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Background: Several genetic variants are associated with chronic liver disease. The role of these variants in outcomes after liver transplantation (LT) is uncertain. The aim of this study was to determine if donor genotype at risk-associated variants in PNPLA3 (rs738409 C>G, p.I148M) and HSD17B13 (rs72613567 T>TA; rs80182459, p.A192Lfs∗8) influences post-LT survival., Methods: In this retrospective cohort study, data on 2346 adults who underwent first-time LT between January 1, 1999 and June 30, 2020 and who had donor DNA samples available at five large Transplant Immunology Laboratories in Texas, USA, were obtained from the United Network for Organ Sharing (UNOS). Duplicates, patients with insufficient donor DNA for genotyping, those who were <18 years of age at the time of transplant, had had a previous transplant or had missing genotype data were excluded. The primary outcomes were patient and graft survival after LT. The association between donor genotype and post-LT survival was examined using Kaplan-Meier method and multivariable-adjusted Cox proportional hazards models., Findings: Median age of LT recipients was 57 [interquartile range (IQR), 50-62] years; 837 (35.7%) were women; 1362 (58.1%) White, 713 (30.4%) Hispanic, 182 (7.8%) Black/African-American. Median follow-up time was 3.95 years. Post-LT survival was not affected by donor PNPLA3 genotype but was significantly reduced among recipients of livers with two HSD17B13 loss-of-function (LoF) variants compared to those receiving livers with no HSD17B13 LoF alleles (unadjusted one-year survival: 82.6% vs 93.9%, P  < 0.0001; five-year survival: 73.1% vs 82.9%, P  = 0.0017; adjusted hazard ratio [HR], 2.25; 95% CI, 1.61-3.15 after adjustment for recipient age, sex, and self-reported ethnicity). Excess mortality was restricted to those receiving steroid induction immunosuppression (crude 90-day post-LT mortality, 9.3% [95% CI, 1.9%-16.1%] vs 1.9% [95% CI, 0.9%-2.9%] in recipients of livers with two vs no HSD17B13 LoF alleles, P  = 0.0012; age, sex, and ethnicity-adjusted HR, 2.85; 95% CI, 1.72-4.71, P  < 0.0001). No reduction was seen among patients who did not receive steroid induction (90-day mortality 3.1% [95% CI, 0%-7.3%] vs 2% [95% CI, 0.9%-3.1%], P  = 0.65; adjusted HR, 1.17; 95% CI, 0.66-2.08, P  = 0.60)., Interpretation: Donor HSD17B13 genotype adversely affects post-LT survival in patients receiving steroid induction. Additional studies are required to confirm this association., Funding: The National Institutes of Health and American Society of Transplant Surgeons Collaborative Scientist Grant., Competing Interests: All authors declare no competing interests., (© 2023 The Authors.)

Despite advances in therapies treating non-Hodgkin's lymphoma (NHL), 20~40% of patients experience relapsed or refractory disease. While solid tumors with homologous recombination deficiencies have been successfully targeted with synthetic lethal agents such as poly-ADP ribose polymerase (PARP) inhibitors, such synthetic lethality strategy has not yet been approved to treat patients with NHL. Here we investigated the mechanism of action (MoA) and therapeutic potential of a new-generation acylfulvene compound, LP-284, in both in vitro and in vivo NHL models. One of LP-284's MoA includes inducing the repair of double-strand DNA break (DSB). We found that LP-284 exerts nanomolar potency in a panel of hematological cancer cell lines including fifteen NHL cell lines. In vivo , LP-284 treatment prolongs the survival of mantle cell lymphoma (MCL) cell line JeKo-1 derived xenograft mice by two-fold and shows increased efficacy over bortezomib and ibrutinib. In addition, LP-284 is capable of inhibiting tumor growth of JeKo-1 xenografts that are refractory to bortezomib or ibrutinib. We further showed that LP-284 is particularly lethal in cells with deficient DNA damage response and repair, a targetable vulnerability in NHL.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a major analytical platform for the determination and localization of lipid metabolites directly from tissue sections. Unlike analysis of lipid extracts, where lipid localizations are lost due to homogenization and/ or solvent extraction, MALDI-MSI analysis is capable of revealing spatial localization of metabolites while simultaneously collecting high chemical resolution mass spectra. Important considerations for obtaining high quality MALDI-MS images include tissue preservation, section preparation, MS data collection and data processing. Errors in any of these steps can lead to poor quality metabolite images and increases the chance for metabolite misidentification and/ or incorrect localization. Here, we present detailed methods and recommendations for specimen preparation, MALDI-MS instrument parameters, software analysis platforms for data processing, and practical considerations for each of these steps to ensure acquisition of high-quality chemical and spatial resolution data for reconstructing MALDI-MS images of plant tissues.

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Background: Chronic spinal cord injury (SCI) portends a low probability of recovery, especially in the most severe subset of motor-complete injuries. Active spinal cord stimulation with or without intensive locomotor training has been reported to restore movement after traumatic SCI. Only three cases have been reported where participants developed restored volitional movement with active stimulation turned off after a period of chronic stimulation and only after intensive rehabilitation with locomotor training. It is unknown whether restoration of movement without stimulation is possible after stimulation alone. Objective: We describe the development of spontaneous volitional movement (SVM) without active stimulation in a subset of participants in the Epidural Stimulation After Neurologic Damage (ESTAND) trial, in which locomotor training is not prescribed as part of the study protocol, and subject's rehabilitation therapies are not modified. Methods: Volitional movement was evaluated with the Brain Motor Control Assessment using sEMG recordings and visual examination at baseline and at follow-up visits with and without stimulation. Additional functional assessment with a motor-assisted bicycle exercise at follow-up with and without stimulation identified generated work with and without effort. Results: The first seven participants had ASIA Impairment Scale (AIS) A or B thoracic SCI, a mean age of 42 years, and 7.7 years post-injury on average. Four patients developed evidence of sustained volitional movement, even in the absence of active stimulation after undergoing chronic epidural spinal cord stimulation (eSCS). Significant increases in volitional power were found between those observed to spontaneously move without stimulation and those unable ( p < 0.0005). The likelihood of recovery of spontaneous volitional control was correlated with spasticity scores prior to the start of eSCS therapy ( p = 0.048). Volitional power progressively improved over time ( p = 0.016). Additionally, cycling was possible without stimulation ( p < 0.005). Conclusion: While some SVM after eSCS has been reported in the literature, this study demonstrates sustained restoration without active stimulation after long-term eSCS stimulation in chronic and complete SCI in a subset of participants. This finding supports previous studies suggesting that "complete" SCI is likely not as common as previously believed, if it exists at all in the absence of transection and that preserved pathways are substrates for eSCS-mediated recovery in clinically motor-complete SCI. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03026816., (Copyright © 2020 Peña Pino, Hoover, Venkatesh, Ahmadi, Sturtevant, Patrick, Freeman, Parr, Samadani, Balser, Krassioukov, Phillips, Netoff and Darrow.)

Seeds of the desert shrub, jojoba ( Simmondsia chinensis ), are an abundant, renewable source of liquid wax esters, which are valued additives in cosmetic products and industrial lubricants. Jojoba is relegated to its own taxonomic family, and there is little genetic information available to elucidate its phylogeny. Here, we report the high-quality, 887-Mb genome of jojoba assembled into 26 chromosomes with 23,490 protein-coding genes. The jojoba genome has only the whole-genome triplication (γ) shared among eudicots and no recent duplications. These genomic resources coupled with extensive transcriptome, proteome, and lipidome data helped to define heterogeneous pathways and machinery for lipid synthesis and storage, provided missing evolutionary history information for this taxonomically segregated dioecious plant species, and will support efforts to improve the agronomic properties of jojoba., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Brassica napus ( B. napus ) is the world's most widely grown temperate oilseed crop. Although breeding for human consumption has led to removal of erucic acid from refined canola oils, there is renewed interest in the industrial uses of erucic acid derived from B. napus , and there is a rich germplasm available for use. Here, low- and high-erucic acid accessions of B. napus seeds were examined for the distribution of erucic acid-containing lipids and the gene transcripts encoding the enzymes involved in pathways for its incorporation into triacylglycerols (TAGs) across the major tissues of the seeds. In general, the results indicate that a heterogeneous distribution of erucic acid across B. napus seed tissues was contributed by two isoforms (out of six) of FATTY ACYL COA ELONGASE ( FAE1) and a combination of phospholipid:diacylglycerol acyltransferase (PDAT)- and diacylglycerol acyltransferase (DGAT)-mediated incorporation of erucic acid into TAGs in cotyledonary tissues. An absence of the expression of these two FAE1 isoforms accounted for the absence of erucic acid in the TAGs of the low-erucic accession., (Copyright © 2020 Lu, Aziz, Sturtevant, Chapman and Guo.)

Objective: Traumatic brain injuries (TBIs) are largely underdiagnosed and may have persistent refractory consequences. Current assessments for acute TBI are limited to physical examination and imaging. Biomarkers such as glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCH-L1), and S100 calcium-binding protein B (S100B) have shown predictive value as indicators of TBI and potential screening tools., Methods: In total, 37 controls and 118 unique trauma subjects who received a clinically ordered head computed tomography (CT) in the emergency department of a level 1 trauma center were evaluated. Blood samples collected at 0-8 hours (initial) and 12-32 hours (delayed) postinjury were analyzed for GFAP, UCH-L1, and S100B concentrations. These were then compared in CT-negative and CT-positive subjects., Results: Median GFAP, UCH-L1, and S100B concentrations were greater in CT-positive subjects at both timepoints compared with CT-negative subjects. In addition, median UCH-L1 and S100B concentrations were lower at the delayed timepoint, whereas median GFAP concentrations were increased. As predictors of a positive CT of the head, GFAP outperformed UCH-L1 and S100B at both timepoints (initial: 0.89 sensitivity, 0.62 specificity; delayed: 0.94 sensitivity, 0.67 specificity). GFAP alone also outperformed all possible combinations of biomarkers., Conclusions: GFAP, UCH-L1, and S100B demonstrated utility for rapid prediction of a CT-positive TBI within 0-8 hours of injury. GFAP exhibited the greatest predictive power at 12-32 hours. Furthermore, these results suggest that GFAP alone has greater utility for predicting a positive CT of the head than UCH-L1, S100B, or any combination of the 3., (Published by Elsevier Inc.)

Introduction: Castor (Ricinus communis L.) seeds are valued for their production of oils which can comprise up to 90% hydroxy-fatty acids (ricinoleic acid). Castor oil contains mono-, di- and tri- ricinoleic acid containing triacylglycerols (TAGs). Although the enzymatic synthesis of ricinoleic acid is well described, the differential compartmentalization of these TAG molecular species has remained undefined., Objectives: To examine the distribution of hydroxy fatty acid accumulation within the endosperm and embryo tissues of castor seeds., Methods: Matrix assisted laser desorption/ionization mass spectrometry imaging was used to map the distribution of triacylglycerols in tissue sections of castor seeds. In addition, the endosperm and embryo (cotyledons and embryonic axis) tissues were dissected and extracted for quantitative lipidomics analysis and Illumina-based RNA deep sequencing., Results: This study revealed an unexpected heterogeneous tissue distribution of mono-, di- and tri- hydroxy-triacylglycerols in the embryo and endosperm tissues of castor seeds. Pathway analysis based on transcript abundance suggested that distinct embryo- and endosperm-specific mechanisms may exist for the shuttling of ricinoleic acid away from phosphatidylcholine (PC) and into hydroxy TAG production. The embryo-biased mechanism appears to favor removal of ricinoleic acid from PC through phophatidylcholine: diacylglycerol acyltransferase while the endosperm pathway appears to remove ricinoleic acid from the PC pool by preferences of phospholipase A (PLA 2 α) and/or phosphatidylcholine: diacylglycerol cholinephosphotransferase., Conclusions: Collectively, a combination of lipidomics and transcriptomics analyses revealed previously undefined spatial aspects of hydroxy fatty acid metabolism in castor seeds. These studies underscore a need for tissue-specific studies as a means to better understand the regulation of triacylglycerol accumulation in oilseeds.

Despite the importance of oilseeds to worldwide human nutrition, and more recently to the production of bio-based diesel fuels, the detailed mechanisms regulating seed oil biosynthesis remain only partly understood, especially from a tissue-specific perspective. Here, we investigated the spatial distributions of lipid metabolites and transcripts involved in oil biosynthesis from seeds of two low-erucic acid genotypes of Brassica napus with high and low seed-oil content. Integrated results from matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of lipids in situ, lipidome profiling of extracts from seed tissues, and tissue-specific transcriptome analysis revealed complex spatial distribution patterns of lipids and transcripts. In general, it appeared that many triacylglycerol and phosphatidylcholine species distributed heterogeneously throughout the embryos. Tissue-specific transcriptome analysis identified key genes involved in de novo fatty acid biosynthesis in plastid, triacylglycerols assembly and lipid droplet packaging in the endoplasmic reticulum (ER) that may contribute to the high or low oil phenotype and heterogeneity of lipid distribution. Our results imply that transcriptional regulation represents an important means of impacting lipid compartmentalization in oil seeds. While much information remains to be learned about the intricacies of seed oil accumulation and distribution, these studies highlight the advances that come from evaluating lipid metabolism within a spatial context and with multiple omics level datasets., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

There is considerable interest in the de novo production of omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), not least of all given the importance of these fatty acids in both aquaculture and human nutrition. Previously we have demonstrated the feasibility of using metabolic engineering in transgenic plants (Camelina sativa) to modify the seed oil composition to now include EPA and/or DHA. In this study, we further tailored the seed oil profile to reduce the omega-6 content, and evaluated the performance of such GM plants under field conditions (i.e. environmental releases), in terms of agronomic performance and also the lipidomic profile of seed oil. We used MALDI- mass spectrometry imaging to identify discrete tissue-types in the seed in which these non-native fatty acids preferentially accumulated. Collectively, these data provide new insights into the complexity of plant lipid metabolism and the challenges associated with predictive manipulation of these pathways. However, this study identified the likely dispensable nature of a Δ12-desturase activity in our omega-3 metabolic engineering rationales for Camelina.

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Urushiols are the allergenic components of Toxicodendron radicans (poison ivy) as well as other Toxicodendron species. They are alk-(en)-yl catechol derivatives with a 15- or 17-carbon side chain having different degrees of unsaturation. Although several methods have been developed for analysis of urushiols in plant tissues, the in situ localization of the different urushiol congeners has not been reported. Here, we report on the first analysis of urushiols in poison ivy stems by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Our results show that the urushiol congeners with 15-carbon side chains are mainly localized to the resin ducts, while those with 17-carbon side chains are widely distributed in cortex and vascular tissues. The presence of these urushiols in stem extracts of poison ivy seedlings was confirmed by GC-MS. These novel findings provide new insights into the spatial tissue distribution of urushiols that might be biosynthetically or functionally relevant.

Rationale: Refined cottonseed oil has widespread applications in the food and chemical industries. Although the major lipids comprising cottonseed oil (triacylglycerols) are well known, there are many diverse lipid species in cotton seeds that occur at much lower levels and have important nutritional or anti-nutritional properties., Methods: The lipid technical samples were prepared in chloroform. The biological samples were extracted using a mixture of isopropanol/chloroform/H 2 O (2:1:0.45). The data were collected using high and low collision energy with simultaneous data collection on a time-of-flight (TOF) mass spectrometer which allowed the characterization of lipids by precursor and product ion alignment. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation. The comprehensive method was used to screen seed lipid extracts from several cotton genotypes using multivariate statistical analysis., Results: Method variables influencing the peak integrity and chromatographic separation for a mixture of lipids with different degrees of polarity were explored. The experiments were designed to understand the chromatographic behavior of lipids in a controlled setting using a variety of lipid extracts. Influences of acyl chain length and numbers of double bonds were investigated using single moiety standards., Conclusions: The methodology parameters were examined using single moiety lipid standards and standard mixtures. The method conditions were applied to biological lipid extracts, and adjustments were investigated to manipulate the chromatography. Insights from these method variable manipulations will help to frame the development of targeted lipid profiling and screening protocols. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 ( DGAT1 ) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE ( PDAT ) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3 n -3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3 n -3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2 n -6 at the expense of 18:3 n -3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C. sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2 n -6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3 n -3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2 n -6. The higher levels of 18:3 n -3 in DGAT1 -silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape ( Brassica napus ) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality., (© 2017 The author(s). All Rights Reserved.)

Main Conclusion: Genetically diverse cottonseeds show altered compositions and spatial distributions of phosphatidylcholines and triacylglycerols. Lipidomics profiling led to the discovery of a novel FAD2 - 1 allele, fad2 - 1D - 1 , resulting in a high oleic phenotype. The domestication and breeding of cotton for elite, high-fiber cultivars have led to reduced variation of seed constituents within currently cultivated upland cotton genotypes. However, a recent screen of the genetically diverse U.S. National Cotton Germplasm Collection identified Gossypium accessions with marked differences in seed oil and protein content. Here, several of these accessions representing substantial variation in seed oil content were analyzed for quantitative and spatial differences in lipid compositions by mass spectrometric approaches. Results indicate considerable variation in amount and spatial distribution of pathway metabolites for triacylglycerol biosynthesis in embryos across Gossypium accessions, suggesting that this variation might be exploited by breeders for seed composition traits. By way of example, these lipid metabolite differences led to the identification of a mutant allele of the D-subgenome homolog of the delta-12 desaturase (fad2-1D-1) in a wild accession of G. barbadense that has a high oil and high oleic seed phenotype. This mutation is a 90-bp insertion in the 3' end of the FAD2-1D coding sequence and a modification of the 3' end of the gene beyond the coding sequence leading to the introduction of a premature stop codon. Given the large amounts of cottonseed produced around the world that is currently not processed into higher value products, these efforts might be one avenue to raise the overall value of the cotton crop for producers.

Arabidopsis thaliana has been widely used as a model plant to study acyl lipid metabolism. Seeds of A. thaliana are quite small (approximately 500×300μm and weigh ~20μg), making lipid compositional analyses of single seeds difficult to achieve. Here we have used matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to map and visualize the three-dimensional spatial distributions of two common membrane phospholipid classes, phosphatidylcholine (PC) and phosphatidylinositol (PI), in single A. thaliana seeds. The 3D images revealed distinct differences in distribution of several molecular species of both phospholipids among different seed tissues. Using data from these 3D reconstructions, the PC and PI mol% lipid profiles were calculated for the embryonic axis, cotyledons, and peripheral endosperm, and these data agreed well with overall quantification of these lipids in bulk seed extracts analyzed by conventional electrospray ionization-mass spectrometry (ESI-MS). In addition, MALDI-MSI was used to profile PC and PI molecular species in seeds of wild type, fad2-1, fad3-2, fad6-1, and fae1-1 acyl lipid mutants. The resulting distributions revealed previously unobserved changes in spatial distribution of several lipid molecular species, and were used to suggest new insights into biochemical heterogeneity of seed lipid metabolism. These studies highlight the value of mass spectrometry imaging to provide unprecedented spatial and chemical resolution of metabolites directly in samples even as small as a single A. thaliana seeds, and allow for expanded imaging of plant metabolites to improve our understanding of plant lipid metabolism from a spatial perspective., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern. Graphical Abstract ᅟ.

Direct visualization of plant tissues by matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has revealed key insights into the localization of metabolites in situ. Recent efforts have determined the spatial distribution of primary and secondary metabolites in plant tissues and cells. Strategies have been applied in many areas of metabolism including isotope flux analyses, plant interactions, and transcriptional regulation of metabolite accumulation. Technological advances have pushed achievable spatial resolution to subcellular levels and increased instrument sensitivity by several orders of magnitude. It is anticipated that MALDI-MSI and other MSI approaches will bring a new level of understanding to metabolomics as scientists will be encouraged to consider spatial heterogeneity of metabolites in descriptions of metabolic pathway regulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Summary: Monitoring and localizing molecules on living plants is critical for understanding their growth, development and disease. However, current techniques for molecular imaging of living plants often lack spatial information or require tedious pre‐labelling. Here, we proposed a novel molecular imaging platform that combines sliver nanowire‐doped Ti3C2 MXene (Ag NWs@MXene) flexible film substrate with laser desorption/ionization mass spectrometry imaging (AMF‐LDI‐MSI) to study the spatial distribution of biomolecules on the surface of living plants. This platform overcomes the MSI challenges posed by difficult‐to‐slice plant tissues (e.g., tough or water‐rich roots and fragile flowers) and enables precisely transfer and visualize the molecule. Comparisons of the measurement results to those from matrix‐assisted LDI‐MSI (MALDI‐MSI) technology demonstrate the accuracy and reliability of the platform. Biocompatibility evaluations indicated that the platform without observable adverse effects on the health of living plants. The distribution of growth and disease‐associated signalling molecules, such as choline, organic acids and carbohydrates, can be in situ non‐destructively detected on the surfaces of living plants, which is important for tracking the health of plants and their diseased areas. AMF‐LDI‐MSI platform can serve as a promising tool for label‐free, in situ and non‐destructive monitoring of functional biomolecules and plant growth from a spatial perspective. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigates the set of proteins that comprise a Vibrio ordalii strain Vo-LM-18 and its outer membrane vesicles (OMVs), which are involved in the pathogen's interaction with fish hosts, especially salmonids. Vibrio ordalii is a major cause of vibriosis, a disease that results in significant mortality in fish farms, a relevant industry in Chile. By analyzing the proteins expressed by both the bacteria and their OMVs, this research identifies key proteins linked to virulence, iron uptake, and cellular communication. The findings suggest that OMVs carry virulence factors that could aid in the pathogen's ability to infect and persist in fish. These vesicles may also contribute to the pathogen's survival in harsh environments, such as during host immune responses. This study provides new insights into bacterial pathogenesis and highlights potential targets for developing treatments or vaccines to combat infections in aquaculture. Vibrio ordalii is the causative agent of atypical vibriosis in salmonids cultured in Chile. While extensive research provides insights into V. ordalii through phenotypic, antigenic, and genetic typing, as well as various virulence mechanisms, proteomic characterization remains largely unexplored. This study aimed to advance the proteomic knowledge of Chilean V. ordalii Vo-LM-18 and its OMVs, which have known virulence. Using Nano-UHPLC-LC-MS/MS, we identified 2242 proteins and 1755 proteins in its OMVs. Of these, 644 unique proteins were detected in V. ordalii Vo-LM-18, namely 156 unique proteins in its OMVs and 1596 shared proteins. The major categories for the OMVs were like those in the bacteria (i.e., cytoplasmic and cytoplasmic membrane proteins). Functional annotation identified 37 biological pathways in V. ordalii Vo-LM-18 and 28 in its OMVs. Proteins associated with transport, transcription, and virulence were predominant in both. Evident differences in protein expression were found. OMVs expressed a higher number of virulence-associated proteins, including those related to iron- and heme-uptake mechanisms. Notable pathways in the bacteria included flagellum assembly, heme group-associated proteins, and protein biosynthesis. This proteomic analysis is the first to detect the RTX toxin in a V. ordalii strain (Vo-LM-18) and its vesicles. Our results highlight the crucial role of OMVs in the pathogenesis and adaptation of V. ordalii, suggesting use as potential diagnostic biomarkers and therapeutic targets for bacterial infections. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is a leading cause of death, especially among the elderly. This bacterium produces several surface membrane proteins, with staphylococcal protein A (SpA) being particularly important. Despite its prevalence, there are no targeted treatments available for geriatric patients. In homeopathy, Rhus toxicodendron (RT) is frequently used in various dilutions-Mother Tincture (MT), 6CH, 30CH, and 200CH- for conditions like skin infections, soft tissue disorders, and joint ailments. Methods: In this study, we evaluated the effects of Rhus toxicodendron (RT) at different concentrations on Staphylococcus aureus through bacterial plate cultures and compared the outcomes with Nano-RT. Notably, Nano-RT MT is not commercially available. This research is the first to showcase both the efficacy and biosafety of the innovative nano-ZnO RT MT homeopathic formulation. Results: This study examined the inhibitory effects of Rhus toxicodendron in its Mother Tincture (MT) form and in 6CH, 30CH, and 200CH dilutions against Staphylococcus aureus. Nano-ZnO was synthesized from Rhus toxicodendron MT and combined to create Nano-MT. Both Rhus Tox MT and Nano-RT MT demonstrated significant inhibition of Staphylococcus aureus within 24 h of application. Conclusions: Rhus Tox MT and Nano-RT MT present promising new options for treating Staphylococcus aureus infections in elderly patients. Furthermore, studies have demonstrated that Nano-RT MT is completely biologically safe in mice. [ABSTRACT FROM AUTHOR]

This review examines the role of Pseudomonas spp. bacteria as biocontrol agents against crop diseases, focusing on their mechanisms of action, efficacy, and potential applications in sustainable agriculture. Pseudomonas spp., ubiquitous in soil ecosystems and root microbiomes, have attracted attention for their ability to suppress phytopathogens and enhance plant health through various mechanisms. These include direct competition for nutrients, production of antimicrobial compounds and volatile organic compounds, competition using type VI secretion systems, and indirect induction of systemic resistance. Our review shows that Pseudomonas strains effectively control a wide range of diseases across diverse plant species, with some strains demonstrating efficacy comparable to chemical fungicides. However, the review also highlights challenges in achieving consistent performance when using Pseudomonas inoculants under field conditions due to various biotic and abiotic factors. Strategies to optimize biocontrol potential, such as formulation techniques, application methods, and integration with other management practices, are discussed. The advantages of Pseudomonas -based biocontrol for sustainable agriculture include reduced reliance on chemical pesticides, enhanced crop productivity, and improved environmental sustainability. Future research directions should focus on understanding the complex interactions within the plant microbiome, optimizing delivery systems, and addressing regulatory hurdles for commercial deployment. This review underscores the significant potential of Pseudomonas spp. in sustainable crop protection while acknowledging the need for further research to fully harness their capabilities in agricultural systems. [ABSTRACT FROM AUTHOR]

Jojoba is an important tropical oil crop, and jojoba oil is widely used in the aerospace lubricant and cosmetic industries. Jojoba exhibits high tolerance to droughts and high temperatures. However, there is currently a lack of rapid and effective methods for identifying stress-tolerant genes in jojoba. Here, an efficient hairy root genetic transformation system of jojoba (Simmondisa chinensis) mediated by Agrobacterium rhizogenes was established and used for the functional evaluation of ScGolS1, a putative stress-tolerant gene. First, using the leaves of jojoba as explants, transgenic jojoba hairy roots carrying the RUBY gene were obtained under sterile conditions using the "soaking co-cultivation method". Second, we optimized the four conditions affecting hairy root genetic transformations, namely, the strains of A. rhizogenes, co-cultivation under light or dark conditions, the infection time, and the OD600 value of the bacterial suspension. The following best transformation conditions were determined, A. rhizogenes K599, light during co-cultivation, an infection time of 10 min, and bacterial suspension OD600 = 0.6, under which the transformation rate could reach 27%. Third, based on the "soaking co-cultivation method", a new method called the "wrapping co-cultivation method" was developed, which does not require tissue cultures and can induce transgenic hairy roots of jojoba in two months. Using the "wrapping co-cultivation method", we successfully obtained transgenic hairy roots overexpressing the ScGolS1 gene, which exhibited higher tolerance to low-temperature stress. A hairy root-based genetic transformation system of jojoba will promote the functional genomics and molecular breeding of jojoba. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae, a major opportunistic pathogen, causes severe infections in both community and healthcare settings, especially in intensive care units (ICUs), where multidrug-resistant (MDR) strains, such as carbapenem-resistant K. pneumoniae (CRKP), pose significant treatment challenges. The rise in hypervirulent K. pneumoniae (hvKP) with enhanced virulence factors complicates management further. The ST11 clone, prevalent in China, exhibits both resistance and virulence traits, contributing to hospital outbreaks. ICU patients, particularly those with comorbidities or prior antibiotic exposure, are at higher risk. Treatment is complicated by limited antibiotic options and the increasing prevalence of polymicrobial infections, which involve resistant pathogens like Pseudomonas aeruginosa and Acinetobacter baumannii. Combination therapies offer some promise, but mortality rates remain high, and resistance to last-resort antibiotics is growing. Infection control measures and personalized treatment plans are critical, alongside the urgent need for vaccine development to combat the rising threat of K. pneumoniae, particularly in vulnerable populations. Effective management requires improved diagnostic tools, antimicrobial stewardship, and innovative treatment strategies to reduce the burden of this pathogen, especially in resource-limited settings. This review aims to provide a comprehensive analysis of the virulence, resistance, and epidemiological risks of K. pneumoniae in ICUs over the past decade, highlighting the ongoing challenges and the need for continued efforts to combat this growing threat. [ABSTRACT FROM AUTHOR]

Diacylglycerol acyltransferase (DGAT) plays a pivotal role in lipid metabolism, particularly in the biosynthesis of triacylglycerols (TAGs), which are essential for energy storage and cellular membrane formation. Xanthoceras sorbifolium, commonly known as yellowhorn, is notable for the high oil content of its seeds and its potential in biodiesel production. However, its DGATs have not been thoroughly studied. In this study, we conducted the bioinformatics analysis of the XsDGAT gene family in X. sorbifolium. The XsDGAT homologs, sequence features, domain architecture, and evolutionary relationships were identified, the expression patterns across different tissues and their potential regulatory elements of XsDGATs were also displayed, providing valuable insights into their potential roles in lipid biosynthesis and accumulation. This comprehensive bioinformatics analysis offers valuable resources for further experimental investigations and potential applications aimed at enhancing lipid productivity in X. sorbifolium. [ABSTRACT FROM AUTHOR]

Type VI secretion systems (T6SSs) are macromolecular assemblies that deliver toxic effector proteins between adjacent bacteria. These effectors span a wide range of protein families that all lack canonical signal sequences that would target them for export. Consequently, it remains incompletely understood how conserved structural components of the T6SS apparatus recognize a diverse repertoire of effectors. Here, we characterize a widespread family of adaptor proteins, containing the domain of unknown function DUF4123, that enable the recognition and export of evolutionarily unrelated effectors. By examining two nearly identical paralogs of the conserved T6SS spike protein, VgrG, we demonstrate that each spike protein exports a structurally unique effector. We further show that the recruitment of each effector to its respective spike protein requires a cognate adaptor protein. Protein–protein interaction experiments demonstrate that these adaptor proteins specifically tether an effector to a structurally conserved but sequence divergent helix-turn-helix motif found at the C-terminus of its cognate VgrG. Using structural predictions and mutagenesis analyses, we elucidate the molecular contacts required for these interactions and discover that these adaptor proteins contain a structurally conserved N-terminal lobe that has evolved to bind VgrG helix-turn-helix motifs and a structurally variable C-terminal lobe that recognizes diverse effector families. Overall, our work provides molecular insight into a mechanism by which conserved T6SS components recognize structurally diverse effectors.It is not clear how the conserved components of bacterial type VI secretion systems (T6SSs) recognize and deliver a diverse repertoire of toxic effector proteins. Here, Colautti et al. characterize a widespread family of adaptor proteins that enable the recognition and export of structurally diverse effectors. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus is a significant opportunistic pathogen with the highest fatality rate among foodborne microbes. However, due to a lack of comprehensive surveillance, the characteristics of isolates in China remain poorly understood. This study analyzed 60 strains of V. vulnificus isolated from diverse sources in Shanghai, including shellfish, crabs, shrimps, throat swabs of migratory birds, as well as seafood farming water and seawater. Identification of the genotypes was performed using PCR, and cytotoxicity was determined using an LDH assay. DNA was sequenced using Illumina NovaSeq followed by a bioinformatic analysis. The results demonstrated that a majority of the strains belonged to the 16S rRNA B-vcgC genotype. All strains carried five antibiotic resistance genes (ARGs), with some strains carrying over ten ARGs, mediating resistance to multiple antibiotics. Five strains possessed a highly abundant effector delivery system, which further investigations revealed to be a type III secretion system II (T3SS2), marking the first description of T3SS2 in V. vulnificus. Phylogenetic analysis indicated that it belonged to a different genetic lineage from T3SS2α and T3SS2β of V. parahaemolyticus. Bacteria with T3SS2 sequences were concentrated in coastal areas and mostly within the genus Vibrio in the global prevalence survey. Our study provides essential baseline information for non-clinical V. vulnificus and discovers the existence of T3SS2 in several strains which may be more virulent, thereby posing a new threat to human health. [ABSTRACT FROM AUTHOR]

Vibrio harveyi is a major bacterial pathogen that causes disease in aquaculture animals worldwide. Although V. harveyi consistently harbors a range of traditional virulence genes, it remains unclear which specific genes are crucial for virulence at different infection stages. Dual RNA-seq is a cutting-edge RNA sequencing technology that is ideal for investigating the gene expression patterns of pathogens within the host, which is highly effective in identifying key virulence genes. In previous artificial infection experiments, we have identified the liver of hybrid grouper (♀ Epinephelus polyphekadion × ♂ E. fuscoguttatus) as the main target organ for pathogenic V. harveyi GDH11385 during the initial infection phase. To further explore the key virulence factors of V. harveyi at the early stage of infection, the liver of the hybrid grouper infected with strain GDH11385 was analyzed here by dual RNA-seq. The transcriptome data were compared with that of in vitro cultured bacteria. The results showed that 326 and 1140 DEGs (differentially expressed genes) were significantly up- and down-regulated, respectively, at 4 h post-infection (hpi). Further pathway enrichment analyses revealed that these up-regulated DEGs in vivo were mainly enriched in siderophore biosynthesis and transport, type VI secretion system (T6SS), flagellar assembly, glycolysis/gluconeogenesis, and ribosome. Notably, all genes involved in the metabolism and utilization of vibrioferrin (a carboxylate class of siderophore produced by Vibrio), and most of the genes within one of three T6SSs, were significantly up-regulated in vivo. This indicates that siderophore-dependent iron competition and T6SS-mediated delivery of virulence factors are vital for the successful colonization of V. harveyi at the early stage of infection. This study provides more precise clues to reveal the virulence mechanism of V. harveyi during the initial phase of infection. [ABSTRACT FROM AUTHOR]

Rapeseed is an important commercial crop globally, used for both animal fodder and human consumption. Varied insolation duration and intensity are among the main factors affecting the seed yield and quality of Brassica napus (B. napus) worldwide. In this study, the high-oil-content rapeseed cultivar "Qingyou 3" was subjected to a light supplementation trial during both the vegetative growth period and the seed productive stage. Different light intensity conditions were stimulated using light-emitting diodes (LEDs). The main plot factor was land condition, with LED treatment (Treatment) and without LED treatment (Control) under natural conditions. The results showed that the leaf size and thickness, photosynthesis efficiency, and seed oil content of B. napus increased significantly after light supplementation. Then, 18 cDNA libraries were constructed from leaf segments (30 days after transplanting—DAT) and seeds 30 and 40 days after pollination (DPA) for RNA transcriptome sequencing. It was found that genes encoding lipid transfer protein, phenylpropanoid biosynthesis, photosynthesis, and plant hormone signal transduction were enriched in differentially expressed genes (DEGs). The qRT-PCR analysis showed that eight key genes had significant variations, a finding also consistent with the RNA-seq results. The aim of this study was to identify the DEGs and signaling pathways in the leaves and seeds of B. napus during the vegetative and seed productive stages under different light intensities. The results provide insight into how sufficient light plays a critical role in promoting photosynthesis and serves as the foundation for material accumulation and yield formation. [ABSTRACT FROM AUTHOR]

To date, the public health system has been impacted by the increasing costs of many diagnostic and therapeutic pathways due to limited resources. At the same time, we are constantly seeking to improve these paths through approaches aimed at personalized medicine. To achieve the required levels of diagnostic and therapeutic precision, it is necessary to integrate data from different sources and simulation platforms. Today, artificial intelligence (AI), machine learning (ML), and predictive computer models are more efficient at guiding decisions regarding better therapies and medical procedures. The evolution of these multiparametric and multimodal systems has led to the creation of digital twins (DTs). The goal of our review is to summarize AI applications in discovering new immunotherapies and developing predictive models for more precise immunotherapeutic decision-making. The findings from this literature review highlight that DTs, particularly predictive mathematical models, will be pivotal in advancing healthcare outcomes. Over time, DTs will indeed bring the benefits of diagnostic precision and personalized treatment to a broader spectrum of patients. [ABSTRACT FROM AUTHOR]

The HIV/AIDS epidemic in Zambia threatens maternal survival and jeopardizes the ability for families to care for their children. The Christian Alliance for Children in Zambia (CACZ) operates a program called Milk and Medicine (M&M) that distributes food, formula, and medicine at churches in the compounds. This article reports on a mixed methods study to evaluate the outcomes of the M&M program. On-site interviews with families combined with an analysis of a longitudinal data set were the methods used. The results of the study showed families face continuous hardship including hunger, unemployment, disease, and loss. Families expressed appreciation for the program and its staff and suggested improvements. The longitudinal data review helped researchers to recommend an improved protocol for data management. Improved data will assist researchers in an on-going evaluation to compare the growth rates of children in the study to the Zambian normal growth charts. Lessons learned from this evaluation validated the use of mixed methods design for exploratory research on an emerging program. Lessons were also learned about the difficulty of working in natural settings with political and cultural variations. Future evaluations of the M&M program are expected to shed light on more specific program impacts., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Let F be a collection of k-element sets with the property that the intersection of no two should be included in a third. We show that such a collection of maximum size satisfies .2715 k+ o( k)≦≦log | F|≦.7549 k+ o( k) settling a question raised by Erdős. The lower bound is probabilistic, the upper bound is deduced via an entropy argument. Some open questions are posed. [ABSTRACT FROM AUTHOR]

Targeted increases in monounsaturated (oleic acid) fatty acid content of refined cottonseed oil could support improved human nutrition and cardiovascular health. Genetic modifications of cottonseed fatty acid composition have been accomplished using several different molecular strategies. Modification of oleic acid content in cottonseed embryos using a dominant-negative protein approach, while successful in effecting change in the desired fatty acid composition, resulted in reduced oil content and seed viability. Here these changes in fatty acid composition were associated with changes in dominant molecular species of triacylglycerols (TAGs) and their spatial distributions within embryo tissues. A combination of mass spectrometry (MS)-based lipidomics approaches, including MS imaging of seed cryo-sections, revealed that cotton embryos expressing a non-functional allele of a Brassica napus delta-12 desaturase showed altered accumulation of TAG species, especially within cotyledonary tissues. While lipid analysis of seed extracts could demonstrate detailed quantitative changes in TAG species in transgenics, the spatial contribution of metabolite compartmentation could only be visualized by MS imaging. Our results suggest tissue-specific differences in TAG biosynthetic pathways within cotton embryos, and indicate the importance of considering the location of metabolites in tissues in addition to their identification and quantification when developing a detailed view of cellular metabolism., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)