Towards large-scale production of human-induced pluripotent stem cell-derived extracellular vesicles in stirred-tank bioreactors

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Control of the root lesion Pratylenchus penetrans - the effect of nematocidal activity of plant-derived compounds

The root lesion nematode, Pratylenchus penetrans, is one of the most harmful plant parasites, responsible for worldwide productivity losses in a significant number of plant hosts. Generally, chemical control relies on synthetic compounds used through fumigation or direct contact, which offers a systemic protection. These control methods are costly and hazardous to the environment and to humans. Phytochemicals may play an important role in nematode control. The nematicidal activity of eight compounds that occur naturaly in plants, from two classes of compounds, was assessed at 2 mg/mL, for 24 h. Bioassays were performed following the standard direct contact methodology. P. penetrans was remarkably tolerant to the tested compounds, with mortality range between 1.0 and 5.8 %. To the best of our knowledge, the nematicidal activity of 4 phenolic compounds (catechin, caffeic acid, gallic acid and gentisic acid) was evaluated for the first time for P. penetrans

Plant-nematode co-cultures in the screening of sustainable nematicides against soil-dwelling parasitic nematodes of plants

The diseases caused by plant parasitic nematodes are still a serious constraint to modern global crop production. An increasing number of active compounds in commercial nematicidal formulations is being banned from use by common policies of pest management. Farmer communities report a low efficiency for the replacement pesticides, which reflects on crop yield and productivity. Novel sustainable biopesticides are urgently needed to cope with global food demands while respecting the most recent environmental policies. Plant-nematode co-cultures offer a stable biotechnological screening tool able to assess the active compound’s nematicidal activity and its effect on host tissues, simultaneously, in an easily accessible system that simulates natural infection. These systems are being developed and optimized at the Nematology laboratory of INIAV. Preliminary results were obtained for co-cultures of Solanum lycopersicum with the nematode Meloidogyne ethiopica and S. tuberosum with Globodera pallida. Future studies will target other plant parasitic nematodes, e.g., the root lesion nematodes (Pratylenchus penetrans) in transgenic roots of potato and the pinewood nematode (Bursaphelenchus xylophilus) on in vitro pine shoots (Pinus sp.)

PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis

<p>Abstract</p> <p>Background</p> <p>Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer <it>Streptomyces natalensis</it>, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from <it>S. natalensis </it>in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.</p> <p>Results</p> <p>Adjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.</p> <p>Conclusion</p> <p>This report describes the involvement of an amino acid exporter (encoded by <it>pimT </it>in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.</p

Inulin potential for encapsulation and controlled delivery of Oregano essential oil

The ability of inulin, a prebiotic material, as encapsulation matrix was explored. Microcapsules of Raftiline were produced by spray drying inulin solutions at different solids content (5, 15 and 25%) at 120, 155 and 190 °C, according to a Central Composite Rotatable design. Produced capsules were analysed for morphology and size by SEM and physiochemical characterized by DSC, IR and RAMAN. Oregano essential oil was incorporated in the inulin solutions at 15% solids basis and the emulsions dried at the same conditions. The above mentioned methodologies were applied to evaluate the encapsulation ability and the changes induced by the presence of the EO in capsules morphology and structure. Furthermore the kinetics and amount of release was assessed by a spectrophotometric method. Results showed that it was possible to produce regular spherical inulin microcapsules (3–4.5 μm) for all the tested experimental conditions. According to IR and Raman results mainly drying temperature affected the structure of the capsules, three groups being clearly formed. These groups could be related to the morphology of inulin crystals. The EO was successfully encapsulated in the system as demonstrated by IR and Raman analysis. The differences found in the EO releasing amount, make clear that different degrees of core material retention is achieved, what should be related to structural changes in the matrix wall, denoting in some processing conditions interactions phenomena among inulin and EO. Those different releasing profiles patterns may be quite useful in finding different potential uses for the encapsulates.Thanks are due for the financial support given by Portuguese Foundation for Science and Technology (FCT) through the project PTDC/AGR/ALI/67194/2006 and through a post-doctoral grant of first author (SFRH/BPD/44200/2008) supported by programme QREN - POPH - Tipologia 4.1. The authors also express their gratitude to Mr Octavio Chaveiro from INIAV for the given support in microscopy studies

The effect of the matrix system in the delivery and in-vitro bioactivity of microencapsulated oregano essential oil

Microencapsulation allows bioactive compounds protection from external factors. Innovation in food industry often requires adding functional ingredients, to tailor flavour and texture, to improve preservation, to control bioactive compounds stability and controlled release during processing/storage. Oregano, besides richness in aroma compounds, is also known by potential antioxidant and antimicrobial activities. These sensitive compounds need protection in order to allow their use in a wider range of processes. In this study, oregano essential oil (EO) was microencapsulated by spray/freeze drying in: rice starch (with/without bonding agents), gelatine/sucrose and inulin, dried at different temperatures. Microencapsulates were analysed for morphology and structure (SEM, CLSM, X-ray diffraction and FTIR). Releasing ability of entrapped EO (UV–VIS spectroscopy) was evaluated by diffusion coefficient (D). Antioxidant activity (AA) - ORAC and HORAC- and antimicrobial activities against pathogens were evaluated. Rice starch spherules, presenting interconnecting cavities, were formed. Spray-dried inulin and gelatine/sucrose systems formed continuous walled and smooth surface spherical capsules (3-4.5 and 0.9-10m, respectively). EO was uniformly distributed inside the structures (CLSM) and its presence confirmed by FTIR. Depending on the system, D varied among 10-13 (starch), 10-13-10-15, (gelatine/sucrose) and 10-16 m2/s (inulin). In starch system, D was mainly influenced by the gelatin concentration, increasing with it. X- ray diffraction and FTIR results suggest some kind of linkage between gelatine and starch. Spray-dried gelatine/sucrose system, revealed to be unsuitable for EO encapsulation due to capsules disintegration but freeze-drying was effective. The D of EO from inulin capsules decreases when these are produced above 140 ºC. The impact of encapsulation method on EO bioactivity and product stability during 6 months, was verified through the determination of microcapsules AA, using free EO value as reference. The results obtained provide information on the release/stability of oregano EO from different matrices, relevant for functional ingredients microencapsulation

How parasitism genes are regulated: a motif to search for genes regulators in the plant parasitic nematode Bursaphelenchus xylophilus

Plant-parasitic nematodes threaten global agricultural and forestry systems. The search for new control strategies in line with the EU’s sustainability goals highlight significant knowledge gaps. Like all other plant pathogens, plant-parasitic nematodes deliver several parasitism proteins (effectors) into the host plant to cause disease. NemaWAARS project focuses on mechanism(s) of regulation and gene control expression of parasitism genes in pinewood nematode, Bursaphelenchus xylophilus. From the previous transcriptomic data derived from the pharyngeal gland cells (considered a specialized tissue potentially related to parasitism) we have identified a non-coding DNA motif - STATAWAARS - associated in the promotor region of highly abundant and secreted expressed genes. Given that this non-coding genetic signature unifies many sequences of unrelated parasitism genes, it implies the existence of a potential major regulator(s), that binds to this sequence to control the expression of downstream genes. We hypothesize that by disrupting this regulator(s), it would be possible to simultaneously disrupt the expression of many associated parasitism-related genes. To test the hypothesis the project aims to identify proteins (or complex of proteins) that bind in the promoter regions of parasitism-related genes (in vivo) or identify other regulatory candidates for master regulators of parasitism-related genes expression that are enriched in the pharyngeal gland cell tissues. For the best candidate regulatory proteins, an RNAi approach will target the selected gene candidates and evaluate the regulatory role in effector genes expression and in interaction with the host (in planta). Under an ongoing national and international collaborative network, the strategy in NemaWAARS will include innovative approaches to explore the regulators that govern effector gene expression applied in B. xylophilus research

Low frequency of CD4+CD25+ Treg in SLE patients: a heritable trait associated with CTLA4 and TGFβ gene variants

<p>Abstract</p> <p>Background</p> <p>CD4⁺CD25⁺regulatory T cells play an essential role in maintaining immune homeostasis and preventing autoimmunity. Therefore, defects in Treg development, maintenance or function have been associated with several human autoimmune diseases including Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease characterized by loss of tolerance to nuclear components and significantly more frequent in females.</p> <p>Results</p> <p>To investigate the involvement of Treg in SLE pathogenesis, we determined the frequency of CD4⁺CD25⁺CD45RO⁺T cells, which encompass the majority of Treg activity, in the PBMC of 148 SLE patients (76 patients were part of 54 families), 166 relatives and 117 controls. SLE patients and their relatives were recruited in several Portuguese hospitals and through the Portuguese Lupus Association. Control individuals were blood donors recruited from several regional blood donor centers. Treg frequency was significantly lower in SLE patients than healthy controls (z = -6.161, <it>P </it>< 0.00001) and intermediate in the relatives' group. Remarkably, this T cell subset was also lower in females, most strikingly in the control population (z = 4.121, <it>P </it>< 0.001). We further ascertained that the decreased frequency of Treg in SLE patients resulted from the specific reduction of <it>bona fide </it>FOXP3⁺CD4⁺CD25⁺Treg. Treg frequency was negatively correlated with SLE activity index (SLEDAI) and titers of serum anti-dsDNA antibodies. Both Treg frequency and disease activity were modulated by IVIg treatment in a documented SLE case. The segregation of Treg frequency within the SLE families was indicative of a genetic trait. Candidate gene analysis revealed that specific variants of <it>CTLA4 </it>and <it>TGFβ </it>were associated with the decreased frequency of Treg in PBMC, while <it>FOXP3 </it>gene variants were associated with affection status, but not with Treg frequency.</p> <p>Conclusion</p> <p>SLE patients have impaired Treg production or maintenance, a trait strongly associated with SLE disease activity and autoantibody titers, and possibly resulting from the inability to convert FOXP3⁺CD25^-into FOXP3⁺CD25⁺T cells. Treg frequency is highly heritable within SLE families, with specific variants of the <it>CTLA4 </it>and <it>TGFβ </it>genes contributing to this trait, while <it>FOXP3 </it>contributes to SLE through mechanisms not involving a modulation of Treg frequency. These findings establish that the genetic components in SLE pathogenesis include genes related to Treg generation or maintenance.</p

The Neuroprotective Action of Amidated-Kyotorphin on Amyloid β Peptide-Induced Alzheimer’s Disease Pathophysiology

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous dipeptide initially described to have analgesic properties. Recently, KTP was suggested to be an endogenous neuroprotective agent, namely for Alzheimer’s disease (AD). In fact, KTP levels were shown to be decreased in the cerebrospinal fluid of patients with AD, and recent data showed that intracerebroventricular (i.c.v.) injection of KTP ameliorates memory impairments in a sporadic rat model of AD. However, this administration route is far from being a suitable therapeutic strategy. Here, we evaluated if the blood-brain permeant KTP-derivative, KTP-NH2, when systemically administered, would be effective in preventing memory deficits in a sporadic AD animal model and if so, which would be the synaptic correlates of that action. The sporadic AD model was induced in male Wistar rats through i.c.v. injection of amyloid β peptide (Aβ). Animals were treated for 20 days with KTP-NH2 (32.3 mg/kg, intraperitoneally (i.p.), starting at day 3 after Aβ administration) before memory testing (Novel object recognition (NOR) and Y-maze (YM) tests). Animals were then sacrificed, and markers for gliosis were assessed by immunohistochemistry and Western blot analysis. Synaptic correlates were assessed by evaluating theta-burst induced long term potentiation (LTP) of field excitatory synaptic potentials (fEPSPs) recorded from hippocampal slices and cortical spine density analysis. In the absence of KTP-NH2 treatment, Aβ-injected rats had clear memory deficits, as assessed through NOR or YM tests. Importantly, these memory deficits were absent in Aβ-injected rats that had been treated with KTP-NH2, which scored in memory tests as control (sham i.c.v. injected) rats. No signs of gliosis could be detected at the end of the treatment in any group of animals. LTP magnitude was significantly impaired in hippocampal slices that had been incubated with Aβ oligomers (200 nM) in the absence of KTP-NH2. Co-incubation with KTP-NH2 (50 nM) rescued LTP toward control values. Similarly, Aβ caused a significant decrease in spine density in cortical neuronal cultures, and this was prevented by co-incubation with KTP-NH2 (50 nM). In conclusion, the present data demonstrate that i.p. KTP-NH2 treatment counteracts Aβ-induced memory impairments in an AD sporadic model, possibly through the rescuing of synaptic plasticity mechanisms.publishersversionpublishe