High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

Reticulate evolution of the tertiary relict Osmanthus.

When interspecific gene flow is common, species relationships are more accurately represented by a phylogenetic network than by a bifurcating tree. This study aimed to uncover the role of introgression in the evolution of Osmanthus, the only genus of the subtribe Oleinae (Oleaceae) with its distribution center in East Asia. We built species trees, detected introgression, and constructed networks using multiple kinds of sequencing data (whole genome resequencing, transcriptome sequencing, and Sanger sequencing of nrDNA) combined with concatenation and coalescence approaches. Then, based on well-understood species relationships, historical biogeographic analyses and diversification rate estimates were employed to reveal the history of Osmanthus. Osmanthus originated in mid-Miocene Europe and dispersed to the eastern Tibetan Plateau in the late Miocene. Thereafter, it continued to spread eastwards. Phylogenetic conflict is common within the 'Core Osmanthus' clade and is seen at both early and late stages of diversification, leading to hypotheses of net-like species relationships. Incomplete lineage sorting proved ineffective in explaining phylogenetic conflicts and thus supported introgression as the main cause of conflicts. This study elucidates the diversification history of a relict genus in the subtropical regions of eastern Asia and reveals that introgression had profound effects on its evolutionary history.

High-quality genome assembly enables prediction of allele-specific gene expression in hybrid poplar.

Poplar (Populus) is a well-established model system for tree genomics and molecular breeding, and hybrid poplar is widely used in forest plantations. However, distinguishing its diploid homologous chromosomes is difficult, complicating advanced functional studies on specific alleles. In this study, we applied a trio-binning design and PacBio high-fidelity long-read sequencing to obtain haplotype-phased telomere-to-telomere genome assemblies for the 2 parents of the well-studied F1 hybrid "84K" (Populus alba × Populus tremula var. glandulosa). Almost all chromosomes, including the telomeres and centromeres, were completely assembled for each haplotype subgenome apart from 2 small gaps on one chromosome. By incorporating information from these haplotype assemblies and extensive RNA-seq data, we analyzed gene expression patterns between the 2 subgenomes and alleles. Transcription bias at the subgenome level was not uncovered, but extensive-expression differences were detected between alleles. We developed machine-learning (ML) models to predict allele-specific expression (ASE) with high accuracy and identified underlying genome features most highly influencing ASE. One of our models with 15 predictor variables achieved 77% accuracy on the training set and 74% accuracy on the testing set. ML models identified gene body CHG methylation, sequence divergence, and transposon occupancy both upstream and downstream of alleles as important factors for ASE. Our haplotype-phased genome assemblies and ML strategy highlight an avenue for functional studies in Populus and provide additional tools for studying ASE and heterosis in hybrids.

Neural Encoding for Spatial Release from Informational Masking and its Correlation with Behavioral Metrics.

The central auditory system encompasses two primary functions: identification and localization. Spatial release from masking (SRM) highlights speech recognition in competing noise and improves the listening experience when a spatial cue is introduced between noise and target speech. This assessment focuses on the integrity of auditory function and holds clinical significance. However, infants or pre-lingual subjects sometimes provide less reliable results. This study investigates the value of cortical auditory evoked potentials (CAEPs) onset and acoustic change complex (ACC) as an objective measurement of SRM. Thirty normal-hearing young adults (11 males) were recruited. We found the spatial separation of signals and noise (±90 degrees symmetrically) resulted in a signal-to-noise ratio (SNR) improvement of 9.00 ± 1.71 dB behaviorally. It significantly enhanced cortical processing at all SNR levels, shortened CAEPs latencies, and increased amplitudes, resulting in a greater number of measurable peaks for ACC. SRM showed mild to moderate correlations with the differences between the two conditions in CAEP measures. The regression model combining N1'-P2' amplitude at 5 dB SNR (R2 = 0.26), P1 amplitude at 0 dB SNR (R2 = 0.14), and P1 latency at -5 dB SNR (R2 = 0.15), explained 45.3% of the variance in SRM. Our study demonstrates that introducing spatial cues can improve speech perception and enhance central auditory processing in normal-hearing young adults. CAEPs may contribute to predictions about SRM and hold potential for practical application.

Controlling diurnal flower-opening time by manipulating the jasmonate pathway accelerates development of indica-japonica hybrid rice breeding.

Inter-subspecific indica-japonica hybrid rice (Oryza sativa) has the potential for increased yields over traditional indica intra-subspecies hybrid rice, but limited yield of F1 hybrid seed production (FHSP) hinders the development of indica-japonica hybrid rice breeding. Diurnal flower-opening time (DFOT) divergence between indica and japonica rice has been a major contributing factor to this issue, but few DFOT genes have been cloned. Here, we found that manipulating the expression of jasmonate (JA) pathway genes can effectively modulate DFOT to improve the yield of FHSP in rice. Treating japonica cultivar Zhonghua 11 (ZH11) with methyl jasmonate (MeJA) substantially advanced DFOT. Furthermore, overexpressing the JA biosynthesis gene OPDA REDUCTASE 7 (OsOPR7) and knocking out the JA inactivation gene CHILLING TOLERANCE 1 (OsHAN1) in ZH11 advanced DFOT by 1- and 2-h respectively; and knockout of the JA signal suppressor genes JASMONATE ZIM-DOMAIN PROTEIN 7 (OsJAZ7) and OsJAZ9 resulted in 50-min and 1.5-h earlier DFOT respectively. The yields of FHSP using japonica male-sterile lines GAZS with manipulated JA pathway genes were significantly higher than that of GAZS wildtype. Transcriptome analysis, cytological observations, measurements of elastic modulus and determination of cell wall components indicated that the JA pathway could affect the loosening of the lodicule cell walls by regulating their composition through controlling sugar metabolism, which in turn influences DFOT. This research has vital implications for breeding japonica rice cultivars with early DFOT to facilitate indica-japonica hybrid rice breeding.

Structure activity relationship and target prediction for ABX464 analogues in Caenorhabditis elegans.

Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the 'Pandemic Response Box' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.

The acoustic change complex evoked by location-change sounds in the elderly.

OBJECTIVE: We have shown that the acoustic change complex (ACC) can be elicited by changing the horizontal sound location in young individuals. In this study, we aimed to evaluate the application of ACC within the elderly and its relationship with behavioural results. DESIGN: The minimum audible angle (MAA), as well as onset cortical auditory evoked potentials (onset-CAEPs) and ACC elicited by the stimuli of location-change white noise (±45 to ±2 degrees) were recorded. Latencies and amplitudes were analysed using repeated-measures ANOVA. Pearson correlation analysis was conducted to examine the relationship between ACC and MAA. STUDY SAMPLE: Ten older adults with normal hearing (NH) and twenty with presbycusis. RESULTS: The ACC was effectively elicited with angular variations in elderly participants. The onset-CAEP N1 latency, ACC N1'-P2' amplitude, and N1' latency were all associated with the angle shifts, with the N1' latency being the most predictive factor for angle discrimination. The consistency between MAA and ACC made them complementary for the clinical evaluation of sound localisation. CONCLUSIONS: The utilisation of ACC, evoked by location-change sounds, presented a promising clinical objective measure for evaluating sound localisation abilities in the elderly.

Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

Longitudinal spatial mapping of lipid metabolites reveals pre-symptomatic changes in the hippocampi of Huntington's disease transgenic mice.

In Huntington's disease (HD), a key pathological feature includes the development of inclusion-bodies of fragments of the mutant huntingtin protein in the neurons of the striatum and hippocampus. To examine the molecular changes associated with inclusion-body formation, we applied MALDI-mass spectrometry imaging and deuterium pulse labelling to determine lipid levels and synthesis rates in the hippocampus of a transgenic mouse model of HD (R6/1 line). The R6/1 HD mice lacked inclusions in the hippocampus at 6 weeks of age (pre-symptomatic), whereas inclusions were pervasive by 16 weeks of age (symptomatic). Hippocampal subfields (CA1, CA3 and DG), which formed the highest density of inclusion formation in the mouse brain showed a reduction in the relative abundance of neuron-enriched lipids that have roles in neurotransmission, synaptic plasticity, neurogenesis, and ER-stress protection. Lipids involved in the adaptive response to ER stress (phosphatidylinositol, phosphatidic acid, and ganglioside classes) displayed increased rates of synthesis in HD mice relative to WT mice at all the ages examined, including prior to the formation of the inclusion bodies. Our findings, therefore, support a role for ER stress occurring pre-symptomatically and potentially contributing to pathological mechanisms underlying HD.

Thermostable allergens in canned fish: Evaluating risks for fish allergy.

BACKGROUND: Major fish allergens, including parvalbumin (PV), are heat stable and can withstand extensive cooking processes. Thus, the management of fish allergy generally relies on complete avoidance. Fish-allergic patients may be advised to consume canned fish, as some fish-allergic individuals have reported tolerance to canned fish. However, the safety of consuming canned fish has not been evaluated with comprehensive immunological and molecular analysis of canned fish products. METHODS: We characterized the in vitro immunoreactivity of serum obtained from fish-allergic subjects to canned fish. Seventeen canned fish products (salmon n = 8; tuna n = 7; sardine n = 2) were assessed for the content and integrity of PV using allergen-specific antibodies. Subsequently, the sIgE binding of five selected products was evaluated for individual fish-allergic patients (n = 53). Finally, sIgE-binding proteins were identified by mass spectrometry. RESULTS: The canned fish showed a markedly reduced PV content and binding to PV-specific antibodies compared with conventionally cooked fish. However, PV and other heat-stable fish allergens, including tropomyosin and collagen, still maintained their sIgE-binding capacity. Of 53 patients, 66% showed sIgE binding to canned fish proteins. The canned sardine contained proteins bound to sIgE from 51% of patients, followed by canned salmon (43%-45%) and tuna (8%-17%). PV was the major allergen in canned salmon and sardine. Tropomyosin and/or collagen also showed sIgE binding. CONCLUSION: We showed that canned fish products may not be safe for all fish-allergic patients. Canned fish products should only be considered into the diet of individuals with fish allergy, after detailed evaluation which may include in vitro diagnostics to various heat-stable fish allergens and food challenge conducted in suitable environments.

Widespread remodeling of proteome solubility in response to different protein homeostasis stresses.

The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington's disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.

Liquid chromatography coupled with tandem mass spectrometry for simultaneous quantification of valproate, valproate-glucuronide and lamotrigine in various biological matrices of rat.

Valproate and lamotrigine are commonly used as antiepileptic drugs even in pregnant and breastfeeding women. The extent and effects of drug exposure on the developing brain of the offspring are not well understood. Animal models can be utilised to investigate the transfer of substances into fetal brain with the ultimate aim of providing insights to aid clinical decisions. In the present study, an LC-MS/MS method was developed and validated for quantification of valproate (VPA), valproate-glucuronide (VPA-Gluc, a major metabolite of valproate) and lamotrigine (LTG) in rat blood plasma, cerebrospinal fluid and brain tissue. A 10 µl sample was spiked with stable isotope-labelled internal standards and extracted by methanol. An Agilent RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm) was used. The MS/MS transitions were 143.1016-143.1016 (VPA), 319.1392-143.0978 (VPA-Gluc) and 256.0157-210.9826 (LTG). The linear ranges of VPA, VPA-Gluc and LTG were 30-250, 10-140 and 0.3-1 µg/ml, respectively. The intra- and inter-day accuracy and precision, carryover, sensitivity and recovery were evaluated according to the US Food and Drug Administration guidance for bioanalytical method validation. Finally, the validated method was applied to a set of experimental animal samples and produced results highly comparable with those from an orthogonal analytical method.

Study on the Recognition of Coal Miners' Unsafe Behavior and Status in the Hoist Cage Based on Machine Vision.

The hoist cage is used to lift miners in a coal mine's auxiliary shaft. Monitoring miners' unsafe behaviors and their status in the hoist cage is crucial to production safety in coal mines. In this study, a visual detection model is proposed to estimate the number and categories of miners, and to identify whether the miners are wearing helmets and whether they have fallen in the hoist cage. A dataset with eight categories of miners' statuses in hoist cages was developed for training and validating the model. Using the dataset, the classical models were trained for comparison, from which the YOLOv5s model was selected to be the basic model. Due to small-sized targets, poor lighting conditions, and coal dust and shelter, the detection accuracy of the Yolov5s model was only 89.2%. To obtain better detection accuracy, k-means++ clustering algorithm, a BiFPN-based feature fusion network, the convolutional block attention module (CBAM), and a CIoU loss function were proposed to improve the YOLOv5s model, and an attentional multi-scale cascaded feature fusion-based YOLOv5s model (AMCFF-YOLOv5s) was subsequently developed. The training results on the self-built dataset indicate that its detection accuracy increased to 97.6%. Moreover, the AMCFF-YOLOv5s model was proven to be robust to noise and light.

The Proteome and Lipidome of Extracellular Vesicles from Haemonchus contortus to Underpin Explorations of Host-Parasite Cross-Talk.

Many parasitic worms have a major adverse impact on human and animal populations worldwide due to the chronicity of their infections. There is a growing body of evidence indicating that extracellular vesicles (EVs) are intimately involved in modulating (suppressing) inflammatory/immune host responses and parasitism. As one of the most pathogenic nematodes of livestock animals, Haemonchus contortus is an ideal model system for EV exploration. Here, employing a multi-step enrichment process (in vitro culture, followed by ultracentrifugation, size exclusion and filtration), we enriched EVs from H. contortus and undertook the first comprehensive (qualitative and quantitative) multi-omic investigation of EV proteins and lipids using advanced liquid chromatography-mass spectrometry and informatics methods. We identified and quantified 561 proteins and 446 lipids in EVs and compared these molecules with those of adult worms. We identified unique molecules in EVs, such as proteins linked to lipid transportation and lipid species (i.e., sphingolipids) associated with signalling, indicating the involvement of these molecules in parasite-host cross-talk. This work provides a solid starting point to explore the functional roles of EV-specific proteins and lipids in modulating parasite-host cross-talk, and the prospect of finding ways of disrupting or interrupting this relationship to suppress or eliminate parasite infection.

A Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress.

Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.

Phosphomatics: interactive interrogation of substrate-kinase networks in global phosphoproteomics datasets.

MOTIVATION: Mass spectrometry-based phosphoproteomics can routinely identify and quantify thousands of phosphorylated peptides from a single experiment. However interrogating possible upstream kinases and identifying key literature for phosphorylation sites is laborious and time-consuming. RESULTS: Here, we present Phosphomatics-a publicly available web resource for interrogating phosphoproteomics data. Phosphomatics allows researchers to upload phosphoproteomics data and interrogate possible relationships from a substrate-, kinase- or pathway-centric viewpoint. AVAILABILITY AND IMPLEMENTATION: Phosphomatics is freely available via the internet at: https://phosphomatics.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Polymorphic Microsatellite Development, Genetic Diversity, Population Differentiation and Sexual State of Phytophthora capsici on Commercial Peppers in Three Provinces of Southwest China.

Pepper blight, caused by the oomycete pathogen Phytophthora capsici (P. capsici), is one of the most destructive soilborne diseases worldwide. Between 2019 and 2020, 235 single spore isolates of P. capsici were collected from 36 commercial pepper planting areas in Sichuan, Chongqing, and Guizhou provinces in China. A novel full set of 323 high-quality polymorphic microsatellites was obtained by resequencing 10 isolates. In total, 163 isolates with two alleles per microsatellite locus were used for population analysis and resulted in 156 genotypes on 10 microsatellite loci. The genetic diversity, population differentiation, principal component, genetic structure, and genetic relationships analyses showed an extensive variety of the P. capsici in Sichuan and Guizhou with clonal lineages, two shared genotypes, and no geographic differentiation. The population from Chongqing was differentiated from that of Sichuan and Guizhou and had the highest genetic diversity. There was no significant distinction between the populations of the two sampling years, but there was a small differentiation between the populations from bell peppers and hot peppers. The isolates from Southwest China were largely distant from the two reference isolates from the USA. The analysis of molecular variance showed that the major variance of the populations was within populations. The linkage equilibrium test, mating type composition, and oospore detection indicated that only P. capsici from the Jiulongpo district of Chongqing had appeared in sexual recombination. Overall, this study revealed that the high and complex genetic diversity population of P. capsici in Sichuan, Chongqing, and Guizhou with uneven geographic variation and limited sexual reproductive behavior in Chongqing, potentially driven by differences in the geographical environment, reproductive patterns, different cultivars, and artificial long-distance transfers. IMPORTANCE Phytophthora capsici, a notorious soilborne and rapidly evolving pathogen with a wide range of hosts, is a huge threat to pepper production worldwide. However, the detailed genetic structure and dynamics of P. capsici in most Chinese provinces are still unclear, even though China is the world's largest producer and consumer of peppers. Here, a novel full set of high-quality polymorphic microsatellites, obtained by genome resequencing data of 10 isolates from Southwest China, was provided for future population analyses. In this study, we further investigated and established the genetic structure, sexual recombination, geographic subdivisions, interannual stability, differentiation in different types of host peppers, and member relationships of P. capsici from three provinces in Southwest China. These results reveal the genetic structure and dynamics of P. capsici in three provinces of Southwest China and help us to execute more effective management strategies in the future.

Respiratory strategy at birth initiates distinct lung injury phenotypes in the preterm lamb lung.

BACKGROUND: A lack of clear trial evidence often hampers clinical decision-making during support of the preterm lung at birth. Protein biomarkers have been used to define acute lung injury phenotypes and improve patient selection for specific interventions in adult respiratory distress syndrome. The objective of the study was to use proteomics to provide a deeper biological understanding of acute lung injury phenotypes resulting from different aeration strategies at birth in the preterm lung. METHODS: Changes in protein abundance against an unventilated group (n = 7) were identified via mass spectrometry in a biobank of gravity dependent and non-dependent lung tissue from preterm lambs managed with either a Sustained Inflation (SI, n = 20), Dynamic PEEP (DynPEEP, n = 19) or static PEEP (StatPEEP, n = 11). Ventilation strategy-specific pathways and functions were identified (PANTHER and WebGestalt Tool) and phenotypes defined using integrated analysis of proteome, physiological and clinical datasets (MixOmics package). RESULTS: 2372 proteins were identified. More altered proteins were identified in the non-dependent lung, and in SI group than StatPEEP and DynPEEP. Different inflammation, immune system, apoptosis and cytokine pathway enrichment were identified for each strategy and lung region. Specific integration maps of clinical and physiological outcomes to specific proteins could be generated for each strategy. CONCLUSIONS: Proteomics mapped the molecular events initiating acute lung injury and identified detailed strategy-specific phenotypes. This study demonstrates the potential to characterise preterm lung injury by the direct aetiology and response to lung injury; the first step towards true precision medicine in neonatology.

Hexosaminidase A (HEXA) regulates hepatic sphingolipid and lipoprotein metabolism in mice.

Hexosaminidase A (HexA), a heterodimer consisting of HEXA and HEXB, converts the ganglioside sphingolipid GM2 to GM3 by removing a terminal N-acetyl-d-galactosamine. HexA enzyme deficiency in humans leads to GM2 accumulation in cells, particularly in neurons, and is associated with neurodegeneration. While HexA and sphingolipid metabolism have been extensively investigated in the context of neuronal lipid metabolism, little is known about the metabolic impact of HexA and ganglioside degradation in other tissues. Here, we focussed on the role of HexA in the liver, which is a major regulator of systemic lipid metabolism. We find that hepatic Hexa expression is induced by lipid availability and increased in the presence of hepatic steatosis, which is associated with increased hepatic GM3 content. To assess the impact of HEXA on hepatic lipid metabolism, we used an adeno-associated virus to overexpress HEXA in the livers of high-fat diet fed mice. HEXA overexpression was associated with increased hepatic GM3 content and increased expression of enzymes involved in the degradation of glycated sphingolipids, ultimately driving sphingomyelin accumulation in the liver. In addition, HEXA overexpression led to substantial proteome remodeling in cell surface lipid rafts, which was associated with increased VLDL processing and secretion, hypertriglyceridemia and ectopic lipid accumulation in peripheral tissues. This study established an important role of HEXA in modulating hepatic sphingolipid and lipoprotein metabolism.

What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses.

Hydrophilic interaction liquid chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterization of glycoproteomes. Despite its utility, HILIC enrichment is associated with a number of shortcomings, including requiring large amounts of starting materials, potentially introducing chemical artifacts such as formylation when high concentrations of formic acid are used, and biasing/undersampling specific classes of glycopeptides. Here, we investigate HILIC enrichment-independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments, yet are readily identified in whole proteome samples. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation, we show that at high compensation voltages (CVs), short aliphatic glycopeptides can be enriched from complex samples, providing an alternative means to identify glycopeptide recalcitrant to hydrophilic-based enrichment. Combining whole proteome and FAIMS analyses, we show that the observable glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought. Excitingly, the ability to enrich glycopeptides using FAIMS appears generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.