Membrane Glycolipids Research Articles

Ganglioside expression delineates human mesenchymal stem/stromal cell populations derived from different tissue sources.

Prior studies have indicated that human embryonic stem cells can be distinguished from those of other mammals based on variable expression of a class of membrane glycolipids known as glycosphingolipids (GSLs), raising the question as to whether GSL display could be utilized to phenotypically define subsets of human adult stem cell populations. Adult stem cells known as "mesenchymal stem/stromal cells" (MSCs) have shown immense promise in therapeutic applications for a variety of clinical indications. Most commonly, these cells are harnessed and then culture-expanded from bone-marrow (BM-MSCs) or from adipose tissue (A-MSCs) sources. Though operational differences exist between human BM-MSCs and A-MSCs, no surface markers have been characterized to date that distinguish these as distinct subsets of culture-expanded human adult stem cells. Accordingly, we isolated GSLs from primary cultures of marrow- and adipose-derived human MSCs and an unbiased screen was performed by mass spectrometry (via matrix-assisted laser desorption/ionization (MALDI)-quadrupole ion trap (QIT)-time-of-flight (TOF), hence, via "MALDI-QIT-TOF") to analyze all component glycans. Flow cytometry was then undertaken to assess the relative levels of expression of MS-defined glycan determinants, followed by RT-qPCR to measure transcripts of genes encoding key enzymes involved in glycolipid biosynthesis. Notably, our data indicate that neither BM- nor A-MSCs display any significant level of either lacto-series or neolacto-series GSLs, but distinct differences exist in GSL species among A-MSCs and BM-MSCs: while both cell types express GSLs of the ganglio- and the globo-series, the ganglio-series GSLs GD3 and GD2 and the globo-series GSL SSEA-4 (also known as sialylGb5) are dominantly expressed only among human BM-MSCs. These structural features are shaped by divergent patterns of glycosyltransferase gene expression, with striking differences between BM- and A-MSCs in the expression of transcripts encoding GD3 synthase, GM2/GD2 synthase, and Gb5 synthase. Importantly, expression of GD3, GD2, and SSEA-4 is markedly diminished on differentiation of BM-MSCs, and co-cultures of A-MSCs and BM-MSCs show that the expression of GD3, GD2, and SSEA-4 is a cell-intrinsic feature of BM-MSCs. These data stratify the glycosignature(s) of human MSCs derived from different tissue sources, provide direct evidence that expression of these structures is cell stage-/lineage-specific, unveil the mechanistic basis of the differential expression of these glycan determinants, and draw attention to how knowledge of the MSC glycosignature can impact cytotherapeutic strategies.

Molecular insights reveal how the glycolipids in cell membrane mitigates nanomaterial's invasion

Nanomaterial-cellular membrane interaction is crucial for the cytotoxicity of such materials in theoretical investigations. However, previous research often used cellular membrane models with one or few lipid types, which deviates significantly from realistic membrane compositions. Here, employing molecular dynamics (MD) simulations, we investigate the impact of a typical nanomaterial, boron nitride (BN), on a cellular membrane model based on the realistic small intestinal epithelial cell (SIEC) membrane. This membrane contains a complex composition, including abundant glycolipids. Our MD simulations reveal that BN nanosheet can partially insert into the SIEC membrane, maintaining a stable binding conformation without causing obvious structural changes. Dynamic analyses suggest that van der Waals (vdW) interactions drive the binding process between BN and the SIEC membrane. Further simulation of the interaction between BN nanosheet and deglycosylated SIEC membrane confirms that BN nanosheet cause significant structural damage to deglycosylated SIEC membranes, completely inserting into the membrane, extracting lipids, and burying some lipid hydrophilic heads within the membrane interior. Quantitative analyses of mean squared displacements (MSD) of membranes, membrane thicknesses, area per lipid, and order parameters indicate that BN nanosheet causes more substantial damage to deglycosylated SIEC membrane than to intact SIEC membrane. This comparison suggests the molecular mechanism involved in mitigating BN invasion by SIEC membrane that the polysaccharide heads of glycolipids in the SIEC membrane form a significant steric hindrance on membrane surface, not only hindering the insertion of BN, but also resisting the lipid extraction by BN. Free energy calculations further support this conclusion. Overall, our MD simulations not only shed new light into the reduced impact of BN nanosheet on the realistic SIEC membrane but also highlight the importance of glycolipids in protecting cell membranes from nanomaterial invasion, contributing to a deeper understanding of nanomaterial-realistic cell membrane interactions.

Investigation Of Lectin Binding On Rabbit Spleen Cell Membrane Infected With Proteus vulgaris

This study investigated the effects of Proteus vulgaris OX19 infection on the carbohydrate composition of spleen cell membranes in New Zealand adult male rabbits. Rabbits were injected with increasing doses of P. vulgaris OX19 (0.5 ml, 1 ml, 2 ml, 4 ml, 5 ml) at five-day intervals over the course of one month. Following the treatment period, spleen tissues were collected from both the control and infected groups. Tissue sections were stained using the Avidin-Biotin-Peroxidase method with five different lectins: Concavalia ensiformis (Con A), Arachis hypogaea agglutinin (PNA), Bauhinia purpurea agglutinin (BPA), Griffonia simplicifolia I (GS-I), and Ulex europaeus agglutinin I (UEA-I). The stained sections were examined by light microscopy to evaluate lectin binding. Among the lectins used, Con A showed strong binding (+++) to spleen cell membranes of the Proteus-infected group, while moderate binding (++) was observed in the control group. UEA-I exhibited weak binding in the control group but demonstrated moderate binding in the Proteus-infected group. In contrast, PNA, BPA, and GS-I exhibited strong binding (+++) to spleen cell membranes in the control group and moderate binding (++) in the infected group. These findings suggest that P. vulgaris OX19 infection induces alterations in the carbohydrate moieties of glycoproteins and glycolipids in the spleen cell membranes of infected rabbits. It is hypothesized that P. vulgaris modifies the terminal carbohydrates of glycoproteins and/or glycolipids in spleen cell membranes, contributing to the observed changes in lectin binding patterns.

Enzyme-Linked Lipid Nanocarriers for Coping Pseudomonal Pulmonary Infection. Would Nanocarriers Complement Biofilm Disruption or Pave Its Road?

Cystic fibrosis (CF) is associated with pulmonary Pseudomonas aeruginosa infections persistent to antibiotics. To eradicate pseudomonal biofilms, solid lipid nanoparticles (SLNs) loaded with quorum-sensing-inhibitor (QSI, disrupting bacterial crosstalk), coated with chitosan (CS, improving internalization) and immobilized with alginate lyase (AL, destroying alginate biofilms) were developed. SLNs (140-205 nm) showed prolonged release of QSI with no sign of acute toxicity to A549 and Calu-3 cells. The CS coating improved uptake, whereas immobilized-AL ensured >1.5-fold higher uptake and doubled SLN diffusion across the artificial biofilm sputum model. Respirable microparticles comprising SLNs in carbohydrate matrix elicited aerodynamic diameters MMAD (3.54, 2.48 µm) and fine-particle-fraction FPF (65, 48%) for anionic and cationic SLNs, respectively. The antimicrobial and/or antibiofilm activity of SLNs was explored in Pseudomonas aeruginosa reference mucoid/nonmucoid strains as well as clinical isolates. The full growth inhibition of planktonic bacteria was dependent on SLN type, concentration, growth medium, and strain. OD measurements and live/dead staining proved that anionic SLNs efficiently ceased biofilm formation and eradicated established biofilms, whereas cationic SLNs unexpectedly promoted biofilm progression. AL immobilization increased biofilm vulnerability; instead, CS coating increased biofilm formation confirmed by 3D-time lapse confocal imaging. Incubation of SLNs with mature biofilms of P. aeruginosa isolates increased biofilm density by an average of 1.5-fold. CLSM further confirmed the binding and uptake of the labeled SLNs in P. aeruginosa biofilms. Considerable uptake of CS-coated SLNs in non-mucoid strains could be observed presumably due to interaction of chitosan with LPS glycolipids in the outer cell membrane of P. aeruginosa. The biofilm-destructive potential of QSI/SLNs/AL inhalation is promising for site-specific biofilm-targeted interventional CF therapy. Nevertheless, the intrinsic/extrinsic fundamentals of nanocarrier-biofilm interactions require further investigation.

Machine Learning Approach to Identify Dysregulation in the Plasma Proteome of CADASIL

AbstractBackgroundThe plasma proteome provides information to identify dysregulated molecular pathways underlying disease. Cerebrovascular diseases often manifest a significantly different plasma proteomic signature. Cerebral‐Autosomal‐Dominant‐Arteriopathy‐Subcortical‐Infarcts‐and‐Leukoencephalopathy (CADASIL) is one such vascular brain disease. The CADASIL plasma proteome has yet to be investigated.MethodTo investigate the plasma proteome in the context of CADASIL, we used large‐scale proteomics, measuring over 7,000 proteins via aptamer‐based technology (SomaLogic) from 53 study participants (nCADASIL = 25; nControl = 28). We employed machine learning (ML) methods as an unbiased approach to uncover disease‐associated change in proteomic networks. This approach is based on the premise that protein sets that best classify disease state could be important biological drivers of disease. We developed a novel ML method: coupling recursive feature extraction with logistic regression (LR) and XGBoost evaluators, as well maximum‐relevance‐minimum‐redundancy with Random‐Forest and F‐Statistic evaluators. The results of all four models were selectively aggregated in order to delineate the CADASIL plasma proteome signature while minimizing overfitting to the low sample size data. We developed a 45 protein model of the CADASIL proteomic signature from these results. To evaluate the classification ability, we tested the Repeated‐Stratified‐10‐fold‐Cross‐Validation accuracy of a LR classifier. Next, we investigated whether the CADASIL proteomic signature was relevant for other neurodegenerative diseases with vascular components; the classifier was applied to an Alzheimer’s Disease (AD) dataset. Finally, the functional signature of the 45 proteins was investigated through gene ontology (GO).ResultThe LR classifier in CADASIL is 99.8% accurate. In AD, the accuracy is 71.2%. This implies the CADASIL signature shares commonalities as well as pertinent differences with other neurodegenerative diseases. The GO of the 45 proteins resulted in GO terms corresponding to extracellular matrix and collagen, cellular membrane lipoproteins and glycolipids, and proteasome biological pathways.ConclusionThe proposed method allows for an initial unbiased discovery of important proteomic changes associated with disease state. The identified proteins would provide starting points for mechanistic studies. In addition, a panel of proteins could be used for screening at population level for risk stratification and more in‐depth phenotyping. Next steps include investigating the biological relevance of findings in mechanistic models for therapeutic developments.

Machine Learning Approach to Identify Dysregulation in the Plasma Proteome of CADASIL

AbstractBackgroundThe plasma proteome provides information to identify dysregulated molecular pathways underlying disease. Cerebrovascular diseases often manifest a significantly different plasma proteomic signature. Cerebral‐Autosomal‐Dominant‐Arteriopathy‐Subcortical‐Infarcts‐and‐Leukoencephalopathy (CADASIL) is one such vascular brain disease. The CADASIL plasma proteome has yet to be investigated.MethodTo investigate the plasma proteome in the context of CADASIL, we used large‐scale proteomics, measuring over 7,000 proteins via aptamer‐based technology (SomaLogic) from 53 study participants (nCADASIL = 25; nControl = 28).We employed machine learning (ML) methods as an unbiased approach to uncover disease‐associated change in proteomic networks. This approach is based on the premise that protein sets that best classify disease state could be important biological drivers of disease. We developed a novel ML method: coupling recursive feature extraction with logistic regression (LR) and XGBoost evaluators, as well maximum‐relevance‐minimum‐redundancy with Random‐Forest and F‐Statistic evaluators.The results of all four models were selectively aggregated in order to delineate the CADASIL plasma proteome signature while minimizing overfitting to the low sample size data. We developed a 45 protein model of the CADASIL proteomic signature from these results. To evaluate the classification ability, we tested the Repeated‐Stratified‐10‐fold‐Cross‐Validation accuracy of a LR classifier. Next, we investigated whether the CADASIL proteomic signature was relevant for other neurodegenerative diseases with vascular components; the classifier was applied to an Alzheimer’s Disease (AD) dataset. Finally, the functional signature of the 45 proteins was investigated through gene ontology (GO).ResultsThe LR classifier in CADASIL is 99.8% accurate. In AD, the accuracy is 71.2%. This implies the CADASIL signature shares commonalities as well as pertinent differences with other neurodegenerative diseases.The GO of the 45 proteins resulted in GO terms corresponding to extracellular matrix and collagen, cellular membrane lipoproteins and glycolipids, and proteasome biological pathways.ConclusionThe proposed method allows for an initial unbiased discovery of important proteomic changes associated with disease state. The identified proteins would provide starting points for mechanistic studies. In addition, a panel of proteins could be used for screening at population level for risk stratification and more in‐depth phenotyping. Next steps include investigating the biological relevance of findings in mechanistic models for therapeutic developments.

Trehalose as a Stabilizer of the Lipid Composition of Membranes and the Composition of the Cytosol of Frozen/Thawed Rooster Spermatozoa

Low-temperature semen storage technologies are already being used in poultry conservation programs, but the quality of reproductive material stored in cryobanks varies greatly and cannot always be successfully used for practical purposes. Therefore, it is necessary to improve the compositions of cryoprotective media to improve their quality. This study aimed to investigate the composition of membrane lipids and carbohydrates in the cytosol of rooster spermatozoa, to explain the dose-dependent effect of a combination of trehalose and fructose in cryoprotective media on the preservation of their morphological and kinetic parameters during freezing/thawing, and to determine the most effective diluent composition. Ejaculates were collected from Rhode Island Red roosters (n = 10). The effectiveness of three diluents containing trehalose was evaluated: LCM-control (0 mM), Treh20 (9.5 mM), and Treh30 (13.4 mM). Chromatographic analysis of membrane lipids, carbohydrates, and polyols of the spermatozoa cytosol was performed. A decrease in the content of glycolipids in the plasma membranes of spermatozoa from 2.0% in native spermatozoa to 1.1–1.4% (frozen/thawed) and phospholipids from 71.2% (native) to 70.5% (frozen/thawed) reduced the progressive sperm motility from 65.7% in native spermatozoa to 12.6–27.6% (frozen/thawed). The same dynamics were observed for the viability parameter of 90.4% (native) and 27.0–41.2% (frozen/thawed). The Treh20 diluent, using a combination of fructose (36 mM) and trehalose (9.5 mM) saccharides, maximally preserved the lipid profile of plasma membranes and the composition of the cytosol of frozen/thawed rooster spermatozoa, which positively affected the indicators of general and progressive mobility and viability.

Antibody binding reports spatial heterogeneities in cell membrane organization

The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane. However, we currently do not have the methods to quantify the spatial heterogeneities of macromolecular crowding on live cell surfaces. In this work, we combine experiment and simulation to report crowding heterogeneities on reconstituted membranes and live cell membranes with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to engineered antigen sensors, we discover sharp gradients in crowding within a few nanometers of the crowded membrane surface. Our measurements on human cancer cells support the hypothesis that raft-like membrane domains exclude bulky membrane proteins and glycoproteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.

PREVALENCE OF BLOOD GROUPA2 AMONG GROUPAAND AB DONORS

The ABO blood group antigens are clinically signicant and their discovery by Landsteiner in 1900 paved way for the performance of safe blood transfusions. These antigens are characterized by their distinct carbohydrate structures attached to the glycoprotein or glycolipid membranes of red blood cells (RBCs). Their expression can also be detected on various human cell and tissue surfaces including sensory neurons, vascular endothelium and platelets. [1] Subgroups of the ABO antigens occur and are characterized by reduced quantities of the antigens on the RBCs and saliva of secretors. [2] Landsteiner & Levine subdivided blood group A into their principal subgroups, A1 and A2. The presence of Asubgroups is due to genetic variations in the coding region which leads to diminished amounts of A substance and higher amounts of H substance on red blood cells. There are weaker subgroups than A2 which do not frequently occur but when present; they show a characteristic decreased Aantigen sites on their RBC and a corresponding increased H antigen activity. [3] The difference between subgroups A1 and A2 is seen in their reactivity with the Dolichos biflorus lectin. The Dolichos lectin specically agglutinates A1 cells, leaving the A2 cells unagglutinated. The ABO antibodies occur naturally in the sera of individuals who do not have the corresponding antigens and can cause haemolysis in-vivo. They are mostly immunoglobulin M (IgM). These antibodies through complement activation can cause severe, life-threatening transfusion reactions due to incompatible ABO blood transfusions. Anti-A1 antibody appears as an atypical cold agglutinin in the sera of few people with group A2 or A2B who do not have the corresponding antigen.[4] One to eight percent of persons with blood group A2 and about 22-35% of persons with group A2B have serum anti-A1. These antibodies may cause discrepancies in ABO blood grouping which can further result in HTR.[5] Rare cases of haemolytic transfusion reaction due to anti-A1 have been reported.[4] It is estimated that about 80% of individuals with antigen A are A1 while the remaining 20% are A2. [5] Among Indians, the frequency of subgroup A2 among group A individuals is 6.58% whiles that of A2B among group AB is 8.99%.[6] Knowledge of the frequency and distribution of blood subgroup A2 will help to enhance blood compatibility testing, population genetic studies, investigation of transfusion reactions, studying disease associations and resolving some medico-legal issues.[7]

TAY-SACHS DISEASE IN A CHILD: A RARE CASE REPORT ,GGH KURNOOL

The neurodegenerative condition known as Tay-Sachs disease is inherited in an autosomal recessive pattern and is caused by an abnormal accumulation of the cell membrane glycolipid and GM2 ganglioside within the lysosomes of affected cells. A lack of the isoenzyme -hexosaminidase A, which is synthesised in the endoplasmic reticulum, is the root cause of this condition. In patients who have Tay-Sachs disease, the first few months of life are characterised by normal motor development, followed by gradual weakening and loss of motor abilities beginning around 2 to 6 months of life. Due to the irreversible nature of neurodegeneration, death typically occurs between the ages of 4 and 5 years. The hexosaminidase enzyme assay and DNA analysis of the HEXA gene are two methods that can be used to diagnose TaySachs disease. On the other hand, there is no specific treatment that has been established. We discuss here a case of TaySachs disease found in an 9-month-old male child who presented with loss of milestones and seizure symptoms. Upon examination of the fundus, it was found that this patient had cherry red spot in the macula. In the enzymatic assay, the hexosaminidase A activity was zero percent, and DNA analysis revealed a mutation in which glutamine was replaced by a stop codon at position 390

All-in-One Nanowire Assay System for Capture and Analysis of Extracellular Vesicles from an ex Vivo Brain Tumor Model.

Extracellular vesicles (EVs) have promising potential as biomarkers for early cancer diagnosis. The EVs have been widely studied as biological cargo containing essential biological information not only from inside vesicles such as nucleic acids and proteins but also from outside vesicles such as membrane proteins and glycolipids. Although various methods have been developed to isolate EVs with high yields such as captures based on density, size, and immunoaffinity, different measurement systems are needed to analyze EVs after isolation, and a platform that enables all-in-one analysis of EVs from capture to detection in multiple samples is desired. Since a nanowire-based approach has shown an effective capability for capturing EVs via surface charge interaction compared to other conventional methods, here, we upgraded the conventional well plate assay to an all-in-one nanowire-integrated well plate assay system (i.e., a nanowire assay system) that enables charge-based EV capture and EV analysis of membrane proteins. We applied the nanowire assay system to analyze EVs from brain tumor organoids in which tumor environments, including vascular formations, were reconstructed, and we found that the membrane protein expression ratio of CD31/CD63 was 1.42-fold higher in the tumor organoid-derived EVs with a p-value less than 0.05. Furthermore, this ratio for urine samples from glioblastoma patients was 2.25-fold higher than that from noncancer subjects with a p-value less than 0.05 as well. Our results demonstrated that the conventional well plate method integrated with the nanowire-based EV capture approach allows users not only to capture EVs effectively but also to analyze them in one assay system. We anticipate that the all-in-one nanowire assay system will be a powerful tool for elucidating EV-mediated tumor-microenvironment crosstalk.

Distribution of Glycolipids in the Plasma Membrane Monitored by Specific Probes in Combination with Sodium Dodecyl Sulfate-Digested Freeze-Fracture Replica Labeling (SDS-FRL).

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside.

Role of endoplasmic reticulum stress in hepatic glucose and lipid metabolism and therapeutic strategies for metabolic liver disease.

The endoplasmic reticulum is the most abundant membrane organelle in eukaryotic cells. It is an important site of membrane and secretory protein folding and glycolipid synthesis and transport, and it is responsible for regulating intracellular calcium homeostasis. When the load of protein synthesis and folding exceeds the processing capacity of the endoplasmic reticulum, the excessive accumulation of misfolded proteins leads to endoplasmic reticulum stress and activates the cellular unfolded protein response. Hepatocytes contain an abundance of smooth and rough endoplasmic reticulum, which can sense changes in various nutritional metabolic and external stimuli and mediate the regulation of glucose and lipid metabolism by activating the unfolded protein response signaling pathways. Endoplasmic reticulum stress plays a very important role in metabolic regulation and in the occurrence and development of liver diseases. This review summarizes the recent research progress on the unfolded protein response and hepatic glucose and lipid metabolism and discusses the mechanism linking endoplasmic reticulum stress with glucose and lipid metabolism disorders and related metabolic liver diseases to improve the understanding of the molecular pathological basis of major chronic diseases such as obesity, type II diabetes and nonalcoholic fatty liver disease.

Membrane Fusion Mediated by Non-covalent Binding of Re-engineered Cholera Toxin Assemblies to Glycolipids.

Membrane fusion is essential for the transport of macromolecules and viruses across membranes. While glycan-binding proteins (lectins) often initiate cellular adhesion, subsequent fusion events require additional protein machinery. No mechanism for membrane fusion arising from simply a protein binding to membrane glycolipids has been described thus far. Herein, we report that a biotinylated protein derived from cholera toxin becomes a fusogenic lectin upon cross-linking with streptavidin. This novel reengineered protein brings about hemifusion and fusion of vesicles as demonstrated by mixing of fluorescently labeled lipids between vesicles as well as content mixing of liposomes filled with fluorescently labeled dextran. Exclusion of the complex at vesicle-vesicle interfaces could also be observed, indicating the formation of hemifusion diaphragms. Discovery of this fusogenic lectin complex demonstrates that new emergent properties can arise from simple changes in protein architecture and provides insights into new mechanisms of lipid-driven fusion.

Disialoganglioside GD2-Targeted Near-Infrared Photoimmunotherapy (NIR-PIT) in Tumors of Neuroectodermal Origin.

Disialoganglioside (GD2) is a subtype of glycolipids that is highly expressed in tumors of neuroectodermal origins, such as neuroblastoma and osteosarcoma. Its limited expression in normal tissues makes GD2 a potential target for precision therapy. Several anti-GD2 monoclonal antibodies are currently in clinical use and have had moderate success. Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that arms antibodies with IRDye700DX (IR700) and then exposes this antibody–dye conjugate (ADC) to NIR light at a wavelength of 690 nm. NIR light irradiation induces a profound photochemical response in IR700, resulting in protein aggregates that lead to cell membrane damage and death. In this study, we examined the feasibility of GD2-targeted NIR-PIT. Although GD2, like other glycolipids, is only located in the outer leaflet of the cell membrane, the aggregates formation exerted sufficient physical force to disrupt the cell membrane and kill target cells in vitro. In in vivo studies, tumor growth was significantly inhibited after GD2-targeted NIR-PIT, resulting in prolonged survival. Following GD2-targeted NIR-PIT, activation of host immunity was observed. In conclusion, GD2-targeted NIR-PIT was similarly effective to the conventional protein-targeted NIR-PIT. This study demonstrates that membrane glycolipid can be a new target of NIR-PIT.

Inositol acylation of phosphatidylinositol mannosides: a rapid mass response to membrane fluidization in mycobacteria

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.

Position-Specific Secondary Acylation Determines Detection of Lipid A by Murine TLR4 and Caspase-11.

Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against bacterial infection and a contributor to the hyperinflammatory response, potentially leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor Toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, the precise features of lipid A that determine the differential activation of each receptor remain poorly defined, as direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we found that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4 or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4 or Casp11. Our findings indicate that TLR4 and Casp11 sense nonoverlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.

Gangliosidome of a Human Hippocampus in Temporal Lobe Epilepsy Resolved by High-Resolution Tandem Mass Spectrometry.

In this study, we developed a high-resolution tandem mass spectrometry (HR MS) approach to assess presumed changes in gangliosidome of a human hippocampus affected by temporal lobe epilepsy (TLE) in comparison with a normal hippocampus. Gangliosides, membrane glycolipids, are particularly diverse and abundant in the human brain, and participate in ion transport and modulation of neuronal excitability. Changes in structural ganglioside pattern potentially linked to TLE molecular pathogenesis have not been explored in detail. Aiming to characterize TLE-specific gangliosidome, we analyzed the native gangliosides purified from a human hippocampal tissue sample affected by TLE and a control hippocampus using HR MS. Marked differences of ganglioside expression were shown in TLE vs. control, particularly with respect to the sialylation degree of components, discovered as a characteristic feature of TLE. Another major finding is the occurrence of tetrasialofucogangliosides in TLE and species modified by either O-acetylation or CH3COO−. Structural analysis by higher-energy collisional dissociation (HCD) MS/MS gave rise to fragmentation patterns implying that the GQ1b (d18:1/18:0) isomer is specifically associated with TLE. Further investigation in a larger sample is needed in order to confirm the discovery of ganglioside structures specifically expressed in human TLE and to provide information on the probable role of gangliosides in the molecular events underlying seizures.

The Effects of Bacterial Lipopolysaccharide (LPS) on Turkey Poults: Assessment of Biochemical Parameters and Histopathological Changes.

A lipopolysaccharide (LPS) is a large molecule and an outer membrane glycolipid found in Gram-negative bacteria, including Escherichia coli (E. coli). These molecules (LPS) target acute inflammatory responses and significant physiological changes. Importantly, E. coli is considered one of the most important bacterial causes of avian colibacillosis that affect domestic turkey industry. However, little information is available about the potential influence of LPS on the biochemical parameters and histopathological changes in turkey poults. Therefore, this study aimed to evaluate the influence of bacterial lipopolysaccharide (LPS) molecules on serum biomarkers and histopathological changes in turkey poults. The birds were randomly divided into five groups, as follows: group I did not receive any inoculation; group II was inoculated with sterile saline; and groups III, IV, and V were inoculated intraperitoneally with LPS at 0.01, 0.1, and 1 mg/kg of body weight (BW), respectively. The biochemical parameters and the histopathology of different organs were examined in all birds one day post-inoculation. Our results revealed hypolipidemia, hypoglycemia, a significant decrease in uric acid, and a significant increase in serum activities of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and creatine kinase (CK), as well as cardiac troponin T concentrations in treated groups. Moreover, there was a significant increase in α1-, β-, and γ-globulin concentrations and a decrease in albumin and α2-globulin concentrations in group V. However, a significant increase in α2- and γ-globulin levels and a decrease in albumin levels were detected in groups III and IV. In addition, significant decreases in the albumin/globulin ratio were recorded in all LPS-treated groups. Hepatocellular and cardiac muscle necrosis, slight renal changes, and massive pulmonary inflammatory reactions were recorded. This study provides valuable information about serum biomarkers, protein fractions, and histopathological changes in turkey poults treated with LPS for further investigations of pathophysiological mechanisms in avian medicine along with biomedical research.

Vps13-like proteins provide phosphatidylethanolamine for GPI anchor synthesis in the ER.

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.