Drosophila Models Uncover Substrate Channeling Effects on Phospholipids and Sphingolipids in Peroxisomal Biogenesis Disorders.

Peroxisomal Biogenesis Disorders Zellweger Spectrum (PBD-ZSD) disorders are a group of autosomal recessive defects in peroxisome formation that produce a multi-systemic disease presenting at birth or in childhood. Well documented clinical biomarkers such as elevated very long chain fatty acids (VLCFA) are key biochemical diagnostic findings in these conditions. Additional, secondary biochemical alterations such as elevated very long chain lysophosphatidylcholines are allowing newborn screening for peroxisomal disease. In addition, a more widespread impact on metabolism and lipids is increasingly being documented by metabolomic and lipidomic studies. Here we utilize Drosophila models of pex2 and pex16 as well as human plasma from individuals with PEX1 mutations. We identify phospholipid abnormalities in Drosophila larvae and brain characterized by differences in the quantities of phosphatidylcholine (PC) and phosphatidylethanolamines (PE) with long chain lengths and reduced levels of intermediate chain lengths. For diacylglycerol (DAG) the precursor of PE and PC through the Kennedy pathway, the intermediate chain lengths are increased suggesting an imbalance between DAGs and PE and PC that suggests the two acyl chain pools are not in equilibrium. Altered acyl chain lengths are also observed in PE ceramides in the fly models. Interestingly, plasma from human subjects exhibit phospholipid alterations similar to the fly model. Moreover, human plasma shows reduced levels of sphingomyelin with 18 and 22 carbon lengths but normal levels of C24. Our results suggest that peroxisomal biogenesis defects alter shuttling of the acyl chains of multiple phospholipid and ceramide lipid classes, whereas DAG species with intermediate fatty acids are more abundant. These data suggest an imbalance between de novo synthesis of PC and PE through the Kennedy pathway and remodeling of existing PC and PE through the Lands cycle. This imbalance is likely due to overabundance of very long and long acyl chains in PBD and a subsequent imbalance due to substrate channeling effects. Given the fundamental role of phospholipid and sphingolipids in nervous system functions, these observations suggest PBD-ZSD are diseases characterized by widespread cell membrane lipid abnormalities.

Correction: Vu et al. Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance. Metabolites 2022, 12, 385.

There was an error in the original publication [...].

Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species.

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.

Exploring the potential effect of phospholipase A2 antibody to extend beef shelf-life in a beef liposome model system.

The objective of this study was to elucidate the effect of phospholipase A2 (PLA2) and a PLA2 antibody (aPLA2) on phospholipid (PL) hydrolysis in beef and to understand how the altered PL composition may affect lipid oxidation and antioxidant capacity of beef in an in vitro system. Various combinations of PLA2 and aPLA2 were introduced to a beef liposome model system and exposed to a retail display. The PL and free fatty acid (FFA) profiles, antioxidant capacity and lipid oxidation were measured for the liposome system. Key PL classes were reduced and the release of polyunsaturated FFAs was increased with the inclusion of PLA2 in the treatments (P < 0.05). There was no inhibition of PL hydrolysis with the addition of aPLA2. PLA2 showed strong antioxidant capacity in the liposome system (P < 0.01), but lipid oxidation still increased in samples treated with PLA2 throughout the retail display (P < 0.01). Finally, aPLA2 treatments demonstrated potential to decrease lipid oxidation (P < 0.01).

Lipid modulation contributes to heat stress adaptation in peanut.

At the cellular level, membrane damage is a fundamental cause of yield loss at high temperatures (HT). We report our investigations on a subset of a peanut (Arachis hypogaea) recombinant inbred line population, demonstrating that the membrane lipid remodeling occurring at HT is consistent with homeoviscous adaptation to maintain membrane fluidity. A major alteration in the leaf lipidome at HT was the reduction in the unsaturation levels, primarily through reductions of 18:3 fatty acid chains, of the plastidic and extra-plastidic diacyl membrane lipids. In contrast, levels of 18:3-containing triacylglycerols (TGs) increased at HT, consistent with a role for TGs in sequestering fatty acids when membrane lipids undergo remodeling during plant stress. Polyunsaturated acyl chains from membrane diacyl lipids were also sequestered as sterol esters (SEs). The removal of 18:3 chains from the membrane lipids decreased the availability of susceptible molecules for oxidation, thereby minimizing oxidative damage in membranes. Our results suggest that transferring 18:3 chains from membrane diacyl lipids to TGs and SEs is a key feature of lipid remodeling for HT adaptation in peanut. Finally, QTL-seq allowed the identification of a genomic region associated with heat-adaptive lipid remodeling, which would be useful for identifying molecular markers for heat tolerance.

A Water Extract from Chlorella sorokiniana Cell Walls Stimulates Growth of Bone Marrow Cells and Splenocytes.

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C.sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells.

Lipidomic Analysis of Arabidopsis T-DNA Insertion Lines Leads to Identification and Characterization of C-Terminal Alterations in FATTY ACID DESATURASE 6.

Mass-spectrometry-based screening of lipid extracts of wounded and unwounded leaves from a collection of 364 Arabidopsis thaliana T-DNA insertion lines produced lipid profiles that were scored on the number and significance of their differences from the leaf lipid profiles of wild-type plants. The analysis identified Salk_109175C, which displayed alterations in leaf chloroplast glycerolipid composition, including a decreased ratio between two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3). Salk_109175C has a confirmed insertion in the At5g64790 locus; the insertion did not co-segregate with the recessive lipid phenotype in the F2 generation of a wild-type (Columbia-0) × Salk_109175C cross. The altered lipid compositional phenotype mapped to the At4g30950 locus, which encodes the plastidial ω-6 desaturase FATTY ACID DESATURASE 6 (FAD6). Sequencing revealed a splice-site mutation, leading to the in-frame deletion of 13 amino acids near the C-terminal end of the 448 amino acid protein. Heterologous expression in yeast showed that this deletion eliminates desaturase activity and reduces protein stability. Sequence comparison across species revealed that several amino acids within the deletion are conserved in plants and cyanobacteria. Individual point mutations in four conserved residues resulted in 77-97% reductions in desaturase activity, while a construct with all four alanine substitutions lacked activity. The data suggest that the deleted region of FAD6, which is on the C-terminal side of the four putative transmembrane segments and the histidine boxes putatively involved in catalysis, is critical for FAD6 function.

Specific Changes in Arabidopsis thaliana Rosette Lipids during Freezing Can Be Associated with Freezing Tolerance.

While the roles of a few specific lipids in plant freezing tolerance are understood, the effect of many plant lipids remains to be determined. Acclimation of plants to non-freezing cold before exposure to freezing temperatures improves the outcome of plants, compared to plants exposed to freezing without acclimation. Arabidopsis thaliana plants were subjected to one of three treatments: (1) "control", i.e., growth at 21 °C, (2) "non-acclimated", i.e., 3 days at 21 °C, 2 h at -8 °C, and 24 h recovery at 21 °C, and (3) "acclimated", i.e., 3 days at 4 °C, 2 h at -8 °C, and 24 h recovery at 21 °C. Plants were harvested at seven time points during the treatments, and lipid levels were measured by direct-infusion electrospray ionization tandem mass spectrometry. Ion leakage was measured at the same time points. To examine the function of lipid species in relation to freezing tolerance, the lipid levels in plants immediately following the freezing treatment were correlated with the outcome, i.e., ion leakage 24-h post-freezing. Based on the correlations, hypotheses about the functions of specific lipids were generated. Additionally, analysis of the lipid levels in plants with mutations in genes encoding patatin-like phospholipases, lipoxygenases, and 12-oxophytodienoic acid reductase 3 (opr3), under the same treatments as the wild-type plants, identified only the opr3-2 mutant as having major lipid compositional differences compared to wild-type plants.

Nanoparticles are linked to polar lipids in xylem sap of temperate angiosperm species.

In previous research, xylem sap of angiosperms has been found to include low concentrations of nanoparticles and polar lipids. A major goal of this study was to test predictions arising from the hypothesis that the nanoparticles consist largely of polar lipids from the original cell content of vessel elements. These predictions included that polar lipid and nanoparticle concentrations would be correlated, that they both do not pass through pit membranes and that they do not vary seasonally because they originate from living vessel element cells. We collected xylem sap of six temperate angiosperm species over the whole year to consider seasonal variation. Concentrations of nanoparticles and lipids in xylem sap and contamination control samples were measured with a NanoSight device and mass spectrometry. We found that the concentration of nanoparticles and polar lipids was (i) diluted when an increasing amount of sap was extracted, (ii) significantly correlated to each other for three species, (iii) affected by vessel anatomy, (iv) very low and largely different in chemical composition from contamination controls and (v) hardly variable among seasons. Moreover, there was a minor freezing-thawing effect with respect to nanoparticle amount and size. Xylem sap lipids included polar galactolipids and phospholipids in all species and neutral triacylglycerols in two species. These findings support the predictions and, by implication, the underlying hypothesis that nanoparticles in xylem sap consist of polar lipids from the original cell content of living vessel element cells. Further research is needed to examine the formation and stability of nanoparticles concerning lipid composition and multiphase interactions among gas, liquid and solid phases in xylem conduits of living plants.

Association of colchicine use for acute gout with clinical outcomes in acute decompensated heart failure.

BACKGROUND: Gout is a common comorbidity in heart failure (HF) patients and is frequently associated with acute exacerbations during treatment for decompensated HF. Although colchicine is often used to manage acute gout in HF patients, its impact on clinical outcomes when used during acute decompensated HF is unknown. METHODS: This was a single center, retrospective study of hospitalized patients treated for an acute HF exacerbation with and without acute gout flare between March 2011 and December 2020. We assessed clinical outcomes in patients treated with colchicine for a gout flare compared to those who did not experience a gout flare or receive colchicine. The primary outcome was in-hospital all-cause mortality. RESULTS: Among 1047 patient encounters for acute HF during the study period, there were 237 encounters (22.7%) where the patient also received colchicine for acute gout during admission. In-hospital all-cause mortality was significantly reduced in the colchicine group compared with the control group (2.1% vs. 6.5%, p = .009). The colchicine group had increased length of stay (9.93 vs. 7.96 days, p < .001) but no significant difference in 30-day readmissions (21.5% vs. 19.5%, p = .495). In a Cox proportional hazards model adjusted for age, inpatient colchicine use was associated with improved survival to discharge (hazards ratio [HR] 0.163, 95% confidence interval [CI] 0.051-0.525, p = .002) and a reduced rate of in-hospital CV mortality (HR 0.184, 95% CI 0.044-0.770, p = .021). CONCLUSION: Among patients with a HF exacerbation, treatment with colchicine for a gout flare was associated with significantly lower in-hospital mortality compared with those not treated for acute gout.

Functional Genomics Platform, A Cloud-Based Platform for Studying Microbial Life at Scale.

The rapid growth in biological sequence data is revolutionizing our understanding of genotypic diversity and challenging conventional approaches to informatics. With the increasing availability of genomic data, traditional bioinformatic tools require substantial computational time and the creation of ever-larger indices each time a researcher seeks to gain insight from the data. To address these challenges, we pre-computed important relationships between biological entities spanning the Central Dogma of Molecular Biology and captured this information in a relational database. The database can be queried across hundreds of millions of entities and returns results in a fraction of the time required by traditional methods. In this paper, we describe Functional Genomics Platform (formerly known as OMXWare), a comprehensive database relating genotype to phenotype for bacterial life. Continually updated, the Functional Genomics Platform today contains data derived from 200,000 curated, self-consistently assembled genomes. The database stores functional data for over 68 million genes, 52 million proteins, and 239 million domains with associated biological activity annotations from Gene Ontology, KEGG, MetaCyc, and Reactome. The Functional Genomics Platform maps all of the many-to-many connections between each biological entity including the originating genome, gene, protein, and protein domain. Various microbial studies, from infectious disease to environmental health, can benefit from the rich data and connections. We describe the data selection, the pipeline to create and update the Functional Genomics Platform, and the developer tools (Python SDK and REST APIs)which allow researchers to efficiently study microbial life at scale.

Teaching and learning under COVID-19 public health edicts: the role of household lockdowns and prior technology usage.

Public health edicts necessitated by COVID-19 prompted a rapid pivot to remote online teaching and learning. Two major consequences followed: households became students' main learning space, and technology became the sole medium of instructional delivery. We use the ideas of "digital disconnect" and "digital divide" to examine, for students and faculty, their prior experience with, and proficiency in using, learning technology. We also explore, for students, how household lockdowns and digital capacity impacted learning. Our findings are drawn from 3806 students and 283 faculty instructors from nine higher education institutions across Asia, Australia, Europe, and North America. For instructors, we find little evidence of a digital divide but some evidence of a digital disconnect. However, neither made a difference to self-reported success in transitioning courses. Faculty instructors were impacted in a myriad of diverse ways. For students, we show that closure and confinement measures which created difficult living situations were associated with lower levels of confidence in learning. The digital divide that did exist among students was less influential than were household lockdown measures in undermining student learning.

Phenelzine and Morphine Drug-Drug Interaction? A Literature Review.

The objectives of this manuscript are to describe a case report of a patient whose phenelzine maintenance therapy was discontinued due to concern for a phenelzine-morphine drug interaction, to review the available literature regarding the potential for this drug-drug interaction, and provide recommendations for this clinical scenario. A PubMed/MEDLINE literature search was conducted and all publications determined to be relevant to this case report were included. Literature describing in vitro data, case reports/human studies, and review articles concerning the interaction between morphine and monoamine oxidase inhibitors (MAOIs) were included. A total of 14 publications pertinent to the potential phenelzine-morphine interaction were included in this review including 5 in vitro studies, 4 human studies, and 6 review articles detailing the drug interaction profile between opioids and antidepressants. Of these publications, only a single case report of a potential drug interaction between morphine and phenelzine was identified. The literature suggesting a drug interaction between morphine and phenelzine is limited. The combination of phenelzine and morphine, with close monitoring for signs and symptoms of serotonin syndrome, is reasonable for patients with appropriate indications for both agents.

Lipids in xylem sap of woody plants across the angiosperm phylogeny.

Lipids have been observed attached to lumen-facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots and eudicots, to characterize and quantify phospholipids, galactolipids and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using transmission electron microscopy and confocal microscopy. Xylem sap contained the galactolipids di- and monogalactosyldiacylglycerol, as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol ml-1 across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared with wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen-facing vessel surfaces of L. nobilis, and lipids were highly concentrated in inter-vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion-tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem.

Leaf Lipid Alterations in Response to Heat Stress of Arabidopsis thaliana.

In response to elevated temperatures, plants alter the activities of enzymes that affect lipid composition. While it has long been known that plant leaf membrane lipids become less unsaturated in response to heat, other changes, including polygalactosylation of galactolipids, head group acylation of galactolipids, increases in phosphatidic acid and triacylglycerols, and formation of sterol glucosides and acyl sterol glucosides, have been observed more recently. In this work, by measuring lipid levels with mass spectrometry, we confirm the previously observed changes in Arabidopsis thaliana leaf lipids under three heat stress regimens. Additionally, in response to heat, increased oxidation of the fatty acyl chains of leaf galactolipids, sulfoquinovosyldiacylglycerols, and phosphatidylglycerols, and incorporation of oxidized acyl chains into acylated monogalactosyldiacylglycerols are shown. We also observed increased levels of digalactosylmonoacylglycerols and monogalactosylmonoacylglycerols. The hypothesis that a defect in sterol glycosylation would adversely affect regrowth of plants after a severe heat stress regimen was tested, but differences between wild-type and sterol glycosylation-defective plants were not detected.

A Lipidomic Approach to Identify Cold-Induced Changes in Arabidopsis Membrane Lipid Composition.

Lipid changes that occur in leaves of plants (e.g., Arabidopsis thaliana), during cold and freezing stress can be analyzed with electrospray ionization triple quadrupole mass spectrometry, using high-throughput multiple reaction monitoring (MRM). An online tool, LipidomeDB Data Calculation Environment, is employed for mass spectral data processing.

A pharmacy liaison-patient navigation intervention to reduce inpatient and emergency department utilization among primary care patients in a Medicaid accountable care organization: A pragmatic trial protocol.

OBJECTIVE: To determine whether employing the services of a pharmacy liaison to promote medication adherence (usual care), relative to a pharmacy liaison with training in motivational interviewing and as a patient navigator who systematically screens for health-related social needs and provides targeted navigation services to connect patients with appropriate community resources in partnership with a community-based organization (enhanced usual care), will reduce inpatient hospital admissions and emergency department visits among patients who are members of a Medicaid ACO and receive primary care at a large urban safety-net hospital. BACKGROUND: Prior studies have demonstrated only modest effects in reducing utilization among safety-net patient populations. Interventions that address health-related social needs have the potential to reduce utilization in these populations. DESIGN/METHODS: Assignment to treatment condition is by medical record number (odd vs. even) and is unblinded (NCT03919084). Adults age 18-64 within the 3rd to 10th percentile for health care utilization and cost among Medicaid Accountable Care Organization membership attending a primary care visit in the general internal medicine practice at Boston Medical Center enrolled. DISCUSSION: Our study will advance the field in two ways: 1) by providing evidence about the effectiveness of pharmacy liaisons who also function as patient navigators; and 2) by de-implementing patient navigators. Patients in the enhanced usual care arm will no longer receive the services of a clinic-based patient navigator. In addition, our study includes a novel collaboration with a community-based organization, and focuses on an intermediate-cost patient population, rather than the most costly patient population.

Characteristics of High-Performing Interprofessional Health Care Teams Involving Student Pharmacists.

Objective. To identify key themes of interprofessional models of care that offer experiential education opportunities for pharmacy learners. Methods. Six pharmacists from four Area Health Education Centers in North Carolina participated in individual, 60-minute interviews. Using two pre-established frameworks, the data were analyzed qualitatively by two members of the research team to identify the characteristics of interdisciplinary care teams. Results. At the level of the organization or health care system, the theme of appropriate resources and procedures emerged. At the level of the team, the themes of appropriate resources and procedures, communication, appropriate skill mix, climate, quality and outcomes of care, and respecting and understanding roles emerged. At the level of the individual, the themes of communication, respecting and understanding roles, and individual characteristics emerged. Three themes identified in a previous study failed to emerge in the interviews: leadership and management; personal rewards, training and development; and clarity of vision. Conclusion. Although a growing body of evidence highlights the importance of designing practice models to achieve interdisciplinary care that is patient-centered and effective, capacity to support learners and effectively educate them in the principles and practices of team-based care is limited. This study provides critical insight into characterizations of interprofessional models that integrate pharmacy learners. Further research is needed to better understand the barriers to designing and implementing IPE in experiential settings.

Identifying the Presence of Cognitive Apprenticeship in the Layered Learning Practice Model.

Objective. To identify the presence of cognitive apprenticeship themes in the layered learning practice model (LLPM). Methods. Attending pharmacists who had implemented an LLPM completed an individual 90-minute face-to-face semi-structured interview. Three researchers independently reviewed transcripts to identify cognitive apprenticeship themes according to the framework's dimensions and sub-dimensions. Results. Of 25 eligible attending pharmacists, 24 (96%) agreed to participate. All core dimensions of the cognitive apprenticeship framework emerged during the interviews; however, preceptors varied in how they used the framework in the training of pharmacy learners at different levels. This variability was especially apparent within the sub-dimensions of the content and method domains. Conclusion. This study demonstrates that all four cognitive apprenticeship principles are being used in the clinical environments operationalizing the LLPM. These findings suggest that cognitive apprenticeship is an applicable and relevant educational framework when engaging multiple learners in clinical education environments.

An efficient modified method for plant leaf lipid extraction results in improved recovery of phosphatidic acid.

BACKGROUND: Lipidomics plays an important role in understanding plant adaptation to different stresses and improving our knowledge of the genes underlying lipid metabolism. Lipidomics involves lipid extraction, sample preparation, mass spectrometry analysis, and data interpretation. One of the practical challenges for large-scale lipidomics studies on plant leaves is the requirement of an efficient and rapid extraction method. RESULTS: A single-extraction method with a polar solvent mixture gives results comparable to a widely used, multi-extraction method when tested on both Arabidopsis thaliana and Sorghum bicolor leaf tissue. This single-extraction method uses a mixture of 30 parts chloroform, 25 parts isopropanol, 41.5 parts methanol, and 3.5 parts water (v/v/v/v) and a 24-h extraction time. Neither inclusion of ammonium acetate nor inclusion of acetic acid increased extraction efficiency. CONCLUSIONS: The extract produced by this method can be used for analysis by mass spectrometry without a solvent evaporation step. The amount of lipid extracted, including phosphatidic acid, is comparable to widely used, more labor-intensive methods. The single-extraction protocol is less laborious, reducing the potential for human error.