Engineering of AceTr membrane transporters to improve organic acid production in yeast

Organic acids are industrially relevant chemicals obtainable from renewable feedstocks via microbial cell factories. Microbially produced organic acids have a wide variety of applications, including bioplastic synthesis. Thus, they possess the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for microbial production of organic acids, namely due to their tolerance of low pH environments. Such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in low pH conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Our current objective is to deepen the knowledge on organic acid transporters from the AceTR family (1,2,3). We performed functional characterization by studying transporter kinetics, energetics and specificity as well as site-directed mutagenesis to acquire insight into the structural features of transporters. Finally, we aim to improve organic acid production in S. cerevisiae cell factories via expression of engineered AceTR transporters with altered activity and substrate specificity.UID/BIA/04050/2013(POCI-01-0145-FEDER-007569) and TransAcids(PTDC/BIAMIC/5184/2014) funded by national funds, FCT-IP and ERDF by COMPETE 2020-POCI; EcoAgriFood(NORTE-01-0145-FEDER-000009), supported by NORTE-2020, under the PORTUGAL 2020 Partnership Agreement.TCacknowledgesYeastdocEuropean Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curie grant agreement No 76492

Glycerolized reticular dermis as a new human acellular dermal matrix: An exploratory study

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco's Modified Eagle's Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties

Exploring plasma membrane transporters to improve organic acid production in yeast – Characterization and engineering

Organic acids are industrially relevant building-block chemicals obtainable from renewable feedstocks by utilization of microbial cell factories. With a wide variety of applications, including bioplastics synthesis, microbially produced organic acids have the potential to replace petroleum-derived commodity chemicals that are obtained through unsustainable production processes. Yeasts commonly represent the organisms of choice for production of organic acids, namely due to their tolerance of low pH environments, since such production conditions allow for direct formation of the desired protonated form of the acid and thus cut downstream processing costs. Efficient product export over the plasma membrane in such conditions is particularly demanding, therefore expression of membrane transporters with adequate substrate specificity and transport mechanism is often the determining factor at acquiring competitive product titres. Here, we are characterizing and engineering plasma membrane transporters with the final aim to improve production of dicarboxylic acids, namely succinic acid, in yeast. This includes transporters that have already been described as efficient dicarboxylate transporters, as well as promising transporters from the AceTr family. First, we perform functional characterization by studying transporter kinetics, energetics and specificity, as well as site-directed mutagenesis, to acquire insight into functional-structural relationship of transporters. This insight further uncovers engineering targets that can lead to improved transporter activity as well as altered substrate specificity. Finally, the performance of these transporters can be assessed via their expression in S. cerevisiae that is engineered for succinic acid production.This work was supported by the strategic programme UID/BIA/ 04050 2019 funded by Portuguese funds through the FCT IP, the project TransAcids (PTDC/ 5184 2014 funded by FCT IP and ERDF by COMPETE 2020 POCI and the project EcoAgriFood (NORTE 01 0145 FEDER 000009 supported by NORTE 2020 under the PORTUGAL 2020 Partnership Agreemen

The Inborn Errors of Immunity—Virtual Consultation System Platform in Service for the Italian Primary Immunodeficiency Network: Results from the Validation Phase

purposeInborn errors of immunity (IEI) represent a heterogeneous group of rare genetically determined diseases. In some cases, patients present with complex or atypical phenotypes, not fulfilling the accepted diagnostic criteria for IEI and, thus, at high risk of misdiagnosis or diagnostic delay. this study aimed to validate a platform that, through the opinion of immunologist experts, improves the diagnostic process and the level of care of patients with atypical/complex IEI.MethodsHere, we describe the functioning of the IEI-Virtual consultation system (VCS), an innovative platform created by the italian Immunodeficiency network (IPINet). resultsIn the validation phase, from January 2020 to June 2021, 68 cases were entered on the IEI-VCS platform. a final diagnosis was achieved in 35/68 cases (51%, 95% CI 38.7 to 64.2). In 22 out of 35 solved cases, the diagnosis was confirmed by genetic analysis. In 3/35 cases, a diagnosis of secondary immunodeficiency was made. In the remaining 10 cases, an unequivocal clinical and immunological diagnosis was obtained, even though not substantiated by genetic analysis. conclusionFrom our preliminary study, the VCS represents an innovative and useful system to improve the diagnostic process of patients with complex unsolved IEI disorders, with benefits both in terms of reduction of time of diagnosis and access to the required therapies. these results may help the functioning of other international platforms for the management of complex cases

Protocols for drug allergy desensitization in children

International audienc

Exploring the function and structure of the acetate transporter family

The AceTr transporter family is a group of Acetate Transporters with six predicted transmembrane segments. It has homologues in fungi, bacteria, archaea and protozoa. As acetate transporters, these proteins play a crucial role on cell metabolism being involved in the capacity of cells to adapt to nutrient availability. As these membrane transporters also transport other substrates, they may be an important asset for biotechnological purposes. To better understand the mechanism of acetate transport, we have investigated functional and structural features of the AceTr family. We have performed site-directed mutagenesis in residues highly conserved, or possibly involved in substrate binding, from the Saccharomyces cerevisiae Ady2. Functional assays determined the influence of these residues on the transport capacity and protein localization. Another goal of this work is to determine the structure of the AceTr family. Accordingly, several of its members were cloned in different expression vectors and their expression was evaluated in different E. coli expression strains. SatP from E. coli and its AceP from Methanosarcina acetivorans presented satisfactory levels of protein production and are currently being tested in production, purification and crystallization trials

MTT test for evaluation of dermal viability on reticular dermis.

<p>Results of the vitality index assessment showed the absence of viable cells in the HADM after glycerolization and decellularization processes.</p

Histochemical reactions showing histological features of human acellular reticular dermal matrices at different incubation times (20X magnification).

<p>In comparison with control (T0) samples (<b>A</b>: hematoxylin-eosin; <b>D</b>: trichrome staining), after 5 weeks of treatment (T5), specimens showed the presence of stromal shrinkage and tissue fragmentation, edema and focal condensations with spongy patterns; these denaturation artifacts were rare in samples treated with DMEM (<b>B</b>: hematoxylin-eosin; <b>E</b>: trichrome staining) and more evident in those treated with NaOH (<b>C</b>: hematoxylin-eosin; <b>F</b>: trichrome staining). Elastic fibers, stained in black using Elastic Von Gieson histochemical reaction, showed no significant alterations in terms of length, diameter and mean number comparing T0 samples (<b>G</b>) with T5 specimens treated with DMEM (<b>H</b>) or NaOH (<b>I</b>).</p