Analysis of temperature‐mediated changes in the wine yeast Saccharomyces bayanus var uvarum. An oenological study of how the protein content influences wine quality

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D‐DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC–MS was performed. The proteomic and “aromatomic” results are discussed in relation to the oenological properties of S. bayanus var. uvarum.     

Synthetic Cyclolipopeptides Selective against Microbial,Plant and Animal Cell Targets by Incorporation of D-Amino Acids or Histidine

Cyclolipopeptides derived from the antimicrobial peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln) (BPC194) were prepared on solid-phase and screened against four plant pathogens. The incorporation at Lys⁵ of fatty acids of 4 to 9 carbon atoms led to active cyclolipopeptides. The influence on the antimicrobial activity of the Lys residue that is derivatized was also evaluated. In general, acylation of Lys¹, Lys² or Lys⁵ rendered the sequences with the highest activity. Incorporation of a D-amino acid maintained the antimicrobial activity while significantly reduced the hemolysis. Replacement of Phe with a His also yielded cyclolipopeptides with low hemolytic activity. Derivatives exhibiting low phytotoxicity in tobacco leaves were also found. Interestingly, sequences with or without significant activity against phytopathogenic bacteria and fungi, but with differential hemolysis and phytotoxicity were identified. Therefore, this study represents an approach to the development of bioactive peptides with selective activity against microbial, plant and animal cell targets. These selective cyclolipopeptides are candidates useful not only to combat plant pathogens but also to be applied in other fields.     

Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

One of the hallmarks of HIV-1 disease is the association of heightened CD4⁺ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4⁺ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4⁺ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4⁺ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4⁺ Th-cells induced by exposed DCs.     

B Cell,Th17, and Neutrophil Related Cerebrospinal Fluid Cytokine/Chemokines Are Elevated in MOG Antibody Associated Demyelination

Background

Myelin oligodendrocyte glycoprotein antibody (MOG Ab) associated demyelination represents a subgroup of autoimmune demyelination that is separate from multiple sclerosis and aquaporin 4 IgG-positive NMO, and can have a relapsing course. Unlike NMO and MS, there is a paucity of literature on immunopathology and CSF cytokine/chemokines in MOG Ab associated demyelination.

Aim

To study the differences in immunopathogenesis based on cytokine/chemokine profile in MOG Ab-positive (POS) and -negative (NEG) groups.

Methods

We measured 34 cytokines/chemokines using multiplex immunoassay in CSF collected from paediatric patients with serum MOG Ab POS [acute disseminated encephalomyelitis (ADEM = 8), transverse myelitis (TM = 2) n = 10] and serum MOG Ab NEG (ADEM = 5, TM = 4, n = 9) demyelination. We generated normative data using CSF from 20 non-inflammatory neurological controls.

Results

The CSF cytokine and chemokine levels were higher in both MOG Ab POS and MOG Ab NEG demyelination groups compared to controls. The CSF in MOG Ab POS patients showed predominant elevation of B cell related cytokines/chemokines (CXCL13, APRIL, BAFF and CCL19) as well as some of Th17 related cytokines (IL-6 AND G-CSF) compared to MOG Ab NEG group (all p<0.01). In addition, patients with elevated CSF MOG antibodies had higher CSF CXCL13, CXCL12, CCL19, IL-17A and G-CSF than patients without CSF MOG antibodies.

Conclusion

Our findings suggest that MOG Ab POS patients have a more pronounced CNS inflammatory response with elevation of predominant humoral associated cytokines/chemokines, as well as some Th 17 and neutrophil related cytokines/chemokines suggesting a differential inflammatory pathogenesis associated with MOG antibody seropositivity. This cytokine/chemokine profiling provides new insight into disease pathogenesis, and improves our ability to monitor inflammation and response to treatment. In addition, some of these molecules may represent potential immunomodulatory targets.     

Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling,Inflammation, Adaptive Thermogenesis and Lipolysis

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE₂ levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE₂ as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE₂ significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-β. In addition, PGE₂ inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE₂ anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE₂ induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE₂ might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE₂ inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE₂ as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.     

Use of the SAMe-TT2R2 Score to Predict Good Anticoagulation Control with Warfarin in Chinese Patients with Atrial Fibrillation: Relationship to Ischemic Stroke Incidence

Background

The efficacy and safety of warfarin therapy for stroke prevention in atrial fibrillation (AF) depends on the time in therapeutic range (TTR). We aimed to assess the predictive ability of SAMe-TT₂R₂ score in Chinese AF patients on warfarin, whose TTR is notoriously poor.

Methods and Results

This is a single-centre retrospective study. Patients with non-valvular AF on warfarin diagnosed between 1997 and 2011 were stratified according to SAMe-TT₂R₂ score, and TTR was calculated using Rosendaal method. The predictive power of SAMe-TT₂R₂ scores for good TTR i.e. >70% was assessed. We included 1,428 Chinese patients (mean age 76.2±8.7 years, 47.5% male) with non-valvular AF on warfarin. The mean and median TTR were 38.2±24.4% and 38.8% (interquartile range: 17.9% and 56.2%) respectively. TTR decreased progressively with increasing SAMe-TT₂R₂ score (p = 0.016). When the cut-off value of SAMe-TT₂R₂ score was set to 2, the sensitivity and specificity to predict TTR<70% were 85.7% and 17.8%, respectively. The corresponding positive and negative predictive values were 10.1% and 92.0%. After a mean follow-up of 4.7±3.6 years, 338 patients developed an ischemic stroke (4.96%/year). Patients with TTR≥70% had a lower annual risk of ischemic stroke of 3.67%/year compared with than those with TTR<70% (5.13%/year)(p = 0.08). Patients with SAMe-TT₂R₂ score ≤2 had the lowest risk of annual risk of ischemic stroke (3.49%/year) compared with those with SAMe-TT₂R₂ score = 3 (4.56%/year), and those with SAMe-TT₂R₂ score ≥4 (6.41%/year)(p<0.001). There was also a non-significant trend towards more intracranial hemorrhage with increasing SAMe-TT₂R₂ score.

Conclusions

The SAMe-TT₂R₂ score correlates well with TTR in Chinese AF patients, with a score >2 having high sensitivity and negative predictive values for poor TTR. Ischemic stroke risk increased progressively with increasing SAMe-TT₂R₂ score, consistent with poorer TTRs at high SAMe-TT₂R₂ scores.     

MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC₅₀s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.     

Measuring Hair Cortisol Concentrations to Assess the Effect of Anthropogenic Impacts on Wild Chimpanzees (Pan troglodytes)

Non-human primates face major environmental changes due to increased human impacts all over the world. Although some species are able to survive in certain landscapes with anthropogenic impact, their long-term viability and fitness may be decreased due to chronic stress. Here we assessed long-term stress levels through cortisol analysis in chimpanzee hair obtained from sleeping nests in northwestern Uganda, in order to estimate welfare in the context of ecotourism, forest fragmentation with human-wildlife conflicts, and illegal logging with hunting activity (albeit not of primates), compared with a control without human contact or conflict. Concerning methodological issues, season [F(2,129) = 37.4, p < 0.0001, r² = 0.18] and the age of nests [F(2,178) = 20.3, p < 0.0001, r² = 0.11] significantly predicted hair cortisol concentrations (HCC). With regard to effects of anthropogenic impacts, our results neither showed elevation of HCC due to ecotourism, nor due to illegal logging compared to their control groups. We did, however, find significantly increased HCC in the fragment group compared to chimpanzees living in a nearby intact forest [F(1,88) = 5.0, p = 0.03, r² = 0.20]. In conclusion, our results suggest that hair cortisol analysis is a powerful tool that can help understanding the impact of anthropogenic disturbances on chimpanzee well-being and could be applied to other great ape species.     

Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke

Purpose

Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.

Methods

Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3–7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.

Results

Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.

Conclusions

Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.     

Identification of the phospholipid lysobisphosphatidic acid in the protozoan Entamoeba histolytica: An active molecule in endocytosis

Phospholipids are essential for vesicle fusion and fission and both are fundamental events for Entamoeba histolytica phagocytosis. Our aim was to identify the lysobisphosphatidic acid (LBPA) in trophozoites and investigate its cellular fate during endocytosis. LBPA was detected by TLC in a 0.5 R_f spot of total lipids, which co-migrated with the LBPA standard. The 6C4 antibody, against LBPA recognized phospholipids extracted from this spot. Reverse phase LC-ESI-MS and MS/MS mass spectrometry revealed six LBPA species of m/z 772.58–802.68. LBPA was associated to pinosomes and phagosomes. Intriguingly, during pinocytosis, whole cell fluorescence quantification showed that LBPA dropped 84% after 15 min incubation with FITC-Dextran, and after 60 min, it increased at levels close to steady state conditions. Similarly, during erythrophagocytosis, after 15 min, LBPA also dropped in 36% and increased after 60 and 90 min. EhRab7A protein appeared in some vesicles with LBPA in steady state conditions, but after phagocytosis co-localization of both molecules increased and in late phases of erythrophagocytosis they were found in huge phagosomes or multivesicular bodies with many intraluminal vacuoles, and surrounding ingested erythrocytes and phagosomes. The 6C4 and anti-EhADH (EhADH is an ALIX family protein) antibodies and Lysotracker merged in about 50% of the vesicles in steady state conditions and throughout phagocytosis. LBPA and EhADH were also inside huge phagosomes. These results demonstrated that E. histolytica LBPA is associated to pinosomes and phagosomes during endocytosis and suggested differences of LBPA requirements during pinocytosis and phagocytosis.