Multicentric study of the effect of pre-analytical variables in the quality of plasma samples stored in biobanks using different complementary proteomic methods

12 páginas, 7 figuras.-- Jesús Mateos ... et al.Analytical proteomics has experienced exponential progress in the last decade and can be expected to lead research studies on diagnostic and therapeutic biomarkers in the near future. Because the development of this type of analysis requires the use of a large number of human samples with a minimum of quality requirements, our objective was to identify appropriate indicators for quality control of plasma samples stored in biobanks for research in proteomics. To accomplish this, plasma samples from 100 healthy donors were obtained and processed according to the pre-analytical variables of: a) time delay for the first centrifugation of the original blood sample (4 or 24 h) and b) number of freeze/thaw cycles (1, 2 or 3) of the processed plasma samples. The analyses of samples were performed by different and complementary methods such as SPE MALDI-TOF, DIGE, shotgun (iTRAQ, nLC MALDI TOF/TOF) and targeted nLC MS/MS proteomic techniques (SRM). In general, because the distribution of proteins in all samples was found to be very similar, the results shown that delayed processing of blood samples and the number of freeze/thaw cycles has little or no effect on the integrity of proteins in the plasma samplesThis work was supported by the grant numbers PI12/02670 and PT13/0001/0034, integrated in the National Plan for Scientific Research, Development and Technological Innovation 2008–2011 and 2013–2016 and funded by the ISCIII-General Subdirection of Assesment and Promotion of the Research-European Regional Development Fund (FEDER) “A way of making Europe”Peer reviewe

AFM study of strawberry pectin nanostructure and its relevance on fruit texture

Atomic force microscopy (AFM) has been used to characterize the nanostructure of cell wall pectins during strawberry fruit growth and ripening, as well as in transgenic fruits with pectinase genes downregulated. This technique allows the imaging of individual polymers at high magnification with minimal sample preparation. AFM studies during fruit development show that pectin size, ramification and aggregation is reduced in ripe fruits. Additionally, transgenic lines with different pectinase genes downregulated (polygalacturonase, pectate lyase and B-galactosidase) also show a more complex pectin nanostructure, including longer chains, higher branching degree and larger presence of aggregates. In all those cases the higher pectin complexity at nanoscale correlates with a reduced softening in strawberry fruits at macroscale level. Globally, our results support the key role of pectins in fruit structure and highlights the use of AFM as a powerful tool to gain insights about the bases of textural fruit quality not only in strawberry, but also in other commercial crops.AGL2017-86531-C2-1-R, Ministerio de Economía, Industria y Competitividad of Spain and FEDER EU funds. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Serum levels of anti-alphaGalactosyl antibodies predict survival and peritoneal dialysis-related enteric peritonitis rates in patients undergoing renal replacement therapy

[Abstract] Background: Anti-Galα1-3Gal antibodies (anti-αGal) represent a significant fraction of natural antibodies and were implicated in several disease states, yet their origin and physiological significance remain largely undisclosed. Methods: Under a prospective observational design, we estimated anti-αGal immunoglobulin G (IgG)/IgM and antipig hemolytic antibody (APA) levels in 133 patients starting dialysis therapy and again after a 1-year follow-up. We used baseline data to show correlations with demographic, nutritional, inflammatory, and anemia markers and analyzed their correlation with outcomes by using univariate and multivariate strategies of survival analysis. Results: Serum anti-αGal and APA levels showed wide baseline variability, but remained relatively stable in time. Both were measurable in dialysate of peritoneal dialysis (PD) patients, showing close correlation to serum levels. We observed no association between levels of anti-αGal/APA and nutritional markers, but showed direct correlations of anti-αGal IgM (P = 0.005) and APA levels (P = 0.001) with tumor necrosis factor α (TNF-α) levels. High APA levels also were associated with severe anemia (P = 0.006). High baseline anti-αGal IgM (P = 0.03) and APA levels (P = 0.045) predicted later risk for enteric peritonitis in PD patients. Finally, univariate and multivariate analyses showed a consistent association between high baseline anti-αGal IgM (P = 0.014) and APA (P = 0.021) levels and global risk for mortality during follow-up. Conclusion: Anti-αGal IgM and APA levels at the start of dialysis therapy are significant predictors of later risk for mortality and, in PD patients, enteric peritonitis. Both correlate directly with TNF-α levels and, in the case of APA, severity of anemia in these patients

Intraneural IFG-1 in cryopreserved nerve isografts increase neural regeneration and functional recovery in the rat sciatic nerve

[Abstract] Background: Insulin-like growth factor 1 (IGF-1) was found to stimulate Schwann cell mitosis. Exogenous IGF-1 may improve nerve regeneration after cryopreservation. Objective: To evaulate the effect of intraneural administration of IGF-1 in cryopreserved nerve isografts. Methods: Eighteen millimeter grafts were used for bridging an 18-mm defect in the rat sciatic nerve. A total of 57 rats were randomly divided into three groups: (1) autograft (Group 1); (2) cryopreserved isograft (Group 2); (3) cryopreserved isograft with intraneural IGF-1 administration (Group 3). 12 weeks after surgery, functional recovery (Sciatic functional index [SFI], Swing speed [SS], nerve conduction velocity [NCV], amplitude of compound motor action potentials [CMAP], and gastrocnemius muscle index [GMI]) and nerve regeneration (myelin sheath area, total fiber counts, fiber density, and fiber width) were all evaluated. Results: The intraneural injection of IGF-1 significantly improved SFI and SS at weeks 10 and 12. There were no statistical differences between Groups 1 and 3 in any of the SFI or SS evaluations. CMAP and NCV in Group 1 were significantly higher than in Groups 2 and 3, and Group 3 had significantly higher CMAP and NCV compared to Group 2. No significant differences were found in fiber width. The number of nerve fibers, percentage of myelinated fibers, fiber density, and GMI was significantly higher in Group 1 compared to Group 2, but no significant differences were found between Groups 1 and 3. Conclusion: The results show that intraneural injection of IGF-1 in an 18 mm cryopreserved isograft improve axonal regeneration and functional recovery

Mitochondrial DNA haplogroup H as a risk factor for idiopathic dilated cardiomyopathy in Spanish population

[Abstract] Idiopathic dilated cardiomyopathy (IDC) is a structural heart disease with strong genetic background. The different single nucleotide polymorphisms (SNPs) that constitute mitochondrial haplogroups could play an important role in IDC progression. The aim of this study was to test frequencies of mitochondrial haplogroups in healthy controls (n = 422) and IDC patients (n = 304) of a Caucasian Spanish population. To achieve this, ten major European haplogroups were identified. Frequencies and Odds Ratios for the association between IDC and haplogroups were calculated in both groups. We found that compared to healthy controls, the prevalence of haplogroup H was significantly higher in IDC patients (40.0% vs 50.7%, p-value = 0.040).Instituto de Salud Carlos III; PS09/0084

Mitochondrial haplogroups H and J: risk and protective factors for ischemic cardiomyopathy

[Abstract] Background. Since mitochondria are the principal source of reactive oxygen species (ROS), these organelles may play an important role in ischemic cardiomyopathy (IC) development. The mitochondrial genome may influence this disease. The aim of the present study was to test the relationship between IC development and the impact of single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) defining the mitochondrial haplogroups in a population study. Methodology and principal findings. Ten major European haplogroups were identified by using the single base extension technique and by polymerase chain reaction-restriction fragment length polymorphism. Frequencies and Odds Ratios for the association between IC patients (n = 358) and healthy controls (n = 423) were calculated. No convincing associations between classical risk factors for ischemic cardiomyopathy development and haplogroups were found. However, compared to healthy controls, the prevalence of haplogroup H was significantly higher in IC patients (40.0% vs 50.0%, p-value  = 0.039) while the frequency of haplogroup J was significantly lower (11.1% vs 5.6%, p-value  = 0.048). The analysis of the SNPs characterizing the European mtDNA haplogroups showed that the m.7028C allele (40.0% vs 50.0%, p-value  = 0.005) and m.14766C allele (43.0% vs 54.2%, p-value  = 0.002) were overrepresented in IC patients, meanwhile the m.10398G allele (19.8% vs 13.1%, p-value  = 0.015) and m.4216C allele (22.2% vs 16.5%, p-value  = 0.044) were found as protective factors against IC. Conclusions and significance. Our results showed that the haplogroups H and J were found as a risk and protective factors for ischemic cardiomyopathy development, respectively.Instituto de Salud Carlos III; PS09/0084

The addition of albumin improves Schwann cells viability in nerve cryopreservation

[Abstract] The purpose of the current study was to establish a valid protocol for nerve cryopreservation, and to evaluate if the addition of albumin supposed any advantage in the procedure. We compared a traditional cryopreservation method that uses dimethyl sulfoxide (DMSO) as cryoprotectant, to an alternative method that uses DMSO and albumin. Six Wistar Lewis rats were used to obtain twelve 20 mm fragments of sciatic nerve. In the first group, six fragments were cryopreserved in 199 media with 10% DMSO, with a temperature decreasing rate of 1 °C per minute. In the second group, six fragments were cryopreserved adding 4% human albumin. The unfreezing process consisted of sequential washings with saline in the first group, and saline and 20% albumin in the second group at 37 °C until the crioprotectant was removed. Structural evaluation was performed through histological analysis and electronic microscopy. The viability was assessed with the calcein-AM (CAM) and 4′,6-diamino-2-fenilindol (DAPI) staining. Histological results showed a correct preservation of peripheral nerve architecture and no significant differences were found between the two groups. However, Schwann cells viability showed in the CAM-DAPI staining was significantly superior in the albumin group. The viability of Schwann cells was significantly increased when albumin was added to the nerve cryopreservation protocol. However, no significant structural differences were found between groups. Further studies need to be performed to assess the cryopreserved nerve functionality using this new method

Galectina-3 circulante tras el trasplante cardiaco: dinámica a largo plazo y valor pronóstico

[Abstract] Introduction and objectives: Circulating galectin-3 (Gal-3) is elevated and significantly correlates with all-cause and cardiovascular mortality in patients with heart failure. However, the relationship between serum Gal-3 and heart transplant (HT) outcomes is unclear. The aim of this study was to describe the longitudinal trend and prognostic value of Gal-3 levels after HT. Methods: Banked serum samples were available from 122 HT recipients, collected before transplant and at 1, 3, 6, and 12 months posttransplant. Gal-3 levels in these serum samples were measured by enzyme immune assay. Multivariable Cox regression was performed to determine the prognostic value of 12-month posttransplant Gal-3 serum levels. The primary endpoint was the composite variable all-cause death or graft failure over long-term posttransplant follow-up. Results: Circulating Gal-3 concentration steadily decreased during the first year after HT (median values: pretransplant, 19.1 ng/mL; 1-year posttransplant, 14.6 ng/mL; P<.001). Circulating Gal-3 levels 1-year posttransplant were associated with an increased risk of all-cause death or graft failure (adjusted HR per 1 ng/mL, 1.04; 95%CI, 1.01-1.08; P=.008). The predictive accuracy of this biomarker was moderate: area under the ROC curve, 0.72 (95%CI, 0.60-0.82; P<.001). Conclusions: Circulating Gal-3 steadily decreased during the first year after HT. However, 1-year posttransplant Gal-3 serum levels that remained elevated were associated with increased long-term risk of death and graft failure.[Resumen] Introducción y objetivos. Los valores plasmáticos de galectina-3 (Gal-3) están elevados y se correlacionan con la mortalidad total y cardiovascular en pacientes con insuficiencia cardiaca, pero su correlación con el pronóstico tras el trasplante cardiaco (TxC) es desconocida. El objetivo fue describir la tendencia evolutiva y el valor pronóstico de este biomarcador tras el TxC. Métodos. Mediante enzimoinmunoensayo, se midieron las concentraciones plasmáticas de Gal-3 en muestras de suero de 122 receptores de TxC, antes y 1, 3, 6 y 12 meses después de este. Mediante regresión de Cox se analizó el valor pronóstico del valor plasmático de Gal-3 a los 12 meses del TxC. El objetivo primario del estudio fue la variable combinada muerte o disfunción del injerto. Resultados. Las concentraciones de Gal-3 disminuyeron progresivamente durante el primer año tras el TxC (medianas: pretrasplante, 19,1 ng/ml; 1 año postrasplante, 14,6 ng/ml; p < 0,001). Los valores de Gal-3 1 año tras el TxC se asociaron con mayor riesgo de muerte o disfunción del injerto (HR por 1 ng/ml: 1.04; IC95%: 1,01-1,08; p = 0,008). La capacidad predictiva del biomarcardor fue moderada: área bajo la curva ROC, 0,72 (IC95%: 0,60-0,82; p < 0,001). Conclusiones. Las concentraciones plasmáticas de Gal-3 disminuyeron progresivamente durante el primer año tras el TxC. Un valor plasmático elevado de Gal-3 1 año tras el TxC se correlacionó con un pronóstico adverso.Instituto de Salud Carlos III; PI12/0267

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling

[Abstract] Background: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. Methods: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. Results: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile. Conclusions: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.Dr Barallobre-Barreiro is a British Heart Foundation Intermediate Fellow (FS/19/33/34328). Drs Mayr and Shah are British Heart Foundation Chair Holders (CH/16/3/32406 and CH/1999001/11735, respectively) and received support from the British Heart Foundation Center for Vascular Regeneration With Edinburgh/Bristol (RM/17/3/33381). Dr Doménech’s work was supported by Project PI16/02049 integrated in the National Plan for Scientific Research, Development and Technological Innovation, 2013–2016, and funded by the ISCIII–General Subdirection of Assessment and Promotion of Research–European Regional Development Fund. Dr Merkely’s work was funded by the National Research, Development and Innovation Fund (NVKP_16-1–2016-0017) and the Thematic Excellence Program of the Ministry for Innovation and Technology (2020-4.1.1.-TKP2020), Hungary. Dr Radovits is supported by the National Research, Development and Innovation Office of Hungary (K134939)British Heart Foundation; FS/19/33/34328British Heart Foundation; CH/16/3/32406British Heart Foundation; CH/1999001/11735British Heart Foundation Center for Vascular Regeneration; RM/17/3/33381Hungría. Ministry for Innovation and Technology; NVKP_16-1–2016-0017Hungría. Ministry for Innovation and Technology; 2020-4.1.1.-TKP2020Hungría. National Research, Development and Innovation Office; K13493