Total Membrane Protein Research Articles

Sevoflurane-induced overexpression of extrasynaptic α5-GABAAR via the RhoA/ROCK2 pathway impairs cognitive function in aged mice.

Perioperative neurocognitive disorder (PND) is a serious neurologic complication in aged patients and might be associated with sevoflurane exposure. However, the specific pathogenesis is still unclear. The distribution of α5-GABAAR, a γ-aminobutyric acid type A receptor (GABAAR) subtype, at extrasynaptic sites is influenced by the anchor protein radixin, whose phosphorylation is regulated via the RhoA/ROCK2 signaling pathway and plays a crucial role in cognition. However, whether sevoflurane affects the ability of radixin phosphorylation to alter extrasynaptic receptor expression is unknown. Aged mice were exposed to sevoflurane to induce cognitive impairment. Both total proteins and membrane proteins were extracted for analysis. Cognitive function was evaluated using the Morris water maze and fear conditioning test. Western blotting was used to determine the expression of ROCK2 and the phosphorylation of radixin. Furthermore, the colocalization of p-radixin and α5-GABAAR was observed. To inhibit ROCK2 activity, either an adeno-associated virus (AAV) or fasudil hydrochloride was administered. Aged mice treated with sevoflurane exhibited significant cognitive impairment accompanied by increased membrane expression of α5-GABAAR. Moreover, the colocalization of α5-GABAAR and p-radixin increased after treatment with sevoflurane, and this change was accompanied by an increase in ROCK2 expression and radixin phosphorylation. Notably, inhibiting the RhoA/ROCK2 pathway significantly decreased the distribution of extrasynaptic α5-GABAAR and improved cognitive function. Sevoflurane activates the RhoA/ROCK2 pathway and increases the phosphorylation of radixin. Excess α5-GABAAR is anchored to extrasynaptic sites and impairs cognitive ability in aged mice. Fasudil hydrochloride administration improves cognitive function.

Comparison of the Lipid Composition of Milk Fat Globules in Goat (Capra hircus) Milk during Different Lactations and Human Milk.

Goat milk is considered the optimal substitute for human milk and is characterized by variations in the lipid composition of its fat globules across lactation phases. Therefore, the objective of this study was to thoroughly analyze the differences between goat milk during different lactations and human milk, aiming to offer scientific guidance for the production of functional dairy products. Compared with transitional and mature milk, the findings indicated that the total membrane protein content in goat colostrum exhibited greater similarity to that found in human milk. Additionally, goat milk exhibited higher milk fat globule size, as well as a higher total lipid and protein content than human milk. A total of 1461 lipid molecules across 61 subclasses were identified in goat milk and human milk. The contents of glycerides and glycerophospholipids were higher in goat colostrum, whereas sphingolipids and fatty acids were more abundant in human milk. Meanwhile, the compositions of lipid subclasses were inconsistent. There were 584 differentially expressed lipids identified between human and goat milk, including 47 subclasses that were primarily involved in the metabolism of glycerophospholipids, sphingolipids, and triglycerides. In summary, for both the membrane protein and the lipid composition, there were differences between the milk of different goat lactations and human milk.

Alterations in HCN1 expression and distribution during epileptogenesis in rats

BackgroundThe hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear. ObjectiveThe aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis. MethodsWe utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques. ResultsIn the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression. ConclusionsAlterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.

Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

Alcohol-induced hormonal and metabolic alterations in plasma and erythrocytes—a gender-based study

Purpose This study aimed to understand the gender-specific alcohol-induced biochemical changes and TBARS association with the endocrine system. Methods Human male and female subjects ranging from 35 ± 10 years old with an 8-10-year drinking history were included in the study. Results The results demonstrated that testosterone levels were lower in male alcoholics and higher in female alcoholics, as well as higher estrogen and cortisol levels in both genders. In addition, we found lower T3, T4, and thyroid-stimulating hormone (TSH) levels in alcoholics of both sexes. Furthermore, plasma TBARS, protein carbonyls, nitrite, and nitrate levels increased significantly with concomitant decrease in reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in both male and female alcoholics. Furthermore, erythrocyte lysate nitrite and nitrate levels membrane total cholesterol, phospholipid and cholesterol/phospholipid (C/P) ratio with lower total membrane proteins in both genders of alcoholics. SDS-PAGE analysis of erythrocyte membrane proteins revealed increased density of band 3, protein 4.1, 4.2, 4.9 and glycophorins, whereas decreases in spectrin (α and β) were observed in both genders of alcoholics. Besides, alcoholics of both sexes had a lower ability to resist osmotic hemolysis. Plasma TBARS was negatively correlated with testosterone, TSH, T3 and T4 in male alcoholics, moreover, estradiol and cortisol were positively correlated in males and females respectively. Conclusion Female alcoholics may be more susceptible to osmotic hemolysis due to increased erythrocyte membrane lipid peroxidation with decreased antioxidant status, which results in an altered membrane C/P ratio and membrane protein composition.

Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics.

The plasma membrane proteome is a rich resource of functionally important and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here, we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our "Cys-Surf" platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of low-density lipoprotein (LDL) particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

A systemic approach to identify non-abundant immunogenic proteins in Lyme disease pathogens.

Borrelia burgdorferi, the pathogen of Lyme disease, differentially produces many outer surface proteins (Osp), some of which represent the most abundant membrane proteins, such as OspA, OspB, and OspC. In cultured bacteria, these proteins can account for a substantial fraction of the total cellular or membrane proteins, posing challenges to the identification and analysis of non-abundant proteins, which could serve as novel pathogen detection markers or as vaccine candidates. Herein, we introduced serial mutations to remove these abundant Osps and generated a B. burgdorferi mutant deficient in OspA, OspB, and OspC in an infectious 297-isolate background, designated as OspABC- mutant. Compared to parental isolate, the mutant did not reflect growth defects in the cultured medium but showed differential mRNA expression of representative tested genes, in addition to gross changes in cellular and membrane protein profiles. The analysis of differentially detectable protein contents of the OspABC- mutant, as compared to the wild type, by two-dimensional gel electrophoresis followed by liquid chromatography-mass spectrometry, identified several spirochete proteins that are dominated by proteins of unknown functions, as well as membrane transporters, chaperons, and metabolic enzymes. We produced recombinant forms of two of these represented proteins, BBA34 and BB0238, and showed that these proteins are detectable during spirochete infection in the tick-borne murine model of Lyme borreliosis and thus serve as potential antigenic markers of the infection.IMPORTANCEThe present manuscript employed a systemic approach to identify non-abundant proteins in cultured Borrelia burgdorferi that are otherwise masked or hidden due to the overwhelming presence of abundant Osps like OspA, OspB, and OspC. As these Osps are either absent or transiently expressed in mammals, we performed a proof-of-concept study in which their removal allowed the analysis of otherwise less abundant antigens in OspABC-deficient mutants and identified several immunogenic proteins, including BBA34 and BB0238. These antigens could serve as novel vaccine candidates and/or genetic markers of Lyme borreliosis, promoting new research in the clinical diagnosis and prevention of Lyme disease.

Abstract 13049: Electrophysiological Mechanism of Potassium Channel KCNQ1 p.V162M Mutation Induced Atrial Tachycardia and Epilepsy

Objective: Arrhythmia and epilepsy are the causes of syncope and sudden death. Both of them are associated with abnormal function of potassium ion channels. Delayed rectified potassium current (IKs) is generated by combining the α subunit of potassium ion channel encoded by KCNQ1 gene with the β subunit encoded by KCNE1 gene. It plays an important role in electrical excitation and K+ circulation of nerve cells. In this study, a patient with atrial arrhythmia and epilepsy with a history of syncope was identified by genetic testing as having a mutation in the KCNQ1 p.V162M gene. The purpose of this study was to investigate the effect of KCNQ1 p.V162M mutation on the structure and function of potassium ion channels. Methods: The mutant plasmid KCNQ1-p.V162M was constructed for cell transfection. The expression of protein and mRNA were analyzed by western blotting and real-time quantitative PCR. The IKs of the two groups were detected by whole-cell patch clamp technique. Results: Whole exon gene sequencing revealed that the patient carried the KCNQ1 p.V162M mutation. The results of molecular biology experiment showed that mRNA, KCNQ1 total protein and membrane protein expressions in mutant group were significantly lower than those in wild group. Whole-cell patch clamp results showed that the peak current density and tail current of IKs in the mutant group were significantly decreased compared with those in the wild group, indicating that the mutation of KCNQ1 p.V162M could lead to decreased function of potassium ion channels. Conclusions: KCNQ1 p.V162M mutation can lead to decreased expression of KCNQ1 protein and decreased function of potassium ion channel, which may be the potential mechanism of atrial tachycardia and epilepsy induced by this mutation.

A Novel in silico SELEX Method to Screen and Identify Aptamers against Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, has been responsible for global epidemics and many other problems over the centuries. It is one of the main public health issues in less-developed and developing countries and is considered one of the deadliest infectious agents. Therefore, precise and susceptible detection of V. cholerae</i> from environmental and biological samples is critical. Aptamers provide a rapid, sensitive, highly specific, and inexpensive alternative to traditional methods. The present study develops a new protocol inspired by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to implement an in silico</i> aptamer selection against V. cholerae</i>, which can also be employed in the case of other pathogenic microorganisms. First, we built an oligonucleotide pool and screened it based on the secondary structure. Following that, we modeled the tertiary structures of filtered sequences and performed RNAprotein dockings to assess binding affinities between RNA sequences and Outer Membrane Protein U (OmpU), an effective marker in distinguishing epidemic strains of V. cholerae</i>, which constitute up to 60% of the total outer membrane protein. Finally, we used molecular dynamics simulation to validate the results. Three sequences (ChOmpUapta) were proposed as final aptameric candidates. Analysis of the top-ranked docking results revealed that these candidate aptamers bound to all subunits of OmpU at the extracellular side with high affinity. Moreover, ChOmpUapta-3 and ChOmpUapta-2 were fully stable and formed strong bonds under dynamic conditions. We propose incorporating these candidate sequences into aptasensors for V. cholerae</i> detection.

Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acids are specific for the methanotrophic genera Methylacidiphilum and Methylacidmicrobium, while the last 53 amino acids are present only in lipoproteins of members from the phylum Verrucomicrobiota (Hedlund). Heterologous expression of WP_009060351 in Escherichia coli revealed a 25-kDa dimeric protein and a 60-kDa tetrameric protein. Immunoblotting showed that WP_009060351 was present in the total membrane protein and peptidoglycan fractions of M. fumariolicum SolV. The results suggest an involvement of lipoprotein WP_009060351 in the linkage between the outer membrane and the peptidoglycan.

The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells

To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.

Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant

SummaryEpilepsy and neurodevelopmental disorders can arise from pathogenic variants of KCNQ (Kv7) channels. A patient with developmental and epileptic encephalopathy exhibited an in-frame deletion of histidine 260 on Kv7.2. Coexpression of Kv7.2 mutant (mut) subunits with Kv7.3 invoked a decrease in current density, a depolarizing shift in voltage for activation, and a decrease in membrane conductance. Biotinylation revealed an increased level of surface Kv7.2mut compared to Kv7.3 with no change in total membrane protein expression. Super-resolution and FRET imaging confirmed heteromeric channel formation and a higher expression density of Kv7.2mut. Cannabidiol (1 μM) offset the effects of Kv7.2mut by inducing a hyperpolarizing shift in voltage for activation independent of CB1 or CB2 receptors. These data reveal that the ability for cannabidiol to reduce the effects of a pathogenic Kv7.2 variant supports its use as a potential therapeutic to reduce seizure activity.

Quantitative Expression of Drug, Phospholipid and Nucleoside Transporters in the Lung Tissues across Species

ObjectiveIt has been proven that transporters play a pivotal role in organ distribution of drugs, and subsequently in drug efficacy and toxicity. Active transport processes may affect the local pulmonary disposition of drugs administered directly to the lung by the inhaled route as well as the uptake of drugs to the lung from the systemic circulation. While drug transporter expressions and their roles in drug disposition and elimination in the major elimination organs including intestine, liver and kidney are well‐characterized, the expression of drug transporters and the comparison across species in the lung remain overall poorly understood. For example, azithromycin levels in pulmonary tissue remained to be high for up to 5 days after a single oral dose, in contrast to much lower plasma levels. However, the mechanisms associated with azithromycin lung loading and efficacy remain unknown. The aim of this study was to comprehensively determine the mRNA and protein expression levels of drug transporters in the lung in human and preclinical species using quantitative targeted proteomics approaches. The findings can be useful to further explore potential mechanisms in lung specific disposition.MethodsTotal cellular mRNA was isolated from human lower lung tissues using a Qiagen mRNA isolation kit. qRT‐PCR was performed, and β‐actin mRNA expression was used to normalize target gene expression. The mRNA levels were expressed as a fold change using the 2‐ΔΔCt method. The total membrane proteins of lung tissues from human, monkey, dog and rat were extracted by Native Membrane Protein Extraction Kit, and subsequently digested by trypsin followed by the treatment of DTT and iodoacetamide (IAA). The levels of transporter proteins were quantified using a Sciex 7500 triple‐quadruple mass spectrometer coupled to a Shimadzu LC (SLC‐30A) system with optimized ionization conditions. The Na K‐ATPase level in the lung was measured as the membrane protein marker.Results and conclusionIn the current investigation, thirteen ABC efflux transporters and twenty‐three solute carriers (SLC) were characterized. Among those, mRNAs of eleven ABC transporters and fifteen SLC transporters were detected by the RT‐PCR method. In addition, eight ABC transporter and eleven SLC transporter proteins were quantified using the targeted proteomics LC‐MS/MS method. The mRNA expression pattern agrees with the protein expression; however, no quantitative correlation can be drawn between mRNA and protein expression. While OATP2B1 was only detected in human lung tissues, OATP2A1 and 3A1 are expressed in the lungs of human, monkey dog and rat. The expressions of three equilibrative nucleoside transporters (ENT1/2/3) in human lung tissues were higher than those in monkey. The expression of ABCA1, ABCA3 and ABCG1, the ABC transporters that are the regulators of cellular cholesterol and phospholipid homeostasis, were detected in all tested species, and found to be high in rat. Collectively, the comparison of transporter expression profiles can contribute to a better understanding of drug distribution and transport in the lung and aid pulmonary drug discovery and development.

Expression characteristic of 4Ig B7-H3 and 2Ig B7-H3 in acute myeloid leukemia

ABSTRACT 4IgB7-H3 (4Ig) and 2IgB7-H3 (2Ig) expression characteristics in acute myeloid leukemia (AML) remain unknown. This study investigated mRNA and membrane protein expression of two B7-H3 isoforms in AML cell lines and de novo patients by using RT-PCR and flow cytometry, and analyzed the B7-H3 promoter methylation state by utilizing RQ-MSP. 4Ig was the dominant isoform. 2Ig mRNA expression rate and abundance were elevated in AML in comparison with controls (P = 0.000 and 0.000), while no significant difference in 4Ig (P = 0.802, P = 0.398). Membrane protein levels of B7-H3 isoforms in AML was higher than controls, detected by total B7-H3 expression rate (P = 0.002), total B7-H3 mean fluorescence intensity (MFI) ratio of blast cells and lymphocytes (MFI ratio) (P = 0.000), and 4Ig B7-H3 MFI ratio (P = 0.005). Compared with 2Ig low group, 2Ig high patients had older age, lower NPM1 mutation, higher FLT3-ITD mutation, and declining complete remission (CR) rates (P = 0.026, 0.012, 0.047, and 0.028). In B7-H3 high group, there was a trend toward older age, M4 and M5, worse karyotype, and lower CR rates, although with no marked difference (P > 0.05). The overall survival (OS) of 2Ig high and B7-H3 high groups were shorter than that of 2Ig low and B7-H3 low groups in the whole and non-M3 AML cohorts (P = 0.006 and 0.046; P = 0.003 and 0.032). Besides, an unmethylated B7-H3 promoter was identified. In conclusion, 2Ig mRNA and total B7-H3 membrane protein tended to have potential diagnostic value for AML. Specifically, high 2Ig mRNA and total B7-H3 membrane protein expression indicate worse OS. 4Ig and 2Ig expression are methylation-independent.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple ‘glycogenes’ that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses.

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple 'glycogenes' that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.

Differential Membrane Protein Profile in Bovine X- and Y-Sperm.

The aim of this study was to understand the molecular mechanisms behind the biological differences of X- and Y-sperm and to screen the sex-specific candidate antigen proteins for sexed semen production. To this end, we investigated differential expression of total membrane proteins of the two sperm types by using high-purity X- and Y-sperm from 20 Holstein bulls and applying the label-free proteomic technique; 1521 proteins were identified. In the X-sperm group, 8 and 23 proteins were significantly up- and down-regulated, respectively. In the X- and the Y-sperm group, 151 and 88 proteins were specifically expressed, respectively. These were overexpressed in the dynamic changes of the actin cytoskeleton, and cell senescence/apoptosis induced by the immune response, and could result in differences in the state, size, and immune sensitivity of the X-/Y-sperm membranes. The prediction of transmembrane structure, subcellular localization, and Western blotting validation results showed that the CLRN3 and SCAMP1 proteins were cell surface specific antigens of X- and Y-sperm, respectively. Our findings help explain the molecular mechanism behind the biological differences of X-/Y-sperm and lay the foundation for application of immunological methods to produce sex-sorted semen and control livestock sex. Data are available via ProteomeXchange with identifier PXD019435.

A Systematic Review of Campylobacter jejuni Vaccine Candidates for Chickens.

Campylobacter jejuni infection linked to the consumption of contaminated poultry products is one of the leading causes of human enteric illness worldwide. Vaccination of chickens is one of the potential strategies that could be used to control C. jejuni colonization. To date, various C. jejuni vaccines using potential antigens have been evaluated, but a challenge in identifying the most effective formulation is the wide variability in vaccine efficacies reported. A systematic review was undertaken to compare C. jejuni vaccine studies. Based upon specific selection criteria eligible papers were identified and included in the analysis. Vaccine efficacy reported from different C. jejuni antigens, vaccine types, and vaccination regimens reported in these papers were reviewed. Our analysis shows that total outer membrane proteins and cysteine ABC transporter substrate-binding protein were among the most efficacious vaccine antigen candidates reported. This review also highlights the importance of the need for increased consistency in the way C. jejuni vaccine studies in poultry are designed and reported in order to be able to undertake a robust comparison of C. jejuni vaccine candidates.

Proteomics analysis of erythrocyte membrane in rats with high altitude polycythemia before and after intervention with salidroside

To investigate the effect of salidroside on the proteomics of erythrocyte membrane in high altitude erythrocytosis(HAPC) rats, in order to explore the mechanism of salidroside in improving HAPC based on the proteomics analysis. First, HPAC rat models were established, and 16 rats were randomly divided into HAPC model group and salidroside(100 mg·kg~(-1)) treatment group(8 rats per group). Saline was administered to the HAPC model group, while salidroside treatment group was given 100 mg·kg~(-1) salidroside once a day. After continuous oral administration with salidroside for 40 days(once a day), blood was collected from the femoral artery to obtain total red blood cell membrane proteins. Two-dimensional electrophoresis was used to separate total proteins. The two-dimensional electrophoresis of erythrocyte membrane proteins was analyzed before and after salidroside intervention, and the proteins with significant differences were identified by mass spectrometry. Finally, biological functions were analyzed using bioinformatics. A two-dimensional electrophoresis method was used to establish a protein expression profile with a high resolution and reproducibility of erythrocyte membranes in HAPC rats. Salidroside treatment significantly changed 18 protein spots in the 2-DE map of erythrocyte membranes, of which 13 proteins were up-regulated and 5 proteins were down-regulated. Eight differential proteins were successfully identified by mass spectrometry. Moreover, bioinformatics analysis found that these differential proteins were involved in such biological processes as oxidative stress, redox, and peroxisome pathway, which are mainly associated with peroxisome and MAPK signaling pathways. Therefore, salidroside could significantly change the expressions of erythrocyte membrane proteins in HAPC rats. Eight differential proteins were identified by a proteomic-based approach. The differential proteins were involved in such biological processes as oxidative stress, redox, peroxisome pathway.

Omega 3 and Azadirachta Indica J. Seed Oil Inhibit Hemoglobin Polymerization and Modulate Erythrocyte Membrane Atpases in Sickle Cell Disease (SCD)

Back ground: Functional foods/Nutraceuticals efficacy depends on both its composition and the physicochemical properties of the components that used in their formulation. Vegetable oil can play an important role. Several studies are shown that omega 3 (0.2%) and Azadirachta indica J. seed oil (0.4%) have antisickling, antioxidant and anti-haemolytic properties. Objective: The purpose of this present study was conducted to determine effects of omega 3 and Azadirachta indica J. seed oil on hemoglobin polymerization and erythrocyte membrane ATPases in Sickle Cell Disease (SCD). Method: The ability for the omega 3 and Azadirachta indica J. seed oil to inhibit sickle cell hemoglobin polymerization and improve the Fe2+/Fe3+ ratio of HbS blood were assed using spectroscopic method. The activities of three ATPases (Na+/K+; Ca2+; and Mg2+-ATPases ) were investigated by the kinetic method following the release of inorganic phosphate (Pi) by ATP taking into account the determination of the total membrane proteins of erythrocytes by Bradford’s method. Results: The lowest potential was obtained with Azadirachta indica J. seed oil (AO: 0.4% v/v) corresponding significantly (p &lt;0.05) to the highest potential of inhibition of polymerization (83.11 ± 8%) compared to that obtained with omega 3 (ω3 0.2% v/v) estimated at 68.15±7%. The greatest Fe2+Fe3+ ratio was obtained (6.65±1.68) with AO (0.4% v/v) corresponding significantly (p &lt;0.05) to the greatest rate of increase of oxyhemoglobin (79.05±3%) compared to that obtained with ω3 (0.2% v/v) estimated at 70.58±5%. As for the activities of Na+/K+, Ca2+ and Mg2+-ATPases expressed in μmole Pi/mg protein/hour x 10-3, there was an increase in the activities of Na+/K+ and Ca2+-ATPases (203.82±41 and 190.48±32) with AO (0.4% v/v), but without any significant difference (p&gt;0.05) compared to those obtained with ω3 (0.2% v/v) (188.66±9 and 235.61±52). However, for Mg2+-ATPase, there was rather a significant decrease (p &lt;0.05) in these activities ranging from 181.9±32 for ω3 (0.2% v/v) to 124.00±21 for AO (0.4% v/v). Conclusion: The Azadirachta indica J. seed oil at a concentration of 0.4% v/v offered the best potential inhibition of hemoglobin S polymerization, oxyhemoglobin increase rate and the best ATPastic activities. Keywords: Omega 3 - Azadirachta indica J. seed oil - sickle cell disease - polymerization - Fe2+/Fe3+ ratio - ATPase activities.