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Newborn screening (NBS) for sickle cell disease (SCD) requires a robust, high-throughput method to detect hemoglobin S (HbS). Screening for SCD is performed by qualitative methods, such as isoelectric focusing (IEF), and both qualitative and quantitative methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and tandem mass spectrometry (MS/MS). All these methods detect HbS, as well as low-level or absent HbA, and also other variants of hemoglobin. HPLC is considered as a reference method for NBS, because of its high sensitivity and specificity in detecting HbS. NeoSickle®, a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, combined with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation, has increased the throughput of SCD testing. The purpose of this study was to compare the performances of NeoSickle® and HPLC. A prospective study was conducted including 9,571 samples from the NBS program to compare MALDI-MS using NeoSickle® with an HPLC method. Correlation between the two methods was studied. For the MALDI-MS method, sensitivity, specificity, NPV, and PPV were calculated. We found over 99.4 % correlation between the HPLC and MALDI-MS results. NeoSickle® showed 100 % of sensitivity and specificity in detecting SCD syndrome, leading to positive and negative predictive values of 100 %. NeoSickle® is adapted to NBS for SCD, and can be used in first-line high-throughput screening to detect HbS, and beta-thalassemia major warning. When HbS is detected, second-line use of another specific method as HPLC is necessary. [ABSTRACT FROM AUTHOR]

Abstract: Human defensins, which are small cationic peptides produced by neutrophils and epithelial cells, form two genetically distinct alpha and beta subfamilies. They are involved in innate immunity through killing microbial pathogens or neutralizing bacterial toxins and in adaptive immunity by serving as chemoattractants and activators of immune cells. α-defensins are mainly packaged in neutrophil granules (HNP1, HNP2, HNP3) or secreted by intestinal Paneth cells (HD5, HD6), while β-defensins are expressed in mucosa and epithelial cells. Using surface enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry (MS), α-defensins were found to be expressed in a variety of human tumours, either in tumour cells or at their surface. HNP1–3 peptides are also secreted and their accumulation in biological fluids was proposed as a tumour biomarker. Conversely, β-defensin-1 (HBD-1) is down-regulated in some tumour types in which it could behave as a tumour suppressor protein. Alpha-defensins promote tumour cell growth or, at higher concentration, provoke cell death. These peptides also inhibit angiogenesis, which, in addition to immunomodulation, indicates a complex role in tumour development. This review summarizes current knowledge of defensins to discuss their role in tumour growth, tumour monitoring and cancer treatment. [Copyright &y& Elsevier]

Summary: Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two‐day Pan‐European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus‐based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes. [ABSTRACT FROM AUTHOR]

Identifying objective markers of diet would be beneficial to research fields such as nutritional epidemiology. As a preliminary study on the validity of using saliva for this purpose, and in order to explore the relationship between saliva and diet, we focused on clearly contrasted groups of children: children with eating difficulties (ED) receiving at least 50% of their energy intake through artificial nutrition vs healthy controls (C). Saliva of ED and C children was analyzed by various methods (targeted biochemical analyses, 2-D electrophoresis coupled to MS, 1 H NMR) and their diet was characterized using food frequency questionnaires, considering 148 food items grouped into 13 categories. Complete datasets were obtained for 16 ED and 16 C subjects (median age 4.7 y and 5.0 y, respectively) and the statistical link between salivary and dietary characteristics was studied by Multiple Factor Analysis (MFA). Overall, ED children showed as expected lower consumption frequency scores and higher food selectivity. The two groups of children differed in “diet/saliva” associations. Some distinctive salivary variables were common to both groups of children. For example, carbonic anhydrase 6 and the consumption frequency of biscuits & sweets and drinks were positively associated with the MFA axis 1 in C children, but oppositely associated in ED children. Specifically for ED children, abundant salivary proteins (cystatins, amylase, amylase fragments) and some metabolites (amino acids, galactose, lactate) correlated with axis 1, together with the consumption frequency of sauces & seasonings, bread & cereal products, ready-to-eat meals, fish, biscuits & sweets, drinks and potatoes. Specifically for C children, several proteins (serum albumin, haptoglobin, Igκ, apolipoprotein A-1, α-1 antitrypsin) correlated with axis 1, together with the consumption frequency of biscuits & sweets, milk & dairy products, drinks, fruit, meat and vegetables. This study demonstrates that the qualitative aspect of diet is linked to saliva composition, and that the associations between dietary consumption and salivary composition differ between groups of subjects with contrasted diets. [ABSTRACT FROM AUTHOR]

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane. [ABSTRACT FROM AUTHOR]

Background and aims Diabetic patients are at high risk of stroke and coronary artery disease. Recent data suggest that arachidonic acid metabolism is altered in diabetic conditions and that these alterations contribute to accelerated atherosclerosis. Little is known about how these alterations affect the metabolism and the proportions of different lipid species within the atherosclerotic plaque. The aim of our study was to perform a targeted lipidomic analysis of human atherosclerotic lesions, with a specific focus on PUFA-containing lipid species, to reveal differences in the fatty-acid composition of plaque in diabetic patients compared with non-diabetic controls. Methods Carotid atheroma plaque samples were obtained from 31 diabetic and 48 non-diabetic patients undergoing carotid endarterectomy. Targeted lipidomic analysis was then performed to determine the fatty acid composition of major glycerophospholipids and cholesteryl ester species by liquid chromatography-tandem mass spectrometry. Results Atheroma plaques from diabetic patients were significantly enriched with 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) (2.3 ± 0.8% Vs . 1.8 ± 0.6% p = 0.0002). Multivariable logistic regression showed that an increased 2-AA-LPC level was independently associated with diabetes. Finally, a positive relationship was found between 2-AA-LPC and HbA1c levels. Interestingly, endothelial lipase and calcium independent PLA2 gamma which could account for the production of 2-AA-LPC were detected in carotid plaques by immunohistochemistry. Conclusions 2 -AA-LPC stands at the crossroads of major metabolic pathways that lead to the synthesis of bioactive molecules such as AA-derived eicosanoids, 2-AA-lysophosphatidic acid and 2-AA-glycerol. 2-AA-LPC therefore appears to be a promising molecule to investigate in the context of diabetes. [ABSTRACT FROM AUTHOR]

Prolonged enteral or parenteral nutrition in neonatal periods sometimes results in eating difficulties persisting for years, with reduced food intake through the oral route and thereby reduced stimulation of the oral cavity. Aiming at describing the consequences on oral physiology, saliva of 21 children with eating difficulties (ED) was compared to that of 23 healthy controls, using various omics and targeted methods. Overall, despite heterogeneity within the groups (age, medication etc.), the three spectral methods (MALDI–TOF, SELDI–TOF, 1 H NMR) allowed discriminating ED and controls, confirming that oral stimulation by food intake plays a role in shaping the composition of saliva. Saliva of ED patients exhibited a lower antioxidant status and lower levels of the salivary protease inhibitors cystatins. Other discriminant features (IgA1, dimethylamine) may relate to modified oral and/or intestinal microbial ecology. Finally, salivary profiles of ED patients were partly comparable to those of subjects with exacerbated gustatory sensitivities, in particular with reduced abundance of cystatin SN and higher abundance of zinc-alpha-2-glycoprotein. Whether this translates taste hypersensitivity and contributes to the eating difficulties deserves further attention. [ABSTRACT FROM AUTHOR]

High plasma concentrations of nonesterified fatty acids (NEFAs), transported bound to serum albumin, are associated with type 2 diabetes (T2D). The effects of albumin on platelet function were investigated in vitro. Modifications of albumin, such as those due to glycoxidation, were found in patients with T2D, and the consequences of these modifications on biological mechanisms related to NEFA handling were investigated. Mass spectrometry profiles of albumin from patients with T2D differed from those from healthy control subjects. Diabetic albumin showed impaired NEFA binding capacity, and both structural and functional alterations could be reproduced in vitro by incubating native albumin with glucose and methylglyoxal. Platelets incubated with albumin isolated from patients with T2D aggregated approximately twice as much as platelets incubated with albumin isolated from healthy control subjects. Accordingly, platelets incubated with modified albumin produced significantly higher amounts of arachidonate metabolites than did platelets incubated with control albumin. We concluded that higher amounts of free arachidonate are made available for the generation of active metabolites in platelets when the NEFA binding capacity of albumin is blunted by glycoxidation. This newly described mechanism, in addition to hypoalbuminemia, may contribute to platelet hyperactivity and increased thrombosis, known to occur in patients with T2D. [ABSTRACT FROM AUTHOR]

High plasma concentrations of nonesterified fatty acids (NEFAs), transported bound to serum albumin, are associated with type 2 diabetes (T2D). The effects of albumin on platelet function were investigated in vitro. Modifications of albumin, such as those due to glycoxidation, were found in patients with T2D, and the consequences of these modifications on biological mechanisms related to NEFA handling were investigated. Mass spectrometry profiles of albumin from patients with T2D differed from those from healthy control subjects. Diabetic albumin showed impaired NEFA binding capacity, and both structural and functional alterations could be reproduced in vitro by incubating native albumin with glucose and methylglyoxal. Platelets incubated with albumin isolated from patients with T2D aggregated approximately twice as much as platelets incubated with albumin isolated from healthy control subjects. Accordingly, platelets incubated with modified albumin produced significantly higher amounts of arachidonate metabolites than did platelets incubated with control albumin. We concluded that higher amounts of free arachidonate are made available for the generation of active metabolites in platelets when the NEFA binding capacity of albumin is blunted by glycoxidation. This newly described mechanism, in addition to hypoalbuminemia, may contribute to platelet hyperactivity and increased thrombosis, known to occur in patients with T2D. [ABSTRACT FROM AUTHOR]

Saliva has different functions in the mouth and is involved, for example, in taste perception. Saliva composition can also be modified rapidly by taste stimulation. It remains unclear, however, whether the perceived intensity of a tastant may modulate this response. Based on increasing evidence that fat can be perceived by the taste system and that fat taste perception may be associated with fat intake, the aim of this work was to study if stimulation by a fatty acid (oleic acid) modifies saliva composition differently in subjects highly (sensitive+) or weakly (sensitive−) sensitive to that taste. For that purpose, saliva of two groups of subjects was collected after stimulation by either a control emulsion or an emulsion containing 5.61 mM oleic acid. Saliva was analyzed by 2D electrophoresis and 1H NMR spectroscopy. The results show that sensitive+ and sensitive− subjects differ in their salivary response in terms of proteome and metabolome composition. Oppositely to sensitive− subjects, sensitive+ subjects responded to oleic acid by increased abundance of polymeric immunoglobulin receptor, rab GDP dissociation inhibitor beta, and organic acids, and decreased abundance of metabolites characteristic of mucins. The results highlight that modification of saliva composition by taste stimulation may be modulated by taste perception. [ABSTRACT FROM AUTHOR]

Background: The identification of new diagnostic or prognostic biomarkers is one of the main aims of clinical cancer research. Technologies like mass spectrometry are commonly being used in proteomic research. Mass spectrometry signals show the proteomic profiles of the individuals under study at a given time. These profiles correspond to the recording of a large number of proteins, much larger than the number of individuals. These variables come in addition to or to complete classical clinical variables. The objective of this study is to evaluate and compare the predictive ability of new and existing models combining mass spectrometry data and classical clinical variables. This study was conducted in the context of binary prediction. Results: To achieve this goal, simulated data as well as a real dataset dedicated to the selection of proteomic markers of steatosis were used to evaluate the methods. The proposed methods meet the challenge of high-dimensional data and the selection of predictive markers by using penalization methods (Ridge, Lasso) and dimension reduction techniques (PLS), as well as a combination of both strategies through sparse PLS in the context of a binary class prediction. The methods were compared in terms of mean classification rate and their ability to select the true predictive values. These comparisons were done on clinical-only models, mass-spectrometry-only models and combined models. Conclusions: It was shown that models which combine both types of data can be more efficient than models that use only clinical or mass spectrometry data when the sample size of the dataset is large enough. [ABSTRACT FROM AUTHOR]

Saliva is a biological fluid playing numerous roles in the oral cavity and increasingly considered as a source of markers. The role of saliva in sensory perception has been known for years but it is only recently that its potential role in oral fatty acids (FA) perception has been suggested. The aim of the present work was to study the relationships between taste sensitivity to oleic acid and the salivary proteome (2D electrophoresis) and metabolome (H NMR). This was achieved by comparing saliva from two groups of subjects, highly (sensitive+) and weakly sensitive (sensitive−) to the taste of oleic acid. Partial least squares-discriminant analyses (PLS-DA) were used to model the relationship between sensitivity to C18:1, and the proteome and metabolome data. The two groups could be discriminated by ten spots. In particular, cystatin SN, cystatin D, zinc-alpha-2-glycoprotein and carbonic anhydrase 6 were overexpressed in the sensitive+ group. The latter result was confirmed by ELISA. The overexpression of these proteins, which have been associated to taste perception, supports the argument that C18:1 is perceived by the taste system. The two groups could also be discriminated on the basis of eight metabolites, with FA, FA/proline, lysine and FA/pyruvate overexpressed in the sensitive+ group and acetate, leucine/isoleucine and butyrate overexpressed in the sensitive− group. The overexpression of these metabolites suggests a higher bacterial load in this group which could be implicated in perception of FAs. [ABSTRACT FROM AUTHOR]

Abstract: MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin. [Copyright &y& Elsevier]

Widely used in microelectronics and optoelectronics; Gallium Arsenide (GaAs) is a III-V crystal with several interesting properties for microsystem and biosensor applications. Among these; its piezoelectric properties and the ability to directly biofunctionalize the bare surface, offer an opportunity to combine a highly sensitive transducer with a specific bio-interface; which are the two essential parts of a biosensor. To optimize the biorecognition part; it is necessary to control protein coverage and the binding affinity of the protein layer on the GaAs surface. In this paper; we investigate the potential of a specific chemical interface composed of thiolate molecules with different chain lengths; possessing hydroxyl (MUDO; for 11-mercapto-1-undecanol (HS(CH2)11OH)) or carboxyl (MHDA; for mercaptohexadecanoic acid (HS(CH2)15CO2H)) end groups; to reconstitute a dense and homogeneous albumin (Rat Serum Albumin; RSA) protein layer on the GaAs (100) surface. The protein monolayer formation and the covalent binding existing between RSA proteins and carboxyl end groups were characterized by atomic force microscopy (AFM) analysis. Characterization in terms of topography; protein layer thickness and stability lead us to propose the 10% MHDA/MUDO interface as the optimal chemical layer to efficiently graft proteins. This analysis was coupled with in situ MALDI-TOF mass spectrometry measurements; which proved the presence of a dense and uniform grafted protein layer on the 10% MHDA/MUDO interface. We show in this study that a critical number of carboxylic docking sites (10%) is required to obtain homogeneous and dense protein coverage on GaAs. Such a protein bio-interface is of fundamental importance to ensure a highly specific and sensitive biosensor. [ABSTRACT FROM AUTHOR]

Abstract: Immuno-SPR-MS is the combination of immuno-sensors in biochip format with mass spectrometry. This association of instrumentation allows the detection and the quantification of proteins of interest by SPR and their molecular characterization by additional MS analysis. However, two major bottlenecks must be overcome for a wide diffusion of the SPR-MS analytical platform: (i) To warrant all the potentialities of MS, an enzymatic digestion step must be developed taking into account the spot formats on the biochip and (ii) the biological relevancy of such an analytical solution requires that biosensing must be performed in complex media. In this study, we developed a procedure for the detection and the characterization at ~1 µg/mL of the LAG3 protein spiked in human plasma. The analytical performances of this new method was established, particularly its specificity (S/N > 9) and sensitivity (100% of LAG3 identification with high significant mascot score >68 at the femtomole level). The collective and automated on-chip MALDI-MS imaging and analysis based on peptidic fragments opens numerous applications in the fields of proteomics and diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6months of age. Data on teeth eruption and diet at the age of 6months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF–TOF and LC ESI MS–MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) than teeth eruption (overexpression of one peak). Differential peaks corresponded to fragments of acidic and basic PRPs, statherin and histatin. Comparison with existing knowledge on adult saliva peptidome revealed that proteolytic processing of salivary proteins is qualitatively quite comparable in infants and in adults. However, age and diet are modulators of salivary peptidome in human infants. [Copyright &y& Elsevier]

Abstract: GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABAA or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome. [Copyright &y& Elsevier]

The interindividual variation in the sensitivity to bitterness is attributed in part to genetic polymorphism at the taste receptor level, but other factors, such as saliva composition, might be involved. In order to investigate this, 2 groups of subjects (hyposensitive, hypersensitive) were selected from 29 healthy male volunteers based on their detection thresholds for caffeine, and their salivary proteome composition was compared. Abundance of 26 of the 255 spots detected on saliva electrophoretic patterns was significantly different between hypo- and hypersensitive subjects. Saliva of hypersensitive subjects contained higher levels of amylase fragments, immunoglobulins, and serum albumin and/or serum albumin fragments. It also contained lower levels of cystatin SN, an inhibitor of protease. The results suggest that proteolysis occurring within the oral cavity is an important perireceptor factor associated to the sensitivity to the bitter taste of caffeine. [ABSTRACT FROM AUTHOR]

Abstract: In order to document inter-individual variability in salivary protein patterns, unstimulated whole saliva was obtained from 12 subjects at 10 am and 3 pm of the same day. Saliva proteins were separated using two-dimensional gel electrophoresis, and semi-quantified using image analysis. One-way ANOVA was used to test the effects “time of sampling” and “subject”. Data were further explored by multivariate analyses (PCA, hierarchical clustering). Spots of interest were identified by mass spectrometry (MALDI-TOF MS/MS and nanoLC ESI-IT MS/MS). A dataset of 509 spots matched in all gels was obtained. There was no diurnal statistical effect on salivary patterns while inter-individual variability was high with 47 spots differentially expressed between subjects (p <1%). Clustering of these spots revealed that subjects could be discriminated first based on several proteins participating to the non-specific immune response (cystatins, lipocalin 1, parotid-secretory protein and prolactin-induced protein). Independently, subjects were also differentiated by their level of proteins originating from serum and involved in the immune system (complement C3, transferrin, IgG2), as well as the relative abundance of enzymes involved in carbohydrates metabolism (amylase and glycolytic enzymes). Inter-individual variability should be accounted for when searching for salivary biomarkers or when studying in-mouth biochemical mechanisms. [Copyright &y& Elsevier]

The NeoSickle SP ® sp solution based on the detection by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of -, - and -globin is used in routine clinical practice to classify newborns according to their profile SS, AA, AS, S/ SP + sp -thal. Dried blood spot (DBS) samples stored for more than 6 months demonstrated high stability of the globin chain and the low impact of storage conditions with MALDI-MS analysis. B KEYWORDS: b Matrix-assisted laser desorption/ionization (MALDI); mass spectrometry (MS); thalassemia; sickle cell disease; screening; hemoglobin variant. [Extracted from the article]