The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis.

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using 2-DE of extracted proteins from Human Brain Frontal Cortex with SB constituents (7M Urea, 2M Thiourea and 100mM DTT) were made, using different detergent compositions in the buffer. SB preparations in combination with CHAPS and ASB-14 as well as with ASB-16 (reported for the first time in 2-DE experiments) have been tested. Our results confirm that the most efficient solubilizing solution for 2-DE analysis of cytosolic and membrane Human Brain Proteins is SB combined with 4% CHAPS and 2% ASB-14.     

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose R_e ^sat—the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C₁₂E₈ and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.     

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10⁻²⁰⁴) and 10 loci for sphingolipids (smallest P-value = 3.10×10⁻⁵⁷). After a correction for multiple comparisons (P-value<2.2×10⁻⁹), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.     

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.     

Intrapulmonary Vascular Dilatation Evaluated by 99mTc-MAA Scintigraphy and Its Association with Portal Hypertension in Schistosomiasis

Background

Portal hypertension is responsible for various complications in patients with schistosomiasis, among them intrapulmonary vascular dilations (IPVD). In cirrhotic patients the presence of IPVD is a sign of poor prognosis, but in patients with hepatosplenic schistosomiasis (HSS) there are no studies assessing the significance of this change. The aim of this study was to evaluate the occurrence of IPVD through ^99mTc-MAA scintigraphy in patients with HSS and its relationship with clinical, laboratory, endoscopic and ultrasound parameters.

Methods

Cross-sectional study evaluating 51 patients with HSS. Patients were diagnosed with IPVD when the brain uptake of ^99mTc-MAA was higher than 6%. Subsequently, they were divided according to presence (G1) or absence (G2) of IPVD and variables were compared between groups.

Results

Overall, 51 patients with mean age of 56±12 years were assessed. IPVD was observed in 31 patients (60%). There was no statistically significant differences between groups when clinical, laboratory and endoscopic parameters were compared. Regarding ultrasound parameters, the splenic vein diameter was smaller in G1 (0.9±0.3 cm) compared to G2 (1.2±0.4 cm), p = 0.029.

Conclusion

In patients with HSS, the occurrence of IPVD by ^99mTc-MAA scintigraphy was high and was associated with lower splenic vein diameter, which can be a mechanism of vascular protection against portal hypertension. However, more studies are needed to determine the clinical significance of the early diagnosis and natural evolution of IPVD in this population.     

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*O01 was the most common allele found, followed by ABO*O22 and by ABO*A103. We identified 22 new ABO*variants in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.