Nrf2 and Heme Oxygenase-1 Involvement in Atherosclerosis Related Oxidative Stress

Atherosclerosis remains the underlying process responsible for cardiovascular diseases and the high mortality rates associated. This chronic inflammatory disease progresses with the formation of occlusive atherosclerotic plaques over the inner walls of vascular vessels, with oxidative stress being an important element of this pathology. Oxidation of low-density lipoproteins (ox-LDL) induces endothelial dysfunction, foam cell activation, and inflammatory response, resulting in the formation of fatty streaks in the atherosclerotic wall. With this in mind, different approaches aim to reduce oxidative damage as a strategy to tackle the progression of atherosclerosis. Special attention has been paid in recent years to the transcription factor Nrf2 and its downstream-regulated protein heme oxygenase-1 (HO-1), both known to provide protection against atherosclerotic injury. In the current review, we summarize the involvement of oxidative stress in atherosclerosis, focusing on the role that these antioxidant molecules exert, as well as the potential therapeutic strategies applied to enhance their antioxidant and antiatherogenic properties

Use of Sensory Analysis to Investigate the Influence of Climate Chambers and Other Process Variables in the Production of Sweet Wines.

In this study, a climate chamber, as an alternative method, has been used to dry raisins and the sensory profiles of the sweet sherry wines obtained have been evaluated. Other important factors, namely grape variety, vintage, vinification conditions, as well as the ageing method and its length of time, have also been considered. When heavy rainfall had been registered, the musts extracted from grapes dried under controlled conditions in a climate chamber showed a lower intensity of the musty off-odor compared to those elaborated with sun-dried grapes. The wine fermented at low temperature with Saccharomyces bayanus scored the highest in citric and floral notes, and this was preferred over all the other wines that were evaluated. The wines aged in oak barrels were preferred to both, wines aged in the presence of oak chips as well as those aged without any wood contact. The use of climate chambers to dry the grapes that are going to be used for the elaboration of sweet wines appears to be an advantageous alternative to the traditional method, since it allows a more precise control of the process and highly valued sweet wines from a sensory point of view are obtained thereby

Generation of Highly Antioxidant Submicron Particles from Myrtus communis Leaf Extract by Supercritical Antisolvent Extraction Process,

Se han producido partículas submicrónicas a partir de un extracto etanólico de hojas de myrthus communnis utilizando la tecnología del dióxido de carbono supercrítico, en lo sucesivo denominada extracción antisolvente supercrítica (SAE). Se ha investigado la influencia de la presión (9-20 MPa), la temperatura (308 y 328 K) y la velocidad de inyección (3 y 8 ml/min) en la precipitación de las partículas y se ha confirmado que los aumentos de presión y temperatura conducían a partículas de menor tamaño. Las partículas obtenidas tenían una forma casi esférica con tamaños comprendidos entre 0,42 y 1,32 µm. Además, se evaluó la bioactividad de las partículas generadas y se midieron grandes contenidos de compuestos fenólicos con una elevada actividad antioxidante. Las partículas también se sometieron a estudios de liberación in vitro. Las partículas de mirto demostraron propiedades citoprotectoras cuando se aplicaron a bajas concentraciones (1 µM) a líneas celulares de macrófagos.Submicron particles have been produced from an ethanolic extract of myrthus communnis leaves using supercritical carbon dioxide technology, hereinafter referred to as Supercritical Antisol-vent Extraction (SAE). The influence of pressure (9–20 MPa), temperature (308 and 328 K) and in-jection rate (3 and 8 ml/min) on the particles precipitation has been investigated and it has been confirmed that increases in pressure and temperature led to smaller particle sizes. The obtained particles had a quasi-spherical shape with sizes ranging from 0.42 to 1.32 µm. Moreover, the bioactivity of the generated particles was assessed and large contents of phenolic compounds with a high antioxidant activity were measured. The particles were also subjected to in vitro release studies. The myrtle particles demonstrated cytoprotective properties when applied at low concentrations (1 µM) to macrophage cell lines

Effects from the Freezing of Either Whole or Crushed Grapes on the Volatile Compounds Contents in Muscat Wines

The transfer of aromatic compounds from the grape skins to the musts has been studied using a process involving freezing whole bunches or crushed grapes for winemaking the Muscat of Alexandria variety (white wine). Subsequently, a prefermentative maceration has been applied to some of the samples. The aromatic profiles of the final wines have been determined using gas chromatography coupled to mass spectrophotometry (GC-MS). The results revealed that, in the trials in which whole grapes were frozen, the final wines had a higher aromatic concentration compared to that of wines obtained by either freezing crushed grapes or obtained with traditional winemaking techniques. Thus, the wines produced from frozen whole grapes were found to exhibit different characteristics from the rest of the wines. The compounds affected by the freezing either of the whole bunches or the crushed grapes were terpenes, acids, and esters. Lower differences were found for wines produced applying prefermentative maceration after the freezing process

N-glycosylation profile analysis of Trastuzumab biosimilar candidates by Normal Phase Liquid Chromatography and MALDI-TOF MS approaches

The pharmaceutical market has entered an era in which the production of new therapeutics is being often replaced by “biosimilars”, copies of already commercialized products waiting for the patents to expire in order to be distributed in a more competitive and affordable manners. Due to its relevance, the ErbB2-targeted monoclonal antibody Trastuzumab (Herceptin) used as breast cancer therapy is one of the main targets in the production of biosimilars. A major challenge is to produce antibodies with the same or the closest N-glycosylation pattern seen in the commercialized drug. Several factors, such as growing conditions or cell types employed, can determine the final composition and structure of the glycans, significantly affecting the properties of the generated antibodies. Therefore, an appropriate characterization is essential. In the present study, we describe two different but complementary strategies to characterize the N-glycosylation of two biosimilar candidates of Trastuzumab. In the first case, N-glycans are fluorescently labeled and separated by Normal Phase HPLC. Different sugars will elute at different times and can be identified using specific oligosaccharide standards. In the second approach, released glycans are permethylated and analyzed by MALDI-TOF MS, being able to determine the structure because of the differential sugar masses. Biological significance The characterization of the N-glycosylation sites of therapeutic recombinant monoclonal antibodies (mAbs) is usually one of the most critical and time consuming steps in the developing process of biosimilars or any other glycosylated drug. Herein we describe two different but complementary approaches to characterize mAbs glycosylation patterns, the use of glycan fluorescence labeling coupled to HPLC and MALDI-TOF MS profile analysis. This article is part of a Special Issue entitled: HUPO 2014

Role of long non-coding RNAs in adipose tissue metabolism and associated pathologies

The incidence of obesity and its related disorders has increased dramatically in recent years and has become a pandemic. Adipose tissue is a crucial regulator of these diseases due to its endocrine capacity. Thus, understanding adipose tissue metabolism is essential to finding new effective therapeutic approaches. The "omic" revolution has identified new concepts about the complexity of the signaling pathways involved in the pathophysiology of adipose tissue-associated disorders. Specifically, advances in transcriptomics have allowed its application in clinical practice and primary or secondary prevention. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of adipose tissue since they can modulate gene expression at the epigenetic, transcriptional, and post-transcriptional levels. They interact with DNA, RNA, protein complexes, other noncoding RNAs, and microRNAs to regulate a wide range of physiological and pathological processes. Here, we review the emerging field of lncRNAs, including how they regulate adipose tissue biology, and discuss circulating lncRNAs, which may represent a turning point in the diagnosis and treatment of adipose tissue-associated disorders. We also highlight potential biomarkers of obesity and diabetes that could be considered as therapeutic targets.This study was supported by the Spanish Ministry of Science and Innovation (MCIN) (PID2020-114953RB-C21 to LH and DS, co-funded by the European Regional Development Fund [ERDF], PID2020-114343GA-100 to MA, and PID2019-110063RA-I00 to JP and MC), the Biomedical Research Centre in Pathophysiology of Obesity and Nutrition (CIBEROBN) (Grant CB06/03/0001 to LH), and the Merck Health Foundation (to LH). AC is a recipient of the Formación de Personal Investigador (FPI) doctoral fellowship from the MCIN.‬‬‬‬‬‬ We thank MCarmen Soler-Vázquez for her expert assistance in the preparation of Fig. 1

ZNF330/NOA36 interacts with HSPA1 and HSPA8 and modulates cell cycle and proliferation in response to heat shock in HEK293 cells

Background: The human genome contains nearly 20.000 protein-coding genes, but there are still more than 6,000 proteins poorly characterized. Among them, ZNF330/NOA36 stand out because it is a highly evolutionarily conserved nucleolar zinc-finger protein found in the genome of ancient animal phyla like sponges or cnidarians, up to humans. Firstly described as a human autoantigen, NOA36 is expressed in all tissues and human cell lines, and it has been related to apoptosis in human cells as well as in muscle morphogenesis and hematopoiesis in Drosophila. Nevertheless, further research is required to better understand the roles of this highly conserved protein. Results: Here, we have investigated possible interactors of human ZNF330/NOA36 through affinity-purification mass spectrometry (AP-MS). Among them, NOA36 interaction with HSPA1 and HSPA8 heat shock proteins was disclosed and further validated by co-immunoprecipitation. Also, “Enhancer of Rudimentary Homolog” (ERH), a protein involved in cell cycle regulation, was detected in the AP-MS approach. Furthermore, we developed a NOA36 knockout cell line using CRISPR/Cas9n in HEK293, and we found that the cell cycle profile was modified, and proliferation decreased after heat shock in the knocked-out cells. These differences were not due to a different expression of the HSPs genes detected in the AP-MS after inducing stress. Conclusions: Our results indicate that NOA36 is necessary for proliferation recovery in response to thermal stress to achieve a regular cell cycle profile, likely by interaction with HSPA1 and HSPA8. Further studies would be required to disclose the relevance of NOA36-EHR interaction in this context.14 página

REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT-Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT-Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.Consejería de Salud de Andalucía, Junta de Andalucía (grants PI-0365-2013 and PI-0055-2017); Instituto de Salud Carlos III (grant PI16-00784 from the “Plan Estatal de I+D+I 2013–2016/FEDER”