MM mouse model compared to controls. Protein and mRNA arrays showed that both rmCst6 and ZA significantly inhibit OVX-induced expression of inflammatory cytokines. For OVX mice, ERα protein expression in bone was brought to sham surgery level by only rmCst6 treatments. rmCst6 significantly increased mRNA and protein levels of ERα and significantly increased total intracellular estrogen concentrations for ex vivo osteoclast precursor cell cultures. Based on these results, we conclude that CST6 improves MM or OVX bone loss models by increasing the expression of estrogen receptors as well as the intracellular estrogen concentration in osteoclast precursors, inhibiting their maturation., Competing Interests: Competing interests The authors declare no competing interests. Conflict of Interest: The authors have declared that no conflict of interest exists.

Phenolic acids, such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA), can be produced from microbiome digestion of polyphenols. Previously it was found that HA and 3-3-PPA facilitate bone formation and suppress bone resorption. However, the mechanism of action by which HA and 3-3-PPA protect bone from degeneration is currently unknown. In this report, we present that HA and 3-3-PPA suppression of bone resorption is able to ameliorate bone loss in an ovariectomy (OVX) osteopenic mouse model though not to the extent of Zoledronic acid (ZA). HA and 3-3-PPA treatments were shown to significantly decrease bone marrow adipocyte-like cell formation and inhibited gene expression of key adipogenesis regulator peroxisome proliferator activated receptor gamma (PPARγ) and lipoprotein lipase (Lpl) in bone from OVX mice. In addition, ChIP experiments showed that the association between PPARγ and Lpl promoter region in preadipocyte-like cells was significantly suppressed following HA or 3-3-PPA treatment. Contrasting HA and 3-3-PPA, ZA significantly increased TRAP activity in the area close to growth plate and significantly suppressed bone cell proliferation. These data suggest that phenolics acids such as HA or 3-3-PPA may prevent bone degeneration after OVX through suppression of inflammatory milieu in the bone., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lipidomics generates large data that makes manual annotation and interpretation challenging. Lipid chemical and structural diversity with structural isomers further complicates annotation. Although, several commercial and open-source software for targeted lipid identification exists, it lacks automated method generation workflows and integration with statistical and bioinformatics tools. We have developed the Comprehensive Lipidomic Automated Workflow (CLAW) platform with integrated workflow for parsing, detailed statistical analysis and lipid annotations based on custom multiple reaction monitoring (MRM) precursor and product ion pair transitions. CLAW contains several modules including identification of carbon-carbon double bond position(s) in unsaturated lipids when combined with ozone electrospray ionization (OzESI)-MRM methodology. To demonstrate the utility of the automated workflow in CLAW, large-scale lipidomics data was collected with traditional and OzESI-MRM profiling on biological and non-biological samples. Specifically, a total of 1497 transitions organized into 10 MRM-based mass spectrometry methods were used to profile lipid droplets isolated from different brain regions of 18-24 month-old Alzheimer's disease mice and age-matched wild-type controls. Additionally, triacyclglycerols (TGs) profiles with carbon-carbon double bond specificity were generated from canola oil samples using OzESI-MRM profiling. We also developed an integrated language user interface with large language models using artificially intelligent (AI) agents that permits users to interact with the CLAW platform using a chatbot terminal to perform statistical and bioinformatic analyses. We envision CLAW pipeline to be used in high-throughput lipid structural identification tasks aiding users to generate automated lipidomics workflows ranging from data acquisition to AI agent-based bioinformatic analysis.

Spared regions of the damaged central nervous system undergo dynamic remodeling and exhibit a remarkable potential for therapeutic exploitation. Here, lesion-remote astrocytes (LRAs), which interact with viable neurons, glia and neural circuitry, undergo reactive transformations whose molecular and functional properties are poorly understood. Using multiple transcriptional profiling methods, we interrogated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury (SCI). We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after SCI. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fueling this LRA functional adaptation is Ccn1 , which encodes for a secreted matricellular protein. Loss of astrocyte CCN1 leads to excessive, aberrant activation of local microglia with (i) abnormal molecular specification, (ii) dysfunctional myelin debris processing, and (iii) impaired lipid metabolism, culminating in blunted debris clearance and attenuated neurological recovery from SCI. Ccn1 -expressing white matter astrocytes are specifically induced by local myelin damage and generated in diverse demyelinating disorders in mouse and human, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that LRAs assume regionally divergent reactivity states with functional adaptations that are induced by local context-specific triggers and influence disorder outcome. Astrocytes tile the central nervous system (CNS) where they serve vital roles that uphold healthy nervous system function, including regulation of synapse development, buffering of neurotransmitters and ions, and provision of metabolic substrates 1 . In response to diverse CNS insults, astrocytes exhibit disorder-context specific transformations that are collectively referred to as reactivity 2-5 . The characteristics of regionally and molecularly distinct reactivity states are incompletely understood. The mechanisms through which distinct reactivity states arise, how they evolve or resolve over time, and their consequences for local cell function and CNS disorder progression remain enigmatic. Immediately adjacent to CNS lesions, border-forming astrocytes (BFAs) undergo transcriptional reprogramming and proliferation to form a neuroprotective barrier that restricts inflammation and supports axon regeneration 6-9 . Beyond the lesion, spared but dynamic regions of the injured CNS exhibit varying degrees of synaptic circuit remodeling and progressive cellular responses to secondary damage that have profound consequences for neural repair and recovery 10,11 . Throughout these cytoarchitecturally intact, but injury-reactive regions, lesion-remote astrocytes (LRAs) intermingle with neurons and glia, undergo little to no proliferation, and exhibit varying degrees of cellular hypertrophy 7,12,13 . The molecular and functional properties of LRAs remain grossly undefined. Therapeutically harnessing spared regions of the injured CNS will require a clearer understanding of the accompanying cellular and molecular landscape. Here, we leveraged integrative transcriptional profiling methodologies to identify multiple spatiotemporally resolved, molecularly distinct states of LRA reactivity within the injured spinal cord. Computational modeling of LRA-mediated heterotypic cell interactions, astrocyte-specific conditional gene deletion, and multiple mouse models of acute and chronic CNS white matter degeneration were used to interrogate a newly identified white matter degeneration-reactive astrocyte subtype. We define how this reactivity state is induced and its role in governing the molecular and functional specification of local microglia that clear myelin debris from the degenerating white matter to promote repair.

Introduction: Colonocyte oxidation of bacterial-derived butyrate has been reported to maintain synergistic obligate anaerobe populations by reducing colonocyte oxygen levels; however, it is not known whether this process is disrupted during the progression of type 2 diabetes. Our aim was to determine whether diabetes influences colonocyte oxygen levels in the University of California Davis type 2 diabetes mellitus (UCD-T2DM) rat model., Research Design and Methods: Age-matched male UCD-T2DM rats (174±4 days) prior to the onset of diabetes (PD, n=15), within 1 month post-onset (RD, n=12), and 3 months post-onset (D3M, n=12) were included in this study. Rats were administered an intraperitoneal injection of pimonidazole (60 mg/kg body weight) 1 hour prior to euthanasia and tissue collection to estimate colonic oxygen levels. Colon tissue was fixed in 10% formalin, embedded in paraffin, and processed for immunohistochemical detection of pimonidazole. The colonic microbiome was assessed by 16S gene rRNA amplicon sequencing and content of short-chain fatty acids was measured by liquid chromatography-mass spectrometry., Results: HbA1c % increased linearly across the PD (5.9±0.1), RD (7.6±0.4), and D3M (11.5±0.6) groups, confirming the progression of diabetes in this cohort. D3M rats had a 2.5% increase in known facultative anaerobes, Escherichia-Shigella , and Streptococcus (false discovery rate <0.05) genera in colon contents. The intensity of pimonidazole staining of colonic epithelia did not differ across groups (p=0.37). Colon content concentrations of acetate and propionate also did not differ across UCD-T2DM groups; however, colonic butyric acid levels were higher in D3M rats relative to PD rats (p<0.01)., Conclusions: The advancement of diabetes in UCD-T2DM rats was associated with an increase in facultative anaerobes; however, this was not explained by changes in colonocyte oxygen levels. The mechanisms underlying shifts in gut microbe populations associated with the progression of diabetes in the UCD-T2DM rat model remain to be identified., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Statistical analysis and modeling of mass spectrometry (MS) data have a long and rich history with several modern MS-based applications using statistical and chemometric methods. Recently, machine learning (ML) has experienced a renaissance due to advents in computational hardware and the development of new algorithms for artificial neural networks (ANN) and deep learning architectures. Moreover, recent successes of new ANN and deep learning architectures in several areas of science, engineering, and society have further strengthened the ML field. Importantly, modern ML methods and architectures have enabled new approaches for tasks related to MS that are now widely adopted in several popular MS-based subdisciplines, such as mass spectrometry imaging and proteomics. Herein, we aim to provide an introductory summary of the practical aspects of ML methodology relevant to MS. Additionally, we seek to provide an up-to-date review of the most recent developments in ML integration with MS-based techniques while also providing critical insights into the future direction of the field., Competing Interests: The authors declare the following competing financial interest(s): G.C. is the Director of the Merck-Purdue Center for Measurement Science funded by Merck Sharp & Dohme, a subsidiary of Merck and the co-founder of Meditati Inc and BrainGnosis Inc., (© 2024 The Authors. Published by American Chemical Society.)

While various mass spectrometric approaches have been applied to lipid analysis, unraveling the extensive structural diversity of lipids remains a significant challenge. Notably, these approaches often fail to differentiate between isomeric lipids─a challenge that is particularly acute for branched-chain fatty acids (FAs) that often share similar (or identical) mass spectra to their straight-chain isomers. Here, we utilize charge-switching strategies that combine ligated magnesium dications with deprotonated fatty acid anions. Subsequent activation of these charge inverted anions yields mass spectra that differentiate anteiso -branched- from straight-chain and iso -branched-chain FA isomers with the predictable fragmentation enabling de novo assignment of anteiso branch points. The application of these charge-inversion chemistries in both gas- and solution-phase modalities is demonstrated to assign the position of anteiso -methyl branch-points in FAs and, with the aid of liquid chromatography, can be extended to de novo assignment of additional branching sites via predictable fragmentation patterns as methyl branching site(s) move closer to the carboxyl carbon. The gas-phase approach is shown to be compatible with top-down structure elucidation of complex lipids such as phosphatidylcholines, while the integration of solution-phase charge-inversion with reversed phase liquid chromatography enables separation and unambiguous identification of FA structures within isomeric mixtures. Taken together, the presented charge-switching MS-based technique, in combination with liquid chromatography, enables the structural identification of branched-chain FA without the requirement of authentic methyl-branched FA reference standards.

Heterotrimeric G proteins couple activated G protein-coupled receptors (GPCRs) to intracellular signaling pathways. They can also function independently of GPCR activation upon acquiring mutations that prevent GTPase activity and result in constitutive signaling, as occurs with the αqQ209L mutation in uveal melanoma. YM-254890 (YM) can inhibit signaling by both GPCR-activated WT αq and GPCR-independent αqQ209L. Although YM inhibits WT αq by binding to αq-GDP and preventing GDP/GTP exchange, the mechanism of YM inhibition of cellular αqQ209L remains to be fully understood. Here, we show that YM promotes a subcellular redistribution of αqQ209L from the plasma membrane (PM) to the cytoplasm. To test if this loss of PM localization could contribute to the mechanism of inhibition of αqQ209L by YM, we developed and examined N-terminal mutants of αqQ209L, termed PM-restricted αqQ209L, in which the addition of membrane-binding motifs enhanced PM localization and prevented YM-promoted redistribution. Treatment of cells with YM failed to inhibit signaling by these PM-restricted αqQ209L. Additionally, pull-down experiments demonstrated that YM promotes similar conformational changes in both αqQ209L and PM-restricted αqQ209L, resulting in increased binding to βγ and decreased binding to regulator RGS2, and effectors p63RhoGEF-DH/PH and phospholipase C-β. GPCR-dependent signaling by PM-restricted WT αq is strongly inhibited by YM, demonstrating that resistance to YM inhibition by membrane-binding mutants is specific to constitutively active αqQ209L. Together, these results indicate that changes in membrane binding impact the ability of YM to inhibit αqQ209L and suggest that YM contributes to inhibition of αqQ209L by promoting its relocalization., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4 -/- ) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4 fl/fl ), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4 -/- mice compared with Nox4 fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4 -/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4 -/- mice compared with control mice, and significantly lower in 32-week old Nox4 -/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation., (© 2022. The Author(s).)

Deterioration in glucose homeostasis has been associated with intestinal dysbiosis, but it is not known how metabolic dysregulation alters the gastrointestinal environment. We investigated how the progression of diabetes alters ileal and colonic epithelial mucosal structure, microbial abundance, and transcript expression in the University of California Davis Type 2 Diabetes Mellitus (UCD-T2DM) rat model. Male UCD-T2DM rats (age ~170 days) were included if <1-month (n = 6, D1M) or 3-month (n = 6, D3M) post-onset of diabetes. Younger nondiabetic UCD-T2DM rats were included as a nondiabetic comparison (n = 6, ND, age ~70 days). Ileum villi height/crypt depths and colon crypt depths were assessed by histology. Microbial abundance of colon content was measured with 16S rRNA sequencing. Ileum and colon transcriptional abundances were analyzed using RNA sequencing. Ileum villi height and crypt depth were greater in D3M rats compared to ND. Colon crypt depth was greatest in D3M rats compared to both ND and D1M rats. Colon abundances of Akkermansia and Muribaculaceae were lower in D3M rats relative to D1M, while Oscillospirales, Phascolarctobacterium, and an unidentified genus of Lachnospiraceae were higher. Only two transcripts were altered by diabetes advancement within the colon; however, 2039 ileal transcripts were altered. Only colonic abundances of Sptlc3, Enpp7, Slc7a15, and Kctd14 had more than twofold changes between D1M and D3M rats. The advancement of diabetes in the UCD-T2DM rat results in a trophic effect on the mucosal epithelia and was associated with regulation of gastrointestinal tract RNA expression, which appears more pronounced in the ileum relative to the colon., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Rationally sequencing and combining PD-1/L1-and MAPK-targeted therapies may overcome innate and acquired resistance. Since increased clinical benefit of MAPK inhibitors (MAPKi) is associated with previous immune checkpoint therapy, we compare the efficacies of sequential and/or combinatorial regimens in subcutaneous murine models of melanoma driven by Braf V600 , Nras, or Nf1 mutations as well as colorectal and pancreatic carcinoma driven by Kras G12C . Anti-PD-1/L1 lead-in preceding MAPKi combination optimizes response durability by promoting pro-inflammatory polarization of macrophages and clonal expansion of interferon-γ hi , and CD8 + cytotoxic and proliferative (versus CD4 + regulatory) T cells that highly express activation genes. Since therapeutic resistance of melanoma brain metastasis (MBM) limits patient survival, we demonstrate that sequencing anti-PD-1/L1 therapy before MAPKi combination suppresses MBM and improves mouse survival with robust T cell clonal expansion in both intracranial and extracranial metastatic sites. We propose clinically testing brief anti-PD-1/L1 (± anti-CTLA-4) dosing before MAPKi co-treatment to suppress therapeutic resistance., Competing Interests: Declaration of interests R.S.L.: research support from Merck, Pfizer, BMS, and OncoSec. Consultant for Amgen, Novartis, Array BioPharma, Genentech, and Merck. A.R.: consultant for Amgen, BMS, Chugai, Genentech, Merck, Novartis, Roche, Sanofi, and Vedanta. Advisory board member for and stock shareholder of Advaxis, Apricity, Arcus, Compugen, CytomX, Five Prime, Highlight, ImaginAb, Isoplexis, Kalthera, Kite-Gilead, Merus, PACT Pharma, RAPT, Rgenix, and Tango. Research funding from Agilent and BMS through SU2C. A.P.A.: research support, advisory board member, consultant, shareholder and honorarium recipient, OncoSec. Advisory board member for and stock shareholder in Valitor Biosciences. Advisory board member and honorarium recipient for Regeneron and Array. Research support from Acerta, Amgen, AstraZeneca, BMS, Dynavax, Genentech, Idera, Incyte, ISA, LOXO, Merck, Novartis, Sensei, and Tessa. M.O.: consultant for Merck, Biosight, and Daiichi Sankyo; independent data safety monitoring committee member for Celgene and Glycomimetics. C.Y.: consultant for QED Therapeutics and Riptide Biosciences and owner of stocks in Riptide Biosciences. H.L.: co-founder and Executive Vice President of Riptide Biosciences. M.W.B.: consultant for Eli Lilly and research support from AstraZeneca. D.B.S.: consultant for Pfizer, Loxo/Lilly Oncology, Vividion Therapeutics, Fore Therapeutics, and BridgeBio., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Most congenital heart defects (CHDs) result from complex interactions among genetic susceptibilities, epigenetic modifications, and maternal environmental exposures. Characterizing the complex relationship between genetic, epigenetic, and transcriptomic variation will enhance our understanding of pathogenesis in this important type of congenital disorder. We investigated cis-acting effects of genetic single nucleotide polymorphisms (SNPs) on local DNA methylation patterns within 83 cardiac tissue samples and prioritized their contributions to CHD risk by leveraging results of CHD genome-wide association studies (GWAS) and their effects on cardiac gene expression., Results: We identified 13,901 potential methylation quantitative trait loci (mQTLs) with a false discovery threshold of 5%. Further co-localization analyses and Mendelian randomization indicated that genetic variants near the HLA-DRB6 gene on chromosome 6 may contribute to CHD risk by regulating the methylation status of nearby CpG sites. Additional SNPs in genomic regions on chromosome 10 (TNKS2-AS1 gene) and chromosome 14 (LINC01629 gene) may simultaneously influence epigenetic and transcriptomic variations within cardiac tissues., Conclusions: Our results support the hypothesis that genetic variants may influence the risk of CHDs through regulating the changes of DNA methylation and gene expression. Our results can serve as an important source of information that can be integrated with other genetic studies of heart diseases, especially CHDs.

MAPK targeting in cancer often fails due to MAPK reactivation. MEK inhibitor (MEKi) monotherapy provides limited clinical benefits but may serve as a foundation for combination therapies. Here, we showed that combining a type II RAF inhibitor (RAFi) with an allosteric MEKi durably prevents and overcomes acquired resistance among cancers with KRAS, NRAS, NF1, BRAF non-V600 , and BRAF V600 mutations. Tumor cell-intrinsically, type II RAFi plus MEKi sequester MEK in RAF complexes, reduce MEK/MEK dimerization, and uncouple MEK from ERK in acquired-resistant tumor subpopulations. Immunologically, this combination expands memory and activated/exhausted CD8 + T cells, and durable tumor regression elicited by this combination requires CD8 + T cells, which can be reinvigorated by anti-PD-L1 therapy. Whereas MEKi reduces dominant intratumoral T-cell clones, type II RAFi cotreatment reverses this effect and promotes T-cell clonotypic expansion. These findings rationalize the clinical development of type II RAFi plus MEKi and their further combination with PD-1/L1-targeted therapy. SIGNIFICANCE: Type I RAFi + MEKi are indicated only in certain BRAF V600MUT cancers. In contrast, type II RAFi + MEKi are durably active against acquired MEKi resistance across broad cancer indications, which reveals exquisite MAPK addiction. Allosteric modulation of MAPK protein/protein interactions and temporal preservation of intratumoral CD8 + T cells are mechanisms that may be further exploited. This article is highlighted in the In This Issue feature, p. 521 ., (©2020 American Association for Cancer Research.)

Cardiolipins (CLs) are comprised of two phosphatic acid moieties bound to a central glycerol backbone and are substituted with four acyl chains. Consequently, a vast number of distinct CL structures are possible in different biological contexts, representing a significant analytical challenge. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has become a widely used approach for the detection, characterization, and quantitation of complex lipids, including CLs. Central to this approach is fragmentation of the [CLs - H] - or [CL - 2H] 2- anions by collision-induced dissociation (CID). Product ions in the resulting tandem mass spectra confirm the CL subclass assignment and reveal the numbers of carbons and degrees of unsaturation in each of the acyl chains. Conventional CID, however, affords limited structural elucidation of the fatty acyl chains, failing to discriminate isomers arising from different site(s) of unsaturation or cyclopropanation and potentially obscuring their metabolic origins. Here, we report the application of charge inversion ion/ion chemistry in the gas phase to enhance the structural elucidation of CLs. Briefly, CID of [CL - H] 2- anions generated via negative ion ESI allowed for the assignment of individual fatty acyl substituents and phosphatidic acid moieties. Next, gas-phase derivatization of the resulting CL product ions, including fatty acyl carboxylate anions, was effected with gas-phase ion/ion charge inversion reactions with tris-phenanthroline magnesium reagent dications. Subsequent isolation and activation of the charge-inverted fatty acyl complex cations permitted the localization of both carbon-carbon double bond and cyclopropane motifs within each of the four acyl chains of CLs. This approach was applied to the de novo elucidation of unknown CLs in a biological extract revealing distinct isomeric populations and regiochemical relationships between double bonds and carbocyles.

Heightened awareness regarding the implication of disturbances in lipid metabolism with respect to prevalent human-related pathologies demands analytical techniques that provide unambiguous structural characterization and accurate quantitation of lipids in complex biological samples. The diversity in molecular structures of lipids along with their wide range of concentrations in biological matrices present formidable analytical challenges. Modern mass spectrometry (MS) offers an unprecedented level of analytical power in lipid analysis, as many advancements in the field of lipidomics have been facilitated through novel applications of and developments in electrospray ionization tandem mass spectrometry (ESI-MS/MS). ESI allows for the formation of intact lipid ions with little to no fragmentation and has become widely used in contemporary lipidomics experiments due to its sensitivity, reproducibility, and compatibility with condensed-phase modes of separation, such as liquid chromatography (LC). Owing to variations in lipid functional groups, ESI enables partial chemical separation of the lipidome, yet the preferred ion-type is not always formed, impacting lipid detection, characterization, and quantitation. Moreover, conventional ESI-MS/MS approaches often fail to expose diverse subtle structural features like the sites of unsaturation in fatty acyl constituents or acyl chain regiochemistry along the glycerol backbone, representing a significant challenge for ESI-MS/MS. To overcome these shortcomings, various charge manipulation strategies, including charge-switching, have been developed to transform ion-type and charge state, with aims of increasing sensitivity and selectivity of ESI-MS/MS approaches. Importantly, charge manipulation approaches afford enhanced ionization efficiency, improved mixture analysis performance, and access to informative fragmentation channels. Herein, we present a critical review of the current suite of solution-based and gas-phase strategies for the manipulation of lipid ion charge and type relevant to ESI-MS/MS., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS 2 product ion spectra of [FAHFA - H]¯ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen) 3 2+ , to selectively and efficiently derivatize [FAHFA - H]¯ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA - H + MgPhen 2 ] + cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Exclusive breastfeeding impacts the intestinal microbiome and is associated with a better immune function than is seen with milk formula (MF) feeding in infants and yet with mechanisms poorly defined. The porcine model was used to evaluate the impact of MF on ileum microbial communities and gene expression relative to human milk (HM)-fed piglets. Fifty-two Dutch Landrace male piglets were fed an isocaloric diet of either HM ( n  = 26) or MF ( n  = 26) from day 2 through day 21 of age and weaned to a solid diet until day 51. Eleven piglets from each group were euthanized at day 21, while the remaining piglets (HM, n  = 15; MF, n  = 15) were euthanized at day 51 to collect ileal epithelium (EP) scrapings and ileal (IL) tissues. The epithelial mucosa was subjected to shotgun metagenome sequencing, and EP and IL tissues were used for transcriptome analysis. On day 21, transcriptome data revealed that the levels of pathways involved in inflammation and apoptosis were significantly higher in MF piglets than in HM piglets, whereas the levels of tight junctions and pathogen detection systems were lower in MF piglets than in HM piglets. The MF impacts on the small intestine were maintained over the postweaning period (day 51) as indicated by higher levels of Dialister invisus bacteria and higher levels of expression of genes associated with inflammation and apoptosis pathways relative to HM group. The current study demonstrated that MF might impact local intestinal inflammation, apoptosis, and tight junctions and might suppress pathogen recognition in the small intestine compared with HM. IMPORTANCE Exclusive human milk (HM) breastfeeding for the first 6 months of age in infants is recommended to improve health outcomes during early life and beyond. When women are unable to provide sufficient HM, milk formula (MF) is often recommended as a complementary or alternative source of nutrition. Previous studies in piglets demonstrated that MF alters the gut microbiome and induces inflammatory cytokine production. The links between MF feeding, gut microbiome, and inflammation status are unclear due to challenges associated with the collection of intestinal samples from human infants. The current report provides the first insight into MF-microbiome-inflammation connections in the small intestine compared with HM feeding using a porcine model. The present results showed that, compared with HM, MF might impact immune function through the induction of ileal inflammation, apoptosis, and tight junction disruptions and likely compromised immune defense against pathogen detection in the small intestine relative to piglets that were fed HM., (Copyright © 2020 Elolimy et al.)

Cardiolipin (CL) analysis demands high specificity, due to the extensive diversity of CL structures, and high sensitivity, due to their low relative abundance within the lipidome. While electrospray ionization mass spectrometry (ESI-MS) is the most widely used technology in lipidomics, the potential for multiple charging presents unique challenges for CL identification and quantification. Depending on the conditions, ESI-MS of lipid extracts in negative ion mode can give rise to cardiolipins ionized as both singly and doubly deprotonated anions. This signal degeneracy diminishes the signal-to-noise ratio, while in addition (for direct infusion), the dianion population falls within a m / z range already heavily congested with monoanions from more abundant glycerophospholipid subclasses. Herein, we describe a direct infusion strategy for CL profiling from total lipid extracts utilizing gas-phase proton-transfer ion/ion reactions. In this approach, lipid extracts are ionized by negative ion ESI generating both singly deprotonated phospholipids and doubly deprotonated CL anions. Charge reduction of the negative ion population by ion/ion reactions leads to an enhancement in singly deprotonated [CL - H] - species via proton transfer to the corresponding [CL - 2H] 2- ̅ dianions. To concentrate the [CL - H] - anion signal, multiple iterations of ion accumulation and proton-transfer ion/ion reaction can be performed prior to subsequent interrogation. Mass selection and collisional activation of the enriched population of [CL - H] - anions facilitates the assignment of individual fatty acyl substituents and phosphatidic acid moieties. Demonstrated advantages of this new approach derive from the improved performance in complex mixture analysis affording detailed characterization of low abundant CLs directly from a total biological extract.

microRNAs (miRNAs) are conserved non-coding small nucleotide molecules found in nearly all species and breastmilk. miRNAs present in breastmilk are very stable to freeze-thaw, RNase treatment, and low pH as they are protected inside exosomes. They are involved in regulating several physiologic and pathologic processes, including immunologic pathways, and we have demonstrated better immune response to vaccines in piglets fed with human milk (HM) in comparison to dairy-based formula (MF). To understand if neonatal diet impacts circulatory miRNA expression, serum miRNA expression was evaluated in piglets fed HM or MF while on their neonatal diet at postnatal day (PND) 21 and post-weaning to solid diet at PND 35 and 51. MF fed piglets showed increased expression of 14 miRNAs and decreased expression of 10 miRNAs, relative to HM fed piglets at PND 21. At PND 35, 9 miRNAs were downregulated in the MF compared to the HM group. At PND 51, 10 miRNAs were decreased and 17 were increased in the MF relative to HM suggesting the persistent effect of neonatal diet. miR-148 and miR-181 were decreased in MF compared to HM at PND 21. Let-7 was decreased at PND 35 while miR-199a and miR-199b were increased at PND 51 in MF compared to HM. Pathway analysis suggested that many of the miRNAs are involved in immune function. In conclusion, we observed differential expression of blood miRNAs at both PND 21 and PND 51. miRNA found in breastmilk were decreased in the serum of the MF group, suggesting that diet impacts circulating miRNA profiles at PND 21. The miRNAs continue to be altered at PND 51 suggesting a persistent effect of the neonatal diet. The sources of miRNAs in circulation need to be evaluated, as the piglets were fed the same solid diet leading up to PND 51 collections. In conclusion, the HM diet appears to have an immediate and persistent effect on the miRNA profile and likely regulates the pathways that impact the immune system and pose benefits to breastfed infants., (Copyright © 2020 Carr, Bowlin, Elolimy, Byrum, Washam, Randolph, MacLeod and Yeruva.)

Ether lipids represent a unique subclass of glycerophospholipid (GPL) that possesses a 1- O -alkyl (i.e., plasmanyl subclass) or a 1- O -alk-1'-enyl (i.e., plasmenyl subclass) group linked at the sn -1 position of the glycerol backbone. As changes in ether GPL composition and abundance are associated with numerous human pathologies, analytical strategies capable of providing high-level structural detail are desirable. While mass spectrometry (MS) has emerged as a prominent tool for lipid structural elucidation in biological extracts, distinctions between the various isomeric forms of ether-linked GPLs have remained a significant challenge for tandem MS, principally due to similarities in the conventional tandem mass spectra obtained from the two ether-linked subclasses. To distinguish plasmanyl and plasmenyl GPLs, a multistage (i.e., MS n where n = 3 or 4) mass spectrometric approach reliant on low-energy collision-induced dissociation (CID) is required. While this method facilitates assignment of the sn -1 bond type (i.e., 1- O -alkyl versus 1- O -alk-1'-enyl), a composite distribution of isomers is left unresolved, as carbon-carbon double-bond (C=C) positions cannot be localized in the sn -2 fatty acyl substituent. In this study, we combine a systematic MS n approach with two unique gas-phase charge inversion ion/ion chemistries to elucidate ether GPL structures with high-level detail. Ultimately, we assign both the sn -1 bond type and sites of unsaturation in the sn -2 fatty acyl substituent using an entirely gas-phase MS-based workflow. Application of this workflow to human blood plasma extract permitted isomeric resolution and in-depth structural identification of major and, in some cases, minor isomeric contributors to ether GPLs that have been previously unresolved when examined via conventional methods.

Shotgun lipidomics has recently gained popularity for lipid analysis. Conventionally, shotgun analysis of glycerophospholipids via direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides glycerophospholipid (GPL) class (i.e., headgroup composition) and fatty acyl composition. Reliant on low-energy collision-induced dissociation (CID), traditional ESI-MS/MS fails to define fatty acyl regiochemistry along the glycerol backbone or carbon-carbon double bond position(s) in unsaturated fatty acyl substituents. Therefore, isomeric GPLs are often unresolved, representing a significant challenge for shotgun-MS approaches. We developed a top-down shotgun-MS method utilizing gas-phase ion/ion charge inversion chemistry that provides near-complete GPL structural identification. First, in negative ion mode, CID of mass-selected GPL anions generates fatty acyl carboxylate anions via fragmentation of ester bonds linking the fatty acyl substituents at the sn -1 and sn -2 positions of the glycerol backbone. Product anions, including fatty acyl carboxylate ions, were then derivatized in the mass spectrometer via an ion/ion charge inversion reaction with tris-phenanthroline magnesium dications. Subsequent CID of charge-inverted fatty acyl complex cations yielded isomer-specific product ion spectra that permit (i) unambiguous assignment of carbon-carbon double bond position(s) and (ii) relative quantitation of isomeric fatty acyl substituents. The outlined strategy was applied to the analysis of targeted GPLs extracted from human plasma, including several proposed plasma biomarkers. A single experiment thus facilitates assignment of the GPL headgroup, fatty acyl composition, carbon-carbon double bond position(s) in unsaturated fatty acyl chains, and, in some cases, fatty acyl sn -position and relative abundances for isomeric fatty acyl substituents. Ultimately, this MS n platform paired with ion/ion chemistry permitted identification of major, and some minor, isomeric contributors that are unresolved using conventional ESI-MS/MS.

Representing the most fundamental lipid class, fatty acids (FA) play vital biological roles serving as energy sources, cellular signaling molecules, and key architectural components of complex lipids. Direct infusion electrospray ionization spectrometry, also known as shotgun lipidomics, has emerged as a rapid and powerful toolbox for lipid analysis. While shotgun lipidomics can be a sensitive approach to FA detection, the diverse molecular structure of FA presents challenges for unambiguous identification and the relative quantification of isomeric contributors. In particular, pinpointing double bond position(s) in unsaturated FA and determining the relative contribution of double bond isomers has limited the application of the shotgun approach. Recently, we reported the use of gas-phase ion/ion reactions to facilitate the identification of FA. Briefly, singly deprotonated FA anions undergo charge inversion when reacted in the gas phase with tris-phenanthroline magnesium dications by forming [FA - H + MgPhen] + complex ions. These charge-inverted FA complex cations fragment upon ion-trap collision-induced dissociation (CID) to generate product ion spectra unique to individual FA isomers. Herein, we report the development of a mass spectral library comprised of [FA - H + MgPhen] + product ion spectra. The developed FA library permits confident FA identification, including polyunsaturated FA isomers. Furthermore, we demonstrate the ability to determine relative contributions of isomeric FA using multiple linear regression analysis paired with gas-phase ion/ion reactions. We successfully applied the presented method to generate a FA profile for bovine liver phospholipidome based entirely on gas-phase chemistries.

Shotgun lipid analysis based on electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in lipidomic studies. One challenge for the shotgun approach is the discrimination of lipid isomers and isobars. Gas-phase charge inversion via ion/ion reactions has been used as an effective method to identify multiple isomeric/isobaric components in a single MS peak by exploiting the distinctive functionality of different lipid classes. In doing so, fatty acyl chain information can be obtained without recourse to condensed-phase separations or derivatization. This method alone, however, cannot provide carbon-carbon double bond (C=C) location information from fatty acyl chains. Herein, we provide an enhanced method pairing photochemical derivatization of C=C via the Paternò-Büchi reaction with charge inversion ion/ion tandem mass spectrometry. This method was able to provide gas-phase separation of phosphatidylcholines and phosphatidylethanolamines, the fatty acyl compositions, and the C=C location within each fatty acyl chain. We have successfully applied this method to bovine liver lipid extracts and identified 40 molecular species of glycerophospholipids with detailed structural information including head group, fatty acyl composition, and C=C location. Graphical Abstract ᅟ.

Uveal melanoma is the most common intraocular tumor in adults and often metastasizes to the liver, leaving patients with few options. Recurrent activating mutations in the G proteins, Gα q and Gα 11 , are observed in approximately 93% of all uveal melanomas. Although therapeutic intervention of downstream Gα q/11 targets has been unsuccessful in treating uveal melanoma, we have found that the Gα q/11 inhibitor, FR900359 (FR), effectively inhibits oncogenic Gα q/11 signaling in uveal melanoma cells expressing either mutant Gα q or Gα 11 . Inhibition of oncogenic Gα q/11 by FR results in cell-cycle arrest and induction of apoptosis. Furthermore, colony formation is prevented by FR treatment of uveal melanoma cells in 3D-cell culture, providing promise for future in vivo studies. This suggests direct inhibition of activating Gα q/11 mutants may be a potential means of treating uveal melanoma. IMPLICATIONS: Oncogenic Gα q/11 inhibition by FR900359 may be a potential treatment option for those with uveal melanoma., (©2018 American Association for Cancer Research.)

Fatty acids (FA) play vital biological roles as energy sources, signaling molecules and key building blocks of complex lipids in cell membranes. Modifications to FA structure and composition are associated with the onset and progression of a number of chronic diseases. Consequently, the sensitive detection and unambiguous structure elucidation of FA is integral to the advancement of biomedical sciences. Recent advances in FA analysis have taken advantage of wet chemical derivatization to enhance detection and drive unique fragmentation in tandem mass spectrometry protocols. Here, we significantly further this approach through demonstrating gas-phase charge inversion of singly deprotonated FA ions, [M - H] - , using doubly charged tris-phenanthroline alkaline earth metal complexes, [Cat(Phen) 3 ] 2+ (Cat = Mg 2+ , Ca 2+ , Sr 2+ , or Ba 2+ ). Metal cationized FA, [M - H + Cat] + are obtained after the gas-phase ion/ion reaction. Low-energy collision-induced dissociation (CID) of the [M - H + Cat] + cations facilitates double bond localization for a variety of monounsaturated and polyunsaturated FAs. Ultimately, detailed characterization presented unambiguous distinction among FA double bond positional isomers, such as n-3 and n-6 isomers. The method was successfully used to identify the FA profile of corn oil, including double bond localization for unsaturated FAs present.

The air temperatures inside uninsulated and insulated huts were recorded on an outdoor pig unit in the south of England between September 1997 and September 1998, and the herd's production parameters were also recorded. During the summer the temperatures inside some of the uninsulated huts exceeded 45 degrees C, but the temperatures inside the insulated huts were lower and fluctuated less. Despite the high temperatures, the weaning weight of piglets reared in the uninsulated huts were often higher than those of the piglets reared in the insulated huts, possibly as a result of the higher mortality of small piglets in the uninsulated huts, especially during the winter. The weaning weights of the piglets were higher during the summer.