Spatial Proteomics Reveals Alcohol-Induced Damages to the Crypts and Villi of the Mouse Small Intestine.

Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are perturbed in this process. In this study, we employed laser capture microdissection and a label-free proteomics approach to investigate the consequences of alcohol exposure to the proteomes of crypts and villi in the proximal small intestine. Intestinal tissues from alcohol-fed and pair-fed mice were microdissected to selectively capture cells in the crypts and villi regions, followed by one-pot protein digestion and data-independent LC-MS/MS analysis. We successfully identified over 3000 proteins from each of the crypt or villi regions equivalent to ∼3000 cells. Analysis of alcohol-treated tissues indicated an enhanced alcohol metabolism and reduced levels of α-defensins in crypts, alongside increased lipid metabolism and apoptosis in villi. Immunofluorescence imaging further corroborated the proteomic findings. Our work provides a detailed profiling of the proteomic changes in the compartments of the mouse small intestine and aids in molecular-level understanding of alcohol-induced tissue damage.

Conventional type 1 dendritic cells protect against gut barrier disruption via maintaining Akkermansia muciniphila in alcoholic steatohepatitis.

BACKGROUND AND AIMS: Alcohol-perturbed gut immune homeostasis is associated with the development of alcoholic liver disease (ALD). However, the role of intestinal dendritic cells (DCs) in ALD progression is still unknown. This study aimed to investigate the cellular and molecular mechanisms through which intestinal DCs respond to alcohol exposure and contribute to the pathogenesis of ALD. APPROACH AND RESULTS: After 8 weeks of alcohol consumption, the number of basic leucine zipper transcription factor ATF-like 3 ( Batf3 )-dependent conventional type 1 DCs (cDC1s) was dramatically decreased in the intestine but not the liver. cDC1 deficient Batf3 knockout mice along with wild-type mice were subjected to chronic-binge ethanol feeding to determine the role of intestinal cDC1s reduction in ALD. cDC1s deficiency exacerbated alcohol-induced gut barrier disruption, bacterial endotoxin translocation into the circulation, and liver injury. Adoptive transfer of cDC1s to alcohol-fed mice ameliorated alcohol-mediated gut barrier dysfunction and liver injury. Further studies revealed that intestinal cDC1s serve as a positive regulator of Akkermansia muciniphila ( A. muciniphila ). Oral administration of A. muciniphila markedly reversed alcoholic steatohepatitis in mice. Mechanistic studies revealed that cDC1s depletion exacerbated alcohol-downregulated intestinal antimicrobial peptides which play a crucial role in maintaining A. muciniphila abundance, by disrupting the IL-12-interferon gamma signaling pathway. Lastly, we identified that intestinal cDC1s were required for the protective role of Lactobacillus reuteri in alcoholic steatohepatitis. CONCLUSIONS: This study demonstrated that cDC1s protect alcohol-induced liver injury by maintaining A. muciniphila abundance in mice. Targeting cDC1s may serve as a promising therapeutic approach for treating ALD.

Integrated oral microgel system ameliorates renal fibrosis by hitchhiking co-delivery and targeted gut flora modulation.

BACKGROUND: Renal fibrosis is a progressive process associated with chronic kidney disease (CKD), contributing to impaired kidney function. Active constituents in traditional Chinese herbs, such as emodin (EMO) and asiatic acid (AA), exhibit potent anti-fibrotic properties. However, the oral administration of EMO and AA results in low bioavailability and limited kidney accumulation. Additionally, while oral probiotics have been accepted for CKD treatment through gut microbiota modulation, a significant challenge lies in ensuring their viability upon administration. Therefore, our study aims to address both renal fibrosis and gut microbiota imbalance through innovative co-delivery strategies. RESULTS: In this study, we developed yeast cell wall particles (YCWPs) encapsulating EMO and AA self-assembled nanoparticles (NPYs) and embedded them, along with Lactobacillus casei Zhang, in chitosan/sodium alginate (CS/SA) microgels. The developed microgels showed significant controlled release properties for the loaded NPYs and prolonged the retention time of Lactobacillus casei Zhang (L. casei Zhang) in the intestine. Furthermore, in vivo biodistribution showed that the microgel-carried NPYs significantly accumulated in the obstructed kidneys of rats, thereby substantially increasing the accumulation of EMO and AA in the impaired kidneys. More importantly, through hitchhiking delivery based on yeast cell wall and positive modulation of gut microbiota, our microgels with this synergistic strategy of therapeutic and modulatory interactions could regulate the TGF-ß/Smad signaling pathway and thus effectively ameliorate renal fibrosis in unilateral ureteral obstruction (UUO) rats. CONCLUSION: In conclusion, our work provides a new strategy for the treatment of renal fibrosis based on hitchhiking co-delivery of nanodrugs and probiotics to achieve synergistic effects of disease treatment and targeted gut flora modulation.

Effects of multiple stressors on pancreatic human islets proteome reveal new insights into the pathways involved.

Pancreatic ß-cell dysfunction is an early hallmark of type 1 diabetes mellitus. Among the potentially critical factors that cause ß-cell dysfunction are cytokine attack, glucotoxicity, induction of endoplasmic reticulum (ER) or mitochondria stress. However, the exact molecular mechanism underlying ß-cell's inability to maintain glucose homeostasis under severe stresses is unknown. This study used proinflammatory cytokines, thapsigargin, and rotenone in the presence of high concentration glucose to mimicking the conditions experienced by dysfunctional ß-cells in human pancreatic islets, and profiled the alterations to the islet proteome with TMT-based proteomics. The results were further verified with label-free quantitative proteomics. The differentially expressed proteins under stress conditions reveal that immune related pathways are mostly perturbed by cytokines, while the respiratory electron transport chains and protein processing in ER pathways by rotenone. Thapsigargin together with high glucose induces dramatic increases of proteins in lipid synthesis and peroxisomal protein import pathways, with energy metabolism and vesicle secretion related pathways downregulated. High concentration glucose, on the other hand, alleviated complex I inhibition induced by rotenone. Our results contribute to a more comprehensive understanding of the molecular events involved in ß-cell dysfunction.

Hypervirulent Klebsiella pneumoniae detection methods: a minireview.

Hypervirulent Klebsiella pneumoniae (hvKp), characterized by high virulence and epidemic potential, has become a global public health challenge. Therefore, improving the identification of hvKp and enabling earlier and faster detection in the community to support subsequent effective treatment and prevention of hvKp are an urgent issue. To address these issues, a number of assays have emerged, such as String test, Galleria mellonella infection test, PCR, isothermal exponential amplification, and so on. In this paper, we have collected articles on the detection methods of hvKp and conducted a retrospective review based on two aspects: traditional detection technology and biomarker-based detection technology. We summarize the advantages and limitations of these detection methods and discuss the challenges as well as future directions, hoping to provide new insights and references for the rapid detection of hvKp in the future. The aim of this study is to focus on the research papers related to Hypervirulent Klebsiella pneumoniae involving the period from 2012 to 2022. We conducted searches using the keywords "Hypervirulent Klebsiella pneumoniae, biomarkers, detection techniques" on ScienceDirect and Google Scholar. Additionally, we also searched on PubMed, using MeSH terms associated with the keywords (such as Klebsiella pneumoniae, Klebsiella Infections, Virulence, Biomarkers, diagnosis, etc.).

Region-specific mouse brain ganglioside distribution revealed by an improved isobaric aminoxyTMT labeling strategy with automated data processing.

Gangliosides are specialized glycosphingolipids most abundant in the central nervous system. Their complex amphiphilic structure is essential to the formation of membrane lipid rafts and for molecular recognition. Dysfunction of lipid rafts and ganglioside metabolism has been linked to cancer, metabolic disorders, and neurodegenerative disorders. Changes in ganglioside concentration and diversity during the progression of disease have made them potential biomarkers for early detection and shed light on disease mechanisms. Chemical derivatization facilitates whole ion analysis of gangliosides while improving ionization, providing rich fragmentation spectra, and enabling multiplexed analysis schemes such as stable isotope labeling. In this work, we report improvement to our previously reported isobaric labeling methodology for ganglioside analysis by increasing buffer concentration and removing solid-phase extraction desalting for a more complete and quantitative reaction. Identification and quantification of gangliosides are automated through MS-DIAL with an in-house ganglioside derivatives library. We have applied the updated methodology to relative quantification of gangliosides in six mouse brain regions (cerebellum, pons/medulla, midbrain, thalamus/hypothalamus, cortex, and basal ganglia) with 2 mg tissue per sample, and region-specific distributions of 88 ganglioside molecular species are described with ceramide isomers resolved. This method is promising for application to comparative analysis of gangliosides in biological samples.

HLA Allele-Specific Quantitative Profiling of Type 1 Diabetic B Lymphocyte Immunopeptidome.

Peptide ligands presented by human leukocyte antigen (HLA) molecules on the cell surface represent the immunopeptidome that could be utilized for identification of antigenic peptides for immunotherapy and prevention of autoimmune diseases. Although T-cells are well-known key players in the destruction of pancreatic beta-cells in type 1 diabetes (T1D), increasing evidence points toward a role for B-cells in disease pathogenesis. However, as antigen presenting cells, little is known about the comprehensive immunopeptidome of B cells and their changes in the context of T1D. We performed HLA allele-specific quantitative immunopeptidomics using B lymphocytes derived from T1D patients and healthy controls. Hundreds of HLA-I and HLA-II immunopeptides were identified as differentially regulated in T1D per HLA allele for B cells sharing identical HLA alleles. The results were further validated using additional T1D and healthy B cells with partially overlapped HLA alleles. Differentially expressed immunopeptides were confirmed with targeted proteomics and for reactivity using known T-cell assays in the immune epitope database. Considering samples with identical HLA alleles are difficult to obtain for T1D and other similar HLA-restricted diseases, our work represents a viable approach to better understand HLA allele-specific antigen presentation and may facilitate identification of immunopeptides for therapeutic applications in autoimmune diseases. Data are available via ProteomeXchange with identifier PXD026184.

Mouse Paneth Cell-Enriched Proteome Enabled by Laser Capture Microdissection.

Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their coexistence with stem cells, making it difficult to culture Paneth cells alone in vitro. Using a simplified toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region, and surfactant-assisted one-pot protein digestion, we identified more than 1300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes, and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues: a comparable proteomic coverage can be achieved with 3600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplified workflow enables profiling of Paneth cell-associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.

Iminosugar Glucosidase Inhibitors Reduce Hepatic Inflammation in Hepatitis A Virus-Infected Ifnar1-/- Mice.

Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.

Pancreatic INS-1 ß-Cell Response to Thapsigargin and Rotenone: A Comparative Proteomics Analysis Uncovers Key Pathways of ß-Cell Dysfunction.

Insulin-secreting ß-cells in the pancreatic islets are exposed to various endogenous and exogenous stressing conditions, which may lead to ß-cell dysfunction or apoptosis and ultimately to diabetes mellitus. However, the detailed molecular mechanisms underlying ß-cell's inability to survive under severe stresses remain to be explored. This study used two common chemical stressors, thapsigargin and rotenone, to induce endoplasmic reticulum (ER) and mitochondria stress in a rat insuloma INS-1 832/13 ß-cell line, mimicking the conditions experienced by dysfunctional ß-cells. Proteomic changes of cells upon treatment with stressors at IC50 were profiled with TMT-based quantitative proteomics and further verified using label-free quantitive proteomics. The differentially expressed proteins under stress conditions were selected for in-depth bioinformatic analysis. Thapsigargin treatment specifically perturbed unfolded protein response (UPR) related pathways; in addition, 58 proteins not previously linked to the UPR related pathways were identified with consistent upregulation under stress induced by thapsigargin. Conversely, rotenone treatment resulted in significant proteome changes in key mitochondria regulatory pathways such as fatty acid ß-oxidation, cellular respiration, citric acid cycle, and respiratory electron transport. Our data also demonstrated that both stressors increased reactive oxygen species production and depleted adenosine triphosphate synthesis, resulting in significant dysregulation of oxidative phosphorylation signaling pathways. These novel dysregulated proteins may suggest an alternative mechanism of action in ß-cell dysfunction and provide potential targets for probing ER- and mitochondria stress-induced ß-cell death.

Paneth Cell Dysfunction Mediates Alcohol-related Steatohepatitis Through Promoting Bacterial Translocation in Mice: Role of Zinc Deficiency.

BACKGROUND AND AIMS: Microbial dysbiosis is associated with alcohol-related hepatitis (AH), with the mechanisms yet to be elucidated. The present study aimed to determine the effects of alcohol and zinc deficiency on Paneth cell (PC) antimicrobial peptides, α-defensins, and to define the link between PC dysfunction and AH. APPROACH AND RESULTS: Translocation of pathogen-associated molecular patterns (PAMPs) was determined in patients with severe AH and in a mouse model of alcoholic steatohepatitis. Microbial composition and PC function were examined in mice. The link between α-defensin dysfunction and AH was investigated in α-defensin-deficient mice. Synthetic human α-defensin 5 (HD5) was orally given to alcohol-fed mice to test the therapeutic potential. The role of zinc deficiency in α-defensin was evaluated in acute and chronic mouse models of zinc deprivation. Hepatic inflammation was associated with PAMP translocation and lipocalin-2 (LCN2) and chemokine (C-X-C motif) ligand 1 (CXCL1) elevation in patients with AH. Antibiotic treatment, lipopolysaccharide injection to mice, and in vitro experiments showed that PAMPs, but not alcohol, directly induced LCN2 and CXCL1. Chronic alcohol feeding caused systemic dysbiosis and PC α-defensin reduction in mice. Knockout of functional α-defensins synergistically affected alcohol-perturbed bacterial composition and the gut barrier and exaggerated PAMP translocation and liver damage. Administration of HD5 effectively altered cecal microbial composition, especially increased Akkermansia muciniphila, and reversed the alcohol-induced deleterious effects. Zinc-regulated PC homeostasis and α-defensins function at multiple levels, and dietary zinc deficiency exaggerated the deleterious effect of alcohol on PC bactericidal activity. CONCLUSIONS: Taken together, the study suggests that alcohol-induced PC α-defensin dysfunction is mediated by zinc deficiency and involved in the pathogenesis of AH. HD5 administration may represent a promising therapeutic approach for treating AH.

Ganglioside isomer analysis using ion polarity switching liquid chromatography-tandem mass spectrometry.

Gangliosides are ubiquitously present on cell surface. They are more abundantly expressed in nerve cells and tissues and involved in pathology of various diseases. Diversity of molecular structures in the carbohydrate head group, fatty acyl, and long chain base increases the complexity of analyzing gangliosides. In this study, an ultrahigh-performance liquid chromatography-tandem mass spectrometry method is developed for analysis of the co-eluting ganglioside isomers, which uses ion polarity switching to integrate glycan head isomer identification, ceramide isomer differentiation, and quantification of ganglioside into one analysis. The method is facilitated with an extensive ganglioside target list by combining the various glycan head groups, long chain bases, and the experimentally determined fatty acyls. Correlation between the retention time of ganglioside and its ceramide total carbon number is experimentally validated and used to predict retention time of ganglioside target list for scheduling the final multiple reaction monitoring method. This method was validated according to the FDA guidelines: 96.5% of gangliosides with good accuracy (80-120%), precision (< 15%), and linearity R2 > 0.99. The authenticated gangliosides were quantified from mouse brain by isotope dilution. Overall, 165 gangliosides were quantified using 10 mg mouse brain tissue, including 100 isomers of GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, and GT1b.

A UPLC-MRM-MS method for comprehensive profiling of Amadori compound-modified phosphatidylethanolamines in human plasma.

Phosphatidylethanolamines (PEs) are targets of non-enzymatic glycation, a chemical process that occurs between glucose and primary amine-containing biomolecules. As the early-stage non-enzymatic glycation products of PE, Amadori-PEs are implicated in the pathogenesis of various diseases. However, only a few Amadori-PE molecular species have been identified so far; a comprehensive profiling of these glycated PE species is needed to establish their roles in disease pathology. Herein, based on our previous work using liquid chromatography-coupled neutral loss scanning and product ion scanning tandem mass spectrometry (LC-NLS-MS and LC-PIS-MS) in tandem, we extend identification of Amadori-PE to the low-abundance species, which is facilitated by using plasma lipids glycated in vitro. The confidence of identification is improved by high-resolution tandem mass spectrometry and chromatographic retention time regression. A LC-coupled multiple reaction monitoring mass spectrometry (LC-MRM-MS) assay is further developed for more sensitive quantitation of the Amadori compound-modified lipids. Using synthesized stable isotope-labeled Amadori lipids as internal standards, levels of 142 Amadori-PEs and 33 Amadori-LysoPEs are determined in the NIST human plasma standard reference material. These values may serve as an important reference for future investigations of Amadori-modified lipids in human diseases.

Human GDPD3 overexpression promotes liver steatosis by increasing lysophosphatidic acid production and fatty acid uptake.

The glycerol phosphate pathway produces more than 90% of the liver triacylglycerol (TAG). LysoPA, an intermediate in this pathway, is produced by glycerol-3-phosphate acyltransferase. Glycerophosphodiester phosphodiesterase domain containing 3 (GDPD3), whose gene was recently cloned, contains lysophospholipase D activity, which produces LysoPA from lysophospholipids. Whether human GDPD3 plays a role in hepatic TAG homeostasis is unknown. We hypothesized that human GDPD3 increases LysoPA production and availability in the glycerol phosphate pathway, promoting TAG biosynthesis. To test our hypothesis, we infected C57BL/6J mice with adeno-associated virus encoding a hepatocyte-specific albumin promoter that drives GFP (control) or FLAG-tagged human GDPD3 overexpression and fed the mice chow or a Western diet to induce hepatosteatosis. Hepatic human GDPD3 overexpression induced LysoPA production and increased FA uptake and incorporation into TAG in mouse hepatocytes and livers, ultimately exacerbating Western diet-induced liver steatosis. Our results also showed that individuals with hepatic steatosis have increased GDPD3 mRNA levels compared with individuals without steatosis. Collectively, these findings indicate that upregulation of GDPD3 expression may play a key role in hepatic TAG accumulation and may represent a molecular target for managing hepatic steatosis.

Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography-triple quadrupole mass spectrometry.

Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.

Differential Isotope Labeling by Permethylation and Reversed-Phase Liquid Chromatography-Mass Spectrometry for Relative Quantification of Intact Neutral Glycolipids in Mammalian Cells.

Probing the role of glycolipids in health and disease warrants development of practical strategies to determine these molecules at the intact structural level, namely to simultaneously characterize and quantify the glycan and lipid moieties without breaking the linkage between them. Herein we present such an approach utilizing differential isotope labeling and reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) for structural characterization and relative quantification of intact neutral glycolipids. In this approach, each individual sample and a pooled aliquot of each sample were permethylated using 12CH3I and 13CH3I, respectively, with the latter one serving as internal reference standard. The individual 12C-permethylated samples were spiked with equal amounts of the 13C-permethylated pooled sample and analyzed by RPLC-MS/MS. Permethylation not only increased the ionization efficiency of glycolipids but also facilitated structural characterization of both moieties. The ratio of the peak areas between the 12C- and 13C-labeled glycolipids served as surrogate measure of their relative concentrations. The coefficient of variation of the method was <6% measured across four representative glycolipids in five different ratios and triplicate experiments, after correction of natural isotopic distribution. When analyzing the low abundant glycolipids in total lipid extract, permethylation can dramatically reduce the analytical background by depleting most of the highly abundant ester-linked lipids. Application to conduritol B epoxide-, a ß-glucocerebrosidase inhibitor, treated RAW 264.7 cells demonstrated the practical utility of this method in profiling the temporal accumulation of different glycolipids. Overall, this methodology offers a practical LC-MS based identification and quantification strategy to advance intact glycolipids analysis in mammalian cells.

Tree resilience to drought increases in the Tibetan Plateau.

Forests in the Tibetan Plateau are thought to be vulnerable to climate extremes, yet they also tend to exhibit resilience contributing to the maintenance of ecosystem services in and beyond the plateau. So far the spatiotemporal pattern in tree resilience in the Tibetan Plateau remains largely unquantified and the influence of specific factors on the resilience is poorly understood. Here, we study ring-width data from 849 trees at 28 sites in the Tibetan Plateau with the aim to quantify tree resilience and determine their diving forces. Three extreme drought events in years 1969, 1979, and 1995 are detected from metrological records. Regional tree resistance to the three extreme droughts shows a decreasing trend with the proportion of trees having high resistance ranging from 71.9%, 55.2%, to 39.7%. Regional tree recovery is increasing with the proportion of trees having high recovery ranging from 28.3%, 52.2%, to 64.2%. The area with high resistance is contracting and that of high recovery is expanding. The spatiotemporal resistance and recovery are associated with moisture availability and diurnal temperature range, respectively. In addition, they are both associated with forest internal factor represented by growth consistence among trees. We conclude that juniper trees in the Tibetan Plateau have increased resilience to extreme droughts in the study period. We highlight pervasive resilience in juniper trees. The results have implications for predicting tree resilience and identifying areas vulnerable to future climate extremes.

Underestimated ecosystem carbon turnover time and sequestration under the steady state assumption: A perspective from long-term data assimilation.

It is critical to accurately estimate carbon (C) turnover time as it dominates the uncertainty in ecosystem C sinks and their response to future climate change. In the absence of direct observations of ecosystem C losses, C turnover times are commonly estimated under the steady state assumption (SSA), which has been applied across a large range of temporal and spatial scales including many at which the validity of the assumption is likely to be violated. However, the errors associated with improperly applying SSA to estimate C turnover time and its covariance with climate as well as ecosystem C sequestrations have yet to be fully quantified. Here, we developed a novel model-data fusion framework and systematically analyzed the SSA-induced biases using time-series data collected from 10 permanent forest plots in the eastern China monsoon region. The results showed that (a) the SSA significantly underestimated mean turnover times (MTTs) by 29%, thereby leading to a 4.83-fold underestimation of the net ecosystem productivity (NEP) in these forest ecosystems, a major C sink globally; (b) the SSA-induced bias in MTT and NEP correlates negatively with forest age, which provides a significant caveat for applying the SSA to young-aged ecosystems; and (c) the sensitivity of MTT to temperature and precipitation was 22% and 42% lower, respectively, under the SSA. Thus, under the expected climate change, spatiotemporal changes in MTT are likely to be underestimated, thereby resulting in large errors in the variability of predicted global NEP. With the development of observation technology and the accumulation of spatiotemporal data, we suggest estimating MTTs at the disequilibrium state via long-term data assimilation, thereby effectively reducing the uncertainty in ecosystem C sequestration estimations and providing a better understanding of regional or global C cycle dynamics and C-climate feedback.

Comprehensive Identification of Amadori Compound-Modified Phosphatidylethanolamines in Human Plasma.

Amadori compound modified lipids are the result of nonenzymatic glycation and play an important role in several physiological and pathological processes. However, glycation of phosphatidylethanolamine (PE), the most abundant amine-containing lipid in blood plasma, is underexplored and so far only a few glycated PEs have been reported. Herein, we report comprehensive profiling of Amadori-PE and -LysoPE species in human plasma. Using synthetic standards, we first optimized the enrichment procedure for extracting Amadori-PE/LysoPE from plasma. On the basis of the characteristic neutral losses of 303 Da in positive and 162 Da in negative ionization mode, we then applied neural loss scanning-liquid chromatography tandem mass spectrometry (LC-NLS-MS) to identify potentially glycated PE and LysoPE, which was followed by targeted product ion scanning (LC-PIS-MS) to confidently confirm the fatty acyl substitutions of the modified lipids. A total of 20 Amadori-LysoPE and 62 Amadori-PE species, including diacyl, plasmanyl, and plasmenyl, were identified. Among them, the concentrations of 12 Amarodi-LysoPE and 54 Amadori-PE were also quantified in native human plasma, using stable isotope labeled Amadori lipids as internal standards.

Comprehensive analysis of oxylipins in human plasma using reversed-phase liquid chromatography-triple quadrupole mass spectrometry with heatmap-assisted selection of transitions.

Oxylipins, a subclass of lipid mediators, are metabolites of various polyunsaturated fatty acids with crucial functions in regulation of systemic inflammation. Elucidation of their roles in pathological conditions requires accurate quantification of their levels in biological samples. We refined an ultra-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UPLC-MRM-MS)-based workflow for comprehensive and specific quantification of 131 endogenous oxylipins in human plasma, in which we optimized LC mobile phase additives, column, and gradient conditions. We employed heatmap-assisted strategy to identify unique transitions to improve the assay selectivity and optimized solid phase extraction procedures to achieve better analyte recovery. The method was validated according to FDA guidelines. Overall, 94.4% and 95.7% of analytes at tested concentrations were within acceptable accuracy (80-120%) and precision (CV < 15%), respectively. Good linearity for most analytes was obtained with R2 > 0.99. The method was also validated using a standard reference material-SRM 1950 frozen human plasma to demonstrate inter-lab compatibility. Graphical abstract ᅟ.