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Objectives Women of low socioeconomic status have been described as having suboptimal prenatal care, which in turn has been associated with poor pregnancy outcomes. Many types of conditional cash transfer (CCT) programmes have been developed, including programmes to improve prenatal care or smoking cessation during pregnancy, and their effects demonstrated. However, ethical critiques have included paternalism and lack of informed choice. Our objective was to determine if women and healthcare professionals (HPs) shared these concerns.Design Prospective qualitative research.Setting We included economically disadvantaged women, as defined by health insurance data, who participated in the French NAITRE randomised trial assessing a CCT programme during prenatal follow-up to improve pregnancy outcomes. The HP worked in some maternities participating in this trial.Participants 26 women, 14 who received CCT and 12 who did not, mostly unemployed (20/26), and - 7 HPs.Interventions We conducted a multicentre cross-sectional qualitative study among women and HPs who participated in the NAITRE Study to assess their views on CCT. The women were interviewed after childbirth.Results Women did not perceive CCT negatively. They did not mention feeling stigmatised. They described CCT as a significant source of aid for women with limited financial resources. HP described the CCT in less positive terms, for example, expressing concern about discussing cash transfer at their first medical consultation with women. Though they emphasised ethical concerns about the basis of the trial, they recognised the importance of evaluating CCT.Conclusions In France, a high-income country where prenatal follow-up is free, HPs were concerned that the CCT programme would change their relationship with patients and wondered if it was the best use of funding. However, women who received a cash incentive said they did not feel stigmatised and indicated that these payments helped them prepare for their baby’s birth.Trial registration number NCT02402855

Autosomal recessive mutations in genes required for cytotoxicity are causative of a life-threatening, early-onset hyperinflammatory syndrome termed familial hemophagocytic lymphohistiocytosis (FHL). Mutations in UNC13D cause FHL type 3. UNC13D encodes Munc13-4, a member of the Unc13 protein family which control SNARE complex formation and vesicle fusion. We have previously identified FHL3-associated mutations in the first intron of UNC13D which control transcription from an alternative transcriptional start site. Using isoform specific antibodies, we demonstrate that this alternative Munc13-4 isoform with a unique N-terminus is preferentially expressed in human lymphocytes and platelets, as compared to the conventional isoform that was mostly expressed in monocytes and neutrophils. The distinct N-terminal of the two isoforms did not impact on Munc13-4 localization or trafficking to the immunological synapse of cytotoxic T cells. Moreover, ectopic expression of both isoforms efficiently restored exocytosis by FHL3 patient-derived Munc13-4 deficient T cells. Thus, we demonstrate that the conventional and alternative Munc13-4 isoforms have different expression pattern in hematopoietic cell subsets, but display similar localization and contribution to T cell exocytosis. The use of an alternative transcriptional starting site (TSS) in lymphocytes and platelets could be selected for increasing the overall levels of Munc13-4 expression for efficient secretory granule release.

Autologous transplantation of hematopoietic stem cells transduced with a lentiviral vector (LV) expressing an anti-sickling HBB variant is a potential treatment for sickle cell disease (SCD). With a clinical trial as our ultimate goal, we generated LV constructs containing an anti-sickling HBB transgene (HBBAS3), a minimal HBB promoter, and different combinations of DNase I hypersensitive sites (HSs) from the locus control region (LCR). Hematopoietic stem progenitor cells (HSPCs) from SCD patients were transduced with LVs containing either HS2 and HS3 (β-AS3) or HS2, HS3, and HS4 (β-AS3 HS4). The inclusion of the HS4 element drastically reduced vector titer and infectivity in HSPCs, with negligible improvement of transgene expression. Conversely, the LV containing only HS2 and HS3 was able to efficiently transduce SCD bone marrow and Plerixafor-mobilized HSPCs, with anti-sickling HBB representing up to ∼60% of the total HBB-like chains. The expression of the anti-sickling HBB and the reduced incorporation of the βS-chain in hemoglobin tetramers allowed up to 50% reduction in the frequency of RBC sickling under hypoxic conditions. Together, these results demonstrate the ability of a high-titer LV to express elevated levels of a potent anti-sickling HBB transgene ameliorating the SCD cell phenotype. Keywords: sickle cell disease, lentiviral vectors, gene therapy

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic. Keywords: murine CD4+ T cells, murine CD8+ T cells, murine Sca1+ cells, lentiviral transduction, Lentiboost, gene therapy

We report outcomes after hematopoietic stem cell transplant for three patients with X-MAID, including 1 patient from the originally described cohort and two brothers with positive TREC newborn screening for SCID who were found to have a T-B-NK+ SCID phenotype attributable to X-linked moesin associated immunodeficiency (X-MAID). A c.511C>T variant in moesin was identified via exome sequencing in the older of these siblings in the setting of low lymphocyte counts and poor proliferative responses consistent with SCID. He received reduced intensity conditioning due to CMV, and was transplanted with a T-depleted haploidentical (maternal) donor. His post-transplant course was complicated by hemolytic anemia, neutropenia, and sepsis. He had poor engraftment, requiring a 2nd transplant. His younger brother presented with the same clinical phenotype and was treated with umbilical cord blood transplant following myeloablative conditioning, has engrafted and is doing well. The third case also presented with severe lymphopenia in infancy, received a matched related bone marrow transplant following myeloablative conditioning, has engrafted and is doing well. These cases represent a novel manifestation of non-radiosensitive X-linked form of T-B-NK+ SCID that is able to be detected by TREC based newborn screening and effectively treated with HCT.

In this work we address the issue of sustainable cities by focusing on one of their very central components: daily mobility. Indeed, if cities can be interpreted as spatial organizations allowing social interactions, the number of daily movements needed to reach this goal is continuously increasing. Therefore, improving urban accessibility merely results in increasing traffic and its negative externalities (congestion, accidents, pollution, noise, etc.), while eventually reducing the quality of life of people in the city. This is why several urban-transport policies are implemented in order to reduce individual mobility impacts while maintaining equitable access to the city. This challenge is however non-trivial and therefore we propose to investigate this issue from the complex systems point of view. The real spatial-temporal urban accessibility of citizens cannot be approximated just by focusing on space and implies taking into account the space-time activity patterns of individuals, in a more dynamic way. Thus, given the importance of local interactions in such a perspective, an agent based approach seems to be a relevant solution. This kind of individual based and “interactionist” approach allows us to explore the possible impact of individual behaviors on the overall dynamics of the city but also the possible impact of global measures on individual behaviors. In this paper, we give an overview of the Miro Project and then focus on the GaMiroD model design from real data analysis to model exploration tuned by transportation-oriented scenarios. Among them, we start with the the impact of a LEZ (Low Emission Zone) in the city center.

Yellow-headed Blackbirds (Xanthocephalus xanthocephalus) in Manitoba breed in dense colonies in cattail marshes. Their reproductive success is affected mainly by predation. The most important predator on blackbird nests is the Marsh Wren (Cistothorus palustris), which breaks blackbird eggs and kills small nestlings. We examined whether colonial nesting in Yellow-headed Blackbirds may represent an adaptation to reduce Marsh Wren predation. Marsh Wren predation may be reduced by (1) mutual nest defense by adult blackbirds, (2) predator satiation or dilution, or (3) selfish-herd effects. We tested these hypotheses using experimental nests and found that their safety increased with decreasing distance to the nearest blackbird nest and with increasing density of simultaneously active blackbird nests located nearby. Safety also was higher for nests placed inside a blackbird colony rather than outside. These findings support the nest-defense hypothesis. We also found that Marsh Wrens are capable of destroying a whole blackbird colony in a few days, and that colony size is not correlated with nest safety. These results suggest that the satiation or dilution benefits are negligible. Finally, we found that central nests are safer than peripheral nests in a blackbird colony, but not in an artificial colony, providing weak support for the selfish-herd hypothesis. We conclude that nest predation is reduced mainly by mutual nest defense of adult birds and may represent an important selective force favoring colonial nesting in this species. Received 14 February 2000, accepted 7 December 2001.

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34+ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID.

Retrosynthetic pathway design using promiscuous enzymes can provide a solution to the biosynthetic production of natural products. Here, the authors design a pathway for the production of cis-α-irone with a promiscuous methyltransferase using structure-guided enzyme engineering strategies.

Summary: Mutations of the transcription factor FoxP3 in patients with “IPEX” (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) disrupt regulatory T cells (Treg), causing an array of multiorgan autoimmunity. To understand the functional impact of mutations across FoxP3 domains, without genetic and environmental confounders, six human FOXP3 missense mutations are engineered into mice. Two classes of mutations emerge from combined immunologic and genomic analyses. A mutation in the DNA-binding domain shows the same lymphoproliferation and multiorgan infiltration as complete FoxP3 knockouts but delayed by months. Tregs expressing this mutant FoxP3 are destabilized by normal Tregs in heterozygous females compared with hemizygous males. Mutations in other domains affect chromatin opening differently, involving different cofactors and provoking more specific autoimmune pathology (dermatitis, colitis, diabetes), unmasked by immunological challenges or incrossing NOD autoimmune-susceptibility alleles. This work establishes that IPEX disease heterogeneity results from the actual mutations, combined with genetic and environmental perturbations, explaining then the intra-familial variation in IPEX.

Chimeric antigen receptor engineering in T cells has been shown to be of great potential therapeutic benefit in a range of immune pathologies, although the functionality of such cell therapies can be limited due to tonic signalling and the induction of dysfunction. Here the authors show transient inhibition of mTOR can rescue their 41-BB-CAR-Tregs from tonic signalling-induced dysfunction.

Abstract Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active $$\mathrm{\alpha }$$ α -transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of preference for numerous malignant and non-malignant hemopathies. The outcome of this approach is significantly hampered by not only graft-versus-host disease (GvHD), but also infections and relapses that may occur because of persistent T-cell immunodeficiency following transplantation. Reconstitution of a functional T-cell repertoire can take more than 1 year. Thus, the major challenge in the management of allogeneic HSCT relies on the possibility of shortening the window of immune deficiency through the acceleration of T-cell recovery, with diverse, self-tolerant, and naïve T cells resulting from de novo thymopoiesis from the donor cells. In this context, adoptive transfer of cell populations that can give rise to mature T cells faster than HSCs while maintaining a safety profile compatible with clinical use is of major interest. In this review, we summarize current advances in the characterization of thymus seeding progenitors, and their ex vivo generated counterparts, T-cell progenitors. Transplantation of the latter has been identified as a worthwhile approach to shorten the period of immune deficiency in patients following allogeneic HSCT, and to fulfill the clinical objective of reducing morbimortality due to infections and relapses. We further discuss current opportunities for T-cell progenitor-based therapy manufacturing, including iPSC cell sources and off-the-shelf strategies. These opportunities will be analyzed in the light of results from ongoing clinical studies involving T-cell progenitors.

Abstract Background Ultrasonography is an emerging non-invasive bedside tool for muscle quantity/quality assessment; Bioelectrical Impedance Analysis (BIA) is an alternative non-invasive bedside measure of body composition, recommended for evaluation of sarcopenia in clinical practice. We set out to assess impact of position and exercise upon measures towards protocol standardisation. Methods Healthy volunteers aged 18–35 were recruited. Bilateral Anterior Thigh Thickness (BATT; rectus femoris and vastus intermedius), BATT: Subcutaneous Ratio (BATT:SCR), and rectus femoris echogenicity were measured using ultrasound and BIA was performed; 1) lying with upper body at 45° (Reclined), 2) lying fully supine at 180o (Supine), 3) sat in a chair with upper body at 90o (Sitting), and 4) after exercise Reclined. Variability of Skeletal Muscle Mass (SMM) by two different equations from BIA (SMM-Janssen, SMM-Sergi), phase angle, fat percentage, and total body (TBW), extracellular (ECW), and intracellular water (ICW) were assessed. Results Forty-four participants (52% female; mean 25.7 years-old (SD 5.0)) were recruited. BATT increased from Reclined to Sitting (+ 1.45 cm, 1.27–1.63), and after exercise (+ 0.51, 0.29–0.73). Echogenicity reduced from Reclined to Sitting (− 2.1, − 3.9 – -0.26). SMM-Sergi declined from Reclined to Supine (− 0.65 kg, − 1.08 – − 0.23) and after exercise (− 0.70 kg, − 1.27 – -0.14). ECW increased from Reclined to Sitting (+ 1.19 L, 0.04–2.35). There were no other statistically significant changes. Conclusion Standardisation of protocols is especially important for assessment of muscle quantity by ultrasonography; BIA measurements may also vary dependent on the equations used. Where possible, participants should be rested prior to muscle ultrasonography and BIA, and flexion of the knees should be avoided.

Abstract The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

Ubiquinone (UQ) is a polyisoprenoid lipid found in the membranes of bacteria and eukaryotes. UQ has important roles, notably in respiratory metabolisms which sustain cellular bioenergetics. Most steps of UQ biosynthesis take place in the cytosol of E. coli within a multiprotein complex called the Ubi metabolon, that contains five enzymes and two accessory proteins, UbiJ and UbiK. The SCP2 domain of UbiJ was proposed to bind the hydrophobic polyisoprenoid tail of UQ biosynthetic intermediates in the Ubi metabolon. How the newly synthesised UQ might be released in the membrane is currently unknown. In this paper, we focused on better understanding the role of the UbiJ-UbiK2 heterotrimer forming part of the metabolon. Given the difficulties to gain functional insights using biophysical techniques, we applied a multiscale molecular modelling approach to study the UbiJ-UbiK2 heterotrimer. Our data show that UbiJ-UbiK2 interacts closely with the membrane and suggests possible pathways to enable the release of UQ into the membrane. This study highlights the UbiJ-UbiK2 complex as the likely interface between the membrane and the enzymes of the Ubi metabolon and supports that the heterotrimer is key to the biosynthesis of UQ8 and its release into the membrane of E. coli.

Activated PI3-kinase-δ syndrome 2 (APDS2) is caused by autosomal dominant mutations in the PIK3R1 gene encoding the p85α, p55α, and p50α regulatory subunits. Most diagnosed APDS2 patients carry mutations affecting either the splice donor or splice acceptor sites of exon 11 of the PIK3R1 gene responsible for an alternative splice product and a shortened protein. The clinical presentation of APDS2 patients is highly variable, ranging from mild to profound combined immunodeficiency features as massive lymphoproliferation, increased susceptibility to bacterial and viral infections, bronchiectasis, autoimmune manifestations, and occurrence of cancer. Non-immunological features such as growth retardation and neurodevelopmental delay have been reported for APDS2 patients. Here, we describe a patient suffering from an APDS2 associated with a Smith–Magenis syndrome (SMS), a complex genetic disorder affecting, among others, neurological manifestations and review the literature describing neurodevelopmental impacts in APDS2 and other PIDs/monogenetic disorders associated with dysregulated PI3K signaling.

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for a large number of malignant and nonmalignant (inherited) diseases of the hematopoietic system. Nevertheless, non‐HLA identical transplantations are complicated by a severe T‐cell immunodeficiency associated with a high rate of infection, relapse and graft‐versus‐host disease. Initial recovery of T‐cell immunity following HSCT relies on peripheral expansion of memory T cells mostly driven by cytokines. The reconstitution of a diverse, self‐tolerant, and naive T‐cell repertoire, however, may take up to 2 years and crucially relies on the interaction of T‐cell progenitors with the host thymic epithelium, which may be altered by GvHD, age or transplant‐related toxicities. In this review, we summarize current concepts to stimulate reconstitution of a peripheral and polyclonal T‐cell compartment following allogeneic transplantation such as graft manipulation (i.e., T‐cell depletion), transfusion of ex vivo manipulated donor T cells or the exogenous administration of cytokines and growth factors to stimulate host‐thymopoiesis with emphasis on approaches which have led to clinical trials. Particular attention will be given to the development of cellular therapies such as the ex vivo generation of T‐cell precursors to fasten generation of a polyclonal and functional host‐derived T‐cell repertoire. Having been tested so far only in preclinical mouse models, clinical studies are now on the way to validate the efficacy of such T‐cell progenitors in enhancing immune reconstitution following HSCT in various clinical settings. Stem Cells Translational Medicine 2019;00:1–8

Plant α-galactosides belonging to the raffinose family oligosaccharides (RFOs) and considered as prebiotics, are commonly degraded by α-galactosidases produced by the human gut microbiome. In this environment, the Ruminococcus gnavus E1 symbiont–well-known for various benefit–is able to produce an original RgAgaSK bifunctional enzyme. This enzyme contains an hydrolytic α-galactosidase domain linked to an ATP dependent extra-domain, specifically involved in the α-galactoside hydrolysis and the phosphorylation of the glucose, respectively. However, the multi-modular relationships between both catalytic domains remained hitherto unexplored and has been, consequently, herein investigated. Biochemical characterization of heterologously expressed enzymes either in full-form or in separated domains revealed similar kinetic parameters. These results were supported by molecular modeling studies performed on the whole enzyme in complex with different RFOs. Further enzymatic analysis associated with kinetic degradation of various substrates followed by high pressure anionic exchange chromatography revealed that catalytic efficiency decreased as the number of D-galactosyl moieties branched onto the oligosaccharide increased, suggesting a preference of RgAgaSK for RFO’s short chains. A wide prevalence and abundance study on a human metagenomic library showed a high prevalence of the RgAgaSK encoding gene whatever the health status of the individuals. Finally, phylogeny and synteny studies suggested a limited spread by horizontal transfer of the clusters’ containing RgAgaSK to only few species of Firmicutes, highlighting the importance of these undispersed tandem activities in the human gut microbiome.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism

Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. They are defined by the absence of autologous T cells and the presence of an intrinsic or extrinsic defect in the B-cell compartment. In three newborns presenting with frequent infections and profound leukopenia, we identified a private, heterozygous mutation in the RAC2 gene (p.G12R). This mutation was de novo in the index case, who had been cured by hematopoietic stem cell transplantation but had transmitted the mutation to her sick daughter. Biochemical assays showed that the mutation was associated with a gain of function. The results of in vitro differentiation assays showed that RAC2 is essential for the survival and differentiation of hematopoietic stem/progenitor cells. Therefore, screening for RAC2 gain-of-function mutations should be considered in patients with a SCID phenotype and who lack a molecular diagnosis.

Abstract: Patients with mutations in the UNC13D gene (coding for Munc13-4 protein) suffer from familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a life-threatening immune and hyperinflammatory disorder. The only curative treatment is allogeneic hematopoietic stem cell (HSC) transplantation, although the posttreatment survival rate is not satisfactory. Here, we demonstrate the curative potential of UNC13D gene correction of HSCs in a murine model of FHL3. We generated a self-inactivating lentiviral vector, used it to complement HSCs from Unc13d-deficient (Jinx) mice, and transplanted the cells back into the irradiated Jinx recipients. This procedure led to complete reconstitution of the immune system (ie, to wild-type levels). The recipients were then challenged with lymphocytic choriomeningitis virus to induce hemophagocytic lymphohistiocytosis (HLH)–like manifestations. All the clinical and biological signs of HLH were significantly reduced in mice having undergone HSC UNC13D gene correction than in nontreated animals. This beneficial effect was evidenced by the correction of blood cytopenia, body weight gain, normalization of the body temperature, decreased serum interferon-γ level, recovery of liver damage, and decreased viral load. These improvements can be explained by the restoration of the CD8+ T lymphocytes' cytotoxic function (as demonstrated here in an in vitro degranulation assay). Overall, our results demonstrate the efficacy of HSC gene therapy in an FHL-like setting of immune dysregulation.

2,4-Dihydroxybutyric acid has potential to be a precursor to a range of industrially important products, however a natural metabolic pathway for its synthesis does not exist. Here the authors rationally design a synthetic pathway inE. coliby engineering enzymes from malate metabolism.

By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of Neisseria polysaccharea amylosucrase (NpAS), a member of GH13 family. Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted 4C1 conformation, as inferred from structural studies, or a distorted 1S3-like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs). We show that, even though the first step of the reaction (glycosylation) results in a 4C1 conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step (deglycosylation), in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon. The two conformations (4C1 ad E3) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the E3 state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.

Sickle cell disease is characterized by chronic anemia and vaso-occlusive crises, which eventually lead to multi-organ damage and premature death. Hematopoietic stem cell transplantation is the only curative treatment but it is limited by toxicity and poor availability of HLA-compatible donors. A gene therapy approach based on the autologous transplantation of lentiviral-corrected hematopoietic stem and progenitor cells was shown to be efficacious in one patient. However, alterations of the bone marrow environment and properties of the red blood cells hamper the harvesting and immunoselection of patients’ stem cells from bone marrow. The use of Filgrastim to mobilize large numbers of hematopoietic stem and progenitor cells into the circulation has been associated with severe adverse events in sickle cell patients. Thus, broader application of the gene therapy approach requires the development of alternative mobilization methods. We set up a phase I/II clinical trial whose primary objective was to assess the safety of a single injection of Plerixafor in sickle cell patients undergoing red blood cell exchange to decrease the hemoglobin S level to below 30%. The secondary objective was to measure the efficiency of mobilization and isolation of hematopoietic stem and progenitor cells. No adverse events were observed. Large numbers of CD34+ cells were mobilized extremely quickly. Importantly, the mobilized cells contained high numbers of hematopoietic stem cells, expressed high levels of stemness genes, and engrafted very efficiently in immunodeficient mice. Thus, Plerixafor can be safely used to mobilize hematopoietic stem cells in sickle cell patients; this finding opens up new avenues for treatment approaches based on gene addition and genome editing. Clinicaltrials.gov identifier: NCT02212535.

An end-point ADP/NAD+ acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD+ co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD+ into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A:E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (kcat/Km)malate/(kcat/Km)aspartate ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site.

Among α-transglucosylases from Glycoside-Hydrolase family 70, the ΔN123-GB-CD2 enzyme derived from the bifunctional DSR-E from L. citreum NRRL B-1299 is particularly interesting as it was the first described engineered Branching Sucrase, not able to elongate glucan polymers from sucrose substrate. The previously reported overall structural organization of this multi-domain enzyme is an intricate U-shape fold conserved among GH70 enzymes which showed a certain conformational variability of the so-called domain V, assumed to play a role in the control of product structures, in available X-ray structures. Understanding the role of functional dynamics on enzyme reaction and substrate recognition is of utmost interest although it remains a challenge for biophysical methods. By combining long molecular dynamics simulation (1μs) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of ΔN123-GB-CD2 alone and in interaction with sucrose substrate. Overall, our results provide the detailed picture at atomic level of the hierarchy of motions occurring along different timescales and how they are correlated, in agreement with experimental structural data. In particular, detailed analysis of the different structural domains revealed cooperative dynamic behaviors such as twisting, bending and wobbling through anti- and correlated motions, and also two structural hinge regions, of which one was unreported. Several highly flexible loops surrounding the catalytic pocket were also highlighted, suggesting a potential role in the acceptor promiscuity of ΔN123-GBD-CD2. Normal modes and essential dynamics underlined an interesting two-fold dynamic of the catalytic domain A, pivoting about an axis splitting the catalytic gorge in two parts. The comparison of the conformational free energy landscapes using principal component analysis of the enzyme in absence or in presence of sucrose, also revealed a more harmonic basin when sucrose is bound with a shift population of the bending mode, consistent with the substrate binding event.

[This corrects the article DOI: 10.1371/journal.pone.0161209.].

Travel surveys can collect information about practices and behaviours of individual mobility. These surveys and the resulting analyses are necessary for the development and evaluation of public transport policies. Usually, these surveys use traditional methods for data collection with standard survey methods. Today, new survey protocols and data collection methods emerge thanks to the potential of new technologies. Geo-localization technologies, such as GPS, can be an interesting support for new types of so-called "assisted" surveys. However, these technologies may provide new types of challenges in the context of surveys with representative samples of the population. Furthermore, the use of location technologies requires modifying significantly the survey protocols. To identify the potential benefit of geo-localization technologies for travel surveys, we conducted a comparative study between a classic travel survey (Household Travel Survey) and a GPS assisted travel survey. This comparative evaluation also allowed us to identify the main technological and methodological limitations of GPS assisted travel surveys.

An improved production and purification method for Alzheimer's disease related methionine-modified amyloid-β 1-40 and 1-42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies.

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin-CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24- progenitor subpopulations. CD34+lin-CD10+CD24- lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin-CD10+CD24- cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.

We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.10(4) clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide (460)DYVHT(464) in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity.

This article examines the circulation of people between Lisbon and four other European cities (Berlin, Brussels, London, Paris) in terms of the difficulties that it can cause. The proposed approach articulates three dimensions, namely the construction of practices of circulation between cities at a biographical scale, the way families deal with the circulation of one or more of its members, and the daily experience of places frequented in each of the cities. Taking into account these three dimensions offers a means to characterize the people who circulate in terms of mobility situations through which they conceive of the wearisome dimension of circulation. Mobility situations that are well appreciated seem to reveal a relatively balanced way of life associated with circulation. However, the article identifies a number of factors that may contribute to weaken or qualify these situations: weariness, lack of space of identification and project, management within family of the absence of those who circulate, changes in relation to places of healing, and limitations in practicing the places that structure the space of circulation. Overall, there is no clear segmentation between these situations of mobility in terms of difficulty. Situations of mobility that are very different can be experienced in many varied ways. The tiring dimension of circulation ultimately depends on a combination of factors which, taken separately, have no mechanical effect.

The aim of this contribution is to present a summary of the changes in property ownership that have occurred in a tourist area in the high mountains. Priority is given in the text to the analysis of data by taking advantage of a database recently made available on the knowledge market by the Notarial Office of France (Notariat de France). The geographer will find in it matters to reflect upon. First of all, a communal pattern that is to be explained by social qualitative variables (geographical origin of the individuals) emerges from the mapping of the total transactions. The socio-spatial fragmentation is the result of exchange systems for properties that reveal two competing residential processes. The article aims to demonstrate how a selective sorting process operates between communes, one group specialising in welcoming local populations, the other group rejecting the same populations at their outskirts. The analysis of property transactions on this scale indicates the obvious adjustment that exists between potential local demand and accessible supply. These are among the many aspects subject to the wisdom of politicians since they are preoccupied with the question of access of the local population to property (land and buildings) and the cohesion of their region.

The aim of this contribution is to present a summary of the changes in property ownership that have occurred in a tourist area in the high mountains. Priority is given in the text to the analysis of data by taking advantage of a database recently made available on the knowledge market by the Notarial Office of France (Notariat de France). The geographer will find in it matters to reflect upon. First of all, a communal pattern that is to be explained by social qualitative variables (geographical origin of the individuals) emerges from the mapping of the total transactions. The socio-spatial fragmentation is the result of exchange systems for properties that reveal two competing residential processes. The article aims to demonstrate how a selective sorting process operates between communes, one group specialising in welcoming local populations, the other group rejecting the same populations at their outskirts. The analysis of property transactions on this scale indicates the obvious adjustment that exists between potential local demand and accessible supply. These are among the many aspects subject to the wisdom of politicians since they are preoccupied with the question of access of the local population to property (land and buildings) and the cohesion of their region.